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SMEs in Health Research Synopses of projects funded through the SME call for

“Life sciences, genomics and biotechnology for health”

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EUROPEAN COMMISSION

SMEs in Health ResearchSynopses of projects funded through

the SME call for “Life sciences,genomics and biotechnology for health”

(FP6-2005-LIFESCIHEALTH-7)

2008 Directorate-General for Research EUR 23457 ENHealth

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Acknowledgements

This catalogue has been produced thanks to the essential input from all project coordinators. Special thanks goto Séverine Romain for her highly professional and dynamic assistance, pivotal for the catalogue completion. I am very grateful to Rachida Ghalouchi, Christel Jaubert, Charles Kelly, Kristina Kyriakopoulou, and to all the officersin Health Directorate responsible for the projects included in this synopses, for their efficient co-operation. Finally, my warmest thanks to Stéphane Hogan, Head of Unit F1, Horizontal aspects and coordination in the Health Directorate, for the commitment and lead provided.

Ludovica SerafiniEditor

Contact details for FP7 activities

Information on FP7 Health Theme (including calls, available supports and information on SMEs and innovation):http://cordis.europa.eu/fp7/cooperation/health_en.html

The SME Contact Officer in the Health Directorate is Ludovica Serafini, e-mail: [email protected]

SME CALL

Table of content

• Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

• Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 9

• AGLAEA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Development of novel animal modelsof glutamatergic central nervoussystem disorders using in vivosiRNA and transgenic approaches

• ANGIOSTOP . . . . . . . . . . . . . . . . . . . . . . . . 12Novel Anti-angiogenic treatment for Cancer, Arthritis and OcularNeovascularization based onInhibition of Placental Growth Factor (PlGF)

• ARTEMIS. . . . . . . . . . . . . . . . . . . . . . . . . . . . 14In Vitro neural tissue system for replacement of transgenic animals with memory/learningdeficiencies

• AUTOSCREEN . . . . . . . . . . . . . . . . . . . . . . 16AUTOSCREEN for Cell Based High-throughput and High-contentGene Function Analysis and Drug Discovery Screens

• BacAbs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Assessment of StructuralRequirements in Complement-Mediated Bactericidal Events: Towards a Global Approach to the Selection of New VaccineCandidates

• BioBridge. . . . . . . . . . . . . . . . . . . . . . . . . . . 20Integrative Genomics and ChronicDisease Phenotypes: modelling and simulation tools for clinicians

• CancerGrid . . . . . . . . . . . . . . . . . . . . . . . . . 22Grid-aided computer system for rapid anti-cancer drug design

• CAPPELLA . . . . . . . . . . . . . . . . . . . . . . . . . . 26Combating cancer through novelapproaches to protein: proteininteraction inhibitor libraries

• ChILL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Chromatin Immuno-linked ligation: A novel generation ofbiotechnological tools for researchand diagnosis

• CILMALVAC . . . . . . . . . . . . . . . . . . . . . . . . . 30The Tetrahymena system as aninnovative approach to malariaantigene expression

• cNEUPRO . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Clinical Neuroproteomics of Neurodegenerative Diseases

• COBRED . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Colon and breast cancer diagnostics

• COMICS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Comet assay and cell array for fast and efficient genotoxicity testing

• CVDIMMUNE. . . . . . . . . . . . . . . . . . . . . . . 38Immunomodulation and autoimmunity in cardiovasculardisease and atherosclerosis

• DEPPICT . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Designing Therapeutic Protein:ProteinInhibitors for Brain Cancer Treatments

• DeZnIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Design of zinc metalloenzymetargeted drugs using an IntegratedTechnology approach

• Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . 44Development of new and costeffective methods for non-invasivediagnosis of human pathogens

• DIALOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Development of Innovative Assays and Locally acting therapies aiming at critical Kinases in hepatic and renal fibrosis

• DiaNa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Predictive diagnostics for diabetic nephropathy – Novel nanotechnology based test platforms

• Drop-Top. . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Integration of DNA, RNA and proteinmarkers in a tool for the prognosisand diagnosis of human disease

• EACCAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54European approach to combatoutbreaks of clostridium difficileassociated diarrhoea by developmentof new diagnostic tests

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• ENLIGHT . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56New molecular methods and imageanalysis tools for analysis of cancerbiomarkers in situ

• EPIVAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58Development of a multi-step Improved Epidermis Specific VaccineCandidate against HIV/AIDS

• EURO-PHARMACO-GENE . . . . . . . . . 60Design of targeted GenePharmaceutics using self-assemblingfunctional entities

• EXERA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62Development of 3D in vitro models of estrogen-reporter mouse tissues for the pharmaco-toxicologicalanalysis of Estrogen Receptors-Interacting Compounds (ER-ICs)

• FASTEST-TB . . . . . . . . . . . . . . . . . . . . . . . . 66Development and Clinical Evaluation of Fast Tests for Tuberculosis Diagnosis

• FGENTCARD. . . . . . . . . . . . . . . . . . . . . . . . 68Functional GENomic diagnostic Tools for Coronary Artery Disease

• GLYFDIS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70Glycans in Body Fluids- Potential for Disease Diagnostics

• HI-CAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72Development of a high-resolutionAnger camera for diagnosis andstaging of cancer diseases based onstate of the art detector technology

• HighReX . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74High Resolution X-Ray Imaging forImproved Detection and Diagnosis of Breast Cancer

• HIVResInh . . . . . . . . . . . . . . . . . . . . . . . . . . 76Preparation and Identification of New HIV Reverse TranscriptaseInhibitors Targeted Against HIV StrainsResistant to anti-HIV/AIDS drugs

• HIVSTOP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78Development of an Effective RNA Interference-Based Anti-HIV-1 Therapy Using an SV40-Derived Vector

• IBDchip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80Usefulness of a new DNA array(IBDchip) to predict clinical course,development of complications andresponse to therapy in patients withinflammatory bowel disease (IBD)

• IMMUNATH . . . . . . . . . . . . . . . . . . . . . . . . 82Translating innate immune receptor function into diagnostic and therapeutic applications foratherosclerosis

• Immuno-PDT. . . . . . . . . . . . . . . . . . . . . . . 84Immunophotodynamic therapy of cancer: concepts and applications

• INDABIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86Innovative diagnostic approaches for biomarkers in Parkinson disease

• INNOVAC . . . . . . . . . . . . . . . . . . . . . . . . . . . 88Highly innovative strategies forvaccination to poverty relateddiseases

• INTELLIMAZE . . . . . . . . . . . . . . . . . . . . . . 90High-throughput, fully automated and cost-effective behaviouralphenotyping of normal, clinical and genetic mouse models

• InVitroHeart. . . . . . . . . . . . . . . . . . . . . . . . 92Reducing Animal Experimentation inDrug Testing by Human CardiomyocyteIn Vitro Models Derived fromEmbryonic Stem Cells

• LIGHTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94Small ligands to interfere withThymidylate synthase dimer formationas new tools for development ofanticancer agents against ovarian carcinoma

• liintop. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96Optimisation of liver and intestine in vitro models for pharmacokineticsand pharmacodynamics studies

• MagRSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98Fully automated and integratedMicrofluidic Platform for Real-timeMolecular Diagnosis of Methicillin-resistant Staphylococcus Aureus

• MAMMI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100Mammography with molecular imaging

• MANASP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102Development of novel managementstrategies for invasive aspergillosis

• MEGATOOLS . . . . . . . . . . . . . . . . . . . . . . . . 104New tools for Functional Genomicsbased on homologous recombinationinduced by double-strand break andspecific meganucleases

• MEMORIES. . . . . . . . . . . . . . . . . . . . . . . . . . 106Development, characterisation andvalidation of new and original modelsfor Alzheimer’s Disease

• Mimovax . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108Alzheimer’s disease-treatment targetingtruncated Aβ 40/42 by activeimmunisation

• MODEST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110Modular Devices for Ultrahigh-throughput and Small-volumeTransfection

• MUNANOVAC . . . . . . . . . . . . . . . . . . . . . . . . 112Mucosal Nano Vaccine Candidate for HIV

• MYASTAID . . . . . . . . . . . . . . . . . . . . . . . . . . . 114Development of models to improvemanagement of Myasthenia Gravis: Frombasic knowledge to clinical application

• MYOAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116Amplification of human myogenic stem cells in clinical conditions

• NanoSense . . . . . . . . . . . . . . . . . . . . . . . . . . 118Moving sensitive immunoassays from slow and expensive to fast andaffordable nanoparticle-based methods

• NEOBRAIN. . . . . . . . . . . . . . . . . . . . . . . . . . . 120Neonatal estimation of brain damage risk and identification of neuroprotectants

• Net2Drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122From gene regulatory networks to drug prediction

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• NPARI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124Tailoring of Novel Peptide coatingsand therapeutics derived from a newly identified component of the human innate immunity AgainstResistant Infections

• OMVac . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126Novel prevention and treatmentpossibilities for Otitis Media throughthe comprehensive identification ofantigenic proteins

• OptiCryst . . . . . . . . . . . . . . . . . . . . . . . . . . 128Optimisation of protein crystallisationfor european structural genomics

• PHECOMP . . . . . . . . . . . . . . . . . . . . . . . . . 130Phenotypical characterisation ofanimal models for neuropsychiatricdisorders related to compulsivebehaviour

• PHOTOLYSIS . . . . . . . . . . . . . . . . . . . . . . 132Development of flash photolysis for deep uncaging in vivo and high-throughput characterisation of neurotransmitter gated ion channels in drug discovery

• PlasmodiumdUTPase . . . . . . . . . . . . 134Deoxyuridine triphosphatenucleotidohydrolase as a drug target against Malaria

• POC4life . . . . . . . . . . . . . . . . . . . . . . . . . . . 136Multiparametric quantum dot bioassay for point of care diagnosis

• PRIBOMAL . . . . . . . . . . . . . . . . . . . . . . . . 138Preclinical studies towards anaffordable, safe and efficacious two-component paediatric Malaria vaccine

• PRISM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140Phospholipid and glycolipidrecognition, interactions andstructures by magnetic resonance

• PROLIGEN . . . . . . . . . . . . . . . . . . . . . . . . . 142Hypoxic renal proliferation

• QuAGSIC . . . . . . . . . . . . . . . . . . . . . . . . . . 144Quantitative analysis of genes insingle cells

• RATstream™ . . . . . . . . . . . . . . . . . . . . . 146European project on thecharacterisation of transgenic ratmodels for neurodegenerative andpsychiatric diseases: Automated home cage analyses, live imaging and treatment

• RespViruses . . . . . . . . . . . . . . . . . . . . . . 148Immune response to viral respiratory infections and vaccination in theelderly

• SAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150SME-led Antibody Glyco-Engineering

• SERO-TB. . . . . . . . . . . . . . . . . . . . . . . . . . . 152Development of a Specific Serological Kit for the Diagnosis of TB

• SMARTER. . . . . . . . . . . . . . . . . . . . . . . . . . 154Development of small modulators of gene activation and repression by targeting epigenetic regulators

• STEMDIAGNOSTICS. . . . . . . . . . . . . . 156The development of new diagnostictests, new tools and non-invasivemethods for the prevention, early diagnosis and monitoring for haematopoietic stem celltransplantation (HSCT)

• STREPTOMICS. . . . . . . . . . . . . . . . . . . . 158Systems biology strategies andmetabolome engineering for theenhanced production of recombinantproteins in Streptomyces

• SYSCO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160Systematic Functional analysis ofIntracellular Parasitism as a modelof genomes conflict

• SysProt . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162System-wide analysis and modelling of protein modification

• TAMAHUD . . . . . . . . . . . . . . . . . . . . . . . . . 164Identification of early diseasemarkers, novel pharmacologicallytractable targets and small moleculephenotypic modulators inHuntington’s Disease

• TargetHerpes . . . . . . . . . . . . . . . . . . . . . . . 166Molecular intervention strategies targetinglatent and lytic herpesvirus infections

• TargetScreen2 . . . . . . . . . . . . . . . . . . . . . . 168Novel post-genomic cell-based screensfor drug targeting in membrane proteindisorders

• TB-DRUG. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170A SME-STREP for Tuberculosis DrugDevelopment

• TB-trDNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172Evaluation of transrenal-DNA detection to diagnose tuberculosis

• TEMPO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174Temporal Genomics for TailoredChronotherapeutics

• USDEP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176Capture and enrichment of emergingpathogens for multiple and ultra-sensitive diagnostic

• VALAPODYN . . . . . . . . . . . . . . . . . . . . . . . . . 178Validated Predictive Dynamic Model of Complex Intracellular PaCell Deathand Survival

• VASOPLUS . . . . . . . . . . . . . . . . . . . . . . . . . . 180Placental Growth Factor (PlGF): newdiagnostic and therapeutic applicationsin cardiovascular disease

• VITAL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182Development of optimized recombinantidiotypic vaccines for subset-specificimmunotherapy of B cell lymphomas

• ZF-TOOLS . . . . . . . . . . . . . . . . . . . . . . . . . . . 184High-throughput Tools for BiomedicalScreens in Zebrafish

Late submission:• PREGENESYS . . . . . . . . . . . . . . . . . . . . . . . 186

Development of Early Non-InvasiveBiomarkers and Means for the Diagnosisand Progression Monitoring ofPreeclampsia and Tailoring Putative Therapies

• Index by project number . . . . . . . . . . 188

• Index by coordinator. . . . . . . . . . . . . . . 190

5

T he aim of health research under Framework Programmes is to help Europeexploit the unprecedented opportunities for generating new knowledge

and translating it into applications that enhance human health and boost thecompetitiveness of health-related industries and businesses in support of theLisbon Growth and Jobs strategy.

Research intensive SMEs play a key role in the European Union’s economy, interms of competitiveness, innovation, growth and employment. Many of Europe’stop pharmaceutical companies had modest beginnings. Indeed, yesterday’s suc-cessful SMEs are often today’s global giants, employing large workforces andgenerating enormous value.

Hundreds of SMEs are active in health-related R&D and, in recognition of theirinnovative potential, scientific strength and strategic importance, a specialeffort to mobilise research-intensive health and biotech SMEs was made duringthe Sixth Framework Programme (FP6).

In particular, at the end of the Framework Programme, the Thematic Priority enti-tled “Life sciences, genomics and biotechnology for health” published a verysuccessful “SME call” with a budget of € 200 million. This call for proposals wasparticularly aimed at funding projects with high numbers of research intensiveSMEs. The call succeeded in mobilising high-tech companies to participate inFramework Programmes: the 86 projects funded involved 270 SMEs, which wereallocated more than 40 % of the budget.

Through this call, research-intensive SMEs in the health and biotech sectoracceded to a leading role as key players and driving forces in the researchactivities of EU-funded projects.

The momentum achieved through this special call for proposals is expected togenerate an even bigger impact in the Seventh Framework Programme (FP7)projects, with increased participation of SMEs with a key role in research.

This publication presents those 86 projects and aims to illustrate the EU’s com-mitment to health research, bringing together transnational and multidisciplinaryexpertise from both industry and academia.

Manuel HallenActing DirectorHealth Research

SME CALL

Foreword

7

SME CALL

Introduction

interest and potential benefit to one or moreSMEs. The share of the EU contribution going tothe participating SMEs was expected to bebetween 30 and 50 %.

Through this Call, 86 projects, which are the thefocus of this publicationhave been funded fora total contribution of € 200 million, from whicharound 40 % goes to around 270 SMEs. The sci-entific involvement included a wide range ofactivities, such as identification of biomarkers,elucidation of candidate genes and their role,providing microRNA discovery and validationplatforms or bioinformatics and transcriptomicanalysis, developing photonic and electro-optics,designing microchip or magnetic phase platforms,etc. Specialised SMEs with expertise in intellectualproperty management, technology transfer, projectmanagement or training activities also participatedactively in some of these projects.

SMEs are the main economic drivers of health-care, biotechnology and medical technologies.Through EU-funded collaborative research, SMEscan maximise the value of their expertise, networkinternationally, learn from other key players andreduce risk by collaborating with other SMEs andlarger industrial partners and academics from allover Europe and beyond.

At the same time, the often pivotal technologicalinput from SMEs, as well as their business orienta-tion and dynamism, will benefit other project partic-i pants. In this way, the output and the exploitationof the results can be optimised, and the spirit ofentrepreneurship in research supported.

The momentum achieved by this Call to mobiliseresearch intensive SMEs is expected to generatean even bigger impact in the Seventh FrameworkProgramme (FP7) projects on the participation ofSMEs with a key role in research activity.

Through its Framework Programmes, theEuropean Union has provided support to med-

ical research for more than a quarter of a century,striving to prevent and cure diseases, and helpingto improve patients’ chances of survival and theirquality of life. At the same time, the EU has aidedthe efforts of the European healthcare industryin maintaining a leading role in the world.

Between 2002 and 2006, the Sixth FrameworkProgramme (FP6) dedicated € 2.4 billion to theThematic Priority “Life sciences, genomics andbiotechnology for health” to support EuropeanResearch efforts, developing frontier technologies,encouraging excellence in fundamental and clini-cal research, bringing academics, clinicians andindustry together at every stage of the process – from the laboratory bench to the hospital andto the market.

The “Life sciences, genomics and biotechnologyfor health” Thematic Priority emphasised theimportance of innovation and the integration ofindustry, small and medium sized enterprises (1),by ensuring that new knowledge is disseminatedand translated into new therapies, diagnosticsand clinical practice. Overall, of the € 2.4 billion ofthe budget available for ‘health’, some € 335 millionwas awarded to SMEs participating in EU-fundedprojects, in particular through a special “Call forproposals for STREPs dedicated to SMEs”, in thelast year of FP6. These “SME-STREPs” have beenspecifically designed to support SME effortstowards research and innovation. Such projectswere centred on the reinforcement of SME’sscientific and technological knowledge and onthe validation of innovative solutions. Research-intensive SMEs played leading roles – althoughnot necessarily coordinating the projects – andparticipated alongside universities, research cen-tres, other industries and industrial associations.The expected project results had to be of clear

(1) The official EU definition of an SME is an

autonomous company with less than 250 employees,

an annual turnover of not more than € 50 million

and/or an annual balance sheet total of not more

than € 43 million. For more details see:

http://ec.europa.eu/enterprise/enterprise_policy/

sme_definition/index_en.htm

9

Expected results

Access to improved animal models of specific psy-chiatric disorders will enhance the development ofimproved treatments, in particular for disorderssuch as schizophrenia where existing modelsare often based on limited pharmacological andneurobiological information about the disorder.Targeting the glutamatergic system should lead tomodels that more closely resemble the human sit-uation with regard to the symptoms that are atpresent not treated by existing drugs (e.g. cogni-tion in schizophrenia) or when existing drugs haveside effect and/or dependence issues anxiety). Thecreation of these models will also increase fun-damental research into the basic mechanisms ofglutamate function. In addition, the techniquesused will expand the use of siRNA in vivo for thecreation of new models in CNS disease, provid-ing new solutions for the exploration of suchpathologies.

Potential applications

A certain number of CNS disorders, includingschizophrenia, anxiety and cognitive disorderscan be linked to glutamatergic dysfunction. Theexisting treatments for patients suffering fromthese diseases have serious side effects and arenot always efficient. For example, existing drugsare unable to treat the social, emotional andcognitive symptoms of schizophrenia and thereare few marketed treatments for the cognitivedeficits of Alzheimer’s disease. By developingand characterising animal models with hypo-and hyper-glutamatergic states, AGLAEA willprovide valuable data on the impact of gluta-matergic signalling on such disorders, thereforeopening the way for better treatment and care ofpatients.

Background

Modulation of glutamate signalling levels isassociated with many psychiatric disorders.Creation of reliable and practical models ofaltered brain glutamate function has been diffi-cult. Glutamate is the most abundant excitatorytransmitter in the brain and interacts with numer-ous receptors and transporters responsible forboth fast synaptic transmission and modulatoryfunctions. However, development of knockout(KO) rodents is expensive and time consuming,and gene KO is often associated with develop-mental alterations. Inducible and conditionalknockout technologies have helped to alleviatethis problem. However, the gene is nearly com-pletely suppressed when the induction occurs.Because glutamate is a ubiquitous excitatorytransmitter, full knockout of a specific glutamatereceptor or transporter often yields a phenotypeincompatible with behavioural testing.

Aim

AGLAEA is aimed at developing models of selec-tive, partial knockdown of specific componentsof the brain glutamatergic system that will resultin hypo- and hyper-glutamatergic states yield-ing animals amenable to behavioural investiga-tion. The results will then drive the creation ofspecific transgenic lines with targeted knock-down of components of the brain glutamate sys-tem yielding mice that can be studied over longperiods of time.

Development of novel animal models of glutamatergic central nervous system disordersusing in vivo siRNA and transgenic approaches

ACRONYM

AGLAEA

Contract number: LSHM-CT-2006-037554 | EC contribution: € 1 198 900 | Duration: 36 months

Starting date: 1 January 2007

Glutamate is the most abundant excita-tory transmitter in the brain. However,very few models exist to explore thepotential of drugs that modulate gluta-mate transmission. Because alteredglutamate transmission is involved innumerous psychiatric diseases, there isa strong need for such models to charac-terise the effects of hypo- and hyper-glu-tamatergic states on the onset anddevelopment of such diseases. AGLAEAwill develop and characterise models ofselective, partial knockdown of specificcomponents of the brain glutamatergicsystem in mice. This will provide a betterunderstanding of the implication of glu-tamate signalling in diseases such asschizophrenia, anxiety and cognitive dis-orders. AGLAEA will lead to breakthroughresearch on the neurobiological and neu-rochemical bases of psychiatric disordersand will enable the further testing of newdrugs for treatment. In order to selectivelyturn off specific components of the gluta-matergic pathways, an siRNA approachwill be used. The effect of modulation ofglutamate signalling will be characterisedusing functional MRI (fMRI) and micro-dialysis/microsensor analysis. Specificbehavioural tests will be carried outon live animals for the assessment ofglutamate-related psychiatric disorders.Furthermore, the data collected fromsiRNA experiments will be applied to thegeneration of transgenic mice, in whichmodulation of glutamate signalling will beinduced at different stages of develop-ment. Therefore, AGLAEA will provideboth the pharmaceutical and the aca-demic world with potent models. Thesemodels will be used to test novel com-pounds and assess their therapeuticvalue and will benefit researchers investi-gating the neurobiological and neuro-chemical bases of those diseases.

The consortium set up to reach the objec-tives of AGLAEA gathers 3 high tech SMEs,2 academic groups and one large group forthe management of the project (ALMA),altogether representing 4 Europeancountries (FR, HU, UK, NL).

SUMMARY

10

Key words: animal models, glutamate, siRNA, transgenic mice

Scientific coordinator

Bernard LudwigADDEX Pharmaceuticals France SAS Behaviour DepartmentImmeuble Alliance – bat. A&CFR-74160 [email protected]

Partners

Andras DinnyesBioTalentum Ltd.Gödöllo, Hungarywww.biotalentum.hu

Charles MarsdenInstitute of NeuroscienceUniversity of NottinghamNottingham, United Kingdom

Ben WesterinkDepartment of Biomonitoring & SensoringUniversity of Groningen, Brains-On-Line, The Netherlands

Benjamin QuestierALMA Consulting Group Lyon, France

Three SMEs, representing 60 % of the budget, are key contributors to the project. AddexPharmaceuticals, France, will coordinate the project and will study the effects of interfer-ence RNA and genetic manipulation of brain glutamate systems on behaviour in mice. Addexhas extensive expertise in the discovery and development of novel medications for centralnervous system diseases and will also examine the effects of experimental and referencedrugs to further validate the mouse models produced in the project. BioTalentum Ltd., basedin Hungary, has in-depth expertise in animal embryology, micromanipulation, embryonicstem cell transgenesis and cryopreservation, which will be applied to the production oftransgenic mice for the project. BioTalentum will use innovative techniques developed in-house to generate knockdown mice in mouse strains most useful for neurobiological andbehavioural analyses. BioTalentum will also employ the latest techniques in rapid deriva-tion of transgenic mouse lines to provide stable animal models as quickly as possible.Brains On-Line, based in The Netherlands, has broad expertise in the application of micro-dialysis to in vivo neuropharmacology and pharmacokinetic analysis of drugs in the brain.Brains On-Line will study the neuropharmacological consequences of interference RNA andgenetic knockdown of glutamate system genes in brain regions associated with diseases ofinterest, including schizophrenia, anxiety and cognitive disorders. Brains On-Line will alsoperform detailed immunohistochemical analyses of brain tissue to determine the anatomi-cal distribution and extent of genetic knockdown resulting from the interference RNA andtransgenic manipulations. The three SMEs, along with their academic and corporate partners,each bring unique expertise critical to the success of this project.

ROLE OF SMEs

11

| A 2.3 Tesla magnetic resonance imaging (MRI) machineused to investigate the effects of drugs on neuronalactivity in regions of the mouse brain. The method willbe used in the present project to identify the effects ofaltering glutamate activity on brain region function.

Novel Anti-angiogenic treatment for Cancer,Arthritis and Ocular Neovascularization based on Inhibition of Placental Growth Factor (PlGF)

ACRONYM

ANGIOSTOP

SUMMARY

12

Contract number: LSHB-CT-2006-037386 | EC contribution: € 1 993 208 | Duration: 24 months

Starting date: 1 June 2006

Aim

ANGIOSTOP aims to elaborate a comprehensiveapproach to the accelerated development of newefficacious and safer anti-angiogenic medicinesthat reduce the pathological blood vessel growthand can be used for the treatment of major progres-sive disorders such as cancer, ocular neovasculari-sation (as observed in diabetic retinopathy andage-related macular degeneration) and arthritis.The overall objective of ANGIOSTOP is to developan anti-PlGF monoclonal antibody. The roadmapcomprises ‘translational research’ to validate previ-ous proof of concept studies in new therapeuticallyrelevant small animal models, both in terms ofsafety and efficacy, to evaluate PlGF expression andits possible upregulation in cancer patients, and todevelop an industrial production process at theGMP level for critical path development.

The project aims to perform extensive validationstudies of its drug candidate to reduce the risk offailure as the drug advances into clinical trials andto manufacture this product for clinical trials. Theultimate goal of ANGIOSTOP is to develop an anti-PlGF monoclonal antibody for the safe and effec-tive treatment of cancer, ocular disease andarthritis. The research will focus on a selected drugcandidate but the new models and strategies willbe of more general utility for the development ofnew medicines aimed at increasing or reducingblood vessel formation, as well as for the advance-ment of the project’s understanding of pathologicangiogenesis.

Background

Most anti-angiogenic strategies are focused onblocking the interaction between VEGF and itsreceptor VEGFR-2. Despite the success of Avastin,it is unlikely that VEGF-inhibitors alone will be suf-ficient to halt tumour angiogenesis. First, anincreasing number of studies document that block-ing the VEGF pathway leads to the induction ofalternative angiogenic signals. Secondly, it hasbeen reported that treatment of cancer patientswith Avastin significantly upregulates the levels ofPlGF. Finally, the currently available angiogenesisinhibitors have serious side effects, thus mandat-ing the development of additional angiogenesisinhibitors. Due to the potential application ofangiogenesis inhibitors in disorders other thancancer, where the treatment is expected to start atearlier times after the disease onset and continuefor longer periods, safer anti-angiogenic drugswithout the risk of serious side effects are needed.Through gene targeting studies in mice, it has beenshown that loss of PlGF does not cause any vascu-lar defect during development, reproduction ornormal adult life, while it severely impairs angio-genesis and arteriogenesis during pathologicalconditions including ischemia, inflammation andcancer therefore indicating that the ANGIOSTOPanti-angiogenic strategy targeting PlGF couldrepresent a safer and more effective approach.

ANGIOSTOP proposes an approach todevelop a new, safer and more effectiveanti-angiogenic medicine that reducesthe pathological blood vessel formationassociated with solid tumor growth, ocu-lar neovascularization (diabetic retinopathyand macular degeneration) and rheuma-toid arthritis. The proposed drug target isPlacental Growth Factor (PlGF) and thecandidate drug is a humanised neutral-ising monoclonal antibody. This drug tar-get selection is based on recent basicresearch on the role of PlGF in patho-logical angiogenesis and ‘translationalresearch’ that established proof of con-cept in experimental animal models.Using a lead candidate anti-PlGF anti-body, it has been demonstrated that inhi-bition of PlGF reduces solid tumourgrowth, inhibits ocular neovascularisa-tion and alleviates arthritis symptoms.ANGIOSTOP will assure the developmentof an anti-PlGF antibody that may consti-tute a new, safer and efficacious medicinefor the treatment of diseases that dependon PlGF driven angiogenesis such as cancer,ocular disease and arthritis.

Key words: angiogenesis, cancer, ocular disease, arthritis, PlGF

Expected results

• A lead humanised anti-PlGF antibody will be val-idated in appropriate animal models.

• Toxicology studies will identify a safe clinicaldose and document the cross-reactivity profileand any toxic effects of the lead candidate anti-body.

• Process development and industrial GMP manu-facturing of the lead candidate antibody for criti-cal path development will be carried out.

• The protein expression of PlGF will be examinedin patient tumour samples. If PlGF levels corre-late with certain tumour types and associatewith grade and prognosis, such information maybe beneficial for identification of appropriatepatients groups.

• Development of a fully human back-up antibodywith a similar or better pharmacological profileas compared to the lead candidate antibody willbe performed for contingency purposes.


ANGIOSTOP has both strategic and specific deliver-ables and milestones. The new animal models andacquired knowledge on pathologic and therapeuticangiogenesis will transcend the specific aims of thedrug development programs and be of strategicsignificance for angiogenesis research in general.The translational and critical path research pro-gram has a clearly defined ultimate deliverable:a new medicine based on PlGF-neutralizing anti-body for anti-angiogenic treatment of certain solidtumours, ocular diseases and arthritis.

BioInvent is the project coordinator.The purpose of the present consortium is to align a partnership strictly confined to partic-ipants with unique essential assets (uniquely qualified academic research groups, SMEsowning intellectual property as well as essential know-how, and experienced clinical trialexperts) to allow a rapid focused development of new safe and efficacious medicines formajor diseases (i.e. solid tumours, AMD and diabetic retinopathy, and arthritis). The com-plementarities and synergy between the academic groups and SMEs will allow a focusedstreamlined development strategy, avoiding levels of bureaucratic decision making that isan unavoidable handicap of large networks and Big Pharma.

ROLE OF SMEs

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Titti Martinsson-NiskanenBioInvent International ABLund, [email protected]

Partners

Jean Marie StassenThromboGenicsLeuven, Belgium

Peter CarmelietFlanders Interuniversity Institute for Biotechnology VZWLeuven, Belgium

Christian FischerCharité Universitaetsmedizin BerlinBerlin, Germany

Eric Van CutsemKatholieke Universiteit LeuvenLeuven, Belgium

Wen JiangCardiff UniversityCardiff, United Kingdom

| ANGIOSTOP is exploring the therapeutic potential andpleiotropic mechanism of anti-PlGF, antibodies against placental growth factor (PlGF), a VEGF homologue, which regulates the angiogenic switch in disease but not in health.Anti-PlGF antibodies inhibit tumour growth by blockingangiogenesis. Distinct from VEGF inhibitors, however, targetingPlGF has been found to prevent infiltration of angiogenicmacrophages, and thus do not switch on the angiogenic rescue program responsible for resistance to VEGF inhibitors.This mechanism is illustrated with anti-PlGF (depicted in blue)preventing PlGF (green) binding to its receptor VEGFR-1+ (red)expressed on macrophages (orange).

The objective is to use in vitro developed neuraltissues instead of transgenic animals havingmemory and learning deficiencies. For this rea-son, the tissue will be developed in vitro fromembryonic cell lines that have the genes involvedin memory/learning ‘switched-off’, (transgenictissue). The ability of the transgenic tissue tomemorise electrical stimuli will be checked andcompared with this of normal tissue. In this way,the role of genes in memory/learning can bechecked in vitro, providing preliminary informa-tion at the tissue level to orient the design oftransgenic animals towards optimal ones thathave a high probability of having an altered phe-notype, therefore decreasing the number oftransgenic animals currently generated by trial-and-error methods.

Due to the controlled complexity and the ease ofbiochemical analyses in the in vitro system com-pared to in vivo experiments, the system will beused in tests aimed at providing information onthe biochemical mechanisms of memory defectsin neural tissue generated from trangsenic celllines, for which such defects in vivo experimentsfailed to clarify the mechanisms. If this task issuccessfully undertaken, the in vitro systemcould replace the use of transgenic animals insome cases of memory/learning deficiencies.

In addition, the system will be used in neurotoxi-city tests because it has the advantage of meas-uring toxicity end-points at various levels of theorganisation of neural tissue such as sub-cellu-lar, cellular and synaptic network levels, as wellas behavioural-like in memory/learning, whichare currently measured in separate in vitro sys-tems (batteries), with the behavioural onesmeasured only in animal experiments. Specifictests with neurotoxic compounds will be per-formed in the system, to find what set of end-point biochemical measurements are necessaryand sufficient for the prediction of memory dam-age, checking in this way the completeness ofthe complementarity of in vitro system-batteriescurrently in use. The developed in vitro systemis proposed as a system which can integrate

Background

In vitro systems composed of synaptically inter-connected neurons have already been developedand used for pharmacological and toxicologicalstudies, based on the extracellular recording ofcell electrical activity. The neurons used in thesesystems are usually taken from animal tissues,because a readily renewable cell source such asthe tumour-derived neuronal cells, is of limitedapplicability due to their altered physiology.Consequently such approaches do not replacethe use of animals, since the cells they use mustbe taken from animals each time a test or a num-ber of tests is performed. In addition to this limi-tation, the existing in vitro neuronal systemscannot currently be considered as alternatives,but rather as complementary to in vivo proce-dures. This is because they provide only partialanswers to more complex problems, where inter-cellular synaptic network level processes are crit-ical – rather than intracellular ones – in assessinghuman hazards, for example the chemical effecton sensory or cognitive functions.

Aim

The development of an in vitro system of bioarti-ficial neural tissue made from mouse embryonicstem cells with memory/learning capabilities.

The in vitro system consists of a three-dimensionalneural tissue-like construct. It is formed by thesynaptic connections developed among neuronsgenerated from mouse embryonic stem cells. Theneurons are inside a porous biomaterial which con-tains molecules that guide the development of thesynaptic network. The tissue is interfaced on twoopposite sides, with multi-electrode arrays forelectrical stimulation and response recording.During the development of the synaptic connec-tions, an electrical stimulation is applied so that thefinal synaptic connectivity pattern will be stimulus-specific and will generate a stimulus-specific elec-trical response. Based on the signal features of theresponse, it can be evaluated when the tissue has‘memorised’ the electrical signal.

In vitro neural tissue system for replacement of transgenic animals with memory/learning deficiencies

ACRONYM

ARTEMIS

The ARTEMIS project aims to design,develop and optimise an in vitro system toreplace the use of animals in transgenicsand toxicology experiments, and in studiesrelated to the effects of genes, chemicals,and neural tissue structure and function,such as memory and learning. From a sci-entific perspective, the ARTEMIS partnerstarget the development of a three-dimen-sional neural tissue-like construct, which isformed by the synaptic connections devel-oped among neurons produced frommouse embryonic stem cells.

For its operational goal, the consortiumseeks to replace transgenic animals withmemory and learning deficiencies withthe in vitro developed neural tissues.ARTEMIS will develop the tissue in vitrofrom embryonic cell lines with the genesinvolved in memory and learning ‘switchedoff’. The consortium will assess and com-pare the ability of transgenic tissue tomemorise electrical stimuli with that ofnormal tissue.

Assessing the role of genes in memory andlearning in vitro provides preliminary infor-mation at tissue level, so as to determinethe design of the transgenic animalstowards the optimal ones with a higherprobability of having altered phenotypes.This would effectively reduce the numberof transgenic animals that are currentlyproduced by trial-and-error methods.The proposed in vitro system can effec-tively integrate biochemical damage withbehavioural damage.

SUMMARY

14


Starting date: 1 March 2007

Key words: bioartificial neural tissue, embryonic stem cells, multielectrode arrays, in vitro neuronal development, memory acquisition-learning, neurotoxicity, developmental toxicity

biochemical damages with behavioural ones.Based on the results of this project, a proposalfor formal pre-validation studies will be prepared,so that in the near future the system will be vali-dated and used instead of animals, in order topredict behavioural damages.

Expected results

An in vitro neural tissue consisting of a three-dimensional network of synaptically intercon-nected neurons that exhibit higher-level, braintissue-like functionalities in memory acquisitionand learning. This tissue could replace the use ofmemory/learning deficient transgenic animals(in vitro transgenics), and the use of animals inneurotoxicity test related to behavioural-like(memory/learning) end points.


• Assessment of toxic or pharmacological effectson the neuronal functions in direct relation withthe in vivo situation, i.e. the impairment ofmemory or learning instead of end-point bio-chemical markers when it is not known to whatextent they influence higher-level functions.

• Development of in vitro transgenic neural tis-sue as memory/learning disease model sys-tems, using genetically modified instead ofnormal embryonic stem cells.

• Neurotoxicity studies related to the develop-ment with application of neurotoxic substancesat different developmental stages.

• Drug tests directed at deciphering the effect atthe network level; for example, the anticonvul-sant drugs for epilepsy, which is caused by amalfunction of a network of neurons instead ofdefects in particular neurons, or the effects ofagents used in anaesthesia due to the inhibi-tion of the oscillations of the synaptic networkinstead of electrical activities of single neurons.

• Development of intelligent biosensors for thedetection of biohazards training the network toneurotoxic compounds as stimuli, instead ofelectrical signals.

• Development of neuroprostheses of the centralnervous system, intended to replace brain func-tions that have been lost due to disease ortrauma.

• Electronic Technology Team, (eTT)eTT is responsible for the signal analysis of the response of the synaptic network, so thatcriteria to detect the memory/learning ability will be formulated.

• Bio Talentum Ltd., (BIO)BIO develops transgenic cell lines that have deficiencies in neural development andmemory, so that an assessment of the in vitro system will be made as a pre-screeningsystem to switch-off genes before the generation of transgenic animals.

• Histopathology Ltd., (HISTO)HISTO performs all the histochemical analyses of the project for the detection of cellsinside the hydrogels and their state of differentiation.

• BSL Bioservice Scientific Laboratories GmbH, (BSL)BSL participates in the design of the tests and performs the tests and various biochemicalmeasurements and off-line analyses needed for the correlation of the end points.

• Quattromed, (QM)QM develops transgenic cell lines and memory function tests in the in vitro system, in orderto test the ability of the system to be used as transgenic tissue instead of transgenic animals.

ROLE OF SMEs

15


Petros LenasComplutense University of MadridDepartment of Biochemistry and MolecularBiology IV, Veterinary FacultyParque Científico de Madrid Santiago Grisolía, 228760 Tres Cantos, Madrid, [email protected]

Partners

Janusz Marian RosiakInstitute of Applied Radiation Chemistry Division of Applied Radiation Chemistry Politechnika Lodzka Lodz, Polandwww.p.lodz.pl

Daniel HorakDepartment of Bioanalogous and Special Polymers Group of Polymer Particles,Institute of Macromolecular Chemistry, Academy of Sciences of the Czech RepublicPrague, Czech Republicimc.cas.cz

Antonio NovellinoElectronic Technology TeamRome, Italywww.ettsolutions.com

Andras Janos DinnyesBio Talentum Ltd.Gödöllo, Hungarywww.biotalentum.hu

George SzekeresLaboratory of Histopathology Histopathology Ltd.Pecs, Hungarywww.histopat.hu

Thomas HartungEuropean Centre for the Validation of Alternative Methods (ECVAM)Institute for Health and Consumer ProtectionIspra, Italywww.jrc.cec.eu.int

Thomas BeckerBSL Bioservice Scientific Laboratories GmbHPlanegg, Germanywww.bioservice.com

Ernest Arenas Department of Medical Biochemistry andBiophysics Division of Molecular NeurobiologyKarolinska InstituteStockholm, Swedenki.se

Eero VasarTiit TalpsepQuattromed A. S.Tartu, Estoniawww.quattromed.com

José Mendes Department of Physics, University of AveiroCampus Universitario de SantiagoAveiro, Portugalwww.ua.pt

intelligent and efficient high-content screens, butwill also be designed for low cost genetic, medical,chemical and pharmaceutical screens. The integra-tion of novel technologies into a common platformconcept, the development of efficient cell-basedscreens, and the demonstration of the feasibility ofthis approach will constitute a significant competi-tive advantage for the European pharmaceuticaland agrobiotechnological industry.

Expected results

The main expected result of the project is the gen-eration of an innovative screening instrument,called AUTOSCREEN. This instrument, which willconsist of the modular iMIC imaging microscopyplatform as a future microscopy standard, will inte-grate ultra-sensitive CCD-technology and novelsoftware concepts that allow an adaptive, i.e.results-based, shaping of the ongoing experiment.This will facilitate the reduction of an otherwiseuninterpretable datastream. An ultra-sensitive flu-orescence-based scanning device for single-mole-cule measurements and a fully automated platefeeder station for automated sample handling andtracking will increase the flexibility and wide utilityof AUTOSCREEN. This system will be tested ina large number of applications for performance andexcellence.

The project is expected to permit the qualitativeand quantitative monitoring of cellular con-stituents (RNA, proteins, and metabolites) in liv-ing cells at the highest possible cellular andsubcellular resolution and with maximal sensi-tivity and specificity. This will allow quantifyingprotein expression and monitoring its subcellu-lar localisation, its state of modification and itsassociation with other proteins and ligands.Furthermore, it will allow measuring of the changeof these processes over time. Characterisation of the toponome will overcome major limitationsof contempory functional genomic and proteometechnologies, which do not provide cellular orsubcellular resolution, and will permit high-content screens for pharmacological substanceswith fewer side reactions.

Background

As more and more genomes are being sequenced,efficient methods to elucidate the functions of themany unknown genes need to be developed. Suchmethods will be crucial in future attempts to aligngenes of unknown functions in biochemical path-ways and networks that carry out the essentialprocesses in all living organisms. These methodswill also be an essential prerequisite for turningbiology from a qualitative, mostly descriptive sci-ence, into a quantitative, ultimately predictive,science.

Although quantitative tools such as DNA microar-rays for transcriptome analysis of biological sys-tems have been available for some years, they havenot yet been used to their full potential due to theoverwhelming complexity and indeterminate com-position of biological systems. Such intricacy hasoften prevented the integration of this informationinto comprehensive and cohesive models.

Cells are built from thousands of different pro-teins that are expressed, both temporally andspatially, over an extremely wide dynamic range.Proteins and other cellular components are regu-lated through variations of their location, theiractivity and their state of modification. AlthoughDNA microarrays have proved to be importanttools for gene discovery on the tissue level, and,moreover, hold great promise for diagnosticapplications, they have major shortcomings intheir lack of cellular resolution. In order to obtainqualitative and quantitative data on cellular path-ways, equipment that allows recording of tran-scripts and proteins at the high precision and athigh-throughput needs to be developed.

Aim

The main goal of the project is to develop an inno-vative screening platform suitable for high-throughput and high-content cell-based assaysand to demonstrate its suitability for high-resolu-tion in situ techniques. This instrument, calledAUTOSCREEN, will not only provide the basis for

AUTOSCREEN for Cell Based High-throughput and High-content Gene Function Analysis and Drug Discovery Screens

ACRONYM

AUTOSCREEN

The overall objective of the AUTOSCREENproject is the establishment of an innova-tive and automated screening instrumentfor high-throughput and high-contentscreens. This instrument will allow stan-dardised, robust, automated and ultra-sensitive high-resolution analysis of RNAsand proteins at cellular and subcellularresolution. The AUTOSCREEN consortiumis composed of six academic partners(Research Institutes and Universities) andfour SMEs.

SUMMARY

16

Contract number: LSHG-CT-2006-037897 | EC contribution: € 3 217 280 | Duration: 60 months


Key words: high throughput analysis, imaging, screening, genomics, proteomics, drug screening


AUTOSCREEN will have a strategic impact on func-tional genomic, biotechnological and biomedicalresearch by permitting qualitative and quantita-tive monitoring of cellular constituents in cells atthe highest possible cellular and subcellular reso-lution and with maximal sensitivity and speci-ficity. This will allow, for example, quantifyingprotein expression, monitoring of its subcellularlocalisation and state of modification, and charac-terisation of the toponome.

The demonstration of the wide applicability ofAUTOSCREEN to the quantitative monitoring ofbiological processes, within living cells, at highestcurrently possible resolution and with sensitivityto the limit set by the laws of physics, will havea significant impact on biomedical research ingeneral. By combining interdisciplinary activitiesfrom academic and industrial sources and byinterfacing biological research with nanotechnol-ogy, computing and engineering, the team expectto create an important tool for biomedicalresearch. Moreover, AUTOSCREEN-based assayswill allow the monitoring of cellular networks andincorporate in vivo protein interaction assays and

features like protein concentration and kineticparameters. Thus, available information on geneexpression networks will not only be useful foridentifying points of the network affected by thedrugs and for simulations of cellular processes,but will also allow the assessment of drug sidereactions at an early stage and facilitate thedesign of novel, less toxic compounds.

The project will reveal new, faster and better waysto determine gene functions and regulatory net-works in a much shorter period of time. The imple-mentation of these technologies will lead to ahigher competitiveness of European biomedicalSMEs, in the sense that the instrumentation to bedeveloped and assembled in this project willenable many European SMEs to efficiently per-form their screens. The estimated low cost of thisinstrument is expected to be of great benefit forSMEs as it will promote their market success. Theproject will also provide the opportunity for estab-lishing a new industry standard for automatedmicroscopy and thus provide opportunities notonly on an individual basis, but also for largermanufacturers who will benefit from the innova-tive approach for which currently there is no equalon the world market.

Within the AUTOSCREEN project, the partner SME companies will establish a widelyapplicable robot-automated screening tool by conceptualising and assembling a firstAUTOSCREEN prototype. The SMEs will develop a full automation of the prototype,improve the innovative iMIC microscope platform in iterative improvement steps, anddevelop a variety of optimisations, assays and applications. The following partners areinvolved in AUTOSCREEN as SMEs: TILL, MANZ, ANDOR and ARO.

The contribution of TILL will be the development of novel concepts for overcoming the currentlimitations of high throughput imaging, and to contribute to the development of new adap-tive soft- and firm-ware concepts for automated imaging. Later, TILL will integrate new devel-opments both from within and outside the consortium, evaluating the overall performance ofall evolutionary stages of the process and specifying future activities. TILL is also involved inthe exploitation and dissemination of results and in the management of the project.

MANZ will concentrate on defining substrates, handling objects and system specificationsand on automated handling of substrates.

ANDOR will be responsible for further enhancing the new price/performance, single photonsensitive Luca EMCCD camera and will cooperate with TILL in developing novel readout patterns.

ARO will be involved in several WPs to assess the quality of AUTOSCREEN under a broad vari-ety of experimental conditions, specifically with respect to proteome visualisation capabilities.

ROLE OF SMEs

17


Klaus PalmeUniversity of Freiburg Center for Applied BiosciencesInstitute for Biology IIFaculty of BiologySchanzlestr. 179104 Freiburg, [email protected]

Partners

Stefanie KlemmTILL ID GmbHMunich, Germanywww.till-photonics.com

Andras Filep Manz KftDebrecen, Hungarymanzautomation-c.cegbongeszo.hu/nyito-en.htm

Jan Hesse Upper Austrian Research GmbHLinz, Austriawww.uar.at

Colin Coates Andor Technology Plc Belfast, United Kingdomwww.andor.com

Carmen Plasencia Aromics S.L. Barcelona, Spain www.aromics.es/eng/index.htm

Martin OheimInstitut National de la Santé et de la Recherche Médicale (INSERM) Neurophysiology and New Microscopy LaboratoryParis, Francewww.biomedicale.univ-paris5.fr/neurophysiologie/labo/oheim.php

Hartmann Harz Ludwig-Maximilians-Universität BioImaging Zentrum Munich, Germanywww.biz.uni-muenchen.de

Benedetto Ruperti University of Padova Padova, Italywww.unipd.it

Ladislav Nedbal Institute for Systems Biology Nove Hrady, Czech Republicwww.greentech.cz

are also able to predict which antigens can lead tothe production of bactericidal antibodies.

In principle, the capacity of a protein antigen to raise bactericidal antibodies may depend on(a combination of):• size, shape, structural complexity, abundance,

solubility, and propensity to oligomerization; and • sequence, structure, dynamics and location of

specific protein regions (epitopes).

These factors may in turn modulate the propertiesof the Ag-Ab complex and its susceptibility to berecognised by the C1q component. The BacAbsproject aims at deciphering possible correlationsbetween these factors and bactericidity.

Aim

Following the framework outlined above, theBacAbs project is concerned with the identifica-tion of (surface-exposed or exported) proteinantigens that may elicit complement-mediatedbactericidity in vitro. This includes early discrimi-nation of antigens that may induce production ofnon-bactericidal antibodies. To achieve this goal,the Consortium will investigate the requirementsfor productive Ag-Ab-C1q complex formation andproposes to find relevant answers by studyingwith a multidisciplinary and comparative approachthe structure of a number of these complexes,taking the MenB vaccine-development project ofNovartis Vaccines & Diagnostics as a model andsource of useful data and reagent molecules. Tosingle out possible structural determinants,focus will be put on groups of antigens with simi-lar size, abundance, solubility, etc. (eliciting andnot eliciting bactericidal antibodies).

Although initially centred around group-BN. meningitidis, the specific target of the projectis the development of tools that can be effectivelyapplied to genome-wide identification of vac-cine candidates against any bacterial pathogensusceptible of complement-mediated lysis.

Background

The development of antibiotic resistance in patho-genic bacteria is potentially one of the most seriousthreats in modern medicine(1). One approach tominimize the use of antibiotics is to vaccinateagainst pathogenic strains of bacteria. A clear can-didate to this approach is Neisseria meningitidis,a major cause of bacterial septicemia and meningi-tis. N. meningitidis is a Gram-negative bacterium,capsulated in its invasive form, classified into fivemajor pathogenic serogroups on the basis of chem-ical composition of distinctive capsular polysac-charides (2,3). Although a promising candidate ison clinical trials (4), there is not yet an effective vac-cine against serogroup-B N. meningitidis (MenB),responsible for over 50% of all meningococcal dis-ease in Europe(5). The capsular polysaccharide ofMenB is identical to a widely distributed human car-bohydrate, making its use as the basis of a vaccinefor prevention of MenB diseases problematic(6). Asa consequence, most efforts have turned to devel-opment of vaccines based on surface-exposed orexported proteins(7).

A bactericidal response, i.e. one which leads to bac-terial-cell death, can be triggered through a varietyof mechanisms. For meningococcal infections, invitro bactericidity assays with immune sera corre-late with protection in humans(8). Although in vivoprotection against MenB may not be solely achievedby complement-dependent bacteriolysis(10,11), anantigen that elicits (murine) antibodies capable oftriggering bacterial-cell death in vitro in a comple-ment-dependent manner is normally considered acandidate for human vaccine development(11,12).

In this context, one major obstacle to vaccine devel-opment, besides sequence and antigenic variabil-ity(13), is the difficulty to identify antigens that willgenerate a bactericidal response. Typically, onlya very small fraction of the antibodies raised inlarge-scale antigen-screening studies are bacterici-dal(14). Thus, while potential antigens can be readilyidentified this information is of little use unless we

Assessment of Structural Requirements inComplement-Mediated Bactericidal Events: Towards a Global Approach to the Selection of New Vaccine Candidates

ACRONYM

BacAbswww.bacabs.org

High throughput cloning and expressionof large sets of genomic ORFs hasbecome a preferred industrial strategyfor genome-wide searches of new vac-cine candidates. For invasive infectionsin particular, the aim is to find proteinseliciting antibodies capable of binding tothe bacterial cell surface and, throughinteraction with the complement system,effectively kill the bacteria. However,current data accumulating from reversevaccinology studies (targeting of possi-ble vaccine candidates starting fromgenomic information) show that onlya small fraction of surface-exposed pro-teins appears to elicit antibodies withbactericidal activity. By using informa-tion generated by reverse vaccinologyprojects within the Consortium, theBacAbs project will apply a novel multi -disciplinary approach to single out thestructural requirements for viable bacte-ricidal vaccine candidates and willdevelop bioinformatics tools to predictcompliance with such structural require-ments. To this end, a systematic analysisof sequence, structure, dynamics andinteractions of selected protein targetswill be undertaken using model systemsof medical interest such as serogroup-BNeisseria meningitidis, a pathogencausing septicemia and meningitis forwhich no effective vaccine exists. TheConsortium comprises an industrialpartner with extensive experience onvaccine development, three SMEs withstrong expertises on several of the keytechnological aspects of the project, andfive academic partners with internation-ally recognised tracks on experimentaland theoretical studies of protein struc-ture and interactions.

SUMMARY

18



Key words: vaccines, structural biology, computational biology, antigen, monoclonal antibody, complement system, bactericidity, proteincrystallogenesis, X-ray crystallography, NMR, mass spectrometry, molecular modelling, molecular-dynamics simulation, molecular docking

Expected results

The BacAbs project shall provide:

• Structural information on a set of proteins thatare components of the cell surface (the bacterialorgan for interaction with eukaryotic host cells)of a major human pathogen.

• Improved experimental protocols and tech-niques, bioinformatics tools and databases toassist the development of vaccines againsthuman bacterial pathogens in general, andgroup-B Neisseria meningitidis in particular.

• A framework in which experimental and in silicomethods for determining protein structure andstudying macromolecular recognition, immuno-logical response mechanisms, and sequence-structure-function relationships can be furtherdeveloped.

• A web-based technological platform integratingthis knowledge, with a potential to improving theeffectiveness and reducing the costs of vaccine-candidate searches.


The results of the BacAbs project have potentialapplication in the selection of new vaccine candi-dates against group-B Neisseria meningitidis andother bacterial pathogens.

References

(1) Borchardt, Drug. News Perspect. 2004, 17, 219; (2) Gotschlich

et al., J. Exp. Med. 1969, 129, 1349; (3) Gotschlich et al., J. Exp. Med.

1969, 129, 1367; (4) Giuliani et al., Proc. Natl. Acad. Sci. USA 2006,

103, 10834; (5) Cartwright et al., Vaccine 2001, 19, 4347;

(6) Hayrinen et al., J. Infect. Dis. 1995, 171, 1481; (7) Jodar et al.,

Lancet 2002, 359, 1499; (8) Goldschneider et al., J. Exp. Med.

1969, 129, 1307; (9) Welsch et al., J. Infect. Dis. 2003, 188, 1730;

(10) Vermont & Dobbelsteen, FEMS Immunol. Med. Microbiol.

2002, 34, 89; (11) Pizza et al., Science 2000, 287, 1816; (12) Welsch

et al., J. Immunol. 2004, 172, 5606; (13) Poolman, Infect. Agents

Dis. 1995, 4, 13; (14) Rappuoli, Science 2003, 302, 602.

The three SMEs involved in the BacAbs project have a principal technological role. The ITcompany INFOCIENCIA S.L. will be implementing the management and dissemination webservers of the Consortium, performing bioinformatics analysis of antigen and epitopesequences, implementing algorithms, protocols and data emerging from the Consortium’swork into computational tools and databases within a web-based technological platform,and evaluating the commercial interest of this platform via demonstration. Two biotechs willbe working on sample preparation and protein-structure determination. ASLA Biotech Ltd.will be performing protein expression and labelling, screening and optimisation of sampleconditions for NMR analysis, monoclonal antibody generation, and sequential backboneassignment and structure determination via NMR. Bio-Xtal S.A. will be performing proteinexpression and purification, especially in connection to derivatives (seleno-methioninelabelled) for X-ray analysis, expression and solubilization screens on highly hydrophobic orpoorly soluble targets, exploration of protein crystallogenesis using nanodrop scale roboticstechniques based on commercially available and proprietary screening approaches, devel-opment and optimization of robotic processes for crystallization plate storage and drop visu-alization, refinement of crystal growth conditions for selected hits to yield diffraction qualitycrystals, and X-ray data collection and structure solution.

ROLE OF SMEs

19


Xavier DauraUniversitat Autònoma de BarcelonaCampus UAB s/n08193 Bellaterra (Cerdanyola del Vallès)[email protected]

Partners

Guido Grandi Novartis Vaccines and DiagnosticsSiena, Italywww.novartisvaccines.com

Anatoly Sharipo ASLA BIOTECH, Ltd.Riga, Latviawww.asla-biotech.com

Etienne L’hermiteBio-Xtal S.A.Mundolsheim, Francewww.bioxtal.com

Giorgio Colombo Consiglio Nazionale delle RicercheIstituto di Chimica del Riconoscimento MolecolareMilano, Italywww.icrm.cnr.it

Martin ZachariasJacobs University Bremen GmbHSchool of Engineering and ScienceBremen, Germanywww.jacobs-university.de

Martino Bolognesi Università degli Studi di MilanoDipartimento di Scienze Biomolecolari e BiotecnologieMilano, Italywww.unimi.it

Alexandre Bonvin Universiteit UtrechtDepartment of ChemistryUtrecht, The Netherlandswww.uu.nl

Jose Manuel Mas INFOCIENCIA S.L.Barcelona, Spainwww.infociencia.com

| Solution structure of the antigenic domain of GNA1870,a 28-kDa surface-exposed lipoprotein of Neisseriameningitidis. This is one of the most potent antigensof Meningococcus discovered by reverse vaccinology(Protein-Data-Bank entry 1YS5).

disease (COPD) and type II diabetes. The availablefacts strongly indicate that these diseases com-prise a cluster of chronic conditions, all of whichare associated with nitroso-redox imbalance. Theintegration of data into a dynamic framework willenable the development of the first kinetic modelof the metabolism shared by COPD, CHF and typeII diabetes, thereby revealing the common andindividual traits of these three complex diseases.

Expected results

After 30 months, BioBridge will have achievedthe following goals:• creation of a structured database for the collec-

tion of clinical information relating to COPD, CHFand type II diabetes;

• identification of the metabolic pathways impli-cated in the target diseases;

• recording of genomic, proteomic, metabolomicand kinetic information onto the relevant struc-tured databases;

• development of a software product designed forspecific disease-related data searching;

• development of standards for the different levelsof data, which will be useful for their integrationfrom genomic and metabolomic databases, andfrom specific proteomics and metabolomics pro-filing experiments, including microarray analysisand stable isotope tracer data. These will bemainly Bayesian networks and multivariateanalysis tools;

• development of protocols for transferring datafrom the structured databases into dynamicmodels;

• using a differential equation approach, thedesign and development of an innovative simula-tion environment that will accommodate thedynamic behaviour of complex networks, and inparticular the metabolic pathways that arealtered by the target diseases;

• development of generic tools that will be clini callyuseful beyond the target diseases addressedduring the lifetime of the project. BioBridge willalso focus on interfacing with end-users, in par-ticular clinical researchers and clinicians.

Background

Chronic diseases are usually the result of interac-tions between individual susceptibility and differ-ent environmental and/or lifestyle factors, and areoften modulated by multiple genes. The interplaybetween these factors determines disease pheno-type and hence, the prognostic and therapeuticimplications of the disease. This interplay betweengenetically predetermined susceptibility and dis-ease phenotype can, in turn, be revealed by com-puter analysis integrating clinical and biomedicaldata. Computer analysis related to clinical prob-lems is currently in a phase of accelerated growth.

Some examples of the application of computeranalysis to clinical practice are the classificationand prognosis of ovarian cancer(1), the analysis ofmyocardial perfusion images and cardiograms (2)and the development of a screening device for thediagnosis of heart murmurs(3). In addition, severalprojects in the European Union are implementinginformation technology-based services for dia-betes management(4).

However, all the approaches currently implementedin clinical practice use very limited datasets, despitethe availability of vast amounts of data from variouslife science disciplines since the ‘-omics’ revolution.Only by integrating genomic, proteomic andmetabolomic data, can knowledge that is useful forthe understanding and treatment of complexhuman pathologies begin to be obtained. This is thegoal of the BioBridge project.

Aim

The BioBridge objectives are twofold. First, a bioin-formatic aspect will involve the development ofsoftware for integrated genomic, proteomic,metabolomic and kinetic data analysis, in order tobuild a bridge between basic science and clinicalpractice. Secondly, a biomedical aspect will focuson understanding the distortion of cellular metab-olism that is associated with certain target dis-eases. The diseases in question are congestiveheart failure (CHF), chronic obstructive pulmonary

Integrative Genomics and Chronic DiseasePhenotypes: modelling and simulation tools for clinicians

ACRONYM

BioBridge

BioBridge will focus on the application ofsimulation techniques on top of multi-level data, in order to create models forunderstanding how molecular mecha-nisms are dynamically related to complexdiseases at the systemic level. The proj-ect will explore and identify gaps in infor-mation, and develop and apply standardsfor the transfer and filtering of data fromexisting molecular biology databasesand new high-throughput experiments(microarray, in vivo metabolic profilingand proteomics data) into metabolicmodels of complex diseases. SMEs areinvolved in BioBridge from its inception,so that newly developed protocols will becommercially exploitable. The project willtherefore drive the standardisation ofanalysis of relevant aspects of disease.

SUMMARY

20


Starting date: 1 December 2006

Key words: diabetes, chronic obstructive pulmonary diseases, chronic heart failure, systemic effects, genomics, proteomics, metabolomics, modelling, bioinformatic


The main outcome of the project will be a protocolfor organising multilevel data related to the targetdiseases into a convenient form for use in the con-struction and refinement of kinetic models ofintracellular metabolic pathways. The softwaredeveloped will be applicable to more generalcases of multilevel data integration.

In helping to provide insights into the key molec-ular mechanisms that determine poor prognosisin the CHF/COPD/type II diabetes disease cluster,BioBridge will generate novel strategies for per-sonalised prevention and enhanced delivery ofpatient care.

Existing computational models have alreadyproved powerful in this context. For example, oneof the BioBridge partners has recently developeda statistical framework for analysis of multivariatemodels from large-scale datasets. This softwareenvironment (GALGO) uses a genetic algorithmsearch procedure, coupled with statistical model-ling methods, for supervised classification andregression. GALGO is relatively easy to use, canmanage parallel searches and has a toolset forthe analysis of models. Another partner hasdeveloped methodologies for biologically-driven

variable selection, also using a genetic algorithmsearch strategy. BioBridge will build on andimprove these and other computational models.

References

(1) Wu et al, 2003; (2) Fletcher et al, 1978); (3) (Bhatikar et al,

2005); (4) (Bellazzi et al, 2004).

The Biobridge consortium brings together 3 selected SMEs with complementary skills inthe domains of semantic interoperability, heterogeneous data integration and simulationtechnologies.

MathCore Engineering AB develops integrated tools in modelling, simulation, control, andvisualization of complex systems. This expertise is fundamental in the project tasks relatedto the creation of Mathmodelica Metabolic Pathway simulator. This tool will positionMathCore very solidly in the biological simulations field.

Biomax Informatics AG provides state-of-the-art software for the biopharmaceutical indus-try with a focus on data integration, data retrieval, knowledge aggregation and generation.The Biobridge project uses its BioXMTM Knowledge Management Environment to integrateand organize heterogeneous data types. Additionally, its software BioRSTM allows ubiquitousretrieval of data for project participants.

Genfit is a leading European drug discovery company in the field of cardiovascular, meta-bolic, inflammatory and CNS disorders. Genfit brings its LSGraph® technology to the proj-ect that facilitates the handling of information individual graph nodes and integration ofinformation from NCBI Gene, PubChem, Uniprot, GO, KEGG etc. This technology also fea-tures: data mining capabilities, tracking of relations and evidence and an associated searchengine to elucidate the regulatory mechanism(s) behind the observed effects.

ROLE OF SMEs

21


Josep RocaDepartment of PneumologyInstitut d’Investigacions Biomèdiques August Pi i SunyerBarcelona, [email protected]

Partners

Peter AronssonMathCore Engineering ABLinköping, Swedenwww.mathcore.com

John BrozekGenfit LaboratoriesGenfit S.A.Loos, Francewww.genfit.com

Andrea RamgeDieter MaierBiomax Informatics AGMartinsried, Germanywww.biomax.com/company/company.php

Jordi Villà i FreixaComputational Biochemistry and Biophysics Laboratory, Universitat Pompeu FabraBarcelona, Spain

Pranav SinhaInstitut für Medizinische und Chemische Labordiagnostik, Landeskrankenhaus Klagenfurt Klagenfurt, Austria

Francesco FalcianiSchool of BiosciencesUniversity of BirminghamBirmingham, United Kingdom

| The increasing size and complexity ofbiological database and the capacity ofnew experimental designs to determinehigh throughput data for specificemphasises the significance of the threelevels in the figure: a) the need forstructured databases related with theproblem, b) the need for formats, likeSBML, able to translate the structureddata into dynamical models, and c) thedevelopment of powerful modelingtechniques ultimately able to make use ofand to interpret clinical data. Note thatthe connecting SBML spans both thestructure of the database and themodeling environment, gluing togetherall the pieces of the puzzle.

least, ‘hits’ (that are active in micromolar con-centrations) which may subsequently be trans-formed to ‘leads’ (with affinities in thenanomolar range and with reasonable drug-likeproperties) and finally to drug candidates.Combinatorial chemistry has also been on theside of HTS, presenting the ability to synthesisehuge amounts of derivatives based on specific‘scaffolds’.

• Rational drug design approaches such as struc-ture-based design and ligand-based design.The first takes into consideration the detailedatomic structure of the target and the possibili-ties for forming physical interactions (i.e.,hydrogen bonds, Van der Waals interactions,electrostatic complementarity, hydrophobicity,etc.) between small molecules and specificsites on the targets, while the second dependsmore on properties of known active moleculesand uses similarity ideas (including ‘pharma-cophore’ searches) to discover new active mole-cules. The substantial reduction in discoveringnew chemical entities by big pharma in recentyears has been in part attributed to the fail-ures due to very low hit rate in both the HTS andCombichem, on the one hand, and on theinability to properly taking into account thepharmacokinetic (ADME/Tox) effects as well asfor entropy, solvation and target flexibility instructure- and ligand-based designs.

A landmark in introducing pharmacokinetic con-siderations to drug design and developmenthas been the ‘Rule of 5’ of Lipinski. This idea,which is now less than a decade old, also pro-vided an immediate tool to reduce the size ofcombinatorial libraries and of HTS candidates by‘filtering’, i.e., requiring that all molecules mustpass the Lipinski rule (three out of four conditionsfor the limiting of molecular weight, calculatedlipophilicity, and the numbers of H-bond donorsand acceptors) in order to be in the properbioavailability range.

Background

After the completion of the sequencing stage ofthe human genome project, the major focus ofdiscovery efforts turned to the identification ofthe ‘druggable’ portion of the genome that islinked to pathological states and is able to inter-act with the drug-like chemical space, restoringnormal functions.

Apparently, the druggable genome is a subset ofthe 30 000 genes in the human genome thatexpress proteins and represent, in many ways, anunprecedented gift and exceptional opportunity fordrug discovery scientists and for patients who arehoping for therapies of diseases currently uncured.That subset (estimated as ca. 3 000 proteins) isable to bind drug-like molecules as characterisedby the Lipinski’s rule-of-5 criteria.

In order to find more rapidly small molecule mod-ulators to the newly emerging validated targets,the high-throughput screening provides a reason-able solution to screen large compound libraries.However, it seems most of the targets can be clas-sified into large target families such as kinasesand GPCRs: thus, development of target focusedlibraries could dramatically increase the hit rateas well as open the way to identify selectiveinhibitors/antagonists within the target families.

The idea of ‘focused libraries’ or ‘targetedlibraries’ of molecules emerged in recent yearsas a ‘compromise’, or as an attempt to bridgebetween two seemingly conflicting approachesto drug discovery:

• High Throughput Screening (HTS), by whichhundreds of thousands of compounds, mainlyin big pharma, were tested against a (hopefullyvalidated) biological target such as a protein ora cellular system. The basic assumption of HTSis that large numbers and diversity shouldcover chemical space well enough to find, at

Grid-aided computer system for rapid anti-cancer drug design

ACRONYM

CancerGridwww.cancergrid.eu

In the three years of this multidisciplinaryresearch project, the 10-member Consor -tium plans to develop and refine methodsfor the enrichment of molecular librariesto facilitate discovery of poten tial anti-cancer agents. Using grid-aided computertechnology, the likelihood of findinganti-cancer novel leads will sub stantiallyincrease the translation of basic knowledgeto application stage.

In particular, through the interaction withnovel technologies and biology, the R&Dconsortium aims at:• developing focused libraries with a high

content of anti-cancer leads; • building models for prediction of disease-

related cytotoxicity and of kinase/HDAC/MMP and other enzyme (i.e.HSP90)inhibition or receptor antagonism usingHTS results;

• developing a computer system based ongrid technology, which helps to accelerateand automate the in silico design oflibraries for drug discovery processes,and which is also suitable for futuredesign of libraries for drug discoveryprocesses that have different biologicaltargets (the result is a new marketabletechnology).

SUMMARY

22

Contract number: LSHC-CT-2006-037559 | EC contribution: € 2 804 075 | Duration: 36 months


Key words: ??

The molecules that passed the Lipinski filter werethus targeted on oral bioavailability, and theirnumbers were much smaller than those for the ini-tially planned experiments. The idea of ‘filters’thus gained momentum, and additional filterssuch as those of Veber (limiting the number ofrotatable bonds and the size of polar surfacearea), also for bioavilability, were suggested.Both Lipinski and Veber rules did not considerdirectly any conformational aspects (3-dimen-sional descriptors of the molecules to be tested,but pharmacophore searches (ligand-baseddesign) and virtual docking and scoring (structurebased design) serve as subsequent filteringprocesses in 3D that cover the ‘affinity’ part ofdrug action, while the other filters mostly dealwith ‘drug transport’ issues.

These two properties are, to a large extend, orthog-onal. Thus, one may regard the filtering process asbeginning with huge numbers of molecules, which

are reduced to a smaller set by chemical descrip-tors. This smaller set may then be studied withmore detailed conformations at the pharma-cophore level, reducing it further to a group ofmolecules which may be docked virtually to theassumed target, finally leaving a small set of sub-stantially ‘focused’ or ‘targeted’ lead candidates.The main ‘focusing’ activity has been howeverconcentrating on molecular scaffolds that are use-ful for probing families of targets such as GPCRs,tyrosine kinases, MMPs etc.

But, even that approach suffers from many draw-backs. Lipinski and Veber rules can not distin-guish well between drugs and non-drugs, and areclearly not appropriate indicators of ‘drug-like-ness’. Neural networks have been applied specif-ically to this problem and managed to distinguishproperly between drugs and non-drugs, but havethe disadvantage of ‘hidden layers’ which do notenable to plan and design novel molecules.

Inte:Ligand (Austria) will provide in silico screening of compound databases of commer-cially available small drug-like molecules, consulting in compound profiling, and decisionsupport tools for medicinal chemistry. The company will generate virtual libraries for theconsortium partners and will distribute its library generation software tools to the membersof the consortium. Inte:Ligand will also market the enhanced versions of its software andexploit its enlarged competences in ligand target interaction modelling.

GKI Economic Research Co. (Hungary) gained experience in leading international consortiaby acknowledging the importance of interactivity and proactive communication in facilitat-ing projects with a variety of key actors. In CancerGrid, a similar approach will be taken.From the social science standpoint, it is also important to raise awareness towards theobjectives and anticipated results of CancerGrid. With this in mind, GKI will prepare andpublish a case study of the project, targeting a wide audience beyond the direct stakehold-ers. GKI will also be responsible for organising the involvement of direct or indirect stake-holders in the area of CancerGrid (such as healthcare providers and physicians, industry,regulatory authorities, experts in ethics, law and social sciences, health economics, publichealth, patient organisations, policy-makers etc.) as appropriate.

DAC (Italy) is a biopharmaceutical company, founded in 2004, specialized in the identifi-cation of new anti-tumour drugs. In particular, DAC is developing new proprietary small-molecule compounds with inhibitory activity against chromatin remodeling enzymes(histone deacetylases) and against HSP90, key players in cancer and non-cancer diseases.As a partner of the CancerGrid consortium, DAC will initially develop target-based assaysin order to perform screenings of compounds previously identified as hits in the cell-based screening. Moreover, DAC Srl will design, synthesize and weigh-out a focusedlibrary (1500 compounds) for which the target based screening will be repeated.

ROLE OF SMEs

23

Key words: bioinformatics, pharmacology, grid technology, library design, in-silico prediction of drug-like properties, prediction of ADME parameters, predictive toxicology, creation of virtual libraries

| A Typical fold of matrix metalloproteinases structured in 3 α-helices (red) and 4 parallel and1 antiparallel β-sheets (yellow). The binding siteis represented by a white surface while the zincion is shown as a light-gray sphere and the threecatalytic histidines are rendered as ball-and-stick.

| B Coordination between zinc and the three catalytic histidines and the phosphonate group ofthe ligand extracted from the 1ZS0 is highlighted.

|B

|A

ACRONYM

CancerGrid

24

A drug-like index has been suggested but is basedon fragment identification and therefore limited inits ability to discover novel structures. Structure-based approaches can consider small moleculeflexibility, but are still inappropriate for dealing withthe flexibility of the protein targets, especially withthe flexibility of backbone and of larger loops. Thescorings in docking methods have recently beenexposed to much criticism. Using single conforma-tions in pharmacophore searches is clearly inappro-priate, because it has been shown that smallmolecules bind to proteins in conformations thatare higher in energy than their global minima.Toxicity predictions have not yet reached enoughreliability to prevent major toxicity threats by drugs.The need for selectivity has not yet been properlyaddressed in the preparation of focused libraries.

Therefore, although many companies nowadaysare offering focused libraries for kinases, GPCRsand other families of molecules, there is a greatneed to improve the production of such librariesin order to shorten the time for discovery and tosave enormous expense. A main stumbling blockon the way to solving such issues is the complexcombinatorial nature of the problem of libraryconstruction and drug design.

In this proposal, we include methods that dealdirectly with the combinatorial nature of the prob-lems, that have been shown to solve combinatorialproblems in a highly satisfactory manner, that dis-cover the global minimum in most cases andretain a large set of best results, many of themexcellent alternatives to the global minimum.

Expected results

Novelties and added values of the IT part of theproject:• virtual focused libraries of anti-cancer agents;• potential anti-cancer agents;• HTS technology;• data for model building purposes;• models able to predict anti-cancer properties;• CancerGrid System: a grid-based computer aided

tool that able to provide anti-cancer candidatesfaster and in a more efficient way, also suitable todevelop candidates for other targets.


The models developed within the frame of thisproject can be used for filtering large discoverylibraries to find anti-cancer drug candidates,and to design anti-cancer focused libraries. TheCancerGrid computer system will be able to sup-port the design of lead compounds in general, notonly in the anti-cancer field, but in any otheractivity area. Thanks to its grid-based architec-ture, the system will be able to predict moleculardescriptors for compound libraries, containinga large number of molecular structures, in a shorttime. When calculating 3D molecular descriptors,the system will take all major conformers intoaccount. This enables the calculation of informa-tion-rich molecular descriptors, and the develop-ment of reliable linear and non-linear models.The system will also be able to apply these mod-els to predict the biological activity or chemical/physical property of the compounds.

25


György DormánAssistant Director of Science and TechnoloyAMRY HungaryZahony u. 7 Budapest HU 1031, [email protected]

Project manager

István BágyiManager, Grants & IPAMRY [email protected]

Partners

Thierry LangerInte:Ligand Software-Entwicklungs und Consulting GmbHMaria Enzersdorf, Austriawww.inteligand.com

Mati KarelsonTallinn University of TechnologyTallin, Estonia

Mart SaarmaUniversity of HelsinkiHelsinki, Finland

Balazs BorsiGKI Economic Research Co.Budapest, Hungarywww.gki.hu/en/index.html

Peter KacsukComputer and Automation Research InstituteHungarian Academy of SciencesBudapest, Hungary

Amiram GoldblumUniversity of JerusalemJerusalem, Israel

Saverio MinucciDAC S.R.L.Milano, Italywww.genextra.it/dac.htmlwww.dacresearch.it

Angelo CarottiUniversity of BariBari, Italy

Ferran SanzUniversity Pompeu FabraBarcelona, Spain

| Workflow Scheme of Computer Aided Drug Discovery in CancerGrid.

| Grid Based IT Support for Drug Discovery.

Expected results

Innovative tools for designing PPI inhibitors

• Five different PPI-inhibitor library creation tools,based on five complementary approaches: – in silico;– genetic chemistry; – advanced natural product technologies; – retro-synthesis of natural scaffolds and; – ADME improvement.

• Cross-fertilisation of approaches so that each ofthe five approaches learns lessons from the oth-ers and incorporates relevant leanings into itsapproach.

• Three high-content assay systems for threeimportant PPI cancer targets (p53-Mdm2, Betacatenin-TCF4, BRCA2-RAD51).

• Design rules for PPI inhibitor compound libraries(mass, diversity composition, lipophilicity, com-pound class etc.) generated from 15 complementarydata sets.

Novel small-ligand libraries and pre-clinical candidates

• Several ‘PPI inhibitor’ compound libraries.

• Different candidate compound families fromwithin these libraries that can subsequently betaken forward into pre-clinical testing by theSME partners.

Background

Most protein:protein interactions occur withinthe cell and thus can only be targeted by smallmolecules. Furthermore, PPI differ structurallyfrom more classic drug targets such as enzymesand receptors, and consequently existing com-pounds have generally delivered disappointingresults. Therefore, new approaches are neededto develop novel small molecules which inhibitPPI in cancer.

Aim

The objective of this project is to develop a seriesof innovative small-ligand tools and libraries thatallow new approaches to the inhibition of protein-protein interactions in cancer. A key theme is theutilisation of structural motifs found in naturalPPI-inhibitor compounds. This is coupled withhigh content testing of the resultant structures onthree distinct PPI targets relevant to differenttypes of cancer, to allow compound rule-sets tobe developed and improved. We want to developsmall-ligand libraries focused on PPI inhibitorsof relevance to cancer. Furthermore, we willdevelop innovative tools that allow improvedlibrary design in this area by integrating in silicoapproaches, bio-informatics, new approaches tocompound synthesis and pharmacology. The proj-ect will also cover the scientific areas such as insilico prediction of drug-like properties, predic-tion of ADME parameters, predictive toxicologyand creation of virtual libraries.

Combating cancer through novel approaches to protein: protein interaction inhibitor libraries

ACRONYM

CAPPELLAwww.cappellabio.eu

The inhibition of protein:protein inter-actions (PPI) is one of the most prom-ising approaches to the developmentof novel cancer therapies. This projectbrings together some of Europe’s leadingbiotech SMEs and several highly recog-nised academic institutes. By combiningfive distinct chemical approaches andtesting them on three different targets(all from different partners) a series ofinnovative small-ligand tools and librariesthat allow new approaches to the inhi-bition of PPI in cancer will be devel-oped. The project is a unique opportunityto integrate novel in silico, chemical,genetic and ADME-based approaches tothe design, synthesis and optimisationof libraries and compounds.

SUMMARY

26



Key words: identification of novel compounds, natural products, inhibition of protein:protein interactions, high throughput screening

There are five SMEs within the CAPPELLA project consortium. Generally speaking, the SMEsprovide very specific scientific know-how and technological platforms to the project, with-out which the project would be rendered incoherent. SMEs bring to the project the results oftheir in-house developments. By combining the know-how of these different companies, theproject has made a pharmaceutical development which would not have been possible byone of the companies acting alone.

With regard to the SMEs involved in the CAPPELLA project:

PharmaMar is a pharmaceutical company specializing in the detection and development ofpharmaceutical products from extracts obtained from marine organisms. The company pos-sesses a huge collection of marine specimens from which previously unknown natural com-pounds are extracted and characterized. Structural data on such new natural compounds actas a very important database for the CAPPELLA project.

Analyticon is a provider of methods for the fractionation and analysis of highly complexextracts from biological samples and concomitantly a provider of computer-based methodsfor the processing of data obtained.

Inte:Ligand contributes to the project with a computer-based in-silico analysis of potentialpharmaceutically relevant drugs. The analysis will permit the prediction of drug behaviourin toxicological or pharmacokinetic studies and will allow the computer-based evaluation ofpotential drug analogues.

BioLigands is the provider of a highly efficient in vitro assay for the interaction betweena tumor suppressor protein and its antagonist. The screening assay provides compounddata which will be incorporated into a computer-based drug evaluation.

Evolva contributes its chemical evolution platform, which permits chemically evolved meta-bolic pathways to be expressed in yeast. It is expected that chemically evolved expressionlibraries will give access to new pharmaceutically active small molecular weight com-pounds. The elucidated chemical structure obtained from such compounds will be passedthrough the in-silico development procedure developed by Inte:Ligant.

ROLE OF SMEs

27


Sanne JensenEvolva S.A.Hagmattstrasse 64123 Basel, Switzerland

Project manager

Tanja [email protected]

Partners

Jens Peter MullerAnalytiCon Discovery GmbHPosdam, Germanywww.ac-discovery.de/english/go.html

Karsten KristiansenBioLigands ApSOdense, Denmark

Thierry LangerInte:Ligand Software-Entwicklungs und Consulting GmbHVienna, Austriawww.inteligand.com

Birger Lindberg MollerRoyal Veterinary & Agricultural UniversityUniversity of CopenhagenCopenhagen, Denmark

Simon MuntPharmaMar S.A.Madrid, Spainwww.pharmamar.com/language.cfm

Ashok VenkitaramanUniversity of CambridgeCambridge, United Kingdom

Ariel Ruiz i AltabaUniversity of GenevaGeneva, Switzerland

Juhan SedmanUniversity of TartuTarku, Estonia

Aim

The objective of this project is to develop andvalidate a new technology which has the potentialto replace the various ChIP technologies, and totransform the way the molecular analysis ofchromatin is performed. The ChILL technique hasbeen patented by one of the partners of this proj-ect, leaving the consortium free to operate withregard to intellectual property rights. The ChILLmethod is based on specific ligations which occurbetween DNA stretches under diluted conditions.In this environment, ligation partners can onlyinteract if they are in close proximity.

This proximity is created by new oligonucleotide-antibody conjugates (nucleoproteic probes, oroligo-ab), which physically place the target DNAin contact with the oligonucleotide reportersequences. The ligation products are then ampli-fied by the polymerase chain reaction and analysedwith real-time instruments and/or classical gelelectrophoresis.

Due to the ligation step taking place under dilutedconditions, the ChILL method will generate datacomparable to those obtained with ChIP, but withincreased sensitivity and a simplified protocolthat omits the tedious immuno-precipitation step.As a proof of principle, the ChILL method hasalready been shown to be at least 100 times moresensitive than the regular ChIP assay.

ChILL will not only facilitate analysis of very smallsamples, such as early embryos or diagnosticsamples from patients, but will also radicallyimprove the resolution of the epigenetic marks.In addition, strong detergents used in the ChILLassay open up the chromatin structure, renderingit more accessible to antibodies than in conven-tional ChIP assays.

Another major advantage of ChILL will be its abil-ity to interrogate several parameters in a singlesample. For this purpose, a variant of ChILL called

Background

The sequence of an organism’s genome does notdirectly determine how the genome is used tobuild the organism. A second, more complex reg-ulatory code – the epigenetic code – is encryptedin the chromatin structure and the 3D nuclearorganisation of chromosomes. Epigenetic infor-mation is encoded in DNA modifications (namelymethylation), chromatin composition and modifi-cation, and nuclear topology, or the dynamicorganisation of the genome within the nucleus.

Epigenetic information not only provides the firstcue to allow a cell to interpret the genome, it canalso be heritably transmitted through cell divi-sion to maintain cellular identity. Moreover, whilemany heritable disorders in humans are causedby DNA sequence changes (mutations) that abol-ish gene expression, a number of diseases arecaused by inappropriate gene silencing, broughtabout by epigenetic modifications. Indeed, mostcancers involve the epigenetic silencing of genesthat normally control cell proliferation. The princi-pal forms of epigenetic modification are DNA andhistone methylation.

A challenge that is central to modern biology is theidentification of the spatial and temporal dynamicsof epigenetic factors in a number of physiologicalsituations. The Chromatin Immuno-Precipitation(ChIP) assay has played a pivotal role in decipher-ing patterns of epigenetic marks that govern genetranscription. Besides 'classical’ ChIP, several sim-ilar techniques have been described in the litera-ture. Recently, new technologies designed toimprove on the existing ChIP and native ChIP(NChIP) technologies, have emerged.

In addition, low resolution and reproducibility prob-lems are often encountered. These severe limita-tions of the ChIP method are overcome by theChromatin Immuno-Linked Ligation (ChILL) method,which could provide the foundation for a new gen-eration of biotechnology tools and methods.

Chromatin Immuno-linked ligation: A novel generation of biotechnological tools for research and diagnosis

ACRONYM

ChILL

The objective of this project is to developand validate a new technology based onthe Chromatin Immuno-Linked Ligation(ChILL) method that will transform theway the molecular analysis of chromatinis performed and will not only facilitateanalysis of very small sample sizes, suchas early embryos or diagnostic samplesfrom patients suffering from a range ofdiseases, but also radically improve theresolution of the epigenetic marks.

SUMMARY

28


Starting date: 1 October 2006

Key words: chromatin remodeling, transcription regulation, epigenetic, ChIP assay, histones, DNA methylation

combinatorial ChILL will be developed. This willrepresent a major breakthrough, because it willmean that several epigenetic marks can be col-lected from a single tube, making it easier to buildup what might be called an ‘epigenetic profile’ ofthe biological material in question.

Expected results

The Chromatin Immuno-Precipitation (ChIP) assayplays an absolutely pivotal role in decipheringpatterns of epigenetic marks that govern genetranscription. While the ChIP assay is a versatiletool, it suffers from low resolution and low sensi-tivity. These strong limitations of the ChIP methodare overcome by the Chromatin Immuno-LinkedLigation (ChILL) method. ChILL will not onlyfacilitate analysis of very small sample sizes, suchas early embryos or diagnostic samples frompatients suffering from a range of diseases, butalso radically improve the resolution of the epi-genetic marks. The ChILL approach also offers

opportunities to examine simultaneous co-locali-sation of two or more factors on the same chro-matin template, and the epigenetic marks will beresolved in unprecedented detail.

The expected results of the programme wouldbe to make ChILL technology accessible to allEuropean research laboratories via validated pro-cedures, reagents or kits. The project also expectsto launch diagnostic kits using ChILL technologyfor the diagnosis of diseases linked to epigeneticdisorders.


The immediate impact of ChILL will be a betterunderstanding of the epigenetic code. The longerterm commercial impact of the ChILL methodmight also be very important for Diagenode withpossible development of tools for the research ordiagnostic market.

For the ChILL project, Diagenode has three main objectives:

• First, as coordinator of the research project, the company aims to create strong scientificsynergies among the participants.

• As the SME participant, Diagenode ensures that research is oriented towards possiblefinal commercial applications. In the case of ChILL, the interest is concerned with betterunderstanding of how an innovative technology can be translated into a diagnostic tool.

• Last but not least, one of Diagenode’s roles is to create ‘added value’ within the SME,by gaining scientific experience and know-how in a ‘high-tech’ field. The new scientificexpertise created by the EU funded research projects is also recognised and acceptedoutside the company, by the scientific community and, in a broader sense, by thecommercial community.

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Didier AllaerDiagenode S.A.Avenue de L’Hopital, 1 Tour Giga B344000 (Sart Tilman) Liège, [email protected]

Partners

Rolf I. OhlssonUppsala UniversityDept. of Development & Genetics Evolution Biology CentreUppsala, Sweden

Henk G. Stunnenberg Radbout University NijmegenDept. of Molecular BiologyNijmegen, The Netherlands

F. FuksUniversité Libre de BruxellesFaculty of Medicine Laboratory of Molecular VirologyBrussels, Belgium

D. Clark GeneSys Ltd.Camberley, United Kingdom

Expected results

The main objective of this STREP is to producewithin three years at least two candidate vaccineantigens against the blood stage of the malaria par-asite Plasmodium falciparum, ready to use for pre-clinical evaluation. The candidate antigens (MSP-1and VAR2CSA) will be expressed by the novelexpression platform Tetrahymena thermophila,a free-living Ciliate, which expresses Plasmodiumfalciparum antigens efficiently. Partial results willbe: vector constructs that are suitable for expres-sion; appropriate reproducible lab scale purification protocols; optimised host strains; a firstup- and downstream process up to pilot scale; andimplementation of the first steps of a quality man-agement for potential later GMP production.


There are no effective vaccines for malaria cur-rently available. Vaccination trials conducted inhumans with existing candidates have been metwith limited success. This stresses the need forcontinuing efforts towards testing novel candi-dates. This project offers a novel biotechnologicalapproach to vaccine development and respondsto the demands of developing countries, non-profit organisations and charity foundations, totechnologically contribute to actions targeted atthe major poverty-related diseases, includingmalaria. One goal of the approach is to apply theunderlying technological concept of this project tothe later industrial development and productionof new and highly effective vaccine candidatesagainst malaria.

Background

Malaria kills over one million children in Africaalone each year, with an addition of up to 500 mil-lion episodes of clinically significant illness due tomalaria annually. Worldwide deaths are esti-mated at between 2 and 3 million per annum. Fewother infectious diseases place such a burden onthe social, economic and healthcare systems ofdeveloping countries. Therefore there is a press-ing need for the development of a vaccine againstmalaria, to ease at least part of this overwhelmingburden on the continent of Africa, which suffersthe majority of the deaths and illness caused bythe malaria parasite.

Aim

The aim of this project is to use an innovative newexpression platform based on the CiliateTetrahymena thermophila in conjunction with twonovel malaria vaccine candidates, as a method ofadvancing these antigens to pre-clinical testing,prior to testing in humans. It will produce novelTetrahymena organisms expressing Plasmodiumfalciparum antigens at pilot-scale production lev-els. The two antigens selected are novel yet prom-ising candidates based on the N-terminal regionof MSP-1 and the Var2 CSA variant of the PfEMP-1antigen family.

The Tetrahymena system as an innovativeapproach to malaria antigene expression

ACRONYM

CILMALVAC

This STREP, co-ordinated and led by anSME, will focus on the development of newmalaria vaccine candidates with the aim oftaking at least one product to the stage ofpilot scale. Products showing most promise,once expressed in the Tetrahymena system(or Ciliate system), will proceed to antigentesting in vitro and in vivo, to assess theirstructure and antigenic integrity. At leastone product is expected to reach lead opti-misation, safety and toxicology testing.

Combining the expertise of the two aca-demic malaria research laboratories withthe novel Ciliate-based expression sys-tem under development by the SME willgenerate recombinant Tetrahymena ther-mophila strains expressing malaria anti-gens based primarily upon MSP-1 and thevar2CSA genes of Plasmodium falciparum.To utilise the capabilities of the partici-pants to greatest efficiency, this STREPwill comprise, besides the ProjectManagement, work packages dealingwith the generation of antigen expressingCiliate strains, pilot scale production andpurification of the antigens and the test-ing and validation of candidate vaccines.

This STREP application, co-ordinated byCilian AG, will provide an expression plat-form that can easily combine antigens todevelop new combination vaccines thatmay be more promising than vaccines cur-rently under development. Ciliate-basedexpression of Plasmodium falciparumproteins in secreted, membrane bound orcytosolic forms will complement the workdone under the larger European malariavaccine development project.

SUMMARY

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Contract number: LSHP-CT-2007-037304 | EC contribution: € 1 271 664 | Duration: 36 months


Key words: Malaria, Plasmodium, vaccines, Ciliates, Tetrahymena, MSP-1, VAR2CSA, biotechnology, bioproduction

This project is driven by the coordinating German SME Cilian AG which has developeda novel protein expression platform based on recombinant eukaryotic ciliate cells whichexcrete the desired product either membrane-bound or in cytosolic form. Under this projectthe quality and immunogenicity of the secreted proteins will be tested for two currentmalaria vaccine antigens. The project will thus allow the SME to validate the utility of theirnovel expression system and, at the same time, deliver GLP scaled-up processes for twopromising malaria vaccine candidates, provided by the European partners of the project.

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Marcus HartmannRemco Brandt Cilian AGJohann-Krane Weg 42 D-48149 Muenster, [email protected]

Partners

David CavanaghInstitute of Cell, Animal and Population BiologyUniversity of EdinburghEdinburgh, United Kingdom homepages.ed.ac.uk/eang15

Thor TheanderCentre for Medical Parasitoolgy University of CopenhagenCopehagen, Denmark www.euromalvac.org/profiles/theander.htm

© Shutterstock

ACRONYM

cNEUPRO

Clinical Neuroproteomics of Neurodegenerative Diseases

SUMMARY

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Starting date: 1 Janvier 2007

Aim

cNEUPRO is designed to discover innovative pro-tein biomarkers and to develop novel CSF andblood multiplex assays for the accurate early, dif-ferential and possibly predictive diagnosis of AD.Moreover, cNEURPO will develop first EuropeanSOPs for CSF-based NDD.

Expected results

cNEUPRO consists of two R&D modules:

Module I is dedicated to the discovery of novelprotein biomarkers, the generation of high-affin-ity ligands and the implementation of biomarkersinto novel ELISA or Multiplex Assays. The discov-ery of biomarkers is differentiated into the devel-opment activities: definition of high-qualitysamples, identification of candidate biomarkersin blood and CSF by multi-dimensional state-of-the-art proteomics, and validation of the candi-date biomarkers (� biomarker). The validation ofa biomarker comprises its protein chemical (struc-tural) validation by advanced mass spectrometryand its clinical validation within a re-test study.

For validated biomarkers we will devise novelpoly- and monoclonal antibodies as well as high-affinity molecules. The latter highly stable smallbinding proteins are designed using a proprietarytechnology and may possibly also be used astherapeutic tools or scaffolds for molecular neu-roimaging. Finally, the biomarkers will be inte-grated into novel ELISA and, most importantly,Multiplex-Assays. Applying advanced bioinformat-ics tools (proprietary technology), cNEUPRO willdesign a prototype predictor system for AD.

Background

Europe has to face a threatening increase in theprevalence of the most common dementia, i.e.Alzheimer’s Dementia (AD). There is an urgentneed for novel disease- modifying treatments,which will be most effective if offered at a veryearly dementia stage, preferentially as preventivetherapy during the prodromal stage of mild cogni-tive impairment (MCI). However, the reliable clini-cal differential diagnosis of very early AD is notsatisfactory and conventional diagnostic tools donot offer predictive diagnostics for AD. Here,recent research has clearly demonstrated thatmultiparametric neurochemical dementia diag-nostics (NDD) in cerebrospinal fluid (CSF) clearlyimproves the early and differential diagnosis ofAD. Moreover, recent studies have also indicatedthat incipient AD can be predicted several yearsprior to dementia onset. However, these promis-ing results of CSF-based NDD need to be validatedby additional studies. Furthermore, additionalneurochemical biomarkers have to be identifiedfor the improved early differential diagnosis ofdementias and, most importantly, first assaysfor blood-based NDD need to be developed.Moreover, only blood-based NDD will allowa widely applicable monitoring of therapy efficacy.

Meanwhile, CSF-based NDD has entered neuro-chemical routine diagnostics of dementias inEuropean expert centres. However, there isa strong need to develop first standard operationprocedures (SOPs) for CSF-based NDD, which sig-nificantly depends on the quality of preanalyticalsample handling, storage conditions and samplestorage. It is noteworthy that the success of thisnovel approach for the improved diagnosis ofvery early dementias has also stimulated strongresearch activities in the US.

Alzheimer’s Dementia (AD) is one of themost common brain diseases in the elderly.Dementias pose a major health problem inEuropean countries, with currently 5.7 mil-lion affected dementia patients in the EU25.No curative therapies are currently avail-able for these dementias; however, firstdis ease-modifying treatment strategiessuch as Aβ-immunisation have enteredclinical trials. These novel treatments willbe most effective if they are offered ata very early dementia stage. Currently,however, the reliable clinical differentialdiagnosis of very early dementia stages isnot satisfactory. Here, recent research hasclearly demonstrated that multipara-metric neurochemical dementia diagnos-tics (NDD) in cerebrospinal fluid (CSF)clearly improves the early and differentialdiagnosis of dementias.

cNEUPRO will apply advanced proteomictools to discover novel neurochemicaldementia markers (BIOMARKERS) in bloodand CSF for the improved early, as well aspredictive, diagnosis of AD. A predictivedementia diagnosis is essential in orderto optimise the effectivity of forthcomingpreventive therapeutic strategies. Ourinitiative will establish European standardoperating procedures (SOPs) for currentneurochemical dementia diagnostics(NDD) and we will establish the first NDDreference centres in Portugal and Hungary.

A strong methodological impact is placedon the quality of pre-analytical sample han-dling and clinical phenotyping, which hasbeen neglected in industry-driven discov-ery studies. cNEUPRO integrates innovativebiotech and bioinformatic companies withleading clinical and proteomic dementiaresearch centres. cNEUPRO will also sup-port the discovery of new diagnostic targetsand, furthermore, will provide a promisingapproach to identify novel scaffolds foradvanced molecular neuroimaging.

Accordingly, cNEUPRO will contributeconsiderably to the welfare, health andquality of life in Europe, and concomitantlyit will reinforce the competitiveness of theEuropean biotech industry.

Key words: Alzheimer’s Dementia, biomarker, neurochemical dementia diagnostics, cerebrospinal fluid, medical proteomics, blood-based bioassays, multiplex assays, predictive diagnostics for preventive therapy, quality control


Jens WiltfangUniversity of Erlangen-NurembergSchwabachanlage 6D-91054 Erlangen, [email protected] www.uni-erlangen.org

Partners

Sylvain LehmannCentre Hospitalier Universitaire de MontpellierMontpellier, France

Kaj BlennowSahlgrenska Academy at Göteborg UniversityMölndahl, Sweden

Charlotte TeunissenVU University medical centreAmsterdam, The Netherlands

Connie JimenezVrije Universiteit AmsterdamAmsterdam, The Netherlands

Markus OttoUniversitätsklinik UlmUlm, Germany

David Burn (clinics), Chris Morris (proteomics)University of Newcastle upon TyneNewcastle, United Kingdom

Martin WiesenfeldtMatrix Advanced Solutions Germany GmbHLondon, United Kingdomwww.matrix-as.com

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Edgar F. da Cruz e Silva (Proteomics), Odete A.B. da Cruz e Silva (NDD reference center Portugal)University of AveiroAveiro, Portugal

Laszlo VesceiUniversity of SzegedSzeged, Hungary

Johannes SchuchhardtMicroDiscovery GmbHBerlin, Germanywww.microdiscovery.de

Lucilla ParnettiUniversity of PerugiaPerugia, Italy

Helmut E. Meyer, Katrin MarcusRuhr-Universitaet BochumBochum, Germany

Michel BerghFarallone Therapeutics B.V.Amsterdam, The Netherlandswww.farallone.com

Carsten KorthHeinrich Heine University of DuesseldorfDüsseldorf, Germany

Tuula PirttilaUniversity of KuopioKuopio, Finland

Luc BueeInstitut de la Santé et de la Recherche MédicaleLille, France

Stefan MüllnerProtagen A.G. Dortmund, Germanywww.protagen.de

Module II is dedicated to improving the perform-ance of current CSF-based NDD, by definingEuropean SOPs for pre-analytical sample han-dling, sample storage and assay conditions. Aspart of module II, CSF quality surveys will be con-ducted, ‘gold standard’ CSF samples devised, andwe will establish two new NDD reference centresin Hungary and Portugal.


• Improved early and predictive diagnosis ofAlzheimer’s Dementia.

• Development of first blood-based NDD fordementia diagnostics and therapy monitoring.

• Identification of potential therapy respondersby indicative biomarker phenotypes.

• Discovery of novel scaffolds, which may possi-bly also be used as therapeutic tools or scaf-folds for molecular neuroimaging.

• Elucidation of the molecular mechanisms involvedin AD.

• Implementation of first European SOPs for CSF-based NDD.

The SME Protagen (Germany), Farallone Therapeutics B.V. (The Netherlands), MicroDiscovery(Germany) and Matrix Advanced Solutions (Germany) are partners in cNEUPRO. They con-tribute commercially tuned management skills, as well as specialist scientific experience toassist in mass spectrometry-driven candidate biomarker identification (Protagen), develop-ment of high-avidity and highly stable small binding proteins (Farrallone Therapeutics B.V.)and sophisticated multidimensional bioinformatic data analysis (Microdiscovery, MatrixAdvanced Solutions). In several areas of the project’s research SME involvement is crucialsince it provides unique proprietary technology, which is essential to the success of theresearch initiative. The cNEUPRO project has realised a close and synergistic networkbetween the SME and academic partners of the research initiative. Moreover, the SME partnersare centrally involved in the scientific management of the consortium.

| Core facility mass spectrometry unit of one of the laboratories of cNEUPRO partners for identification ofbiomarkers.© 2007 Dr. Oliver Ratajczak – strukturwelt.de

There is ample, but only circumstantial, evidencederiving from survival data of patients with earlystages of cancer, suggesting that earlier diagno-sis would allow a 10-20 % survival rate increase. Infact, the potential benefits of early CRC and BCdiagnosis are so high that a wide range of com-munity and governmental efforts have beenimplemented for population wide screening.

Biomarkers are substances found in the blood,other bodily fluids (e.g. urine) or tissues that eitheralone or in combination may signal the presence ofcancer or the risk of cancer. Diagnostics based onbiomarkers have the potential to significantlyimprove current cancer diagnostic means, provid-ing a higher sensitivity (i.e. much smaller tumourscan be detected), more easily, faster and at a muchlower cost (5).

Biomarker discovery and validation, in the sameway as drug discovery and validation, is a longprocess with a high rate (60-80%) of attrition ofcandidate biomarkers along the major steps ofqualification that ultimately ends in the approvalby the Food and Drug Administration (FDA) in theUS and the European Agency for the Evaluation ofMedicinal Products (EMEA) in the EU. Often,seemingly good candidates that have been identi-fied and found valuable in one study do not showthe expected predictive values in the secondstudy. In fact, the number of new diagnosticsapproved per year is decreasing, in sharp contrastto the intensifying efforts to discover biomarkers.Therefore, despite having the highest potentialvalue, COBRED chose not to pursue the discoveryof screening markers because of economic andlogistic impracticalities of a large scale screening-marker validation in BC and CRC. Instead, the proj-ect focuses on the second largest clinical need,namely the improvement of patient follow-up,through the discovery of monitoring markers,which are expected to report relapse, metastasisand minimal residual disease at earlier stages,which are more amenable to surgical andchemotherapy treatment, and which are morelikely to improve cancer patient survival.

Background

An apparent paradox of current cancer epidemiol-ogy is that while new therapies and diagnosticsimprove survival rates in common cancers, e.g.colon and breast cancer, the incidence rates arealso increasing and thus the net effect is negative.

Colorectal cancer (CRC) is the third most commoncancer type worldwide; in the year 2000 theglobal incidence was around 1 million, close to10 % of all cancers, resulting in about 0.5 milliondeaths, totalling almost 8 % of all cancer mortal-ity (1). Lifetime risk of developing colorectal ade-nocarcinoma is one of the highest of all cancers,approximately 6%, and of colorectal adenoma,the benign but precancerous lesion, is approxi-mately 50%. The risk of CRC rises with age, partic-ularly after the age of 60.

Breast cancer (BC) is the most common canceramong Western women. In these patients, it is notthe primary tumour, but rather its distant metas-tases that are the main cause of mortality. Theyearly incidence rate is over 0.5 million (630 000new breast cancer cases) which result in around0.2 million deaths. Recently, the rates of metasta-sis and mortality in BC patients have decreased asa result of early diagnosis by mammographicscreening and the implementation of systemicadjuvant therapy similarly to CRC. However, asthe population ages, the incidence of breast can-cer increases (2).

Continuous improvements in the treatment ofanother major life threatening illness, namelycardiovascular disease, leads to an increase inthe overall life expectancy. This contributes tothe increase of the incidence rate, in CRC and BCamong others, due to population ageing. Thespeed of population ageing is higher than thedecreased mortality due to the accumulatedtreatment benefits from new cancer diagnos-tics (3) and therapies (4) thus leading to a para-doxically negative net effect.

Colon and breast cancer diagnostics

ACRONYM

COBREDwww.cobred.eu

COBRED aims at discovering colon cancer(CRC) and breast cancer (BC) biomarkersfor patient follow-up (monitoring markers),by exploiting the capacity of three state-of-the-art high-throughput technologies inan integrated systems biology approach.The specific RTD objectives are to:• design a clinical protocol for prospective

clinical CRC and BC collections that fitthe needs of the three high-throughput‘omics’ technologies used, namely trans -criptomics, proteomics and metabolomics;

• identify biomarker candidates (metabo-lites, proteins, PBL derived mRNAs)capable of detecting and assessing thestatus of minimal residual disease,metastases and recurrence after surgeryand chemotherapy;

• develop a centralised database to inte-grate the data generated by the threetechnology platforms, to include the clini-cal and pathological information on thecollections;

• discover biomarkers with better speci -ficity and sensitivity, using cross-platformadvanced data-mining techniques on thecombined data from the consolidateddatabase; and

• validate the biological relevance and diag-nostic potential of the identified biomark-ers by testing their specificity on tissuearrays and in relevant preclinical models.

COBRED gathers the expertise and RTDresources of three biotech SMEs, leadingacademic partners and two leading cancertreatment centres renowned for theirexpertise in BC and CRC treatment. After3 years, COBRED will deliver a set of bio-marker candidates verified in preclinicalstudies and ready for large scale clinicalvalidation and further development forcommer ciali sation by the respective SMEpartners. Furthermore, COBRED will havedemonstrated the potential to exploreconsolidated data resulting from differenthigh-throughput technologies and clinicalprofiles with advanced data mining tech-nologies for enhanced biomarker discov-ery. Although within the project scope,COBRED focuses on biomarkers for fol-low-up diagnostics, which have the poten-tial to evolve into early cancer detection &screening tools.

SUMMARY

34



Key words: cancer, biomarker, monitoring markers, breast, colon, transcriptomics, proteomics, genomics, metabolomics, datamining

Aim

COBRED aims at discovering colon cancer andbreast cancer biomarkers for patient follow-up(monitoring markers) by exploiting the capacity ofthree state-of-the-art high-throughput technolo-gies in an integrated system biology approach.The goal is to identify biomarker candidates(metabolites, proteins, PBL derived mRNAs) capa-ble of detecting and assessing the status of mini-mal residual disease, metastases and recurrenceafter surgery and chemotherapy.

Expected results

COBRED will deliver candidate protein, metabo-lite and mRNA biomarkers tested in preclinicalstudies, ready for large-scale clinical validationand further development for commercialisationby the respective SME partners: Ipsogen formRNA derived markers, BioSystems Internationalfor protein markers and Biocrates Life Sciencesfor metabolomics markers.

Specific project results will include:• sets of biomarkers (gene signatures, proteins,

metabolites, or a combination of these) that willbe considered clinically relevant for early diag-nosis of primary BC and CRC and relapses;

• central repository system hosting the resultsfrom the technological platforms and the rele-vant clinical data;

• prospective clinical collection of BC and CRC;• clinical validation for the diagnostic potential of

subsets of the identified biomarkers in compari-son to existing biomarkers and to currentlyavailable imaging techniques;

• preclinical models for the biomarker evaluationand biological studies.


Diagnostic kits for BC and CRC patient follow-up.

References

(1) Midgley R. et. al. Nat Clin Pract Oncol. Jul; 2(7):364-9 (2005);

(2) Parkin D.M, et. al. Int J Cancer 1999; 80: 827-841. (1999),

Elmore J.G. et. al. JAMA. 293:1245-1256 (2005); (3) Shen Y, J et. al.

Natl Cancer Inst. 17;97(16):1195-203 (2005); (4) Nygren P et. al.

Acta Oncol. 44(3):203-17. (2005); (5) Baker M. Nat Biotechnol.

23(3):297-304. (2005).

BioSystems International, a French SME, is the coordinator of COBRED.

The three SMEs involved (BioSystems International, Biocrates Life Sciences and Ipsogen)are research-intensive SMEs who play leading roles given their expertise in ‘omics’(genomics, proteomics and metabolomics) technologies, an issue which is at the heart ofthe COBRED project and the technological basis for the achievement of the project’s objec-tives. The targeted project results are clearly of interest and potential benefit to SMEs, sincebusiness opportunities will be created for them in the field of diagnostic tools and methods(and related IPR).

A fourth SME (ARTTIC) is responsible for the project management.

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Laszlo TakacsBioSystems International S.A.S.4 rue Pierre Fontaine, 91058 Evry cedex, [email protected]

Partners

Klaus WeinbergerBiocrates Life Sciences A.G.Innsbruck, Austriawww.biocrates.com

Fabienne HermitteIpsogenLuminy Biotech EntreprisesMarseille, Francewww.ipsogen.com

Xavier Sastre-GarauInstitut CurieParis, Francewww.curie.fr

David MalkaInstitut Gustave RoussyVillejuif, Francewww.igr.fr

László FésüsUniversity of DebrecenDebrecen, Hungarywww.unideb.hu

Andras GuttmanUniversität InnsbruckInnsbruck, Austriawww.hlbs.org

Jaak ViloUniversity of TartuTartu, Estoniabiit.cs.ut.ee

Bruno CucinelliARTTIC S.A.S.Paris, Francewww.arttic.com

• to increase the speed of scoring of comets;

• to use lesion-specific enzymes and inhibitors tomeasure different kinds of DNA damage;

• to develop and compare methods for measuringDNA repair activity;

• to develop an approach to measure gene-spe-cific DNA damage and repair;

• to validate the comet assay in its various forms;

• to develop reference and internal standards foruse in the comet assay;

• to make the various innovative products avail-able for use by companies and researchersinvestigating DNA damage and repair.

Expected results

Validated assays for DNA damage and repair thatwill be accepted by regulatory authorities for usein genotoxicity testing and will thereby reducereliance on experimental animals.


Commercial genotoxicity testing, biomonitoring inhuman population studies, basic research in DNAdamage and repair.

Background

The comet assay for DNA damage is widely used,but has a major drawback as it is labour-intensive,and therefore can be used only in studies in whichthe numbers of samples are relatively small. Thedevelopment of high-throughput variants willincrease its applicability in both genotoxicity test-ing and population biomonitoring. DNA damage isa good marker of exposure to genotoxic chemi-cals; measuring cellular repair of this damagegives additional information on the mode ofaction of genotoxic agents and on individual sus-ceptibility to carcinogens.

Aim

The overall objective is to develop reliable andtested genotoxicity and cytotoxicity assays that,combined, will reduce the need for animal experi-ments in assessing the safety of chemicals.Specific aims include:

• to increase the throughput of the comet assayup to 20-fold;

• to develop further the cell array system as a par-allel assay for cytotoxicity;

• to seek optimal cell types for use in genotoxicityand cytotoxicity testing;

Comet assay and cell array for fast and efficient genotoxicity testing

ACRONYM

COMICScomics.vitamib.com

A battery of reliable and validated in vitroassays is needed to test for genotoxic andcytotoxic effects of chemicals, withoutresorting to animal experiments. Thecomet assay, a sensitive indicator of DNAdamage, will be combined in this projectwith the Cell Array system, to establishand validate high capacity assays suitablefor chemical testing. Up to 800 cell sam-ples will be processed for comets on a sin-gle microscope slide. Arrays will use cellswith different metabolic capabilities, withdata on cytotoxicity obtained in parallelwith DNA damage. A medium-throughputassay will also be developed. Cometanalysis by differential staining of dam-aged/undamaged DNA using establishedand novel dyes combined with automatedimage analysis will be faster and morereliable than at present. A crucial aspectof the cellular response to DNA damage isDNA repair; variations between peoplecan affect cancer risk, while genotoxicchemicals can act by interfering withrepair. Two methods for measuring repair,one based on the comet assay, the otherusing a ‘Repair Chip’ approach, will becompared. In addition, fluorescent probeswhich lock onto the DNA after hybridis-ation (‘padlock probes’) will be appliedto study gene-specific DNA repair. Themodified comet assay methods will beassessed for reproducibility and sensitiv-ity in an inter-laboratory prevalidationtrial, using a coded set of standard chem-icals, to satisfy regulatory bodies, as wellas industrial users of the technology. Theresult will be robust, validated, high-throughput in vitro genotoxicity tests.This proposal brings together academicpartners and SMEs, together with a largeindustrial concern, and the EuropeanCentre for the Validation of AlternativeMethods (ECVAM). Dissemination throughprofessional and trade publications, regu-latory channels, and scientific conferenceswill lead to widespread adoption of thenew methods.

SUMMARY

36



Key words: DNA damage, DNA repair, genotoxicity, comet assay, biomonitoring

Out of the seven industrial participants to this project, five are SMEs and they contribute tothe key activities in the project, devising new software for image analysis as required by thenovel methods of scoring of comets (IMSTAR); providing suitable cell lines (Biopredic); pro-viding custom-made DNA-damaging chemicals for calibration purposes (Severn Biotech);providing dyes for differential staining of intact and damaged DNA plus running trainingworkshops (TATAA Biocenter); pre-market product development and testing (Thistle).

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Andrew CollinsDepartment of NutritionUniversity of OsloPB 1046 Blindern0316 Oslo, [email protected]

Partners

Brigitte Fouque Commissariat à l’Énergie AtomiqueGrenoble, France

Françoise SoussalineIMSTAR S.A.Paris, Francewww.imstarsa.com

Thomas HartungJoint Research CentreIspra, Italy

Gunnar BrunborgNorwegian Institute of Public HealthOslo, Norway

Christiane GuillouzoInstitut National de la Santé et de la Recherche MédicaleRennes, France

Katarina VolkovovaSlovak Medical UniversityBratislava, Slovakia

Jon Eigill JohansenChiron ASTrondheim, Norway

Neven ZoricTATAA Biocenter ABGöteborg, Swedenwww.tataa.com

Elizabeth MartinAstraZenecaMacclesfield, United Kingdom

Galen MilneThistle Scientific LimitedGlasgow, United Kingdomwww.thistlescientific.co.uk

Andrew SmartSevern Biotech Ltd.Kidderminster, United Kingdomwww.severnbiotech.com

Christophe ChesneBiopredic InternationalRennes, Francewww.biopredic.com

Mats NilssonUppsala UniversityUppsala, Sweden

Karel AngelisInstitute of Experimental BotanyPrague, Czech Rep.

| The principle of the ‘padlock probe’. The probe(left panel) has at its ends sequences complementaryto adjacent parts of the target DNA. After hybridisation,these ends are ligated and the probe thus locked ontothe target. These probes can be designed to lock ontodefined gene sequences, and so permit an examinationof damage and repair in those specific genes. [Figure provided by Prof. Mats Nilsson.]

In contrast to natural antibodies (which are germline encoded), antiphospholipid antibodies (aPL)are risk markers for both arterial and venousthrombosis, and the main focus is novel types ofaPL against platelet activating factor (PAF) or PAF-like lipids (aPAF). These novel risk markers (aPAF)will be tested in the same cohorts as aPC and, inboth cases, robust kits will be developed. Thegenetic background of aPC and aPAF will be stud-ied, e.g. in the Swedish Twin Registry. In a proto-typic autoimmune disease, namely SLE, where therisk of CVD is very high, such aPL are raised.Annexin A5 is a plasma protein, interfering withphospholipid surfaces and acting as an anticoag-ulant. The project has demonstrated that aPLinhibit Annexin A5 binding to endothelium, aneffect neutralised by intravenous immunoglobu-lins (IVIG) and that Annexin A5 is present in ather-osclerotic lesions, especially at sites prone torupture. Based on these findings, the project hashypothesised that raising Annexin A5 binding,either by administration of neutralising antibod-ies from IVIG in individuals with high aPL levels, orby administration of Annexin A5 per se, couldprevent plaque rupture and atherothrombosis.

Taken together, the project combines diagnosticand therapeutic projects and is focused on therole of the immune system, especially autoanti-bodies, in atherosclerosis and atherothrombosis.

This project combines academic and industrialexpertise and objectives, aiming at developing:• a novel protection marker for atherosclerosis,

natural IgM antibodies against phosphoryl-choline (PC) and novel risk markers, IgG autoan-tibodies against phospholipids (aPL): plateletactivating factor (PAF) and PAF-like lipids (aPAF);

• these markers will be investigated in uniquepatient cohorts. Documentation for registrationof diagnostic ELISA kits in EU and the U.S. will bedeveloped;

• novel immunomodulation therapy against ath-erosclerosis and CVD in direct association withthese factors will be developed, along with the

Background

Cardiovascular disease, mainly caused by athero-sclerosis, is the main cause of death in theWestern world and also increasingly in develop-ing countries. Even though progress has beenmade in prevention of the disease, the inflamma-tory and immunological nature of atherosclerosisis not reflected by available diagnostic measures.Furthermore, treatment has been improved bylipid lowering drugs, e.g. statins, but novel treat-ment modalities aiming at the pathologicalinflammatory and immune reactions characteris-ing atherosclerosis are yet to be developed.Likewise, the understanding of the mechanismsleading to atherosclerosis and causing the immunereactions are poorly characterised. This applies tothe general population but also to SLE, an autoim-mune disease where the risk of CVD is very high.

Aim

Atherosclerosis, the major cause of cardiovascu-lar disease (CVD) is an inflammatory disease,where the immune system plays an importantrole. Autoantibodies as protection or risk mark-ers, and therapy through immunomodulationcould be a major advance in the prevention andtreatment of CVD and is a focus of this project.One type of antibodies are natural antibodiesagainst phosphorylcholine (aPC), an antigenexposed in some bacteria and in phospholipidswith platelet activating factor (PAF)-activity, e.g.in oxidised LDL. The project’s clinical and experi-mental studies indicate that aPC, mainly of IgMtype, are protection factors against human ather-osclerosis and CVD. The project’s plan is thereforeto develop aPC as a diagnostic tool, which will betested in several unique cohorts, includinghealthy individuals and high risk individuals. Theproject also thinks aPC could represent a noveltherapeutic modality, where both polyclonal andmonoclonal aPC are possibilities. Both kinds ofaPC antibodies will be developed and tested inmouse animal models for atherosclerosis.

Immunomodulation and autoimmunity in cardiovascular disease and atherosclerosis

ACRONYM

CVDIMMUNEwww.cvdimmune.com

The CVDIMMUNE project aims at: Investi -gating novel risk markers for CVD inEuropean patient cohorts of in about15 000 patients including unique cohorts asthe Swedish Twin registry, the MORGAM-study and SLE. Investigating immunomod-ulation as a therapeutic strategy for ofCVD. Developing documentation for regis-tration of diagnostic ELISA kits in EU andthe US. Developing proof of concept for atleast one pharmaceutical product candi-date. Providing a strong research foundationand knowledge on mechanisms.

SUMMARY

38


Starting date: 1 August 2006

Key words: atherosclerosis, atherothrombosis, natural antibodies, antiphospholipid antibodies, SLE, immunomodulation

administration of atheroprotective aPC and theincrease of Annexin A5 binding to endotheliumthrough the neutralisation of aPAF and other aPLor administration of Annexin A5 per se, a plasmaprotein that may prevent rupture of atheroscleroticplaques and CVD;

• proof of concept for at least one of these phar-maceutical product candidates.

Expected results

• Development of robust and quantitative test kitsin ELISA format for analysis of IgM antibodies toPC and IgG antibodies to PAF or PAF-like lipids inserum.

• Epidemiological data on aPC and aPAF from sev-eral well characterised and, in several cases,unique cohorts of healthy and high-risk individuals.

• Assessment of genetic variability of aPC andaPAF and identification of candidate genes.

• Establishment of in vitro and in vivo models forstudies of aPC and Annexin A5 relating to ather-osclerosis and atherothrombosis.

• In vivo data for drug candidates for atheroscle-rosis and atherothrombosis and data compiledto submit an IND application from one of thedrug candidates investigated in the project.


The project expects its novel protection and riskmarkers to reflect inflammatory and immunologi-cal factors in plaques and to be useful as tools forrisk assessment for CVD, as a complement to clas-sical risk factors as dyslipidemia, hypertension,diabetes, and smoking. The project also proposesthat treatment relating to these factors could bea major step forward, in addition to establishedones such as lipid lowering drugs.

SMEs play a pivotal role in CVDIMMUNE. Athera Biotechnologies AB is in collaboration withKarolinska Institutet, the originator of the majority of the project ideas and product candidatesexplored in the project. Athera develops the two diagnostic risk markers and therapeutic candi-dates to be investigated. Furthermore, Athera is the main party responsible for the commercial-isation of the results of these efforts. The second SME in the project, IsoSep AB, plays an integralrole by developing the chemistry solutions vital to several of the programmes in the project.

ROLE OF SMEs

39


Johan FrostegårdDepartment of Medicine, Karolinska University Hospital, Huddinge Center for Infectious Medicine and Rheumatology Unit, 141 86 Stockholm, Sweden. [email protected]

Project manager

Narinder [email protected]

Partners

Hans GrönlundAthera Biotechnologies AB Stockholm, Swedenwww.athera.se

Peter SeverImperial College LondonLondon, United Kingdom

Stefan Blankenberg Johannes Gutenberg-University Mainz Mainz, Germany

Ewa Ninio Institut National de la Sante et de la Recherche Medicale, U525Paris, France

Paul Quax TNO Netherlands Organization for Applied Scientific ResearchLeiden, The Netherlands

Marta E. Alarcón-RiquelmeUppsala Universitet Uppsala, Sweden

Thomas NorbergIsoSep ABTullinge, Swedenwww.isosep.com/home.shtml

Wouter Jukema Leiden University Medical CenterLeiden, The Netherlands

Jörg BlaeserPhadia GmbHFrieburgh, Germany www.phadia.com

| Resting levels of EC intracellular calcium before leukocytes were allowed to interactwith ECs is shown in individual cells through continuous registration of fluorescenceintensity with a widefield microscope. Endothelial cell borders are shown by labellingECs with Alexa 488-conjugated anti VE Cadherins antibodies. ECs (GREEN) wereloaded with Calcium sensitive probe.

| Leukocytes, freshly isolated from venous blood of healthy donors on Ficoll/Hypaquegradients were allowed to settle on the monolayers at 37 °C were allowed to transmi-grate through IL-1 β treated EC at 37 °C. Leukocytes,labeled (Red) and are at differentstages of diapedesis process.

| Changes in EC intracellular calcium were measured during leukocytes extravasationon individual cells through continuous registration of fluorescence intensity witha widefield microscope. Leukocytes were allowed to transmigrate through IL-1 βtreated EC monolayers at 37 °C. A transient increase in Enthothelial cells- [Ca2+]i isshown. ECs (GREEN) were loaded with Calcium sensitive probe. Localized increase inEC Calcium in individual cells is shown (GLOWING GREEN).

Aim

The main objective of the proposed project is toprovide more effective anti-tumour therapies bydeveloping targeted small ligand libraries withappropriate physico-chemical properties for ther-apeutic effect targeted against protein:proteininteractions implicated in various tumour types.The research activities will concentrate on knowl-edge-based approaches supporting translationalresearch aimed at bringing basic knowledgethrough to applications in clinical practice andpublic health. The project will thus focus on pro-viding small molecule ligands with minimal sideeffects as treatments for the brain tumoursglioblastoma (GBM) and medulloblastoma.

Expected results

The successful integration of the various aspectsof this proposal will provide a robust knowledge-based strategy for exploiting protein:proteininteractions as drug targets in the treatment ofbrain tumours. The strategy should however besufficiently generic to be transferable to other dis-ease areas, especially within the CNS, where pro-tein:protein interactions provide an entry pointinto disease-modifying therapies.


The design of selective small molecule modula-tors of protein:protein interactions should pro-vide the basis for developing new therapeuticstrategies against brain cancers. They will allowfor improving current libraries and for buildingpredictive databases for library design of newspecific drugs and therapeutic agents with highcentral nervous system penetration, necessary toreach a higher efficacy, selectivity, responsivenessand lower toxicity. In addition these approacheswill represent a powerful system for preclinicaldata collection, leading to an optimisation ofclinical trials setting and development.

Background

Amongst the range of cancer types, brain andperhaps pancreatic cancers are especially lack-ing in effective treatments. In particular braintumours are:• the leading cause of death from childhood can-

cers among persons under 19;• the second leading cause of cancer-related

deaths in males aged 20-39;• the fifth leading cause of cancer-related deaths

in women aged 20-39.

Although onset of disease varies with tumourtype, it can occur at a relatively young age causingadditional and significant social and economicproblems for both patients and their families.

Current standard treatments include surgery,radiation therapy and chemotherapy. These maybe used either individually or typically in combi-nation. Brain cancers however present uniqueproblems due to the location of the tumours: sur-gery and radiotherapy carry considerable risk tothe patient and resection is not always possible.Chemotherapy is faced with the problem of pene-tration of drugs across the blood-brain barrier(BBB) and of lack of specificity. The focus forthese patients is therefore on more effective ther-apies to prevent relapse, and on more efficientscreening and diagnosis to halt the disease at anearly stage.

Designing Therapeutic Protein:Protein Inhibitors for Brain Cancer Treatments

ACRONYM

DEPPICTwww.deppict.eu

Protein:protein interactions (PPI) are cen-tral elements in cellular processes andimportant targets for selective therapeu-tic agents. They constitute a rich area fordiscovery of novel small ligand-basedtherapies. This proposal seeks to utilisesuch interactions, in particular those fea-turing a α-helix binding groove such asp53-MDM2, or more novel targets, e.g.nm23-prune, to develop targeted small-molecule libraries with physico-chemicalproperties appropriate for therapeuticeffect against various tumour types suchas the brain cancers of glioblastoma andmedulloblastoma.Combination of the concepts below shouldprovide an opportunity to unlock the poten-tial of protein interactions as key compo-nents in signalling pathways via designof selective small-molecule modulatorstargeting the kinase-effector interactioninstead of the ATP active site.• Develop an understanding of the ele-

ments controlling selectivity in protein:protein signalling networks by develop-ing approaches for design of small mole-cules that target α-helix binding grooveinteractions through use of structure-based and fragment-based approaches.

• Data-mining of ADME and drug-druginteractions to build a predictive data-base for library design.

• Develop quantitative structure/propertyrelationships, with an emphasis on CYP-mediated metabolism, ABC transportersat the blood/brain and brain/tumourinterfaces, mutagenicity, solubility, pKa,and passive permeability, and predictivetools for mutagenicity and other genetictoxicology end-points.

• Develop predictive PK and PBPK modelsto improve understanding of BBB andtumour penetration.

• In vitro and in vivo PK/PD and TK/TD char-acterisation of compounds, to increaseunderstanding of their mechanism ofaction and reduce the use of laboratoryanimals.

Such knowledge-based approaches willalso be applicable to design of small mol-ecules for other protein:protein interac-tions utilising a α-helix binding grooveboth for peripheral tumours and othertherapeutic areas.

SUMMARY

40



Key words: protein:protein interactions, small molecule inhibitors, knowledge driven drug design, blood/brain, tumour penetrant

The project is specifically designed to increase SME efforts towards research and innova-tion, to encourage collaborations between SMEs and to expand their scientific and techno-logical knowledge base. The four SMEs involved in the project, Sienabiotech S.p.A. (SIBI),Molecular Discovery (MD), Crystax (Crystax), and Aureus Pharma (AUR) take leading rolesin driving and managing the planned library design and drug discovery programme. Theactive and serious involvement of a SME working in drug discovery such as SIBI guaranteesthe rapid application of the research results. Specifically, SIBI researchers will be involvedin library design, construction of in silico paradigms, setting up of experimental metabolismmodels and screening of libraries for ADME attributes. Molecular Discovery will providehigh quality software tools and chemoinformatics procedures aimed at the prediction of thephysicochemical profiling of the investigated compounds with potential anti-tumour activity.Crystax will perform all the necessary processes (cloning, expression, purification and crys-tallisation) to obtain the 3D structure of the pharmacological targets proposed in this proj-ect. This will allow the correct characterisation of the active sites, where small moleculescan bind and have a pharmacological effect. The Aureus Pharma team will set up a databasefrom scientific literature in order to perform data-mining of ADME and drug-drug interac-tions to build predictive tool sets; design and implement predictive models based ondatasets from the knowledgebase developed; and collect pertinent data from literature tobuild predictive toxicology models and filters. In combination, the four SMEs carry out crucialroles in the DEPPICT project.

ROLE OF SMEs

41


Graeme RobertsonSiena Biotech S.p.A.Fiorentina, 153100 Siena, Italy [email protected]/index/index.jsp

Partners

Gabriele CrucianiMolecular Discovery Ltd.Pinner,Middlesex, United Kingdomwww.moldiscovery.com/index.php

Massimo ValotiUniversity of SienaSiena, Italy

Juan AymamiCrystax Pharmaceuticals S.L.Barcelona, Spainwww.crystax.com

Roberto PellicciariUniversity of PerugiaPerugia, Italy

Sophie OllivierAureus PharmaParis, Francewww.aureus-pharma.com

Prof. Herbie NewellUniversity of NewcastleNewcastle, United Kingdom

| Iterative Research Cycles where data will be used to construct robusttestable hypotheses in the context of the pathophysiology of braintumours. To achieve this it is necessary to look for trends – StructureActivity Relationships. The discovery and exploitation of site/mechanismof action and developing knowledge at multiple levels – Target, Cell, and Pathway will be key to achieving this.

Expected results

DeZnIT is highly focused on the identification ofnovel and more effective drug candidates, whichwill be further developed for serious human dis-eases. The other outcomes of DeZnIT are improvedmethods for computational drug design and otherplatform technologies such as crystallography,molecular biology (including development of bio-markers) and chemical synthesis. The success ofDeZnIT would lead to significantly reduced costsand timelines for drug discovery processes, withconsequent health and economic impacts.


In addition to the specific applications outlinedabove in the area of zinc metalloenzymes, anymethods developed during the course of DeZnITmay be applicable to other classes of metal con-taining enzymes of therapeutic importance.

Background

Zinc-containing metalloenzymes have a widerange of specific biological activities. They areimplicated in several types of diseases such ascancer, atherosclerosis, neuronal degenerativediseases, glaucoma and inflammation. In this pro-posal, the project concentrates on the therapeuti-cally important zinc-containing enzyme families ofcarbonic anhydrases, histone deacetylases and theADAM (A Disintegrin And Metalloprotease domain)family of proteinases, in particular ADAM17, alsoknown as TACE (TNF-α Converting Enzyme).

Whilst many computer-assisted drug designmethods have been developed over the past sev-eral decades, their application to the medicinallyimportant class of zinc-containing enzymes hasbeen challenging. The nature of the interactionbetween the potential drug molecule and a metalsuch as zinc requires a very time consuming cal-culation and obtaining accurate results is difficultto achieve in a real-world setting.

Aim

• To develop modern, sophisticated comput-erised methodology for high-throughput drugscreening in silico, focussing on Zn-containingenzymes.

• To isolate and characterise target proteins withinthe Zn-containing enzyme families (includingnewly identified members) for the screening ofmore efficient drugs.

• To design and create new classes of enzymeinhibitors.

Design of zinc metalloenzyme targeted drugsusing an Integrated Technology approach

ACRONYM

DeZnIT

The objective of DeZnIT is to develop andapply new methodology for the rationaland accelerated design of novel drugs tar-geted against zinc-containing enzymes.These enzymes are key modulators ofmany serious human diseases includingcancer, glaucoma, obesity, and rheuma-toid arthritis. Despite some successes,many major technological challengesremain in the development of effectivedrug therapies against this enzyme family.

DeZnIT will address these challenges bydeveloping and integrating key technolo-gies from pharmacogenomics, computermodelling, structural and molecular biol-ogy and chemistry, combined with drugdiscovery infrastructure and expertise. Inparticular, highly novel computationalapproaches taken from the field of com-puter science will be applied to drugdesign for the first time.

The consortium members combine all thenecessary competencies to achieve thegoals and objectives of DeZnIT.

SUMMARY

42



Key words: zinc metalloenzymes, drug design, computer-assisted drug design, cancer, inflammation

There are three SME participants in DeZnIT, each playing a key role. One of the SMEs,InhibOx, is the project co-ordinator and, in addition, will focus on the delivery of novel com-putational methodology. KeyDP will provide molecular and structural biology expertise,whilst TopoTarget will provide drug screening technology. Together, the SMEs provide sig-nificant expertise in the commercial development of small molecule therapeutics in a highlysynergistic manner with the academic partners.

ROLE OF SMEs

43


Paul FinnInhibOx Ltd. Pembroke house36-37 Pembroke street Oxford OX1 1BP, United [email protected]

Partners

Steven ButcherTopoTarget A/SCopenhagen, Denmarkwww.topotarget.com

Flip HoedemaekerKey Drug Prototyping B.V.Amsterdam, The Netherlandswww.keydp.com

Peteris TapencierisLatvian Institute of Organic SynthesisRiga, Latvia

Seppo ParkkilaUniversity of TampereTampere, Finland

Andrea ScozzafavaUniversity of FlorenceFlorence, Italy

William Graham RichardsUniversity of Oxford Oxford, United Kingdom

| A detail from the crystal structure of carbonic anhydrase II, a key enzyme of interest to the project and the first crystal structure produced by the consortium.

Expected results

Novel technologies• New principles for NA separation and

purification• New sorbent methods and materials• Protocols for sample preparation • Protocols for samples target concentration• Chemistry for oligonucleotides synthesis• Multiplex PCR• Protocols for diagnostic assays• Protocols for POC (point of care) sample

preparation

New products• Kits for ultra-rapid separation and purification

of DNA and RNA• Kits for separation and purification of NA from

difficult (inhibitor rich) samples• Kits for separation of different classes of NA• Kits for non-cultural enrichment of

micro-organisms• Kits for diagnostic assays of critical pathogens• Prototype instrument for fluorescent detection• Portable PCR laboratory• Kits for sample preparation outside the

laboratory


The application workpackage will concentrate butnot limit its activities to the following groups ofpathogens and critical sample sources for multi-plex PCR systems with fluorescence detection,thus approving and demonstrating the broadand beneficial universal applicability of the tech-nological development in the work packagestechnological work packages:• mycobacterium complex:in sputum;• difficult culturable fastidious periodontal

pathogens in gingival crevicular fluid;• predominant food-borne human pathogens;• gut flora pathogens in stool samples for analyses

of intestinal microbiota associated with inflam-matory bowel disease;

• fungal pathogens in hair, skin and nail samples.

Background

Infectious diseases are a global concern. Of theannual 12 million deaths attributable to infectiousdiseases on the planet, 95% occur in the developingworld, particularly in the most impoverished areaswhere individual and general hygiene standardsremain very low and prevention policies are non-existent, poorly adapted or insufficiently funded. Onthe other hand, economic and industrial develop-ment also accounts for the emergence of infections,such as food-borne pathogens that take advantageof the increasing industrialisation of the food chain,nosocomial infections in the increasingly complexhospital environment and travel-related infections.In addition, the demographic trend towards anageing population in Europe enhances the risk ofincreasing infection as elderly people are prone toinvasive medical procedures and are, in general,more susceptible to infectious diseases.European science must play a major role in this fightto curb infectious diseases, particularly throughthe establishment of a stronger and more coher-ent surveillance and control system and througha substantially increased investment in researchto underpin this endeavour.Only with this investment will Europe be able tomanage infectious diseases within its own bound-aries. In addition, Europe will also be able to helpprevent the emergence and spread of infectionsprevailing elsewhere on the globe and to pursueits historical tradition of giving assistance to thepoorest countries.

Aim

The main strategic objective of DIAGNOSIS is to con-tribute to the above need by developing a novel,easy to use, low cost and non invasive biotechnol-ogy for infectious diseases diagnosis, i.e. pathogensdetection.The challenging aim is to reach a short and efficientsample preparation followed by highly sensitive,accurate, cost efficient multiplex diagnosis imple-mented i.a. into a portable PCR fluorescent labora-tory. The concept will be proven on human criticalpathogens but will be applicable also for animals,plants and environmental, and food samples.

Development of new and cost effective methodsfor non-invasive diagnosis of human pathogens

ACRONYM

Diagnosis

Infectious diseases continue to be a seri-ous burden around the world, in bothdeveloping and industrialised countries.

The main objective of DIAGNOSIS is to con-tribute to the above need by developinga novel easy to use, low cost, mostly noninvasive biotechnological platform for thedetection of infectious diseases. The chal-lenging aim is to reach a short and efficientsample treatment implemented into exist-ing and newly developed portable PCR lab-oratory for multiplex fluorescent pathogendetection. The concept will be proven onhuman critical pathogens but will beapplicable also for animals, and plantspathogens. The concept will be applica-ble for samples taken from affectedorganisms as well as from food/feed andenvironments (water, air, soil etc.).

The scientific and technological objec-tives will include the development of: • new principles of nucleic acids sorption/

desorption; • innovative concentrating methods and

materials for the enrichment of viruses,micro-organisms and nucleic acids;

• the adaptation of the phosphoramiditechemistry to alternative fluorescencedyes in order to broaden the labellingassortment for multiplex PCR analyses,a portable non-dependent on externalpower supply PCR laboratory;

• exemplary demonstration of the wholetechnological chain on several groupsof human pathogens, including theMycobacterium complex, the periodontalpathogens, the causal agents of popula-tion shifts within the intestinal microfloraassociated with inflammatory boweldiseases and Fungal skin infections,completed by the diagnosis of mainrepresentatives of food-born pathogens.

SUMMARY

44



Key words: infectious diseases; diagnosis; nucleic acids, sample preparation, monolithic sorbent; DNA, RNA, PCR, real-time; fluorescence detection; multiplex PCR; phosphoramidite chemistry; portable instrument; Mycobacterium; gingival crevicular fluid, food-born pathogens; gut flora Pathogens; Fungal pathogens

DIAGNOSIS is enhancing the competitiveness of Europe’s biotechnology industry by thedevelopment of fast and reliable nucleic acids separation and purification, new methods forseparation from inhibitor enriched samples and new instruments and kits for fast and costeffective detection of critical human pathogens.

Most of the partners are industrial partners, thus taking the outcome of the project directlyto the European biotechnology industry.

• The consortium consists of 73 % SMEs, all of which are based on innovative technologiesand research, with 67 % of the budget allocated to SMEs. The main idea is to promote theirtechnologies and develop new products by enhancing their business plans.

• The project management is undertaken by two SMEs: one an expert with the technologi-cal work, and the other an expert in administrative management and coordination of EUfounded projects. This company is led by a woman.

• DIAGNOSIS is allocating one WP to exploitation and dissemination by its SMEs partners,including internal workshops and staff exchange between the relevant entities. A detailedplan of staff exchange will be implemented during the project.

• International Cooperation is assured through the participation of 3 partners from INCOcountries (Russia and Armenia); one of which is a Russian SME (DNAT), while the othertwo are research institutes which will provide essential know-how for the project.

ROLE OF SMEs

45


Robert-Matthias LeiserAgrobiogen GmbH Thalmannsdorf 25, Larezhausen86567 Hilgertshausen, [email protected]

Partners

Denis RebrikovDNA-Technology jscMoscow, Russiawww.DNA-Technology.com

Claus-Detlev BauermeisterLabor Dr. Bauermeister & CoMoers, Germany

Radovan HaluzaGeneri Biotech s.r.o.Hradec Kralove, Czech Republicwww.generi-biotech.com

Camilla GiammariniDIATHEVA SrlFano, Italywww.diatheva.com

Martin GehriPreentTec AGFribourg, Switzerlandwww.preentec.ch

Ronald Arthur BoschHilbrands Laboratorum B.V.Wijster, The Netherlandswww.hlbbv.nl

Sergey ZavrievM.M. Shemyakin-Yu.A. Ovchinnikov Institute of Bioorganic chemistry of RASMoscow, Russiawww.ibch.com

Hamlet BalayanInstitute of Fine Organic Chemistry of NASYerevan, Republic of Armeniawww.sci.am/resorgs.php?oid=14&langid=1

Ulf GoebelCharité – Universitätsmedizin Berlin Institut für Mikrobiologie und HygieneBerlin, Germanywww.charite.de/imh

Pnina DanOSM-DAN Ltd. Rehovot, Israelwww.osmdan.com

Aim

The DIALOK consortium is dedicated to thedevelopment of innovative drugs that displaya restricted action within specific target cells ororgans. Such locally acting drugs will constituteimproved safety and efficacy, since side-effects inother tissues will be prevented. The project willinvestigate this therapeutic strategy for kinaseinhibitors, a class of drugs that has tremendoustherapeutic potential but that is also associatedwith unacceptable side effects. Kinase inhibitorswill be conjugated in two classes of carrier sys-tems, directed towards either the liver or kidney.In this way, the project will develop locally actingdrugs for the treatment of either hepatic or renalfibrosis, aiming to prove this concept in experi-mental animal models.

An important novel aspect of the project is thelinker used for coupling the drug to the carrier.DIALOK will employ Kreatech’s Universal LinkerSystem (ULS), a novel patented drug-linker whichenables straightforward coupling with mostkinase inhibitors (broadly applicable) withoutchanging the original drug structure. Anotherunique property of ULS is that it provides con-trolled release of the conjugated drug overa period of days, which is of great importancefor effectiveness of the delivered drug.

In parallel to development of locally acting kinaseinhibitors, DIALOK will develop novel assays fordetecting activation of kinases. Such assays willbe of great value to the project, accelerating andstrengthening the validation of kinase inhibitor-based therapeutics. Kreatech’s ULS-based (fluo-rescent) reporter molecules are particularlysuitable for the detection of phosphorylatedkinase substrates, and therefore enable broadlyapplicable, non-radioactive detection of activatedkinases, as compared with traditional kinaseactivity assays, which use radiolabeled ATP orphosphoaminoacid-specific antibodies.

Background

Kinases are a class of enzymes that play a key rolein intracellular responses to stress and growthfactors. Kinases catalyse the transfer of phos-phate from ATP to proteins, which may either beother kinases, transcription factors or other regu-latory proteins. As such, the activation of kinasesleads to a cascade of activating signals that regu-late many physiological processes, e.g. cell divi-sion and differentiation. Aberrant kinase activityis associated with many diseases such as cancer,diabetes, and chronic inflammatory disorders.Kinases have been identified by the pharmaceuti-cal industry as important targets for drug devel-opment, with major investment having been madein basic research and drug design in this area,estimated at 20 % of the total R&D budget in thepharmaceutical industry. Over 500 kinases havebeen identified in the human genome and manynew kinase inhibiting drugs will be developed inthe coming decades. Indeed, some of the mostsuccessful drugs introduced in the past years arekinase inhibitors, such as Gleevec and Irresa.

Within the development of this class of highlypotent drugs, some critical problems have arisen.For example, even kinase inhibitors with highspecificity for a specific kinase sometimes haveadverse effects that prevent the further develop-ment of safe drugs. This problem is inherent to theclass of compounds, since kinases often play animportant role in both pathophysiological andnormal physiological processes.

Development of Innovative Assays and Locally acting therapies aiming at critical Kinases inhepatic and renal fibrosis

ACRONYM

DIALOK

The DIALOK consortium consists of twoSMEs (based in The Netherlands andHungary) and five academic participants(based in The Netherlands, Germany andSpain) and is dedicated to the develop-ment of innovative drugs that displaya restricted action within specific targetcells or organs. Such locally acting drugswill have an improved safety and effi-cacy, as side-effects in other tissues willbe prevented.

SUMMARY

46



Key words: drug delivery, kinase inhibitors, kinases, liver disease, renal disease, drug safety, inflammation, fibrosis

Expected results

The DIALOK consortium proposes the improvedsafety and efficacy of kinase inhibitors by meansof local drug delivery. By restricting the activity ofthe inhibitor to the diseased tissue only, the bal-ance between therapeutic effects and side-effects can be altered dramatically (Fig. 1). Sincethe locally delivered drug will provide adequatedrug levels within the targeted organ, in combi-nation with much lower levels in the other partsof the body, the safety and efficacy of the drugswill be improved.


In this project, participants will explore the deliv-ery of kinase inhibitors for the treatment of bothliver and renal fibrosis, since no adequate phar-macotherapy yet exists for these types of dis-eases. The technology will be also applicable tothe treatment of other diseases in which kinasesplay an important role, e.g. cancer, or inflammatorydiseases such rheumatoid arthritis.

There are two SMEs in this project, namely Kreatech and Vichem Chemie. They are experts,respectively, in linking bio-organic molecules based on coordinative chemistry, and in thedesign and synthesis of kinase inhibitors. Taken together, new cell specific therapeutic com-pounds are developed to be tested for specificity and effectiveness by the RTD partnersof the consortium. Kreatech also plays a key role in managing the project as a whole andsafeguarding intellectual property rights.

ROLE OF SMEs

47


Jack VeuskensKREATECH Biotechnology B.V.Vlierweg 201032 LG Amsterdam, The [email protected] www.kreatech.com

Partners

K. PoelstraGroningen University Institute for Drug Exploration Dept. of Pharmacokinetics and Drug DeliveryGroningen, The Netherlands

G. KériVichem Chemie Ltd.Budapest, Hungarywww.vichem.hu

M. TemplinNatural and Medical Sciences Institute at the University of TuebingenReutlingen, Germany

R. BatallerIDIBAPS (Institut d’Investigacions Biomèdiques August Pi i Sunyer) Laboratory of Liver FibrosisBarcelona, Spain

M. RuizUniversity Autonoma Madrid Vascular and Renal Research LaboratoryMadrid, Spain

RJ KokUtrecht UniversityDept. of PharmaceuticsUtrecht, The Netherlands

| Fig. 1 Schematic depiction of how wewant to improve the safety and therapeuticefficacy of kinase inhibitors.

Local diseases require local drug treatment. This can be effectuated by drug delivery.

Aim

The ultimate goal of the project is to developnovel diagnostic tests, based on the best molec-ular understanding reflections in urine to revealthe early diabetic damage during the pre-microalbuminuric stage of the disease.

Expected results

The ultimate goal of the project is to developnovel diagnostic tests based on the best molec-ular understanding reflections in the urine toreveal the early diabetic damage during the pre-microalbuminuric stage of the disease.


The applications will include novel magnetosen-sors for the detection of diabetes-diabetic kidneydamage.

Background

Diabetic nephropathy is the main cause of end-stage renal failure in the Western world. TheWorld Health Organization (WHO) estimates thatcurrently there are over 200 million people withdiabetes worldwide. The prevalence is constantlyrising and estimated to reach over 300 million bythe year 2025. Overall, up to 40 % of diabeticpatients will develop debilitating kidney compli-cations. Diabetic nephropathy is becoming notonly a severe health problem for individualpatients, but also a major economic burden of allsocieties. The project aims to achieve a betterunderstanding of the pathophysiologic mecha-nisms underpinning the development of diabeticnephropathy, a major complication shared byboth type I and type II diabetes.

Predictive diagnostics for diabetic nephropathy – Novel nanotechnology based test platforms

ACRONYM

DiaNawww.diana-eu.fi

DiaNa combines the accumulated expert-ise from the previous FP5 EU project,Mechanisms of Proteinuria (QLG1-2000-00691) and the FP6 project, ADDNET(LSHB-CT-2003-503364). The latest know -ledge on the pathophysiology of diabeticnephropathy and newly identified uri-nary markers are utilised to developpredictive diagnostic tests to follow dis-ease progression. By using metabolomicapproaches, the project aims to findadditional markers from diabetic urine.In parallel, two separate approaches willbe used to develop diagnostic tests, onebased on nanobead technology, and theother on a multiplexing platform allow-ing the combination of several parame-ters in a single test. This will translateinto early identification of patients athigh risk of rapid loss of kidney function.After validation with clinical material,subsequent steps will include transfer ofthe test into patient use by an SME. Thisapproach, which directly aims at devel-oping a clinical urinary test, will be sup-ported by extensive basic research onthe mechanisms/biomarkers of diabeticnephropathy.

SUMMARY

48



Key words: kidney, cardiovascular disease, diabetes, urinary markers, rapid diagnostics, magnetosensors, disease management

Three SMEs are involved, plus a subsidiary of a large company:

• Philogen SpA utilising their expertise to extract antibodies specific for the moleculesinvolved in the pathogenetic sequelae of diabetic nephropathy and used for diagnostics;

• Future Diagnostics B.V. combining the antibodies produced to novel test platforms, includingthe appropriate packaging into existing and to-be-developed concepts;

• United Laboratories Ltd. developing and screening for appropriateness of the platformsdeveloped for daily routines with incoming patient samples; and

• Philips Electronics Netherlands B.V developing novel magnetosensors for detecting lowamounts of target molecules in patients’ urine.

In addition, market analysis and surveys, IPR strategies, and competitor analysis will beprovided by an additional party as a subcontractor of project partner, University of Helsinki.

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Harry HolthoferHaartman InstituteHaartmaninkatu 3University of HelsinkiFI-00014 [email protected]

National Centre for Sensor Research/BioAnalytical Sciences Dublin City UniversityDublin 9, [email protected]

Partners

Per-Henrik GroopSamfundet FolkhälsanHelsinki, Finlandwww.folkhalsan.fi

Rob van der HeijdenUniversiteit LeidenLeiden, The Netherlandswww.leidenuniv.nl

Dario NeriSwiss Federal Institute of TechnologyZurich, Switzerlandwww.ethz.ch

Reinerio GonzalesPhilogen SpASiena, Italy www.philogen.it

Menno PrinsPhilips Electronics Nederland B.V.Eindhoven, The Netherlandswww.philips.com

Mike MartensFuture Diagnostics B.V.Wijchen, The Netherlandswww.future-diagnostics.nl

Jussi VilpoUnited Laboratories Ltd.Helsinki, Finlandwww.yhtyneetlaboratoriot.fi

© Shutterstock

Aim

DRoP-ToP has two major objectives, one techno-logical and one scientific. Regarding the former,the project proposes to develop a tool for multi-parametric analyses (mRNA levels, large geneticrearrangements, genetic mutations, genetic poly-morphisms, protein levels and post-translationalmodifications) of biological samples, to betterpredict tumour progression and recurrence. Theevaluation of such heterogeneous parameterswill be performed on a single microarray: thetriple microarray, an oligonucleotide microarrayfor simultaneous DNA, RNA and protein assess-ment). The triple microarray constitutes the testsurface of a workstation that integrates technol-ogy for the hybridization, scanning and detectionof biomarkers. Its simplicity should facilitatea wide implementation of this tool in the clinic.As scientific objective, the project proposes toidentify a set of biomarkers with power for theprediction of clinical behaviour of bladder cancer.Selection of such set of biomarkers will be the endpoint of a five-phase endeavour: • identification of candidate biomarkers for blad-

der cancer progression and recurrence from thescientific literature and from existing data gen-erated by two DRoP-ToP partners specialised inbladder cancer;

• pre-selection of biomarkers on the basis of thestrength of their association to tumour behav-iour and on the scientific and technical quality ofthe study;

• measurement and validation of said candidatebiomarkers in a set of samples from patientswith a detailed clinical record and follow-up;

• application of bioinformatics and biostatisticstools for the identification of a set of biomarkerswith a strong association to tumour behaviour.

The DroP-ToP strategy should be applicable to thestudy of any tumour type, and more generally to anydisease with a genetic or gene-expression compo-nent. However, and as a proof of concept, theproject proposes to apply it to bladder carcinomabecause it represents a paradigm of the need foruseful biomarkers in the clinical setting:

Background

With an ever increasing incidence, cancer is thesecond major cause of death in the Westernworld. Although advances in our understanding ofthe mechanisms of tumour onset and progressionhave been enormous, major impact on survivalhas been mainly restricted to sertain kinds oftumours. Besides advances in different therapies,the improvement in survival can also be attributedto improvements in diagnosis and the identifica-tion of high-risk groups, which allow for earlierand better treatment selection.In contrast to therapies and diagnosis, the overallprognosis of the most common cancers, such aslung, colon, prostate, breast and bladder cancersremains poor, particularly when the tumour can-not be cured by surgery. The ‘post-genomic era’has brought many promises that this situation willchange, but it has also given the emphasise to theneed to make appropriate and better use of infor-mation, technology and clinical resources. Theproject believes that resources are often wastedbecause of inappropriate approaches and inade-quate collaboration between clinical, academicand industrial partners.Transcriptome analysis by DNA microarrays hasbeen successfully used for the identification of bio-markers of tumour progression. However, due tovarious inconsistencies from study to study, theirapplication to common clinical practice has not yettaken place. The DRoP-ToP project intends to over-come these limitations by a two-fold approach: • prospective validation of the information

acquired through retrospective studies. For this,a collaborative multicentre effort is essential;

• integration of biomarkers from genome, tran-scriptome and proteome analysis in a single pre-dictor set. Even though each of these threeanalyses by itself will likely contribute, it isexpected that the combination of biomarkertypes will result in an enhanced predictivepower.

Integration of DNA, RNA and protein markers in a tool for the prognosis and diagnosis of human disease

ACRONYM

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The management of patients with superfi-cial bladder cancer is difficult. No reli-able means exist to determine whethera tumour will progress towards an infil-trative form, which requires radical sur-gery (cystectomy), or whether it willremain superficial, which requires onlyconservative surgery (resection). In addi-tion, no dependable marker exists topredict whether a primary tumour willreappear or not during the years follow-ing surgical resection, forcing patientsto undergo constant revisions that reducetheir quality of life and overburden healthcare systems.

Studies have identified biomarkers withpotential for the prognosis (progressionand recurrence) of superficial tumours.However, the analyses have often beenlimited to a single type of marker (e.g. pro-tein or genotypic markers) or even toa single marker. To the best of the pro-ject’s knowledge, no study has attemptedto integrate different types of markers foran increased predictive power.

The main scientific goal of DRoP-ToP is toidentify a set of markers (at protein-, RNA-,and DNA level) with high predictive powerfor tumour progression and recurrence. Inaddition, DRoP-ToP pursues an ambitioustechnological challenge: the developmentof a prognosis microarray, based solely onnucleic acid probes, for the detection ofthe above mentioned predictor set.

SUMMARY

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Key words: ??

The DRoP-ToP consortium is made of 8 multi-disciplinary partners coming from 3 EuropeanMember States (France, Spain, and Germany), 2 Associated States (Israel and Switzerland),and one INCO Country (Former Yugoslav Republic of Macedonia).

Among the partners, the participation of 3 European High-Tech SMEs (Progenika Biopharma,Genewave and NuAce Technologies) must be noted. Progenika Biopharma is the coordinatorof the DRoP-ToP consortium.

Progenika is a leading company in Functional Genomics and has wide expertise in microar-ray technology, which is critical to the success of the project. The company disposes also ofa relevant know-how in genomics technology and has gained an outstanding level of expe-rience in sample preparation from almost all human, plants and animal models, in microarrayprocessing and data mining, both in Expression and Genotyping projects. In particular,beside acting as coordinator for the DRoP-ToP project, Progenika is committed to lead thework on the design, validation and optimization of gene specific probes (for mRNA expres-sion analysis) as well as allele specific probes (for genotyping). Progenika is also responsiblefor the mRNA expression analysis and protocol optimization for the to-be developed probes.The SMEs Progenika and Genewave are together responsible for the production of the finalchip functionalized with the three different detection probes.

Genewave is developing and commercializing new biophotonic instruments and systems forgenomic, proteomic and post-genomic applications. Conscious that the sensitivity of detec-tion constitutes one of the major hurdles preventing biochips from reaching mass applica-tions, Genewave has developed an amplifying substrate for biochip (AmpliSlide™) basedon an original optical design and its associated high-throughput reader (AmpliReader™). Within the scope of this project, Genewave will further develop and optimized these tech-nologies with a main goal to obtain highly sensitive substrates with a coating chemistryoptimized for the efficient simultaneous binding of RNA, DNA and Aptamers and integratedinstruments dedicated to the use in clinical diagnostics (integrated microarray analysisstation). This will represent a major advance as such a tool will allow much more powerfuldiagnostics than available today aiming at less invasive, screening and diagnostic tests.

NuAce has developed a unique technology for synthesizing Dinucleotide-based Aptamers(DBAs), which can be employed as target-specific binders for genomic, proteomics andtherapeutics applications.

The DBAs are highly suitable for creating protein recognition agents that can be used in pro-tein chips. NuAce’s expertise will support the project in building up its basic concept, andprovide technological components including the task to generate DBAs to be immobilizedon the chip that specifically detects protein markers identified within the project.

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Key words: urology, clinical analysis, anticancer therapy, prognosis, microarray

• the number of invasive tests will be stronglyreduced, leading to a reduction in costs bydecreasing hospital admissions and the numberof occupation hours lost;

• reduction in the number of invasive tests will alsodiminish morbidity and improve the quality of lifeof patients;

• a better prognosis will allow more adequate choiceof treatment i.e. avoiding therapy to patients whodo not need it and applying more aggressive ther-apy to patients at risk.

While most patients develop bladder tumours witha relatively good prognosis in terms of survival,their management is very expensive because of themultiple recurrences that most patients suffer, theneed for invasive follow-up procedures, and the fre-quent hospitalisations. Overall, bladder cancerpatients generate the highest cost/patient and life-time among patients with cancer. In conclusion,bladder cancer generates very high costs to society. At the present time, there is no test recommendedor approved to help establish the prognosis ofpatients with bladder cancer. Over the past 10 yearsseveral products have been approved by the FDAfor use in the early detection of bladder cancerrecurrence (i.e. nmp22, BTA, Diagnocure Immunocyt,Vysis). However, none of these tests is yet usedroutinely in the clinical setting because they do notprovide a substantial benefit. Therefore, there isa tremendous need for better tests. Patients with bladder cancer develop multiple (upto 30) tumour recurrences, thus requiring contin-ued follow-up after the initial diagnosis. For thisreason, most patients undergo at least two med-ical visits over the first few years after diagnosis.Subsequently, the frequency of medical examina-tions varies according to the evolution of the dis-ease. Therefore, a test that would allow the earlydetection of recurrences and an improved estab-lishment of prognosis would be applied very fre-quently to the patients. The DRoP-ToP proposedtest could even be used more commonly than cys-toscopies are performed today, given that it wouldnot be invasive. Its availability would allow demon-stration of the concept that early detection oftumour recurrence is associated with improvedoverall outcome.


Diagnosis and prognosis for the bladder cancerand other diseases.

• it is one of the best models of tumour progression; • its incidence ranks fifth among all cancer types

(the fourth most common in males and the ninthin females);

• despite widely variable outcome, the diagnosisand prognosis tools used in the clinic are few andthe same, and they are invasive even for asymp-tomatic patients (cystoscopy);

• it is the most expensive cancer type, as it can recurmany times after treatment;

• its evolution is very difficult to predict, whereasthe therapeutic approach for its two forms is com-pletely different: when invasive at the time ofdiagnosis, it has a poor prognosis and requiresaggressive surgery (cystectomy); when non-inva-sive, prognosis is favourable and it only requiresconservative surgery (resection);

• its recurrence is also difficult to predict, whichleads to unnecessary visits and cystoscopies forabout 50% of patients, whose tumours will neverrecur;

• a number of highly promising biomarker candi-dates have already been identified and reportedin the literature.

Expected results

Cancer is the second leading cause of death world-wide. In the year 2002 there were 10 million newcases of cancer in the world, 6 million deaths andapproximately 22 million people living with cancerworldwide. It is estimated that by year 2020 therewill be 15 million new cases per year, and 10 millionsdeaths. Bladder cancer is a highly common neopla-sia, mainly among men, and its incidence is rising inseveral countries in Europe. Approximately 125 000new cases with bladder cancer are diagnosed eachyear in the EU. Despite continued interest in the development ofnovel tests to better predict bladder cancer progno-sis, there has been very limited progress. This is inpart due to the fact that all tests developed untilpresent are based on the detection of only one typeof biomolecule (i.e. RNA, DNA or protein). Ourapproach is radically different: from a systematicreview of current knowledge on biomarkers of blad-der cancer and existing research results of the par-ticipating academic partners, we propose todevelop a microarray that can detect the 3 majortypes of molecules in human biological samples.This should provide a much more solid basis toidentify molecular predictors of the disease.

The DRoP-ToP proposed technology will bringabout three main improvements:

ACRONYM

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ACRONYM

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Gorka OchoaProgenika Biopharma, S.A.Parque Tecnológico de BizkaiaEdificio 801 BDerio-Vizcaya 48160, [email protected]

Partners

Francois RadvanyiInstitut Curie-CNRS Paris, France

Nuria MalatsInstitut Municipal d’Investigacio MedicaUniversitat Pompeu-FabraBarcelona, Spain

Gordana CerovicGenewave S.A.S.Palaiseau Cedex, Francewww.genewave.com

Melanie Hilanio University of GenevaGeneva, Switzerland

Zivko PopovUniversity Cyril and Methodius-Faculty of MedicineDepartment of UrologySkopje, Former Yugoslav Republic of Macedonia

Hader KlessNuAce Tecnologies, Ltd.Rehovot, Israel

Joerg HoheiselDeutsches KrebsforschungszentrumHeidelberg, Germany

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| Triple microarray and Integrated station for improvedmultiparametric analysis of biological sample.

| Comparison between a standard glass slide and anAmpliSlide™. In a standard glass slide a large portion of the emitted fluorescence by substrate-attached fluorophoresescapes into the glass substrate. Constructive interferenceallows AmpliSlide™ to amplify and redirect this emittedfluorescence towards the reader’s optics. This amplification,up to a 20-fold increase in signal, results in enhancedsensitivity. (Top and middle: illustration and computerisedsimulation of light emission from fluorophore placed on the slide. Bottom: side by side comparison of images fromscanned slides hybridised under identical conditions).

C. difficile genes, or the culture and toxin testing ofthe isolates. Toxins can be detected either by theirbiological properties (cell cytotoxicity assay) or byimmunological methods (latex agglutination,immunoassay). The cell cytotoxicity test is the‘gold standard’. However, there are significantdrawbacks. Laboratories must have access to cul-tured monolayers, and results vary according tothe cell line, dilution factors, reagents used andstorage conditions. Additionally, the turnaroundtime is very slow. Enzyme immunoassaysconsistof conventional enzyme immunoassays and mem-brane immunochromatographic tests. The sensi-tivities and specificities of enzyme immunoassaysare within 85-95 % of the cell cytotoxicity test, andnumerous commercial kits are available. However,their performance relative to each other has notbeen subjected to a rigorous meta-analysis.Immuno-chromatography tests are extremely sim-ple, can be performed at the bedside and givea result within 30 minutes. This may be of crucialimportance in an epidemic situation to recogniseinfected patients. DNA-based tests are focussedon the detection of the C. difficile genes encoding16S RNA, GDH or the toxin genes (tcdA & tcdB).

Aim

The recognition of suitable targets and devel-opment of a commercial rapid test that will dis-tinguish variant hypervirulent and antibioticresistant strains from ordinary C. difficile strains isthe main aim of this project. The objective will beachieved through the complementary skills ofa consortium composed of 3 SMEs and 4 publicsector institutes, each experts in their fields.There will be a close collaboration in the form ofsubcontractors with the European Study Group ofC. difficile (ESGCD). Suitable diagnostic markers(DNA- and antigen-based) will be identified usinga combination of targeted approaches (focusingon toxin and antibiotic resistance determinants),and empirical approaches (DNA arrays and HT-AFLP) to identify unique strain differences. Assaydevelopment will be led by the SMEs, using pro-prietary technologies. Clinical validation will beperformed in a certificated reference laboratory.

Background

C. difficile is resistant to various antibiotics andcapitalises on the ensuing disruption of the nor-mal intestinal flora to colonisation and cause dis-ease. The spectrum of disease ranges fromasymptomatic carriage to a fulminant, relapsing,and increasingly fatal colitis. The effects of CDADare devastating, both in terms of morbidity/mor-tality and the high costs of disease management.Presently, C. difficile may only be treated witheither metronidazole or vancomycin. A number offactors have contributed to the worrying escala-tion in the incidence of CDAD. The elderly andimmuno-compromised are particularly at risk(80 % of cases occur in the over 65s). The propor-tion of the population in these high-risks groupsis rapidly rising. Strains exhibiting greater viru-lence are also beginning to emerge, which in someinstance has been attributed to the production ofadditional virulence factors, e.g. binary toxin. Thesituation has now been exacerbated by the arrivalin Europe during 2004 of a new hypervirulentstrain and antibiotic resistant strain (ribotype027, toxinotype III) previously confined to Canadaand the USA. The occurrence of this strain is asso-ciated with an excessive use of cephalosporinesand quinolone antibiotics, and has been responsi-ble for a massive increase in CDAD incidence in N.America and associated deaths, e.g. the propor-tion of patients with CDAD who died within30 days after diagnosis rose from 4.7 % in 1991-92to 13.8 % in 2003. This epidemic strain isolatedin Canada, USA, UK, Belgium, France and TheNetherlands was characterised as toxinotype III,North American PFGE type 1, restriction-endonu-clease analysis group type BI and PCR-ribotype027. The strain produces toxins A and B and con-tains a 18 bp deletion in the toxin regulator geneTcdC. It is imperative that an overall strategy tocombat this and similar novel variant(s) isdevised. Central to control of epidemics by suchnew C. difficile strains with increased virulence isa strategy that will include not only a rapid detec-tion but also a specific recognition of virulentstrains. Diagnostic assays for CDAD rely either onthe detection of C. difficile products (toxins),

European approach to combat outbreaks ofclostridium difficile associated diarrhoea by development of new diagnostic tests

ACRONYM

EACCADwww.cdiff.nl

Clostridium difficile-associated disease(CDAD) has become the most frequentnosocomial infection in many Europeanhospitals. In 2004, the situation wasexacerbated by the arrival in Europe ofa new hypervirulent strain (PCR ribotype027) previously confined to N. America,where it has been responsible for a mas-sive increase in CDAD incidence andassociated deaths. Central to the controlof epidemics are the deployment ofassays able to rapidly diagnose andmonitor the presence and spread of theorganism. No such tests currently existfor these new hypervirulent C. difficilestrains. It is the overall objective of thisproposal to develop the urgentlyrequired rapid, diagnostic assays inclose collaboration with 3 SME’s.

SUMMARY

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Key words: clostridium difficile, hypervirulent type 027, CDAD, diagnostics, outbreaks

The programme outputs will allow the formulationof a European strategy to combat the consider-able threat now being posed to European qualityof life by C. difficile.

Expected results

• Recognition of targets for new diagnostic testsby characterisation of hypervirulent and drugresistant C. difficile strains. The targets are basedon toxins, toxin coding regions, or unique othergenes of C. difficile.

• The availability of molecular tests and rapidmembrane immunoassays for detection of thetarget in patient material and in bacterial isolates.

• Validation of new developed tests for clinicaldiagnostics and strain characterisation. The newmolecular tests and rapid membrane assays willbe investigated in faeces samples of patientswith various forms of C. difficile associated diar-rhoea. The newly developed tests will also beused to recognise hypervirulent isolates andantibiotic resistance of clinical isolates obtainedby prospective surveillance studies in Europe.


The new diagnostic tests will overcome the lackof sensitivity and specificity of current diagnostictests. The new immunoassay offers an alterna-tive for those laboratories which lack capabilityin molecular biology. Recognition of variant 027is not possible with the currently available diag-nostic tests. Rapid recognition of CDAD and itsvariant 027 will result in appropriate patienttreatment and specific measures to prevent noso-comial spread and the development of outbreaks.The rapid molecular test will provide the first indi-cation of the presence of type 027, allowing ade-quate infection control measures to be undertaken

and prevent an outbreak. The genomic approaches(DNA Array and HT-AFLP) will provide fundamen-tal information on the genetic make-up of hyper-virulent C. difficile strains and will lead to greaterinsight into pathogenesis which will in turn allowthe development of more effective therapeuticcountermeasures and IPR spin-offs. Additionally,European guidelines will be formulated to diag-nose CDAD and to combat outbreaks. The intro-duction of these tests and European guidelinesincrease the awareness of CDAD as an importantnosocomial infection and will be of help to pre-vent the development of large outbreaks by newhypervirulent variants, as currently occur in USAand Canada. Application of the new tests andupdated guidelines will contribute to insights inthe epidemiology of CDAD and will be of generalsupport to prevent over-prescription of antibioticsin hospitals. Application of the new developedtests will provide a platform for automation ofdiagnosis. A further stimulant for effective DNA-based diagnostic tests is to extend testing onthe same faecal sample for other non-bacterialcauses of infectious diarrhoea, such as viral diar-rhoea and parasitic diarrhoea. It is possible to auto-mate nucleic acid extraction and use a commonextraction for all subsequent analyses.

The consortium consists of a balanced mix of public sector scientists that have worked onC. difficile for most of their professional careers (taken from four institutes), together withthree SME’s with highly developed technology on bacterial toxin preparation, moleculardiagnostics and enzyme-immunoassays. Therefore, complementary expertise, and ‘state-of-the-art’ technology is supplied by partners specialised in diagnostics, epidemiology,genomics and drug susceptiblity screening.The three SMEs participating in the project, tgcBiomics, PathoFinder and Coris BioConcept,will mainly contribute to the development of molecular tests and rapid membraneimmunoassays for detection of the target in patient material and in bacterial isolates.

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Ed J. KuijperDepartment of Medical MicrobiologyLeiden University Medical Center PO Box 9600, 2300 RCLeiden, The [email protected]

Partners

Maja RupnikDept. Med. Microbiol. Immunol.University of MariborMaribor, Slovenia

Nigel MintonInstitute of InfectionImmunity and InflammationUniversity of Nottingham Centre for Biomolecular ScienceNottingham, United Kingdom

Paola MastrantonioIstituto Superiore di Sanità, ISSRome, Italy

Cristoph von Eichel-StreibertgcBiomics GmbH, Mainz, Germanywww.tgcbiomics.de

Thierry LeclipteuxCoris BioConceptGembloux, Belgium www.corisbio.com/index.asp

Guus SimonsPathofinder B.VMaastricht, The Netherlandswww.pathofinder.nl

| Toxin A (TcdA) and toxin B (TcdB) areencoded on large chromosomal regionPaLoc, which encompasses two toxingenes (tcdA and tcdB) and threeadditional genes coding for regulatoryand putative transport function(tcdR,E,C). In nontoxigenic strains,PaLoc is replaced by 115 bp sequence.

Finally, the new methods and image analysisprocedures will be used to clarify the role ofmolecular biomarkers in tumourigenesis, prima-rily concerning the AP-1 and HER protein familiesand for mitochondrial DNA. The methods will alsobe tested in a high-throughput microarray analy-sis system. The cancer biomarker will primarily beinvestigated in a research setting, but the utility ofthe markers for diagnostic analyses will also beexplored.

Expected results

This project is expected to provide new means tostudy biomarkers for oncogenesis, and to gener-ate novel insights in cancer biology. It is expectedthat the in situ techniques developed in this proj-ect, and the scientific knowledge created, in thelonger run will lead to improved disease preven-tion, more rapid and accurate cancer diagnosis,and better treatment opportunities.

New analytical means will be developed to analysecancer biomarkers in situ. Relative to state-of-the-art procedures, these methods are expected toprovide significantly improved in situ analyses interms of specificity, sensitivity (single moleculedetection), possibility to study biomarker localisa-tion, analysis of protein interactions and proteinmodifications, and an opportunity for simulta-neous analysis of multiple markers (multiplexanalysis). Automated image analysis procedureswill be developed, i.e. software-based classifica-tion of molecules and their localisation in tissuesor cells. The software will provide a rapid way toanalyse many samples as well as user-independent,unbiased data classification.


Cancer biology and diagnosis.

Background

In situ analysis of cells and tissues has been anessential part of pathological research and diag-nosis primarily within cancer for many years, anda number of specific biomarkers of predictiveand prognostic value for various cancers havebeen identified. In situ analysis of nucleic acidsequences is dominated by in situ hybridisation,while in situ analysis of proteins is dominated byimmunohistochemistry where sections of tissuesare tested for the presence of proteins by specificantibodies. Due to limitations in these technolo-gies there is a significant need in the scientificcommunity for new efficient techniques and pro-cedures for more advanced analyses. A majorchallenge is to develop improved means formore detailed studies of biomolecules in situ, inorder to determine their abundance, sub-cellularlocalisation, and secondary modifications, aswell as how they interact with other moleculesand participate in signalling and control of cellularfunction.

Aim

The first aim is to develop new molecular methodsand assays for the analysis of individual DNA andprotein molecules in situ. The project is based ontwo fundamental technological inventions, pad-lock probing and proximity ligation. These are thefirst technologies to offer the sensitivity and speci-ficity required for studies of single bio-moleculesin situ. The padlock probe technology is used tointerrogate nucleic acids and to distinguish closelysimilar sequence variants, while the proximity lig-ation assay (PLA) is applied to analyse individualproteins, interactions between proteins, and post-translational protein modifications.

Secondly, the project will develop automatedimage analysis procedures to complement themolecular methods. The objective is to achievequantitative information about what molecules ormolecule complexes are present in the sampleand their tissue or sub-cellular localisation (i.e. inwhich cellular substructures).

New molecular methods and image analysis toolsfor analysis of cancer biomarkers in situ

ACRONYM

ENLIGHTwww.olink.com/content/Enlight/Enlight.html

The ENLIGHT project aims to developanalytical procedures with the sensitivityand specificity required to study individ-ual nucleic acid and protein molecules,and also interacting molecules, in theirnormal context in cells and tissues (insitu) and in tissue lysate microarrays.A spectrum of reagents will be developedfor the analysis of specific markers of par-ticular interest in oncology. Furthermore,software and algorithms will be estab-lished for automatic user-independent insitu image analysis of single molecule-events. These methods and algorithmswill be applied to evaluate candidate bio-markers of special relevance for tumourbiology and cancer pathology.

SUMMARY

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Starting date: 1 August 2006

Key words: cancer, biomarker, in situ analysis, technology development, proximity ligation, padlock probing

This project is centred around three European SMEs with high future potential. The SMEsare brought together with academic scientists and one large established company, DakoDenmark, to enable the development of new efficient technical solutions for the analysis ofindividual molecules in tissues or in single cells. The results from this project are expectedto provide important new commercial opportunities for products addressing significant mar-ket needs, thereby allowing the participating European SMEs to build sustainable busi-nesses at the forefront of biotechnology. To ensure that the outcome of the project will beadvantageous for the SMEs, one of them, Olink AB, has the role as project coordinator.DakoDenmark will, as a non-SME, have a role as advisor to the SMEs in matters such asproduct requirements and market needs.

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Björn Ekström Anders AlderbornOlink ABDag Hammarskjölds väg 54A751 83 Uppsala, [email protected]@olink.comwww.olink.com

Partners

Mats GullbergOlink ABUppsala, Swedenwww.olink.com

Ulf Landegren Ewert BengtssonUppsala UniversityUppsala, Swedenwww.uu.se

Henrik Winther Dako Denmark A/SGlostrup, Denmarkwww.dako.com

Anders Larsson Immunsystem I.M.S. ABUppsala, Swedenwww.immunsystem.com

Niels Foged Visiopharm A/SHørsholm, Denmarkwww.visiopharm.com

Olli Kallioniemi VTT Technical Research Centre of Finland Turku, Finlandwww.vtt.fi

John Bartlett The University of EdinburghEdinburgh, United Kingdomwww.ed.ac.uk

Anton Raap Hans van Dam Leiden University Medical CenterLeiden, The Netherlandswww.umc.nl

| Microscopic examination of tissue sections.

Based on this knowledge, the EPIVAC Project pro-poses the concept of combining the advantages ofvarious vaccines by engineering them into singlevectors, i.e. by designing plasmids that harbour thegenetic code necessary for the production of recom-binant viral particles, which further target the skinas a preferential site for genetic immunisation.

Aim

EPIVAC aims to generate a multi-step, improved,efficient and affordable DNA-based preventativeand therapeutic vaccine against HIV.

The first goal of EPIVAC is the development ofa reliable, reproducible and robust delivery sys-tem. This will include micro-needle arrays andan injection device for the controlled delivery ofthe GTU®-based vector system and for cell-type-specific epidermal expression of the genes ofinterest in epidermal differentiating keratinocytes,Langerhans cells and melanocytes.

The plasmids and genes of interest used in thesestudies will allow for the quantitative evaluationof the efficiency and kinetics of delivery of plas-mid DNA into the epidermis and epidermal cells.Further, to follow the process of diffusion, degra-dation and intracellular up-take and onset of bio-logical activity of the delivered expression systemin specific cells of the epidermis will be assessed.The plasmids used will carry the site-specific fluo-rescent labels for the monitoring of DNA deliveryinto the epidermis as well as the diffusion anddegradation of the plasmid.

The second goal of the EPIVAC project will be toanalyse the effect of different HIV1 antigenexpression in different cells of the epidermis onthe nature and breadth of the induced immuneresponse.

One major problem in developing an AIDS vaccineis the lack of knowledge regarding immune mech-anisms for protection against HIV1 infection andfor removing the virus from the body. Another

Background

The Human Immunodeficiency Virus (HIV) induceschronic infection in patients, eventually leading todeterioration of the immune system and the onsetof immune deficiency. HIV induces significant cell-mediated and humoral immune responses, whichconsiderably reduce viral titre after initial burst ofthe HIV1 replication and spread in the body. Thesenaturally induced immune responses only partiallycontrol HIV spread and in fact there are no clearlydocumented cases of true clearance of the viralinfection. Nevertheless, it is possible that the trig-gering of additional immune responses, notablythrough the presentation of modified HIV anti-gens, could generate different types of immuneresponses that could contribute towards bettercontrol of the infection and thus improve clinicalevolution and reduce viral transmissibility.

The quality and intensity of the humoral or cellu-lar immune responses required for efficaciouspreventative or therapeutic vaccinations vary,depending on the target infectious agents.Preventative vaccines against acute viral infec-tions often rely on strong neutralising antibodyresponses, while therapeutic vaccines againstchronic infectious diseases often rely on cytotoxicresponses capable of eliminating infected cells.

Most antiviral vaccines in current use are basedon homologous inactivated or attenuated viralparticles, i.e. attenuated measles virus formeasles vaccination. This establishes that pack-aging antigens in/onto particles is one of the mostefficient ways of triggering efficient antiviralimmune responses. Some of the most promisingnew vaccines have been designed and developedin the field of DNA vaccination. DNA vaccines arelow in cost, stable and easy to produce, whilstoffering the possibility to combine several anti-gens when required. However, it appears thatDNA vaccination, typically induces cellular immu-nity, but rarely or inefficiently neutralising anti-bodies that often play a major role in protectionagainst viruses (1).

Development of a multi-step Improved EpidermisSpecific Vaccine Candidate against HIV/AIDS

ACRONYM

EPIVAC

More than 40 million people are currentlyinfected with HIV1, mostly coming fromdeveloping countries resulting in an urgentneed for effective and affordable treatmentof HIV1 infections. The main objectiveof the project is to develop an efficientgenetic vaccine candidate for therapeuticand preventative use against HIV/AIDS.

The vaccine planned should be capable ofinducing cell-mediated and humoralimmunity against the virus and virallyinfected cells in different phases of theviral life-cycle.

DNA vaccines could fulfil these require-ments if rendered more efficient. To thisend, a combination of complementarytechnologies by four SMEs will be used.The novel DNA vaccination technologywill combine:• the restricted expression of the multi-

epitope/multivalent HIV antigens inspecific cells of the epidermis;

• a micro-needle array-based injectiondevice for reproducible and efficientdelivery into the epidermis;

• EPI-GTU® technology that allowsstrong and long-term expression usingsegregation/partitioning function ofthe Bovine papillomavirus type 1;

• Plasmo-VLP® technology that allows anexpression of the immunogens withinand onto virus-like-particles;

• the adjuvant effect of different cytokines.

The step-by-step combination of thesetechnologies will be evaluated at the labo-ratories of the three academic partners byusing state of the art methods. The initialevaluation of vaccine efficacy will be per-formed on mice and pigs and will be ulti-mately validated in Cynomolgus monkeys.

EPIVAC aims to conduct its preclinicaldevelopment up to the GMP productionphase. Testing of numerous vector batcheswill be aimed towards having enoughsuitable material ready for evaluation inclinical trials.

SUMMARY

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Key words: vaccines, DNA Therapy, epidermal genetic vaccination, HIV infection and AIDS

goal of this project is to contribute towards theidentification of some immune correlates of pro-tection during the evaluation of vaccine efficacy innon-human primates. The induced immuneresponse triggered by the vaccination will be char-acterised qualitatively and quantitatively towardsevery viral target protein. The immunised animalswill be challenged after immunisations with thehybrid SHIV, and analysed for control, clearanceand protection.

Expected results

• New devices and modalities for DNA delivery tothe epidermis.

• Generate improved DNA-based vaccines com-bining the advantages of GTU vectors for long-term expression and plasmo-VLP vectors for thepresentation of antigens onto VLPs.

• Generation of new proprietary HIV antigensbased on the EPIVAC optimised vector.


• Epidermal genetic vaccinations; Improved treat-ment of HIV infection and AIDS by using nano -technology.

• Improved standards for vaccination in large ani-mals, notably by using swines that are not oftenused in vaccine development and which couldbecome interesting alternatives to the use ofnon-human primates for challenge experiments.

References

(1) Tuteja et al., 1999.

EPIVAC comprises four SMEs among the seven partners, including the coordinator FITBiotech Plc., SILEX Microsystems, ESTLA Ltd. and EPIXIS s.a.

FIT Biotech will contribute its GTU® (Gene Transport Unit) platform technology and the appli-cations based on it, including novel vectors, self-replicating vectors for DNA immunisationagainst HIV, AIRE (immunological mechanisms) and DNMT3L (immune diseases, new genes).

SILEX will bring to the table extensive expertise from the development and production ofMicro-Electro-Mechanical-System (MEMS) components. SILEX personnel and the fullyequipped MEMS production facility provide an excellent environment for quick prototyping,development and production of microneedles that will be used in this project. The produc-tion of microneedles is an area of specific manufacturing expertise based on previous workfor various diagnostic and drug delivery applications.

ESTLA Ltd. develops, produces and supplies accessories for research organisations (optics,fine mechanics, special instruments for biophysics), special lasers (excimer, dye, coppervapour) and laser-based systems for science, medicine, industry and entertainment. Thecompany will contribute to the EPIVAC project through studies of the injection process andvaccine distribution by optical methods, and by the design and fabrication of the specialisedinstruments needed for injections with microneedle chips.

EPIXIS s.a. is dedicated to the development of preventive and therapeutic vaccines againstinfectious diseases. The company’s proprietary pseudotyped virus-like particle (VLPs) havethe unique property to express the pseudotyped viral envelops in their native conformation,and can likewise efficiently induce broadly neutralising antibodies. EPIXIS will contribute toEPIVAC with pseudotyped VLPs produced in vivo by vaccination with ‘plasmo-VLPs’.

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Ioana StanescuFIT Biotech PlcBiokatu 8, 33520Tampere, [email protected]

Partners

Henrik Hellqvist Silex Microsystems AB Swedenwww.silexmicrosystems.com

Evgeny BerikEstla Ltd.Tartu, Estoniawww.estla.com

Charlotte DalbaEPIXIS S.A.Paris, Francewww.epixis.com

David KlatzmannUniversity of Pierre and Marie CurieParis, Francewww.upmc.fr

Mart UstavTartu University Institute of TechnologyTartu, Estoniawww.tuit.ut.ee

Roger Le GrandFrench Atomic Energy CommissionParis, Francewww.cea.fr

Expected results

The project combines research with technologicalimplementation in the development of new GenePharmaceutics. Obtaining pharmacologically sat-isfactory gene drug delivery systems has thepotential to impact positively on a range of bothcommon and rare healthcare burdens withinEurope and beyond. Strategies targeting hepatitisB and C, and haemophilia are presented in thecurrent proposal. The expected result of the pro-posal would thus be efficient and productiveprogress in the development of clinically-potentand cost-effective pharmacological formulationsof gene-based medicines.

The market for gene drugs is potentially verylarge, although it is dependent upon the develop-ment of safe and pharmaceutically acceptable for-mulations and protocols. This project, through theexpertise of the partners, aims to prove that thistechnology possesses the appropriate propertiesfor successful application.


The potential applications of the drug-deliverytechnology proposed here are much broaderand could include cancer, vaccine approaches toinfectious disease, arthritic disorders, tissueengineering, and various inherited syndromeswhich, while less common, nevertheless imposehuge burdens on patients, their families andhealthcare providers. The success of this proposalwill create a new pharmaceutical platform anddemonstrate its potential for treatment, whichwould have a strong impact on public health andrelated expenses.

Background

During the last years, developments in genomicshave enabled an unprecedented acquisition ofnew knowledge with relevance for human dis-ease. In parallel, the biotech industry hasinvented novel ways of generating drugs in orderto meet the demands from the healthcare sector.

The development of new pharmaceutics in thefield of genomics and biotechnology is likely to besuccessful, provided that there is an optimal inte-gration of multidisciplinary efforts from both aca-demia and industry. This is a major challenge,since new concepts need to be explored and com-bined into commercially viable innovations. Thedevelopment of a drug from the lab bench toa clinical grade product is very costly, involvingmany translational steps and often in excess ofone hundred million euros. Only major pharma-ceutical companies have the financial resourcesto accomplish this task. This means that small andmedium sized enterprises (SMEs) need to developnovel drug concepts to a stage where the proof ofprinciple can be evaluated by the big pharmaceu-ticals, in order to bring the therapy to the clinic.

Aim

The project has the following objectives:• generation of new forms of DNA-binding syn-

thetic compounds, which will serve as geneticglues;

• enhancing linking chemistry for biologically activeentities coupled to DNA binding compounds;

• optimising the assembly of Gene Pharmaceuticsby in vivo experimental studies;

• developing standard operating proceduresfor the manufacturing and assembly of GenePharmaceutics according to good manufacturingprocedures.

Design of targeted Gene Pharmaceutics using self-assembling functional entities

ACRONYM

EURO-PHARMACO-GENE

The project outlines the development ofa novel Gene Pharmaceutics prototypethrough translational research. This canbe accomplished by the combined effortsof three research-intensive biotech com-panies, together with two academicgroups, representing state-of-the-art skillsand proprietary technologies in comple-mentary areas. These include nucleic acidanalogue chemistry, short interfering RNA(siRNA) technology, gene transfer andgene therapy, as well as imaging instru-ment manufacturing, and methods for self-assembling supramolecular complexes.This will lead to the design of new self-assembling Gene Pharmaceutics and bringthem to the clinical grade stage.The aim is to develop plasmid and siRNA-based drugs that target the liver, since thisorgan is affected in many disorders involv-ing a genetic component. Three diseaseshave been selected for this purpose,namely hepatitis B and C infections andan inherited form of bleeding disorder,haemophilia A (Factor VIII deficiency).Moreover, many aspects of this platformtechnology are likely to be applicable toessentially any liver disorder and may alsobe transferable to other organ systems.Fully developed, this technology will resultin new, safer and more rational drugs.Gene Pharmaceutics technology is basedupon the addition of functional entities tonucleic acids. The schematic representa-tion (Fig. 1) shows three different func-tional entities (blue squares, orangecircles and red triangles, or different grey-shades, when printed in black and white)linked to peptide-nucleic-acid (PNA)anchors, which are glued onto the plasmidthrough hybridisation to PNA anchoringsequences (green, yellow and light blueregions, or different grey-shades, whenprinted in black and white) in the plasmid.Other nucleic acid analogues, such asLNA, or derivatives thereof, can also beused as anchors. The marked sequence inthe lower portion of the plasmid repre-sents the transferred gene. This could bea reporter gene, a therapeutic gene, suchas the one encoding Factor VIII, which isdefective in haemophilia A, or a short hair-pin RNA expression cassette, in which theshRNA is directed against a component ofhepatitis B or C virus.

SUMMARY

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Starting date: 1 February 2007

Key words: gene therapy, hepatitis, haemophilia A, siRNA, PNA, nucleic acid analogues

Of the five participants to this project, two are academic and three are SMEs. The academicresearch lab from Karolinska, in addition to coordinate, will perform assembly and biolog-ical testing of the tool kit for gene transfer. These tools can be described as novel self-assembling supramolecular complexes. While the academic research lab in Denmark willconcentrate onnew chemistry, in particular on novel nucleotide analogue chemistry andnucleic acid functionalisation, especially based on the locked-nucleic-acid (LNA) platformtechnology.

The key role of the SMEs is highlighted below: Eurogentec focuses on advanced, peptide-based synthesis, development of optimised coupling procedures for peptides and nucleicacid analogues. The company is an SME based in Belgium.

Biospace provides state-of-the-art imaging solutions for monitoring of the biological effectsof Gene Pharmaceutics. It is an SME based in France.

Avaris concentrates on development of novel pharmaceutics, in particular providing trans-lational research which brings non-viral gene transfer systems from the research lab benchto the clinic. It is an SME based in Sweden.

The strength of this consortium is in the diversity and complementarity among partners,combining basic research with the applications to human health.

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C. I. Edvard SmithClinical Research CenterDepartment of Laboratory MedicineKarolinska Institutet at NovumHälsovägen 7SE-141 57 Huddinge, [email protected]: www.ki.se

Partners

Jesper WengelUniversity of Southern Denmark Department of Physics and ChemistryOdense M, Denmarkwww.sdu.dk

Daniel MarechalIvo RudloffEUROGENTEC S.A.Seraing, Belgiumwww.eurogentec.com/eu-home.html

Serge MaitrejeanBIOSPACE S.A.Paris, France

Mats LakeAVARIS ABc/o Karolinska Innovations Stockholm, Swedenwww.avaris.se

| Fig. 1 Schematic representation of technology. | Fig. 2 Interaction and complementarity among partners. The arrowsindicate reciprocal relationships, i.e. partners connected will provideknow-how, experience or products bi-directionally.

• Conventional cell cultures often do not expresssuitable easy-to-assay quantifiable markers orthey need transfection procedures that increasevariability of the data.

• The systems used for the in vitro and in vivoanalysis of NR-ICs (mainly estrogens and andro-gens) are generally composed of cells derivedfrom reproductive tissues. The recent knowl-edge of the widespread distribution of nuclear-receptors (in particular steroid receptors) in allthe tissues of the organism and their involve-ment in several diseases, makes the availablesystems inadequate to assess the effects ofNR-ICs on the whole physiology (Villa R, 2004).

• The available models do not easily provide infor-mation on the effects of compounds at differentdevelopmental stages.

Aim

EXERA’s original approach is based upon power-ful evidences:

Use of the transgenic MOUSE-1: This estrogen-reporter mouse model represents a new strategythat allows studying estrogen receptors-medi-ated gene regulation in vivo and in derived invitro systems. The numerous studies performedon this animal model in several laboratorieshave demonstrated its reliability and suitabilityto the study of molecules acting through estrogenreceptors.

3D cultures: Cell-cell and extracellular matrix-cell interactions play a fundamental role inmaintaining the function of numerous organsystems. Hence, tissue engineering representsa good way to overcome limits of monolayer cul-tures and to maintain tissue-like architectureand functionality.

Background

Industries from different fields (Pharmaceuticals,Chemicals, Cosmetics, Foods and Toxicologicals)need reliable, fast and economic in vitro models,which are alternative to animal testing and canprovide predictive data on the actions of NR-ICsand in particular ER-ICs (Estrogen ReceptorsInteracting compounds).

The need for appropriate in vitro models whichcan reproduce features and reactivity of specificmammalian target tissue/organs to ER-ICs is thusbecoming an imperative research priority. The sci-entifically, economically, socially and ethically rel-evant stakes are therefore considerable.

The tissue- and organ-specific in vitro modelswhich have been built so far have several seriouslimitations:

• Most of the available cell lines of mammalianorigin are derived from tumours or have a trans-formed phenotype. Their functional and struc-tural features do not mirror the original tissue.

• When mammalian-derived in vitro models areavailable, they consist of primary cell cultures orof isolated tissue slices: their in vitro survival islimited (time-course and dose- response stud-ies are very difficult). Moreover, if of humanorigin, they have the disadvantage of depend-ing on regular supply from available clinicalsources.

• 2-Dimensional culture conditions may be notoptimal for tissue-like organisation and cellularfunctions (e.g. polarised cells of a parenchymaltissue, which normally require complex cellularinteractions, and cannot behave physiologicallywhen adhering to solid substrates, as in thecase of conventional culture conditions).

Development of 3D in vitro models of estrogen-reporter mouse tissues for thepharmaco-toxicological analysis of EstrogenReceptors-Interacting Compounds (ER-ICs)

ACRONYM

EXERAwww.altaweb.eu/exera

The objective of the EXERA project is todevelop novel 3D in vitro models of mousetissues from five major organs for the phar-maco-toxicological analysis of EstrogenReceptors-Interacting Compounds (ER-ICs):liver, skin and bone (non reproductive sys-tems), ovary and testis (male and femalereproductive systems).

This objective will be pursued througha work programme which allies an inte-grated scientific approach between inno-vative technologies such as the 3D-culturedevice, known as ‘Rotary Cell CultureSystem’ (RCCS Technology adapted to theneeds of this project), established trans-genic mouse lines (estrogen-reportermice, here called MOUSE-1) and genomicplatforms for ER-ICs characterisation.

The strategy is planned around five mainpoints including, besides the mentionedtechnologies, multiple cell cultures qualitycontrols, immortalisation control, cell bank-ing and the use of specific markers of estro-genic action and cell differentiation/health,in order to obtain estrogen -responsive 3D-cultures of well differentiated mouse cells.

The corresponding work programme willbe ensured by an important involvementof the whole partnership and by a highdegree of coordination between 6 privateand 3 public institutions.

SUMMARY

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Key words: ??

Industrial participation is important (some 70 % of the EU contribution will go to SMEs) anddiversified. It is composed of 5 SMEs and one industry. One SME (ALTA S.r.l.) will be involvedin this project for administrative management. The fact that SMEs from different but comple-mentary fields take part in this consortium, not only implements the recommendation of the6th FP, but represent an added value since it ensures the strong commitment of the partici-pants to the success of the study and will guarantee the future exploitation of the project’sresults ensuring the eventual transition from discovery to the market.

Specific role description:

BIOSERV: Establishment of Sertoli, Leydig, peritubular and corresponding Granulosa celllines from tissue isolates of the estrogen reporter model ERE-Luc. Development of fully functional micro test systems in which cells are maintained in chipbased micro-culture environment.

CELLON: Cellon will provide the 3D cell culture systems, Rotary Cell Culture Systems (RCCS).Cellon will benefit from the new application of this technology to pharmaco-toxicologicaltesting of hormonal chemicals and will promote the diffusion and applications of the RCCSin Europe and worldwide.

HORMOS MEDICAL: Hormos operates in the project as an implementation and validationarena, to provide the consortium with data on different compounds with varying SERM andNR related activity in different tissues. Hormos can adapt the cellular systems ‘in house’ andvalidate the cell lines under development with selected compounds, which have known tis-sue selectivity and different pharmacological estrogen profiles in tissues eg. bone, uterus,breast and liver, ovaries.

DNAVISION: The main task of DNAVision is to produce gene expression profiling (by DNAmicroarrays) of the cell systems generated by the other partners of the consortium andcompare the data to the in vivo situation (from tissues of the ERE-Luc mouse).

BioUetikon Ltd.: BioUetikon will provide the technical expertise to manufacture and qualitycontrol cryopreserved cell banks of up to twenty immortalised mouse cell lines suppliedto BioUetikon by the consortium.

ALTA: Working in close contact with the Coordinator, ALTA’s specific contribution to thisproject will be: • to check the progress of the administrative work; • to co-ordinate the administrative bodies of the different participants’ institutions; • to organise technology transfer and follow IPR issues.

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Key words: estrogen receptors, endocrine disruptors, estrogenic drugs, transgenic estrogen-reporter mice, in vitro assays, 3D-cultures, in vivo imaging

| Optical imaging on the estrogen reporter mouseERE-Tk-Luc. The mouse was exposed for 6 hours to 5 μg/kg of 17ß-estradiol.

| Cell-medium interface of Sertoli cell aggregatescultured in RCCS. Cells are strain with DAPI (Mag 40X).

Expected results

The corresponding work programme will beensured by the important involvement of thewhole partnership and by a high degree of coordi-nation between 6 private and 3 public institutions.The expected results that will be scientifically,socially and economically relevant are as follows:• the application/adaptation of new 3D-culture

technologies to cell cultures devoted to thestudy of ER-ICs;

• the constitution of a cell bank; • a battery of differentiated 3D cell-based sys-

tems derived from estrogen-reporter mice forbasic and applied research (e.g. pharmacologyand toxicology), public use and industrial use.


The EXERA project seeks to overcome the limita-tions of conventional in vitro approaches to therisk assessment of endocrine active compounds.In addition to scientific and regulatory advances,EXERA will also promote technological innova-tions by including research on the applicability ofnovel cell based methods and novel end-points tothe assessment of the estrogenic action of ER-ICs. The developed cell-based systems will becomevery useful tools in investigating tissue specificregulatory pathways and hormone-dependentphysiological processes.

This project is the first attempt to apply the RCCStechnology performance to the systemic analysisof hormone-dependent pathways. This will beobtained by using different cell types in standard-ised conditions. Its possible adaptations to indus-trial screening procedures will be intensivelyexploited. The expected achievement is to fulfilthe need of additional practical, new and easilystandardisable end-points for all the estrogenhormone target organs.

Techniques and methodologies: The steps toreach the proposed objectives will involve sev-eral complementary techniques and partnerexpertise: cell isolation, conditional immortalisa-tion, cell banking, 3D-cultures, whole genomeexpression profiles, in vitro imaging, in vivoimaging, and application of 3D-cultures devices(RCCS Technology).

Cell isolation from tissues of MOUSE-1: Reliableprotocols available inside the partnership willbe applied so as to isolate well differentiatedcells from liver, skin, bone, testis and ovariesof MOUSE-1 and establish cell cultures with ‘phys-iological’ estrogen-dependent phenotypes forimmortalisation.

Conditional Immortalisation: This step will be per-formed by transfection methodologies in 3D andby using suitable commercially available vectors,made inducible by specifically modified antibi-otics devoid of hormonal actions. The immortalis-ing gene will be switched on for cell production,and off for characterisation and testing.

Constitution of a cell bank: Immortalised cell cul-tures that will satisfy the following parameterswill be expanded and controlled for banking.

3D-cultures adapted to grow cells with an unal-tered estrogen-dependent phenotype: Estrogen-dependent pathways will be characterised in3D-cultures. Comparison between hormone stim-ulation in vitro and in vivo will be performed withthe use of the transgenic marker (luciferase) andgene expression profiles. Data on the activity ofselected ER-ICs will be produced in each specificcell line.

Assessment of the 3D-culture systems for thepharmaco-toxicological characterisation of ER-ICs.

ACRONYM

EXERA

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65


Diego Di LorenzoLaboratorio di Biotecnologie Ospedale Civile di BresciaP.le Spedali Civili 125123 Brescia, Italydilorenzodiego@yahoo.itwww.spedalicivili.brescia.itwww.med.unibs.it

Partners

Adriana Maggi Center of Pharmacology and BiotechnologyUniversity of MilanMilan, Italy

Kalervo Väänänen University of Turku Turku, Finland

Paul TomkinsBioserv Ltd. Athlone, Ireland

Richard Fry CELLON S.A. Bereldange, Luxembourgwww.cellon.lu

Mikko UnkilaHormos Medical Oy PharmacityTurku, Finland

Jean-Pol DetiffeDNAVision S.A. Charleroi, Belgiumwww.dnavision.be

John MilneBioUetikon Ltd. Dublin City UniversityDublin, Ireland

Aldo TagliabueALTA SRLSiena, Italywww.altaweb.eu

| The RCCS (Rotating Cell Culture System) culturing liver fragments from ERE-tk-Luc mice.

Aim

To develop and evaluate high-speed, POC testsbased on the detection of antibodies and antigensin body fluids, e.g. whole-blood, serum, urine, spu-tum or saliva for the diagnosis of both pulmonaryand extra-pulmonary TB.

Expected results

The outcome of this project will lead to new inex-pensive and fast diagnostic tests that can be per-formed on site without the requirement of anycomplicated or expensive instruments. The testswill provide results within 15-20 minutes and shallbe preferably non-invasive.


Potential applications of this project results are:

• The new Fast tests shall enable accurate TBdiagnosis on the spot within 15-20 minutes.

• Inexpensive TB diagnosis shall be feasible inlow-resource settings highly suitable to TBendemic countries.

• Antigen detection Fast tests will have consider-able advantages over current methods especiallywhen dealing with HIV positive populations,which show reduced immune response.

• Additional significant advancement shall bemade by developing dual HIV-TB tests which areurgently needed in endemic countries in Africaand Asia where HIV infection is increasing daily.

Background

The current methods of diagnosis (microscopy,culture, chest x-ray, PPD, PCR) are inadequate forthe diagnosis of tuberculosis, as these are eithertoo slow, not sensitive enough or too expensive.The usual means of diagnosing TB in the majorityof developing countries where culture facilitiesare not available is by the detection of acid fastbacteria in sputum by direct microscopy. But thistest is laborious and insensitive as only 40-60 %of all adults with pulmonary TB can be identifiedby the current smear test using Ziehl-Neelsenstaining.

In low-resource TB endemic countries, poor accessto high-quality microscopy services and/or pau-cibacillary nature of pulmonary TB in HIV positivepatients results in even lower rates of sensitivityof AFB (acid fast bacilli) detection.

Thus, the two main problems concerning TB diag-nosis are:• sputum microscopy, currently the most widely

used method to detect tuberculosis, which iscumbersome and insensitive, leaving manypatients undetected and;

• bacterial culture, the gold standard, is more sen-sitive, but which takes 4-6 weeks to complete,and is too complex for most settings where TB isendemic.

The HIV pandemic has led to a resurgence of TB asa major public health problem. ImmunodeficientHIV-positive patients are particularly vulnerableto TB and are even more difficult to diagnose thanthose who are diagnosed HIV negative.

Development and Clinical Evaluation of Fast Tests for Tuberculosis Diagnosis

ACRONYM

FASTEST-TB

It is widely accepted that rapid, cost-effective diagnosis of high sensitivity andspecificity is a prerequisite for the pre-vention and control of tuberculosis (TB);a global disease in humans, killing morethan 3 million people annually. Methodsand devices currently in use do not meetthese requirements.

New strategies are urgently needed forcombating the problems of TB diagnosis.First-generation detection tests usinga novel, high-speed device for quantita-tive measurement of antigens in sputumand urine of TB patients have recentlybeen developed. The device also enablessimultaneous measurement of antigensand antibodies within 20 minutes. Themain objectives of this proposal are to: • Identify novel antigens using genomic

and proteomic approaches.• Purify sufficient quantities of antigens

and raise antibodies.• Optimise immobilisation conditions for

the specific antigens and antibodies ondifferent Carriers.

• Manufacture and evaluate different, fasttests using approximately 6 000 clinicalspecimens (sputum, saliva, serum, urine)from TB patients at study sites in Asia,Africa, America and Europe.

Such tests and devices would be a majorbreakthrough in the early diagnosis andprevention of tuberculosis. The Coordi -nator (LIONEX, an SME) in co-operationwith a WHO centre, a lung hospital inGermany and additional expert scientistsfrom low-resource, TB-endemic countries(India, Turkey, Nigeria, Mexico) shall in thisproject, evaluate the clinical potential ofantigen and antibody detection using thehigh speed, cost-effective Point of Care(POC) tests, with which results can beobtained on site within 20 minutes. Finally,the project also aims to develop TB-HIVdual antibody detection POC Fast tests.

The outcome of this project will lead tonew inexpensive and fast diagnostic teststhat can be performed on site without therequirement of any complicated or expen-sive instruments. The tests will provideresults within 15-20 minutes and shall bepreferably non-invasive.

SUMMARY

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Key words: Tuberculosis, TB diagnosis, serology, antigen discovery, TB-Fast test development, clinical evaluation

The project is coordinated by LIONEX GmbH, an SME dedicated to the prevention and con-trol of TB. The project’s efforts are focused towards the development of completely new andfast tests for the diagnosis of TB, which shall be of particular use in developing countrieswhere several million people are infected with TB each year. Several of the project’s partnerscome from developing countries. The coordinating SME will have a pivotal role in enhancingthe communicating with endemic countries.

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Mahavir SinghLIONEX Diagnostics & Therapeutics GmbHInhoffenstraße 738124 Braunschweig, Germany [email protected]

Partners

Françoise PortalesInstitute of Tropical MedicineAntwerp, Belgiumwww.itg.be

Helmut BloeckerHelmholtz Centre for Infection Research (previously GBF) Department of Genome AnalysisBraunschweig, Germanywww.genome.gbf.de

Harald Hoffmann Institute of Microbiology and Laboratory MedicineWHO-reference laboratory for Tuberculosis and MycobacteriaMünchen-Gauting, Germanywww.asklepios.com/Gauting

Olcay YeginAkdeniz University Medical School Department of Pediatric Immunology Antalya, Turkeywww.akdeniz.edu.tr

P. R. Narayan, Alamelu RajaTuberculosis Research CentreChetput, Chennai, Indiawww.trc-chennai.org

Gertrud Biersack Sacred Heart HospitalLantoro, AbeokutaOgun State Nigeriawww.geocities.com/sacredheartabeokuta

Jürgen Knobloch Institut für TropenmedizinUniversitätsklinikum TübingenTübingen, Germanywww.med.uni-tuebingen.de/tropenmedizin/index.html

Iris Estrada Dept. of ImmunologyNational School of Biological SciencesSanto Tomas, Mexico

© Shutterstock

Expected results

FGENTCARD will deliver technological and ana-lytical tools applied to the definition of CAD bio-markers through genetic studies in clinicalcohorts and rodent crosses, which will be used tomap CAD susceptibility loci and genes providingtargets for gene cloning and entry points for drugdevelopment. A set of standard operation proce-dures will be developed for the detection of CADbiomarkers for genetic and clinical studies inother cohorts and other disease areas. This pro-gram will design and utilize methods for multi-modal gene expression profiling and subsequentdata integration that can maximize the power ofgenetic linkage and association analyses.


Advancement in knowledge, technological plat-forms and analytical tools will contribute to a bet-ter understanding of genetic systems thatunderpin multifactorial diseases. Progress in thisarea will play a key role in providing the essentialtools for the development of strategies to identifygenes for epidemiological important disordersand targets for the generation of novel therapies.Overall, the potential wealth of informationobtained on gene expression represents novelchallenges in quantitative genetics and ultimatelysignificant advances for disease diagnosis andprevention. Knowledge of the effects of geneticvariations on metabolic processes and metabo-type regulation will have an important impact inthe field of polypharmacology.

Background

The completion of the genomic sequence ofmany mammalian species and progress in high-throughput genotyping and gene expressionprofiling technologies are amongst the most sig-nificant recent advances in biomedical science.With the growing number of cohorts for case-con-trol genetic studies, these resources and toolshave a wide range of applications for localizingchromosomal regions associated with diseasesusceptibility, identifying disease causative DNAvariants and characterizing mechanisms regulat-ing gene expression in health and disease. A par-ticularly important application is in tackling thegenetic cause of increasingly frequent disordersin western society, including CAD, a growinghealth and societal concern in the general popu-lation. The genetic causes of CAD risk factors,which associate diabetes, hypertension, dyslipi-daemia and obesity, will be addressed throughthe definition of biomarkers derived using func-tional genomic and genotyping technologiesapplied in animal models of CAD and large cohortsof patients.

Aim

The general aim of FGENTCARD is to apply func-tional genomic and genotyping technologiesalong with the wealth of knowledge arising frommammalian genome annotations to definingnovel diagnostic tools for risk factors of glucoseintolerance, insulin resistance, hypertension, dys-lipidaemia and obesity, which are key pathophys-iological elements in CAD. FGENTCARD resultsutilize the power of functional genomic technolo-gies to tackle these increasingly frequent andprevalent inherited diseases. The proposed stud-ies will ultimately generate fundamental knowl-edge on the impact of functional genomics toidentify disease biomarkers and test their use fordisease prediction.

Functional GENomic diagnostic Tools for Coronary Artery Disease

ACRONYM

FGENTCARDwww.fgentcard.eu

The focus of the consortium project isto define biomarkers and novel diag-nostic tools for risk factors for coronaryartery disease (CAD) (glucose intoler-ance, insulin resistance, hypertension,dyslipidaemia and obesity), using func-tional genomic and genotyping technolo-gies along with the wealth of knowledgearising from mammalian genome annota-tions. The consortium is preparing innova-tive infrastructure of both techniques andmaterials that provide strategic supportfor CAD quantitative genetic studies inanimal models and humans. FGENTCARDis using state of the art functionalgenomic and genetic technologies includ-ing NMR metabonomic and automatedproteomic profiling of biofluids and organbiopsies, microarray based gene tran-scription profiling and original technolo-gies for identifying disease susceptibilityloci. Results from this research will pro-vide resources for extension and valida-tion of CAD biomarkers in other modelsand human cohorts, and will exemplifymultidisciplinary strategies which can beapplied to other disease areas.

SUMMARY

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Key words: functional genomics, atherosclerosis, metabonomics, nuclear magnetic resonance, transcriptomics, proteomics, rat, mouse

The project provides SMEs with original biological material and data from collections ofcase control cohorts and genetic crosses between animal models. These can be used totest their technologies in deriving disease biomarkers and CAD susceptibility genes. Animportant aspect of the research project lies in the application of a multidisciplinaryapproach, allowing SMEs, specialised in technologies designed to test a specific level ofgene expression control, to obtain data from a comprehensive screening of patterns ofgene expression regulation in health and disease, ranging from genetic polymorphism, totranscriptome, proteome and metabonome. This approach is designed to promote effortstowards research and innovation and to facilitate collaborations between SMEs andbetween academic groups and SMEs. The two SMEs involved in the project, namelyMetabometrix Ltd. and IntegraGen S.A., combine very different expertise in metabo-nomic technologies (Metabometrix) and disease gene mapping (IntegraGen), which willsynergistically be applied to the chromosomal mapping of CAD biomarkers.

Metabometrix is a spin-off company built on the skills of the world’s pre-eminentmetabonomics research team at Imperial College, London, and experienced scientistsfrom the UK and US pharmaceutical industries. The group’s portfolio of intellectual andtechnical advances is unique and underpins a substantial lead in the field. The companyhas a proprietary platform of metabonomics technologies for generating, classifying andinterpreting metabolic information from biological fluids and tissues.

Methods that IntregraGen has developed for the purpose of genome-wide linkage studiesare particularly well adapted to the research of the genes predisposing to CAD pathophysio-logical components, namely late onset multifactorial diseases. The techniques will beproperly used for the identification of the genes predisposing individuals to CAD andrelated phenotypes. The company will be in the best possible position to estimate therelevance of the results obtained and implement the pertinent molecular diagnostic tests.In combination, the two SMEs play crucial and synergetic roles in the FGENTCARD project.

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Dominique Gauguier University of OxfordThe Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United [email protected] www.well.ox.ac.uk/~gdomi

Partners

Pierre ZallouaAmerican University of Beirut,Department of Internal Medicine Faculty of MedicineBeirut, Lebanon

Mark LathropCentre National de GenotypageEvry, France

Jeremy K NicholsonImperial CollegeChemical and Molecular Systems BiologyLondon, United Kingdom

Ulla Grove SidelmannNovoNordisk A/SMalov, Denmark

Jorg HagerIntegraGen S.A.Evry, Francewww.integragen.com

Frank BonnerMetabometrix Ltd.London, United Kingdom www.metabometrix.com

| The consortium utilisesmodern functional genomicand genetic technologies tomap etiological biomarkers of coronary artery disease inanimal models and humans.

Aim

GLYFDIS’ main objectives are:

• To optimise high-throughput methods of glycananalysis for the diagnosis of stomach andpancreatic cancer by the analysis of glycansin blood.

• Identifying cancer associated glyco-markers inserum samples of stomach and pancreaticcancer patients.

• Developing and validating protocols for lectin-based microarrays intended for large scalescreening of cancer associated glyco-markersin serum samples.

Expected results

• To identify biomarkers using glycomic and pro-teomic methods together with computer basedalgorithms.

• To develop a non-invasive, modest, diagnostickit that will identify specific markers for cancerin the blood.

• Constructing a website and a glycome bio-bankintegrating GLYFDIS results and serving as basisfor a continuously growing public glycome data-bank.

• Dissemination of the information to the scientificcommunity and community at large.


The project will generate knowledge relevant fornon-invasive diagnosis of cancerous states withthe effort in developing a standard protocol fordiagnosis of serum samples.

Background

Cancer is a significant burden on individuals, fam-ilies and society. The economic impact of cancer issubstantial. In 2002, the overall cost of cancer, aspublished by the National Cancer Institute, was172 billion US dollars. This does not account forthe psychological toll that it takes on individualsand families.

Early detection and diagnosis of cancer is basedon the observation that treatment is more effec-tive when the disease is detected earlier in itsnatural history, prior to the development of symp-toms than in an advanced stage. Diagnosis of can-cer in the early stages of the disease influencesmany aspects of life. It can significantly decreasecancer-associated morbidity and mortality and torelieve the burden from patients, their familiesand the society. Accurate monitoring of a cancer-ous state following diagnosis can significantlycontribute to prognosis determination and on-lineevaluation of therapeutic regimens. Developingeffective tools to screen for cancer is an importantendeavour and there is much research takingplace to develop these tools. The goal is to detectthe cancer when it is localised to the organ of ori-gin without invasion of surrounding tissues ordistant organs.

The GLYFDIS project will make use of glycans.Their diversity, compared to genome or proteome,makes the glycans ideal for diagnosis and moni-toring of cancer. Cancer-associated changes in theglycome of the tumoural tissue are very frequent.Currently one of the main obstacles is the lack ofsufficient technology. Glycome-analysis technolo-gies today fall behind the rapidly developinggenome- and proteome-analysing technologies.

The group hopes to identify biomarkers that canbe used to develop a non-invasive method for theearly diagnosis of stomach and pancreatic cancerbased on glycan analysis.

Glycans in Body Fluids- Potential for Disease Diagnostics

ACRONYM

GLYFDISwww.glyfdis.org

Developing effective tools to screen forcancer is an important endeavor and thereis much research taking place to developthese tools. GLYFDIS project’s objective isto develop methods for earlier diagnosticand effective disease screening of stomachand pancreatic cancer that will lead to bet-ter treatment outcomes. Early diagnosis ofcancer is of far greater prognostic impor-tance than any attempts to treat the dis-ease in its late stages. Even in cases wherethe eventual outcome cannot be changed,treatment is simpler and quality of lifeimproved for those cases where earlydiagnosis is achieved. For this purpose,GLYFDIS proposed a method of a simplenoninvasive blood testing. Accuratemonitoring of a cancerous state followingdiagnosis can significantly contributeto prognosis determination and on-lineevaluation of therapeutic regimens.

The most widespread and diverse post-translational modification is glycosylation.The location and variation of glycans placethem in a position to mediate cellularand intracellular signalling events, aswell as participate in different biologicalprocesses including pathology states suchas cancer. Therefore, the project proposesto use analyses of glycans for identifyingnovel biomarkers that can be used forthe diagnostics and monitoring of cancer.

SUMMARY

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Starting date: 1 November 2006

Key words: diagnostics, glycome, blood, glycan, mass spectrometry, disease markers

RNTech RNTech is an SME specialising in early stage diagnosis of digestive tract cancers with spe-cial focus on colorectal and pancreatic cancer. The company operates a dedicated biobankof biological samples selected from patients that have undergone surgery for the removal ofdigestive tract solid tumour.

RNTech’s role in GLYFDIS has three facets:• providing biological samples collected from pancreatic and stomach cancer patients and

healthy control subjects for the discovery and validation phases of the project;• contributing to the project database by providing genomics data on pancreatic cancer

patients as well as clinical data on all selected patients and healthy subjects and partici-pating in the bio-statistical and bio-informatics treatment of such data and researchresults;

• providing with its network of clinical oncologists and cancer biology specialists to thevalidation of identified potential biomarkers.

As any other partner, RNTech will also contribute to the management and the disseminationplan of the project.

ProcogniaProcognia has developed a lectin-array based technology for rapid analysis of glycosylationprofiles of intact glycoproteins. The array contains 25-30 well-characterized lectins (carbo-hydrate binding proteins) with overlapping specificities. The binding of a glycoprotein to thearray results in a characteristic fingerprint that is highly sensitive to changes in the protein’sglycan composition. The large number of lectins, each with its specific recognition pattern,ensures high sensitivity to changes in the glycosylation pattern. Automatic algorithms wereconstructed for deconvoluting these signals into a glycan profile output. The major advan-tages of this technology in comparison to traditional methods of glycoanalysis are its shortanalysis times, the relatively low protein amount needed for analysis, the possibility toanalyse multiple samples in parallel, and the relatively low costs compared to the classicalanalysis methods (HPLC/MS).

A highly sophisticated, automated platform (GlycoScope) was first developed for use in thebiopharmaceutical industry for the analysis of recombinant glycoprotein drugs. By tailor-ing algorithms for various protein families, this product provides accurate, quantitativeglycoanalysis for single proteins.

In addition, Procognia is developing a line of products for the life science and academicresearch market. The products are a line of off-the-shelf kits for glycoanalysis, distributedby QIAGEN. The first product, Qproteome™ GlycoArray, launched in 2006, provides a rapidand simple tool for glycoanalysis of glycoproteins. This kit can be used without sophisticatedequipment, and generic interpretation algorithms provide semi-quantitative glycoanalysisfor purified glycoproteins.

The second product will be launched in 2007 for analysis of global glycosylation patterns ofmembrane protein extracts. The kit is intended for analysing global changes in glycosyla-tion patterns in extracts of cell membrane proteins of cultured mammalian cells, with theaim of enabling characterisation of glycosylation-related biological effects.

As a partner in GLYFDIS, Procognia will optimise the existing technology for analysis ofglobal glycosylation pattern to allow the characterisation of complex protein mixtures inserum from healthy donors and donors with pancreatic or stomach cancer.

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Angel PorgadorBen-Gurion University of the NegevBeer-Sheva, [email protected]

Partners

Rakefet RosenfeldProcognia Ltd. Ashdod, Israelwww.procognia.com

Pauline RuddNIBRTDublin, Irelandwww.nibrt.ie

Jasna Peter-KatalinicMuenster UniversityMuenster, Germanywww.uni-muenster.de/en

Julien TaiebRNTech Diagnostics SPRLCharleroi-Gosselies, Belgiumwww.rntech.com

| Vision of MALDI-TOF-MS. © Danny Machlys, BGU.

higher than 20cpm/uCi. Pinhole collimators willbe also realised;

• a very compact geometry of the detection mod-ule, based on a thin substrate where the SDDsarray and VLSI readout circuits will be assem-bled, and a single CsI(Tl) crystal (potentiallysubstituted by the more recently introducedLaBr3:Ce) will be coupled to the photodetectorarray;

• the introduction of a thermoelectric system toattain moderate cooling (~ -20 °C) during opera-tion of the gamma camera;

• the development of dedicated VLSI electronicsfor amplification and filtering of the detector sig-nals, followed by processing electronics basedon FPGA for the event reconstruction;

• the design of a compact assembly of the com-plete Anger Camera. The compactness of theassembly will allow ease of positioning of theinstrument close to the patient’s body surface;

• the realisation of image-reconstruction algo-rithms and user interface software running ona common personal computer.

Expected results

• Development of large-areas low-noise SiliconDrift Detectors.

• Development of high-resolution collimators.

• Development of a high-resolution and compactAnger Camera based on state-of-the-art tech-nologies.

• Improved diagnostic capabilities thanks to theuse of the camera.


• Improved possibility to implement an effectivetherapy with higher capability to detect as smallas possible concentrations of tumour cells.

• Effective imaging on reduced volume of thebiological tissues: less than 10 x 10 cm2 area ofthe planar view (in scintigraphic investigations

Background

The state-of-the-art in the field is represented bya range of commercial systems, usually havinglarge field detectors (~ 40x50 cm2). These sys-tems are best exploited while performing whole-body SPECT studies since their large, heavyPMT-based detector heads and bulky gantriespresent difficulties in operating close to thepatient’s skin for dedicated studies of specific,small tissues such as in parathyroid imaging,brain scanning or investigation of kidney cancerin infants. In a realistic clinical setting, at animaging distance usually rather greater than20 cm, the overall effective spatial resolution istypically 10-16 mm (7-10 mm for brain studies).When a single detector head is used for dedi-cated scintigraphic studies of small organs, per-mitting a closer imaging distance, the overalleffective spatial resolution is limited by both theintrinsic spatial resolution of the system and thecollimator.

Aim

The aim of the project is therefore the develop-ment of a new compact and high position resolu-tion (< 1 mm) gamma camera based on the newSDD photodetector technology. The first techno-logical objective is the development of anextended array of SDDs with large cell size (1 cm2),characterised by high detection efficiency to thescintillation light and low electronic noise. Thelow noise level is a result of specialised advancedsemiconductor processing, as well as of the inte-gration of an on-chip JFET in the detector chip,which allows us to fully exploit the intrinsic lowcapacitance of the SDD, by the minimisation ofparasitic capacitances of the connection betweendetector and electronics.

The other key technological objectives addressedby the project are:• the realisation of a high-resolution collimator.

The aim is to obtain a parallel hole collimatorwhose spatial resolution equals ~2 mm at animaging distance of 5 cm with a sensitivity kept

Development of a high-resolution Anger camera for diagnosis and staging of cancer diseasesbased on state of the art detector technology

ACRONYM

HI-CAMwww.hi-cam.org

The purpose of the project is the develop-ment of a compact and high-resolutionAnger camera to be used in clinical andresearch environments and which allowsearlier and more reliable diagnosis andtherapy planning of cancer diseases inspecific applications where high overallspatial resolution (less than 3 mm) andsystem compactness (less than 10x10 cm2

field of view) are required.

The gamma camera is based on the well-established Anger architecture, wherea collimator acts as a mechanical sieve forincoming gamma photons, a continuousscintillator uses the energy of each selec-tively passed gamma photon to generatevisible photons, and an array of photode-tectors emits electric signals in responseto the absorption of the visible photons.The improvement of performances isbased on the use of a particular type ofphotodetector, the Silicon Drift Detector(SDD), which has recently demonstratedits ability to provide better performancwith respect to the commonly usedphotomultiplier tubes.

The camera is intended for use both sin-gle-handedly for planar scintigraphic stud-ies and inserted in an annular holder(gantry) of small diameter for SPECT imag-ing. Thanks to its compactness and highspatial resolution, it offers potential appli-cations in early diagnosis of cancer dis-eases affecting areas of the human bodywhich can be hardly imaged with the largeand heavy imaging heads and gantries ofcommercial Anger cameras. The camerato be developed in the present projectalso offers promising perspectives of inte-gration at the system level with MRIinstrumentation, thanks to the relativeinsensitivity of the SDD photodetectorsto large magnetic fields.

The research activity will be organised asfollows: the first two years of the three-year project will be dedicated to thedevelopment of the SDD-based Angercamera, while the third year will be dedi-cated to the experimentation of the cam-era in selected imaging applicationsrelated to cancer diagnosis and research.

SUMMARY

72



Key words: technological sciences, health sciences, physical sciences, medical imaging

with one single imaging head); less than 10 cmtotal extent on the coronal plane of the patient’sbody being imaged by the acquired scans (afterappropriate rotation of the camera/s on theannular holder in the tomographic arrangement).

• Measurements on anatomical sites of the targetwhich usually lead to severe space constraintsduring acquisition of the scan.

• Improved use of imaging instrumentation inthose conditions where the patient’s age, condi-tion or physical disabilities (e.g. patient onwheelchair) prevent an effective use of largedetector heads and gantries without compro-mising patient comfort.

• Brain tumours.

• Thyroid cancer, particularly differentiated carci-nomas, with 99mTc-perthecnetate.

• Parathyroid cancer with 99mTc-sestamibi forpreoperative localisation of parathyroid ade-nomas with the aim of surgical planning in MIP interventions (minimally invasive parathy-roidectomy).

• Breast cancer with 99mTc-labelled lipophiliccations (SestaMIBI or tetrofosmin). The mostpromising application of the proposed systemconcern its use in preoperative or postoperativesites.

In addition, the project offers innovativeapproaches to the diagnosis of tumours in infantsand children.

Two SMEs, L’accessorio Nucleare S.R.L. and Nuclear Field Holland B.V., are involved withkey technical aspects of the development of the HICAM gamma camera.

L’ACN is a company currently operating in the market of instrumentation for nuclear medi-cine and will be particularly involved with the development of the gamma camera instru-mentation, taking as a start-point the gamma-ray detector developed by the other partners.Adapting the technology currently employed to the results of the present project, L’ACN willbe ready to release a commercial system within a very short time for planar scintigraphyapplications.

The HI-CAM project will also produce a potential development and market impact for theNuclear Field Holland (NUFI) company, which is particularly interested in launching a veryhigh resolution (VHR) collimator, similar to the one developed within HI-CAM, onto theworld market for use in diagnostic medical imaging. With the potential to create an intrinsicresolution of 1 mm, which is the aim of the HI-CAM project, the goal of NUFI is to createa 0.5 mm hole collimator which would suit the resolution of the HI-CAM camera. In doing so,the project will develop the next generation commercial, low cost collimators, giving OEMcompanies the possibility to move in this direction and begin redesigning their systems.

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Carlo FioriniPolitecnico di MilanoVia Golgi 4020133 Milano, [email protected]

Partners

Carla FinocchiaroCF consulting Finanziamenti Unione europea srlMilano, Italywww.cf-consulting.it

Leonardo PepeL’accessorio Nucleare S.R.L. (L’ACN)Cerro Maggiore (MI), Italywww.acn.it

Pieter van MullekomNuclear Fields International B.V.Vortum Mullem, The Netherlandswww.nuclearfields.com

Lothar StrüderMax-Planck-Gesellschaft zur Foerderung der Wissenschaften eVMünich, Germanywww.mpg.de

Brian HuttonUniversity College LondonLondon, United Kingdomwww.uclh.nhs.uk

Ugo GuerraOspedali Riuniti di BergamoBergamo, Italywww.ospedaliriuniti.bergamo.it

Ignasi CarrióInstitut de Recerca de l’Hospital de la Santa Creu i Sant PauBarcelona, Spainwww.santpau.es

Giovanni LucignaniUniversità degli Studi di MilanoMilano, Italywww.unimi.it

| Principle scheme of the Anger camerabased on Silicon Drift Detectors technologydeveloped in HICAM.

their age, are significantly more radiation sensi-tive compared to older women. Due to the limi -tations of the current technology, in many EUcountries breast cancer screening is presentlyonly offered to women older than 50.

Aim

The HighRex project intends to solve the currentdilemma in mammography by increasing theimage quality in terms of contrast and spatial res-olution while lowering the radiation dose. This willbe achieved by using results from fundamentalresearch in nano-technology, x-ray optics anddetector technology obtained during the lastyears. The currently only European detector plat-form commercially available for digital mammog-raphy will be drastically improved and developedinto a detector system for the next generationbreast imaging equipment.

Expected results

An increase in breast cancer detection rate inscreening of just 1 % in Europe would mean thatan amount of cases in the order of 500 otherwiseundetected cases would be diagnosed annually,with a potential of 100 to 300 lives saved. It may,however, be expected that the increase in detec-tion rate is significantly higher than 1 %, maybeeven exceeding 10 %.


Competitiveness of the Highrex European tech-nology platform will manifest itself stronger inthe second generation of mammography and theproposed project will be able to deliver a stan-dard for 3D mammography that will be unsur-passed for quite some time. reason to believethat the photon counting advantages in mam-mography would also be beneficial in other imag-ing applications such as CT and chest x-rayimaging, and this project has the potential to pro-vide the example needed for the technology tospread also to those areas.

Background

The incidence of breast cancer currently increasesin all European countries and according to theEuropean Breast Cancer Network (EBCN), everyyear 50 000 women are diagnosed with breastcancer. Around 40 % of these women will die fromthe disease, making it the second most commoncause of death for women between the age of 20and 70. The most efficient weapon against breastcancer is currently early detection through mam-mography screening. An early detection makesthe subsequent therapy more successful and alsomitigates bi-effects and facilitates breast con-serving surgery in contrary to mastectomy. Thereis currently scientific evidence for a decrease ofmortality between 30 and 40 % in the screenedpopulation.

Currently, film is the most common image recep-tor in mammography, but this is now beingreplaced by digital mammography.

In mammography screening 70-90 % of the can-cers are detected. The undetected cancers aremainly in women with dense breasts where thecontrast resolution of state-of-the-art equipmentis limited by overlapping tissue. Recent resultsfrom the so called ACRIN trial show that this chal-lenge can to some extent be met with the adventof digital mammography. The improvements are,however, modest and the problem remains. Whatmakes the problem worse is that the risk forbreast cancer is almost a factor three higher forwomen with dense breasts.

One way of solving the problem would be toincrease the radiation dose to increase contrastresolution. However, in dense breasts, this wouldalso increase the noise caused by overlying tis-sue. Moreover, the radiation dose in mammogra-phy is a growing concern, and increasing theradiation dose would mean an increased risk forradiation induced cancers. This is particularly truefor women below the age of 50 who on averagehave much denser breasts and who, because of

High Resolution X-Ray Imaging for ImprovedDetection and Diagnosis of Breast Cancer

ACRONYM

HighReXwww.sectra.se/medical/mammography/highrex

Breast cancer is currently the most com-mon cause of death from cancer forwomen below seventy years of age andcurrently over 100 million Europeanwomen are screened every year for earlydetection through mammography. Theobjective of the proposal is to increasethe efficiency in detection and diagnosisof breast cancer and thus to decrease themortality in breast cancer. To achieve this,we will develop novel imaging methodsbased on recent results in the researchfields of nanotechnology, x-ray optics,detector technology and integratedelectronics. The new modality will bedesigned by leading European industryand SMEs in these areas and develop theonly commercially available Europeandetector platform for digital mammogra-phy today into a leading technology plat-form for tomorrow. The novel method willprovide significantly increased contrastand spatial resolution compared to cur-rent state-of-the art breast imagingthrough elimination of noise from elec-tronics as well as from overlapping tissueand by way of utilizing the signal moreefficiently through fast single photoncounting integrated circuits. To makesure that the project targets the rightissues in breast imaging, experiencedmammography doctors from severalEuropean breast imaging centres areinvolved in the project and they will alsotest and evaluate the new imaging sys-tem and compare it to current state-of-artmammography as well as ultrasound andMR imaging of the breast. The clinical tri-als will involve an enriched populationof symptomatic women and the poten-tial impact on European screening forand diagnosis of breast cancer will beestimated from the results.

SUMMARY

74



Key words: mammography, breast imaging, photon counting, tomosynthesis, contrast mammography, breast cancer, dual-energy

The three participating SMEs, Detection Technology (DEETEE), Silex Microsystems AB(SILEX), and Artinis Medical Systems (ARTINIS) will be heavily involved in the design andassembly of every crucial part of the proof-of-principle prototypes. DEETEE will be respon-sible for designing the required application specific circuits as well as the X-ray sensors;SILEX will design the X-ray optics while ARTINIS will design phantoms for evaluation ofthe prototypes. If the proposed research is successful, the proof-of-principle prototypeswill be developed into real products and the participating SMEs will be in an excellentposition to take part in volume production as well as in continuous development of theproduct. The competitiveness of the SMEs will further be strengthened through theincreased capacities developed within the project, as well as through the internationalcontacts created and a network that will facilitate problem solving in the future and forma basis for other cooperation projects. The know-how gained in the project is likely to besufficiently generic to have the possibility of being applied also in other areas, e.g. indeveloping imaging modalities for other examinations than mammography.

ROLE OF SMEs

75


Mats DanielssonSectra Mamea ABKistagången 2SE-164 40 Kista, Sweden [email protected]/medical

Partners

Mikko Juntunen Detection Technology Inc.Ii, Finlandwww.deetee.com

Edvard Kälvesten Silex Microsystems AB Järfälla, Swedenwww.silexmicrosystems.com/pages

Roeland van der BurghtArtinis Medical Systems B.V.Zetten, The Netherlandswww.artinis.com

Matthew WallisAddenbrooke’s HospitalUniversity of CambridgeCambridge, United Kingdom

Kenneth Young Royal Surrey County Hospital NHS Trust Guildford, United Kingdom

Walter Heindel Münster Universität Münster, Germany

Ulrich Bick Charité Universitätsmedizin BerlinHumboldt UniversityBerlin, Germany

Brigitte Séradour Association pour la Recherche et le Dépistage des Cancers du SeinMarseille, France

Nico Karssemeijer Radboud UniversityNijmegen, The Netherlands

Ruben van Engen Stichting Landelijk Referentie Centrum voor BevolkingsonderzoekNijmegen, The Netherlands

Karin Leifland Capio Diagnostics Radiology SwedenStockholm, Sweden

Ulf Strand Avalon Product Development Helsingborg, Sweden

| Vision of clinical application for breastcancer detection with 3D photon countingtomosynthesis based on research in theHighrex project.

Aim

The general aim of this project is to identify andprepare and identify new potential drugs from theclass of new modified 2’,3’-dideoxynucleosides,active against HIV-1 drug-resistant laboratory andclinical strains.

Expected results

To obtain new modified 2’,3’-dideoxynucleosidesand their phosphates, highly active drugs againstdrug-resistant HIV strains, and to detect theirphysico-chemical and biological properties. Resultswill be disseminated through patenting.


After obtaining positive biological results,patents and publications will be submitted toa pharmaceutical firm (SME) for further investiga-tions in animals and in the clinic, allowing poten-tial outlicensing of the above-mentioned drugs.

Background

Human immunodeficiency virus (HIV) encodes foran RNA-dependent DNA polymerase (reversetranscriptase, RT), but not the specific enzymesrequired for the phosphorylation of 2’,3’-dideoxy-pyrymidine and purine nucleosides. To exertantiviral activity, these analogues must undergoa three-step phosphorylation by host cell kinasesand/or be metabolised by other enzymes. Their5’-triphosphates act as RT competitive inhibitorsand/or DNA terminators. Since RT is essential toHIV replication, the development of RT inhibitorsis a key strategy in the fight against AIDS.However, drug resistance emerges rapidly withthese nucleoside-based inhibitors (NRTIs).

At present, the emergence of HIV-1 variants resist-ant to standard drugs is one of the major obsta-cles to chemotherapy of HIV-1 infection. HIV-1 candevelop multidrug resistance in patients receivingvarious combination chemotherapies. Thereforethe development of novel compounds especiallymodified 2’,3’-dideoxynucleosides, which areactive against wild-type as well as multidrug-resistant variants, is urgently needed.

Preparation and Identification of New HIV Reverse Transcriptase Inhibitors Targeted AgainstHIV Strains Resistant to anti-HIV/AIDS drugs

ACRONYM

HIVResInh

HIV-1 can develop multidrug resistance inpatients receiving various combinationchemotherapies. This is one of main prob-lems in anti-HIV/AIDS chemotherapy. Toidentify compounds active against HIV-1drug-resistant strains, project partnersplan to synthesise and investigate thestructure, conformation and selectedphysicochemical and biological proper-ties of a range of new and known, modi-fied 2’, 3’-dideoxynucleoside analogues.

Compounds of this type should act ascompetitive drug-resistant reverse tran-scriptase (RT-Res) inhibitors. Slow releas-ing forms of these compounds (prodrugs)will also be prepared. Drug-resistantreverse transcriptases will be obtainedfrom engineered HIV-1 drug-resistantmutants, as well as by recombinant tech-niques. Interactions of synthesized com-pounds with the wild type HIV-1 RT, aswell as with HIV-1-Res RT, will be investi-gated and potent inhibitors will be sub-jected to antiviral activity determinationsin cell cultures and in vitro cytotoxicity.

Structure-activity relationships will beperformed and selected RT inhibitors willbe subjected to determination of com-plete cross-resistant profiles in labo-ratory and clinical HIV-1 strains fromdocumented clinical context of resistance.

Investigation of in vitro viral escape willalso be determined. Long-term anti-retroviral therapy often results in toxicadverse events attributable to mitochon-drial damage due to the inhibition of DNApolymerase γ synthesis and the reductionof cellular energy production.

The toxicity of the new drugs will be evalu-ated by determining the inhibitory proper-ties (IC50) and insertion and exonucle olyticremoval of new nucleoside analogues byDNA polymerase γ. Finally, selected activecompounds with low in vitro cytotoxicity willbe subjected to in vivo (in mice and/or rats)pharmaco-toxicological investigations.

SUMMARY

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Starting date: 1 April 2007

Key words: chronic diseases, virology, anti-HIV and AIDS drugs

This project comprises one SME, InPheno AG, which plays a key scientific role in the project.Researchers at InPheno have a solid background in pre-clinical profiling of anti-HIV drugs.InPheno will mainly contribute to the determination of a complete cross resistance profileof new HIV inhibitors in laboratory strains and variants from clinical context, as well asinvestigating whether the new inhibitors provoke viral escape in vitro. InPheno expertise iscentred around preclinical profiling of HIV resistance in diagnostics and drug discovery,which will provide an important contribution to the critical mass of the project.

ROLE OF SMEs

77


Tadeusz KulikowskiInstytut Biochemii iBiofizyki PAN5a Pawinskiego Street02-106 Warsaw, [email protected]

Partners

Kazimierz KitaInstytut Przemyslu Organicznego Oddzia w PszczyniePszczyna, Polandwww.ipo.pszczyna.pl

Marek FiglerowiczInstytut Chemii Bioorganicznej PANPoznan, Polandwww.ibch.poznan.pl

Patricia Laquel-RobertUMR 5097 CNRS/Université Bordeaux 2Bordeaux, Francewww.cnrs.fr

Andrzej PiasekInstytut Medycyny Doswiadczalnej i Klinicznej im. M. Mossakowskiego PASWarsaw, Polandwww.cmdik.pan.pl

Francois HamyIn Pheno AGBasel, Switzerlandwww.inpheno.com

Kazimierz KitaInstytut Przemyslu Organicznego Oddzial w PszczyniePszczyna, Polandwww.ipo.pszczyna.pl

© Shutterstock

Aim

To develop a novel therapy for the treatment ofindividuals infected with HIV-1. This therapyinvolves the application of RNA interference(RNAi) to prevent productive infection of new cellswith HIV-1 and so eventually cure infection.

An SV40-based vector will be used, and the costsand complexity will be kept low. Therefore a pro-ducer cell line will be generated, to produce viralvector particles at high titres and focus ona single-administration, long-lasting therapeuticmolecular vaccination.

Expected results

• Proof of safety and efficacy of the developedtherapeutic vaccine tested in vitro and subse-quently in vivo using mouse and simian immun-odeficiency virus (SIV)/Cynomolgus macaquemodels.

• A producer cell line to produce viral vector parti-cles at high titres.


Therapeutic anti-HIV-1 Vaccine.

Background

HAART can be effective; however, resistant viralstrains do emerge. Eventually, these resistantvariants can cause AIDS in treatment-resistantpatients. A novel therapy that involves the appli-cation of RNA interference (RNAi) to prevent pro-ductive infection of new cells with HIV-1 and thuseventually cure infection is the aim of this project.So far, RNAi-based inhibition of HIV-1 replicationhas been accomplished through the introductionof virus-specific, synthetic short double-strandedRNAs. These are short interfering RNAs or DNAconstructs encoding short hairpin RNAs. However,their use as therapeutic antiviral against HIV-1 islimited because of the rapid emergence of virusescape variants.

In order to solve this durability problem, DNA con-structs encoding virus-specific long hairpin RNAs(lhRNAs) were developed. It was demonstratedrecently that expression of such lhRNAs in targetcells provides durable, sequence-specific andbroad-spectrum inhibition of HIV-1 replication.

Development of an Effective RNA Interference-Based Anti-HIV-1 Therapy Using an SV40-Derived Vector

ACRONYM

HIVSTOP

The Acquired Immunodeficiency Syndrome(AIDS) caused by infection with the humanimmunodeficiency virus type 1 (HIV-1) isa pandemic continuing to grow at analarming rate, despite the availability ofhighly active anti-retroviral chemotherapy(HAART). The World Health Organisation(WHO) and European Union (EU) thereforelaunched a co-ordinated action program tocombat poverty-related communicablediseases, including AIDS.

In this project a novel therapy for thetreatment of individuals infected withHIV-1 is to be developed. This therapyinvolves the application of RNA interfer-ence (RNAi) to prevent productive infec-tion of new cells with HIV-1 and thereforeeventually cure infection. An SV40-basedvector will be used to transfer the thera-peutic anti-HIV-1 sequence to T-cells ofHIV-1 infected individuals in order toresult in long-lasting improvements oftheir condition. SV40 vectors are intrinsi-cally safe, transfecting both non-dividingand dividing cells. In order to use thistherapy in developing countries it isessential to keep the costs and complex-ity low. A producer cell line will thereforebe generated in order to produce viralvector particles at high titres and focuson a single-administration, long-lastingtherapeutic molecular vaccination.

The safety and efficacy of the developedtherapeutic vaccine will be tested in vitroand subsequently in vivo using mouseand simian challenge models.

SUMMARY

78



Key words: therapeutic molecular vaccination, HIV-1, AIDS, SV40-Vector and RNA interference

The SME involved in this project, Viruvation B.V., will be responsible for the co-ordinationof the project. Viruvation is a young biotechnology company which focuses on RNAi-basedantiviral strategies and will be responsible for the development of SV40 production tech-nology. The company will further contribute with know-how on virology and proprietarytechnology of importance for the project’s core tasks.

ROLE OF SMEs

79


Gerrit-Jan van HolstViruvation B.V.Wassenaarseweg 72, PO box 10482302 BA Leiden, The [email protected]

Partners

Ben BerkhoutAcademisch Medisch Centrum (AMC)Amsterdam, The Netherlands www.amc.nl.

Puri FortesFundación para la Investigación Médica Aplicada (FIMA)Pamplona, Spainwww.cima.es

Neil AlmondNational Institute for Biological Standards and Control (NIBSC)Potters Bar (Hertfordshire), United Kingdomwww.nibsc.ac.uk

© Shutterstock

• A clear understanding of the pathways to clini-cal service, ethical and legal issues, and cost-effectiveness of the IBDchip, which will resultin maximum uptake of the IBDchip in routineclinical practice.

• The results for the academic partners will benew knowledge derived from the researchundertaken in the project and published ina range of leading academic publications.


The IBDchip will have very wide application acrossthe EU and beyond. The team has the workingobjective of the IDBchip being used for 15 % ofboth UC and CD patients (a total of approximately320 000 people) within three years of the endof the project (i.e. in 2011). These illnesses areincreasing and the project team expects that theIBDchip and new reader will be embedded asa routine part of treatment over the coming decade.

It is also probable the IBDchip project R&Dprocesses and the resulting technologies can beadapted to address problems in the predictionand treatment of other polygenic inflammatoryconditions such as rheumatoid arthritis. The teamintends to use the consortium and its work overthe next three years as the foundation for futureprojects to explore other potential applications ofthe technology.

Background

Inflammatory bowel disease (IBD) includes Crohndisease (CD) and ulcerative colitis (UC). Both areincreasingly common, chronic illnesses, currentlyaffecting nearly 1 million patients in Europe. CDand UC affect patients early in life, seriouslyimpairing their quality of life and resulting in enor-mous personal, social, and economic costs.

There is evidence suggesting that genetic factorsplay a key role in IBD pathogenesis, pointingtowards a polygenic mode of inheritance for CDand UC. However, to date studies have onlyaddressed the influence of single mutations onIBD, resulting in a poor prediction of clinical courseor response to therapy in individual patients.

Aim

The main aim of this project is to provide doctors,for the first time, with a non-invasive, predictivetool to optimise treatment in IBD patients, thusresulting in better clinical outcomes and improvedcost-effectiveness of treatment.

Expected results

• A fully validated innovative prototype IBDchipwhich will give doctors vastly improved capaci-ties to make more accurate individualised pre-dictions of clinical outcomes of IBD and choosethe optimum and most cost effective therapy foreach patient.

• A new DNA array reader which will be faster andone fifth of the price of existing machines tooptimise the reading of the IBDchip and helpmake it ubiquitous.

Usefulness of a new DNA array (IBDchip) to predictclinical course, development of complications andresponse to therapy in patients with inflammatorybowel disease (IBD)

ACRONYM

IBDchip

The IBDchip Project will develop an easyto use DNA array and accompanying inno-vative chip reading device. The IBDchipwill be a non-invasive tool with thecapacity for the simultaneous analysis ofaround 100 relevant mutations to predictthe clinical evolution, the risk of develop-ing IBD-related complications, and thelikelihood of responding to certain drugsfor each IBD patient.

SUMMARY

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Key words: inflammatory bowel disease, IBD, Crohn’s disease, ulcerative colitis, genetic factors, DNA array, scanner, pathways to clinical service

Progenika Biopharma S.A., one of the two SMEs involved in the project, has been the mainproject driver, having originated the idea, identified and engaged the partners and will per-form tasks that use a major part of the EC contribution to the budget. Building on develop-ment work done for their existing products, Progenika will use this project to validate newtechnology and knowledge and create a prototype IBDchip that has been tested in clinicaluse and, therefore, is close to final commercialisation. The innovative prototype chip willgive Progenika a clear advantage over competitors in becoming first to market with theirnew knowledge intensive product.

Innopsys is the second SME involved in the project and will develop a slide reader to becommercialised alongside the IBDchip, which will be smaller and much cheaper than exist-ing machines and will take the company into a new market place (the reading of DNA arrays)while reinforcing their scientific and technological capacity for future innovations.

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Miquel SansGastroenterology DepartmentHospital Clínic/IDIBAPSBarcelona, [email protected]

Partners

Marta ArtiedaProgenika Biopharma S.A.Derio, Spainwww.progenika.com

Stéphane Le BrunInnopsys S.A.Carbonne, Francewww.innopsys.fr

Derek JewellUniversity of OxfordNuffield Department of Medicine and Oxford GKPRadcliffe InfirmaryOxford, United Kingdomwww.ndm.ox.ac.ukwww.oxfordgkp.org

Severine VermeireThe University Hospital in LeuvenLeuven, Belgiumwww.kuleuven.be

Salvador PeñaThe Laboratory of Immunogenics Department of PathologyVU University Medical CenterAmsterdam, The Netherlandswww.vumc.nl

Stefan SchreiberUniversity Hospital Schleswig-HolsteinThe Institute for Clinical Molecular Biology (ICMB)Kiel, Germanywww.uk-sh.de

Milan LukasThe University Hospital in PraguePrague, Czech Republicwww.cuni.cz

Silvio DaneseIstituto Clinico HumanitasRozzano Milan, Italywww.humanitas.it

also by immunisation in mouse models. In ath-erosclerotic mice, it has been shown that thevaccination approach may also be applied toatherosclerotic disease. A successful vaccina-tion strategy to prevent adverse outcomes dueto atherosclerotic disease will have enormousimpact on healthcare.

Aim

General project objective: to develop and validatetherapeutic approaches that modulate the innateand adaptive immunological responses in athero-sclerotic disease.

This general objective can be specified accordingto 4 Work packages:

WP 1 – Receptors. To identify targets for interven-tion and test antagonizing compounds in the sig-nalling cascade of the innate receptors, TLR andNOD, to inhibit atherosclerotic lesion development.

WP 2 – Ligands. To identify endogenous ligandsfor TLR and NOD that are involved in athero- ge nesis and assess diagnostic value of ligandexpression and receptor responsiveness followingligation.

WP 3 – Innate Immunity. To inhibit atheroscleroticlesion progression by leading compounds, target-ing TLR ligation or signalling and TNFalpha thathave been developed and licensed by participat-ing SMEs.

WP 4 – Adaptive immunity. To inhibit atheroscleroticlesion progression by immunisation targetinglipoproteins that have been shown to induce a vas-cular inflammatory response and plaque formation.

Background

Atherosclerotic cardiovascular disease remainsas number one killer of the aging population inWestern Society and numbers of cardiovascularevents are strongly increasing in developingcountries. Worldwide about 17 million deaths arecaused by this inflammatory disease and thecosts for health care and loss of productivityexceeds $ 169 billion Euro a year in Europe.Fortunately, increasing knowledge on the mecha-nisms of atherosclerotic disease resulted in pre-ventive strategies and a subsequent decrease inrate of mortality in industrialized countries since1950. However, the increasing prevalence of typeII Diabetes in developed and developing coun-tries deserves careful consideration since thiswill surely influence morbidity and mortality ratesdue to cardiovascular disease. Improvement ofour understanding of the mechanisms that leadto atherosclerotic disease has resulted in innova-tive modalities that may help diagnose, preventand treat this life threatening disease.

We now know that atherosclerosis is an inflam-matory disease and that the body’s immune sys-tem plays a central role in the initiation andprogression of atherosclerotic lesion develop-ment. The recent insights in how the immune sys-tem recognizes endogenous and exogenousligands and how ligation of innate immune recep-tors results in a local inflammatory response, hasopened exciting new therapeutic avenues whichwe would like to implement. Atherosclerosisbears many similarities to other inflammatory dis-eases like rheumatoid arthritis or Crohn disease.Experimental studies revealed that such diseasesmay be treated by different approaches in humans:blockade of innate immunity (anti-TNFalpha) and

Translating innate immune receptor function into diagnostic and therapeutic applications for atherosclerosis

ACRONYM

IMMUNATH

The immune system has a major role in ath-erosclerosis and its innate and adaptivearms jointly and separately co-determineatherosclerotic disease initiation and pro-gression. The search for approaches tomodulate the inflammatory response inatherosclerotic disease is still in its infancy.Inflammatory diseases like rheumatoidarthritis (RA) have much longer been rec-ognized as immune disorders and henceare much ahead in development of thera-peutics targeting inflammation. A multi-disciplinary approach will stimulate thediscovery of immune modulating com-pounds to treat atherosclerotic disease.Thereto, the current project joins forces ofthree SMEs owning unique proprietarycomplementary technology together withfour academic groups, which are activelyinvolved in the field of cardiovascular dis-ease and immune modulation. Researchgroups’ work is dedicated to SMEs todevelop new and test pre-existing leadcompounds that modulate the innate oradaptive immune response and subse-quent atherosclerotic disease. In addition,diagnostic markers will be exploited thatreflect the severity of atherosclerotic dis-ease based on innate receptor ligands andresponsiveness, focused on TLR and NODreceptors.

In work packages (WP) 1 and 2, targets forintervention that have been discoveredusing an integrated genomics or pro-teomics approach, will be validated andantagonists constructed using both smallmolecule and antibody technology. WP 1will focus on innate receptor signalling,WP 2 will address the therapeutic anddiagnostic value of TLR ligands andresponsiveness. In WP 3 and 4, the prop-erties of therapeutic lead compounds anddiagnostic modalities that are based onmodification of the innate and adaptiveimmune response will be assessed usingstate of the art in vitro and animal modelsystems. This STREP will significantlyadvance the position of three SMEs basedon a multidisciplinary approach, and willpromote therapy and diagnosis for a dis-ease with high socio-economic impact.

SUMMARY

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Key words: Atherosclerosis, immune system, toll like receptor, vaccination

Expected results

It is expected to detect:• molecules/proteins that serve as a therapeuti-

cal target in atherosclerotic disease;• targets that may serve as surrogate endpoints

for adverse cardiovascular events in clinical tri-als or as a prognostic marker for clinical eventsdue to atherosclerotic disease;

• therapeutic interventions to prevent initiation orprogression of atherosclerotic disease by: – antagonising receptors, – antagonising ligands, – altering receptor response, – immunisation.


• Therapeutical interventions based on immunomodulation that may prevent initiation of andcomplications by pre-existent atheroscleroticdisease.

• Prognostic measures that may help diagnosingthe patients prone to develop acute myocardialinfarction.

Three SMEs will participate in this consortium: OPSONA (Dublin), Bio-Invent (Lund) andAlloksys (Utrecht). All three companies have specific expertise that does not overlap: pro-duction of TLR agonists/antagonists, immune-therapy based on passive immunisation andToll Like receptor ligand recognition, respectively. These companies will provide tools andcompounds that will be used in experimental models in order to examine applicability totreat atherosclerotic disease. In addition, the academic institutes will search for new lig-ands and proteins that play a role in TLR signalling in atherosclerotic disease. The SMEs willthen try to develop antagonising therapies to prevent or stabilise atherosclerotic disease.These will be evaluated in experimental models in the participating academic centres.

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Gerard PasterkampUniversity Medical Center UtrechtExperimental CardiologyRoom G02-5123Heidelberglaan 1003584CX UtrechtThe [email protected]

Project manager

Arnold HeijkampInteruniversity Cardiology Institute of The NetherlandsPostbox 192583501 DG UtrechtThe [email protected]

Partners

Mark HeffernanChrisopher LocherOPSONA Therapeutics Ltd.Dublin, Irelandwww.opsona.com

Marc FeldmannClaudia MonacoKennedy Reserach Institute Imperial CollegeLondon, United Kingdom

R. CarlssonBo JanssonBioinvent International ABLund, Swedenwww.bioinvent.com

J NilssonHarry BjorkbackaUniversity of LundLund, Sweden

G. HanssonZhong-Qun YanKarolinska InstituteStockholm, Sweden

Jon Daniel LamanErasmus UniversityRotterdam, The Netherlands

Ruud BrandsAlloksys Lifescience B.V.Utrecht, The Netherlandswww.alloksys.eu/php/index.php

Gerard PasterkampDominique de KleijnUtrecht University Medical CentreUtrecht, The Netherlands

Medium TNF10 ng/ml TNF + Remicade Sups + Remicade

Sups IL-1 10 ng/ml IL-1 + IL-1 Ra Sups + IL -1 Ra

| Monaco C. et al, 2007 (unpublished data).

In detail, the objectives are:• the synthesis of novel infrared photosensitizers

with sufficient water solubility (i.e., not sticky tounwanted cells and tissues), which absorb inthe near-infrared light spectrum and which effi-ciently generate singlet oxygen and/or otherreactive oxygen species;

• screening of phage display libraries to find themost suitable antibodies for the project;

• investigation of a novel method for the conjuga-tion of the antibody and photosensitizer mole-cules. The project proposes to investigate thecoupling of photosensitizers to antibodies,based on the non-covalent but stable interac-tion of photosensitizing molecules with specificantibody fragments or suitable single domainbinders;

• evaluation of the conjugates in vitro and in vivo.The project’s novel PDT agents will be exten-sively tested in vitro, in order to ascertainwhether non-covalently bound photosensitizerscan retain singlet oxygen production activityupon irradiation.

The agents will be tested in rodent models of can-cer and, if successful, will open novel therapeuticopportunities for the selective treatment of super-ficial tumours in accessible body cavities.

Expected results

Most importantly, there is a reasonable expecta-tion of a medical benefit for cancer patients stem-ming both directly and indirectly from this Project:• directly, since immuno-PDT procedures promise

to be invaluable for the selective ablation ofinoperable superficial neoplastic lesions, suchas certain head and neck, gastrointestinal, uro-genital and gynecological tumours;

• indirectly, since the knowledge generated bythe validation of novel antibodies for vasculartargeting applications is likely to have an impactin other forms of immunotherapy, including theuse of full IgGs and antibody-cytokine fusionsfor cancer therapy.

Background

Cancer chemotherapy is generally accompaniedby severe side effects, mainly due to unspecificcytotoxicity of classic antineoplastic treatments.Photodynamic Therapy contributes to a signifi-cant efficacy in the treatment of neoplasticand abnormal tissues, using a combination ofphotosensitizer, such as porphyrin, chlorin, bac-terio chlorin or phthalocyanine derivatives, andtissue-penetrating visible laser light. Laser lightpromotes the photosensitizer into its excitedstate. The photosensitizer, in turn, interacts withmolecular oxygen and returns to its ground state,resulting in the generation of the highly localisedcytotoxic agent, singlet oxygen, which ultimatelyaffords tumour destruction.

PDT is a modality of cancer treatment that causescytotoxic action only locally in the region of expo-sure to laser light with a specific wavelengthmatching the absorption profile of the photosen-sitizer, thus leading to very site specific toxicity.The targeted delivery of photosensitizers to suit-able neoplastic sites is likely to increase the scopeand the efficacy of PDT therapy still further. Theantibody-mediated targeted delivery of photosen-sitizers to the tumour neo-vasculature mediates arapid occlusion of blood vessels, thus deprivingtumour cells of oxygen and nutrients and trigger-ing an avalanche of tumour cell deaths. As anadditional benefit, lower doses of photosensitizercan be administered, thus reducing problems ofskin photosensitivity.

Aim

The present project has as objectives the synthe-sis and conjugation of novel infrared photosensi-tizers to the most promising antibodies againstvascular tumour antigens obtained by humanantibody technology, the immunohistochemicalcharacterisation, the biodistribution and imagingtargeting in vivo, in order to select the best anti-body-photosensitizer conjugates to be taken for-ward into clinical trials as a final objective.

Immunophotodynamic therapy of cancer: concepts and applications

ACRONYM

Immuno-PDTwww.immunopdt.net

Photodynamic therapy of cancer, i.e. thegeneration of reactive oxygen species inthe tumour environment which followsthe irradiation of suitable photosensitiz-ing molecules, is an attractive modalityfor the selective ablation of inoperablesuperficial neoplastic lesions. This Proj -ect puts together a network of academicresearch groups and companies for thedevelopment of antibody-based targetedphotodynamic therapy modalities. Theplanned research activity starts with thesynthesis of novel photosensitizing mol-ecules suitable for conjugation to anti-bodies, and with the identification ofnovel human monoclonal antibodies,capable of a selective targeting of thetumour neovasculature for immuno-PDTapplications. Following an extensive invitro characterisation of the most prom-ising antibody-photosensitizer conju-gates, the therapeutic potential of thebest conjugates will be tested in rodentmodels of cancer, paving the way forfuture clinical applications.

SUMMARY

84



Key words: photodynamic therapy, antibody-photosensitizer conjugates, tumour neovasculature

Potential applicationsWhen considering immuno-PDT applications inoncology in a strict sense, the potential market isdirectly determined by the incidence of inopera-ble superficial neoplastic lesions, such as certainhead and neck, gastrointestinal, urogenital andgynecological tumours, which would benefit froma PDT-based ablation.

In a broader sense, discoveries in terms of newtumour targets, new antibodies, new couplingmethodologies, and new photosensitizers willbenefit several areas of biomedical development,including non-oncological indications such aspotentially-blinding angiogenesis-related oculardisorders (such as age-related macular degenera-tion, diabetic retinopathy, etc.).

Immuno-PDT has put together a network of academic research groups and companies forthe development of antibody-based targeted photodynamic therapy modalities. The threeSMEs – Philogen, Photobiotics and Trojantec – are crucial partners in Immuno-PDT andshare 52 % of the budget.

Philogen (Italy) will produce antibodies against vascular tumour antigens by humanantibody technology, with the final aim of selecting the best antibody-photosensitiserconjugates to be taken forward into clinical trials.

Photobiotics (UK) will contribute with the synthesis of novel photosensitizing molecules,suitable for conjugation to antibodies. The wide range of immuno-conjugates made will havediffering physico-chemical properties, which may lead to different internalisation rates.

Trojantec (Cyprus) will use low levels of cell permeating peptides (antennapedia) co-coupled toantibody fragments to enhance cellular delivery. This technology will be used to supplementphotosensitiser delivery to tumour cells, by facilitating photosensitiser delivery to tumour cellssurrounding the targeted vasculature.

The collaborations will establish the feasibility and biomedical potential of a novel class oftargeted PDT applications, and will provide invaluable information about their anti-cancerpotential for selective destruction of tumour neo-vasculature. The three SMEs will ensurethat all necessary expertise and resources are in place, to assist translational activities suchas patent filing, Material Transfer Agreements, licensing deals, GMP manufacture activitiesand clinical development.

ROLE OF SMEs

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Chiara Falciani Reinerio GonzalezPhilogen S.p.A.Siena, [email protected]@philogen.itwww.philogen.it

Partners

Ross BoyleUniversity of HullHull, United Kingdom

Gokhan YahiogluPhotobiotics Ltd.London, United Kingdomwww.photobiotics.com

Luciano ZardiCentro Biotecnologie AvanzateGenova, Italy www.biotecnologie.it

Dario NeriSwiss Federal Institute of TechnologyZurich, Switzerlandwww.pharma.ethz.ch/people/nerid

Peter VajkoczyUniversity of HeidelbergHeidelberg, Germanywww.uni-heidelberg.de/index_e.html

Mahendra DeonarainImperial College LondonLondon, United Kingdomwww3.imperial.ac.uk

Christina KousparouTrojantecNicosia, Cypruswww.trojantec.com

To study the development and evaluate potentialtargets and therapies for Parkinson disease, bio-logical models are needed, in which the eventsthat occur in the patient are mimetized. A target isany molecular entity (transcript, protein, …) thatcan be interfered with to interrupt the chain ofevents leading to the onset or development of thedisease. We will develop novel in vitro models forParkinson disease based on cell cultures for thedevelopment of a high throughput screening plat-form, and use these for in vitro target validationexperiments.

Finally, we will initiate a screen for potential drugsinteracting with selected molecular targets.

Expected results

The INDABIP project aims to deliver an early diag-nostic assay for Parkinson disease validated inthree independent laboratories. We will workclosely together with Parkinson disease PatientsOrganizations to discuss and promote the possi-ble future implantation of this diagnostic assay inthe national health systems.


Early diagnostics becomes really helpful whenappropriate treatments become available thatcan prevent the further development of the incip-ient disease. The INDABIP project will thereforedevelop the tools to screen for drugs that can pre-vent the further development of Parkinson diseaseand initiate the process of drug development.

Background

Parkinson disease (PD), a specific type ofa group of neurodegenerative disorders calledsynucleinopathies, is the second most commonneurodegenerative disorder worldwide. Manyneuro degenerative disorders are preceded bya pre-symptomatic phase, probably lasting years,during which degeneration and death of neuronsoccurs before any clinical symptoms appear. Onemajor challenge of clinical research is to improveearly detection of these diseases by developingtools to move diagnosis backward in the neurode-generation temporal course. Thus, the centralobjective of this INDABIP proposal is the identifi-cation of molecular markers that hallmark theonset of a cellular dysfunction in the brain areasinvolved in PD, and that enable to identify at-riskgroups, both for disease onset and progressionduring the pre-clinical period.

Aim

While biomarkers can take many forms, the IND-ABIP project aims to identify relevant proteins,mRNA, miRNAs, differentially matured RNAs andmethylated DNA, whose analysis can be trans-formed in diagnostic tests. The brain damage inParkinson disease and other neurodegenerativediseases is progressive and, by opening a windowfor therapeutic treatment and prevention, the earlydetection of biomarkers may help to identify indi-viduals that have started to develop the disease.

While most biomarkers are mere reporters of thedisease state, others may actually be key factors inthe processes that lead to the onset and develop-ment of the disease. The second aim of the INDABIPproject is therefore to identify these key factors andto evaluate their potential as drug targets.

Innovative diagnostic approaches for biomarkers in Parkinson disease

ACRONYM

INDABIP

The INDABIP project aims to identify biomarkers for the early diagnostics ofParkinson disease. Biomarkers are anytype of biological molecules that arepresent in people suffering a specific dis-ease, even when the symptoms are mild,but that are not present in people freefrom the disease or suffering from otherdiseases. The identification of the earlyindications of the development of a neu-rodegenerative disease is the basis forpreventive treatment strategies, whichaim to control the disease at early stagesrather than when irreparable neurologicaldamage has been caused.

SUMMARY

86



Key words: Parkinson disease, diagnostics, drug target identification

INDABIP is a SME driven and SME coordinated project. Two companies with complemen-tary interests will receive about 50 % of the project financing: Oryzon Genomics, a biotechcompany based in Barcelona (Spain) and Genfit, a biopharmaceutical company located inLille (France). Oryzon Genomics contributes to the Project with experience in DNA chiptechnologies and bioinformatics applied to the discovery of biomarkers in neurodegener-ative and oncological diseases. Oryzon Genomics will perform analysis of gene expres-sion, splicing and methylation analysis in the project. Genfit has solid knowledge in thefield of gene regulation. The Company has gained a solid experience in the analysis of thefamily of transcription factors known as ‘nuclear receptors’ and has developed innovativestrategies to screen for drug candidates that target and modulate these receptors. Genfitwill also bring its experience in animal models to the project. Both companies believe inthe power of the symbiosis between academic and SME partners and are determined toadd a long term product-oriented vision to the project.

ROLE OF SMEs

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Tamara MaesC. S. O. Oryzon genomics Oryzon genomics S.A.Parc Cintífic de BarcelonaJosé Samitier 1-508028 Barcelona, [email protected]

Partners

Sakina Sayah-JeanneGenfit S.A.Lille, Francewww.genfit.com

Isidro FerrerFundació Privada Institut d’InvestigacióBiomèdica de Bellvitge Bellvitge, Spain

Hans KretschmarLudwig-Maximilians-University of MunichZentrum für Neuropathologie und PrionforschungMünich, Germany

Irina AlafuzoffKuopio UniversityDepartment of Neurology and NeuroscienceKuopio, Finland

Regis BordetDepartment of PharmacologyFaculty of Medicine, UL2leUniversity HospitalLille, France

| LEWY BODIES Spherical intracytoplasmic inclusions withconcentric eosinophilic core and peripheralunique or multilayered narrow, pale-stained,hallo. Diameter ranging from 8 to 30 μm. May bwe unique or multiple. The core iscomposed of densely packed filaments andgranular material. The hallo is formed by radially-arranged 7-20 nm intermediate filaments associated with granular electron-dense coating material and vesicular structures.

PD

Lewy bodies: optical and e.m.

DLBD

Expected results

It is not expected that a ‘ready-to-go’ vaccine willbe developed during the lifetime of this project.The aims are more realistic, namely the valida-tion of one or possibly more of the platform tech-nologies in development. In principle, the projectmay generate encouraging results with one ormore vaccine candidates against either malariaor TB. This could lead to preclinical studies andlong-term clinical studies, in collaboration withindustrial partners.


Heat stable vaccines are of particular interest todeveloping countries enabling more stable androbust products. Invasive delivery systems, ifvalidated, could be efficient at targeting andpreventing infection of not only Plasmodiumand M. tuberculosis but also other intracellularpathogens.

Background

The partners have a background in vaccinedevelopment (Cutting; Cobra Biomanufacturing &Nano-S), vaccine manufacture (Vabiotech), malaria(Heussler) and tuberculosis (Manganelli andDelogu). They have formed a collaborative group-ing through which they will exploit novel intellec-tual property (IP) with the aim of demonstratingproof of principle and commercial feasibility.

Aim

The aim of this project will be to develop new andnovel vaccines that could be developed for vacci-nation against malaria and/or TB. Proof-of-principle that can enhance the value of existing IPwill be the primary aim. The technologies underdevelopment are particularly novel and includeheat-stable bacterial spores for antigen deliveryas well as bacterial systems that can deliver anti-gens inside the host cell (intracellular delivery).Malaria and TB are of concern to both developingand developed countries. One of the partners inthe project, Vabiotech, is a commercial partnerwith a long history in vaccine production froma developing country, namely Vietnam. It isexpected that this company will facilitate thedevelopment of novel vaccine technology fordeveloping countries.

Highly innovative strategies for vaccination to poverty related diseases

ACRONYM

INNOVAC

The INNOVAC project comprises sevenpartners, three of whom are SMEs. Theconsortium will develop three platformtechnologies that will be used for noveland highly innovative methods for vacci-nation against two of the most importantpoverty-related diseases, namely tuber-culosis (TB) and malaria. An importantaspect to this project is the inclusion ofa vaccine-producing SME, Vabiotech,from a developing country. The threeR&D platforms are:• bacterial spores. Robust and heat-stable

bioparticles with proven efficacy asmucosal vaccines;

• intracellular & Invasive bacteria includ-ing E. coli strains and Mycobacteriumbovis (rBCG);

• S-layer protein conjugates and S-layerprotein coated liposomes.

INNOVAC will focus on discovery activi-ties including proof-of-principle studiesto show Ag expression, testing of vac-cines in vitro as well as challenge experi-ments in vivo. The project will test andevaluate highly innovative strategies forvaccination using recombinant systems,some in their infancy and others at a moreadvanced stage of development. Thisproject will include construction of vac-cine vehicles, their evaluation in animalmodels, challenge experiments andfinally safety tests, where appropriate, inorder to take potential vaccines to thestage of clinical evaluation. Inclusion ofa partner SME from Vietnam will enabletechnology transfer to disease-endemiccountries.

SUMMARY

88



Key words: Bacillus, bacterial spores, spore vaccines, heat-stable vaccines, 2nd generation vaccines, mucosal vaccines, oral vaccines, edible vaccines, malaria, tuberculosis, nanobiotechnology, S-layers, M. bovis, M. tuberculosis

The INNOVAC consortium carries 2 SMEs, Nano-S, and Cobra that have IP rights to novel vac-cination strategies, vectors and strains. A further SME is Vabiotech in Hanoi, Vietnam, whowill collaborate with the European partners. Its inclusion is important to introducing andtransferring technology rights to disease-endemic countries.

Cobra, will manage pilot scale production of spore vaccines. This will include the design andproduction of GM spores that are unable to proliferate; GMP production methods for sporeproduction and optimised methods for large scale production.

Nano-S will be responsible for optimisation of S-layer vesicles for mucosal immunization.

ROLE OF SMEs

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Simon M. CuttingSchool of Biological SciencesRoyal Holloway University of LondonEgham, Surrey, TW20 0EX, United [email protected]

Partners

Rocky CranenburghCobra Biomanufacturing plcKeele (Staffordshire), United Kingdomwww.cobrabio.com

Alexander MatisNano S Biotechnologie GmbHVienna, Austria www.nano-s.com

Riccardo ManganelliDepartment of HistologyMicrobiology and Medical BiotechnologiesUniversity of PadovaPadova, Italywww.unipd.it

Giovanni DeloguIstituto di MicrobiologicaFacolta’ di Medicina e Chirurgia “A. Gemelli”Universita Cattolica del Sacro CuoreRoma, Italywww.unicatt.it

Volker HeusslerBernhard Nocht Institute for Tropical MedicineDepartment of Molecular ParasitologyHamburg, Germanywww.bni-hamburg.de

Nguyen Thu VanVabiotechNational Institute of Hygiene & EpidemiologyHanoi, Vietnamwww.vabiotechvn.com

© Shutterstock

• there is limited knowledge about the behav-ioural biology of mice;

• species-pecific functions of structures in themouse brain are poorly known;

• the development of clinical mouse models forpsychopathology faces problems in recognisinghow a behavioural impairment relevant to thehuman disorder would manifest itself in a mouse;

• all these problems are compounded by the lackof standardisation and comparability of testresults.

Animal welfare. For behavioural testing, mice areoften kept isolated over prolonged periods. Yet,mice are social animals and isolation in an impov-erished environment can cause many physiologicaland neurochemical changes producing con-founded results. Moreover, European legislation islikely to ban animal housing under isolated condi-tions. This calls for social in-cage alternatives inbehavioural testing.

Aim

This SME project allies three SME’s and four aca-demic partners for developing and validatinga compact, economic and fully automated modu-lar platform, INTELLIMAZE, that will permit bothhigh-throughput and detailed behavioural char-acterisation of current and future mouse modelsin biomedicine. In accordance with legislativetrends in Europe, the system will operate withmice living in social groups in a home cage.

The technical development will be done by twoSMEs (NewBehavior, and Frank Buschmann Inter -national) and one academic partner (Laboratoryof Systemogenesis at the Anokhin Institute ofNormal Physiology of the Russian Medical Academyof Science).

The commercial objective is to obtain a significantmarket share of the developing market of auto-mated test systems, expand the present market toinclude new categories of customers, and to tailorsuch systems for small biotech SMEs in need ofbehavioural characterisation.

Background

Too many mice. During the past 15 years, molecu-lar biology and ENU mutagenesis programmeshave led to an explosion of mouse models, manyof them needing behavioural characterisation.Behavioural characterisation (‘phenotyping’) oflaboratory animals is an established tool in neu-robiology and neuropharmacology, but may findmuch wider application in drug development, eco-toxicology and other fields of biomedicine dealingwith changes in body physiology. However, thereis no appropriate technology to deal with the largenumber of mice. High-throughput behaviouralphenotyping (HTBP) would be needed, either formass screening of mice, or, equally importantly,for the application of numerous tests to selectedsamples.

Simplistic technology. Traditionally, animals aresubjected manually to a battery of tests designedfor demonstrating changes in spatial or fear-related memory, motivation, exploration or learn-ing abilities, thought to reflect the operation ofspecific brain system. In addition, tests have beendesigned to model processes observed in humandiseases of the CNS. Thus, every laboratory spe-cialising in behavioural analysis must maintain anexpensive collection of test set-ups, all of thembeing conducted with single mice.

Economic limits. A standardised preliminary behav-ioural screen of a knockout mouse requires testingof about 100 mice in 4-6 different tests, equallingthree person-months; a more detailed characteri-sation requires up to 6 person-months, even whenusing highly standardised procedures and auto-mated data analysis. Thus, the main obstacles forhigh-throughput behavioural phenotyping arecosts in terms of space, salaries and expertise.

Hidden problems in mouse testing. Traditionalbehavioural characterisation of mice faces a num-ber of problems well known to experts but rarelydiscussed publicly:• mice are far less cooperative than rats and other

laboratory animals;

High-throughput, fully automated and cost-effectivebehavioural phenotyping of normal, clinical andgenetic mouse models

ACRONYM

INTELLIMAZEwww.intellimaze.org

Behavioural change is the most sensitive bio-logical end-point signalling any alteration inthe organism of a mouse. However, large-scale bio-assays of behaviour face limita-tions: outdated technology, need for spaceand specialised manpower, lack of standard-isation, and increasing legal demands on ani-mal husbandry. This has limited the use ofbehavioural methods to specialised labora-tories, also limiting the market.In order to advance scientific progress andexpand the market, 2 SMEs and 1 academicpartner will combine their existing expert-ise and products for behavioural pheno-typing to generate a compact modularsystem, INTELLIMAZE, based on a recentlydeveloped technology of in-cage testing ofmicrochipped mice, INTELLICAGE. This sys-tem should fit into a single small mouseroom, where it will: • assess home cage activity and learning of

transponder-tagged mice living in socialgroups;

• automatically analyse social behaviour;• guide individual mice to a battery of tra-

ditionally used tests;• show ongoing learning on-line to a super-

visor working in his office;• analyse data according to expert knowl-

edge-based rules;• provide a web-based analysis of results

for user groups.The bottleneck of such technological devel-opment is functional validation and compar-ison with traditional tests. Three reputedacademic partners will use the novel tech-nology for: • generating new mouse models of depres-

sion;• profiling malfunctions of specific brain sys-

tems; and • monitoring the effects of age-dependent

neurodegeneration in existing and newmouse models. Finally, an SME partner inneed of efficient behavioural phenotypingwill validate the novel systems for drug dis-covery and development.

It is expected that the availability of simpli-fied, rapid and thorough behavioural testingof mice without need for specialised per-sonnel will open new and much larger mar-kets for the SMEs. Moreover, significantscientific discoveries in the fields of drugdevelopment, psychopharmacology, neu-rodegeneration, neural plasticity and repair,and genetic engineering are predicted.

SUMMARY

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Key words: mouse phenotyping, testing apparatus, high-throughput testing, transponder technology, automation, Alzheimer’s disease, depression, anxiety, brain lesions, neurology, transgenic mice

The joint scientific objectives of the academicpartners are validation of this novel technologyand inter-laboratory standardisation. Accordingto their expertise, they will also implement thenovel technology specifically for:• in-depth behavioural profiling of brain lesions in

mice, focussing on hippocampus, striatum andprefrontal (University of Zurich);

• development of novel behavioural paradigmsfor mouse models of anxiety and depression(Istituto Superiore di Sanita in Rome);

• monitoring the progress of brain malfunction inmouse models of Alzheimer’s disease and pre-mature ageing (EVOTEC Hamburg and NEUROTECat Karolinska Institute Stockholm).

Expected results

What is proposed here is almost a dream for mousebehavioural biologists: a fully automated behav-ioural test system for mice inside a home cage, andoutside in traditional test arenas, working withoutsupervision, and outputting data in familiar format

and ready for statistics. Scientifically, it will permitto conduct almost all those studies that shouldhave been done but were never realised. Moreover,cross-laboratory standardisation is achieved withminimal efforts, and animal welfare guaranteed.Commercially, it will certainly penetrate the market,and has the potential of opening new markets inbiotechnology and pharmaceutical industry havingbeen hesitant in using costly behavioural assess-ment. Finally, as the products combine state-of-the-art technology with expert behavioural knowledge,it will offer a sustainable perspective for the SMEs,providing a technological edge over the US, andkeeping production competence in Europe.


It is expected that these systems will be employedby the pharmaceutical industry, by small biotechcompanies producing and testing their own com -pounds, by companies offering phenotypingservices for mice, and by academic behaviouralresearch laboratories.

Three SMEs, sharing 45% of the project budget, are key players of the project. NewBehavioracts as coordinator and is responsible for the development of hard- and software for novelautomated systems evolving from current products centred on automated assessment ofbehaviour and learning in home cage systems. Frank Buschmann International adds comple-mentary expertise in construction of behavioural apparatus of single animal testing, togetherwith expertise derived from its products in web-based management of experimental protocolsand mouse colony supervision. Thus, the resulting INTELLICAGE and INTELLIMAZE system willnot only operate in a single laboratory but can be used for parallel multi-user testing inEuropean projects and large pharmaceutical companies. Finally, the company Evotec, actingas a validating user, brings its experience in industrial drug development and testing. This willensure that the developed systems do comply with both the needs of academic research lab-oratories and those of pharmaceutical and biotech companies. It is anticipated that thisapproach will substantially enlarge the market for behavioural testing systems for mice.

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Hans-Peter Lipp NewBehavior AGHardurmstrasse 76CH-8005 Zürich, [email protected]

Project manager

Toni Lipp [email protected]

Partners

Frank Buschmann Frank Buschmann International GmbH Bochum, Germanywww.fbiscience.com

Enrico AllevaIstituto Superiore di SanitàDipartimento di Biologia Cellulare e NeuroscienzeRoma, Italy

Abdul Mohammed Karolinska InstituteDepartment of NeurobiologyHealth Care Sciences and Society (Neurotec)Karolinska University HospitalStockholm, Sweden

David P. Wolfer University of ZürichInstitute of Anatomy Division of Functional NeuroanatomyZürich, Switzerland

Konstatin Anokhin Russian Academy of Medical ScienceAnokhin Institute of Normal PhysiologyDepartment of SystemogenesisMoscow, Russia

Antje Willuweit EVOTEC Neurosciences GmbHIn vivo Pharmacology GroupHamburg, Germany

| Assessing learning abilities of individualtransponder-tagged mice in an automated home-cage (INTELLICAGE). Each cage house upto 16 mice and contains 4 operant conditioningunits allowing for assessment of spatial, temporal and operant learning abilities.

Clinical cardiotoxic effects are defined as symp-toms of clinical heart failure, and subclinical car-diotoxic effects such as cardiac abnormalitiesdetected in asymptomatic persons by means ofvarious methods. One of the problems is the avail-ability of preclinical models able to screen rapidlya large library of substances and to providerational bases for clinical trials. A significant bot-tleneck in the development of novel assays hasbeen the lack of research purposes. Animal mod-els have been invaluable for risk assessment ofcompound safety; however, critical limitationsremain in these models for robust prediction ofcertain toxic outcomes in humans.

Aim

The overall aim of the project can be summarisedas follows:• to replace animals with human cell culture sys-

tems in preclinical pharmaceutical developmentand chemical substance toxicity testing;

• to support the predictability of the drug discov-ery and development process of cardiovascularpharmaceuticals by allowing more reliable andrelevant testing in the preclinical phase andhinder weak lead candidates to enter clinicalphases with innovative human cardiomyocytecell systems;

• to deliver an in vitro testing system with adja-cent methodology pertinent for validation inGLP/SOPs environment for cardiac safety;

• to deliver in vitro testing systems with adjacentmethodology pertinent for chemical substancetoxicity testing within REACH;

• ultimate aim: to reduce or even totally abolishthe use of animals in drug and chemical sub-stance testing, refine the model system underconsideration and to replace the animal modelscurrently used.

Specific technology related objectives:• to establish relevant hES cell derived cardiomy-

ocytes cultures that allow a more predictable pre-clinical lead testing program to be carried out;

Background

In the pharmaceutical industry, reliable in vitrocell models would contribute to replace currenttechniques with animal experimentation in theselection and optimisation of lead compoundsand in documentation of a selected drug candi-date before it enters clinical phases. In the toxic-ity testing of chemical substances, replacement ofanimal testing methods can be attained as well.The means to accomplish the objective, in addi-tion to new stable hES cell derived cardiomy-ocytes, are as follows:• state of the art methods for electrophysiological

cardiac cell monitoring;• optical micro-sensor monitoring in micro-culti-

vation systems for in vitro screening;• a multi-micro-bioreactor platform for high-

throughput screening of drugs and chemicals.

Invitroheart will carry out comparative studies ofcardiomyocytes derived from hES cells withestablished in vitro models in order to validate thenew models and methods. The outcome of theproject is new efficient in vitro pre-validationmodels which will significantly reduce the use ofanimal experimentation for cardiotoxicity testingby 60-80 %. Furthermore, it will strengthen thepossibility for the participating SMEs to marketnew potential products in the areas of in vitroassay methods and in vitro compound screening.

Studies of toxicity and safety pharmacology ingeneral, and cardiotoxicity in particular, are keyactivities throughout the drug discovery programsin the pharmaceutical industry. Such activities areinitiated to detect detrimental compound effects.For example, electrophysiological changes such asQT prolongation, which leads to delayed ventricu-lar repolarisation and cardiac arrhythmia, areinduced by a vast range of chemical entities formultiple therapeutic areas. Unintended QT effectsof new drugs are the most common cause of drugwithdrawal from the market and delays in or lackof regulatory approval for marketing.

Reducing Animal Experimentation in Drug Testingby Human Cardiomyocyte In Vitro Models Derivedfrom Embryonic Stem Cells

ACRONYM

InVitroHearter-projects.gf.liu.se/~invitroheart

Invitroheart seeks to establish stable celllines that reliably reflect human cardiomy-ocyte properties through the develop-ment of models derived from humanembryonic stem (hES) cells. Its aim is todeliver reliable in vitro models that couldbe used by the pharmaceutical industryto replace experimental animals in: • investigations on pharmacological toxi-

city and safety of compounds in the drugdiscovery and development processes;

• the testing of toxic effects of chemicalsaccording to the new system of the Com -munity on the Registration, Evaluationand Authorisation of Chemicals (REACH).

The Invitroheart consortium is composedof nine participants and four of them areSMEs. The SMEs contribution is crucial tothe project as regards the generation ofcells and assay technologies, and asexperts in life science and micro-sensortechnologies, they are the key providersof state-of-the-art technology.

SUMMARY

92



Key words: embryonic stem cells, cardiomyocyte, animal experimentation, safety assessment, in vitro models, cardiac toxicity, electrophysiology, QT prolongation, optical sensor, high-throughput screening, bioassays

• to develop a real-time sensor based in vitromodel for short-term studies of cardiac side-effects that mimics the function and complexityof the cardiomyocyte tissue in vivo;

• to develop a real-time sensor based in vitromodel for long-term studies of cardiac side-effects that mimics the function and complexityof the cardiomyocyte tissue in vivo;

• to establish a versatile cell lab platform basedon the developed cell lines and cultivated inadvanced miniaturised bioreactor systems withnon-invasive measurement techniques for invitro testing of cardiotoxicity.

Expected results

Successful results of the project will, if adopted bythe European pharmaceutical community, lead to:• the preservation of significant numbers of

experimental animals;• reduced work carried out during pharmaceuti-

cal drug discovery and development in thepreclinical and early clinic phases;

• improved predictability of quality of lead candi-dates increases the chances for passing theentire clinical trials process;

• reduced total development time of leads;• attainment of better quality and safety in docu-

mentation filed to regulatory bodies.

Out of nine partners in Invitroheart, four are SMEs (Cellartis, Multi Channel Systems,Pharmacelsus and PreSens) and their share of the requested funding from the Commissionamounts to 59 %. They provide state of the art expertise in key research activities.

In particular:

Cellartis brings to the project a strong expertise in the field on hES cells. They will establishappropriate protocols for the production and characterisation of cardiac cells, for qualitycontrol of these cells according to GMP-standards, and for development of innovative cellpreservation methods. They will provide to partners well characterised human cardiomy-ocytes for the planned research. A future goal is to investigate in detail differentiationprocesses and to provide large quantities of cardiomyocytes to customers.

Multi Channel Systems (MCS) provides functional electrophysiological monitoring duringthe development of the hES cell derived cardiomyocytes. Furthermore, MCS will developa hardware platform optimised for cardiomyocyte electrophysiology screening, includingdevelopment of software to automate the data acquisition and analysis. Also, a prototype ofan integrated automated screening system will be manufactured. MCS provides access tothe worldwide distribution network to market the complete assay.

Pharmacelsus will design and build new in vitro assay systems for hES cell derived car-diomyocytes in order to support predictive in vitro models of the consortium. Furthermore,Pharamacelsus will provide its scientific experience of a broad range of in vitro models forpharmaceutical testing and will integrate consortium knowledge in statistical validationinto the developed new assay systems. Pharmacelsus’ excellent in-house facilities foranalysis (Fluorimetry, Spectroscopy, HPLC, LC-MS/MS) will support partners to carry outspecific assay testing with the different systems developed.

PreSens will integrate chemical optical sensors for pH and oxygen measurements in theassay technologies developed, adapt existing sensors and develop new sensors accord-ing to the special demands of the new methods. PreSens will integrate optoelectronicinstruments based on its sensor technology and will deliver these to the partners of theconsortium. Furthermore, it will investigate the implementation of fluorescence intensitymeasurements in the existing instruments and will give support in data interpretation.

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Carl-Fredrik MandeniusLinköping University, IFM, SE-581 83Linköping, [email protected]/biotech/

Project manager

Magnus [email protected]

Partners

Peter SartipyCellartis ABGöteborg, Swedenwww.cellartis.com

Thomas MeyerMulti Channel Systems GmbHReutlingen, Germanywww.multichannelsystems.com

Christine Batzl-HartmannPharmacelsus GmbHSaarbrücken, Germanywww.pharmacelsus.com

Christian KrausePreSens GmbHRegensburg, Germany www.presens.de

Morten LaursenLundbeck A/SDept. of Safety PharmacologyValby, Denmarkwww.lundbeck.com

A. LindahlGöteborg UniversityDept. of Clinical Chemistry/Transfusion MedicineGöteborg, Swedenwww.biomedicine.gu.se/avdelningar/avdelning_4

Elmar HeinzleSaarland UniversityDept. of Biochemical EngineeringSaarbrücken, Germanywww.uni-saarland.de

Susanne BremerECVAM (European Centre for the Validation of Alternative Methods)Joint Research Center Institute for Health and Consumer ProtectionIspra, Italyecvam.jrc.it/index.htm

medicinal chemistry issues that can critically influ-ence the time schedule for obtaining an investiga-tional drug candidate. Nevertheless it is alsoexpected as a byproduct of the project that poten-tial drug candidate(s) with high quality in vitroactivity profile can be obtained ready for in vivopharmacology profiling.

In particular LIGHTS objectives are:• derivation of small-ligand libraries with ligands

design to bind to the Thymidylate synthasemonomer/monomer interface affecting dimerformation and TS- TSmRNA interactions;

• validation of the integrated, multidisciplinarydrug design strategy necessary to achieveobjective 1, which poses a highly challengingdesign problem. The strategy including proteincysteine SH-labelling to identify low-affinityligands, peptide mimetic design, and filteringfor ADME properties;

• identification of small-ligands identified ina chemical-biology approach as effective per-turbing agents to investigate the mechanismof resistance against a panel of cis-platinumresistant ovarian carcinoma cell lines;

• provide potential drug candidate(s) with newmechanism of action for further development assafer therapeutic agent(s) for the treatment ofovarian carcinoma.

Expected results

The project provides the integration of differentscientific areas that are culturally well separated intomore established medical approaches to impacthealth determinant in ovarian cancer disease.

It supports the discovery of new potential anti-cancer drugs with non-cross resistance profiles;the new potential drug candidate will be ready toenter the pharmacological phase. If the new identi-fied molecules will be moderately/very active withgood bioavailability in vitro against ovarian cancer,these products will be very interesting to largerpharmacompany and could provide a successfultechnological transfer outcome.

Background

Ovarian cancer is the fifth most common cause ofdeath from cancer and the most common cause ofdeath from gynecologic cancer in women of allages in the Western world. Single-agent carbo-platin (cDDP) has been considered a reasonableoption for first-line chemotherapy for ovarian can-cer. However, the occurrence of resistant cell pop-ulations in the tumour, limiting the usefulness ofthe platinum drug represents a growing problem.Resistant cells often became refractory to the ini-tially used drugs and extremely difficult to eradi-cate. Therefore the use of drug combinations isnecessary. The combination of cDDP and antifo-lates such as azidothymidine (AZT), a deoxythymi-dine analogue, or more recently pemetrexed(Alimta), which inhibits three enzymes in the denovo purine and pyrimidine pathways, has beenshown to synergistically affect the growth ofhuman ovarian carcinoma cells resistant to DDP.Due to the role played by the enzymes of DNAsynthesis and repair in the occurrence of cDDP-resistance, it seems of great priority to developclinical reagents designed to limit the intracellu-lar level of TS protein, which is associated withclinical resistance, thus sensitising even resist-ant cells to the effects of anticancer drugs. Theultimate aim of LIGHTS is to directly halt thetumour progression and interfere with the devel-opment of drug resistance upon treatment withplatinum derived drugs by inhibiting the proteinregulatory function of TS through small moleculecellular perturbation.

Aim

The project is clearly oriented to directly halt theprogression of ovarian cancer and interfere withthe development of drug resistance upon treat-ment with platinum-derived drugs by inhibiting theprotein regulatory function of monomeric TS. Theintermediate objectives are based on employingnovel medicinal chemistry strategies to identifypotential drug candidates with new mechanisms ofaction. LIGHTS specifically addresses early phase

Small ligands to interfere with Thymidylate synthase dimer formation as new tools fordevelopment of anticancer agents against ovarian carcinoma

ACRONYM

LIGHTSwww.lights-EU.org

Ovarian cancer is the fifth most commoncause of death from cancer in women. Thestandard first-line treatment is a combina-tion of paclitaxel and carboplatin (DDP) orcarboplatin alone. In the case of progres-sive disease or drug resistance treatmentwith platinum, either alone or in combi-nation, especially investigational com-pounds should be used. The mechanismsbehind acquired resistance to cDDP andits derivatives are not clear yet, althoughit is evident that the process is multifacto-rial, including enhanced DNA repair. In thehuman ovarian carcinoma cell line A2780,a 3-fold-DDP-resistance was associatedwith cross-resistance to the thymidylatesynthase (TS) inhibitor 5-fluorouracil andto methotrexate, a 2.5-fold increase in TS,and an increase in the intracellular poolsof the TS cofactor 5, 10-methylentetrahy-drofolate and of tetrahydrofolate. The ulti-mate goal of LIGHTS is to directly halttumour progression and the developmentof drug resistance upon treatment withplatinum derived drugs, by inhibiting theprotein regulatory function of monomericTS through small molecule cellular per-turbation. The scientific and technologicalobjectives will be to design small-ligandlibraries to bind to the TS monomer (dimerinterface) and thereby disrupt TS. Thestrategy will include systems pathwayanalysis, protein SH-labelling to identifylow-affinity ligands, peptide mimic design& synthesis, and filtering for ADME prop-erties. The multidisciplinary approach willbe carried out by a consortium integratingMolecular modelling, Chemistry, Chemoin -formatics, Structural Biology and Pharma -cology, and will apply the knowledgebeing created by genomics and otherfields of basic research to the problem ofdiscovery of anticancer agents. The con-sortium consists of six groups from fivedifferent countries, including three SMEs.

SUMMARY

94

Contract number: LSHC-CT-2006- 037852 | EC contribution: € 1 902 150 | Duration: 36 months


Key words: ovarian cancer, thymidylate synthase, drug resistance, drug discovery, protein-protein interaction

The proposed research could provide the technicaldevelopments and innovation, which could havea large impact on Biotech industry. The selection ofthe participating SME partners guarantees that thenew knowledge (methods, potential drug(s) candi-date(s)) might be transformed into new technologyand new products.


The proposed research could lead to technicaldevelopments and innovation, which could havea large impact on Biotech industry. The selectionof the participating SME partners guaranteesthat the new knowledge (methods, potentialdrug(s) candidate(s)) might be transformed intonew technology and new products. The consor-tium, in addition to its contribution to the pro-ject’s basic research by providing a suitablediscovery chemistry programme, will be respon-sible for further development of the selectedpromising new chemical entities comprisingintellectual property protection, chemical andpharmaceutical development.

Out of the 6 partners, three are SMEs, highly committed to the project research objectivesand outcome. In fact, the involvement of the SME Naxospharma, Molecular Discovery andEML provide expertise in discovery and synthetic chemistry support, lead development,intellectual property issues, searching for out-licensing and/or co-operative opportunitiesfor the inventive aspects of the project.

In particular:

EML Research will contribute by setting up and simulating biochemical network models forthe cycles involving TS, protein design, protein structure-based ligand and peptide design,ligand optimisation, and mechanistic studies of effects of ligands on TS dimerisation andRNA binding.

Naxospharma encompasses discovery and synthetic chemistry support, lead development,intellectual property issues, searching for out-licensing and/or co-operative opportunitiesfor the inventive aspects of LIGHTS.

Molecular Discovery will provide high quality software tools and chemoinformatics proce-dures aimed at the prediction of the physicochemical profiling of the investigated com-pounds with potential anti-tumour activity. This data is crucial for the discovery planningand flow-chart LIGHTS. IT procedures, models building and applications will be the maintask of Molecular Discovery. The software will be developed and implemented by intro-ducing the data obtained by the other partners in LIGHTS. Particular focus will be givento ADMEtox profiles.

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Costi Maria PaolaInstitution, University of Modena and Reggio Emilia, ItalyVia Campi 18341100 Modena, [email protected]/home/dipfarm/costi.mariapaola

Partners

Rebecca WadeEML Research GmbH Heidelberg, Germanywww.eml-r.org/english/index.php

Paolo LombardiInstitution, Naxospharma srlCesate (MI), Italywww.naxospharma.com

Hannu MyllykallioInstitut de Génétique et Microbiologie Université Paris-SudOrsay, Francewww.igmors.u-psud.fr/MYLLYKALLIO/MYLLYKALLIO-eng.htm

Massimo BaroniMolecular Discovery Ltd.Ponte San Giovanni (PG), Italywww.moldiscovery.com

Robert StroudUCSF-Genentech Hall, UCSFSan Francisco, USAmsg.ucsf.edu/stroud

| Detail of the X-ray source of the European Synchrotron Radiation Facility (ESRF), Grenoble, France.

from industrial enterprises. In vitro systems complywith the request of reducing animal experiments,thus satisfying a widely shared ethical concern. Onthe other hand, they offer economic advantages interms of reduced time consumption and lower costsin safety assessment of novel drugs.

Aim

The main aim of the project is to optimise and pro-vide established protocols and experimental invitro models for testing intestinal and liverabsorption, metabolism and toxicity of moleculesof pharmacological interest. Moreover, from a sci-entific point of view, the project representsa unique effort to link, by an in vitro approach, dif-ferent systems (i.e. gastrointestinal tract andliver) and pathways involved in vivo in the absorp-tion and metabolism of orally ingested sub-stances. This kind of approach is important forfurther development of tests for chronic exposurein which the interrelation between differentorgans is a key factor and where at present nearlyno in vitro data are available. Such integratedmodels will be developed to the point of enteringpre-validation procedures to be submitted toregulatory boards.

Expected results

• To provide standardised cellular models ofhuman hepatocytes and enterocytes reliablefor prediction of drug absorption, metabolismand toxicity. Sequential procedures, easilyamenable to validation studies, possibly byminiaturised and automated technology will bedeveloped.

• To obtain on these models a database concern-ing in vitro absorption, metabolism and toxicityof selected drugs including the characterisationof the regulation of relevant genes.

Background

In Europe, three Directives regulate the testingof chemicals: Council Directive 67/548/EEC andits subsequent amendments; Council Directive88/379/EEC and subsequent amendments, andCouncil Directive 76/769/EEC. Moreover, theCouncil Regulation No. 793/93 on the evaluationand control of risk of existing substances alsodeals with the same topic. The main new aspect isthat a distinction has been made between the newsubstances notified since 1981, and those notifiedbefore then. For the latter (about 100 000), it hasbeen estimated that insufficient data are availableconcerning their safety. Thus, the White Paper pro-poses a harmonisation of testing requirements fornew and existing substances, by introducing a newsystem for the Registration, Evaluation and Authori -sation of new and existing Chemical Substances(REACH) (COM-2001/88 Final; COM-2003/644).

This implies, on the one hand, a cumbersome planof testing, and on the other hand, the use ofa huge number of animals. The European Centrefor Validation of Alternative Methods (ECVAM)has already addressed the possibility of usingalternative methods, according to the 3Rs model,in order to reduce this number, or at least the ani-mal suffering associated with certain kind of tests.The EU has addressed this issue in the directive86/609. The use of non-validated alternatives hasalso been suggested based on the ‘weight of evi-dence’, i.e. widely used and well consolidated pro-cedures. This project may well contribute to thoseaspects, by providing new procedures submittedto optimisation.

A successful outcome of the project, in fact, willhave a strong and diversified impact on social andeconomic issues. The main effect will be related tothe expanded use of in vitro systems, meetingexisting expectations from the public at large and

Optimisation of liver and intestine in vitro models for pharmacokinetics and pharmacodynamics studies

ACRONYM

liintopwww.liintop.cnr.it

The main aim of the project is to optimiseand provide established protocols andexperimental in vitro models for testingintestinal and liver absorption, metabo-lism and toxicity of molecules of pharma-cological interest. The added value of theproject, with respect to the existingexperimental approaches in this field, isto provide optimised sequential proce-dures, easily amenable to validationstudies for screening and testing of newdrugs, possibly by miniaturised andautomated technology. A consortium of15 participants includes seven compa-nies, out of which six are SMEs. Thedirect participation of SMEs in theresearch activities will assure that thoseprocedures will meet the requirements ofindustrial application.

The project is, therefore, articulated indifferent approaches, which will integrateat various levels. A first basic approachwill be the optimisation of in vitro liverand intestinal models for their use intransport and toxicity of structurallydiverse reference drugs chosen with thehelp of a steering committee of relevantstakeholders. A parallel approach willdeal with the identification of the trans-port and metabolic pathways and possi-ble cytotoxic effects of these drugs, inorder to develop appropriate monitoringprocedures. New advanced technologies(genomics, proteomics, metabolomics)will be used in order to develop highthroughput models related to the specificarea of intestine-liver absorption andbiotransformation. Each approach willtake care of the reliability of the protocolsused and of the relevance of the wholeprocedure to the in vitro/in vivo extrapo-lation of drug effects. To this end, a unit incharge of computer-based studies willsupport and pilot the project throughout.

SUMMARY

96



Key words: in vitro human hepatic and intestinal models, metabolism, absorption pharmacokinetic and pharmacodynamics, in silico modelling

• To develop prediction mathematical models ofpharmacokinetics and pharmacodynamics basedon the available in vivo data and on the in vitrodata from the cellular models used in the project.

Best case scenario:

• to transfer the best hepatocyte and enterocytemodels and relevant testing procedures to theindustrial setting for high throughput applica-tions in the development of new drugs;

• establishing the relevance of the proposed invitro models to human in vivo situation.


Optimised sequential procedures and referencestandards concerning in vitro models of hepa-tocytes and enterocytes, easily amenable tovalidation studies will allow a new molecule ofpharmacological activity to be tested for:• potential toxicity at the level of the intestinal

mucosal barrier;• intestinal absorption;• metabolic modification in the intestinal cells;• absorption and metabolism in the hepatocytes;• hepatotoxicity;• optimal pharmacokinetic behaviour.


Flavia ZuccoCNR, Istituto di Neurobiologia e Medicina MolecolareVia del Fosso di Fiorano 6400143 Roma, [email protected] www.cnr.it/sitocnr/home.html

Partners

Pascale AnderleEnte Ospedaliero Cantonale/Istituto Oncologico Svizzera Italiana (EOC/IOSI)Bellinzona, Switserlandwww.eoc.ch/

Jose V. CastellDep.de Bioquímica Fac. Medicina/Centro de InvestigacionHospital Universitario La FeValencia, Spainwww.fundacionlafe.org/indexE.htm

Christophe Chesne BIOPREDIC InternationalRennes, Francewww.biopredic.com

Andrè GuillouzoINSERM U62OFaculté de Pharmacie, Université de Rennes 1Rennes, Francewww.inserm.fr/en/home.html

Jouni HirvonenFaculty of Pharmacy, University of HelsinkiHelsinki, Finlandwww.helsinki.fi/university/index.html

Brian HoustonSchool of Pharmacy and Pharmaceutical SciencesUniversity of ManchesterManchester, United Kingdomwww.manchester.ac.uk

Among the 15 consortium participants, seven are industrial companies, namely six SMEsplus a large company, NV Organon. This project presents a strong contribution from high-tech SMEs, like Biopredic International, Advancell, Siena Biotech that will convey into theproject leading and innovative core technologies and services. Their direct participation inthe research activities (particularly on transporters and metabolites identification) willassure that the optimised sequential procedures, for screening and testing of new drugs willmeet the requirements of industrial application.

This proposal addresses an issue of great relevance in the drug discovery process, i.e.absorption and entero-hepatic bio-disposition of drugs, which presently still relies onin vivo experimentation. The development of integrated in vitro predictive tools capable ofaddressing these issues, possibly sustained by miniaturised and automated technology,will have a positive outcome in competitiveness and success of pharmaceutical industry,since decisions taken on partially characterised compounds (or with questionable humanrelevance) may lead to great economic losses at later stages.

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Maria Laura ScarinoIstituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN)Roma, Italywww.inran.it

Vera RogiersVrije Universiteit BrusselDept. ToxicologyBrussels, Belgiumwww.vub.ac.be/english/index.php

Jouko UusitaloNovamass Analytical Ltd. Oulu, Finlandwww.novamass.net

Sjeng Horbach NV OrganonDepartment of PharmacologyOss, The Netherlandswww.organon.com/authfiles/index.asp

Dr. Karen Rowland -YeoSimcyp Limited Blades Enterprise CentreSheffield, United Kingdomwww.simcyp.com

Ugo ZanelliSienaBiotech SpADrug Profiling – MetabolismSiena, Italywww.sienabiotech.com/index/index.jsp

Myriam FabreAdvancell Barcelona, Spainwww.advancell.net/index.html

Peter KrajcsiSOLVOBudaörs, Hungarywww.solvo.hu

| In vitro intestinal cells, immunostained with a thight junctions marker.

Aim

The MagRSA project aims at the development ofa new diagnostics platform that will provide a fast,simple, automated and accurate identification ofMRSA from clinical samples.

The diagnostic protocol that is proposed relies ona new and clinically validated procedure that con-sists of a direct one-step enrichment of MRSApresent in either nasal or inguinal swabs, followedby DNA extraction of immunocaptured bacteriaand their identification by multiplex sequenceamplification, using real-time quantitative PCR.This protocol will be implemented with a simple‘hands-off’ system based on: • novel strategies for the integration of full opera-

tions required for the entire nucleic acid analy-sis chain in a microfluidic platform; and

• advanced microfluidic magnetic nanoparticlesmanipulation technology allowing efficient cap-ture and extraction of target bacteria andnucleic acids. The separate steps of samplepreparation, signal amplification by multiplexPCR, and simultaneous detection of multiplegenes, will be performed as one single stepusing a ready-to-use disposable fluidic chip.

In light of the above, this project aims to providehospitals and care units with a fast, easy andautomated test for the rapid diagnostic of MRSA.Moreover, the simplicity of the proposed technol-ogy concept, integrating cost effective and widelyavailable components, allows for the provision oflow cost systems, a prerequisite condition for thelarge adoption of molecular tests by hospitals.

Background

Methicillin-resistant Staphylococcus aureus (MRSA),an organism resistant to many drugs, is seen withincreasing frequency in hospitals and long-termcare facilities. It can cause life-threatening disease,and treatment options are limited. MRSA infectionsare indeed associated with a 40% mortality whenfound in the blood of patients suffering fromsevere staphylococcal infection. According to theWorld Health Organization (WHO), resistance ofStaphylococcus aureus (Staph A) to methicillin, itsusual antibiotic, increased from 2% in 1975 to 60%today and no new antibiotic is expected on the mar-ket for many years. Whereas MRSA is considered asa nosocomial pathogen, recent reports showed anincreasing number of outbreaks in the community,despite the absence of known risk factors (priorhospitalisation, antibiotic use or household con-tacts from the healthcare system). Indeed, a rela-tively large spread of MRSA strains within the gaycommunity was recently reported in severalEuropean countries and the United States. Suchatypical MRSA is known to produce a potent toxincausing severe skin infections and necrotisingpneumonia in both immunocompromised andimmunocompetent individuals.

With such critical health issues, an early detectionof MRSA carriers is crucial for infection controlstrategies but also to take appropriate therapeuticdecisions, avoiding non-appropriate utilisation oflast barrier antimicrobial agents. Strategies to fightMRSA transmission are indeed well-documented,and can efficiently reduce subsequent colonisationand infection. However, for cost issues and betterpatient management, these strategies need to befocused on patients with confirmed MRSA anddefined resistance phenotype.

Fully Automated and Integrated MicrofluidicPlatform for Real-Time Molecular Diagnosis of Methicillin-Resistant Staphylococcus Aureus

ACRONYM

MagRSA www.MagRSA.org

Methicillin-resistant Staphylococcus aureus(MRSA), a virulent organism resistantto many drugs, is responsible for mostnosocomial and community-acquiredinfections. It can cause life-threateningdisease, and treatment options are lim-ited. Effective diagnostics is a strategic keyelement in the campaign against thespread of MRSA, allowing better infectionsurveillance and control measures as wellas more efficient patient treatment and/orisolation options. The MagRSA projectaims at the development of a new diag-nostics platform that will provide a fast,simple and accurate identification of MRSAfrom clinical samples.

SUMMARY

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Key words: magnetic nanoparticles, microfluidics, quantitative PCR, antimicrobial resistance, MRSA, molecular diagnostics

Expected results

The MagRSA project measurable and quantifiableobjectives can be classified in three categories:• new molecular diagnostics protocol allowing

efficient and reliable MRSA diagnostics andgenotyping;

• new assay reagents including magnetic nanopar-ticles for sample preparation and quantitativePCR (Q-PCR) related reagents;

• fully automated systems, mainly based onadvanced microfluidics and nanoparticles han-dling technologies, for MRSA diagnostics andgenotyping.


The MagRSA project will address the unmet needfor new diagnostics tools for management andcontrol of antimicrobial resistance in generaland Methicillin-resistant Staphylococcus aureus(MRSA) in particular. Moreover, MagRSA projectwill provide a diagnostics platform with poten-tial applications in molecular diagnostics as themost growing segment within the global in vitrodiagnostics market.

The MagRSA project Consortium is composed of 6 partners, among which are 3 high-techSMEs with complementary expertise. Ademtech is an established company with largeexpertise in magnetic nanoparticles manufacturing for in vitro diagnostics and life sci-ences applications; Spinomix is a start-up company specialized in the development of fullyautomated and miniaturized solutions based on a proprietary magnetic nanoparticleshandling technology – termed MagPhase™ – in a micro fluidic environment. The tech-nology offers cost-efficient, rapid, automated and compact sample handling systems formultitude of applications in life-science and in vitro diagnostics (IVD) market; and TATAA isa leading service provider in tailor-made real-time quantitative PCR (RT-qPCR) geneexpression analysis in Europe.

The project presents therefore a strong contribution from high-tech SMEs with leadingand innovative core technologies and services. With complementary competencies andcore activities, all SMEs expect from this project to expand further their technologies andactivities portfolio. Moreover, these SMEs consider the project as a technological andcommercial opportunity that may open large market perspectives to their existing coretechnology and products.

In addition another SME, a young Swiss company called SCIPROM, takes care of the projectmanagement.

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Jacques SchrenzelGeneva University HospitalsDivision of Infectious Diseases – GenomicResearch LabRue Micheli-du-Crest 24CH-1211 Geneva, Switzerland [email protected] www.genomic.ch

Project manager

Kirsten LeufgenSCIPROMrue du Centre, 70CH-1025 St-Sulpice, [email protected]

Partners

Amar RidaSpinomix S.A.Lausanne, Switzerland [email protected]

Neven Zoric TATAA Biocenter ABGöteborg, [email protected]

Felix von Stetten Department of Microsystems Engineering Faculty of Applied Sciences Laboratory for MEMS ApplicationsFreiburg, [email protected]

Manuel Gaboyard ADEMTECH S.A. Pessac, [email protected]

| Staphylococci growing on agar plates (yellowcolonies are Staphylococcus aureus, and white ones are Staphylococcus epidermidis).

Expected results

MAMMI proposes a new PET device specificallydesigned for breast cancer diagnosis and evalua-tion of therapy response. The dedicated breastcancer PET camera will improve the position reso-lution of current whole-body PET cameras (about5mm) and will push it to the physical limit (slightlybelow 1mm).

The new detector design will also have the abilityof detecting the depth of interaction of the gammaray interactions within the crystal with a resolu-tion better than 3mm. This is an essential featuresince it allows for an improvement of the finalimage resolution by almost eliminating the paral-lax error present in current PET detectors. This isessential in the case of breast examination sincethe detector cameras are placed close to thebody, to increase the sensitivity.

The advantages of the new generation of photo-detectors (silicon photo-multipliers), such astheir compactness, will be explored. The designof the electronics, including an ASIC, will allow anacquisition rate capability of the order of 1 MHz,with minimum dead time, to cope with the highersensitivity.

Moreover MAMMI will develop and study morespecific than FDG radio-tracers for breast cancerdiagnostic and therapy monitoring, based onhuman amino acids (such as FLT) and fatty acids(such as FAS).


The main application will be early breast cancerdiagnosis and evaluation of chemotherapyresponse. New radio-tracer molecules will besearched for the detection and visualisation of thepharmacokinetics of breast tumours, more spe-cific than glucose (FDG) for breast cancer, andbased on human amino and fatty acids.

Background

Breast cancer is the most common non-skin can-cer and the leading cause of cancer death inwomen. The best condition for successful breastcancer treatment is early detection. Some stud-ies indicate that early breast cancer detectionhas reduced the disease mortality by about29 %. The ability to define the extent of disease,to monitor response, and to predict tumourbehaviour in patients with breast cancer aretherefore important public health problems.

Conventional methods for breast cancer imag-ing like X-ray mammography, ultra-sound andMagnetic Resonance Imaging (MRI) produce mor-phologic and structural images, show lesions(like micro-calcifications), but not cancers. On thecontrary, imaging methods based on MolecularImaging show functional images, metabolism.This implies that they are much more sensitiveand hence these devices can be used to detectand locate malign tumours at an early stage. Forinstance, PET (Positron Emission Tomography) isa powerful tool for non-invasive molecular imag-ing diagnostic based on gamma ray (emitted byan isotope compound, previously administeredto the patient) detection.

Aim

• Design and development of a dedicated low costPET camera prototype for breast examinationwith an intrinsic resolution of less than 1 mm,high sensitivity, and tomographic 3D recon-struction.

• Study of new and more specific radio-pharma-ceuticals for breast cancer detection and ther-apy monitoring (FLT, FAS, etc.). Perform phase Iclinical trials of the radio-tracers.

• Clinical multi-centric validation of the new PEMTprototype.

Mammography with molecular imaging

ACRONYM

MAMMIevalin12.ific.uv.es/ep_mammi

The proposed project focuses on thedevelopment of a PET prototype dedi-cated to the examination of breast can-cer, using a gamma ray sensor based onan innovative design and the new gener-ation of photo-detectors and scintillatingcrystals. The innovative features of thePEMT (Positron Emission Mammo Tomo -graphy) system proposed will implya high resolution (pushed to the physicallimit), higher sensitivity and lower costs.It includes integrated analogue and digi-tal electronics through the design of anASIC chip. The main application will beearly breast cancer diagnosis and evalua-tion of chemotherapy response. Newradio-tracer molecules will be searchedfor the detection and visualisation ofthe pharmacokinetics of breast tumours,more specific than glucose (FDG) forbreast cancer, and based on human aminoand fatty acids. Phase I Clinical trials willbe performed with the new radio-tracers.A clinical multi-centric validation will beperformed for the PEMT prototypes.

SUMMARY

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Key words: PEMT, PET mammography, molecular imaging, breast cancer diagnosis, chemotherapy

Each of the three SMEs participating in MAMMI, namely General Equipment for MedicalImaging S.L. (GEM-Imaging), DIGI UTOPIKA Ltd. (DUT) and Ray Therapy Imaging AB (C-RAD),brings specific expertise into this consortium, complementing and strengthening the over-all group. GEM-Imaging has experience in the construction of gamma ray detectors and istherefore crucial to the overall project. It will not only help constructing the prototype, butalso be instrumental in the eventual exploitation at the end of the project. For this purpose,GEM-Imaging has already conducted a market research study and set up a preliminarybusiness plan. C-RAD is involved in the manufacturing of associated electronic parts andplays an important role in the building of the prototype. DUT has extensive experience withsoftware development, and bring this knowledge to bear on the project for tailor-madesoftware solutions essential for the operation of the final machine.

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Jose Ma Benlloch Consejo Superior de Investigaciones científicasIFIC- Instituto de Física CorpuscularEdificio Institutos de Investigación46071 Valencia, [email protected]

Partners

Sibylle ZieglerKlinikum Rechts der Isar der Technischen Universität München Munich, Germany portal.mytum.de/navigation_view

Angels BernabeuGeneral Equipment for Medical Imaging, S. L.Paterna, Spainwww.gem-imaging.com

Johann HauerFrauhnofer-Institut für Integrierte Schaltungen (IIS)Erlangen, Germanywww.iis.fraunhofer.de

Pedro BrancoDIGI-UTOPIKA Ltd.Lourinhã, Portugalwww.utopika.net/utopika/EN

Norberg GunnarC-Rad Imaging ABFrösön, Sweden www.c-rad.se

Anders BrahmeKarolinska InstitutetStockholm, Swedenki.se

Renato A. Valdês OlmosNetherlands Cancer InstituteAmsterdam, The Netherlandswww.nki.nl

© Shutterstock

Aim

The overall goal of this project is to develop moreeffective management strategies for IA with thefollowing aims: • development of immunotherapeutic strategies for

Invasive Aspergillosis. This will be achieved byidentifying the T-cell clones that react with spe-cific Aspergillus targets and evaluating their effi-cacy for prevention and treatment of IA. Successwill be measured by characterisation of the gener-ated T-cells, and demonstration of their functionalproperties against Aspergillus;

• development of improved diagnostic tests for IAwith commercial potential. This will be achievedby identifying appropriate nucleic acid targets,extraction and amplification protocols and demon-strating their sensitivity and specificity in clinicalsamples;

• validation of a dendritic cell based vaccineimmunotherapy strategy in animal model to gen-erate protective immunity against Aspergillus;

• use of genomic and proteomic techniques to iden-tify new Aspergillus targets that interact with thehost’s immune system.

Expected results

• Identification of different pattern recognitionreceptors in response to A. fumigatus and theirrole in activating DCs.

• Identification of PAMPs of A. fumigatus usefulfor immunotherapy strategies.

• Identification of murine DC subsets capable ofinducing antifungal Th responses.

• Adoptive transfer of manipulated DC into micesuffering from IA.

• Characterisation of Aspergillus-specific T-cellresponse in healthy individuals.

• Identification of a protective T-cell response inpatients surviving IA.

• Development of a clinical protocol under currentcGMP-conditions for treatment of patients with IA.

• Modulation of Aspergillus-specific immuneresponse by transduced T-cells.

• Preparation of a bank of monoclonal antibodiesagainst A. fumigatus.

Background

Over the past decade, invasive aspergillosis (IA)has emerged as the most serious life-threateninginfectious complication of intensive remission-induction chemotherapy and allogeneic HSCT inpatients with a variety of haematological malignan-cies. Aspergillus fumigatus is the most commonlyisolated species from cases of IA and is the focus ofproject research although it is believed that theintended outcomes will act as a paradigm for man-agement of IA due to less common species such asA flavus and the emerging A terreus. Despite thelimited improvements that have been made withpreventative strategies and the developmentof new antifungal drugs, IA has an incidence of10-30% and is still associated with high mortalityrates as high as 90% in some surveys. T lympho-cytes provide a critical secondary defense againstthis and other fungal pathogens. Therefore,Aspergillus-specific T-cell immunity, transferredthrough the infusion of ex vivo-generated, donor-derived, Aspergillus-specific T-cells might be bene-ficial for recipients of allogeneic HSCT.

There are only raw data regarding immunotherapyof patients with invasive fungal infection, and min-imal data relating to IA. This might be due, at leastin part, to the complex antigenic properties ofA. fumigatus, which have been less well charac-terised compared to viruses such as CMV or EBV.Only a minority of the hundreds of (glyco)proteinsof A. fumigatus reported in the literature havebeen characterised at either a molecular and/orbiochemical level.The diagnosis of IA is a particular challenge.Conventional microbiological methods fail to detectmost cases so that current diagnostic categoriesare based on a combination of clinical, imaging,and histopathological (often post-mortem) criteria.There have been some advances with non-culturetechniques based on PCR based amplification offungal DNA or detection of Aspergillus antigens inblood samples. However, these have not beenvalidated as tools for identifying patients at risk orfor monitoring responses to treatment by deter-mination of fungal loads.

Development of novel management strategies for invasive aspergillosis

ACRONYM

MANASPwww.manasp.org

• The goal of the project is to develop new treatment strategies for InvasiveAspergillosis (IA) which has become themajor infectious complication of treatinghaematological malignancies with inten-sive chemotherapy or haematopoieticstem cell transplantation (HSCT).

• Ineffective host immune responsesfacilitate fungal invasion of the pul-monary system and other vital organsleading to death. In order to redressthis immunological imbalance, it is pro-posed that new immunotherapeuticstrategies are developed, which willaugment current antifungal treatmentsand reduce morbidity and mortality.

• Facilitated by recent publication of theA. fumigatus (the principal pathogen ofthe genus) genome sequence the projectwill exploit new knowledge and tech-niques in genomics and post-genomics.The project will identify the immuno-logically important fungal molecules todesign immunotherapies based on vac-cines using fungus-primed innate immunecells, monoclonal antibodies or adoptivetransfer of specific T lymphocyte clones.

• The project will also generate newnucleic acid based diagnostic tests toinform when and how immunotherapycan be optimally applied.

The outcomes will have strong commer-cial applications which will be deliveredby three leading European SMEs withinthe consortium.

SUMMARY

102

Contract number: LSHE-CT-2006-037899 | EC contribution: € 2 914 000 | Duration: 36 months


Key words: immunotherapy, vaccine, antigens, T cells, dendritic cells, A. fumigatus, PCR diagnostics, monoclonal antibodies, GMP-conditions, aspergillosis, transplantation

• Selection of the most efficient antibodies by invitro and in vivo analysis.

• Development of an assay to detect AspergillusDNA with high specificity.

• Development of an assay to detect AspergillusRNA with high sensitivity.

• Clinical evaluation to confirm sensitivity in detect-ing Aspergillus infection using standardised defi-nitions produced by International consensus andcomparison to different well established assays.

• Commercialisation of the assay into an afford-able and rapid diagnostic test.


• Surveillance of patients being treated for haema-tological malignancies by chemotherapy or HSCTusing DNA/RNA based molecular diagnostics.

This will generate greater ascertainment of possi-ble and probable cases of IA and earlier therapeu-tic intervention leading to improved survival rates.

• Incorporation of these diagnostic tests into inter-nationally accepted EORTC/MSG criteria for clin-ical application in categorising cases of IA tofacilitate research trials of new antifungal agentsor other novel therapies.

• Development of immunotherapeutic strategiesbased on demonstrated efficacy against Asper -gillus in vitro and in animal models. This will eitherbe through adoptive transfer of donor T-cells, oradministration of monoclonal antibodies tar-geted against immunologically relevant Aspergillusantigens/epitopes.

• Wider application of this technology to the treat-ment of other groups of patients (outside theHaematological Malignancy field).

MANASP focuses on the commercialisation of assays for molecular diagnosis and immunetherapy of invasive aspergillosis. Three out of nine partners from the consortium are SMEs,while one Work Package leader comes from an SME. Translational strategies for commercialexploitation of the results exist, including an IPR Advisory Committee.

Cepheid AB, who acquired the diagnostic company Sangtec in 2007, already producesmolecular diagnostic kits for the detection of viral infections. They have 11 years of experi-ence within the field of molecular diagnosis and are certified according to ISO-9001/2000.Their activities comprise research and development, manufacturing and the sale ofCE-labelled PCR-based molecular diagnostics. Cepheid has many patents relevant to MANASP,including a non-exclusive worldwide PCR license, a non-exclusive license for real-time PCR andalso a non-exclusive worldwide UDG sterilisation license. Currently, they are working on theoptimisation of different fungal PCR assays and on automated fungal DNA extraction. MAT Biotech is a private biotech company founded in 1999. The company has two activities,namely the development of therapeutics and an antibody high throughput screening plat-form. With the former, the company focuses on the development of antibody therapies totreat haematological cancers. MAT’s product portfolio comprises a 90Y-anti-ferritin poly-clonal (Ferritarg) antibody to treat refractory Hodgkin’s disease, and a 90Y-AMB8LK mono-clonal antibody-radioisotope product to treat refractory Hodgkin’s disease, liver cancer, andpancreas cancer, which is in advanced pre-clinical trials. Currently, they perform screeningassays to identify reactive monoclonal antibodies against A. fumigatus.Miltenyi Biotec GmbH is a leading, international biotech company in the field of magneticcell separation (MACS®technology). Miltenyi Biotec develops, produces and sells world-wide magnetic cell separation reagents and equipment for biomedical research and clinicalapplications, particularly in the fields of haematology and immunology. The CliniMACS®system is an automated cell selection device. It is approved for clinical stem cell graft engi-neering using CD34 and CD133 selection and permits large-scale cell isolation of other cel-lular products such as dendritic cell precursors, NK cells and T-cell subsets. In addition,pioneering technologies are made available, such as MACSiBead™ particles for activationand expansion of T-cells, or the Cytokine Secretion Assay for isolation of cytokine-secretingcells, e.g. antigen-specific T-cells. Reagents and tools for the GMP-grade generation of den-dritic cells and T cells for immune therapy against invasive aspergillosis are also available.

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Hermann EinseleUniversity of WuerzburgMedizinische Klinik IIKlinikstrasse 6 – 8 97070 Wuerzburg, [email protected]

Project manager

Juergen [email protected]

Partners

Birger JanssenCepheid ABBromma, Swedenwww.sangtec.com

Tom RogersTrinity CollegeDepartment of Clinical MicrobiologyDublin, Ireland

Jean Paul LatgéInstitute PasteurUnité AspergillusParis, France

Luigina RomaniUniversity of PerugiaDepartment of Experimental Medicine and Biochemical Science Microbiology SectionPerugia, Italy

Jean KadoucheMonoclonal Antibody Therapeutics (MAT)Evry, Francewww.matbiopharma.fr

Axel BrakhageLeibniz Institute for Natural Product Research and Infection BiologyJena, Germany

Georg RauserMiltenyi Biotec GmbHBergisch Gladbach, Germanywww.miltenyibiotec.com/en/default.aspx

Jean-Marie FrançoisInstitut National des Sciences AppliquéesDépartement de Génie Biochimique et AlimentaireToulouse, France

studies should reveal the laws governing theseinteractions, and this data could in turn be usedin a predictive way, for the design of novelmeganucleases;

• the methods, procedures and quality standardsto make these meganucleases widely usable asresearch tools;

• a refined method to use these meganucleases incells. The focus will be on mouse cells for func-tional genomics, providing a direct validation.

Expected results

• Protein engineering: Partner 1 has establishedthe basis of a combinatorial process to assem-ble functional engineered meganucleases. It isexpected that this combinatorial strategy willprovide a functional meganuclease for mostchosen genes.

• Computational biology: The FoldX algorithmcan successfully predict the effect of proteinmutation on the specificity of protein-DNArecognition specificities. Subsequent versionsof FoldX should allow for more efficient designof meganucleases combinatorial process.

• Structural Biology: A continuous flow of novelstructures that will contribute to the computa-tional steps is expected.

• Standardisation of protein storage and use:An efficient purification and characterisationprocess for each engineered protein is expected.

• Validation of the general approach: The wholeapproach should eventually be validated bythe use of engineered meganucleases on realchromosomal targets in rodent cells. A general,standard protocol for rodent cells is expected.


The possibility of correcting errors in a genomethrough targeted homologous recombination ormodifying at will any DNA sequence is clearlyenormously attractive to the scientific community.

Background

The genome sequence programmes have con-tributed a huge amount of information, and cre-ated many possibilities. An exhaustive catalogueof genes is now available for many organisms,but the real meaning of this information remainsto be deciphered. Thus, the success of functionalgenomics definitively lies in the development ofnovel tools breaking through the practical limits itsuffers today. Meganucleases-induced recombi-nation could provide a practical alternative to cur-rent approaches.

Meganucleases are, by definition, sequence-specific endonucleases with large (� 14 bp)recognition sites. However, the inactivation ormodification of any and all known genes, orgenomic sequence, depends on the availability ofMeganucleases that cleave within or in the vicin-ity of each gene sequences. This issue would beaddressed if it was possible to rapidly engineerthe specificity of natural Meganucleases.

Aim

The first objective of the project is to provide themeans to modify a large number of sequences inrodent genomes. The second is to develop thetools to engineer a large number of rodent genes,for functional genomic purposes.

Since meganuclease-induced recombination rep-resents an extremely powerful tool for gene alter-ation, MEGATOOL will focus on the generation offour kinds of results:• a large collection of novel meganucleases. This

collection of novel proteins should greatlyenhance the repertoire of natural meganucle-ases and thus allow for the targeting of a largenumber of genes in organisms whose genomehas been sequenced, with a strong focus onrodent genomes;

• the means to exponentially increase this collec-tion. The collection of novel meganucleasesshould provide a unique database of charac-terised DNA binders. Structural and statistical

New tools for Functional Genomics based on homologous recombination induced by double-strand break and specific meganucleases

ACRONYM

MEGATOOLSwww.cellectis.com/megatools

The MEGATOOL project aims to developa large number of new sequence-specificendonucleases to recognise and targetalmost any possible DNA sequence inany living cell or organism, as well as tooptimise homologous recombination, toprovide scientists with a powerful toolto undertake functional genomics.

SUMMARY

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Key words: functional genomics, genome engineering, protein engineering, meganucleases, gene targeting, computational biology

Although it is clearly valuable to understand howgenomic information is translated into function,rational modification of the DNA sequence of anorganism has been limited by the time consumingprocess it requires, despite the development ofnew tools for the construction of targeting vectors.

Thus, the possibility of having new tools that willallow targeting of any DNA sequence for insertion,deletion or repair could introduce a new revolu-tion in the field of functional genomics and couldalso bring a new paradigm and a new momentumto human gene therapy.

The project involves two SMEs (Cellectis S.A. and Fermentas UAB) and two academic labo-ratories (CRG and CNIO).

Cellectis S.A. is a SME biotech company leading the field of genome engineering. Its uniquetechnology and strong intellectual property are centered on homologous recombinationinduced by double-strand breaks using meganucleases and its applications. Its aim is todevelop new tools for functional genomics, gene therapy and genome modification.Cellectis has entered into more than 40 deals with industrial partners using living strains ororganisms in their processes. It is also pursuing applications of its technologies to humantherapy. Cellectis has developed specific skills in the field of genome engineering and pro-tein engineering. One of the great achievements of the company lies in a High-ThroughputScreening platform. Using cell-based functional assays for the identification of novelendonucleases, this platform has delivered hundreds of novel meganucleases. MEGATOOLSwill allow a broad dissemination of Cellectis’ genome engineering tools to the research com-munity. As a SME demonstrating very strong innovative activity, Cellectis is at the crossroadof the upstream research that feed the project and of downstream development steps. Assuch, Cellectis has a pivotal role in the development of novel products and process withinMEGATOOLS. Cellectis will use its High-Throughput Screening platform to deliver custommeganucleases cleaving sequences in the mouse genome. Cellectis will also contribute itsexpertise in the field of genome engineering and, more specifically, in gene targeting andknock-out rodent genes with a panel of novel meganucleases, thereby validating the gen-eral approach and refining the methods for using these novel tools for functional genomics.

Fermentas UAB, an ISO09001, ISO140001 certified company, is a world leading SME biotechcompany in the discovery, manufacturing and marketing of quality molecular biologicalsand in the providing of services to the international research community. Fermentas hasstrong expertise in nucleases and in their production, implementation and enzymology. Thecompany’s product range consists of approximately 400 different molecular biology prod-ucts, of which 200 are nucleases. Products under the Fermentas brand name are marketedvia a distributor network covering 74 countries in Europe, North and South America, Asia,Australia and Africa. The key commercial target for Fermentas in MEGATOOLS is to produceand to make meganucleases developed during the project available to the research com-munity. Meganucleases, like other restriction enzymes, perform site-specific cleavage ofdouble-stranded DNA. In fact, these rare-cutting enzymes could be used in many in vitroapplications where restriction enzymes are commonly used. Meganucleases should con-form to all quality requirements that are applicable to traditional restriction enzymes. Theprimary function of the SME in the MEGATOOLS project is to test whether mutant meganu-cleases created during the project can be used as molecular tools for the rearrangement ofDNA molecules not only in vivo, but also in vitro. After receiving meganuclease mutants,Fermentas will follow the traditional implementation route of restriction enzymes, includingpreliminary evaluation of properties in crude cell extracts, purification, quality controls,basic enzymology studies and stability tests. These studies will show if and how thesemutant enzymes could be used as analytical tools.

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Frédéric PâquesCELLECTIS S A102 Route de Noisy92 235 Romainville [email protected]

Partners

Centro Nacional de Investigaciones Oncologicas (CNIO)Structural Biology – MacromolecularCristolography GroupMadrid, Spainwww.cnio.es/ing

Centro de Regulacio GenomicaSystem Biology LaboratoryBarcelona, Spainwww.crg.es

Fermentas UABVilnius, Lithuaniawww.fermentas.com

advances in understanding the biology and subse-quent diagnosis of AD, such research has not beentranslated into a disease-modifying treatment.

One limit in translating basic research findingsinto therapeutical agents is the lack of suitableanimal models fully reproducing the AD neurode-generation. Having animal models reduces therisk, and thus the cost, of developing drugs.

In the past decade, AD research has been funda-mentally influenced by the development of geneti-cally modified animal models of amyloid-driven ortau-driven neurodegeneration. These in vivo mod-els – exploited on the basis of early-onset diseasedetermined by rare, inherited mutations – areimportant in understanding the involvement of amy-loid or tau in the onset of the disease, but failed toreproduce the hallmarks of the AD pathology fully.

One other potential pitfall in these transgenicmice is that these models might not be applicableto sporadic AD. Indeed sporadic forms of AD,which account for more than 95 % of the popula-tion afflicted by AD, are multifactorial diseases,the progression of which is influenced by genderand epigenetic factors (such as neuroinflamma-tion, growth factor deficits, autoimmunity/auto-toxicity). The use of animal models derived fromthe genetic forms of AD to screen drugs, insteadof more appropriate animal models, revealed thelimitations of their use for sporadic AD. Indeed,the vaccine approach seemed to work well inmice, but brain inflammation in a few patientstriggered an abrupt halt to the clinical trial.

Aim

The MEMORIES hypothesis-driven project gatherstogether eight partners from five different coun-tries towards the aim of developing, characteris-ing and validating new animal models that havea real potential for becoming a gold standard inthe AD field.

Background

Alzheimer’s disease (AD) is a neurological disor-der and is the most common form of dementia inlater life. It is estimated that, by 2050, the numberof people aged 80 years or older will approach370 million worldwide and that 50 % of thoseaged 85 years and older will be affected by AD.

AD is clinically characterised by short-term mem-ory loss and cognitive dementia, associated withlanguage and behavioural impairments. Thepathological hallmarks of AD include the presenceof extracellular amyloid plaques, intracellularneurofibrillary tangles (NFT), neurodegenerationand cell loss. One severely affected region of theAD brain is the basal forebrain (which includes thenucleus basalis of Meynert, the medial septumand the diagonal band of Broca), a group ofcholinergic neurons that are connected to areas ofthe neocortex and hippocampus and that areimportant for learning, memory and attention.

The complex degeneration in AD has been fertileground for the formulation of hypotheses on thepathogenesis of the disorder. Methodologicaladvances allowedshedding light on alterations ofthe various neurotransmitter systems in the ADbrain. The discovery of the degeneration in thebasal forebrain, in the context of experimental evi-dence for the role of acetylcholine in memory, haveled to the development of a symptomatic therapyfor AD, based on enhanced acetylcholine availabil-ity determined by cholinesterase inhibitors.

Recently, memantine, an uncompetitive antago-nist of the N-methyl-D-aspartate receptor, wasapproved for the treatment of moderate-to-severeAD. These agents can benefit some AD patients fora limited period of time. However, due to the exten-sive and multifocal nature of AD degeneration, theeffects of these neurotransmitter modulators aremodest and the need of a truly disease-modifyingdrug persists. Although there have been significant

Development, characterisation and validation ofnew and original models for Alzheimer’s Disease

ACRONYM

MEMORIES

Alzheimer’s disease (AD) is a progressivebrain disorder that gradually destroysa person’s memory and ability to learn,reason, make judgments, communicateand carry out daily activities. The diseaseis the major kind of dementia affectingelderly people, and the percentage ofpatients is going to increase exponen-tially over the next few years. Currently, noaetiology and cure has been found. Onemajor hurdle in drug screening and targetdiscovery in AD is the lack of a suitableanimal model, as these fail to fully repro-duce the characteristics of the disease.The MEMORIES project aims to developnew mouse models based on deficit ofneurotrophic signalling, potentially use-ful for developing new therapeutic tools.

SUMMARY

106



Key words: Alzheimer’s disease, transgenic mouse model, neurotrophins

Expected results

Using a multidisciplinary approach, a panel ofmouse models will be produced and analysedfor the presence of neurodegeneration. AD11 anti-NGF, which already represent a good model forsporadic AD, will be crossed to mice in whichpro-convertases or SorLA receptors are knockedout. Additional mice will be created, expressingmutated form of pro-NGF or in which knockoutof TrkB, TrkA and NGF will be achieved.

Mice will be analysed using standardised method-ology for neuroanatomy and behavioural analy-sis. We anticipate that blocking differentsignalling pathways will help in ameliorating thecurrently available experimental mouse models,and will also be useful for developing new thera-peutic tools for this disease and strengtheningEuropean competitiveness in the war against AD.


The creation of a mouse model that by fully repro-ducing the hallmarks of the disease, will not onlyprovide insights into the neurobiology of the dis-ease, but will also allow the evaluation of theresponse to drugs, especially in relation to theeffects on pathology is one of the most challengingaims of AD research.

The potential medical benefits that will derive fromthe production of this mouse model are immense.The identification of protein linked to specific cel-lular pathways will provide the possibility todevelop new diagnostic assays and new drug tar-gets for the treatment of AD. Mouse models willalso make it possible to visualise neural circuits intheir normal and abnormal states, which is likelyto have a large impact on the diagnosis of diseaseand the evaluation of the effectiveness of therapy.

Three European SMEs are partners in MEMORIES, and are based in Italy and France.These SMEs will receive approximately 35 % of the project budget. Lay Line Genomics isan integrated antibody company, focused on the therapeutic area of neurodegenerativediseases, whose mission is the discovery of drugs for Alzheimer’s and other neurologicaldiseases, leveraging on a unique antibody based technology platform. LLG has an exclu-sive licence covering any use of the AD11 anti-NGF mouse model. During the MEMORIESproject, LLG will exploit the AD11 mouse model and produce new antibody based mousemodels with the aim of creating new experimental models, also useful in the develop-ment of new therapeutic tools for this disease. The involvement of the other two SMEswill be crucial for the standardisation of the procedures to be used in the different labo-ratories and the management of the project. Neureva conducts drug discovery and drugdevelopment for diseases of the central nervous system. Its research activity is centredon anatomical and behavioural characterisation of mouse models for CNS pathologies,the choice of mice models being driven by the availability of a large number of transgenicmice which allows alternative ways for testing molecular or cellular hypotheses. Neurevawill add competencies to standardised protocols and validate mouse models. ACIES isexpert in managing International Management projects and in optimising the financingof innovation for enterprises working in various industrial and technical sectors. Since1990, over 500 major companies, research organisations and very innovative small andmedium-sized businesses, have put their confidence in ACIES to implement financingsystems, valorise innovation or to set up and manage their International projects.ACIES’s methodology, based on the principles of Total Quality Management (TQM),allows managing multidisciplinary consortiums. ACIES will provide high quality manage-ment services (including knowledge) throughout the project, in accordance with theISO 10006 (guidelines for quality management in projects) standard.

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Antonino CattaneoEuropean Brain research InstituteRita Levi-Montalcini FoundationVia del Fosso di Fiorano 64/6500143 Roma, [email protected]

Partners

Manuel GaviriaNeuréva Inc.Montpellier, Francewww.neureva.com

Thomas E. WillnowMax Delbrück Centrum Für Molekulare MedizinBerlin, Germany

Liliana MinichielloEuropean Molecular Biology LaboratoryMonterotondo, Italy

Eero CastrenUniversity of HelsinkiHelsinki, Finland

Daniel Constam Swiss Federal institute of Technology – “Daniel Constam” École Polytechnique Fédérale de LausanneLausanne, Switzerland

David KoubiACIESLyon, Francewww.acies.fr

Anders NykjaerNeuronIcon A/SAarhus, Denmark

Aim

The novel technology presented in the MimoVaxproject has been developed to create antigensmimicking the structure of neo-epitopes which donot contain sequences of the native Aβ peptide.A Mimotope-based AD vaccine would thereforeinduce antibody responses, exclusively reactingwith the pathological Aβ molecules but notwith parental structures like APP. Furthermore,Mimotopes do not contain potential T-cell self-epitopes and avoid induction of detrimental auto -reactive T-cells. Thus, the goal of the MimoVaxproject is the development of a safe and effica-cious Alzheimer vaccine which is predicted toavoid the development of immunological compli-cations due to autoreactive T-cells. The develop-ment of such innovative AD vaccines targeting Aβcould therefore be a safe treatment regimen toefficiently fight AD in patients. In addition, newdiagnostic methods will be developed in thecourse of the MimoVax project in order to monitortreatment efficacy.

Expected results

Successful preclinical evaluation of a Mimotope-based AD vaccine is expected to demonstratereduction of amyloid plaque load and alleviationof the pathologic hallmarks in the brain of animalmodels for AD. Additionally, Mimotope-based ADvaccines will provide means to target the solubleoligomers of Aβ which are thought to be a majorcause of the synaptic alterations and cognitivemalfunctions typical of AD.

Once these vaccines have proven their efficacy inreducing Alzheimer-like pathology in our animalmodels, an initial clinical trial will be initiated todemonstrate safety of the identified peptide vac-cines in patients. The development of such inno-vative AD vaccines targeting Aβ could thereforebe a safe treatment regimen to efficiently fight ADin patients and successful completion of initialclinical testing will be followed by further productdevelopment.

Background

Alzheimer’s disease (AD) is the most commonform of dementia in humans. According to theAlzheimer Association, there are currently 12 mil-lion patients worldwide with estimated socialcosts for every patient reaching € 40 000 per year.At present, no effective treatment is available tostop the progressive neuro-degeneration andassociated cognitive decline in AD patients. Alltreatment strategies applied today are focusingon the use of small molecular drugs inhibiting theactivity of Cholinesterase to alleviate diseasesymptoms. These drugs however have not provento effectively halt or revert disease progressionafter prolonged treatment.

AD is characterised by the abnormal accumulationof amyloid plaques in the brain. These plaquesmainly consist of the Amyloid- (A) peptidesAβ40/42 derived from the Amyloid PrecursorProtein (APP). In humans, the majority of amyloidplaque material is formed by Aβ40/42 derivativeswhich are frequently truncated and modified. Aβpeptides are considered to be directly involved inthe pathogenesis and progression of AD.

Immunotherapeutic treatment targeting Aβ ledto amyloid plaque reduction and had beneficialimpact on disease progression in animal models.However, the first phase II clinical vaccination trialin AD patients using full length Aβ42 as antigenhad to be discontinued due to severe neuroin-flammatory side effects including brain infiltrationby autoreactive T-cells.

Similar to full length Aβ, truncated and modifiedAβ peptides also seem to be involved in thepathogenesis and progression of AD. They aretherefore also a suitable point of attack for noveltreatment strategies, but no relevant develop-ment programme has been started to date.

Alzheimer’s disease-treatment targeting truncatedAβ 40/42 by active immunisation

ACRONYM

Mimovaxwww.mimovax.eu

Alzheimer’s disease (AD) is characterisedby the abnormal accumulation of amy-loid plaques in the brain. These plaquesmainly consist of the Amyloid- (A) pep-tides Aβ40/42 derived from the AmyloidPrecursor Protein (APP). In light of thecharacteristics of amyloid composition inpatients, Aβ peptides and its truncatedand modified derivatives are highlyattractive targets providing neo-epitopesnot present on parental APP. A Mimotope-based AD vaccine targeting truncatedforms of Aβwould therefore induce a safeantibody response exclusively reactingwith the pathological Aβ molecules butnot with parental APP and would avoidthe induction of autoreactive T-cells. Thisvaccine will be evaluated in a preclinicalAD animal model and tested in a clinicaltrial in patients. Thus, the MimoVax vac-cine will provide an innovative, safe, costeffective and efficient approach to suc-cessfully treat AD patients and to limit thesevere personal and economic impact ofAD on patients, their families and society.

SUMMARY

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Key words: Alzheimer’s disease, neurodegeneration, dementia, amyloid plaque, truncated Aβ 40/42, mimotope, active immunotherapy, preclinical characterisation, animal models, clinical trial


Currently 12 million patients worldwide are suffer-ing from AD. This number, however, will increase upto 22 million by 2025, according to the AlzheimerAssociation. There is, however, no effective treat-ment available to stop the progressive neuro-degeneration and associated cognitive decline inhuman patients, thus creating an enormous socialproblem for all European societies as well as for therest of the western world.

The novel peptide vaccine developed in theMimoVax STREP therefore aims at the potentialsubsequent use in treatment of Alzheimer’s dis-ease in human patients. Vaccination will be a costeffective and powerful way to reduce the econom-ical and psychological burden exerted by this dis-ease. It would significantly reduce the high costsassociated with this disease, which will soonexceed US$ 4 billion annually.

The project consortium is constituted by 5 SMEs and 2 academic institutions.

MimoVax is coordinated by an SME, AFFiRiS GmbH, expert in the development of vaccineson the basis of peptides to treat Alzheimer’s and atherosclerosis. It is supported a dedicatedmanagement partner (Biolution Grünert & co keg) also in charge of knowledge transfer andpublic relations. JSW-Research’s competences comprise R&D and contract research as wellas the performance of clinical research complemented by histological and biochemical eval-uation methods. Its staff possesses long standing experience with a quality assurance sys-tem and behavioural studies. piCHEM has been working in the field of peptide chemistry formore than 10 years. This enterprise puts its main stress on developing and producing pep-tides for all kinds of research purposes worldwide. EuroEspes, S.A. expertise includesregular clinical and lab evaluations of patients as well as clinical trials, preclinical pharma-cology and genetic diagnosis. Having focused on biochemical research of disorders ofthe Central Nervous System, EuroEspes has already performed more than 20 clinical trialsconcerning Alzheimer’s disease.

The tight collaborations between academia and participating SMEs and the financing of theclinical trial will provide the basis for the financial exploitation of any IPR generated withinthe project by participating SMEs and academic institutions and will lead to potentialbenefit for the participating SMEs and University Institutes.

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Markus MandlerAffiris GmbHCampus Vienna BiocenterViehmarktgasse 2a1030 Vienna, [email protected]

Partners

Iris GrünertBiolution gruenert & co KEGVienna, Austriawww.biolution.net

Antón Alvarez EUROESPES S.A.Department of NeuropharmacologyBergondoA Coruna, Spainwww.euroespes.com

Manfred WindischJSW – Research GmbHGraz, Austriawww.jswresearch.com

Fritz Andreae piCHEM research developmentGraz, Austriawww.pichem.at

Richard Dodel Philipps-Universität MarburgMarburg, Germanywww.med.uni-marburg.de/d-einrichtungen/neurologie

Alexander Drzezga Technische Universität MünchenKlinikum rechts der IsarMunich, Germanywww.nuk.med.tu-muenchen.de

| Amyloid plaque staining in the brain of an AD mouseOne of the hallmarks of Alzheimer’s disease is theaccumulation of amyloid plaques in the brain. Theseamyloid plaques can be visualised on sections of thesebrains by staining with amyloid specific antibodies. Thisstaining reaction results in a red amyloid plaque (indicatedby a white arrow) surrounded by blue nuclei of surroundingcells like neurons or astrocytes. A Mimotope-based ADvaccine as described in this abstract would induce suchspecific antibody responses reacting with the pathologicalAβ molecules and could therefore be a safe treatmentregimen to efficiently fight AD in patients.

as neuronal disorders and liver metastasis tofacilitate investigations of possible mechanismsof intervention.

Expected results

On the basis of the Nucleofector® 96-wellShuttle® device and 96-well Nucleocuvette®plates and modules which were launched byAMAXA in 2006, the Consortium will developultrahigh-throughput devices and plates. In par-allel, protocols for cultivating, differentiation,nucleofection®, and functional screens of pri-mary cells in small volumes and with low cellsnumbers will be elaborated and, in a further step,adapted to the devices.


Innumerable opportunities, e.g., in discovery ofnew regulatory pathways, novel targets, andpotential drug candidates as well as clarificationof signal transduction are addressed by thedevices that shall be developed in the frame ofthis project. Researchers will make rapid stridesin the analysis of the biology of cells among oth-ers for the development of therapeutics, and newtypes of treatments as well as possible cures fora variety of diseases and injuries.

Background

Cell transfection belongs to the well establishedtechnologies in biological, medical, and pharma-ceutical research but delivery to relevant cellssuch as primary cells and adult stem cells is stilla major bottleneck. While conventional transfection methods, such as lipofection or electro-poration, usually yield satisfactory results forstandard cell lines, many other cell lines as wellas most primary cells are difficult or even impos-sible to transfect with these methods. Viral vec-tors – as alternative for DNA delivery – work wellin some cases, but are labour-intensive, not ver-satile, and remain connected with significantsafety issues. In consequence, most primary cellswere considered non-transfectable, representinga tremendous disadvantage in highly relevantresearch areas as primary cells most closelyresemble the situation in the living organism.Unlike cell lines, which often have been culturedfor decades, primary cells are freshly isolatedfrom the organism’s tissue, and have not gonethrough any transformations, the prerequisite forthe unlimited growth of conventional cell lines.Against this background, it goes without sayingthat the pharmaceutical industry is highly inter-ested in using primary cells instead of cell lines forcell-based screening campaigns in drug develop-ment. With more predictive screens – in terms ofboth the relevance of a target and the pharma-cokinetics/-dynamics of a drug compound – itbecomes much easier to take adequate decisionsas to which targets or compounds to focus forfurther development. Consequently, the use ofprimary cells in preclinical R&D will positivelyimpact attrition rates and reduce the significanttime and capital involved in developing a drug.

Aim

The main objectives are:• the development of devices for ultrahigh-

throughput nucleofection® of primary cells inmulti-well plates; and

• the application of this technology to thehighly relevant area of immunological as well

Modular Devices for Ultrahigh-throughput and Small-volume Transfection

ACRONYM

MODEST

Devices for ultra high-throughput trans-fection and screening of primary cells, foruse in the study of e.g., immunological,neuronal and liver disorders shall bedeveloped. Modification of cells is a cen-tral topic in pharmaceutical and medicalsciences as well as in basic research. Thedominant consideration for cell manipula-tion is the type of cells used. Until now,most often conventional, immortal celllines, being cultured for years or evendecades in flasks, have been assigned tothis specified use, although they aremainly irrelevant in physiological andmedical respects. The optimal choicewould be primary cells, since they are dis-tinctly closer to the physiological situa-tion than cell lines. However, primary cellsuntil recently were hard or often evenimpossible to transfect by non-viral meth-ods, and consequently, many researchersstill hesitate to adopt these cells despitetheir unquestionable advantages. Withthe advent of the nucleofection® tech-nology, a unique method for the highlyefficient transfection of primary cellswas recently made available to the lifescience community.

SUMMARY

110



Key words: nucleofection, primary cells, hard-to-transfect cell lines, RNAi, siRNA, ultra high throughput transfection, adult stem cells, neuronal cells, apoptosis, lead, gene silencing/knockdown, screening

SMEs play a major role in MODEST, since five of the eight partners are SMEs. Moreover, themedium-sized enterprise AMAXA acts as co-coordinator of the whole project.

AMAXA is the only institution worldwide which has the necessary knowledge and experi-ence to adapt the technical needs of device development to the conditions used for the effi-cient transfection of primary cells and hard-to-transfect cell lines. After performing anextensive market study, AMAXA is responsible for prototype building from concept for thedevices up to testing of software and hardware specifications. As co-ordinator of the proj-ect, AMAXA will ensure that the expertise brought to the table by each partner will enablethe collaboration to achieve the goal of providing powerful new tools for basic research anddrug discovery.

The main emphasis of Fotec is on the development steps in injection molding of the multi-well plates. This partner will develop the production process for a micro-plate with 384 wellsand will develop a process concept for the production process of a micro-plate with1 536 wells (technical study). Together with another partner (HTP Electronics GmbH), Fotecalso designs the necessary injection moulds.

AMAXA will support RNAx, Protobios and Dominion Pharmakine to establish nucleofectionprotocols and functional assays for relevant cells, in order to evaluate the devices inrespect to nucleofection of small volumes and cell numbers up to single cells, respectively.With the expertise of RNAx as a partner of MODEST, RNAi technology will become the idealbasis for a highly efficient high-throughput validation system for the definition of functionsof identified targets. RNAx has extensive experience in the development of automatedRNAi, including the adaptation of the system to various cells and cell lines, as well as in thedevelopment and adaptation of assays to the RNAx automated platform.

Protobios has unique expertise in neural stem cells, nervous system development mecha-nisms and transcription regulation networks. The uniqueness of the input of Protobios liesin the competition advantage of the new method of creating human neural stem cells andinfluencing them to differentiate into various derivates of the nervous system. Protobiosappraises the devices for ultra-high-throughput application, by contributing large cellnumbers of hNCS.

The role of Dominion Pharmakine is to identify new biomarkers related to site-specific devel-opment of metastasis, which may be used as prognosis indicators at the clinics, and to opti-mise the selection of the most likely successful antimetastic targets among large lists ofcandidate genes, ensured by the knock-down functionality assays. Moreover, DominionPharmakine contributes to the MODEST project by providing high-quality primary cell culturesand considerable experience in cell culture techniques.

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111


Herbert Müller-HartmannAmaxa A.G.Research & DevelopmentNattermannallee 150829 Cologne, [email protected]

Project manager

Birgit [email protected]

Partners

Andrej ManteiDeutsches Rheuma Forschungszentrum Berlin Immunomodulation/DRFZ/German Rheumatism Research Centrewww.drfz.de

Jörg PötzschRNAx GmbHBerlin, Germanywww.rnax.de/en/index.html

Kaia PalmProtobios Ltd.Tallinn, Estonia

Helmut LoiblFOTEC Forschungs- und Technologietransfer GmbHWiener Neustadt, Austriawww.fotec.at/pages/indexEN.html

Josef Anton PallanitsHTP Electonics GmbHWiener Neustadt, Austriawww.hti-ag.at/de/geschaftsfelder/kunststoff/beteiligungen/htp_electronics/

Naiara Telleria GarayDominion Pharmakine S.L.Cell Biology and Culture Laboratory/Dominion PharmakineDerio, Bizkaia, Spainwww.pharmakine.com

Thomas SchaumannPrevas ABInstrument DevelopmentVästerås, Swedenwww.prevas.com/product development.html

properties but also the polymer composition andmacromolecular architecture, are critical in induc-ing an effective immune response. In this context,MuNanoVac aims to assess the proof-of-conceptof a new vaccine candidate to prevent HIV-1 infec-tion at portal of entry; based on a primo-vaccina-tion using a biodegradable synthetic colloidalcarrier, made of PLA nanoparticles bearing anyabsorbed HIV-1 antigens on their surface.

Expected results

The target vaccine candidates will be intensivelyevaluated and optimised to reach the best potential.The project will cover the necessary steps to achievea complete proof of concept, including preliminaryprocess development aspects. MuNanoVac willcontribute towards:• bringing new knowledge and innovative tech-

nologies through the HIV vaccine discoveryprocess, and;

• proposing new vaccine candidates for combat-ing and preventing HIV/AIDS.

Additionally, the project results will be promotedas a basis for developing vaccine candidates forother poverty-related diseases. Indeed, the proj-ect aims to establish a proof-of-concept in orderto promote the potential of PLA nanoparticle-based compounds as effective vaccine candidatesto support a new HIV vaccine strategy.


MuNanoVacs aims at an efficient vaccine strategyfor the prevention of HIV infection, with a view totreating also other poverty-related diseases.Moreover, the advanced research activities con-ducted in MuNanoVac will generate novel data oninnovative HIV vaccine vehicles with a view toimproving the efficacy of the proposed vaccine.This data is crucial for publishing scientific results,contributing to sharply estimating the accuracyand pertinence of such new HIV vaccine candi-dates and, therefore, for accelerating vaccinedevelopment.

Background

In order to halt HIV infection spreading, a safe andeffective AIDS vaccine for both developing anddeveloped countries is urgently needed. Indeed,HIV is still spreading worldwide with more than65 million people infected (source: UNAIDS/WHO,2006) and the number of new infections is risingsharply in Asian countries, Eastern Europe and sub-Saharan Africa. The numerous research efforts car-ried out in this domain have not yet produced anefficient anti-HIV vaccine.

Many infectious diseases for which no vaccine isavailable suffer from the absence of a good candi-date that allows efficient T and B cell immuneresponses. In the case of HIV-1 mediated infec-tion, the present postulate is that both arms of theimmune response (humoral and cellular) shouldbe stimulated by any potential vaccine candidate.Moreover, recent data on natural primary HIV-1infection establish that the spreading of the virusin the mucosa is essential for the infection to takeplace. Hence, every vaccination strategy shouldbe able to elicit a strong mucosal immunity at thepotential sites of contamination and preventspreading of HIV-1 virions.

A future goal for vaccine design is therefore toincrease their efficiency by reaching the highestnumber of antigen-presenting cells (APCs) possi-ble and to achieve the high local concentrationsrequired, inducing a potent immune response.Thus, facilitating vaccine compounds penetrationinto immunisation sites that are rich in APCs aswell as specific migration and activation of APCsthat would benefit the efficacy of new vaccines inthe induction of protective immune response.

Aim

Current research in the nanotechnology of bioma-terials is aimed at using nanosystems as vaccinecarriers able to target dendritic cells (DCs) orallow transport through skin or mucosal epithelialbarriers. Results show that not only the colloidal

Mucosal Nano Vaccine Candidate for HIV

ACRONYM

MUNANOVAC

The MuNanoVac STREP project will assessa new vaccine strategy to prevent HIV-1infection based on a primo-vaccinationusing a biodegradable synthetic colloidalcarrier made of poly-lactic acid (PLA)nanoparticles covered with adsorbedantigens. The aim is to demonstrate thatPLA nanoparticles are a perfect transcu-taneous or mucosal vaccine vehicle,immunogenic for both arms of immunityand adaptable to many types of antigensas well as easy and simple to produce.

Such nanoparticle-based vaccine carrierswill allow targeting dentritic cells or trans-porting the vaccine through skin ormucosal epithelial barriers. To amplify themucosal immune response, the projectwill investigate the potential use ofimmunomodulator molecules associatedwith the comparison of two differentimmunisation routes.

Moreover, the MuNanoVac project willcontribute to advancing a promising vac-cine approach for HIV that could alsoprove versatile enough for application toother poverty-related diseases such astuberculosis. With the proposed vaccinecandidate, the project gives Europea tremendous opportunity to gain lead-ership in the use of biodegradablenanoparticles for vaccine carriers.

SUMMARY

112



Key words: vaccines, immunology and Infections, HIV/AIDS, immune response, vaccine strategy, mucosa, skin, poly (lactic acid), PLA, nanoparticle and biodegradable carrier

PHUSIS, as the project coordinator, will be in charge of securing the manufacture of selectedpoly (D, L-lactic acid) – PLA – nanoparticles, with full quality control of the materials, and forthe scaling-up of the production process – polymerisation, purification, and drying. PHUSISwill also take the lead in securing an agreement among all the parties involved, so that theprocess of PLA nanoparticles preparation can be subsequently marketed.

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113


Jacqueline HuetPHUSISEspace Randon, 58 Route du Rivet38330 Saint Ismier, [email protected]

Project manager

Bernard VerrierInstitut de Biologie et Chimie des ProtéinesUMR 5086 CNRS/UCBL7 Passage du vercors 69367 Lyon Cedex 07, France [email protected]

Partners

Robin ShattockSt George’s Hospital Medical SchoolLondon, United Kingdomwww.sgul.ac.uk

Teresa GallartHospital Clínic de BarcelonaBarcelona, Spain

Roger Le GrandCommissariat à l’Énergie Atomique Paris, Francewww.cea.fr

Milan RaskaPalacky University in Olomouc Olomouc, Czech Republicwww.upol.cz

Behazine CombadiereUniversité Pierre et Marie Curie – Paris 6Paris, France www.upmc.fr

Ulrike Blume-Peytavi Charité Universitätsmedizin Berlin Berlin, Germanywww.charite.de

| Uptake of Poly Lactic Acid Nanoparticles loaded with p24 HIV gag protein.by dendritic cells.Illustration by electron microscopy of sub-cellular location in endosomal compartments.

autoantigens? Why is the thymus pathologic inmost cases? Why are females predominant in theearly onset disease? What occurs in the muscleafter the autoimmune attack? And – finally – howcan therapy of MG be improved?

Aim

Myasthenia gravis (MG) is a well-defined antibody-mediated disease, but the pathogenic mecha-nisms at the effector organ (the thymus) and at the target organ (the muscle) are still puzzling. Theaims are to improve knowledge, diagnosis, moni-toring and management of MG patients. The mainscientific and technological objectives of the projectare as follows:

1. To improve knowledge on the mechanisms ofpathogenicity occurring in myasthenia gravis.

• Mechanisms of thymus remodelling in youngpatients. This topic will be addressed bya genomic approach using cDNA microarrays anda post-genomic approach exploring the role ofchemokines previously identified as key mole-cules in the development of the disease. Newtransgenic models overexpressing CXCL13 in thethymus will be established to investigate whethergerminal centers (GCs) will be generated afterinducing the disease in this animal model.

• Determination of the consequences of theautoimmune attack on the muscle organisation,gene expression and function.– The transcriptome analysis of muscle biopsies

should identify a specific signature for the dif-ferent subgroups of patients: patients withanti-AChR antibodies or with anti-Musk anti-bodies, and double negative patients.

– Identification of new genes and proteinsderegulated as a result of the autoimmuneattack by analysis of muscle transcriptome inMG patients, rat EAMG, and in vitro model.

– Investigating the specificity and pathogenicityof each subunit domain. New in vivo modelswill be created by immunising the rodentswith each subunit ectodomain as well as theircombination to evaluate the pathogenicity ofeach subunit.

Background

Acquired Myasthenia Gravis (MG) is a rare autoim-mune disease with a prevalence around 0.1/2000.This disease is due to autoantibodies directedagainst components of the neuromuscular junc-tion and leading to disabling fatigability of mus-cles. MG is mainly (85 % of the patients) caused byanti-acetylcholine receptor (AChR) autoantibodiesand in some patients (about 5 %) by autoantibod-ies against a muscle-specific receptor tyrosinekinase (MuSK). The remaining population forwhich the autoimmune target is not yet identifiedis defined as double negative MG patients.

If the muscle is the target organ in this disease,the thymus is clearly involved in the pathogenesisof MG with anti-AChR antibodies. Indeed, 50-60 %of the patients present a thymic hyperplasia withgerminal centres (GCs) and around 20 % of thepatients have a thymoma. The titre of anti-AChR isnot correlated with the severity of the disease butis associated with thymic abnormalities. Thehyperplastic thymus includes all the componentsof the anti-AChR response: the autoantigen(AChR), B cells producing anti-AChR antibodiesand anti-AChR autoreactive T cells. The thymus isthus clearly involved in the development of MGand thymectomy is often advised for MG patientsimproving slowly but efficiently over a few yearstheir symptoms. The other pharmacological treat-ments proposed are: • symptomatic drugs such as cholinesterase

inhibitors preventing the degradation of acetyl-choline;

• glucocorticoids and immunosuppressor drugswhich are used for many autoimmune diseasesand act in a global way on the immune response;

• plasmapheresis or intravenous human immuno -globulins in crisis period.

Although progress has been made in developingtherapies for MG, this disease is still incapacitat-ing and treatments are not satisfactory. Manyquestions remain unanswered: Why is there nocorrelation between the anti-AChR antibody titerand the disease severity? Which are the other

Development of models to improve management of Myasthenia Gravis: From basic knowledge toclinical application

ACRONYM

MYASTAIDwww.euromyasthenia.eu

Autoimmune Myasthenia Gravis (MG) isa rare, chronic and heterogeneous neuro-muscular disease characterised by severemuscle weakness. In most patients, it isdue to auto-antibodies to the acetylcholinereceptor (AChR). The detrimental effects ofthe autoimmune attack on the muscle arenot fully understood. The current treat-ments of MG induce severe side-effectswith no permanent clinical remission. Theearly-onset patients are mostly femaleswith thymic hyperplasia, while the existingexperimental model for MG (EAMG) doesnot present thymic pathology. This projectintends to develop models to progress inknowledge, monitoring, diagnosis andtherapy of MG: • videomicroscopic models to explore the

motility of lymphocytes involved inthymic remodelling in young females;

• new transgenic models overexpressingthe CXCL13 chemokine in the thymus, tobetter mimic the human disease whichinvolves thymic abnormalities andincreased thymic CXCL13 expression;

• mouse model of estrogen deficiency, todetermine the influence of estrogens inpredisposition and progression of MG;

• humanised NOD/SCID mouse model trans ferred with human MG thymocytes totest therapy by human regulatory T cells;

• T cell receptor-based signature, for bet-ter classification and monitoring of MGpatients;

• rat EAMG models immunised withTorpedo AChR, to test an array of ther-apies (IVIg subfractions, chemokineinhibitors, regulatory cells);

• rat models immunised with recombinanthuman AChR subunits, to determine thecontribution of each subunit to the path-ogenicity and develop immunotherapies;

• monkey models to test protective anti-AChR antibodies.

Since the research teams involved in thisprogramme also participate in a large‘European MG Network” supported by theEC, interactions between the two networkswill promote efficient dissemination of theobtained knowledge.

SUMMARY

114



Key words: Myasthenia gravis, rare disease, chemokines, hormones, transcriptome, thymus, muscle, diagnosis, anti-TCR antibodies, therapy, antibodies, acetylcholine receptor, regulatory cells

• Understanding the female predominance in theearly onset disease by analysing the transcrip-tome of the human thymus in young normalmales and females and by exploring the influ-ence of sexual hormones on the susceptibility ofMG disease in animal models.

2. To improve diagnosis and monitoring ofMyasthenias.

• By developing a new diagnostic assay moreeasy and to develop a monitoring assay of theautoimmune and regulatory status.

• By searching for the autoantigen in the anti-MuSK and anti-AChR negative patients by usingproteomics approach.

• By analysing the complete repertoire of the T cellsand the diversity of the anti-TCR antibodies.

3. To improve therapy for MG using severalapproaches all of which are based on modula-tion of the adverse autoimmune responseand/or activation of the muscle response to theautoimmune attack in animal models.

Expected results

• Identification of genes involved in MG patho-genesis.

• Mechanisms of action of germinal centre devel-opment in the thymus.

• Establishment of transgenic mice overexpress-ing CXCL13 in the thymus and determination ofthe role of CXCL13 in the development of thymicpathology.

• Identification of genes of predisposition specificto females.

• Identification of new autoimmune targets indouble negative MG patients.

• Mechanisms of action of anti-TCR antibodies. • Development of new therapies for MG.


This project aims to improve diagnosis and moni-toring. The collaboration with 6 industrial part-ners included in this project will help developingthe following clinical applications:

A new, easier diagnostic assay for anti-AChR anti-body titers is expected to be developed.

A biological diagnosis is still expected for doubleseronegative MG patients. But in that case, theresearch proposed in this project aims first toidentify this autoantigen.

The use of the technology developed by one of theproject’s industrial partners to analyse the T cellrepertoire in MG patients will provide a pioneer-ing work as an example for studies in other dis-eases therefore increasing the existing market forimmune signatures. This technology is based ona genomic approach, exploiting sequences of theentire TCR locus.

The development of a new tool named antiTracKeR,to analyse the anti-TCR repertoire.

The arrays of therapies to be used in animal mod-els and particularly in the humanised mice modelshould result in novel therapeutic approaches.

The MYASTAID consortium includes 6 SMEs which will have a key role in the development ofnew diagnostic and monitoring assays, and in the development of new therapeuticsapproaches. Therefore, MYASTAID will reinforce the SMEs scientific and technological knowl-edge on the validation of innovative solutions. The highly experienced SMEs participating inthis project aim to exploit the RTD findings for commercial markets and its translation to theclinic. The following table shows the contribution and potential benefits to each of the SMEs.

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Sonia Berrih-Aknin Université Paris Sud (UPS)CNRS UMR 8162 Hôpital Marie LannelongueAvenue de la Résistance 13392350 Le Plessis-Robinson, France [email protected]

Partners

Socrates Tzartos Hellenic Pasteur Institute (HPI)Athens, Greece

Marc De Baets University of Maastricht (UM)Maastricht, The Netherlands

Sara FuchsMiry SouroujonTsvee Lapidot Weizmann Institute of Sciences (WIS)Rehovot, Israel

Amnon PeledOrly eizenbergBiokine Therapeutics Ltd. (BKT)Jerusalem, Israelwww.biokine.com

Eugene Boswans Epsilon Biotech (Ebiot)Antwerp, Belgium

Paul ParrenGenmab B.V. (GMB)Utrecht, The Netherlandswww.genmab.com

Nicolas Pasqual ImmunId Technologies (IDT)Grenoble, Francewww.immunid.com

Orgad LaubOMRIX biopharmaceuticals Ltd.Nes-Ziona, Israelwww.omrix.com/index.asp

Michael Roberts Regulon A.E.Athens, Greecewww.regulon.org

| Thymic Hyperplasia in early onsetmyasthenia gravis patient.The staining in red (anti-keratin) represents theepithelial network all over the thymus,and the staining in green (anti-CD21)labels B cells and follicular dendritic cells visualizing the germinal centers. The picture was obtained by an originaltechnique using a scanner for microarray.

be monitored, as well as a combination of recep-tors known to trigger proliferation, (FGF, IGF1, … ). The stability of the parameters initially examinedin non-GMP conditions, will be checked throughamplification in various conditions, to allow thedefinition of both guidelines for GMP productionand key-parameters to be followed during theGMP amplification.

The number of cells to be injected at each implan-tation, which is purely empirical in many clinicaltrials, will be tested and optimized in a model ofimplantation of human cells in immuno-deficientmice, in order to define the maximum number ofcells to be finally amplified in GMP conditions.

Expected results

The main expected result is to obtain the finalproduct, i.e. protocols to obtain amplified humanstem cells, in a state that will allow an optimizedefficiency in injections in vivo.

In addition to basic knowledge on the amplifica-tion mechanisms, MYOAMP will bring guidelinesand standard operative procedures to obtainthese cells in a reproducible and safe manner thatcan be directly transferred to SMEs and cliniciansfor clinical applications.

These guidelines will therefore address technical,ethical and safety issues in a GMP environment.


Pre-clinical Protocols, Standard Operating Proce -dures to characterize, amplify and assess myo-genic human stem cells for autologous celltherapy treatments in muscle disease.

Ethical and safety procedures to cover the pro-tocols.

Specific culture medium with a defined set ofgrowth factors.

Background

Many clinical trials using muscle cells have beendeveloped in the past for Duchenne MuscularDystrophy with very limited success. The recentemergence of new therapeutic venues, basedupon post-transcriptional genetic correctionscalled ‘exon-skipping’, has raised new hope forthis disease. Using viral transfer approaches ithas given very promising results but cannot reachevery muscle of the body and trigger an immuneresponse to the vector. Autologous cell therapymay bypass this reaction and be used as a com-plement or alternative if the cell type used ful-filled both being an efficient vector and bringinga functional benefit to the diseased muscle.

Autologous muscle cells cannot be used sincethese are already defective in dystrophic muscle,while stem cells from other origins are ideal candi-dates, as long as their myogenic and proliferativepotentials are ensured. In this perspective mesoan-gioblasts, which have already been used ina mouse model of muscular dystrophy, and AC133cells have a therapeutic potential as demonstratedin the mouse, but very little is known about the con-ditions required to amplify in GMP conditions thesestem cells isolated from humans, which is anessential step required before any clinical trial.

Aim

MYOAMP will address the question of the amplifi-cation of these cells used as autologous cell ther-apy vectors and their safety.

The cells transduced with a lentiviral constructallowing exon-skipping will be selected and furtheramplified. It should be noted that transduced cellsmay be cloned so that their integration site is deter-mined), as will be proposed in the part of MYOAMPthat focus on safety and ethics. In vitro and in vivoapproaches will bring understanding on the regula-tion of proliferation, while the telomere length(reflecting the mitotic clock status of the cell) will

Amplification of human myogenic stem cells in clinical conditions

ACRONYM

MYOAMP

Many groups have used animal models toinvestigate the possibilities of usingautologous cell therapy for muscular dys-trophies, but these data are dispersed,not always comparable and little atten-tion has been focused on the transfer ofthis knowledge towards applications fortherapeutic trials.

Data exist on injecting murine cells intomouse muscle, but information regardinghuman cells is sparse. The feasibility ofautologous myoblast transfer therapyhas already been demonstrated for car-diac repair, even if in cardiac therapy,injected cells were mainly used to coun-teract the development of fibrosis inpatients devoid of any defect in skeletalmuscle.

The fact that pre-clinical trials developedin mouse models of muscular dystro-phies have been successful as comparedto clinical trials, which used mostly allo-genic cells and resulted in very limitedclinical benefit for the patients, illus-trates the urgent need for pre-clinicalstudies using human cells.

MYOAMP will aim to define conditionsand guidelines to produce transducedhuman stem cells as vectors for clinicaltrials. MYOAMP will synergize expertisesfrom European leaders in their respectivefield to set up conditions for autologoustransfer of human stem cells in GMP con-ditions for the treatment of DMD byexon-skipping. It will ensure that theseconditions and guidelines are transferredto SME and clinicians, defining efficientintegration through dedicated partnerswithin the 3-years duration of this project.

SUMMARY

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Key words: human stem cells, neuro-muscular diseases, cell proliferation, cell therapy, exon-skipping

3H Biomedical cell provider and responsible for defining standard operating procedures(SOP) for cell handling.

CELLGENIX development of adapted serum-free culture medium and definition of SOP.

GENOSAFE development of safety procedures and assays for the process.

ROLE OF SMEs

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Vincent Mouly INSERM UMR S 787 MyologieInstitut de Myologie105 bd de l’Hôpital75634 Paris Cedex [email protected] www.institut-myologie.org

Partners

Luis GarciaINSERMParis, France

Yvan Torrente University of MilanMilano, Italy

Giulio CossuFondazione Centro San Raffaele del Monte Tabor Milano, Italy

Jenny MorganImperial CollegeLondon, United Kingdom

Otto MertenGénéthon, Evry, Francewww.genethon.fr

Mallen Huang3H BiomedicalUppsala, Swedenwww.3hbiomedical.com

Roland BosseCellgenix GmbhFreiburg, Germanywww.cellgenix.com

Vincent GiulianiGenosafe S.A.Evry, Francewww.genosafe.com

Anton OttaviInserm-TransfertParis, Francewww.inserm-transfert.fr

Expected results

To achieve the goal of this project, methods andtechniques will be used to optimise each compo-nent in the assay. When technology, as describedabove, was developed for small molecule markers15 years ago, a large change in the diagnosticindustry was seen and two small SMEs grew intobig industrial corporations. The project foreseesthat similar effects may occur when such technol-ogy is also developed for large molecule markers.


New high sensitivity high speed assay productson automated clinical chemistry platforms for usein laboratories throughout the healthcare system,for higher throughput and for improved cost-effectiveness.

Background

Traditionally, immunoassays have been separa-tion based, meaning that the analyte of interestgoes through several steps of antibody binding,washing and separation before final detection.This type of assay requires a high use of consum-ables, which is expensive and time consumingdue to all the steps. On the other hand, in anon-separation assay no separation steps areinvolved and the use of consumables is limited,making the assay less expensive and with a muchshorter assay time. A non-separation assay willtypically be run on a clinical chemistry platformintended for high-throughput of analytes, makinghomogeneous non-separation immunoassaysa high potential market growth opportunity.

Aim

The aim of the project is to move immunoassaysfrom slow and expensive methods to fast, high-throughput super-sensitive nanoparticle basedmethods. In addition, the project aims to generateintellectual property for such technology.

Moving sensitive immunoassays from slow and expensive to fast and affordablenanoparticle-based methods

ACRONYM

NanoSensewww.nanosense.eu

There is a need for high sensitivity non-separation immunoassay technology forgeneral clinical chemistry instrumentplatforms. This need is particularly evi-dent for large protein disease markers oflow concentration, such as NT-proBNP,PSA, and a long list of other plasma pro-teins, protein hormones and specificantibodies. Such new technology will sig-nificantly change the diagnostic industryand healthcare providers much greaterefficacy.

SUMMARY

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Key words: nanoparticles, clinical assays, turbidimetry, high sensitivity

There are five participants in this project, with three being SMEs. The co-ordinator role isundertaken by one of the SMEs, Dalen Diagnostics AS in Norway. In addition to the coordi-nation, the company possesses the necessary expertise for improved signal generation inhomogeneous nanoparticle assays. The other two SMEs are: 77 Elektronika Kft in Budapest,Hungary, which specialises in the field of medical electronics and manufactures blood glu-cose meters, urine analysers and rapid test readers; and Getica AB, a small, Swedish biotechcompany specialising in bioorganic coupling chemistries and bioprocessing of proteinconjugates and nanoparticles.

In the consortium, there is also a large industrial company, Merck Chimie SAS, Europe’slargest producer of nanoparticles, and an academic participant, the GroningenUniversity Medical Centre, which provides a reference laboratory for measurements inlarge epidemiological studies.

ROLE OF SMEs

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Guri SkjeltorpDalen Diagnostics ASKolsroedveien 1201599 Moss, [email protected]/dalendiagnostics/vis.php

Partners

Erling SundrehagenIngrid Hulthén Getica ABTösse, Swedenwww.getica.se

Richard VidalCécile Genies Merck Chimie SAS EstaporFontenay-Sous-Bois, Francewww.estapor.com

Uzonka FarkasPéter Jakus 77 Elektronika Muszeripari KftBudapest, Hungarywww.e77.hu

Dick de ZeeuwStephan J L Bakker Department of Clinical PharmacologyUniversity Medical Center Groningen Groningen, The Netherlandswww.umcg.nl

for interventional neuroprotection. The consortiumwill study, in various established animal models,the mechanisms that lead to perinatal brain dam-age in order to identify genomic, proteomic andmetabolomic biomarkers to generate biomarkerprofiles. They will also establish biomarker profilesin human newborns in an observational clinicalstudy involving extremely pre-term infants bornbefore completion of 28 weeks gestation (normalpregnancy duration: 40 weeks).

Once identified biomarkers of damage and poten-tial avenues for neuroprotection, the project willstart developing an intervention, using multipleanimal models to pursue this goal. Only the mostpromising strategies will be considered worthy ofbeing translated from bench to bedside.

Further step of the project will be to implementa clinical platform • to design a biomarker profile of perinatal brain

damage in experimental animals and in humannewborns (see above). Thus, participants willestablish a functional network of institutions car-ing for newborns that can serve as the basis forsuch a clinical study, designed to identify humanbiomarker profiles based on genetic and bio-chemical markers, electroencephalographic (EEG)patterns and magnetic resonance imaging (MRI);

• the consortium will use and expand this platformfor clinical drug testing both within the 36 monthsof NEOBRAIN and thereafter.

Finally, NEOBRAIN will pave the way for clinicaldrug development. In essence, the clinical plat-form will be designed in a way that allows forquick expansion (i.e. recruitment of further cen-tres), so that bench-to-bedside translationalsteps (i.e. a clinical trial) can be taken quickly afterNEOBRAIN is finished.

Expected results/potential applications

NEOBRAIN provides an obvious potential impactin helping to reduce mortality and stamp out devel-opmental disabilities associated with perinatalbrain damage.

Background

Prevention of perinatal brain damage is of majorimportance for public health and obviously forindividual wellbeing. Both white and grey brainmatter are affected in perinatal brain damageobserved in pre-term infants. Long-term conse-quences of extreme prematurity are devastating,and perinatal brain damage clearly plays a role inthis scenario. The current pathogenetic paradigmof perinatal brain damage in pre-term infantshas multiple interrelated aspects and includesinfection/inflammation, hypoxia-ischemia, exci-totoxicity, and free radicals. It is likely that thesemechanisms do not act alone, but in concert.

The absolute number of neurological handicapsof perinatal origin is increasing in western coun-tries due to the increasing survival of pre-terminfants. The major brain lesions associated withcerebral palsy (CP) and cognitive impairment arewhite matter damage (WMD), mostly occurring invery pre-term infants (born in less than 32 weeksof gestation), and cortico-subcortical lesionsmostly observed in term infants. For financial,technical and ethical reasons, the pharmaceuticalindustry has difficulties in making substantialinvestments in this area, and this has left perina-tologists with a limited therapeutic arsenal. At thepresent time, despite major improvements inneonatal care, there are no established therapeu-tic regimens for the prevention or treatment ofperinatal brain lesions that are successful.Nevertheless, epidemiological and experimentaldata have allowed the identification of potentialtargets for neuroprotection. New animal models,such as those employed in NEOBRAIN, clearlyshow the pathophysiology involved in neurode-generation and will help identify neuroprotectivestrategies in the newborn.

Aim

To help reduce the enormous individual, familialand societal burden that perinatal brain damagerepresents, the first objective of this project is toidentify early damage markers and novel pathways

Neonatal estimation of brain damage risk and identification of neuroprotectants

ACRONYM

NEOBRAINwww.neobrain.eu

NEOBRAIN brings together small andmedium-size enterprises (SMEs), largercompanies and academic groups devotedto the diagnosis, management and neu-roprotection in newborns with perinatalbrain damage. The focus of NEOBRAIN isthe prevention of brain damage mainlyobserved in pre-term newborns.

The objectives of NEOBRAIN are: • to generate marker profiles of damage in

multiple animal models and in humanpre-term infants using genomic/genetic,proteomic and metabolomic approaches,as well as imaging and electrophysiologicmodalities;

• to develop neuroprotective strategiesby identifying candidate molecules forintervention in animals;

• to implement a platform for an observa-tional clinical epidemiologic study inhuman infants designed to contributeto objective 1 above, and to transferfrom the animal to the human levelinsights gained in objectives 1 and 2;

• to prepare for drug testing by using theproject structure as a platform for initialsteps in clinical testing of potentialinterventions discovered in NEOBRAIN.

The project further envisages developingthe clinical platform in such a fashion that itcan serve as the basis for subsequent large-scale pan-European perinatal neuropro-tective research initiatives (Euro-Neo-Net,EURAIBI).

SUMMARY

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Key words: newborns, hypoxia/ischemia, inflammation, perinatal brain damage neuroprotectants

The most important source of societal sufferingfrom perinatal brain damage is at the individualand family level. Four out of five pre-term infantsare limited in their everyday activities. Moreover,brain-damage-associated cognitive and learningdifficulties represent a potentially preventablesource of suffering. It is NEOBRAIN’s goal to con-tribute to an improvement of this situation forfuture generations.

Also the economic burden of prematurityimmense, taking into consideration cost of hos-pital inpatient admissions plus non-hospitalinpatient health care costs, such as costsincurred through family practitioners, educationaland social services, considering that approxi-mately 60 000 infants sustaining some sort ofbrain injury.

In addition to the societal and economic impact,the close cooperation and integration of enter-prises and academic centres offers the uniquechance to exploit new business areas and posi-

tion the participating companies as market lead-ers not only in Europe, but also in the USA,Australia and Asia.

New multi-parametric biomarker measurementtools will be developed based on metabolomicand proteomic techniques. These tools, in turn,will help improve clinical diagnostics with regardto perinatal brain injury, and this will contributeto a greater potential for neonatologists to con-sult with parents and within their therapeuticteams about the individual child’s prognosis. Thegrowing medical subdiscipline of ‘neonatology’is a potential market for tools to be developedin NEOBRAIN.

Another potential market among sick and pre-term newborns is called ‘theranostics’, the indi-vidualised surveillance of therapy efficacy andresults. NEOBRAIN will contribute to this fieldby offering improved strategies for biomarkermeasurements, including imaging and electroen-cephalographic markers of infant wellbeing.

Five companies (three of them being SMEs) play crucial roles in the NEOBRAIN project. TwoSMEs are devoted to develop biomarkers in the field of metabolomics (BIOCRATES life sciences,Austria) and proteomics (BIOANALYT, Germany). A larger company, NEUROPHARMA (Spain) andTHERAPTOSIS S.A., an SME (France) are designing and developing innovative neuroprotectivedrugs. BrainZ, a New Zealand based company (but not an SME) producing electroencephalogra-phy (EEG) hard- and software, is interested in the development of EEG-markers of perinatal braindamage. BrainZ has expertise and services not available in Europe and offers important assis-tance at no cost to the NEOBRAIN project. All efforts of the NEOBRAIN research are directedtowards the development of neuroprotective pharmacological strategies that will be directlyexploited by the SMEs participating in the project. The involvement of the companies providesa unique opportunity for using the research results in product development without delay.

ROLE OF SMEs

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Olaf DammannHannover Medical SchoolCarl-Neuberg-Str. 130625 [email protected]

Partners

Medizinische Hochschule HannoverHannover, Germany

BIOCRATES life sciences GmbHInnsbruck, Austriawww.biocrates.at

BrainZ Instruments Ltd.New Zealandwww.brainzinstruments.com/index.asp

Bioanalyt GmbHPotsdam, Germanywww.bioanalyt.com

THERAPTOSIS S.A.Romainville Cedex, Francewww.theraptosis.com/english/home/index.htm

NEUROPHARMA S.A.Tres Cantos, Madrid, Spainwww.neuropharma.es

University Medical Center UtrechtUtrecht, The Netherlandswww.umcutrecht.nl/zorg

Göteborgs universitetGoteborg, Swedenwww.gu.se

Institut National de la santé et de la Recherche MédicaleParis, Francewww.inserm.fr

Université de GenèveHôpitaux Universitaires de GenèveGeneve, Switzerlandwww.unige.ch/medecine/index.html

Charité Universitätsmedizin BerlinBerlin, Germanywww.charite.de

Università degli Studi di SienaSiena, Italywww.unisi.it

Lunds UniversitetLunds, Swedenwww.lu.se

Aim

The main aim of this project is to develop anintegrated technology, comprising cutting edgebioinformatics, chemoinformatics and experi-mental methods, to study the mechanisms thatcause complex diseases. Understanding themechanism of diseases is the key to rationalisingdrug development.

Expected results

The aim of this project is to develop a toolbox thatcombines and puts different high-throughputexperimental methods together under the roof ofbio- and chemo-informatics, to allow the exploita-tion of the full potential of the included methods,as well as the generated data.

This toolbox shall provide a cost- and time-effec-tive, knowledge-based approach that allows iden-tification of signal transduction mediators and/ortranscriptional regulators responsible for certainchanges or responses of the cell, in particularthose which lead to diseases, thereby facilitatingthe discovery of new targets and consequently therational design of target-specific drugs.


Identification of drug targets in breast cancer andother diseases.

Background

Complex diseases such as cancer are caused bythe disregulation of expression of many differentgenes, resulting in the malfunction of complex cel-lular process. Every cellular state is characterisedand precisely organised by differential expres-sions of specific sets of genes. Gene expression ismainly regulated at transcriptional level throughsequence-specific binding of transcription factors(TFs) to their target sites in regulatory regions ofgenes, where the combination of these sites andbound factors provide the required specificity. Forthe whole genome, we might expect millions offunctional sites regulating expression of genesunder different conditions, of which we have theknowledge of only a few per million. Therefore,computational methods for the prediction of TFbinding sites in genomes are needed.

New experimental high-throughput approacheshave been introduced to study the gene regulatory(and other molecular) mechanisms of complex dis-eases. Among them are the ChIP (ChromatinImmuno Precipitation)-on-chip method for the iden-tification of target genes for various transcriptionfactors on a high-throughput scale, and RNAiapproaches (gene silencing by small double-stranded interfering RNAs) for the functional eluci-dation of selected genes. Novel computationmethods are needed for the automatic interpreta-tion of high throughput data in order to formulatea hypothesis of molecular mechanisms of diseases.

From gene regulatory networks to drug prediction

ACRONYM

Net2Drug

New high-throughput methods allow thegeneration of massive amounts of molec-ular biological data. These, mainly phe-nomenological, data are often difficult toascribe to the activation of particular sig-nal transduction pathways and/or tran-scriptional regulators. A way to facilitatedata interpretation is to construct generegulatory networks that include signaltransduction mediators, transcriptionalregulators and target genes. This isa complex task, not only because of thehuge number of molecules involved, butalso because of variations across tissues,developmental stages and physiologicalconditions. However, these networks holdthe key to the understanding of the regu-latory processes within a cell and, thus, tomost life processes in general. The aim ofthis project is, therefore, to developa toolbox that combines the differenthigh-throughput experimental methodstogether under the roof of bio- andchemo-informatics, to allow the exploita-tion of the full potential of the includedmethods, as well as the generated data.The toolbox will comprise a combinationof the most innovative bioinformatictechniques, based on databases andmethods of artificial intelligence, mod-ern methods of chromatin structureanalysis, micro-array technologies,transcription factor-binding site identi-fication, as well as chemoinformaticsmethods for computer-aided predictionof biological activity spectra.

SUMMARY

122


Starting date: 1 February 2007

Key words: transcription factors, ChIP-on-chip method, microarray, analysis of promoters, drug targets

A number of SMEs (BIOBASE, Progenika and ISB), particularly from the bioinformatics sec-tor, take part in the project, thereby strengthening the link between basic research anda possible later application of the developed technology, either in the form of a service forthe pharmaceutical and biotechnological industry, or as a marketable product.

BIOBASE is a coordinator of the project. Among the tasks carried out are: • manual annotation of project relevant data into existing databases, namely TRANSFAC®,

TRANSPATH®, and TRANSCompel®; • providing the data collected to the community;• DNA sequence analysis: search for putative binding sites for TFs;• development of software for the reconstruction of a gene regulatory network (transcription

network) from the microarray experiments;• causal analysis of experimental data provided by other partners of the consortium and

computational identification of drug targets; and• maintaining the workflow for the chemoinformatics group, providing drug targets for

further prediction of perspective chemical compounds.

Progenika carries out the microarray analysis and gene validation by RNA interference.

ISB is responsible for the visualisation of gene regulatory networks, automatic in silicoreconstruction of pathways, and methods of semi-quantitative modelling of gene regula-tory networks. It is involved in the development of a microarray database format and userinterface, composite modules, methods of artificial intelligence and data mining, and theprediction of the most promising drug targets and markers for breast cancer.

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Alexander Kel BIOBASE GmbHHalchtersche Str. 33 38304 Wolfenbüttel Germanyalexander.kel@biobase-international.comwww.biobase-international.com

Partners

Progenika Biopharma S.A.Derio, Spainwww.progenika.com

National Public Health InstituteHelsinki, Finnlandwww.ktl.fi

Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V.Munich, Germanywww.fraunhofer.de

Universitätsmedizin GöttingenGeorg-August-Universität GöttingenStiftung Öffentlichen RechtsGottingen, Germanywww.med.uni-goettingen.de

Institute of Systems Biology, Ltd.Novosibirsk, Russian Federation

Institute of Biomedical Chemistry of Russian Academy of Medical SciencesMoscow, Russian Federation

Karolinska InstitutetStockholm, Swedenwww.ki.se

National Research Council – CNRRome, Italywww.cnr.it

© Shutterstock

Aim

The aims of the NPARI consortium are to fullyexploit the exciting properties of this novel pep-tide class. Specifically the consortium aims totarget peptide sequences into two areas; coatingagents for medical devices and therapeuticsagents.

A major application for the apoE peptides is ascoatings for medical devices such as catheters.ApoE derivative peptides are already in develop-ment as coatings for contact lenses. The projectaims to extend this application to use active pep-tides as coating agents for catheters. Catheterrelated infection causes significant morbidity andmortality across the EU and resistant bacteria andfungi are often responsible.

Another focus for the application of apoE peptidesis as therapeutic agents. The project will focus ontargeting specific resistant bacteria and fungi inorder to rapidly establish in vivo efficacy in a vari-ety of animal models. It will specifically target res-piratory infections caused by Pseudomonas andApsergillus. This will have direct clinical relevanceto the 30 000 European CF patients where resistantPseudomonas aeruginosa infections are a majorcause of mortality. Colonisation with Aspergillus isalso a major problem is this patient group.

Expected results

• The design of a small peptide library which willbe tailored to the proposed exploitable applica-tion of the project.

• Determination of the activity spectrum of activepeptides and the ranking of peptide variants.

• Optimisation and toxicity profiling of activepeptides.

• Efficacy profiles against a panel of resistantorganisms growing as biofilms.

Background

Infectious diseases represent the most commoncause of morbidity in the world (WHO). Over thelast 40 years, major advances have been made inthe development of numerous classes of antimi-crobial agents to treat serious life threateninginfections. This is particularly true for antibacterialagents. However, microorganisms are slowly turn-ing the tide and becoming increasingly resistant tothe agents developed by man. Long term andindiscriminate use of antibacterials has led toresistance developing for all the major classes oftherapeutic agents. Increasingly clinicians arefighting a rearguard action with a dwindlingarmoury of drugs to combat serious life threaten-ing infections. Nosocomial or hospital acquiredinfections (HAIs) represent an increasingly seriousproblem across Europe and the rest of the world.

Data from the SENTRY Antimicrobial Surveillanceprogram of 25 European university hospitals high-lighted the five most common bacterial bloodisolates. The most prevalent organisms (E. coli,S. aureus, P. aeruginosa and K. pneumoniae) arealso the most prevalent CVC associated infections(Clin Inf Dis 30; 3). Candida species were alsocommon organisms isolated from blood and car-ried a crude mortality rate of up to 40 %. Theseorganisms are the same organisms where resist-ance is a major issue. The incidence of resistantbacterial and fungal nosocomial infections ishigh. The European Study Group on NosocomialInfections (ESGNI) reports on blood stream infec-tions indicated 72.8 % of infections were nosoco-mial and mortality due to bacteremia was 7.1 %.The most frequently isolated microorganismsfrom BSI were S. aureus (15.1 %), E. coli (14.5 %),S. epidemidis and coagulase negative staphs(CNS) (17.8 %), P. aeruginosa / K. pneumoniae(both 5.3 %) and Candida spp and enterococci(both 4.6 %).(ESGNI-001, ESGNI-002).

Tailoring of Novel Peptide coatings and therapeuticsderived from a newly identified component of thehuman innate immunity Against Resistant Infections

ACRONYM

NPARIwww.npari.org

The apoE and apoB human proteins haverecently been linked to the innateimmune system. Peptide sequencesderived from these proteins have beenshown to have varied anti-infective prop-erties which can be modified by smallchanges to the core peptide sequence.Thus, the apoE and apoB peptidesexhibit antibacterial, antifungal andantiviral properties and present an excel-lent opportunity to develop novel thera-peutics and medical device coatings.Specifically the exploitation of thesenovel peptides allows for the potentialdevelopment of a new array of agents tar-geting against the growing problems ofantibiotic resistant microorganisms.

SUMMARY

124



Key words: peptide, resistance, infection antibacterial, antifungal, therapeutic agent, coating agent

• Pharmacological and efficacy evaluation ofpeptides in a range of models.

• Efficacy data for candidate coatings in dynamicbiofilm models.


The exploitation of this new class of antimicro-bial peptides offers the potential to develop newtherapeutic against a range of the most resistantand problematic organisms facing EuropeanInfectious Disease Clinicians.

The rate infection by resistant bacteria is increas-ing but new chemical agents are some way fromthe clinic. This project aims to develop candidatepeptide therapeutics which target the most prob-lematic organisms and develop them to the pointwhere they show sufficient potential for furtherdevelopment.

In addition, the project focuses on the preventionof infection by the same resistant organismsthrough the coating of medical devices with activepeptides.

By preventing the colonisation of catheters byresistant microorganisms it is expected that seriouslife threatening infections can be avoided.

A vital component of the project is the participation of several SMEs who each haveexpertise in the field of drug development. This, combined with the academic expertise ofthe remaining partners, allows for an experienced and focused consortium.

Nikem Research is an Italian based company who specialise in drug development. Nikemhas developed a wide range of ADMET and toxicological assays which can be applied tothe project. In addition, Nikem has a range of bioanalytical capabilities which will beemployed to monitor the pK/pD profiles of the peptides.

Similarly, F2G has expertise in the discovery and development of anti-infective agents,specialising in anti-fungals. With a range of HTS screening capabilities, chemistry andpharmacology experience, F2G is able to rapidly assess and develop peptides for use intherapeutic candidates.

The focus of Ai2 is on the use of peptides to prevent colonisation of medical devices, whichvery well complements the expertise within the consortia.

ROLE OF SMEs

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Mike BirchF2G Ltd.Lankro Way, EcclesManchester, United [email protected]

Partners

Christophe d’EnfertInstitut PasteurBiologie et Pathogénicité FongiquesParis, France www.pasteur.fr/ip/index.jsp

Jean-Marc GhigoInstitut PasteurGroupe de Génétique des Biofilms CNRS URA 2172Paris, France www.pasteur.fr/recherche/unites/Ggb

Chiara Bigogno, Giulio DondioNiKem Research [email protected]

Niels HøibyDepartment of Clinical MicrobiologyUniversity Hospital of CopenhagenCopenhagen, Denmark

Curtis DobsonAi2 Ltd.Manchester IncubatorManchester, United Kingdom

Expected results

• Comprehensive identification of antigens fromM. catarrhalis and NTHi by screening of bacterialsurface display libraries.

• Characterisation of the membrane proteomeand surface located proteins from bothpathogens by 2D-PAGE and 2D-LC-MS2 andserological proteome analysis.

• Selection of the most promising vaccine candi-dates by validation in vitro and in animal models.

• Characterisation of protective antigens withrespect to their role in pathogenesis of M. catar-rhalis and NTHi (including biofilm formation).

• Definition of natural immune responses againstthe identified antigens from M. catarrhalis, NTHiand S. pneumoniae using the available sera andIg preparations.


The achievement of the goals will drive the devel-opment of a prophylactic vaccine against OM andthereby will have a large benefit for the health ofchildren in Europe and beyond. Because NTHiand M. catarrhalis not only cause OM in childrenbut also other infections of the respiratory tractlike sinusitis, pneumonia or bronchitis, sucha vaccine could proof useful for prevention ofthese diseases as well.

As the project will contribute to science study-ing these bacteria and their pathogenesis, itmight also offer new approaches for alternativetherapies of OM.

Background

Approximately 80 % of all children undergo anOM episode by 3 years of age. The maincausative agents are the bacterial pathogensStreptococcus pneumoniae, Haemophilus influen-zae and Moraxella catarrhalis which colonise themiddle ear, often after a primary viral infection.After introduction and widespread use of pneu-mococcal vaccines, there is evidence that theimpact of the latter two species increased. Acuteotitis media (AOM) is characterised by the pres-ence of middle ear effusion accompanied by therapid onset of signs of inflammation such as otal-gia, otorrhea or fever. Recurrent OM affects up to40 % of children and may persist for weeks tomonths causing symptoms ranging from hearingloss and tinnitus to anorexia or conjunctivitis. AsAOM is very painful, it results very often in antibi-otic treatment although solid evidence is lackingthat this therapy alters the course of OM diseasein children. Also in case of recurrent OM the fail-ure rate of antibiotic therapy is quite high, oftendue to antibiotic resistances of the pathogens.Considering also the high direct and indirect costsof OM, there is an urgent need for an alternativeand effective therapy.

Aim

The OMVac project addresses as main objec-tives the identification of vaccine candidatesfrom Moraxella catarrhalis and non-typeableHaemophilus influenzae (NTHi) to develop a pro-phylactic vaccine against OM and the compre-hensive characterisation of natural immuneresponses against proteineacous antigens ofthe major three bacterial pathogens causingOM. Moreover, the role of selected antigens dur-ing pathogenesis and biofilm formation will beinvestigated.

Novel prevention and treatment possibilities for Otitis Media through the comprehensive identification of antigenic proteins

ACRONYM

OMVac

Otitis media (OM) is one of the most fre-quent diseases in childhood and themost common indication for the prescrip-tion of antibiotics. Due to the poor evi-dence for the effectiveness of thistreatment and widespread drug-resis-tances, there is a need for a prophylacticvaccine to reduce the misery of childrenby preventing the occurrence of OMaltogether. This study focuses on thecomprehensive identification of antigencandidates from two of the maincausative agents of OM, namely non-typeable Haemophilus influenzae (NTHi)and Moraxella catarrhalis. The antigenomeof both pathogens will be identified byscreening of bacterial surface displaylibraries with human sera from exposedindividuals and complementary pro-teomic approaches. Selected antigenswill be evaluated by testing in animalmodels, characterisation of their role inpathogenesis with special emphasis onbiofilm formation and study of the natu-ral immune response. This approach willenable the project to transfer the mostpromising vaccine candidates to clinicaltrials for the prevention of OM.

The consortium comprises proven andrenowned experts of OM research, pro-teomics/mass spectrometry and vaccinedevelopment. Therefore, this multidisci-plinary approach will go beyond anyeffort that could be undertaken by a sin-gle participant by the complementarityof the technologies.

SUMMARY

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Key words: Otitis Media, vaccine development, Streptococcus pneumoniae, Haemophilus influenzae,Moraxella catarrhalis, biofilm formation

Intercell AG is an international vaccine company founded in 1998 and located in Vienna,Austria and Livingston, Scotland. Intercell’s current activities include the discovery anddevelopment of innovative immunological products and technologies for the prevention andtreatment of infectious diseases. Within the OMVac project, Intercell will lead the antigendiscovery and validation process using proprietary technologies. Moreover, Intercell isresponsible for all project management activities.

AGOWA GmbH is an internationally acting genomics company founded in 1993 and locatedin Berlin, Germany. It is recognised as a competent outsourcing partner for customers in pri-vate and public research, with the company offering a broad spectrum of services rangingfrom library construction, custom DNA sequencing service, genotyping and bioinformaticsup to the complete analysis of genomes. In the course of the OMVac project, AGOWA willsupport the antigen discovery process by high-throughput sequencing.

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Andreas MeinkeIntercell AGCampus Vienna Biocenter 61030 Wien, [email protected]

Partners

Andreas J. KunglInstitute of Pharmaceutical SciencesUniversity of GrazGraz, Austriawww.uni-graz.at

Wolfgang ZimmermannAGOWA GmbHBerlin, Germanywww.agowa.de

Éva Bán2nd Department of PediatricsSemmelweis UniversityBudapest, Hungarywww.sote.hu

John HaysErasmus MCRotterdam, The Netherlandswww.erasmusmc.nl

Peter WM HermansLaboratory of Pediatric Infectious DiseasesRadboud University Nijmegen Medical CentreNijmegen, The Netherlandswww.umcn.nl

Birgitta Henriques-NormarkKarolinska InstitutetSolna, Swedenki.se

Expected results

Moving away from current approaches, and apply-ing methods based on understanding the funda-mental principles of crystallisation, the OptiCrystproject will focus on designing techniques toactively control the crystallisation environment asthe project progresses through its stages.


Structural Genomics is a key discipline in post-genomic biology, and today the pressure to pro-duce diffraction-quality crystals that can yieldnew protein structures is greater than ever. Asa result, the science of crystallisation is becom-ing a rapidly developing field and it is gatheringnew momentum. The work being carried out bythe OptiCryst project will go a significantly longway towards addressing the outstanding needswithin that research area.

Background

The wealth of information obtained by StructuralGenomics has allowed protein structure-baseddrug design to complement screening and combi-natorial chemistry to provide more efficient drugdevelopment. Ultimately, this approach will reducethe time of production cycles and therefore costper drug.

Structural Genomics has coincided with the era ofhigh-throughput, resulting in major advances inthe automation of protein preparation and X-raycrystallographic analysis, and in automating andminiaturising crystallisation trials (thousands perday). Despite this, the success rate in going fromcloned gene to high-resolution protein structureis still relatively low in all current StructuralGenomics projects, with a major bottleneck situa-tion from purified protein to diffracting crystals.This problem clearly needs to be addressed. Thiscan be done through the production of a designthat will offer new and improved optimisationmethods, in order to turn crystal leads into usefuldiffracting crystals.

Aim

The key objective of the OptiCryst project is toaddress the critical post-protein production bot-tleneck area in the field of Structural Genomics.To date, this area has been consistently ignoredby initiatives worldwide. The project proposes toenhance the state-of-the-art in protein crystaloptimisation by increasing the success rate of theproduction of diffraction-quality crystals from thecurrent rate of 21 % to at least 40 %.

Optimisation of protein crystallisation for european structural genomics

ACRONYM

OptiCrystwww.opticryst.org

The OptiCryst project aims to providea support platform for Europe’s StructuralGenomics initiatives. The project aimstohelp such initiatives take a leap forwardscientifically and commercially, in orderto obtain high-quality crystals of proteinswhich are currently proving difficult tocrystallise.

SUMMARY

128



Key words: structural genomics, SME, protein crystallisation, phase diagrams, nucleation, robotics, high throughput, structural genomics

The OptiCryst consortium is composed of seven SMEs and four academic groups. The role ofthe SMEs in OptiCryst is to develop new equipment and tools to aid crystallisation. This willbe achieved by designing new and improved apparatus, based on the joint research results ofthe academic partners and the SMEs. These results will be made available to the communityby commercialising the apparatus and turning new techniques into crystallisation kits.

ROLE OF SMEs

129


Roslyn BillAston University Dept. of Life and Health Sciences Aston TriangleBirmingham B4 7ETUnited Kingdom [email protected]

Project manager

Eric BourguignonAston UniversityDept. of Life and Health SciencesBirmingham, United [email protected]

Partners

Naomi Chayen Imperial CollegeBiological Structure and Function Biomedical Sciences London, United Kingdom

Patrick Shaw Stewart Douglas Instruments Ltd.Hungerford, United Kingdom

Flip Hoedemaeker Key Drug Prototyping B.V.Amsterdam, The Netherlands

Anthony Savill Molecular Dimensions Ltd. Newmarket, United Kingdom

Lea Vaiana Triana Science & Technology S.L.Armilla, Granada, Spain

Rolf HilgenfeldInstitute of Biochemistry University of LübeckLübeck, Germany

Marcus SwannFairfield Scientific LimitedCrewe, United Kingdom

Juan Manuel Garcia-RuizConsejo Superior De Investigaciones Cientificas (Csic)Laboratorio de Estudios CristalograficosPT Ciencias de la Salud Armilla, Granada, Spain

Christian BetzelPLS Design GmbHHamburg, Germany

Lena GustafsonGothia Yeast Solutions ABGothenburg, Sweden

| OptiCryst Kick-Off meeting at the DESY facility in Hamburg (March 2007).

European pharmaceutical companies, in order todevelop new drugs for the treatment of both drugcraving and relapse, core features of the compul-sive components of addictive disorders, or for pro-viding new drugs for treating compulsive eatingleading to obesity and metabolic imbalance.

Expected results

The neurobiological bases of compulsive behav-iours share common mechanisms, as drug-seekingor compulsive food intake promote activation andneuro-adaptations within the common neuronalnetworks. The general strategy of the project is,first, to carry out a complete phenotypical charac-terisation of four animal models, addressing dif-ferent components of compulsive disorders thatare already being studied in partner laboratories,namely: • the modified conflict model (Dr. Piazza’s group at

Bordeaux); • the deprivation model (Dr. Spanagel’s group at

Mannheim); • the reinstatement model (Dr. Maldonado’s group

at Barcelona); and • the compulsive food-seeking/-taking model (Dr.

Heyne’s group at Reutlingen).

Those models will be transferred from rat to mousewhen needed (involving the former groups as wellas that of Dr. Dierssen at Barcelona). The proposalwill investigate selected genes and proteins(Dr. Przewlocka’s group at Krakow) which couldplay a role in compulsive-derived brain plasticityand its pathophysiological response. Moreover,the availability of specific feeding patterns eval-uation tools (Dr. Célérier’s group at Cornellà) andthe set-up of potent neuroimaging technologiesadapted to small experimental animals (Dr. Millán’sgroup at Barcelona) will allow correlating anatomic-functional events with behavioural and genetic par-adigms. Finally, a mechanistically targeted-orientedarray of genetically modified mice (Dr. Tronche’sgroup in Paris) will be used to specifically addressthe elucidation at molecular level of the neurobio-logical basis of compulsive disorders, i.e. the role ofglucocorticoid receptors.

Background

Both drug addiction and food intake disorders con-stitute increasingly serious health care and socialproblems in the EU and are also responsible for theloss of millions of working hours every year.However, there is still a lack of suitable specific ani-mal models to further elucidate the neurobiologi-cal substrate of such disorders. In this context,PHECOMP offers an impact in new animal modelsfor probing these and other related psychiatric dis-orders and compulsive behaviours.

The present proposal is a multidisciplinary projectbringing together new and sophisticated behav-ioural methodologies as well as cutting-edgemolecular and imaging techniques which will beapplied to the phenotypical characterisation of tar-geted and refined animal models. The characteri-sation of new animal models and their transfer tomice when needed will allow the use of theserodent behavioural models for the study of thecompulsive components in addiction and relatedpsychiatric disorders.

Aim

There are three main aims of the specific-targetedresearch project:• deliver four phenotypically well-characterised

animal models, namely the modified conflict, thedeprivation and the reinstatement models ofcompulsive drug intake, and the compulsivefood-seeking/-taking model, addressing differ-ent components of compulsion using rats andmice;

• elucidate the role of selected gene activities andprotein receptors in the neurobiological mecha-nisms involved in compulsive disorders;

• provide complete new structural and functionalillustration of the reward pathways imbalancefound in compulsion, using cutting-edge imagingtechniques (PET).

The ultimate objective of this STREP Project is to provide new knowledge that can be used by the participants, by other research groups or by

Phenotypical characterisation of animal models for neuropsychiatric disorders relatedto compulsive behaviour

ACRONYM

PHECOMP

Compulsive disorders, including drugabuse and compulsive overeating, rep-resent prevalent neuropsychiatric dis-eases that have a large health andsocio-economic impact in the Europeanpopulation. These disorders are pro-duced by an alteration of the capabilityto control seeking for reward, and seemlinked by common neurobiological sub-strates. However, there is an importantgap in the availability of reliable behav-ioural models in animals that permit toinvestigate compulsion towards rewardin the perspective of human pathologies.

The present proposal will use newsophisticated behavioural and neu-roimaging techniques for the characteri-sation of four new and complementaryanimal models of compulsive disorders,allowing to analyse precisely the maincomponents of those behavioural alter-ations. The study will be performed inmice and rats, including the transfer of ratmodels to mice when necessary. Thebehavioural and molecular characterisa-tion of the models, along with parallelneuroimaging (PET), will provide a com-plete anatomical and functional illustra-tion of the reward pathways imbalance inthe above-mentioned pathological situa-tions. Novel behavioural paradigms willbe proposed, tested and validated withinthe project, taking advantage of cutting-edge imaging technologies. Molecularstudies will characterise changes inducedin several key elements of the reward cir-cuits during these behavioural disorders.

After the full characterisation of the mod-els, they will be used on genetically mod-ified mice for glucocorticoid receptors toascertain correlations between behav-ioural and genetic components of com-pulsion in drug addiction and eatingdisorders. Hence, reliable and predictiveanimal models will be fully characterisedand employed to better understandthe mechanisms involved in those alter-ations, and to design new therapeuticstrategies in neuropsychiatric disordersrelated to compulsive behaviour.

SUMMARY

130



Key words: compulsive disorders, animal models, behavioural paradigms, Positron Emission Tomography (PET), drug abuse, eating disorders, glucocorticoid receptors


The optimisation of targeted animal models willrender them suitable for finding new treatmentapproaches for these compulsive disorders andready-to-use in pre-clinical research to testputative anti-obesity and anti-addictive com-pounds. It is recognised that both opioid,dopamine and cannabinoid systems play keyroles in regulating not only addiction but alsoother aspects of limbic and motor function. Thepotential for the use of these animals to investi-gate other CNS diseases as well as to developand test new pharmacotherapies is accordinglyvery high.

Moreover, PHECOMP offers three SMEs, leadersin their fields, to take great advantage of partici-

pating in a project at the European level, thusenabling the possible outputs of the project tofinally benefit the same (and other) SMEs, thusincreasing their know-how and commercial com-petitiveness in and outside Europe. In particular,at the end of the project, medimod (DE) will havea fully-operative food-seeking/food-taking modelin rat ready for the preclinical search for new phar-maco-therapeutic solutions in food intake disor-ders. Also, IAT (ES) will develop and validate newPET built-in methodologies for neuroimaginganalysis of mice, correlated with behavioural andmolecular data. Finally, Panlab (ES) will set-upand validate specific devices that will improve thequality and efficiency of animal feeding patternsevaluation tools.

Three SMEs, leaders in their fields, are crucial partners in PHECOMP and share 42 % of thebudget. The German company medimod will develop a set of novel tools for preclinical test-ing of compounds with putative anti-obesity efficacy. The Spanish manufacturer Panlabwill contribute with an array of technical and software solutions in the precise and accurateanalysis of the pattern of eating and drinking behaviours in rodents. Finally, the SpanishInstitute of Advanced Technologies (IAT) will apply current expertise and know-how toinnovation through adaptation of functional PET neuroimaging of small experimentalanimals to set up new techniques of imaging in mice. Their participation will enable theconsortium to carry out a complete phenotypical characterisation of selected animalmodels of compulsive disorders and their transfer from rat to mice when needed, as wellas a fruitful transfer of knowledge to the market.

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Rafael MaldonadoLaboratory of Neuropharmacology-NeuroPharUniversitat Pompeu FabraBarcelona Biomedical Research Park c/Dr Aiguader 808003 Barcelona, [email protected]

Partners

Mara DierssenCentre for Genomic RegulationBarcelona Biomedical Research ParkBarcelona, Spain

Barbara PrzewlockaInstitute of PharmacologyPolish Academy of science Krakow, Poland

Rainer SpanagelCentral Institute for Mental HealthMannheim, Germany

Pier Vincenzo PiazzaINSERM u588Paris, France

Andrea Heynemedimod pharmacology services GmbH Reutlingen, Germanywww.medimod.com/welcome.html

Olga MillánInstitut d’Alta TecnologiaBarcelona, Spainwww.iat-prbb.com/ingles/index.html

François TroncheCNRS (UMR 7148)Paris, France

Evelyne CélérierPanlab SLUBarcelona, Spainwww.panlab.com

| Alcohol sensitization and reward areinfluenced by circadian genes andrhythm (Abarca et al., Proc Natl AcadSci USA 99, 9026-30, 2002).

Aim

To develop new photochemical probes and opti-cal methods in localising and extending the rangeof photolysis for use in neuroscience; to adaptphotolysis to current parallel patch clamp methodsin the development of new medicines.

Expected results

Improved photochemical properties of probes,particularly making better use of the two- photon effect for localisation and extending therange of synaptic and local messenger systemsto which the method can be applied. Improvedmethods in drug discovery of ligands active atbrain synapses. Specific therapeutic targetingof genetically linked channelopathies and appli-cations in developing somatic cell replacementtherapies.


To investigate neurotransmitter receptors in synaptic transmission in situ. To investigate mediatorsin neurovascular regulation. To improve methodsof deep imaging in vivo. To develop better drugsacting at synaptic receptors in the brain.

Background

Experimentally it is difficult to separate the con-tributions of presynaptic and postsynaptic mech-anisms to the efficiency and strength of asynapticconnection. The method of flash photolysis canprovide a way of determining the properties ofsynapses between neurones in situ that are cur-rently studied only in recombinant receptors invitro, and would allow the testing of putativemediators of local vasoregulation in the brain.These applications of photolysis in neuroscienceare impeded by the poor photochemical proper-ties and stability of caged neurotransmitters, bythe unavailability of many mediators in photo -labile caged form, and by the unsuitability ofpresent optical methods.

Currently, high throughput screening methods tofind ligands acting at brain synapses use conven-tional perfusion methods to apply the activatingneurotransmitter or analogue. These methods aretoo slow to activate receptors on a physiologicaltimescale because of diffusional access to the cellsurface, and for this reason, potential medicinesthat affect the degree of activation of synapsesand act in a use-dependent way will not be opti-mally detected. The application of this method inhigh-throughput screening to test for new medi-cines has the potential to detect use-dependentligands acting at the rapidly desensitising aminoacid receptors that mediate neuronal signalling.

Development of flash photolysis for deep uncaging in vivo and high-throughput characterisation of neurotransmitter gated ion channels in drug discovery

ACRONYM

PHOTOLYSIS

Flash photolysis is widely applied in cellphysiology to initiate neurotransmitterand other ligand-receptor interactions inconditions that are subject to poor diffu-sional access and receptor desensitisa-tion, and for ligands that are particularlylabile. It has the potential to initiate reac-tions on physiological time and spatialscales (sub-msec and sub-micron) in com-plex tissues such as brain slices and invivo, and is often combined with electro-physiology and optical imaging.

However, this potential is unrealised in neu-roscience and medicine in several areas:• photolabile ‘cages’ optimised to make

use of the localisation achievable withexcitation by the two-photon effect;

• wavefront modulation of photolysislight to make z axis location and spot sizereadily changeable;

• application in high-throughput screeningfor drug discovery of ligands acting atrapidly desensitising neurotransmitterreceptors in the brain.

The PHOTOLYSIS consortium comprisesneuro physiologists, photochemists, opticalphysicists, specialists in high-throughputpatch clamp screening and ion channel tar-geted drug discovery, to address theseareas. New photochemistry of cages com-bined with new pulsed lasers and newadaptive optics will optimise the effi-ciency, depth and location of photolysisin whole brain in vivo and in vitro.

These developments will be combinedwith deep imaging to:• identify mediators and cell types in neu-

rovascular coupling of blood perfusionto neuronal activity;

• applied to synaptic transmission to studypostsynaptic channels in situ, identifytheir role in synaptic plasticities; and

• investigate the interactions betweenmetabotropic receptors and fast trans-mitter channels;

• finally, adapt near-UV flash photolysis topatch clamp HTS technology, in order tocharacterise drugs acting at fast activat-ing and desensitising neurotransmitterreceptors, to study the functional phar-macology of genetically-linked chan-nelopathies, and in developing somaticcell replacement therapies.

SUMMARY

132



Key words: photolysis, caged neurotransmitters, synaptic transmission, synaptic plasticity, neurovascular regulation, wave front engineering, optical imaging, high throughput screening, channelopathies

The three high-tech SMEs in the Photolysis project, Flyion, GeneCraft and Tocris-Cookson,will play three important roles. One is to further develop the library of caged neurotrans-mitter ligands available to neuroscience researchers as probes in experiments to investi-gate synaptic transmission and its modification in disease. The second is to develop theapplication of photolysis in drug discovery to improve the detection of ligands that act atfast-responding neurotransmitter receptors. This will be based on existing parallel patchclamp technology modified to introduce a light path for- irradiation with a pulse of near-UVlight to initiate photorelease of the neurotransmitter. Thirdly, through their connectionswithin the community of drug discovery and ion channel therapies, the SMEs will beeffective in translating the proven power of photolysis in investigating synaptic functionto the task of identifying potential drugs.

The company Inserm Transfert takes care of the administrative project management.

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133


David Ogden CNRS UMR 8118Physiologie CérébraleUniversité René Descartes Paris 545 Rue des Saints PeresF-75006 [email protected]

Project manager

Isabelle Geahel Inserm-Transfert Paris, [email protected]

Partners

Serge CharpakInstitut National de la Santé et de la Recherche MédicalINSERM FR 101 Paris, France

Gail McConnellJohn GirkinUniversity of StrathclydeGlasgow, United Kingdom

Michael FejtFlyion GmbHTubingen, Germanywww.flyion.com

Duncan CrawfordTocris-Cookson Ltd.Bristol, United Kingdomwww.tocris.com/UK

Ladislav VyklyckyDept. of Cellular NeurophysiologyInstitute of PhysiologyPraha, Czech Republic

Yair FeldGeneGraftIsrael

Expected results

The expected results are compounds that areadvanced leads or preclinical drug candidates forthe treatment of malaria.


The potential applications of outputs of this proj-ect are compounds, which could go through topreclinical development for the treatment ofmalaria and if successful proceed to clinical trials.

Background

As part of a Framework 5 programme (QLRT-2001-00305), the project team discovered a novel classof inhibitors, which selectively inhibit dUTPaseform Plasmodium falciparum, the causative agentof malaria.

Aim

The aim is to optimise these early lead moleculesin order to generate advanced leads or preclinicaldrug candidates.

Deoxyuridine triphosphate nucleotidohydrolase as a drug target against Malaria

ACRONYM

PlasmodiumdUTPase

There are around 500 million clinicalcases of malaria each year and about 1-2 million people die from this debilitat-ing disease. There is an urgent need forthe development of new drugs becauseof drug resistance issues. New drugsshould have novel mechanisms of actionto prevent cross-resistance with existingdrugs. Some novels, drug-like and selec-tive inhibitors of the enzyme deoxyuri-dine triphosphate nucleotidohydrolase(dUTPase) from Plasmodium falciparum,the causative agent of malaria have beendiscovered. The role of this enzyme is tohydrolyse dUTP to dUMP, maintaininga low dUTP:dTTP ratio. The aim of thisproject is to optimise these early leadmolecules to generate late-stage leadsor preclinical drug candidates.

The project will include:• medicinal chemistry activities for the

preparation and optimisation of leadcompounds;

• ADME-Tox assays to ensure that thecompounds have correct ‘drug-like’properties;

• biological evaluation to determine theefficacy of the compounds;

• mode of action studies;• crystallography of inhibitors with the

enzyme active site to assist in the drugdesign process.

SUMMARY

134



Key words: Malaria, drug discovery

This project is an exciting collaboration between an SME, the Swedish Pharmaceuticalcompany Medivir AB and 4 academic institutions. The group has characterized theenzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) from the protozoanparasite Plasmodium falciparum, which causes malaria, in an approach to develop novelantimalarials. The project is a drug discovery project focusing on the optimization anddevelopment of inhibitors of this promising new malaria drug target. Medivir is able tobring its considerable experience in drug discovery to the project. Medivir is driving theproject towards a preclinical Candidate Drug status and carries a major part of the projectwork, including the synthesis of compounds and provision of a large compound libraryfor testing as potential inhibitors of dUTPase. For this strong Swedish drug developmentSME, the development of an antimalarial drug targeting dUTPase, may in addition constitutea proof of principle for dUTPase as a drug target for treatment of also other diseases.

ROLE OF SMEs

135


Ian GilbertSchool of Life SciencesUniversity of DundeeMSI/WTB/CIR ComplexDow StreetDundee DD1 5EHUnited [email protected]

Partners

Nils Gunnar JohanssonMedivir ABHuddinge, Swedenwww.medivir.se

Dolores González PacanowskaInstituto de Parasitología y Biomedicina “López-Neyra”Consejo Superior de Investigaciones CientíficasArmilla, Granada, Spainwww.ipb.csic.es

Keith WilsonUniversity of York Department of ChemistryStructure Biology LaboratoryHeslington, United Kingdomwww.york.ac.uk

Reto BrunSwiss Tropical InstituteParasite ChemotherapyBasel, Switzerlandwww.sti.ch

© Shutterstock

Aim

Objective 1: Elaborate an innovative medicaldevice: a unique point of care diagnosis platformwhich will help specialists to make earlier diagno-sis and provide more appropriate treatments.

Objective 2: Provide the best possible integrationof parameters by means of a consortium com-posed of public and private partners such asresearch intensive SMEs and academic entitiesEurope-wide.

Objective 3: Elaborate the new device for a spe-cific application: the primary diagnosis of the dif-ferent types of lung cancer. The project will thenhave a huge impact on health by contributing tothe fight against cancer and the development ofgender dimension in research.

The objective of the project is to generalise thisapproach to combinations of immuno-assaymeasurements to deliver a clear diagnosis of thedisease or monitoring information. The generali-sation means first that the diagnosis should beaccessible to various types of medical practices(medical doctor to hospitals) and thus a low cost,easy to use ‘Point of Care’ (POC) device shouldbe developed. However, this should not bedone to the detriment of quality and precision.A homogeneous technology known for high levelprecision can therefore be a judicious choice.Generalisation also means that various patholo-gies could be addressed: typically from 2 to 4 or 5immuno-assays. The project therefore aims toallow the simultaneous measurement of 4 to 5immuno-assays on the POC device with one drawof patient sample (1 droplet). Finally, generalisa-tion means universality of the measurement tech-nique and of the data reduction process. In thisway, fluorescent measurement based on FRET(Fluorescence Resonance Energy Transfer) seemsan excellent choice, since it may be extendedin the future to other diagnostics such as DNAanalysis, coagulation, microbiology etc.

Background

Each year 377 000 new European citizens developlung cancer and 340 000 die from it. Studies showthat early diagnosis and accurate cancer typingcould save a number of lives. Laboratories nowa-days have a wide panel of reproducible diagnostictests at their disposal: these are mostly routinetests realised in centralised laboratories. Theneeds for early diagnosis, multiparametric analy-sis of results and quick monitoring of diseaseprogression or therapeutic sensitivity were pro-gressively left aside, whereas they could be ful-filled with using decentralised diagnostic tools,close to the patient.

Among the possible applications of this new con-cept, the partners have chosen to work on the pri-mary diagnosis of the histological types of lungcancer to help to give an improved initial diagno-sis and to eliminate the 15-20 % problematic orlate diagnosed cases. The study will pay specialcare to women (lung cancer death rates forwomen have been still increasing in Europe sincethe 1990s and marker patterns may be different).It will then have a huge impact on health by con-tributing to the fight against cancer and the devel-opment of gender dimension in research. For thispurpose, the multidisciplinary project will involveacademic researchers (from Germany, Spain andFrance) and SMEs (from Sweden and the UK) gath-ering around the initiators of the project (the SMECEZANNE, the University of Strasbourg in Franceand the University of Potsdam in Germany) whichhave the skills to build a multiparametric device,to develop immunoassays and to design an inter-pretation software.

Multiparametric quantum dot bioassay for point of care diagnosis

ACRONYM

POC4lifewww.poc4life.eu

This project aims to improve the health-care of patients by elaborating a uniquePOC diagnosis platform which will helpspecialists to deliver an earlier diagnosisand to decide on appropriate treatment.The goal is to provide the clinicians withmultiparametric measurement of themain 4/5 essential markers and to sup-port decision with a software tool. Thiswill be a cost-effective breakthrough inthe diagnosis market.

SUMMARY

136



Key words: lung cancer, point of care, quantum dots, uv light source, flash lamp, fluorescence, immuno-diagnosis, multiparameter, blood separation, expert system, molecule coupling, luminescent tags

Expected results

The project should attain the following challeng-ing objectives:• develop a functional prototype of POC multi-

parametric measurement for immuno assays,based on Homogeneous Time Resolved Fluores -cence (HTRF), for which main characteristics are:equivalent to an A4 sheet of paper, cost of lessthan EUR 2 000, works with a sample dropletdeposited on a disposable reagent vessel con-taining dried reagents;

• define the panel of assays and how to combinethem into a decision making software (to bedeveloped).


Multiparametric diagnostics in the followingfields:• cancer; • prenatal diagnostics; • sepsis; • cardiac.

The SMEs will bring to the consortium the complementary and comprehensive expertiserequired by the other public researchers.

CEZANNE S.A. brings competence in two complementary scientific and technical fields:immuno-assay development and scientific instrumentation, which over the years CEZAmanaged efficiently to cross-fertilise.

FUJIREBIO DIAGNOSTICS brings competence related to antibody development and charac terisation (hybridoma technology, phage display, gene expression analysis, c-DNAimmunisation, DNA-shuffling, affinity maturation) and competence in assay development.

EDINBURGH INSTRUMENTS brings experience in flash lamps, laser diodes, fluorescencespectrometry and a prototype nanosecond plate reader.

ROLE OF SMEs

137


Emmanuel BoisCezanne SAS280 allée Graham BellParc Scientifique Georges Besse30035 Nîmes cedex [email protected]

Partners

Olle Nilsson Fujirebio DiagnosticsGöteborg, Sweden

Desmond Smith Edinburgh Instruments2 LivingstonScotland, United Kingdom

Philippe Pieri Centre National de la Recherche ScientifiqueParis, France

Hans-Gerd LöhmannsröbenUniversity Of PotsdamInstitut für Chemie und Interdisziplinäres Zentrum für PhotonikPotsdam – Golm, Germany

Yves Caristan Commissariat à l’Énergie AtomiqueGrenoble, France

Klaus-Dieter WeltmannEckhard KindelInstitute Of Low Temperature Plasma PhysicsGreifswald, Germany

Rafael MolinaHospital Clinic BarcelonaLaboratory of Biochemistry Hospital Clinic CBarcelona, Spain

Petra StieberInstitute For Clinical ChemistryUniversity Hospital Munich-GrosshadernMunich, Germany

© Shutterstock

countries in sub-Saharan Africa do not have theeconomic means to purchase expensive vaccines.The manufacture of rBCG follows the exact sameprotocols and procedures as established for BCGand guarantees that sufficient doses of rBCGmalaria vaccine can be manufactured at a priceper dose similar to BCG. Consortium memberCrucell has extensive expertise in adenoviral vaccine manufacturing, employing a mammalian cell line, coded PER.C6®, which can be cultured in suspension in large volumes. Therefore, thePRIMOBAL consortium members consider the pro-posed two-component paediatric malaria vaccineto be affordable, thus applicable in developingcountries such as sub-Saharan Africa.

AimTo demonstrate feasibility (safety and efficacy) inpreclinical studies of a novel, affordable, two-component, paediatric malaria vaccine.

Expected results

The ultimate deliverable of this programme is anefficacious paediatric malaria vaccine candidatethat will eventually be advanced to GMP develop-ment and clinical trials. Besides reaching this aim,the consortium expects that during the executionof this programme extensive knowledge will begathered regarding immunological features ofdifferent vaccination schedules, in combinationwith information on their protective ability. Thisinformation will help elucidate correlates of pro-tection and facilitate rational design of futuremalaria vaccines.


It can be envisioned that vaccines against otherdisease such as for instance HIV, TB or even can-cer vaccine development can directly benefitfrom the PRIBOMAL research. Like malaria thesediseases are considered to require vaccinesthat elicit strong humoral and cellular immuneresponses against a broad range of epitopes.

Background

It is estimated that more than 300 million individ-uals suffer from acute disease caused by themalaria parasite and that more than 1 million peo-ple succumb to this disease each year. Malaria,which is highly endemic in sub-Saharan Africa,claims its victims predominantly among childrenwith the peak incidence of clinical malaria and90 % of malaria deaths in children younger than5 years.

The challenge faced by the PRIBOMAL consortiumis to design a safe, affordable paediatric malariavaccine that provides long lasting protectionagainst malaria and that fits within the existingWHO Expanded Programme on Immunisation (EPI)so as not to further complicate operational vacci-nation logistics. The first vaccination that childrenwould receive is at birth with Bacille Calmette-Guérin (BCG) a live and attenuated strain ofMycobacterium bovis, which is currently the onlyavailable vaccine against tuberculosis. BCG hasbeen globally used as the TB vaccine for decadesand has proven to be safe in hundreds of millionsof children. In recent years BCG receives additionalattention as a potential vaccine vehicle.

The PRIBOMAL consortium considers that abooster immunisation will be required to achievelong-lasting protection. Therefore, a choice wasmade to boost the rBCG.malaria vaccine, given atbirth either alone or in addition to classical BCG,with a recombinant adenoviral vector, carryingthe identical P. falciparum derived antigens, at14 weeks after birth, thus compliant with WHO EPIschedule. Like rBCG, replication deficient aden-oviral vector has an excellent safety record withtens of thousands of patients receiving recombi-nant adenoviral vectors in diverse gene therapyand vaccination trials without adverse effects.

To ultimately eradicate malaria disease hundredsof millions of vaccine dosages will need to bemanufactured at low cost per dose, given that

Preclinical studies towards an affordable, safe and efficacious two-component paediatricMalaria vaccine

ACRONYM

PRIBOMAL

Malaria is one of the major public healthchallenges in the world, causing morethan one million deaths each year. Thedisease primarily affects children of thedeveloping world. The available meas-ures, such as personal protection ordrugs, have proven to be insufficient tocontrol the disease. A safe, affordableand efficacious paediatric malaria vac-cine, which fits in the existing WHOExpanded Programme on Immunisation,would alleviate tremendous suffering inhuman kind.

Taking up this challenge, the PRIBOMALconsortium proposes to generate and testin preclinical models the safety and effi-cacy of an innovative malaria vaccine. Thevaccine consists of a prime, to be adminis-tered at birth, of a novel recombinant BCGvector carrying preferentially multipleantigens derived from the Plasmodiumfalciparum parasite, the cause of malaria.The priming vaccine is followed at week 14after birth by a booster vaccination usingindustrially developed, recombinant adenoviral vector carrying the identicalPlasmodium falciparum antigens as therBCG-based malaria vaccine.

Generation of these novel vaccine candi-dates, as well as testing in establishedand novel pre-clinical models to deter-mine potency and safety, requires a com-bined European effort to bring togetherthe required expertise on basic parasitebiology, malaria epidemiology, diseaseonset and progression, recombinant vec-tor technology, fundamental immunol-ogy, advanced animal models, andsophisticated proteomics. The interna-tional PRIBOMAL consor tium bundles allrequired experience and as such is wellpositioned to successfully conduct thisresearch programme.

SUMMARY

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Key words: Malaria, rBCG, rAd35, paediatric vaccine

This project forms part of a wider effort by the coordinating Dutch biotech SME Crucell todevelop a malaria vaccine based on several pre-erythrocytic antigens presented bya BCG/adenovirus-based prime-boost approach. This FP6-funded project contributes tothe preclinical testing of this approach in three different animal models, which involvesa number of European partner groups and thus requires support at European level.Crucell’s role in the project is very strong, with a large work-share in the generation of thecandidate product but also in all other work packages, underpinning the importance ofthe project for this SME-based European vaccine development project.

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Jaap GoudsmitCrucell Holland B.V.Archimedesweg 4PO Box 20482301 CA Leiden, The [email protected]

Partners

Stefan H.E. KaufmannMax Planck Institute for Infection BiologyBerlin, Germanywww.mpiib-berlin.mpg.de

Leif LindforsStockholm UniversityDepartment of ImmunologyStockholm, Swedenwww.wgi.su.se

Tom Van der PollAcademic Medical CentreUniversity of AmsterdamLaboratory of Experimental Internal MedicineAmsterdam, The Netherlandswww.amc.uva.nl

Ronald BontropBiomedical Primate Research CentreRijswijk, The Netherlandswww.bprc.nl

© Shutterstock

Expected results

The expected goals of this project are the devel-opment and dissemination of biophysical toolsand structural models for protein membraneinteractions that can be used by other researchersin an industrial or academic environment. Thespecific results include: • tools for studying protein phospholipid inter-

actions; • NMR techniques using tags and DNP; • efficient preparation of membrane proteins

and intracellular probes; • protocols for the preparation of membrane

models and lipid systems; and • new insights into protein-lipid interactions and

dynamics.


The proposed research will be focussed ona selection of model proteins and membranetypes to achieve realistic goals. However, theresults of this project will be of wider importancedue to the provision of basic answers which canlater be applied to other members of the sameclass of proteins, and general physico-chemicalprinciples can be extrapolated to other lipid inter-acting proteins. Broad applicability is guaranteedby the fact that approximately a third of the pro-teome and half of the known drug targets aremembrane-associated proteins. The tools andresults will be used by researchers interested inmembrane-associated proteins, lipidomics, func-tional and structural genomics, biophysical tech-niques including NMR spectroscopy and DNPapplications, and groups interested in membranebiology and signalling mechanisms.

Background

Lipids and the proteins that bind them are of sig-nificant societal value and medical importancedue to their central roles in health and disease,and their relevance to the discovery of novel ther-apeutic agents. Phospholipids such as phospho-inositides and sphingolipids have crucial rolesboth in cancer development and progression.They act as bioactive lipid mediators, affectingfundamental cellular functions which include pro-liferation, differentiation, survival, migration,adhesion, invasion, and morphogenesis. Thesefunctions influence many biological processesincluding neurogenesis, angiogenesis, woundhealing, immunity, and carcinogenesis. While ini-tial discoveries on lipid binding domains havespurred interest among structural biologists, littleis known about the precise control or specificity ofmembrane insertion and recognition. This projectwill therefore have effects on public health by aid-ing in the design of new types of agents that bindto membrane interaction sites and provides newavenues to modulate target protein activities.

Aim

Prism project main objectives are:• test and validate model micelle systems to

mimic properties of cellular membranes forresearch of membrane-associated proteins;

• develop molecular screening methods in orderto define lipid/micelle specificity profiles ofprotein modules;

• apply dynamic nuclear polarization methodsand paramagnetic spin label studies for themeasurement of lipids and protein interactions;

• design, express and screen novel tags andlipid binding domains involved in membranetrafficking and signalling;

• test and evaluate new computer modellingapproaches for membrane:protein complexes.

Phospholipid and glycolipid recognition, interactions and structures by magnetic resonance

ACRONYM

PRISM

In this specific targeted research project,cutting-edge nuclear magnetic resonance(NMR) techniques are used to developa sensitive, rapid and integrated approachto understanding the mechanisms of lipidbinding proteins. The project will gener-alise and accelerate the efforts to charac-terise a range of peripheral membraneproteins, thus providing new insight intoan important class of proteins which playa key role in regulating complex cellularpathways. Model membranes will betested to mimic various types of cellularmembranes for selected protein mod-ules which will be prepared by specificadvanced tailored protein expressionmethods. The generation of lipid librarieswill allow definition of membrane speci-ficity, and spin labels and micelle-formingcompounds will facilitate the meas-urement of the angles and depths ofbilayer insertion. These advances in NMRmethodology will be complemented bynew computer modelling approaches formicelle:protein complexes. Predictiveprinciples of membrane insertion will beused to model membrane orientations ofstructures of proteins. To demonstratethe impact for endocytosis, exocytosisand signal transduction, the protocolsand products will be developed andtested using the signalling domains andmodules involved in the trafficking ofmembranes and modification of lipids.

SUMMARY

140



Key words: membrane protein, signal transduction, phospholipid, glycolipid, NMR spectroscopy, dynamic nuclear polarization,structural biology

The PRISM project includes two SME partners. Oxford Instruments Molecular Biotools Ltd. (OIMBL) offers advanced scientific instrumentation and expert knowledge for biomolecularanalysis. HyperSense™ is OIMBL’s groundbreaking dynamic nuclear polarisation (DNP)polarizer which is capable of amplifying the NMR signal to noise ratio by a factor of up to10 000. This instrument is the first commercial DNP system, and has been installed atHWB•NMR. This project provides PRISM with access to DNP-NMR technology for the studyof proteins and lipids.

ProtaMAX Ltd. offers bespoke mutagenesis, custom randomisation of protein sequences,and efficient saturation mutagenesis to produce DNA libraries and designer proteins includingbiopharmaceuticals. ProtaMAX Platform Technology allows encoding of selected or fullyrandomised amino acids, for example, within binding site residues of a protein structure.The technology was invented and patented at the University of Aston, UK, and is availableto PRISM researchers to obtain and screen for novel protein probes and tags.

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Michael OverduinUniversity of BirminghamEdgbaston, Birmingham, B15 2TTUnited [email protected]

Partners

Harald SchwalbeJohann Wolfgang Goethe-UniversityBMRZ-Center for Biomolecular Magnetic ResonanceFrankfurt, Germany

Kai SimonsMax Planck Institute of Molecular Cell Biology and GeneticsDresden, Germany

Jean GruenbergUniversité de Genève Faculté des SciencesDépartement de BiochimieGeneva, Switzerland

Gerrit van MeerUtrecht UniversityUtrecht, The Netherlands

Andrew SowerbyOxford Instruments Molecular Biotools Ltd. Oxfordshire, United Kingdomwww.oxford-instruments.co.uk/wps/wcm/connect/Oxford+Instruments/Internet/Home

John SlackProtaMAX Ltd.Birmingham, United Kingdomwww.protamax.com

Expected results

• Provide new therapeutic tools able to promoterecovery from acute renal failure (ARF).

• Gain insight into principles of the regeneration.

• Provide the conceptual basis for the develop-ment of new therapeutic strategies.


The resulting gene function and new cell therapyinformation may provide the PROLIGEN consor-tium the necessary tools to deliver new biologicalsand cell-based therapies for kidney regeneration.

Potential applications of the generated know -ledge to improve the regeneration in other tissuesavoiding the use of stem cells may also result.

Background

PROLIGEN differs from previous approaches inthat it splits the regeneration process into thedifferent main biological process (apoptosis, for-mation of pro- versus anti-inflammatory media-tors/macrophage phenotype and cell proliferation),and aims to study regeneration as the result of theirinterdependence. The project takes into accountthat macrophages are at the centre of a complexregulatory network receiving and distributingsignals from and to all biological process, thusaffecting recovery. At variance with previous stud-ies particularly focus is not only on the identifica-tion of markers relevant for regeneration ratherthan the use of functional genomic technologywhich provide a new network of gene function andnew platforms to deliver a new cellular basedtherapy strategy.

Aim

PROLIGEN aims to enhance the endogenousregenerative capacity of injured kidneys based oninformation derived from genomics/proteomicsand functional genomics.

Hypoxic renal proliferation

ACRONYM

PROLIGENproligen.eu

PROLIGEN aims to enhance the endoge-nous regenerative capacity of injuredkidneys based on information derivedfrom genomics/proteomics and functionalgenomics. The approach of PROLIGEN is: • to define and identify set of genes/pro-

teins associated with functional recov-ery from renal injury;

• to build up high throughput test sys-tems to follow the basic biologicalprocess involved in regeneration and touse these read out systems for func-tional genomics; and

• to develop biologicals and cell basedtherapy to foster proliferation.

Currently, studies are focused on evalu-ating single factors as growth factors orstem cell therapy. The project proposes toenhance regeneration by knowing howthe genes/proteins that determine dif-ferent steps in kidney regeneration arerelated, and how these settings can beinfluenced to improve regeneration. Then,the project will provide new therapeutictools able to promote recovery from acuterenal failure (ARF) based on functionalgenomic studies and cell therapy.

SUMMARY

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Key words: acute renal filure, regenerative medicine, kidney regeneration, functional genomics, genomics, proteomics, macrophage therapy

The PROLIGEN consortium brings together selected academics, clinitians and industryexperts. Three SMEs play key roles in the research activities of the project, namelyGalapagos NV provides its expertise on functional genomics, Genedata AG takes care ofbioinformatics and ProtEra s.r.l. contributes to protein synthesis.

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Georgina HotterInstituto de Investigaciones Biomédicas de BarcelonaConsejo Superior de Investigaciones Científicas (IIBB-CSIC)Rosellon 16108036 Barcelona, Spain [email protected] www.csic.es

Partners

Bernhard BrüneUniversity of Frankfurt – Medical SchoolFrankfurt, Germanywww.uni-frankfurt.de

Joan TorrasFundació Privada Institut d’InvestigacióBiomèdica de Bellvitge Lab.4122 Experimental NephrologyL’Hospitalet de LlobregatBarcelona, Spainwww.csub.scs.es

Bob. v.d. WaterLeiden/Amsterdam Center for Drug ResearchLeiden UniversityLeiden, The Netherlandswww.leiden.edu

Jochen KoenigGenedata AGBasel, Switzerlandwww.genedata.com

Richard JanssenGalapagos NVLeiden, The Netherlandswww.glpg.com

Rebecca del ConteProtERA s.r.l.Sesto FiorentinoFirenze, Italywww.protera.it

| PROLIGEN aims to enhancethe endogenous regenerative

capacity of injured kidneysbased on information derived

from genomics/proteomicsand functional genomics.


The technologies developed demand a minimumamount of material to be analysed. Withoutdoubt, the single cell is the basic entity of a livingorganism (or it is even the whole organism).Therefore, applying the methods mentionedabove to single cells is not only a technologicalchallenge but it is also the major important taskin system biology over the coming years, partic-ularly to be able to address medical diagnosticsin the regime of single cells. Single cell analysisbecomes more and more attractive because of lim-ited cell population of interest, cell heterogeneityof samples, or when cells are isolated automati-cally as early dissemination of tumour cells.

It is hoped that the project’s efforts will developbreakthrough analytic tools and systems for pre-vention, diagnosis, or monitoring of a broad rangeof diseases.

Background

Many basic questions in Biology and Medicinedemand methods for the analysis of the basic unitof life, the single cell. The partners will thusdevelop methods and instruments to analyse thegenetic content of single cells, with regard toquantitative variation of sequences (copy numbervariations), as well as qualitative variation ofsequences (single nucleotide polymorphisms).

Aim

Shortly after the end of the project, QuAGSiC willput on the market machines and consumablesthat allow gene measurements down to the singlecell level with high precision, in a parallel format(an Ampligrid can accommodate 48 cells), witha high amount of automation.

Expected results

By the end of the project the partners will be ina position to quickly market systems which allowgene analysis at the single cell level at low cost,with high speed, reliability and throughput.

Quantitative analysis of genes in single cells

ACRONYM

QuAGSIC www.uni-ulm.de/quagsic/index.php

The partners will develop methods andinstruments to analyse the copy numberof nucleic acid sequences down to the sin-gle cell/single molecule level, with thegoal of developing an early diagnosis sys-tem for a children’s disease, namely hemo-phagocytic lymphohistiocytosis (HLH).The underlying technique is amplificationbased counting (ABC), which enablesresearchers to quantify the copy numberof genetic sequences with a resolutionof about 100 base pairs in single cells.The method provides a resolution whichis of a magnitude higher than that ofFluorescence In Situ Hybridisation, andworks quantitatively with much lowersample amounts than quantitative PCR. Toprove ABC’s effectiveness for clinical appli-cations, the partners will develop a singlecell manipulation unit that picks cells froma solution and transfers them onto anintegrated PCR and hybridisation slide(AmpliGrid). The AmpliGrid contains dried-on PCR reagents, as well as hybridisationprobes to detect the presence and speci-ficity of the PCR products. The single cellson the AmpliGrid will then be processedautomatically in an integrated PCR andhybridisation machine (AmpliHyb). Clinicalsamples will be investigated, in whichcopy number deviations are pathologic asin genetic diseases. As a model system,QuAGSiC chose HLH which is hard todiagnose and fatal without specific thera-peutic measures, as well as trisomy 21,which is relevant in prenatal and postnataldiagnostics.

SUMMARY

144


Starting date: 1 September 2006

Key words: single cell analysis, hybridization, amplification based counting, AmpliGrid

The three SMEs in the project have central roles:

• MMI (Molecular Machines & Industries) is developing an automated cell picker from itssemi-automatic two dimensional cell selection tool, capillary based cell handling system,to a fully automated three dimensional cell finding and sorting system (cellector 3D).

• Adavlytix is using its photolithographically structured microscope slide AmpliGrid™ thatis suitable for performing 48 different 1μl PCR reactions on the same substrate. Thespecial AmpliGrid™ surface chemistry will be used to define a physical platform for theintegrated PCR and hybridisation at the single cell level.

• Genewave is developing an integrated system which will allow both the PCR and opticaldetection of hybridisation in an unsupervised single machine.

ROLE OF SMEs

145


Claude WeisbuchGenewave SASXTEC – Bâtiment 404École Polytechnique91128 [email protected]

Partners

Wolfgang MannAdvalytix AGMünchen, Germanywww.advalytix.de

Stefan NiehrenMolecular Machines & Industries AG (MMI)Glattbrugg, Switzerlandwww.molecular-machines.com

Andres MetspaluEstonian BiocentreTartu, Estoniawww.ebc.ee

Marion SchneiderClinical Centre, University of UlmUlm, Germanywww.uni-ulm.de

| Imaging of an Ampligrid.

This phenotyping set up (1-5) will be used todevelop a minimised and essential set of bio-markers, in order to monitor disease progressionreliably in the transgenic rat models of PD, HD,and SCA17. Phenotype data are also correlated toneuropathological features such as protein aggre-gates, neuronal cell loss, and neurotransmitteralteration at different disease stages. The pheno-typing approach will be used to characterise foreach disease model a minimised set of markersbest suited as read-outs in pre-clinical studies,applying novel compounds delaying or preventingneurodegeneration.

The objective of this project will be to provide theproof-of-principle that it is possible in the rat:• to develop, validate, and use standardised auto-

mated home cage systems, and to adapt in vivoimaging techniques for phenotyping neurologi-cal and cognitive function (1st year);

• to harvest large data sets on gene functions andcorresponding phenotypes thereof (2nd year);

• to determine and validate a minimised set ofpredictive parameters (2nd year) and experi-ments as well as of appropriate time slots foreach rat model (low-cost approach); and

• to develop and apply specific quality standardsapplicable for phenotyping tools and models(over the entire project duration);

• furthermore, these rat models will be usedto scrutinise novel experimental, pre-clinicaltreatments (year 1-3), chiefly with regard toeffectiveness, side-effects, applicability, andtransferability.

Expected results

• Validated novel automated behavioural andphysiological test systems.

• Behavioural markers derived from correlationanalysis of data from automated and classicalscreening for transgenic rats.

• Data on MRI (DTI), PET and Microarray expres-sion profiling as regards disease progression intransgenic rats.

Background/Aim

RATstream™ is an ambitious European projectthat aims to characterise and use three transgenicrat models of neurological diseases which – inhumans – present with a wide range of neurologicaland psychiatric phenotypes: • transgenic rat model of HD (1); • transgenic rat model of PD overexpressing

alpha-Synuclein with the A30P mutation; and• transgenic rat model of SCA17 with 64 expanded

CAG repeats in the TATA binding protein. Suchtransgenic rat models are unique worldwide.

The project aims to pursue a completely novelgene-to-function approach resulting in a compre-hensive phenotyping process which will comprisethe following components: • classical phenotyping; • monitoring of behavioural and physiological

performance in fully automated physiologicaland behavioural home cage test systems;

• non-invasive imaging technologies which havebeen adapted to small animals;

• neuropathology;• microarray analysis.

The project will apply this approach to all rat mod-els in order to achieve comprehensive high-qual-ity characterisation of models.Two members of the consortium (TSE, New -Behavior) aim to develop automated home cagetest systems for rat models which do not exist yet,but which are imperative in view of the upcominglarge number of transgenic rat models in both indus-try and academia. Collaboration with the academicpartners FAU and Uni Tübingen will provide an opti-mal environment for development and refinementof home cages which are validated via correlationwith data from classical read-outs and by crosscomparison between two experienced academicpartners. Cage systems will be suitable for contin-uous monitoring of spontaneous, social, cogni-tive, emotional and physiological measures(drinking, feeding, metabolic performance/calorime -try, telemetry for temperature and biopotentials) inhome-cage-like environments for rats.

European project on the characterisation of transgenic rat models for neurodegenerativeand psychiatric diseases: Automated home cageanalyses, live imaging and treatment

ACRONYM

RATstream™www.ratstream.eu

The RATstream Consortium will concen-trate on the comprehensive phenotypicalcharacterisation of rat models of neurode-generative diseases such as Huntington’sdisease (HD), Parkinson’s disease (PD) andspinocerebellar ataxia type 17 (SCA17).Ultimately, the project will deliver a proce-dure for low-cost automated drug screen-ing along with a set of data describing thephenotype for each of the models.

To achieve this goal, automated home cagesystems for behavioural and physiologicalphenotyping will be developed by twoSMEs and validated independently by twoacademic partners, and individual data willbe incorporated into an integrated data-base developed by a third SME. In a jointeffort, the groups will develop a compre-hensive set of behavioural and physiologi-cal phenotyping procedures, including PETand DTI technologies, in order to detectsystematically neuropsychiatric correlatesof neuronal dysfunction and disease pro-gression in rat models of HD, PD, andSCA17. The resulting set of biomarkers willlead to a valid set of minimised experi-ments and markers best suited to provideread-out parameters in pre-clinical studiesapplying novel substances delaying orpreventing neurodegeneration.

SUMMARY

146



Key words: brain research, neurodegeneration, transgenic animal models, comprehensive phenotyping, automated behavioral phenotyping, in vivo imaging, pre-clinical treatment studies

• Full scale phenotyping data for tg rats.

• Standardised progression features of tg rats.

• Minimised parameter sets for pre-clinical trialsin transgenic rat models.

• Power calculation for pre-clinical trials.

• Pharmocokinetic and toxicity data for com-pounds to be applied in pre-clinical trials.

• Study reports on preclinical trials.

• Data base for phenotyping and treatment stud-ies of transgenic rats.


The commercial impact of the project can be esti-mated explicitly for the two SMEs engaged in devel-opment of automated home cage test systems.NewBehavior expects a growing share (10-25%) inthe market for rat behavioural equipment, esti-mated to be about €5 million per annum in Europeand € 15 million worldwide. TSE expects, in 2008,a share in Europe worth €1.5 million and, for therest of the world, worth €0.15 million. In 2009, thisis expected to increase up to €3.75 million inEurope and €1.8 million in the rest of the worldrespectively. While the market itself is not large incomparison with other fields of biotechnology, onecan expect sustainability over a long period, sinceautomated home-cage testing systems will gradu-ally replace existing rat testing equipment in everyuniversity and larger pharmaceutical company.

Big pharmaceutical companies such as BoehringerIngelheim and Novartis have already expressedinterest in the generated transgenic rat models ofPD and HD. With widespread distribution of themodels and their proven validity for therapeuticalstudies, this interest and thus the interest in thebehavioural test systems will be further increased.For instance, based on the publication of the rat HDmodel(2), more than 40 academic and pharmaceu-tical partners were interested in investigating treat-ment tools (stem cells, drugs, viruses) with thehelp of this model. With the publication of the PDand SCA17 models, an even greater interest is fore-seeable (for PD because of the limitations of themouse models and because of the impact in drugdevelopment for pharmaceutical companies).

For Trophos, the commercial gain consists ofa potential reduction in the risk to further clinicaldevelopment of its drug candidate for the indica-tion targeted in this STREP, in case the models arevalidated and found predictive of the human dis-ease. This can lead to significant advantage toother commercial competitors, once outcomescan be approved in clinical trials in humans, andcan point to significant potential earnings.

CrossLinks’ product, namely an integrated data-base and software tool for complex phenotypingof transgenic animals, will bridge a gap in the mar-ket. To our knowledge, such a tool does not exist.

References

(1) von Hörsten et al. 2003, Hum Mol Genet 12:617-624.

(2) Idem.

Four SMEs, sharing 30% of the project’s research budget, play key roles in the project. TSESystems, based in Bad Homburg, Germany and New Behavior, based in Zurich, Switzerland,develop cage systems for automated screening for behavioural, cognitive and physiologicalmalfunctions of transgenic rat models. The aim is to identify minimal parameter sets as regardsnumber of treated animals, study time and number of parameters to be applied in preclinicaltreatment studies. Those systems will be validated against classical methods of screening byacademic partners of the consortium. Trophos S.A., a French biopharmaceutical company, spe-cialising in the discovery and development of drugs for neurodegenerative diseases optimisestwo promising drug candidates for preclinical studies and develops respective analyticalmethods. CrossLinks, a spin-off company of the Department of Bioinformatics of theErasmus University Medical Center in Rotterdam, develops, adjusts, implements and hostsa data collection, storage and analysis system to support high-throughput (HTP) data col-lected from automated cages, genomics data and imaging data. One of the crucial aims is thecalculation of the minimised sets of parameters and markers best suited for treatment studies.

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Olaf Riess

Project manager

Holm GraessnerEberhard-Karls-Universität TübingenDepartment of Medical GeneticsCalwerstr. 772076 Tübingen, [email protected]/klinische_genetik

Partners

Stephan von HoerstenFriedrich-Alexander-Universität ErlangenGermanywww.fpz.uni-erlangen.de/Exp_Biomedizin.htm

Hans-Peter LippNewBehavior AGSwitzerland www.newbehavior.com

Silvia BrendaTSE Systems GmbHGermanywww.newbehavior.com

Bertrand TavitianCommissariat à l’Énergie AtomiqueFrancewww-dsv.cea.fr/en/instituts/institut-d-imagerie-biomedicale-i2bm/unites-de-recherche/service-hospitalier-frederic-joliot-shfj-a-syrota/laboratoire-d-imagerie-moleculaire-experimentale-lime

Rebecca PrussTrophos S.A.France www.trophos.com

Ronald NaningaCrossLinks B.V.The Netherlandswww.crosslinks-it.com

Annemie van der LindenUniversity of AntwerpBelgium webh01.ua.ac.be/biomag

response to the viruses, a defined and uniformpanel of relevant clinical data related to respira-tory infections will be collected. Internationallystandardised prospective data mining and a newmulti-lingual software tool will be developedwithin the project and used by the partners.

Expected results

The project expects to gain detailed knowledgeon the innate and acquired immune responseto emerging respiratory viruses in the elderly.A thorough basic investigation of emerging respi-ratory viruses will provide further handles todevelop antiviral strategies based on siRNA andexternal guide sequences (EGSs). Furthermore,known antivirals will be evaluated with the finalgoal to support the elderly’s immune responsewhilst reducing mortality in the elderly caused byrespiratory infections.


New diagnostic assays; new antiviral therapies;optimised treatment and vaccination strategies;new software tool for surveillance and clinicaldata mining.

Background

During the last few years some new respiratoryviruses (HMPV/hCoV-NL63/SARS/Bocavirus/fluH5N1) have emerged. In addition to other viruses,such as RSV, EBV, and Paramyxoviruses, they areable to induce severe respiratory diseases inhigh-risk patients, in particular young childrenand elderly.

Aim

The first objective of the project will be to studythe innate and acquired immune response of theelderly against the new and known respiratoryviruses, both clinical and basic research will beaddressed, in addition, a new diagnostic assays toevaluate the immune status of the elderly will bedeveloped. Within the project, an animal model forthe investigation of the elderly’s immune responsewill be established. In this model, antiviral agentswill be tested for their ability to support thepatient’s immune response. Up to 40 000 elderlysera collected during the last 9 years will be testedfor antibodies to emerging respiratory viruses(HCoV-NL63/HMPV/RSV/EBV/fluH5N1), provid-ing a wide view of the epidemiology of theseviruses. For a precise view on the elderly’s immune

Immune response to viral respiratory infections and vaccination in the elderly

ACRONYM

RespVirusesmedical-surveillance.com/index.html

The RespViruses project will study theinnate and acquired immune response ofthe elderly against the new and knownrespiratory viruses. Clinical and basicresearch aspects will be addressedequally, and new diagnostic assays toevaluate the immune status of the elderlywill be developed.

SUMMARY

148



Key words: new respiratory viruses, immune response of the elderly, mouse models, diagnosis, siRNA, EGS

Three European SMEs are partners in RespViruses, and are based in Belgium, Germany andSpain. These SMEs will receive approximately 50 % of the project budget. Having as partnerINGENASA skilled in enzyme immunoassays, monoclonal antibodies production, nucleicacid cloning and recombinant protein expression will help to develop new prototypes fordiagnostic assay development in respiratory diseases. INGENASA is a SME Biotechnologycompany dedicated to the research, development, production and commercialisation ofproducts for diagnostic sector with 25 years’ history in this field, mainly for viruses thataffect livestock animals or pets. INGENASA is also active in areas of prevention of animaldiseases (vaccines) and has participated in several funded European projects (BRIDGE,BIOTECH, FAIR, FP5 and FP6 programmes).

The Belgian SME, RNA-TEC, with its profound knowledge and expertise in oligonucleotidechemistry plays a crucial role in the project by designing and synthesizing suitably stabi-lized siRNAs and external guide sequences that target highly conserved RNA sequencesof the respiratory viruses that are the subject of the study. The partners will test thesecompounds in vitro and also in suitable animal models. A long term goal of the project isto identify potent lead compounds that can be further exploited as potential antiviraltherapeutics using appropriate delivery systems that we can access.

As software developer and consulter in IT departments, SME Mattes Hamann will cover twomean parts during the project. One will be the internal communication and the presentationof the project information. In the other part he is responsible for collecting and evaluatingmedical data. Therefore MH will develop multilingual software for acquiring relevantmedical data around respiratory disease. SME MH already made some needful experiencesin surveillance tools. MH will create and develop the project website, a Contact ManagementSystem and a software tool for acquiring project information. He will also consult any teammember if necessary.

ROLE OF SMEs

149


Oliver SchildgenInstitute for Medical MicrobiologyImmunology, and ParasitologyDepartment of VirologyUniversity of Bonn Sigmund-Freud-Strasse 2553105 Bonn, Germany [email protected]/startseite/jsp/index.jsp

Partners

Maria Grazia CusiUniversity of SienaSiena, Italywww.unisi.it

Lia van der HoekAcademic Medical CenterAmsterdam, The Netherlandswww.amc.uva.nl

Catherine ManohaUniversity Hospital DijonDijon, Francewww.chu-dijon.fr

Matthias Hamann HamannGermanywww.hamann-software.de

Brian SproatRNA-TECLeuven, Belgium www.rna-tec.com

Beatriz LazaroINGENASASpainwww.ingenasa.es

Michael KleinesUniversity Hospital AachenAachen, Germanywww.ukaachen.de/content/folder/1202162

The partners, part of an international consortiumof four research organisations, one small andmedium-sized enterprise (SME) and two compa-nies, will produce an antibody as a panel of differ-ent glycoforms, which will be tested for thefollowing:• stability;• efficacy (e.g. in Fc-receptor binding assays,

tumour cell binding assays and tumour grafting);• pharmacokinetic properties, such as serum half

life and antibody-dependent cellular cytoxicity(ADCC).

The project partners will use the results todevelop safer and more active glycoform varietiesfor therapeutic applications.

By using the plant expression systems and BY-2cells to generate the same recombinant antibody,the consortium targets the production of H10,a human full-length immunoglobulin G (IgG) thatrecognises the CEA.

SAGE will also conduct a comprehensive, compar-ative study to establish how the structural, func-tional and clinical properties of the antibody areinfluenced by the glycan structures. The projectpartners will compare the properties of the H10antibodies with those of a control H10 moleculegenerated in Chinese Hamster Ovary (CHO) cells.

Background

The use of plant systems for the production ofpharmaceutical glycoproteins (including antibod-ies) offers an attractive alternative to the currentstate-of-the-art in protein production, given theircost efficiency and overall safety. Producing largequantities of antibodies for use in cancer diagnos-tic and therapeutic systems using plant systemscould therefore prove a viable and financiallysound approach.

However, the differences in glycan structuresadded by plants, in comparison to those found inhumans, pose significant obstacles and in factresearchers recognise that the plant speciesused, as well as the tissue and cell type and theage, have a huge impact on the final glycoform ofan antibody.

Aim

The SAGE project targets an improved plant pro-duction platform for pharmaceutical glycopro-teins. The partners, a high-calibre team of expertsin plant-based production technology andimmunology, will use four plant-based expressionsystems (including transgenic plants, virus-infected plants and transformed plant cell lines)and mammalian cells as a control to generatea therapeutic antibody that recognises the well-characterised carcinoembryonic antigen (CEA),as well as their experience and know-how tolaunch protein therapeutics on the market.

SME-led antibody glyco-engineering

ACRONYM

SAGE

An attractive alternative to the currentstate-of-the-art in protein production, isthe use of plant systems for the produc-tion of pharmaceutical glycoproteins(including antibodies), given their costefficiency and overall safety. The SAGEproject targets an improved plant produc-tion platform for pharmaceutical glyco-proteins, to produce large quantities ofantibodies for use in cancer diagnosticand therapeutic systems.

SUMMARY

150



Key words: glycol-engineering, plant-made pharmaceuticals, recombinant antibody, transgenic plants

Expected results

The use of advanced plant-based expression tech-nologies will help SAGE generate innovative thera-peutic and diagnostic antibodies. The consortiumwill establish which best plant-based expressionplatform should be used to produce therapeuticantibodies, as well as identify the glycan struc-tures that give antibodies superior properties ina clinical setting.

The team will also determine the effects the vari-ous plant-derived glycans have on the physicaland functional properties of antibodies. Thisaction will support SAGE’s intention to produceimproved antibody-based therapeutics with supe-rior performance, as well as to provide treatmentfor many more people who need it. Ultimately,SAGE will be instrumental in improving healthcarefor everyone and in bringing down the costs fortreatment.

With strong business support, SAGE will convertany new knowledge or product developed duringthe duration of the three-year project directlyinto strategic advantage. The project partnerBayer BioScience will utilise IP managementsupport to conduct IP searches and secure extraIP protection, if needed.

SAGE will also transfer the acquired knowledgeto other scientific groups working in the samefield. Results and information will be dissemi-nated via electronic mail, peer-reviewed articles,abstracts and posters. The SAGE consortiumwebsite will also make key findings available tothe public.

greenovation Biotech GmbH has developed innovative technologies for the production ofproteins in mosses. The production technology is based on protein secretion into thesurrounding medium of mosses which are cultivated under liquid conditions in photo-bioreactors. Moreover, greenovation has developed expertise in the genetic modification ofmosses and in plant glyco-engineering. The SME will produce the H10 antibody in mossand the effect of glycan modifications on therapeutic applications will be analysed in vitroand in vivo.

ROLE OF SMEs

151


Stefan SchillbergFraunhofer-Institut für Molekularbiologie und Angewandte Oekologie IME Department of Plant BiotechnologyForckenbeckstrasse 6 52074 Aachen, [email protected]/EN/index.jsp

Partners

Yuri GlebaICON GeneticsHalle, Germanyicongenetics.com/html/home.htm

Gilbert Gorrgreenovation Biotech GmbHFreiburg, Germanywww.greenovation.com/english/index.php

Gerben van EldikBayer BioScienceGhent, Belgiumwww.bayer.be/cms/benelux/byc_cpstd_befr.nsf/

Dirk BoschDion FlorackGerard RouwendalPlant Research International B.V.Wageningen, The Netherlandswww.pri.wur.nl/UK

Eva StogerUniversity of Natural Resources and Applied Life SciencesVienna, Austriawww.boku.ac.at/home.html

Hardev S PandhaUniversity of SurreySurrey, United Kingdomwww.surrey.ac.uk

| Regeneration of transgenicplants producing pharmaceuticalsin petri dishes.

Expected results

By the end of this project, it is expected that theidentification of two or three M. tuberculosis anti-gens, which can form part of a serodiagnosticassay as a tool for detecting active TB in a highand a medium TB incident setting, will have beenachieved. Furthermore, performance data on theICT prototype are also expected. As a part of this,data which highlight the influence of TB incidenceon the performance of an antibody based kit fordiagnosis of active TB are expected.


The development of the proposed ICT assaywould lead to an inexpensive and more accessiblediagnostic TB tool whilst enabling local and familyphysicians in the field to perform the TB diagnosisfrom the bed-side. This will permit an earlier diag-nosis of the disease and therefore an early initia-tion of the treatment of a contagious diseasewhich will lead to a decrease of the transmission.Only by preventing the spread of the disease inthe local community can long-term control of TBbe achieved.

Background

Globally, TB is a severe problem, and the rapidand accurate diagnosis of active TB is the corner-stone of TB control. There is a direct need for:• better TB diagnostics methods in developing

countries;• the development of a simple and rapid test based

on antibody monitoring, which will be of greatimportance in controlling the global epidemic.

There are currently a number of serology-basedcommercial TB tests available, but none with therequired sensitivity and specificity.

The publications of the genomic regions of differ-ence between M. tuberculosis and M. bovis BCGallows identification of genes that are presentonly in M. tuberculsosis. From these gene regions100 proteins have been screened, specific for theM. tuberculosis complex. From these, ten anti-gens have been selected, which are frequentlyrecognised by both HIV negative and HIV positiveTB patients.

Aim

To identify the best combination of two to threeantigens to be incorporated into an immunochro-matographic test, which will be evaluated forperformance in two real life settings.

Development of a Specific Serological Kit for the Diagnosis of TB

ACRONYM

SERO-TB

There is a great need for simple androbust diagnostic methods for the diag-nosis of tuberculosis (TB). From thebasis of a through-screening processten M. tuberculosis antigens have beenidentified, exhibiting potential as sero-diagnostic TB antigens. Two to three ofthese antigens will be selected with theaim of achieving the highest sensitivityand specificity in a setting with a highnumber of latent TB infections and HIVco-infection. The selection will be basedon evaluating patients with TB, latentlyinfected individuals and symptomaticnon-TB patients.

The selected antigens will be incorpo-rated into an immunochromatographictest (ICT) which will be optimised for thedetection of M. tuberculosis-specificantibodies. Prototypes of the test kitwill be produced and in-house proof ofperformance data generated. Finally,the performance of this test kit will beevaluated in a real life scenario in twoindependent hospital settings in Turkeyand Ethiopia.

SUMMARY

152

Contract number: LSHP-CT-2006-037796 | EC contribution: € 827 313 | Duration: 36 months


Key words: tuberculosis, serodiagnosis, antibodies, diagnostics, immunochromatographic test

The SME role is undertaken by Vircell, a company specialised in infectious diseases andmore specifically focused on respiratory infections, but which has previously had no TBspecific activities. This project is regarded as an opportunity to enter the field of TB diagnosis.In addition, this project will establish a new collaboration between SSI and Vircell. Thefinancial support of this project will allow a SME company like Vircell to undertake a projectwith the aim of providing developing countries with a diagnostic kit, regardless of thecommercial compensation obtained. Vircell is part of the Work Package (WP) managementteam in the project and leads WP 2 “Development of a TB immunochromatographic prototype”and WP3 “Demonstration of performance of prototype”.

ROLE OF SMEs

153


Mark DohertyStatens Serum InstitutDepartment of Infectious Disease ImmunologyArtillerivej 5, 2300 Copenhagen [email protected]

Partners

Arantxa CortésVIRCELL P.I. Granada, Spain www.vircell.com

Ismail CeyhanRefik Saydam National Institute of HygieneAnkara, Turkeywww.rshm.saglik.gov.tr

Abraham AseffaArmauer Hansen Research InstituteAddis Ababa, Ethiopia

| Principle of an immunochromatographic test. © Vircell

• establishment of histone modification states asstandard readouts for drugs that target epige-netic modifiers;

• improvement of known epigenetic modulatorsthrough medicinal chemistry;

• identification of target genes that are regulatedby the SMARTER molecules;

• application of the SMARTER molecules in stan-dard animal model systems to verify their activityin living organisms.

Expected results

Our proposal will thereby promote the develop-ment and improvement of a new branch of cancerdrugs and as well support validation of newpotential drug target enzymes. Additionally, toolswill be generated, which allow new insights infundamental mechanisms of gene regulation byepigenetic modification.


In the context of human health, an understandingof gene regulation is central to our understandingof many medical complaints and conditions.Fundamental aspects of chromatin function areincreasingly recognized as important factor in thedevelopment of many severe and often untreat-able diseases. Therefore many proteins that areinvolved in the regulation of chromatin structureare potential drug targets and small moleculesdirected against these factors will play an increas-ingly important role in treating patients that areaffected by one of these maladies.

Background

The epigenetic level of gene regulation is beinganalysed intensively worldwide. However, theknowledge gained from these studies has notbeen transferred to drugs or drug candidates forthe treatment of major diseases. Equally, devel-opment of small molecules targeting epigeneticregulators have so far not been the major focus ofdrug discovery efforts.

To pursue this promising approach it is obviouslyimportant to further improve understanding howthe eukaryotic genome in general, and the humangenome in particular, operates. Therefore knowl-edge about its DNA sequence, its epigenetic con-trol systems and its dynamic structure in relationto gene expression must be integrated.

Aim

The SMARTER project aims at the developmentand improvement of compounds targeting epige-netic regulators. These compounds will be testedin various assays making it possible to collectdata sets of several parameters as histone modi-fications, chromatin states, gene expressionpatterns and physiological characteristics in anintegrative manner for the first time.

Therefore major objectives are:• identification of small molecule inhibitors that

target various histone-modifying enzymes;• validation of these inhibitors through in vivo

analytics of histone modifications states;

Development of small modulators of gene activation and repression by targeting epigeneticregulators

ACRONYM

SMARTERwww.smarter-chromatin.eu

The identity of a given cell within a meta-zoan organism is primarily defined by theexpression pattern of its genes. The acti-vation and repression of genes is tightlyregulated by the concerted action of tran-scription factors that recognise and bindspecific DNA sequences within regulatoryregions.

Work done over the last 20 years revealedthis basic mechanism of gene activationand repression, while recent experimentsexposed an additional layer of regulationinvolving modifications of DNA and boundhistones. These modifications are involvedin cellular inheritance of transcriptionalstates through cell division and develop-ment, and as they are not coupled to DNAsequence, are referred to as epigenetic.

Many factors that impact on epigeneticphenomena are clearly distinct frombasic transcription factors and areinvolved in regulating chromatin struc-ture. Modulation of chromatin structureis frequently achieved by intrinsic enzy-matic activities that either mark partic-ular regions within the genome foractivity or repression, or use the hydrol-ysis of ATP to remodel nucleosomalarrays. This variation of gene expressionpatterns in response to external andinternal signals has a major influence onstem cell differentiation, the mainte-nance of tissue integrity, and the adap-tation of organisms to environmentaldynamics.

Recently, small molecules that target his-tone deacetylases (HDAC) have been usedin the treatment of cancer, opening upnew avenues in therapeutic research.

The SMARTER project aims at the devel-opment and improvement of such com-pounds, which is the primary mission ofChroma, the SME participating in theconsortium.

SUMMARY

154



Key words: epigenetics, small molecules, histone deacetylases, chromatin

The SMARTER project is specifically designed to strengthen the knowledge base ofChroma Therapeutics, a privately-held biotechnology company focused on the discoveryand development of novel small molecule drugs targeting epigenetic modifiers. The col-laboration with leading chromatin laboratories will be of great benefit by facilitating theanalysis of SMARTER inhibitor molecules that have been and will be discovered byChroma in high-throughput screens. A rapid testing in various biological systems willallow Chroma a more directed optimization of the small molecules by their medicinalchemistry specialists. The new knowledge gained from this project will be efficientlytranslated into new therapies and clinical practice through Chroma’s well-establishedR&D pipeline.

ROLE OF SMEs

155


Axel ImhofHistone Modifications Group/Protein Analysis Core FacilityAdolf-Butenandt InstituteLudwig Maximilians University of MunichSchillerstr. 4480336 Munich, [email protected]/groups/imhof

Project manager

Julia HochstatterAdolf-Butenandt-InstitutLMUMunich, [email protected]

Partners

Scott CuthillChroma TherapeuticsOxford, United Kingdomwww.chromatherapeutics.com

Dirk SchuebelerFriedrich-Miescher-InstitutBasel, Switzerlandwww.fmi.ch/html/research/research_groups/epigenetics/Dirk_Schuebeler/Dirk_Schuebeler.html

Manel EstellerCNIOMadrid, Spainwww.cnio.es/ing/grupos/plantillas/presentacion.asp?grupo=50004270

Tony KouzaridesGurdon InstituteCambridge, United Kingdomwww.gurdon.cam.ac.uk/~kouzarideslab/tony.html

MOSAIQUES), ELISA kits (SME APOTECH) andprotein biochip prototypes (SME ORLA), for thedevelopment of fast high throughput technologies;

• development of novel reagents for monitoringgraft versus leukaemia, GvHD and targeted ther-apy (SME MULTIMUNE; SME NASCACELL);

• comparative studies in an autoimmune diseasemodel of inflammation, rheumatoid arthritis.

The SMEs primary specific Aims include:• development of new tools for novel diagnostics,

novel drug targets and therapeutics for use bothin the transplant and autoimmune setting;

• access to important clinical and biological sam-ples and data for use in accurate assessment ofresults and confirmatory studies for the devel-opment of novel diagnostics;

• testing and evaluation of new diagnostics onindependent cohorts and correlation of dataacross HSCT centres;

• testing of new prototypes against currentassays via collaboration between SMEs;

• use of new target molecules in monitoring ofresponse to therapy and during the post trans-plant period to assess acute and chronic GvHD.


• Identification of new diagnostics in the form ofnovel proteins associated with graft versus hostdisease (GvHD) compared to viral disease.

• Development of early diagnostic tools for GvHDand rheumatoid arthritis using gene profiling.

• The fast throughput development of novel mono-clonal antibodies and ELISA kits for research,diagnostics, and potentially therapeutic use.

• Development of novel peptides for use in moni-toring GvHD and graft versus leukaemia effectsin transplant patients.

• Development of prototype biochips (Fig. 1).

• Identification of new single nucleotide polymor-phisms (SNPs) for analysis in prognostic/diag-nostic indices.

Background

Over 7 000 allogeneic haematopoietic stem celltransplants (HSCT) are carried out each year inEurope alone, as a treatment for leukaemia andlymphoma. Techniques and cure rates are improv-ing but the overall survival rate remains between40-60 %.

Aim

This project will develop new proteomic, biologi-cal and genomic tests and tools for early diagno-sis and monitoring of patient response to noveltherapeutics for the most severe complication ofHSCT; graft versus host disease (GvHD) and willbring to the clinic a new generation of diagnosticsthat will significantly improve HSCT therapy andpatient outcome.

The Consortium unites 5 European SMEs withexpertise and markets in genomic and proteomictesting, diagnostic assay development andbiochips, with clinical partners selected for theirworld leading research in HSCT and access to clin-ical samples and patient groups.

The project will focus on the role of relevant genesand biomarkers associated with acute and chronicGvHD, using retrospective samples from estab-lished biobanks and prospective clinical trials to:• identify novel bio and genomic markers for

diagnostics;• develop novel diagnostic tools using genomics,

proteomics, in vitro bioassays and biochips;• test the new diagnostics in animal models & on

clinical samples;• exploit the new tools for commercial use.

Expected results

The above will be realised by:• development of diagnostic tests using single

nucleotide polymorphism (SNP) analyses (SMEIMGM), based on results from previous ECfunded research (EUROBANK, TRANSEUROPE);

• using proteomics via mass spectrometry evalua-tion/development of diagnostic patterns (SME

The development of new diagnostic tests, newtools and non-invasive methods for the prevention,early diagnosis and monitoring for haematopoieticstem cell transplantation (HSCT)

ACRONYM

STEMDIAGNOSTICSwww.stemdiagnostics.com

The StemDiagnostics project aims at thedevelopment of next-generation of med-ical tests and tools for significantlyimproving the survival rate of patientsundergoing haematopoietic stem celltransplant also known as HSCT – a med-ical treatment for life threatening condi-tions such as leukaemia and lymphoma.

SUMMARY

156


Starting date: 1 June 2007

Key words: haematopoeitic stem cell transplantation, graft versus host disease, graft versus leukaemia, proteomics, genomics, pharmacogenomics, clinical trials, early diagnostics

The STEMDIAGNOSTICS Consortium comprises SME’s that are already key players in theEuropean diagnostics market and already have the infrastructure and established market cred-ibility to turn research outputs from the STEMDIAGNOSTICS Project into marketable diagnos-tic products. The SMEs will target not only the European market, but also international healthmarkets including opportunities in the USA, Japan and Asia. The Consortium consists of 5 SMEswith expertise in state of the art technologies (IMGM, MOSAIQUES, mi, APOTECH, ORLA). APOTECH is a life science reagents company discovering, developing and producing new prod-ucts in the field of apoptosis and inflammation for laboratory based diagnostic tests.ORLA has already developed a platform technology, which may form the basis of a clinic basedor bedside assay. Orla Protein Technologies is a leader in the emerging European nanotechnol-ogy sector. The company has developed a ‘surface biology platform’ which is finding applicationin many areas including nanoscale diagnostics.Multimmune GmbH is a biopharmaceutical company dedicated to the discovery and develop-ment of novel products, including antibodies and peptides, for the treatment of cancer throughits innovative manipulation of the immune system.IMGM Laboratories GmbH is one of the leading SMEs for functional genomics and biomedicalresearch in Germany and has extensive experience in molecular diagnostics. IMGM is amongthe top 5 laboratories in Europe that are experienced in the ABI microarray technology.Microarrays, real-time PCR low-density arrays and SNP-detection technologies will be usedthroughout the project.MOSAIQUES’ established protein discovery systems will contribute to discovery of new bio-markers. Mosaiques Diagnostics will define and identifiy peptides and proteins in body fluids,which will enable diagnosis, of graft versus host disease as well as and response to therapybased on patterns of polypeptides in patients urine samples. To this end, MosaiquesDiagnostics has developed proprietary technology and software which enable the diagnosis ofdiseases based on polypeptides.CENAMPS is a not-for-profit technology commercialisation company, established in 2003.Cenamps is dedicated to the development and exploitation of emerging small-scale tech-nologies for applications in healthcare, ambient intelligence and consumer products. The com-pany will play a major role in management and exploitation of the project using state of the artmanagement software and aid in bringing new developments to market and protecting IP.

ROLE OF SMEs

157


Anne DickinsonUniversity of Newcastle upon Tyne, School of Clinical and Laboratory Sciences,Haematological Sciences, The Medical SchoolFramlington Place, Newcastle upon TyneNE2 4HH, United [email protected]

Partners

Ernst HollerKlinikum der Universität RegensburgDept. Hamatology und Internistiche OnkologieRegensburg, Germany

Harald MischakMosaiques Diagnostics GmbHHannover, Germanymosaiques-diagnostics.de

Gabriele Multhoffmultimmune GmbHMunich, Germanywww.multimmune.de

Ralph OehlmannIMGM LaboratoriesMartinstried, Germanywww.gene-expression-center.dewww.imgm.com

Lars FrenchDepartment of DermatologyZurich University HospitalZürich, Switzerland

Olivier DonzéCSO and Operating ManagerApotech Corporation (Headquarters)Epalinges, Switzerlandwww.apotech.com

Hans-Jochem KolbKlinical Cooperation Group Hematopoietic CellTransplantation, Institute of MolecularImmunology, Forschungszentrum fuer Umwelt und GesundheitMuenchen, Germany

Dale AtheyOrla Protein Technologies Ltd., NanotechnologyCentre, University of Newcastle upon TyneNewcastle upon Tyne, United Kingdomwww.orlaproteins.com

Gérard SociéAssociation de Recherche sur la Greffe de CSP, Aplasies et HPNParis, France

Hildegard GreinixBone Marrow Transplantation UnitUniversity Hospital of ViennaVienna, Austria

Ilona Hromadníková3rd Medical Faculty, Charles UniversityPrague, Czech Republic

Shak GohirCENAMPSThe Fabriam Centre, Middle Engine LaneNewcastle upon Tyne, United Kingdomwww.cenamps.com

Host antigen presenting cells (APC)Irradiation/chemotherapy

Host tissues:skinLiverGI tract

Pro-inflammatory cytokinesTNF-alpha, IL-1, IL6-Heat shock protein activationAnti-inflammatory cytokines IL-1Ra, IL-10

Phase 1: Patient conditioning- activation of inflammasome and early events

Phase 2: Donor T cell activation- further activation of inflammasome

Phase 3: Inflammatory effectors

Donor T cell

TCR-MHC

TNF-α IL-2 IFNγ

IL-1 TNF-α, chemokines; IL-8

Host APC, fibroblasts, epithelial/endothelial cells

T

NK

Nitric oxideTNF-α

(IL-1Ra IL-10)

Target cellAPOPTOSIS and tissue damage

Differential cytokine IL-1, IFNγ / chemokine release

IL-10 IL-1Ra TGFβ Immune Modulation

Th1 IL-2, IFNγ Th2 IL-4, IL-6,IL-10

aGvHD cGvHD

| Fig. 1 Phase 1: Patient conditioning leads to tissue damage in host tissuesproducing pro- and anti- inflammatorycytokines influenced by the patientnon-HLA genotype. Phase 2: In coming donor T cells andtheir activation is influenced by donornon- HLA genotype with further releaseof cytokines and upregulation ofsurface HLA Class II and adhesionmolecules on target tissues. Phase 3: Patient and donor non- HLAgenotype may influence theexacerbation or reduction of severity of GvHD. Molecules involved in earlyevents in phases 1 and 2 of cellactivation via activation of theinflammasome.(Reprinted from Dickinson A.M., andCharron, D. 2005. Non-HLAimmunogenetics in hematopoietic stemcell transplantation. Curr. Opin.Immunol., 17, 517. with permission from Elsevier.)

The cytokine storm and involvement of the inflammasome/non HLA genetics

• to analyse metabolic flux control and flux bal-ance with a view to engineering metabolic path-ways found in a Streptomyces background, andhence to exploit cellular pathways which pro-vide improved energy transduction, balancedgrowth and supramolecular assembly;

• to engineer better production/secretion strainsof Streptomyces based on the above, and basedon information about secretion bottlenecks thatwill be identified through the production ofmuteins, either via direct mutation of specificamino acids, or by directed evolution;

• to optimise the protein production process.

Expected results

Based on a better understanding of metabolome-secretome interplay, strategies for improvedprotein secretion will be designed. These willcombine better energy generation and directedenergy consumption for either cell mass pro-duction or heterologous protein secretion. Ulti -mately, a ‘toolbox’ of Streptomyces strains willbe engineered and refined, which optimally over-secrete proteins of interest during fermentation.

Consequently, Streptomics will generate knowl-edge which will assist SMEs in the biotechnol-ogy and other industries to develop new andmore efficient systems for the industrial produc-tion of heterologous proteins, using S. lividansas a cell factory. These systems will be useful inboth red (medical) and white (industrial) areasof biotechnology.


This project aims to increase the number of effi-cient cell factory platforms for the production ofheterologous proteins important in health, bio-catalysis and the environment, using Streptomycesas a host. It will therefore contribute to a competi-tive, knowledge-based economy and sustainabledevelopment in Europe, by serving the needs ofa research-intensive industrial sector in whichmany SMEs have traditionally been involved.

Background

The biotechnology industry is constantly search-ing for better hosts for the production of biophar-maceuticals and enzymes of diverse origin. TheGram-positive soil bacterium Streptomyces hasalready proved an invaluable host for this pur-pose, since it can secrete several heterologousproteins in satisfactory amounts. However, inorder to optimise strain selection, knowledge isrequired concerning the following points: • how protein secretion processes are integrated

within the metabolome, and how they interact;• how heterologous protein secretion stresses

the metabolome and induces negative cellularcascades.

Systems biology, the science of analysing andmodelling genetic, macromolecular and meta-bolic networks, provides the means to addressthese questions. By combining biochemical infor-mation with genetic and molecular data, theStreptomics consortium hopes to gain novelinsights into the functions of genes related to pro-tein secretion, as well as how that protein secre-tion mechanism responds to external and internalstimuli. With a better understanding of this mech-anism at the cellular level, it should be possible tooptimise protein secretion.

Aim

Streptomics aims to enhance the production ofheterologous proteins, using Streptomyces as ahost. More specifically, it has the following goals: • to evaluate Streptomyces lividans as a cell fac-

tory for the production of heterologous proteinsof interest;

• to investigate the transcriptome and proteomeof the host strain under different growth condi-tions, with different expression/secretion vectors,and using different fermentation strategies, inorder to identify the genes important for optimalcell performance, with respect to hetero logousprotein secretion;

Systems biology strategies and metabolome engineering for the enhanced production of recombinant proteins in Streptomyces

ACRONYM

STREPTOMICSwww.streptomics.org

The Gram-positive soil bacterium Strep -tomyces is an invaluable host for thesecretory production of biopharmaceu-ticals and other heterologous proteins.STREPTOMICS aims to further evaluatethis host as a cell factory for the industrialproduction of proteins of diverse originincluding from mammalians, bacteria andarcheae of interest for human health andenvironment.

SUMMARY

158



Key words: systems biology, streptomyces, protein secretion, enzymes, biopharmaceuticals, directed evolution, metabolomics, transcriptomics, proteomics

The four SMEs involved in the project, Prokaria (Matis) (Reykjavik, Iceland), Direvo, (Köln,Germany), Eurogentec (Liège, Belgium) and BioXPr (Namur, Belgium) each have their specifictask, from assessing the Streptomyces protein production platform for the production of pro-teins of their interest (Prokaria and Direvo) to helping to strain improvement of proteins whichplay a role in protein secretion via directed evolution (Direvo). Prokaria will take responsibilityfor cloning, expressing and secretion of various enzymes from extremophiles, primarily frombacterial and archeal thermophiles into Streptomyces. These thermostable enzymes have pre-viously been proven to be difficult expressed or produced in other hosts like E. coli or Thermusthermophilus. Direvo, a leading company in protein engineering by screening-based directedevolution and its application to biomolecules, specifically to biocatalysts for chemical, techni-cal, industrial, scientific and pharmaceutical purposes, will test Streptomyces lividans andevaluate expression/secretion levels and product characteristics for the production of engi-neered proteins/enzymes with market potential in Pharma and Industrial Biotechnology, andevaluate whether SecA mutants could improve secretion characterization. As such, Direvo com-plements the expertise of the other partners with its broad and advanced technologicalportfolio in Directed Evolution and high-throughput protein screening. Eurogentec will takeresponsibility for the transcriptomic analysis of S. lividans. This SME developed previously incollaboration with different members of this project a complete DNA micro array based on longoligo’s representing the entire set of ORFs specific to Streptomyces coelicolor. The use of theS. coelicolor DNA array for S. lividans transcriptomic analysis will be validated and the arraymodified if necessary. Eurogentec will act as service provider for transcriptomic analysis for allpartners of this project. Data will be generated in collaboration with different partners for analy-sis of up and down regulated genes in different experimental conditions. BioXpr will organiseand set-up a data repository system that will allow all the member of the consortium to storethe results of their work packages on a centralized system. The central database used to storethe data will also be the source of information for analysis and visualisation system that willcombine proteomics, transcriptomics and metabolomics information to define the best condi-tions for protein secretion. BioXpr will therefore collect information to combine the sourcesof data into one picture.The successful integration of the various aspects of this project STREPTOMICS will providein-depth knowledge about interactions that may occur at the genetic or proteomic level dur-ing the heterologous protein secretion process in S. lividans of heterologous proteins.Partners will work together to apply a step-by-step approach to analyze the physiologicalstate and performance of the cell, and based on these results, to engineer strains that mayovercome or suppress negative effects.

ROLE OF SMEs

159


Jozef AnnéRega InstituteCatholic University of LeuvenLaboratory of BacteriologyMinderbroedersstraat 103000 Leuven, [email protected]/rega

Partners

Michael HeckerErnst-Moritz-Arndt-UniversityInstitute for MicrobiologyGreifswald, Germanywww.uni-greifswald.de/indexuk.html

Wayne M. CocoDirevo Biotech AG Cologne, Germanywww.direvo.com

Anastassios EconomouFoundation of Research and Technology FORTHInstitute of Molecular Biology and Biotechnology,Iraklio, Crete, Greecewww.forth.gr

Marc DaukandtEurogentec S.A.DNA MicroArray Dept.Seraing, Belgiumwww.eurogentec.com/eu-home.html

Jakob KristjánssonProkaria Ltd.Reykjavik, Icelandwww.prokaria.is

Benjamin DamienBioXpr S.A.Namur, Belgiumwww.bioxpr.be

Roy GoodacreUniversity of Manchester School of ChemistryManchester, United Kingdomwww.chemistry.manchester.ac.uk

Anna Eliasson LantzTechnical University of DenmarkCentre for Microbial BiotechnologyLyngby, Denmarkwww.cmb.dtu.dk/English.aspx

Daniel BadcockGlaxoSmithKlineHarlow, United Kingdomwww.gsk.com

| Automated protein engineering: A precision robot arm retrievesa custom manufactured 1536-well plate in one corner of a room fullof robotics-compatible equipment including nano-liter volume liquidhandlers, single cell sorters, humidified incubators, heating andcooling blocks, centrifuges, confocal laser-based plate readers andother equipment integrated for fully automated high throughputprotein engineering at Direvo Biotech AG in Cologne, Germany.

Expected results

After 36 months, SYSCO will have achieved thefollowing aims: • development of a hybrid, in silico model for the

innate response of macrophages to an intra-cellular pathogen, based on the composition ofinterconnected modules that mimic differentcellular events;

• development of a comprehensive systemsontology;

• experimental investigation and categorisationof four different modules, namely gene regula-tion, gene and protein expression and signaltransduction;

• complementary high throughput analysis ofthe macrophage transcriptome by Affymetrixoligonucleotide arrays and serial analysis ofgene expression, both in parasite-infected andin non-infected cells;

• prediction and validation of the regulatory net-works in macrophages;

• experimental determination of cell regulation byquantitative transcription factor assays and byRNA interference.


Leishmaniasis is one of the world’s major para-sitic diseases, but there is no vaccine for it asyet, and the drugs currently prescribed to treat itare fairly toxic. Millions of people living in devel-oping countries, mainly in southern and east -ern Mediterranean regions and in central andSouth America, are exposed to leishmaniasis. TheLeishmania parasite is also a major co-pathogenin the context of HIV infection in southern Europe.The results of this project will be significant,not only in the context of leishmaniasis, butalso for the understanding and treatment ofinfection by other intracellular pathogens, suchas Mycobacterium tuberculosis, the bacteriumwhich causes tuberculosis.

References

(1) Daar et al, 2002.

Background

A study conducted by an international expertpanel for the University of Toronto, ranked thecomputational examination of host-pathogeninteractions among the top 10 biotechnologiesmost likely to improve global health in the next10 years (1). However, information about funda-mental aspects of the cellular machinery involvedin the interactions between macrophages andintracellular pathogens has not yet been suffi-ciently categorised, particularly with regard tomacrophage function, and there is a need fora systematic and integrative approach to theidentification of interconnected functional mod-ules and salient modifications triggered byintracellular parasitism.

Aim

SYSCO will decipher and modularise the cascadeof intracellular events generated by parasite-cellinteractions, and also how they result in either par-asite elimination or infection in humans. A com-parative analysis with mouse strains expressingdiffering susceptibilities will help identify keydeterminants of natural resistance or susceptibil-ity to parasites acting at the macrophage level.

In a combined strategy of experimental and theo-retical work, the SYSCO consortium will systemat-ically capture data at different levels of cellularinformation, using state-of-the-art, multi-para-metric molecular technologies (both in humanand in mouse). These data will be used to identifyregulatory motifs through systematic promoteranalysis, and to populate computer models withthe relevant motifs and associated signallingpathways. The computer models will be designedas independent modules covering gene regula-tion, gene expression, protein interactions andsignalling. This modular approach will be used tomimic different types of innate macrophageresponses, and to map theoretical predictions toexperimental data.

Systematic Functional analysis of IntracellularParasitism as a model of genomes conflict

ACRONYM

SYSCOwww.biobase-international.com/pages/index.php?id=438

The overall objective of the SYSCO projectis to decipher the intracellular biologicalpathways and basic cellular processesthat act in physiological conditions aswell as in the context of intracellular par-asitism, in order to highlight the alter-ation in gene expression that stems fromthe conflict between the host andpathogen genomes. More specifically,the project will use human and mousemacrophages as cellular targets, and theLeishmania parasite as a prototype forintracellular pathogens. Leishmania isone of the most intensively studied bio-logical models in terms of parasite, hostimmune response and genetics.

SUMMARY

160


Starting date: 1 September 2007

Key words: intracellular parasitism, host-pathogen interaction, functional genomics

BIOBASE is the leading content provider of biological databases, knowledge tools andsoftware for the life science industry. They offer well-structured data, assembled by highlyqualified subject-matter experts, organized in an accessible and easily searchable mannerthat enables researchers to identify connections between disparate pieces of informationand to apply that knowledge to their specific topic of interest.

The tasks of BIOBASE are high quality manual literature annotation of project relevantdata into the proprietary databases TRANSFAC(R), TRANSPATH(R) TRANSCompel(R) andProteome™. They collect data on mechanisms of gene regulation, transcription factors andtheir binding sites and on signal transduction pathways in the human cells in response toLeshmania. The collected data along with the full content of the databases will be providedto the consortia. BIOBASE will also do DNA sequence analysis: search for putative bindingsites for TFs in promoters of genes regulated during cellular response to the parasite attack.Biobase will be involved in development of software for the reconstruction of a gene regu-latory network (transcription network) and in designing artificial promoters which can beused to study mechanisms of gene regulation in various immune responses.

Furthermore, BIOBASE will manage the project. Management activities include communi-cation with the commission, organisation of regular financial audits, dissemination andexploitation of project results, and regular project meetings.

Skuld-Teck (SKT) set up one bioinformatics platform, and two gene profiling technicalplatforms one based on the SAGE™ and one based on real-time quantitative PCR (qPCR).The SAGE platform has two advantages: the exhaustiveness of the results and the networkof its users (who share their gene profiles data). Through its two gene profiling platformsSKT has developed a first thematic dedicated to Leukaemia (end of 2002). This has permit-ted to identify a set of genes allowing for the design of a Leukaemia diagnosis/prognosistool. This tool is being validated within clinical trials to draw up the gene profiles of patientssuffering from Leukaemia and to monitor them during the treatment.

SKT will provide a relational database integrating all public and private SAGE data. SKT willdevelop another relational database integrating the data generated along the project i.e.:the gene profiles data that is being generated using SAGE and those that will be generatedusing Affymetrix. This platform will include two interfaces: a working interface that allowsintroducing annotations and integrating new data (under PostgreSQL/Linux) and anexploitation interface, regularly updated that will be easy to query for non-specialists.Considering the increasing volume of data in public databases, the working interface will beimplemented under Oracle. SKT will also validate the expression of target genes obtainedin WP2 through its Real-Time PCR (qPCR) platform.

ROLE OF SMEs

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Alexander KelBIOBASE GmbH Department of Research and DevelopmentHalchtersche strasse 3338304 Wolfenbüttel, Germany Alexander.kel@biobase-international.comwww.biobase-international.com/pages

Partners

David PiquemalSkuld-Tech SARLMontpellier, Francewww.skuldtech.com

Ralf HerwigMax Planck Institute for Molecular GeneticsBerlin, Germanywww.molgen.mpg.de

Béatrice RegnaultInstitut Pasteur Paris, France www.pasteur.fr/english.html

Patricia RenardFacultés Universitaires Notre-Dame de la PaixUnité de recherche en biologie cellulaireNamur, Belgiumwww.fundp.ac.be

Koussay DellagiInstitut Pasteur de Tunis, Laboratoire d’immunopathologie, vaccinologie et génétique moleculaire (LIVGM)Tunis, Tunisia www.pasteur-international.org

Winston HideUniversity of the Western Cape South African National Bioinformatics Institute Bellville, South Africawww.sanbi.ac.za

Pierre-Andre CazenaveUniversité Pierre et Marie Curie-Paris VI Laboratoire d’immunophysiopathologie infectieuse – URA 1961Paris, Franceenglish.upmc.fr/UK/info/00

genomics platforms developed by and accessibleto the partners. In particular, the consortium aimsto demonstrate newly developed technologies ina proof-of-principle study within an obesity-induced type-2 diabetes mouse model. The project consortium is headed by an SME andincludes four academic partners from threeEuropean countries. This composition of commer-cial and academic interests guarantees high-levelscientific research, as well as a strong focus onthe commercial relevance and exploitation of theproject’s results. As a consequence, the proposedproject will strengthen Europe’s scientific andcommercial competitiveness in the field of sys-tems biology, one of the key technologies infuture medical and pharmacogenetics research.

Expected results

An important feature of the project’s approachwill be the integration of phenotypic and physio-logical parameters with proteomics data andexpression profiles from time course series repre-senting the onset and progression of insulinresistance of type-2 diabetes. Ultimately, the plat-form will enable medical researchers to combineheterogeneous biomolecular data with physiolog-ical and clinically relevant parameters to predictindividual predispositions to obesity-inducedtype-2-diabetes.

The objectives of this project are: • model the knowledge about biological objects

(genes, proteins and protein complexes) inthe context of nutrition and type-2 diabetes inequivalent computer objects;

• integrate heterogeneous data types from pro-teomics and functional genomics approaches;

• develop and use a prototype framework for theautomatic detection and localisation of proteinmodifications on high-accuracy mass spectrom-etry data;

• generate specific proteomics and functionalgenomics data providing the necessary infor-mation for disease model generation with anappropriate animal model;

Background

Bioinformatics methods for diagnostic screeningare a bottleneck in current biomedical research.While exploratory methods – such as statisticalhypotheses testing, clustering of gene expressionprofiles and classification methods – have beensuccessful in the detection of molecular markersfor interesting diseases, these techniques fail tovalidate these markers in their gene regulatorycontext and to integrate other data sources rele-vant for diagnostic purposes. For these tasks,novel modelling techniques, network analyses,and data integration methods are indispensable.The analysis of processes involved in the courseof complex polygenic diseases, such as obesityand type-2 diabetes, is in fact a multi-step proce-dure that has to cope with data from diverseexperimental functional genomics platforms(gene and protein expression), physiologicaldata, environmental factors, and others.

Aim

The project SysProt aims to develop a new para-digm for the integration of proteomics data intosystems biology. The goal is to gain relevantknowledge on the biological processes that areimportant for human health and to use this knowl-edge for the purpose of disease modelling. In order to achieve this objective, an innovative,explorative biological systems approach (on boththe molecular and the physiological level) will beadopted, with a strong focus on protein functionand modification. SysProt will produce pro-teomics data, indispensable for the identificationof novel circulating protein factors, and post-translational protein modifications that are impor-tant for the onset, dynamics, and progression ofcomplex diseases. Data generation will be complemented by thedevelopment of computational analysis methodsfor these novel data types and the creation of ade-quate modelling technology. The project will ben-efit from the utilisation of established mousedisease models, existing benchmarking modulesfor computational analysis, and the functional

System-wide analysis and modelling of protein modification

ACRONYM

SysProtwww.sysprot.eu

SysProt aims at the development of a newparadigm for the integration of pro-teomics data into systems biology. Thegoal is to gain relevant knowledge onbiological processes that are importantfor human health and to use thisknowledge for the purpose of diseasemodelling. The strategy to achieve thisobjective is an innovative, explorativesystems biology approach both on themolecular and physiological level witha strong focus on protein function andmodification.

The consortium aims at demonstratingthe newly developed technologies ina proof-of-principle study in an obesity-induced type-2 diabetes mouse model.

SUMMARY

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Key words: systems biology, fundamental biological processes, proteomics, bioinformatics

• gain new knowledge on the pathways andmarker genes relevant for obesity-inducedtype-2 diabetes disease progression that willlead to the discovery of novel diagnostic bio-markers for disease susceptibility;

• stimulate perturbations of the disease-relevantpathways;

• develop tools and methods for the correlation ofphenotype and genotype;

• accelerate the identification and positionalcloning of disease candidate genes by combin-ing gene expression, proteomics, genotype, andclinical data;

• set up a knowledge base that integrates all avail-able data and methodology as an exploitableproduct for disease modelling.


The main result of the project will be an exploitableprototype that allows medical researchers to drawpredictions on disease-relevant pathways. Thesepredictions will be valuable for diagnosis and drugdevelopment purposes. The platform will enablethe validation of potential drug targets in-silicoand thus give support in explorative data mining of

the expanding body of gene and protein data togain the knowledge necessary to discover betterand safer drugs. In-silico disease modelling willnot only cut the costs of future drug developmentby reducing the number of false drug targets butwill also save development time. Additionally thein-silico systems will reduce the necessary numberof animal trails in drug development explicitly.Thus, the proposed platform has a high commer-cialisation potential in future diagnostics applica-tions as well as in drug development.Systems biology approaches will increasinglyhave an impact on Life Science and Health pro-grammes in general and on drug development inparticular. They provide a huge potential forimproving the quality of life through the creationof highly skilled jobs, improved competitiveness,and economic growth in Europe, as well as betterhealthcare and new tools to address different andimportant challenges of the EC. Through the appli-cation and broadening of systems biologyapproaches, the SysProt project is likely to impacton the scientific understanding of biologicalprocesses, with particular relevance to improvinghuman health and wellbeing.

MicroDiscovery is coordinator of SysProt having as a bioinformatics company a strong focuson systems biology. The company sees a high potential for modelling and simulation tech-nologies in the life science arena. However, currently available methods are far from commer-cial applications and need massive additional scientific and developmental efforts. TheSysProt consortium offers the possibility of an efficient technology transfer from key playersin the nutrigenomics, proteomics and systems biology field into a commercial application.MicroDiscovery intends to develop the resulting knowledgebase into a product ready for theLife Science market. There will be a rising demand for solutions in the area of systems biology.Systems biology will have a tremendous impact on the development of new drugs, diagnos-tics of complex diseases and personal medicine. Pharmaceutical companies, and also toa large extent biotech companies, design and market products which affect, inhibit or excite,biological systems, often via highly specific intervention points. Nevertheless these productstypically change systemic aspects of cells, tissues, entire organs, or organisms. There is nodoubt that new knowledge extending the ability to control biological systems via combinedcomputational and experimental approaches will be key in health care and in particular in thedrug development process in the next 5-10 years. This will for example be highly relevant inthe area of complex diseases, such as obesity induced type-2 diabetes, where many genes areresponsible for a particular, pathogenic phenotype. The commitment of MicroDiscovery to the project guarantees the midterm conversion of theconsortium results into an applicable product. The long-term goal is the establishment of sys-tems biology technologies in the life science arena. There are currently various companies out-side the EU (e.g. Entelos (US), Gene Network Sciences (CAN), BioSeek (US), Genstruct (US),GeneGO (US)) with large financial backing positioning themselves competing into this emerg-ing future market. There is still a large potential in the European academic community in sys-tems biology field with has to be mobilised in order to cope with the efforts outside the EU.

ROLE OF SMEs

163

Project coordinator

Arif MalikMicroDiscovery GmbHNutriSystemicsMarienburger Str. 110405 Berlin, [email protected]

Partners

Hadi Al-HasaniDeutsches Institut fuer Ernaehrungsforschung (DIFE)Department of Pharmacology-German Institute of Human Nutrition Potsdam-RehbrueckeNuthetal (OT Bergholz-Rehbruecke), Germanywww.dife.de/en/index.php

Ralph SchlapbachEidgenössische Technische Hochschuleule, ZürichFunctional Genomics Center ZurichZurich, Switzerlandwww.fgcz.ethz.ch

Rainer CramerThe University of Reading The BiocentreReading, United Kingdomwww.biocentre.reading.ac.uk

Ralf HerwigMax Planck Institute for Molecular GeneticsDepartment Vertebrate GenomicsBerlin, Germanywww.molgen.mpg.de

| C57BL/6J mouse (black), Swiss Jim Lambert (SJL)mouse (white) and New Zealand Obese (NZO) mouseare used as animal model for diabetes and obesity inthe SysProt project. The NZO mouse is an establisheddisease model in diabetes research and characterisedby heredity in obesity and by Typ2-diabetes withInsulin resistance. The SJL mouse never developsobesity and therefore serves as counterpart to the NZO mouse. C57BL/6J mice serve as control strain.

possible relationships (proteins, genes, regulation,and pathology). Therefore, TAMAHUD aims atdelivering novel targets causally associated withaspects of the HD pathology in model systems,where biological target validation is accompaniedby demonstration of target relevance throughsmall-molecule pharmacological modulation. Inparallel, the biomaterial repository made availableto the consortium from a disease-specialist aca-demic partner will be investigated through state-of-the-art metabonomics approaches to identifybiomarkers predictive of disease onset.

Expected results

The expected results can be summarised into:• the identification of novel, tractable targets

causally associated with the pathology, alongwith small molecules capable of modifyingaspects of the pathology in cellular diseasemodels via modulation of target activity; and

• biomarkers of disease onset/progression.


If met with success, the scope of the project goesbeyond the 3.5 year granting period and theresearch outcomes are relevant to:• further development of therapeutic compounds

through preclinical and clinical studies;• reduction of costs in clinical development: the

lack of reliable and predictive biomarkers of dis-ease onset requires clinical trials of higher com-plexity in order to reach statistical significance;

• creation and development of a network of com-petencies aimed at progressing drug discovery insuch a specialist field as the neuroscience area.

TAMAHUD aims to terminate its activities in theconstitution of a solid base for the later comple-tion of pre-clinical development of potentialdrugs, comprising the demonstration of in vivoefficacy of TAMAHUD-derived chemical series alsothrough the use of TAMAHUD-derived biomark-ers. At even later times, clinical studies on candi-date drugs originating from TAMAHUD activitieswill again benefit from the inclusion of TAMAHUDbiomarkers to determine clinical efficacy.

Background

No curative therapy is currently available forpatients with HD and the pathophysiology of HD isstill not well understood. Generally, the currenttreatment of HD combines non-pharmacologicaltherapy with management of the symptoms ofthe disease. Pharmacological treatment of thedisease must be tailored to the specific needs ofthe patient. Physicians may prescribe a number ofmedications to help control emotional and move-ment problems associated with HD, althoughno drug is yet available to stop or reverse the pro-gression of the disease. The significant unmet medical need for HDincludes the following:• better understanding of pathophysiology to

yield more relevant targets;• improved symptomatic treatments;• drugs that slow, halt or reverse disease progres-

sion;• diagnostics of disease onset/progression.

Aim

This project addresses the challenge of identifyingnovel tractable targets causally associated withthe pathology, to support the development of dis-ease-modifying therapeutics for the cure of HDand of discovering novel early biomarkers leadingto the development of new diagnostic tools. High throughput-RNAi, focusing on genes encod-ing pharmacologically tractable proteins ratherthan on a whole-genome approach, will beemployed on a novel and robust HD cellular dis-ease model to identify genes whose inhibition ofexpression is protective against the HD mutation.Following a stringent target validation approach,selected validated targets will be progressed toassay development and primary screening activi-ties, to identify druggable compounds active on thetarget and efficacious against the HD mutation incellular disease models. The complete process willbe accompanied by an extensive data and text min-ing workflow providing background information forthe HD knowledge network. This network will guidethe experts in the generation of hypotheses about

Identification of early disease markers, novel pharmacologically tractable targets and small molecule phenotypic modulatorsin Huntington’s Disease

ACRONYM

TAMAHUDwww.tamahud.eu/home.jsp

Huntington’s Disease (HD) is a devas-tating neurodegenerative disease withmany unmet patient needs. There are noknown ways of slowing or preventing theneurodegeneration associated with thedisease, and clinical trials in humans arehampered by the slow disease progres-sion and the absence of suitable bio-markers of short-term progression. Thegenetics of HD is characterised, andinvolves the expansion of a polygluta-mine tract at the amino-terminus of theHuntingtin gene (HTT). However, thetranslation of this knowledge into thera-peutic and diagnostic approaches is ham-pered by the scarce knowledge of HTTbiology, the paucity of information onhow cellular signalling pathways interactwith the HD mutation, and the lack of sys-tematic and modern approaches aimed atidentifying useful biopredictors of dis-ease progression in individuals diag-nosed with HD. The TAMAHUD projectaddresses key areas of HD patient needs,namely the discovery and developmentof therapeutically meaningful novel tar-gets and biomarkers. More precisely,TAMAHUD project aims to deliver solidlyvalidated, druggable targets accompa-nied by developable small molecule mod-ulators and candidate diagnostics formonitoring of disease progression. Toachieve these aims, a consortium of spe-cialist partners has been assembled, rep-resenting complementary and specificknow-how and expertise which will beintegrated and further developed in thecourse of the project.

SUMMARY

164



Key words: Huntington’s disease, HT-RNAi, functional genomics, validated target, chemical hit, metabonomics, biomarker, text-mining, data-mining, data visualization

TAMAHUD’s aims are designed to meet two unmet needs in Huntington’s Disease (HD): first,the identification of novel small molecule modulators of therapeutically relevant targets andsecondly, the identification of novel biomarkers. The coordinating SME, Siena Biotech, ispivotal to TAMAHUD as it will provide novel potential pharmacological targets of therapeuticrelevance in HD, contribute to their validation and exploit its pervasive drug discovery plat-form to discover small molecule modulators of such targets. A second SME, TCP InnovationsLtd., participates to identify novel biomarkers of HD onset and progression. Once achieved,these aims will form the basis for the development of novel pharmacological therapies andprovide the means to monitor their efficacy in pre-clinical and clinical studies.

ROLE OF SMEs

165


Andrea CaricasoleSiena Biotech SpAvia Fiorentina 153100 Siena, [email protected]/index/index.jsp

Partners

David RubinszteinUniversity of CambridgeCambridge, United Kingdomwww.cimr.cam.ac.uk/index.html

David GraingerTCP Innovations Ltd.Cambridge, United Kingdomwww.tcpinnovations.com

Christian BlaschkeAlma Bioinformatics S.L.Madrid, Spainwww.bioalma.com/index.php

Reinhard SchneiderEMBLHeidelberg, Germanywww.embl.de

• generate synthetic peptides that enable anti-body dependent cellular cytolysis against her-pesviruses;

• define, investigate and apply RNA silencingreagents that block the expression of viral genesthat enhance herpesvirus replication;

• define, investigate and apply RNA silencingreagents that interfere with proviral host genes;

• identify viral and cellular genes involved in her-pesvirus-mediated oncogenesis, and define RNAsilencing reagents and peptide inhibitors; and

• develop approaches to inhibit the reactivationof HSV from latency. This programme of workwill provide innovative technologies for theidentification and development of future prod-ucts targeted at preventive and therapeuticinterventions for human herpesvirus diseases.Moreover, these strategies will be transferableto many other persistent infections.

Expected results

The TargetHerpes project is divided into six exper-imental work packages (WPs). The aim of WP1 isthe development of peptide molecules that willinhibit the functions of herpesvirus glycoproteinsand their roles in entry of the virus particles intocells. Preliminary work has provided proof-of-principle that mimetic peptides to HSV gH inhibitinfection. However, the first-generation peptideswere active only at very high concentrations, andas such they could only be applied to culturedcells. TargetHerpes expects to generate highlybioactive and specific peptides that do not exhibittoxicity to uninfected cells. Such peptides target-ing HSV glycoproteins, will be suitable for futureanimal experimentation and translational researchby partners PRIMM and IBA. WP2 will generate

Background

Herpes viruses cause many serious and life-threat-ening diseases, especially in immunocompro-mised patients, such as transplant recipients andHIV-infected individuals. Even in healthy ones,herpesviruses can result in serious diseases. Forexample, the herpes simplex virus (HSV) remainsone of the most common sexually transmitted dis-eases, while human cytomegalovirus (HCMV) isa leading cause of birth defects, and human her-pes virus 8 (HHV-8) causes a number of cancers.At present, the options for antiviral therapy arelimited and, owing to toxicity, the current anti-herpesvirus drugs cannot be administered topregnant women. There is a continuing need todevelop new treatments, as drug-resistant virusesare constantly evolving.

A principal characteristic of herpesvirus infec-tions, is that after primary infection (usually inchildhood), the viruses establish a latent statethat remains for life. Up to 90 % of the populationmay be latently infected with one or more herpesviruses. The social and psychological conse-quences of the herpesvirus infections are severe.

Aim

TargetHerpes will translate basic knowledge ofviral replication strategies and evasion from hostattack into the concept of a multi-pronged attack,using combinatorial sets of antiviral compoundswith proven antiviral efficacy in cells and a smallanimal model. Specifically, TargetHerpes will per-form the following actions: • develop peptide inhibitors that interfere with

virus entry;

Molecular intervention strategies targeting latent and lytic herpesvirus infections

ACRONYM

TargetHerpeswww.targetherpes.org

Herpesviruses are important humanpathogens. As of today, their infectionscan only be controlled by acyclovir orderivates. The TargetHerpes project willapply novel technologies provided bythree SMEs to design and develop novelclasses of antiviral treatments. Key tothe project is the SMEs connection withfundamental advances being made inEurope’s leading herpesvirus researchlaboratories. The technologies to beapplied to the search of novel, effectivetreatments against a broad spectrum ofherpesvirus diseases include the rationaldesign of peptide-based and siRNA-based herpesviral inhibitors. The steps ofthe viral life cycle to be targeted are virusentry, evasion of host defences, persist-ence in infected individuals, reactivationfrom latency.

SUMMARY

166



Key words: herpes virus, chemiotherapeutics, herpes simplex virus, human cytomegalovirus, human herpesvirus 8, fusion, glycoproteins, siRNA, innate immunity, host response, IFN

synthetic peptides that enable IgG antibodies toexecute cell-mediated cytolysis against HSV andHCMV. Such peptides will be evaluated for theirindividual potency in vitro, then bioactive peptideswill be evaluated with regard to safety and harm-lessness to cells, as well as optimal stability in cul-tured cell systems. WP3, WP4, WP5 and WP6 willidentify suitable molecular targets for antiviralintervention by RNAi. These targets will includeimportant herpes virus gene products that haveknown or suspected roles in promoting viral repli-cation directly or indirectly. In case of WP5, siRNAstargeted at viral genes will be selected based ontheir capacity to interfere with HHV-8 mediatedcell transformation and immortalisation. WP6expects to identify the cellular interaction partnersof ICP0 (the viral protein that is necessary for HSVto reactivate from latency), and to define thoseelements that are required for its activity.


TargetHerpes will identify novel strategies, lead-ing to the development of new approaches toinhibit the replication of, or pathogenesis causedby, HSV, HCMV and HHV-8. Due to the conserva-tion of genes and replication strategies within her-pesviruses, the approaches discovered will beapplicable to other human herpesviruses as well.For example, treatments that target HSV-1 arehighly likely to be effective against HSV-2 and maybe adapted to counteract VZV. Similarly, treat-ments that are effective against HHV-8 may alsobe applicable to EBV. Given the figures on thehealth burden and costs of herpesvirus infections,the potential impact of a successful outcome ofthe TargetHerpes project, is considerable.

Four SMEs are involved in the project. Their respective roles are as follows. BioDec,a young Italian spin-off company born out of University of Bologna, will perform bio -informatics searches, develop algorithms and software for the design of siRNAs orshRNAs and for prediction of protein structures involved in protein:protein interactions;IBA, a small German-based biotec industry will provide tools for si-RNA production aswell as for protein purification and studies of protein:protein interactions. PRIMM, anItalian small biotec company will provide tools for design and synthesis of antagonist andmimetic peptides as well as immunological reagents; ARTTIC, a transnational Europeancompany specializing in the management of large projects will take care of the managementof the project, dissemination of results to public, intellectual property issues.

ROLE OF SMEs

167


Gabriella Campadelli-FiumeUniversity of BolognaCentro Interdipartimentale Galvani (CIG) via S. Giacomo 1240126 Bologna, Italygabriella.campadelli@unibo.itwww.microbiology-virology.unibo.it/persone_en.html

Partners

Roger EverettMedical Research CouncilMRC Virology UnitGlasgow, United Kingdomwww.mrc.co.uk

Hartmut HengelUniversity of DuesseldorfInstitute for VirologyDusseldorf, Germanywww.uni-duesseldorf.de

Joachim BertramIBA GmbHGoettingen, Germanywww.iba-go.com

Frank NeipelUniversity of ErlangenInstitut fuer Klinische und Molekulare VirologieErlangen, Germanywww.viro-med.uni-erlangen.de

Michael NevelsUniversity of RegensburgInstitute for Medical Microbiology and HygieneFaculty of Medicine Molecular Virology UnitRegensburg, Germanywww.uni-regensburg.de

Angela PontilloPRIMM SrlMilan, Italywww.primm.it

Ivan RossiBioDec SrlBologna, Italywww.biodec.com

Wolfgang Laepple-BoettigerARTTIC S.A.Office MannheimSchifferstadt, Germanywww.arttic.com

Expected results

The major scientific and technological milestonesto be achieved within TargetScreen2 are:• development and establishment of robust

immunofluorescence-based microscopy high-throughput assays to study traffic and/or func-tion of membrane proteins;

• development and establishment of a novel pro-tein complementation platform (PCP) and a split-ubiquitin assay to screen for proteins interactingwith a membrane protein of choice;

• identification and validation of novel therapeu-tic targets following the application of the twoabove innovative approaches to the three dis-ease-associated model membrane proteins;

• target-tailored design and pre-clinical validationof novel therapeutic small-molecules.


Potential applications include identification ofnovel target-tailored lead compounds to threemodel membrane proteins associated with humandiseases (obesity, hyperinsulinemia, hypertensionand cystic fibrosis) and possibly other proteintrafficking disorders. As additional potential appli-cations, novel assays for the identification ofprotein-protein functional/physical interactionsinvolving membrane proteins will result fromthis project.

Background

Membrane proteins include antibodies, receptors,channels, carriers, transporters, etc. and constitutesome of the largest families encoded in the humangenome, including the ATP-binding cassette (ABC)transporters superfamily, the G protein-coupledreceptors (GPCRs) as well as a large number ofchannels. Given their major roles in cells and organ-isms (e.g., communication, signalling, response toenvironmental stimuli, immunity, heart rhythm,multiple nervous system functions, etc.), mem-brane proteins are extremely relevant to a highnumber of human disorders, including commonones (like cardiovascular diseases, obesity, canceror diabetes) and rare genetic diseases. GPCRs andion channels currently represent the most attrac-tive target classes for the drug discovery industry.

Aim

The objective of the TargetScreen2 proposal is todemonstrate the identification of novel therapeutictargets for disorders associated with membraneproteins, using as examples three model proteins,namely: one ABC transporter (the cystic fibrosistransmembrane conductance regulator, CFTR), oneoligomeric channel (the epithelial sodium channel,ENaC) and one GPCR (melanocortin receptor 4,MC4R). To achieve this, TargetScreen2 will developtools and standardise protocols for new post-genomic approaches addressing the functionalstudy of membrane proteins. TargetScreen2 thusproposes to develop and establish several newhigh performance techniques which will be usedand fully exploited so as to demonstrate their appli-cability. Hence, the project’s ultimate aim is to facil-itate the generation of new knowledge in functionalgenomics.

Novel post-genomic cell-based screens for drug targeting in membrane protein disorders

ACRONYM

TargetScreen2

Elucidation of protein function is the nextpost-genomic challenge towards theunderstanding of biological processes inhealth and disease. Transcriptomics andproteomics approaches usually deliverlong lists of “candidate” genes that aresupposedly associated with the respec-tive diseases. However, neither functionalinformation nor a direct relationship withthe pathology is established.

Strategies and tools are thus criticallyneeded to distinguish genes and pro-teins with mere pathologic associationfrom those primarily responsible for thebasic cellular defect(s) in such patholo-gies. Membrane proteins, includingreceptors, channels, antibodies, trans-porters, etc. play major roles in cells andorganisms as they are involved in impor-tant cellular mechanisms, such as com-munication, immunity, signalling andresponse to environmental stimuli.

By bringing together experienced aca-demic and industrial (SMEs) partners ina close and balanced collaboration,utilising a multidisciplinary approach,TargetScreen2 proposes to develop cut-ting-edge post-genomics functional cell-based assays aimed at identifying novelgene targets to correct for traffic and/orfunction of three model membrane pro-teins (MC4R, ENaC and CFTR) involved inhuman disorders. TargetScreen2 will alsovalidate such targets in the most appro-priate cellular systems and, throughstructural modelling of targets, novelsmall molecule compounds will be tai-lored-designed, optimised and tested tothe pre-clinical stage.

The expected results will consist of novelcell-based assays to identify relevanttherapeutic targets in a wide number ofdiseases related to membrane proteins.As a proof-of-concept, TargetScreen2 willapply these innovative ‘from the bench tothe bedside’ approaches to ultimatelydeliver novel therapeutic small mole-cules, tested to preclinical stage andapplicable to common diseases such asobesity, hyperinsulinemia, hypertensionand cystic fibrosis.

SUMMARY

168



Key words: functional genomics, membrane proteins, target-tailored drugs, protein trafficking disorders, protein-protein interactions, robotised confocal microscopy

Three SMEs (ECBio, SygnatureChem and Dualsystems) and one additional company participatein TargetScreen2. Their profiles and roles are, respectively:

ECBio, is a biotech SME that established its own animal and human cell R&D laboratory in2002 and carries out its research and development activities towards the development ofvarious cell isolation and culture processes, including media development, cryopreservationand development of cytotoxicity and drug testing using innovative target cells. ECBio has alsomade a strategic alliance with a recently established Portuguese cell therapy GMP company,being responsible for human stem cell isolation, culture, cryopreservation and differentiation.Within TargetScreen2, ECBio will be mostly involved in the establishment/immortalisationof primary cultures for the production of novel cell lines in order to both validate the proteintargets resulting from the screens in more appropriate cellular systems and also test thetherapeutic effects of small molecules in such systems.

SygnatureChem is a young computational-based drug discovery company with capacities incomputational compound design and synthetic medicinal chemistry, with particular experi-ence and focus on GPCRs and kinases. It has in-house experience in automated and semi-automated library preparation of small- and medium-scale compound libraries resulting fromsynthetic work. It also possesses state-of-the-art analytical and purification technology.The role of SygnatureChem within TargetScreen2 is to design and optimise novel small mole-cules of potential therapeutic interest by specifically modulating the action of the targetsidentified in the screens and to be subsequently tested up to the pre-clinical phase.

Dualsystems major focus is to provide tools and services in the field of interactive proteomicsto the life sciences community. Currently, Dualsystems uses two platform technologies toidentify and characterise novel protein-protein interactions. DUALhybrid is an improved yeasttwo-hybrid platform developed in-house, which allows screening for novel interactors of a pro-tein of interest. This technology is aimed mainly at soluble proteins, domains or protein frag-ments. The DUALmembrane system is a unique screening platform aimed at identifyinginteractions involving integral membrane proteins and membrane-associated proteins.Within TargetScreen2, Dualsystems will further develop this technology as novel geneticassays for protein-protein interactions, based on the split-ubiqutin system and other proteincomplementation assays.

OSIS (until recently, OBS – Olympus BioSystem GmbH) develops integrated digital imagingsystems for life-science applications serving biological, pharmaceutical and medical researchinstitutions worldwide. With the launch of the real-time fluorescence imaging station cell inJune 2003 OSIS was setting a new standard for leading edge research imaging systems withhighest precision and optimum synchronisation. Meanwhile a whole family of cell ImagingStations are available. Based on the real-time technology of the cell Imaging Station a fullyautomated high speed image acquisition and image analysis system for screening applica-tions was developed: the Screening Station scan (responsibility of K Joanidopoulos) that willbe launched in February 2006. OSIS is focused on innovative technology development and hasmore than 10 patents pending or granted. OSIS excels in system integration and has the fol-lowing development expertise: optics, mechanics, electronics, firmware development, soft-ware development, image analysis, data analysis. A well equipped workshop allows for fastprototyping and specific adaptations. OSIS can draw on the competence and experience ofa young team of physicists, biologists, chemists that still have close contacts to research lab-oratories, as well as highly qualified mechanical and electronics engineers and technicians.The role of OSIS in TargetScreen2 will be during the development of confocal microscope-based software to monitor the robotised screening assays, aimed at identifying protein tar-gets involved in the traffic and function of three model membrane proteins, which provesuitable for high-throughput screening (HTS) analyses.

ROLE OF SMEs

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Margarida D. AmaralDepartment of Chemistry and BiochemistryFaculty of Sciences, University of LisboaCampo Grande-C81749-016 Lisboa, Portugalwww.fc.ul.pt

Coordinator Cystic Fibrosis Research UnitCentre of Human Genetics National Institute of HealthAv. Padre Cruz,1649-016 Lisboa, Portugal

[email protected]/docentes/mdbotelho/

Partners

Rainer PepperkokEuropean Molecular BiologyLaboratoryHeidelberg, Germanywww.embl-heidelberg.de

Karl KunzelmannUniversity of RegensburgRegensburg, Germanywww.uni-regensburg.de/Universitaet/welcome2.html

John BC FindlayUniversity of LeedsLeeds, United Kingdomwww.leeds.ac.uk

Constança Coelho Investigação e Desenvolvimentoem Biotecnologia, S.A.Oeiras, Portugalwww.ecbio.com

Simon HirtsSygnature Chemical Services LimitedNottingham, United Kingdomwww.sygnaturechem.com

Daniel AuerbachDualsystems Biotech AG Zurich, Switzerlandwww.dualsystems.com

Konstantin JoanidopoulosOlympus Soft Imaging Solutions GmbHPlanegg, Germanywww.soft-imaging.net

• screening for and identification of inhibitors foreach protein for which a suitable assay has beendeveloped;

• assessment of the effectiveness of the bestinhibitors identified against live M. tuberculosisin vitro;

• optimisation of lead compounds by medicinalchemistry.


TB patients in parts of Eastern Europe and CentralAsia have a significant risk of acquiring multidrug-resistant TB (MDR-TB). TB incidence rates alsocontinue to rise at an alarming rate in Africancountries with high HIV prevalence. This rise ofMDR-TB and increased susceptibility to TB causedby co-infection with HIV is driving the worldwideTB epidemic and is likely to worsen in the years tocome. The discovery of novel antitubercular drugsnot only promises to benefit people in the easternEuropean, Asian and African countries who suffermost from TB, but also to fight the spread of MDR-TB that could fuel a TB epidemic in all of Europe.

By bringing together scientists from many differ-ent disciplines and by connecting academics withindustry research, TB-DRUG establishes an inte-grated drug discovery and development process.The range of complementary expertise availablewithin the proposed Project, together with a state-of-the-art discovery technology, facilitates theentire process of preclinical drug discovery. TheTB-DRUG Consortium strives to have a significantimpact in the area of TB drug development andto contribute to the education of young scien-tists by offering exceptional teaching and train-ing opportunities for post-doctoral fellows andPhD students.

Background

Tuberculosis (TB) causes more deaths worldwidethan almost any other infectious disease, withnearly two million deaths per year and has a dev-astating impact on developing countries. Moreeffective means of medical intervention arerequired, both to reduce the number of deathsfrom tuberculosis and to allow for more effectivetreatment of drug-resistant infections.

Aim

The TB-DRUG project aims to discover novel com-pounds against Mycobacterium tuberculosiswhich can be developed into products that canalleviate the global burden of TB, by carefullyselecting proteins to be taken into the drug dis-covery process as novel targets. In anticipation ofthe significant attrition rate of the drug discoveryprocess, it is suggested that the different targetsare included at the earliest stages of drug devel-opment, followed by a more focused approach onthe most promising candidates in the later stages.The project will be carefully managed by takingadvantage of industrial knowledge in planningand management. As a minimal outcome of thisconsortium, the aim is to identify 2-3 attractiveleads to be taken forward into a preclinical drugdevelopment phase.

Expected results

It is expected that the proposed work will result in:• expression and purification of each protein

being targeted;• structure determination of each protein purified

by X-ray crystallography;• development of biochemical assays suitable for

adaptation to HTS for each target purified;

A SME-STREP for Tuberculosis Drug Development

ACRONYM

TB-DRUGwww.tbdrug.eu

The primary objective of this project is toidentify suitable drug targets and to dis-cover lead compounds suitable for devel-opment into new drugs active againsttuberculosis. The plan of action is to: • express and purify each protein being

targeted using available expressionsystems;

• determine the structure of each proteinby X-ray crystallography;

• develop biochemical assays suitable foradaptation to high-throughput screening(HTS);

• screen for and identify inhibitors for eachprotein where a suitable assay has beendeveloped;

• assess the effectiveness of the bestinhibitors identified against Mycobac -terium tuberculosis in vitro, and;

• optimise lead compounds by medicinalchemistry.

SUMMARY

170



Key words: microbiology, infections, pharmaceutical chemistry, drug targets, biochemistry, structural biology, tuberculosis

Within this Consortium, there are two SMEs:

Vichem is the coordinator and contributes with hit finding and lead optimization against thetarget molecules chosen. Following initial development of biochemical and cellular assaysfrom the different partners within this Project, Vichem will proceed to the development ofhigh-through put screenings assays.

R&D Management GmbH organises and chair consortia meetings and gives generaladministrative support to the management of the project.

ROLE OF SMEs

171


György Keri Vichem Chemie Research Ltd.Herman Ottó utca 151022 Budapest, Hungary [email protected]

Partners

Mamadou Daffé Institut de Pharmacologie et Biologie StructuraleUniversité Paul SabatierCNRS UMR 5089Toulouse, Francewww.ipbs.fr

Menico RizziDipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche (DiSCAFF)University of Piemonte Orientale ‘Amedeo Avogadro’Novara, Italywww.discaff.unipmn.it

Peter SanderInstitut für Medizinische MikrobiologieUniversity of ZurichZurich, Switzerlandwww.imm.unizh.ch

Andrea F. Degen IseliR&D Management GmbHZurich, Switzerlandwww.researchmanagement.ch

© Shutterstock

Expected results

TB-trDNA is designed to develop a rapid diagnos-tic procedure for utilising transrenal DNA as a tar-get sample for the identification of Tuberculosispatients. The findings of TB-trDNA will also con-tribute to policy development through knowledgeand awareness of the importance of TB diagnosis,with close association with the respective min-istries of health and international organisationssuch as the World Health Organisation.


Given the significant challenges of Mtb detectionand monitoring in developing countries, theapplication of the Tr-DNA test could providea very useful new diagnostic tool. By simplifyingthe sampling procedure and combining this withimproved molecular detection methods (whichcould eventually lead to simple dip-stick methods)the findings of TB tr-DNA could ensure that simple,cheap, efficacious TB diagnosis is made availableto the developing world to ensure targeted use ofthe available therapy.

Background

Tuberculosis remains among the most prevalentcauses of death from an infectious disease in theworld. While global targets for rates of cure havebeen reached in many areas, case detectionremains a significant bottleneck to effective dis-eases control. Microscopy, the only widely avail-able laboratory diagnostic test for tuberculosis,is both difficult to implement and insensitive.Consequently, the availability of new diagnostictools that are more accurate and accessible maygreatly benefit individual patients and signifi-cantly contribute to the control of the disease.

Aim

TB tr-DNA aims to develop a new and highly inno-vative platform for the detection of poverty-related diseases (TB followed by HIV, malaria).This platform is based on the principle that dyingcells release cell-free DNA into the blood streamthat then passes through the renal barrier and cansubsequently detected in urine.

Evaluation of transrenal-DNA detection to diagnose tuberculosis

ACRONYM

TB-trDNA

Tuberculosis (TB) continues to be a globalthreat to public health of important socialand financial concern to the expandingEuropean Union and a cause of enormousmorbidity and mortality in much of thedeveloping world. Timely and accuratediagnosis is a critical obstacle to TB con-trol and the currently available diagnosticmethods are marked by being insensitive,slow, and/or cumbersome to use. Nucleicacid amplification is the only rapid detec-tion method with proven sensitivity andspecificity, but is difficult to implement inits current format. A method that avoidedcomplex sputum processing and cell lysissteps that was applicable across multipleamplification formats (e.g. in addition toPCR) would be a tremendous advantage.

There is growing evidence that short DNAfragments, arising from human or bacter-ial cells dying throughout the body, passthrough the renal barrier and appear inurine as transrenal DNA (Tr-DNA). Ina preliminary study conducted at theNational Institute of Infectious Diseasesin Rome, it has been shown that Tr-DNAfrom M. tuberculosis was detectable inthe urine by polymerase chain reaction(PCR) in 100 % of patients with pulmonarytuberculosis and that these DNA frag-ments disappeared following anti-TBdrug therapy. TB tr-DNA aims to validatethe diagnostic potential of Tr-DNA detec-tion for TB, to optimise and simplify thesample preparation methods, and toexplore the feasibility of using a diagnos-tic approach based on this method ina developing world setting.

SUMMARY

172



Key words: transrenal DNA, tuberculosis, diagnosis

The SME of the project, FIND, is an independent, not-for-profit, foundation wholly dedicatedto the development, evaluation, and demonstration of diagnostics for infectious diseasesrelevant for the developing world. FIND has a minor role in most of the WPs of the project,but they will coordinate the interface between test development and product evaluationand conduct a project workshop in 2007 and project public health advisory meeting in2009. FIND will provide documentation and technical expertise related to the customerrequirements and design of the product version(s) to be tested and will ensure that clinicalprotocols will yield data that will have the greatest utility to determining the future of thetechnology for the public health sector.

ROLE OF SMEs

173


Jim HuggettCentre for Infectious Diseases and International HealthWindeyer Institute University College London46 Cleveland St. London, W1T 4JF, United [email protected] www.ucl.ac.uk/medicalschool/infection-immunity/

Partners

Peter Mwaba Department of MedicineUNZA-UCLMS projectUniversity Teaching Hospital D Block Lusaka, Zambia

Michael HoelscherDepartment of Infectious Diseases & Tropical MedicineUniversity of MunichMunich, Germany

Leonard MabokoMbeya Medical Research ProgrammeMbeya, Tanzania

Enrico GirardiDipartimento di EpidemiologiaIstituto Nazionale per le Malattie Infettive L. Spallanzani – IRCCSRome, Italy

Giorgio RoscignoFIND, Foundation for Innovative New DiagnosticsCointrin, Switzerlandwww.finddiagnostics.org

Expected results

TEMPO combines functional genomics, pro-teomics, cell signalling, systems biology andpharmacokinetics to optimise the therapeuticindex in patients. This index in turn determines thechronotherapeutics schedules, according to whichtemporal delivery patterns of the same anticancerdrug vary. Each schedule is adjusted to a differentdynamic class of temporal genomics and phe-nomics parameters, relating to interwoven circa-dian and cell division cycles as well as drugmetabolism. The multidisciplinary nature of theconsortium means that in vivo, in vitro and in silicoapproaches will be integrated to achieve this end.


TEMPO epitomises the translation of basicresearch findings into useful clinical applications.Through the identification of nodes in the interplaybetween the circadian timing system, the cell divi-sion cycle and drug pharmacology parameters, itwill provide critically important information for thetargeted development of new anti-cancer drugs.

Background

Non-communicable, chronic diseases representthe bulk of morbidity, disability and prematuredeaths in Europe, and account for 75 % of disabil-ity-adjusted life years. Among those diseases,cancer is the second most important cause ofmorbidity and mortality. Differences in the molec-ular characteristics of tumour cells, as well as dif-ferences in patients’ genetic make-up, gender,age, lifestyle and circadian rhythms, account forlarge variability in the time-course of cancer andin patients’ responses to treatment.

Aim

The general objective of TEMPO is to design mouseand in silico models that reflect this variability andallow the prediction of optimal chronotherapeuticdelivery patterns for anti-cancer drugs.

Temporal Genomics for TailoredChronotherapeutics

ACRONYM

TEMPO

TEMPO will combine functional genomics,proteomics, cell signalling, systems biol-ogy and pharmacokinetics to optimisetherapeutic efficacy. In vivo, in vitro, in sil-ico approaches are integrated through themultidisciplinary excellence in the consor-tium. TEMPO will offer a proof of principleof tailored chronotherapeutics in mousemodels for irinotecan, an active drugagainst colorectal cancers, and for selici-clib, currently in clinical testing. TEMPOwill gather the corresponding human pre-requisites and technology for subsequentapplication to patients.

SUMMARY

174



Key words: cell cycle, circadian clock, chronotherapeutics, anti-cancer drugs

Three SMEs play a pivotal role for the impact of TEMPO on European health, economics andsociety. Novel and complementary in silico dynamic models of coordinated clock, cell cycleand pharmacology pathways will identify new therapeutic targets and delivery schedules ofactive molecules, thus improving drug development processes.

ROLE OF SMEs

175


Francis LéviInstitut National de La Santé et de la Recherche MédicaleINSERM U776 – Rythmes biologiques et cancersHôpital Paul BrousseAvenue Paul-Vaillant Couturier 14Villejuif, [email protected]

Partners

Franck DelaunayCentre National de la Recherche Scientifique (CNRS)Université de Nice – CNRS UMR 6348 – Bâtiment de Sciences NaturellesPhysiologie cellulaire et moléculaire des systèmes intégrés Nice, France

Laurent MeijerCentre National de la Recherche Scientifique (CNRS)Laboratoire Mer et santé UMR7150Station Biologique – Amyloïds and Cell Division Cycle Roscoff Cedex, France

Jean ClairambaultInstitut national de recherche en informatique et automatique INRIA Rocquencourt Research Unit – Teams Bang and ContraintesLe Chesnay, France

Stefano IacobelliConsorzio Interuniversitario Nazionale per la Bio-oncologia (CINBO)Laboratori of molecular oncology center of excellence on aging Ce. S.I.Chieti, Italy

Marco PirovanoH.S. Hospital Services S.p.ATherapeutic deliveryAprilia (Latina), Italy

Todor VujasinovicHelios Biosciences SARLCréteil, France

Christophe ChassagnolePhysiomics PLCThe Magdalen Centre The Oxford Science ParkOxford, United Kingdom

Isabelle GeahelInserm TransfertEuropean Project Management DepartmentParis, France

Metabolism

Proliferation

| Schematic representation of cellular circadian rhythmsThe expression of many genes, proteins and enzymatic activities responsible for cellular metabolism and proliferation display marked 24-hour rhythms inhealthy mammalian tissues. These rhythms are generated by a molecular clockconstituted of 12 specific genes.

Aim

The aim of this research programme is to exploitthe ‘non-self’ recognition and binding proper-ties of human apolipoprotein H (ApoH) for thedevelopment of novel tools to isolate pathogensfrom complex biological mixtures. ApoH bindspathogens enabling their capture and concentra-tion from different biological samples. Magneticbeads coated with ApoH protein can be efficientlyused as a pre-treatment step to greatly improvethe detection threshold and thereby increasing thesensitivity for diagnosis of emerging pathogens,regardless of the molecular or immunologicaltechniques used in the final diagnostic step.

Expected results

The project will focus on:• increasing the sensitivity and specificity of the

currently available detection methods used forpathogenic bacteria and viruses;

• on the development of novel techniques forpathogen detection from clinical and environ-mental samples including those that could beused in bioterrorism.


The industrial partners will develop and standard-ise novel technologies for rapid, multiple and ultra-sensitive pathogen diagnosis such as mini-arraysystems with the objective of commercialisation.

Background

In the 1970’s WHO proclaimed that eradication ofsmallpox should be attempted. This goal was suc-cessfully achieved in 1979. Nonetheless, presentlythere is a general consensus that the list of newlyemerging or re-emerging pathogens is continu-ously growing. Indeed, during the last decadespathogens such as Marburg, Ebola, Hepatitis-C,Hantavirus, HIV and more recently, SARS corona -virus have emerged. Furthermore, the apparentrisk of a new influenza pandemic again highlightsthe global threat of infectious diseases. In addi-tion, the possibility of bioterrorist attacks usinghighly pathogenic viruses and bacteria can not beignored. In consequence, the current require-ments for novel, highly sensitive and specific diag-nostics technologies have increased.

A major obstacle for the detection of pathogens inclinical or environmental samples are false nega-tive results, e.g. for HCV ‘occult infections’. This ismainly due to the lack of a rapid and reliablepathogen concentration methodology, and theinability of most of the currently used tech-nologies to eliminate or neutralize interferingmolecules, ‘natural inhibitors’, present in mostcomplex samples.

Capture and enrichment of emerging pathogens for multiple and ultra-sensitive diagnostic

ACRONYM

USDEPwww.usdep.eu

The threat of emerging or re-emerginginfectious diseases, as well as the risk ofbioterrorism, has enhanced the currentrequirements for novel, highly sensitiveand specific diagnostics technologies. TheUSDEP project will focus on improving thedetection of emerging pathogens, devel-oping novel techniques for pathogeniso lation and detection from clinical andenvironmental samples, including thosethat could be used in bioterrorism, andincreasing the sensitivity and specificity ofthe currently available detection methodsused for pathogenic bacteria and viruses.

SUMMARY

176



Key words: virus diagnostics, ultra sensitive detection, magnetic beads, Apolipoprotein H, emerging pathogens

The USDEP project has a very complementary consortium, where the publicly funded part-ners, the Robert Koch Institut (RKI), the Institut de Recherches pour le Développement(IRD), the privately funded Pontifica Universidad Catolica de Chile, working in virology andpublic health will incorporate ApoH technology into their panel of regular techniques forpathogen detection. It will be the task of the industrial partners to develop and standardisethe new diagnostic technologies. Namely, ApoH-Technologies develop ApoH coated supportsfor diagnostic purposes, GenExpress is specialized in the development and optimisation ofmolecular biology assays, the Institut für Siliziumtechnologie (ISIT) is specialised in thedevelopment and production of microelectronic components and will supply the electronicbiochips, eBiochip Systems is focussed on manufacturing of technology for electronicbiochip applications, SKULD-TECH develops a mini array system for virus detection andIMMUNOCLIN provides strategic direction as well as management and laboratory servicesfor clinical development and pre-clinical contract research.

ROLE OF SMEs

177


Heinz EllerbrokRobert Koch-InstitutCentre for Biological SafetyNordufer 20133353 Berlin, Germany [email protected]

Partners

Dorothy BrayImmunoClin Ltd.London, United Kingdomwww.immunoclin.com

Elias StefasApoH-TechnologiesMontpellier, France

Francisco VeasViral Immuno-Physiopathology LabU178/IRD Viral Emerging DiseasesEA 3755 (Bacteriology-Virology)University of Montpellier 1Montpellier, France

Marcelo Lopez LastraPontificia Universidad Católica de ChileSantiago, Chilewww.puc.cl

Roland Lauster GenExpress Gesellschaft für Proteindesign mbH Berlin, Germany www.genexpress.de

Rainer HintscheFraunhofer Institut für Silizium TechnologieItzehoe, Germanywww.isit.fhg.de

Ralf WörleBiochip Systems GmbHItzehoe, Germanywww.ebiochipsystems.com

Didier RitterStamatis VarsamosSkuld-TechMontpellier, Francewww.skuldtech.com

the development of mathematical models of thecomplex molecular interaction networks (MINs).

Moreover, this approach is emerging also in LifeSciences, with systems pathology guiding an under-standing of the multidimensional aspects of diseasesystem fingerprints and systems pharmacology.This provides important insights into dynamic sys-tem responses upon multiple drug perturbations.Knowledge of the changes of system characteristicsduring the progression of a disease is mandatoryto create a conceptual framework for the design ofnew therapeutic strategies.

Aim

The aim of the VALAPODYN project is to set up thescientific and technological basis, for tasks withinthe following areas:• Pathway analysis: functional annotation of genes

and proteins, investigation of structure anddynamics of signal transduction and transcriptionregulatory networks.

• Predictive bioinformatics platform for dynamicmodelling: use of innovative biomathematics/bioinformatics to integrate experimental MINdata with biological tissue and pathologicalstates data obtained through the use of tran-scriptomic and proteomic approaches.

• Bioinformatics: establishment of a highly spe-cialised database on the genomics and proteomicsof MIN modelling.

• Pathological tissue & animal models: analysis ofvalidated animal models of brain pathologies toevaluate gene/protein expression during initialcell death.

• Microarrays: extensive multi-level global geneexpression profiling using the Affimetrix platform.

• Proteomics: application of advanced quantitativeproteomics technologies (MALDI, ICAT, 2-DPAGE,Heavy Peptides isotopic dilution) for large-scaleproteome screening.

• Neuroprotective molecules: characterization ofmolecules in the MIN of cell death the modulationof which should improve or cure neurodegenerativebrain disease.

Background

Selecting the best therapeutic target(s) is one ofthe major challenges in developing new and effi-cient drugs. In addition to its scientific relevance,it has a profound impact on the health/quality oflife of the European population, as well as thepharmaceutical industry, by decreasing the costof drug development.

Nowadays, although our understanding of individ-ual gene and protein function becomes important,it is not sufficient for unravelling the complexity ofbiological and pathological processes! Indeed,modern high throughput technologies in biologi-cal science, such as genomics, transcriptomicsand proteomics, often generate lists of moleculesof interest. However, they do not always increaseour knowledge of the biological processes or thesubsequent identification of therapeutic targets.It therefore becomes critical to develop our under-standing of biological system’s structure anddynamics.

A biological system is more than an assembly ofgenes and proteins. Its properties cannot be fullyunravelled through static diagrammatic representa-tions of their interconnections. Although such dia-grams represent an important step, we now needinformation on the dynamics of these interconnec-tions and how we can control them. The challenge istherefore, to construct a descriptive model fromthese lists of molecules that could reflect theunderlying biological mechanisms as accurately aspossible and, ultimately, allow the identificationand selection of the best therapeutic targets totreat human disease.

The aim of Systems Biology is to decipher the intri-cacies of complex biological systems/organismsthrough the integration of biological, clinicalchemical, physical, mathematical, and computerknowledge.

Their approach uses theorical concepts, compu-tational modelling and experiment data to allow

Validated Predictive Dynamic Model of Complex Intracellular PaCell Death and Survival

ACRONYM

VALAPODYNwww.valapodyn.eu

The VALAPODYN project seeks to furtherthe development of multidisciplinary func-tional genomics relating to complex bio-logical processes and cellular networks.The project is concerned with both DNAand protein applications, to be followed byinnovative dynamic modelling of pathological disease states, such as epilepsyand cancer, in order to validate the model.The overall aim is to develop an innovativesystems biology approach, in order tomodel the dynamics of Molecular Inter -action Networks (MINs) related to celldeath and survival in the organism.

SUMMARY

178



Key words: functional genomics, predictive dynamic models Molecular Interaction Networks, cell death and survival, neurodegeneration

Expected results

The VALAPODYN network is composed of leadingauthorities in the fields of genomics, proteomics,bioinformatics and neuroscience in Europe. Theyhave decided to join their efforts to develop a newinnovative System Biology approach to model thedynamics of Molecular Interaction Networks (MIN)related to cell death and survival in the brain. Thismodel will be dedicated to the selection of drugtargets for human brain.

The project will first validate dynamic models forcell death through the characterisation of newpotential drug targets in an animal model forepilepsy where neurodegeneration is the initialstep of the development of epileptic seizures.


The development of new and unique dynamic mod-elling tools will allow the consortium to participatein the process of applying integrative biology topathology research. This should significantlyimprove the quality of life for EU citizens, byadvancing the identification of new generations ofmore efficient drug targets; these drugs will beused to treat numerous diseases accounting formortality and several serious illnesses in the EU,such as cancer, cardiovascular diseases, neurolog-ical diseases, etc. Dynamic models will form the

basis for the next generation of biological valida-tions for novel therapeutic targets, instead of themethods currently in use, namely those involvinganimal models for drugs acting symptomatically atend receptors and effectors.

VALAPODYN will also have a significant impact onthe ERA, by creating a new foundation for theexchange of fundamental research and know -ledge. The development of the internationalR&D network of SMEs in the biotechnology sec-tor (HELIOS, BIOBASE and SynapCell throughINSERM during the project) will accelerate theemergence of the EU as a powerful contender inthe global technological market. The VALAPODYNconsortium will also allow for optimal use of theavailable EU resources and human potential.

To disseminate the project results as widely aspossible, several different levels of informationdistribution are proposed. This will involve pre-sentations and forums for discussion, both ina scientific context (doctors, researchers, neuro-science students and collaborators in the field ofneurodegenerative disease research), and alsodirected towards pharmaceutical industrials andEuropean citizens.

There are two SMEs in this consortium Biobase GmbH and Helios Biosciences SARL.

The role of Biobase GmbH in the Valapodyn project is to provide high quality manually anno-tated databases to the consortium and applying them to the particular biological field inorder to systematically produce experimentally testable hypotheses. As Valapodyn isdevoted to study mechanisms of neurodegeneration, Biobase focuses on collecting relevantdata including regulation of cell cycle, apoptosis and mechanisms of neurodegeneration.BIOBASE, in collaboration with the other partners, plans to integrate data coming frompartners, and, provide this information to all partners. This activity is in the frame of WP3.

Instead, Helios BioSciences will select and classify the therapeutic targets or combina-tion of therapeutic to cure neuro-degenerescence. Helios activity is at the cross-road ofthe different work-packages of the project as Helios has to integrate (with the help ofBiobase) Molecular Interaction Network and Biological data to build Dynamic Models ofMolecular Interaction Networks and select therapeutic targets which will be then validatedin vivo. In addition to that Helios will commercialize the validated therapeutic targets.

ROLE OF SMEs

179


Antoine DepaulisGrenoble – Institut des Neurosciences Centre de recherche Inserm U 836Université Joseph FourierBP170, 38042 Grenoble Cedex, [email protected]://neurosciences.ujf-grenoble.fr/

Partners

Olga Kel-MargoulisEdgar WingenderBiobase Wolfenbuettel GmbH Germanywww.biobase-international.com/pages

Todor VujasinovicHELIOS Biosciences SARLCréteil, Francewww.helios-bioscience.com

Despina Sanoudou Foundation of Biomedical Research of the Academy of Athens FBRAA Molecular Biology Division Center for basic research Athens, Greece

Edwin De PauwUniversity of Liege ULG Department of chemistry Mass Spectrometry Laboratory Liège, Belgium

Hermona SoreqHebrew University of Jerusalem Department of Biological ChemistryInstitute of Life Sciences Jerusalem, Israel

Raffaella CatenaAlma Consulting GroupFrancewww.almacg.com

attributed to refractoriness of such diseasegroups to angiogenic growth factors. Thesemodels and strategies will not only be of valuefor the present project but also for the devel-opment of pro-/anti-angiogenic medicines ingeneral.

• Once the pro-angiogenic effect of PlGF will beestablished in rodent models, these effects needto be confirmed and validated in large animalmodels that are physiologically more closelyrelated to the situation in patients. Thereforenewly established models of cardiac ischemia inpigs and in baboons will be used. Demonstrationthat PlGF stimulates vessel growth in diseasesconditions closely related to the situation inpatients will markedly increase the likelihood ofsuccess in humans.

2. Further development of the production process.• The production process has been transferred from

its original site of development to an industrialmanufacturer for further development so as to fitwith final production scale and GMP constraints.

• State of the art application of ‘in process’ and‘final’ quality and stability control will requireadditional validation of new assays.

• Discovery research will be performed on compoundimprovements, involving Cys-to-Gly substitutionfor improved stability and alternative isoforms(glycosylated PlGF-1 and PlGF-2 produced inChinese hamster ovary (CHO) cells).

3. Determine the potential of PlGF as biomarker forcardiovascular disease.

• Population study: A database, recently developedby IGB, holds the description of the health status,the genealogy and the genome-wide microsatellitemarker scan of approximately 4 000 individualsfrom genetically isolated villages in Italy. Thisdatabase will be studied to determine the con-tribution of PlGF and VEGF to specific humancardiovascular diseases and cancer.

• Development of a reliable diagnostic test formeasurement of PlGF concentrations in humanblood samples based on compounds fromGeymonat and ThromboGencics/Thromb-X. It isintended to evaluate the degree of angiogenesis

Background

Ischemic heart disease (IHD) and peripheral arte-rial occlusive disease (PAOD) are the major causeof severe morbidity and mortality in Western soci-eties. Patients who presently survive acute coro-nary events as a result of coronary intervention(angioplasty, stenting or coronary bypass surgery)or pharmacological coronary artery reperfusionoften develop congestive heart failure that is resist-ant to intervention or pharmacological treatment.Thus, the progress in the treatment of cardiovascu-lar disease has converted acute lethal syndromesinto a chronic debilitating disease. Therapeuticangiogenesis is a novel treatment paradigm butongoing clinical trials with the angiogenic proteinsVEGF and bFGF have not achieved convincing pri-mary endpoints of improved tissue perfusion andfunctional recovery. Therefore, current strategiesmust be reconsidered and alternative angiogenictargets validated.The pro-angiogenic drug candidate PlGF, unlikeVEGF, has in proof of concept studies been demon-strated to target pathological angiogenesis and notphysiological angiogenesis and to be devoid ofmost of the systemic side effects associated withadministration of VEGF. In initial proof of conceptstudies, PlGF levels in blood have been shown to bea distinct biomarker for cardiovascular disease.

Aim

The aim is threefold and summarised below.

1. Determine the potential of PLGF as therapeuticfor major chronic progressive ischemic cardio-vascular disorders.

• Determine the pro-angiogenic effects of PlGF on:– Revascularisation of ischemic limbs. This will

be studied in mice, rats and in rabbits.– Revascularisation of ischemic cardiac tissue.

This will be investigated in mice. These modelswill be of critical importance as these diseasesare prevalent in patients eligible for therapeu-tic angiogenesis, and the limited success ofclinical trials with VEGF and basic fibroblastgrowth factor (bFGF) has indeed been in part

Placental Growth Factor (PlGF): new diagnostic and therapeutic applications in cardiovascular disease

ACRONYM

VASOPLUSwww.vasoplus.eu

The Vasoplus project evaluates the poten-tial of PlGF as a pharmaceutical for majorchronic progressive ischemic cardiovas-cular disorders (ischemic heart disease,heart failure) and arterial insufficiency(intermittent claudication and critical limbischemia) and as a biomarker for cardio-vascular disease. The project is based onbasic research on the VEGF-signalingpathway (via PlGF) performed over thelast five years (CTG, VIB-3), prototyperesearch performed with recombinantPlGF for pro-angiogenesis (GT and TG/TX),basic research on the association ofplasma PlGF and VEGF levels and humandiseases (IGB) and the development ofa diagnostic test to evaluate PlGF asa biomarker for cardiovascular disease.

The comprehensive strategy encom-passes the validation of the ‘proof ofconcept’ observations made by the aca-demic partners of the consortium intotherapeutic concepts in new relevantsmall animal models, development ofindustrial scale GLP/GMP quality materi-als and confirmation of their safety andefficacy in new, relevant large animalmodels. The strategy will be applied tothe development of pro-angiogenicrecombinant PlGF in ischemic heart dis-ease models and in peripheral arterialinsufficiency models. In parallel, thevalue of PlGF as a predictive biomarkerand as a diagnostic test for cardiovas-cular disease will be explored in a collab-orative effort with a large diagnosticcompany.

The principal aim of the project is todevelop a new, safe and efficacious med-icine to enhance the formation of bloodvessels and in doing so, to develop newtreatment paradigms for heart failureand critical limb ischemia.

SUMMARY

180



Key words: placental growth factor, PlGF, ischemia, cardiovascular disease

based on PlGF blood levels, together with otherbiomarkers of cardiovascular disorders, in a well-characterised Italian cohort with the intent tostratify patients who might benefit from proan-giogenic treatment.

Expected results

Expected results for the different objectives are asfollows.

1. Determine the potential of PLGF as therapeuticfor major chronic progressive ischemic cardiovas-cular disorders.

• Efficacy analysis of rhPlGF-1 for revascularisationof ischemic myocardium and limb muscle.

• Comparative analysis of rhPlGF-1 efficacy forrevascularisation of ischemic tissue in rat andrabbit ischemia models.

• Comparative analysis of rhPlGF-1 efficacy forrevascularisation of ischemic tissue in a mousemodel of ischemic cardiac failure.

• Data on the percentage of animals that develop adysfunctional and ischemic myocardium versusthose that develop significant transmural necrosiscaused by acute occlusion of the stented seg-ment. This will allow the project to determine thenumber of animals required to enter the study.

• An evaluation of the size of dysfunctional but viablemyocardium by magnetic resonance imaging withdelayed enhancement as well as dobutaminestress echo and quantitation of global LV functionallowing power calculations for the NNT in order toobserve a significant increase in global LV function(increase in Ejection Fraction by 4-6%).

• Perfusion studies to quantify the degree ofmyocardial perfusion both in the endo- and epi-cardial myocardium. The effect of intervention onLV function will be expected within 1.5 years.

2. Further development of the production process.• GLP qualified and GMP qualified rhPlGF-1. • High yield production of the mutein. • Stability of the mutein in solution and gel analysed. • Biological activity of the mutein verified by in vitro

assays. • Small scale preparations available of CHO-pro-

duced rhPlGF-1, rhPlGF-2 and rhPlGF-1CG. • Validation for each assay development. • GLP compliant certification/recognition of the

Belgian Monitoring authorities based on a fictiveGLP stability study with the aim to determine theshelf life and stability on rhPlGF-1.

3. Determine the potential of PlGF as biomarker forcardiovascular disease.

• Correlation of the plasma levels of VEGF and PlGF(-1 and -2) and health status of 2 800 samples byanalysis of the project database.

• Definition of the VEGF and PlGF haplotypes in2 800 individuals.

• Association analysis of VEGF and PlGF variants andhaplotypes with a trait or disease.

• Status of PlGF assay development including pre-analytical conditions.

• Completion of sample measurements using PlGFprototype assay, sCD40L and NT-pro-BNP diag-nostic assays.

• Correlation of plasma levels of PlGF, sCD40L, NT-pro- BNP and health status of the study individuals.

• Multi-variate analysis of the study completed.


Two possible applications are being pursued withthis programme: a new, safe and effective medi-cine to enhance the formation of new blood vesselsin ischemic tissues and a new diagnostic tool forcardiovascular disease.

Three European SMEs based in Ireland, Belgium and Italy are involved. The project is led byThrombogenics/Thromb-X, an Irish biopharmaceutical company focused on the clinicaldevelopment of cardiovascular drugs in-licensed from academic institutions via its R&Daffiliate Thromb-X. Their research focuses on the preclinical development of biopharmaceu-ticals for innovative treatments of severe diseases as well as on the development of improvedtechnologies for transgenesis in mammalian species and for regenerative medicine. An Italianpharmaceutical SME, Geymonat SpA, has experience in translational biopharmaceuticalresearch and development. A Belgian biotech SME, Eurogentec S.A., will provide valuableexpertise in industrial development of recombinant PlGF compounds.These SMEs will receive 58 % of the project budget. There is a well-balanced integration ofthe industrial partners (three SMEs plusl a large company) and academic groups, orientatedtowards industrial exploitation of expected results.

ROLE OF SMEs

181


J.M. StassenThromboGenics Ltd.14 Bridgecourt Office ParkWalkinstown AvenueDublin 12, [email protected]

Partners

Mauro BattistiGeymonat SpAAnagni, Italywww.geymonat.com/profilo.htm

Florence Xhonneux Eurogentec S.A.Liege Science ParkSeraing, Belgiumwww.eurogentec.com/eu-home.html

Stefan JanssensKULeuvenLeuven, Belgium

Maria G. Persico Institute of Genetics and Biophysics A.B.T.Naples, Italy

Philip Badenhorst University of the Free StateBloemfontein, South Africawww.uovs.ac.za

Georg Hess Roche Diagnostics GmbHMannheim, Germanywww.roche.de/diagnostics/index.htm

| More visible collateral vessels after 7 days ofPIGF treatment in rabbit hindlimb ischemia.

Placebo

PIGF treatment

Aim

The objective of VITAL is the development andproduction of optimized recombinant idiotypicvaccines for the treatment of subgroups of lym-phoproliferative disorders expressing molecularlycorrelated idiotypes. These vaccines will beincluded in new formulations for innovative trialsof immunotherapy potentially targeting a largefraction of lymphoma/leukemia patients.

Expected results

• Establishment of a large database includingsequences of idiotypic VH and VL genesexpressed by a variety of lympho-proliferativedisorders, including low grade B-NHL, autoim-munity-associated lympho-proliferations, andchronic lymphocytic leukemia. This will allowthe identification of candidate Id proteins for‘cross-reactive’ immunotherapy.

• Pre-clinical characterization of the immuno-genicity of selected natural Id proteins, with par-ticular regard to their ability to induce immuneresponses against lymphoma cells expressingmolecularly correlated Id proteins. The charac-terization will include the identification of B cellepitopes and HLA Class I-restricted cytotoxic Tcell epitopes using innovative approaches andwill allow the development of dedicated assaysfor immunomonitoring.

• Design and validation of optimized Id vaccines.

• Evaluation and validation of new adjuvants andinnovative delivery systems for improved Idvaccine formulations and administration.

• ‘Clinical-grade’ production and purification ofoptimized Id proteins for patient vaccination.

The SMEs are an integral part in the project inmaking the new diagnostic and therapeutic toolsavailable, not only for Europe but also for theworld market. The close integration between clin-ical and research activities at several university

Background

Non-Hodgkin’s lymphomas (NHL) constitutea heterogeneous group of malignancies whoseincidence has significantly increased in recentdecades. In the year 2000, more than 145 000cases of NHL were diagnosed in developed coun-tries, representing thus the sixth most commoncancer occurring among men and the eighthamong women. Low-grade B-cell NHLs, in par-ticular, are incurable diseases characterized byrelatively slow growth and excellent initial respon-siveness to chemotherapy but also by continuousrelapses. In particular, for patients with follicularlymphoma, median overall survival (7-10 years)has not improved over the past 30 years.Although in the vast majority of patients completeor partial remissions can be obtained with eithersingle agents or combination chemotherapy, theclinical course is characterized by a high relapserate. After relapse, both the response rate andrelapse-free survival after subsequent salvagetreatment regimens steadily decrease, resultingin a median survival of only 4-5 years after the firstrelapse. These clinical findings, coupled with thesubstantial toxicities of standard treatments,have stimulated the search for novel and moretumor-selective therapies. Therapeutic vaccinestargeting B cell lymphoma idiotype (Id) representa promising immunotherapeutic approach fora better clinical control of these malignancies.This strategy is based on the observation thatimmunoglobulins (Ig) expressed by neoplastic Blymphocytes carry unique determinants in theirvariable regions (idiotypes), which can be recog-nized as tumor specific-antigens. Indeed, bothprotein- and dendritic cell-based vaccines thatuse the patient-specific Id have resulted in clini-cally significant tumor-specific cellular responseswith very little toxicity. A broad use of Id-basedvaccination for B cell lymphomas, however, ishampered by the fact that these approaches arepatient-specific so that the vaccine must be indi-vidually produced for each patient. On thesegrounds, new strategies obviating the need toproduce customized vaccines would further sim-plify clinical applications of idiotypic vaccines.

Development of optimized recombinant idiotypicvaccines for subset-specific immunotherapy of B cell lymphomas

ACRONYM

VITALwww.cro.sanita.fvg.it/progetti/vital/index.htm

Therapeutic vaccines targeting B cellnon-Hodgkin lymphoma (NHL) idiotype(Id) represent a promising approachagainst these malignancies. A broad useof Id-based vaccination, however, ishampered by the complexity and costsdue to the individualized production ofthese vaccines. Recent evidence indi-cates that these limitations may be over-come. In fact, distinct sets of stereotypedimmunoglobulins have been identified invarious B-NHL, suggesting that patientsshare Id with a higher frequency thanappreciated previously. Through thecomplementary and synergistic work ofacademic partners and three SMEs, weplan to exploit the molecular featuresof Id proteins of distinct B cell lym-phomas/leukemias, particularly thosepathogenically associated with antigenstimulation and/or selection, to developpre-made, recombinant Id proteins tovaccinate subgroups of lympho-prolifer-ative disorders expressing molecularlycorrelated idiotypes. A database of Idsequences expressed by different B-NHLwill be constructed to identify subgroupsof tumors expressing molecularly corre-lated Id proteins. Selected Id proteins willbe characterized for their immunogenic-ity and, particularly, for the ability toinduce cross-reactive immune responsesagainst related Id proteins. B and T cellepitopes will be identified using innova-tive approaches and dedicated assaysfor immunomonitoring will be devel-oped. Optimized versions of selected Idvaccines will be produced using newstrategies and validated in animal mod-els. New adjuvants and delivery systemsfor improved Id vaccine formulations andadministration will be also evaluated andvalidated. The most promising Id proteinswill be produced and purified accordingto GMP standards and included in newvaccine formulations for innovative trialsof ‘cross-reactive’ immunotherapy.

SUMMARY

182



Key words: lymphoma, leukaemia, vaccine, idiotype, immunotherapy

hospitals and Cancer Centers with the SMEs willform new centres of excellence where EuropeanSMEs will benefit from close collaboration at thesame time as new diagnostic and therapeuticproducts will be developed to the benefit ofpatients with lymphoid malignancies.


The results obtained in the present project willallow the design and activation of phase I/II clin-ical trials aimed at validating the use of opti-mized, pre-made vaccines for the treatment ofa relatively broad spectrum of lymphoid malig-nancies. The proposed Id vaccination may be ben-eficial also for patients with pre-neoplastic B-celllymphoproliferations, such as mixed cryoglobu-linaemia. These vaccines, in fact, may be usedwith the purpose to alleviate the symptoms and,ultimately, to prevent the possible evolutiontowards an overt B cell malignancy. Once vali-dated as drugs, the vaccines will have the advan-tage to be easily distributed to all Hematologyand Oncology Departments, including those ofperipheral Hospitals/Universities. Thus, resultsobtained in the present project will have animportant strategic impact in solving, at least inpart, the dramatic social and health problemrepresented by NHL.

Three SMES, out of seven participants, play a key role in the research activities of the project.

In particular, PEPSCAN will be responsible for design, preparation and screening of syn-thetic peptide libraries and for the reconstruction of interaction sites in the complexesincluded in VITAL. It will coordinate the mapping of B cell epitopes and test the efficacy ofnew adjuvants.

ProImmune Ltd. will be involved in the identification of HLA Class I-restricted CTL epitopes.It will do so by using its Pro5® MHC Pentamers and ProVE™ MHC Pentamer Libraries. In closecollaboration with PEPSCAN, optimised versions of selected Id vaccines will be producedusing different new strategies to enhance both humoral and cellular immune responses.

Areta International will, through process development and manufacturing of tailored-madebatches, produce the most promising Id proteins according to GMP standards. If necessary, itwill define innovative technological approaches for GMP scale-up production and purificationof selected recombinant Id proteins.

ROLE OF SMEs

183


Riccardo DolcettiCentro di Riferimento Oncologico – IRCCSNational Cancer InstituteVia Franco Gallini, 233081 Aviano, [email protected]

Partners

Bjarne BogenUniversity of Oslo and Rikshospitalet University HospitalOslo, Norway

Maria MasucciKarolinska InstitutetStockholm, Sweden

Antonio RosatoDepartment of Oncology and Surgical SciencesUniversity of PadovaPadova, Italy

Hans Petrus Maria LangedijkPepscan Therapeutics B.V.Lelystad, The Netherlandswww.pepscan.nl

Nikolai SchwabeProImmune LimitedOxford, United Kingdomwww.proimmune.com/ecommerce

Maria Luisa NolliAreta International S.r.l.Gerenzano (VA), Italywww.aretaint.com

| Flow chart, outlining the main expectedresults, leading from identificationof shared idiotypes to the developmentof optimized vaccines for the treatmentof B-cell lymphoproliferations.

metastasis in an optically transparent vertebratemodel organism. However, for the realisation of this complex screen-ing system, the identification of relevant diseasemarker genes in zebrafish represents a crucial step.In ZF-TOOLS, different genomics approaches will beused to discover novel markers, which will be suit-able for application in the ZF-TOOLS tumour screen-ing system and will also have a broader utility fordisease research in the zebrafish model. The exper-imental design of this genomics approach isexpected to result in the identification of twoclasses of markers, namely markers correlatingwith growth and metastasis of tumour cells andmarkers correlating with the immune or otherdefence responses of the organism towards tumourcells. The project will concentrate on both classesof markers because the interactions betweendeveloping tumours and the tumour microenviron-ment are decisive for tumour survival or rejection.

Expected results

The strategic aim of ZF-TOOLS is the developmentof a zebrafish embryo screening system as aninnovative genomics tool. This system will beemployed for high-throughput effectiveness test-ing of pharmaceutical compounds that have thepotential to influence disease processes, includingtumour growth, metastasis and immune defenceresponses. This zebrafish screening tool offerssome unique features that make it very attractive incomparison with existing tools. First, it combinesthe power of genomic analysis of disease markergenes with the power of in vivo monitoring of dis-ease processes in a transparent vertebrate modelorganism. Secondly, it has the potential for high-throughput application due to the small size ofzebrafish embryos, to the high numbers with whichembryos can be obtained, and to the choice ofhigh-throughput molecular screening tools thatwill be developed within the project. In order to establish the zebrafish screening tools,the project will undertake a multidisciplinaryfunctional genomics approach which integratesdifferent global expression profiling techniquesand bioinformatics. Based on this approach, the

Background

Human disease research and drug developmentrely heavily on the use of animal models. Amongthese, the mouse model is the most intensivelystudied. However, over the last decade thezebrafish has emerged as an attractive alternativemodel and has progressively gained importance.This is due to the fact that the zebrafish offersexciting novel research opportunities because ofthe optical transparency of its embryos and itsamenability to genetics. To date, the value ofzebrafish in pharmacological studies has not yetbeen extensively explored and exploited. However,findings emphasise the potential of using zebrafishin several phases of drug discovery processes andin toxicological screens.The ZF-TOOLS project is focused on the incorpora-tion of zebrafish into the preclinical drug screeningpipelines. The small size of zebrafish embryos andthe large numbers in which they can be obtainedmake them particularly suitable for high-through-put drug screens. Furthermore, drugs can be easilyapplied to zebrafish embryos through dilution inthe embryo medium and absorption through skinand gills.

Aim

The project aims to develop a case study for an anti-tumour drug screening system, based on theimplantation of fluorescently labelled tumour cellsinto zebrafish embryos. This innovative tumour cellimplantation system is currently being developedby one of the SME partners and has the majoradvantage that it does not involve the use of trans-genic animals. Growth and metastasis properties ofimplanted tumour cells can be efficiently monitoredby fluorescence microscopy during the develop-ment of the transparent zebrafish embryos. Thissystem resembles the natural situation of tumourgrowth, as the tumour cells are derived fromzebrafish cell cultures of embryonic origin andimplanted back into zebrafish embryos. It is envis-aged that a powerful screening system can arise bycombining high-throughput marker analysis withthe possibility to visualise tumour growth and

High-throughput Tools for Biomedical Screens in Zebrafish

ACRONYM

ZF-TOOLSbiology.leidenuniv.nl/ibl/S1/research/ZF-TOOLS

The ZF-TOOLS project comprises the coor-dinated efforts of three research laborato-ries and three SMEs aimed to achievethe following two main objectives: First,a genomic-based marker discovery forbiomedical screens in zebrafish and, sec-ondly, the use of high-throughput markeranalysis and tumour cell implants forthe identification of tumour growth andmetastasis factors and organismaldefence factors.

SUMMARY

184



Key words: zebrafish embryo model, oncogenic cell implants, anti-tumor drug discovery, reporter cell lines, tumor markers,immune response markers, expression profiling

ZF-TOOLS project expects to achieve the followingresults: • knowledge of tumour growth and metastasis

factors and organismal defence factors; • high-throughput tools for quantitative analysis

of disease marker sets; • a collection of constitutive and inducible, onco-

genic and non-oncogenic reporter cell lines use-ful for basic disease research and for applicationin screening systems;

• case study results of a novel anti-tumour drugscreening system, based on the implantation offluorescently labelled tumour cells into zebrafishembryos.


The ZF-TOOLS project aims to reinforce Europeancompetitiveness by generating strategic know -ledge, thanks to its multidisciplinary researchapproach. The developed tools and technologywill be exploited for basic research on vertebratedisease and for strategic research and serviceactivities on behalf of three high-tech SMEs.

The ZF-TOOLS objectives are focused on the incor-poration of the zebrafish embryo model into thepreclinical drug screening pipelines. The use ofmice for in vivo monitoring of disease processessuch as tumourigenesis, metastasis and immuneresponse to tumours is limited by costs andthroughput level. Introducing a high-throughputzebrafish embryo model could potentially con-tribute to cost-effective and more efficient methodsin the anti-tumour drug discovery process.Moreover, acceleration of drug lead time benefitseconomy as well as quality of life of cancer patients. The lack of basic knowledge of disease markergenes is the current bottleneck for biomedicalresearch in zebrafish and for genomics-based com-pound screens in this model organism. The ZF-TOOLS project uses multidisciplinary functionalgenomics approaches to discover novel diseasemarkers. The expected identification of factorsimportant for tumour growth and metastasis andorganismal defence responses will generate funda-mental knowledge relevant to human health andwill open the door to the establishment of zebrafish-based biomedical research and screening tools.

Three research intensive SMEs, ZF-screens B.V, BaseClear B.V and ZenonBio Ltd., play a crucialrole in the ZF-TOOLS project.

The activities of ZF-Screens B.V. are focused on the development and application of the tumourimplantation system, including drug screens and the identification and exploitation of noveltumour factors. Zebrafish embryo-based screens, integrated at an early phase in the drug dis-covery pipeline, can likely increase efficiency of further tests in a rodent system. It is expectedthat a proof-of-principle of the tumour implant screening system would open up a fast market,not only for contract research in screens for new therapeutics but also for licensing of the noveltechnology that ZF-Screens has developed and will further strengthen during this project.

The main role of BaseClear B.V. in the project is the development of high-throughput expressionanalysis tests, based on the use of MLPA (Multiplex Ligation-dependent Probe Amplification)technology. MLPA has been an important breakthrough in DNA diagnostics and is also extremelyvaluable to increase efficiency of mRNA expression profiling (RT-MLPA). BaseClear has beenactive in custom molecular services and contract research projects since 1993. Currently, thecompany serves more than 2 400 customers from 450 national and international companies anduniversity research departments. BaseClear intends to exploit the experience gained from the ZF-TOOLS project to add MLPA and RT-MLPA custom services to their portfolio. In addition,RT-MLPA probe sets developed in the project will be sold for research purposes.

ZenonBio Ltd. is the main bioinformatics partner in the project, responsible for analysis and inte-gration of genomic data sets. The company markets state-of-the-art diagnostic systems inHungary and previously developed software for signal and image processing. Their bioinfor-matics expertise, further strengthened by the project, will be directed towards development ofservices. A potentially good market for new entrepreneurial activities of ZenonBio Ltd. lies in theSouth-Eastern European region, including Hungary, Serbia-Montenegro, Romania and Bulgaria.

ROLE OF SMEs

185


Annemarie H. MeijerLeiden University Institute of Biology Clusius LaboratoryWassenaarseweg 642333 AL Leiden, The Netherlandsa.h.meijer@biology.leidenuniv.nlwww.biology.leidenuniv.nl/ibl/about.shtml

Partners

Herman P. SpainkZF-screens B.V.The Netherlandswww.zf-screens.com

Nicholas Simon FoulkesForschungszentrum KarlsruheKarlsruhe, Germanywww.fzk.de

Bas ReichertBaseClear B.V.The Netherlandswww.baseclear.com

Tamás ForraiZenon Bio Ltd.Hungarywww.zenonbio.hu

Mátyás MinkSzeged UniversityDepartment of Genetics and Molecular BiologyHungarywww.u-szeged.hu/indexe.html

| Transparent zebrafish embryo.

Development of Early Non-Invasive Biomarkers and Means for the Diagnosis and ProgressionMonitoring of Preeclampsia and Tailoring Putative Therapies

ACRONYM

PREGENESYSwww.pregenesys.net/main

Preeclampsia is a multi-system disorderthat complicates 5-7% of all pregnancies,is responsible for 18% (the second largestcause) of maternal deaths during preg-nancy, and for a third of premature births.

This complex disorder is expressed asa newly onset hypertension and proteinloss in urine (proteinuria) that develops inthe mother after the 20th week of gesta-tion, in association with varying degreesof end-organ damage. It may be coupledto potentially life-threatening complica-tions of the kidney, liver, blood systemand brain, and can result in convul-sions/seizures (eclampsia).

Concerning the baby, preeclampsia isresponsible for approximately 7-9 % ofneonatal morbidity and mortality. Majormotor and cognitive disabilities, blind-ness and life-long complications can taketheir toll.

While clinically diagnosed in the secondhalf of pregnancy, the underlying patho-physiology is associated with deleteriousalterations of implantation and placen-tation, which already begin in the firsttrimester.

There is no ‘gold standard’ for an effec-tive preventive therapy and the only cur-rently accepted treatment of establishedpreeclampsia is (premature) delivery. Thisis often done at the expense of foetal well-being (excess neonatal morbidity andmortality).

The Pregenesys research project is co-financed by and carried out within Priority 1of FP6. It aims to develop early (1st trimesterof pregnancy) non-invasive biomarkersand means for the diagnosis and monitor-ing of preeclampsia and its progression,that would help to tailor appropriate puta-tive therapies, thus optimising them forthe prevention of preeclampsia or thereduction of its severity.

The project is undertaken by a consortiumconsisting of 10 members from 9 differentcountries, including universities, hospitals,private research organisations, SMEs anda large industrial company.

SUMMARY

186



Diagnostic Technologies Ltd. (DTL) is the project coordinator. It is a medical diagnostic andbiotechnology company at the forefront of placental protein molecular biology and bio-chemistry incubated at the Technion-Israel Institute of Technology (Haifa, Israel) in 1994and emerged as an independent company in 2001.

ImunoSTAR – Research and Commercialisation of Bio-diagnostic Products S.A. – aims atpromoting and commercialising innovative immunology tests and diagnostic servicesfor application in biomedical and health market. It specialises in the Obstetrics andGynaecology field.

ROLE OF SMEs


Hamutal MeiriDiagnostic Technologies Ltd.2 Ha’Carmel St., Building B, 4th floorYokneam 20692, [email protected]@hotmail.com

| Confocal microscopy of normal term placental villi.Placental villi from a normal term placenta stained for PP13(red) and actin (green) are shown. Nuclei were counterstained with DAPI. Shown is an overlay of a stack of 20single images. (Courtesy of B. Huppertz, Graz.)

SME CALL

Index by projectnumber

• LSHP-CT-2005-037912FASTEST-TB . . . . . . . . . . . . . . . . . 66

• LSHP-CT-2006-036871INNOVAC . . . . . . . . . . . . . . . . . . 88

• LSHM-CT-2006-036534NEOBRAIN . . . . . . . . . . . . . . . . . 120

• LSHB-CT-2006-036813PROLIGEN . . . . . . . . . . . . . . . . . 142

• LSHM-CT-2006-037050INDABIP. . . . . . . . . . . . . . . . . . . . 86

• LHSB-CT-2006-037168EXERA . . . . . . . . . . . . . . . . . . . . 62

• LSHP-CT-2006-037200MUNANOVAC . . . . . . . . . . . . . . . . 112

• LSHB-CT-2006-037212Diagnosis . . . . . . . . . . . . . . . . . . 44

• LSHP-CT-2006-037217TB-DRUG . . . . . . . . . . . . . . . . . . 170

• LSHG-CT-2006-037220ZF-TOOLS . . . . . . . . . . . . . . . . . . 184

• LSHG-CT-2006-037226MEGATOOLS . . . . . . . . . . . . . . . . 104

• LSHM-CT-2006-037227CVDIMMUNE . . . . . . . . . . . . . . . . 38

• LSHG-CT-2006-037231SYSCO . . . . . . . . . . . . . . . . . . . . 160

• LSHB-CT-2006-037244PREGENESYS . . . . . . . . . . . . . . . 186

• LSHB-CT-2006-037245NanoSense . . . . . . . . . . . . . . . . . 118

• LSHC-CT-2006-037251CAPPELLA . . . . . . . . . . . . . . . . . . 26

• LSHM-CT-2006-037254VASOPLUS . . . . . . . . . . . . . . . . . 180

• LSHP-CT-2006-037276RespViruses . . . . . . . . . . . . . . . . 148

• LSHG-CT-2006-037277VALAPODYN . . . . . . . . . . . . . . . 178

• LSHB-CT-2006-037293QuAGSIC . . . . . . . . . . . . . . . . . . 144

• LSHB-CT-2006-037319IBDchip . . . . . . . . . . . . . . . . . . . 80

• LSHB-CT-2006-037325BacAbs . . . . . . . . . . . . . . . . . . . . 18

• LSHB-CT-2006-037386ANGIOSTOP . . . . . . . . . . . . . . . . . 12

• LSHM-CT-2006-037400IMMUNATH . . . . . . . . . . . . . . . . . 82

• LSHG-CT-2006-037415SMARTER . . . . . . . . . . . . . . . . . . 154

• LSHG-CT-2006-037457SysProt . . . . . . . . . . . . . . . . . . . 162

• LSHG-CT-2006-037462ChILL . . . . . . . . . . . . . . . . . . . . . 28

• LSHB-CT-2006-037479MYOAMP . . . . . . . . . . . . . . . . . . 116

• LSHC-CT-2006-037489Immuno-PDT . . . . . . . . . . . . . . . . 84

• LSHP-CT-2006-037494PRIBOMAL . . . . . . . . . . . . . . . . . 138

• LSHB-CT-2006-037499liintop . . . . . . . . . . . . . . . . . . . . 96

• LSHG-CT-2006-037517TargetHerpes . . . . . . . . . . . . . . . 166

• LSHG-CT-2006-037543TEMPO. . . . . . . . . . . . . . . . . . . . 174

• LSHB-CT-2006-037545ENLIGHT . . . . . . . . . . . . . . . . . . . 56

• LSHM-CT-2006-037554AGLAEA . . . . . . . . . . . . . . . . . . . . 10

• LSHC-CT-2006-037555MAMMI . . . . . . . . . . . . . . . . . . . 100

188

• LSHC-CT-2006-037559CancerGrid . . . . . . . . . . . . . . . . . . 22

• LSHB-CT-2006-037560USDEP . . . . . . . . . . . . . . . . . . . . 176

• LSHB-CT-2006-037575COMICS . . . . . . . . . . . . . . . . . . . . 36

• LSHG-CT-2006-037586STREPTOMICS . . . . . . . . . . . . . . 158

• LSHP-CT-2006-037587PlasmodiumdUTPase . . . . . . . . . . 134

• LSHP-CT-2006-037651EPIVAC . . . . . . . . . . . . . . . . . . . . 58

• LSHB-CT-2006-037653OMVac . . . . . . . . . . . . . . . . . . . . 126

• LSHB-CT-2006-037661GLYFDIS . . . . . . . . . . . . . . . . . . . 70

• LSHB-CT-2006-037681DiaNa . . . . . . . . . . . . . . . . . . . . . 48

• LSHG-CT-2006-037683FGENTCARD . . . . . . . . . . . . . . . . 68

• LSHM-CT-2006-037692NPARI . . . . . . . . . . . . . . . . . . . . 124

• LSHB-CT-2006-037702Mimovax . . . . . . . . . . . . . . . . . . 108

• LSHC-CT-2006-037737HI-CAM . . . . . . . . . . . . . . . . . . . . 72

• LSHB-CT-2006-037739Drop-Top . . . . . . . . . . . . . . . . . . 50

• LSHP-CT-2006-037785TB-trDNA . . . . . . . . . . . . . . . . . . 172

• LSHG-CT-2006-037793OptiCryst . . . . . . . . . . . . . . . . . . 128

• LSHP-CT-2006-037796SERO-TB. . . . . . . . . . . . . . . . . . . 152

• LSHM-CT-2006-037833MYASTAID . . . . . . . . . . . . . . . . . 114

• LSHM-CT-2006-037846RATstream™. . . . . . . . . . . . . . . . 146

• LSHC-CT-2006-037852LIGHTS . . . . . . . . . . . . . . . . . . . . 94

• LSHM-CT-2006-037870EACCAD . . . . . . . . . . . . . . . . . . . 54

• LSHC-CT-2006-037874VITAL . . . . . . . . . . . . . . . . . . . . . 182

• LSHG-CT-2006-037897AUTOSCREEN . . . . . . . . . . . . . . . . 16

• LSHE-CT-2006-037899MANASP . . . . . . . . . . . . . . . . . . 102

• LSHG-CT-2006-037939BioBridge . . . . . . . . . . . . . . . . . . 20

• LSHM-CT-2006-037950cNEUPRO . . . . . . . . . . . . . . . . . . 32

• LSHM-CT-2006-037957MagRSA . . . . . . . . . . . . . . . . . . . 98

• LSHM-CT-2006-037965INTELLIMAZE . . . . . . . . . . . . . . . . 90

• LSHB-CT-2007-036644DIALOK . . . . . . . . . . . . . . . . . . . . 46

• LSHB-CT-2007-037241SAGE . . . . . . . . . . . . . . . . . . . . . 150

• LSHB-CT-2007-037283EURO-PHARMACO-GENE . . . . . . . . 60

• LSHG-CT-2007-037291MODEST . . . . . . . . . . . . . . . . . . 110

• LSHP-CT-2007-037301HIVSTOP . . . . . . . . . . . . . . . . . . . 78

• LSHB-CT-2007-037303DeZnIT . . . . . . . . . . . . . . . . . . . . 42

• LSHP-CT-2007-037304CILMALVAC . . . . . . . . . . . . . . . . . 30

• LSHB-CT-2007-037365TargetScreen2 . . . . . . . . . . . . . . 168

• LSHM-CT-2007-037472TAMAHUD . . . . . . . . . . . . . . . . . . 164

• LSHB-CT-2007-037590Net2Drug . . . . . . . . . . . . . . . . . . . 122

• LSHB-CT-2007-037636InVitroHeart . . . . . . . . . . . . . . . . . . 92

• LSHC-CT-2007-037642HighReX . . . . . . . . . . . . . . . . . . . . 74

• LSHM-CT-2007-037669PHECOMP . . . . . . . . . . . . . . . . . . 130

• LSHB-CT-2007-037703STEMDIAGNOSTICS. . . . . . . . . . . . . 156

• LSHB-CT-2007-037730COBRED . . . . . . . . . . . . . . . . . . . . 34

• LSHB-CT-2007-037740PRISM . . . . . . . . . . . . . . . . . . . . . 140

• LSHP-CT-2007-037760HIVResInh. . . . . . . . . . . . . . . . . . . . 76

• LSHM-CT-2007-037765PHOTOLYSIS . . . . . . . . . . . . . . . . . 132

• LSHM-CT-2007-037831MEMORIES . . . . . . . . . . . . . . . . . . 106

• LSHC-CT-2007-037834DEPPICT . . . . . . . . . . . . . . . . . . . . 40

• LSHM-CT-2007-037862ARTEMIS . . . . . . . . . . . . . . . . . . . . . 14

• LSHB-CT-2007-037933POC4life . . . . . . . . . . . . . . . . . . . . 136

189

SME CALL

Index by coordinator

• Allaer Didier . . . . . . . . . . . . . . . . . . . . . . 28Diagenode S.A.

• Amaral Margarida D. . . . . . . . . . . . 168University of Lisboa

• Anné Jozef. . . . . . . . . . . . . . . . . . . . . . . . . 158Catholic University of Leuven

• Bágyi István . . . . . . . . . . . . . . . . . . . . . . . 22AMRI Hungary

• Benlloch Jose Ma . . . . . . . . . . . . . . . . 100Consejo Superior de Investigaciones científicas

• Berrih-Aknin Sonia . . . . . . . . . . . . . . 114Université Paris Sud (UPS)

• Bill Roslyn . . . . . . . . . . . . . . . . . . . . . . . . 128Aston University

• Birch Mike . . . . . . . . . . . . . . . . . . . . . . . . 124F2G Ltd.

• Bois Emmanuel . . . . . . . . . . . . . . . . . . 136Cezanne SAS

• Brandt Remco . . . . . . . . . . . . . . . . . . . . . 30Cilian AG

• Campadelli-Fiume Gabriella . . . 166University of Bologna

• Caricasole Andrea. . . . . . . . . . . . . . . . 164Siena Biotech SpA

• Cattaneo Antonino . . . . . . . . . . . . . . . 106European Brain Research Institute

• Collins Andrew . . . . . . . . . . . . . . . . . . . . 36University of Oslo

• Costi Maria Paola . . . . . . . . . . . . . . . . . 94University of Modena and Reggio Emilia

• Cutting Simon M. . . . . . . . . . . . . . . . . . 88Royal Holloway University of London

• Dammann Olaf . . . . . . . . . . . . . . . . . . . 120Hannover Medical School

• Danielsson Mats . . . . . . . . . . . . . . . . . . 74Sectra Mamea AB

• Daura Xavier . . . . . . . . . . . . . . . . . . . . . . . 18Universitat Autònoma de Barcelona

• Depaulis Antoine . . . . . . . . . . . . . . . . . 178UMR 704 Inserm-Université Joseph Fourier

• Di Lorenzo Diego . . . . . . . . . . . . . . . . . 64Ospedale Civile di Brescia

• Dickinson Anne . . . . . . . . . . . . . . . . . . . 156University of Newcastle upon Tyne

• Dolcetti Riccardo . . . . . . . . . . . . . . . . . 182National Cancer Institute

• Einsele Hermann . . . . . . . . . . . . . . . . 102University of Wuerzburg

• Ekström Björn . . . . . . . . . . . . . . . 56Olink AB

• Ellerbrok Heinz . . . . . . . . . . . . . . . . . . 176Robert Koch-Institut

• Falciani Chiara . . . . . . . . . . . . . . . . . . . . . 66Philogen

• Finn Paul . . . . . . . . . . . . . . . . . . . . . . . . . . . 68InhibOx Ltd.

• Fiorini Carlo . . . . . . . . . . . . . . . . . . . . . . . 20Politecnico di Milano

• Frostegård Johan . . . . . . . . . . . . . . . . . 38Karolinska University Hospital

• Gauguier Dominique . . . . . . . . . . . . . 68University of Oxford

• Gilbert Ian. . . . . . . . . . . . . . . . . . . . . . . . . 134University of Dundee

• Goudsmit Jaap . . . . . . . . . . . . . . . . . . . . 138Crucell Holland B.V.

• Hartmann Marcus . . . . . . . . . . . . . . . . . 30Cilian AG

• Holthofer Harry. . . . . . . . . . . . . . . . . . . . 48Haartman Institute

• Hotter Georgina . . . . . . . . . . . . . . . . . 142Instituto de Investigaciones Biomédicas de Barcelona

190

• Huet Jacqueline . . . . . . . . . . . . . . . . . . 112PHUSIS

• Huggett Jim . . . . . . . . . . . . . . . . . . . . . . . 172University College London

• Imhof Axel . . . . . . . . . . . . . . . . . . . . . . . 154Ludwig Maximilians University of Munich

• Jensen Sanne . . . . . . . . . . . . . . . . . . . . . 26Evolva S.A.

• Kel Alexander . . . . . . . . . . . . . . . 122/160BIOBASE GmbH

• Keri György . . . . . . . . . . . . . . . . . . . . . . . 170Vichem Chemie Research Ltd.

• Kuijper Ed J. . . . . . . . . . . . . . . . . . . . . . . . 54Leiden University Medical Center

• Kulikowski Tadeusz . . . . . . . . . . . . . . . 76Instytut Biochemii iBiofizyki PAN

• Leiser Robert-Matthias . . . . . . . . . . 44Agrobiogen GmbH

• Lenas Petros . . . . . . . . . . . . . . . . . . . . . . . 14Complutense University of Madrid (UCM)

• Lévi Francis. . . . . . . . . . . . . . . . . . . . . . . . 174INSERM U776

• Lipp Hans-Peter . . . . . . . . . . . . . . . . . . . 90NewBehavior AG

• Ludwig Bernard . . . . . . . . . . . . . . . . . . . 10ADDEX Pharmaceuticals France SAS

• Maes Tamara . . . . . . . . . . . . . . . . . . . . . 86C. S. O. Oryzon genomics

• Maldonado Rafael . . . . . . . . . . . . . . 130Universitat Pompeu Fabra (UPF)

• Malik Arif . . . . . . . . . . . . . . . . . . . . . . . . . 162MicroDiscovery GmbH

• Mandenius Carl-Fredrik . . . . . . . . . . 92Linköping University

• Mandler Markus . . . . . . . . . . . . . . . . . 108Affiris GmbH

• Martinsson-Niskanen Titti. . . . . . . . 12BioInvent International AB

• Meijer Annemarie H. . . . . . . . . . . . . 184Leiden University

• Meinke Andreas . . . . . . . . . . . . . . . . . . 126Intercell AG

• Meiri Hamutal . . . . . . . . . . . . . . . . . . . 186Diagnostic Technologies Ltd.

• Mouly Vincent . . . . . . . . . . . . . . . . . . . . 116INSERM

• Müller-Hartmann Herbert . . . . . . . 110AMAXA GmbH

• Ochoa Gorka . . . . . . . . . . . . . . . . . . . . . . . 50Progenika Biopharma

• Ogden David . . . . . . . . . . . . . . . . . . . . . . 132CNRS UMR 8118

• Overduin Michael . . . . . . . . . . . . . . . 140University of Birmingham

• Palme Klaus . . . . . . . . . . . . . . . . . . . . . . . 16University of Freiburg

• Pâques Frédéric . . . . . . . . . . . . . . . . . 104CELLECTIS S A

• Pasterkamp Gerard . . . . . . . . . . . . . . 82University Medical Center Utrecht

• Porgador Angel . . . . . . . . . . . . . . . . . . . . 70Ben-Gurion University of the Negev

• Reinerio Gonzalez. . . . . . . . . . . . . . . . . 84Philogen

• Riess Olaf . . . . . . . . . . . . . . . . . . . . . . . . 146Eberhard-Karls-Universität Tübingen

• Robertson Graeme . . . . . . . . . . . . . . . . 40Siena Biotech SpA

• Roca Josep . . . . . . . . . . . . . . . . . . . . . . . . . 20Institut d’Investigacions Biomèdiques August Pi i Sunyer

• Sans Miquel . . . . . . . . . . . . . . . . . . . . . . 80Hospital Clínic/IDIBAPS

• Schildgen Oliver . . . . . . . . . . . . . . . . . . 148Institute for Medical Microbiology,Immunology, and Parasitology

• Schillberg Stefan . . . . . . . . . . . . . . . . . . . 150Fraunhofer-Institut für Molekularbiologie undAngewandte Oekologie IME

• Schrenzel Jacques . . . . . . . . . . . . . . . . . . 98Geneva University Hospitals

• Singh Mahavir . . . . . . . . . . . . . . . . . . . . . . . 66LIONEX Diagnostics & Therapeutics GmbH

• Skjeltorp Guri . . . . . . . . . . . . . . . . . . . . . . . 118Dalen Diagnostics AS

• Smith C. I. Edvard . . . . . . . . . . . . . . . . . . . 60Clinical Research Center

• Stanescu Ioana . . . . . . . . . . . . . . . . . . . . . 58FIT Biotech Plc

• Stassen J.M. . . . . . . . . . . . . . . . . . . . . . . . . 180ThromboGenics Ltd.

• Takacs Laszlo . . . . . . . . . . . . . . . . . . . . . . . . 34BioSystems International

• van Holst Gerrit-Jan . . . . . . . . . . . . . . . . . 78Viruvation B.V.

• Veuskens Jack . . . . . . . . . . . . . . . . . . . . . . . 46KREATECH Biotechnology B.V.

• Weisbuch Claude . . . . . . . . . . . . . . . . . . . 144Genewave SAS

• Weldingh Karin . . . . . . . . . . . . . . . . . . . . . 152Statens Serum Institut

• Wiltfang Jens . . . . . . . . . . . . . . . . . . . . . . . . . 32University of Erlangen-Nuremberg

• Zucco Flavia . . . . . . . . . . . . . . . . . . . . . . . . . 96Istituto di Neurobiologia e MedicinaMolecolare

191

European Commission

EUR 23457 — Synopses of projects funded through the SME call for “Life sciences, genomics and biotechnology for health”

Luxembourg: Office for Official Publications of the European Communities

2008 — 191 pp. — 21.0 x 29.7 cm

ISBN 978-92-79-08803-2DOI 10.2777/13756

HOW TO OBTAIN EU PUBLICATIONS

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SMEs in Health research, synopses of projects funded through the SME call for “Life sciences, genomics and biotechnology for health”

Hundreds of research-intensive small and medium sized enterprises (SMEs) are active in the health sector. In recognition of their innovative potential, scientifi c strength and strategic importance, a special effort was made to involve them in the Sixth Framework Programme (FP6), where a very successful ‘SME call’ was published with a budget of € 200 million.

As a result of this call, 270 SMEs in the health and biotech sector, acceded to a leading role as key players and driving forces in the research activities of 86 EU-funded projects, receiving more than 40 % of the EU contribution.

This publication presents those 86 projects and aims to illustrate the EU’s commit-ment to health research, bringing together transnational and multidisciplinary expertise from both industry and academia.

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