Poster Session – Immunotherapy Wednesday 19 November 2014 41

the safe clinical profile of adenosine A2A receptor inhibitors (A2ARi) inParkinson’s disease. Monoclonal antibodies (mAb) that block programmeddeath (PD)-1 or cytotoxic T lymphocyte antigen (CTLA-4) receptors havebeen associated with durable clinical responses against a variety of cancertypes and hold great potential as novel cancer therapeutics. Metastasisis the main cause of cancer related deaths worldwide, and therefore wehave studied experimental and spontaneous mouse models of melanomaand breast cancer metastasis to demonstrate the efficacy and mechanismof a combination of A2A receptor inhibitor in combination with variousimmune check point inhibitors. The combination of anti-PD-1 and A2Areceptor inhibitor significantly reduces metastatic burden and prolongs thelife of mice compared with either monotherapy alone. Importantly, thecombination was only effective when the tumor expressed high levels ofCD73, suggesting a tumor biomarker that at a minimum could be used tostratify patients that might receive this combination. The mechanism of thecombination therapy was critically dependent on NK cells and interferongamma, and to a lesser extent, CD8+ T cells and the effector molecule,perforin. Consistent with the anti-metastatic role of NK cells, we observedsignificantly high number of NK cells in the lungs of tumor bearing mice aftercombination immunotherapy. Overall, our preclinical data provide a strongrationale to use A2ARi with anti-PD-1 mAb for the treatment of minimalresidual and metastatic disease.

116 POSTERNovel targets for antibody–drug conjugate therapy

A.G. Grandi1, S.C. Campagnoli1, M.P. Parri1, E.D.C. De Camilli2, B.J. Jin3,P.S. Sarmientos1, G.G. Grandi4, L.T. Terracciano5, P.P. Pileri1, G.V. Viale2,R. Grifantini6. 1Externautics, R&D, Siena, Italy; 2European Institute ofOncology, Pathology, Milan, Italy; 3Fourth Military University, Immunology,XI’an, China; 4Novartis Vaccines, R&D, Siena, Italy; 5Basel MedicalUniversity, Pathology, Basel, Switzerland; 6Externautics, Siena, Italy

The study focuses on two novel potential therapeutic targets identifiedby a systematic immune-histochemistry (IHC) screening with a largecollection of polyclonal antibodies (approximately 1600) raised againstmarginally characterized human proteins. Here we describe the molecularcharacterization of two surface-associated proteins (EXN36 and EXN91)associated to different cancer types. EXN36 is mainly over-expressedin ovary and breast cancers (frequency of approximately 30−40%).Interestingly, it is also over-expressed in triple negative breast cancer.The protein is involved in cell proliferation, migration and invasiveness.Concerning EXN91, it is an adhesion molecule and it acts as a signalingreceptor, likely to be important in developmental processes and cellcommunication. The protein is mainly detected in colon cancer with highfrequency (more than 80%), both in early and advanced stages, in highand low grade cancers. Interestingly, EXN91 is over-expressed in KRASand BRAF mutant colon cancers with significant frequency (approximately50%). Finally, it is also detected in esophagous SCC and ccRCC (10−20%).Murine monoclonal antibodies able to recognize EXN36 and EXN91 on thesurface of cancer cells have been selected and characterized to assesstheir potential for specific therapeutic indications. In particular, five anti-EXN36 mAbs are able to recognize the target protein in breast (Her2+,Er+

and triple negative cells) and ovary cell lines. A murine monoclonalantibody is able to recognize EXN91 on the surface of colon cancer cells.This antibody also shows the ability to inhibit growth of colon cancer inxenograft mouse models. Finally, it specifically binds cancer tissues byIHC, suggesting that it could be also developed as companion diagnostictool for EXN91-based therapies.EXN36 and EXN91 monoclonal antibodies show a high number ofbinding sites on the cancer cell surface, ranging from 10,000 to 100,000sites per cells. The antibody specificity has been confirmed in differentimmunoassays (Western blot, FACS, IHC) by gene silencing experimentsand/or competition with peptides containing the antibody epitopes. Theseantibodies have high affinity for their target epitopes (KD: 10

−9,10−10 nM).Moreover, they show limited IHC reactivity in normal tissues (FDA tissuepanel).Some antibodies are efficiently internalized by cancer cells, suggestingthat they can be exploited for the development of Antibody–Drug Conjugate(ADC). The analysis of their potential for ADC is ongoing. Initial results fromin vitro studies show that these antibodies, indirectly linked to commerciallyavailable drugs (e.g. auristatin-based, DM1, and duocarmycin), showsignificant anti-tumor activity with specific drugs and linker chemistry.Overall, the results indicate that EXN36, EXN91 and their specificmonoclonal antibodies could be developed for the targeted therapy ofcancer indications at high medical need, either alone or in combinatorialstrategies.

117 POSTER“Arming” the chimeric oncolytic adenovirus enadenotucirev to

deliver checkpoint inhibitors and other therapeutics directly to

tumours

B. Champion1, P. Kodialbail1, S. Illingworth1, N. Rasiah1, D. Cochrane1,J. Beadle1, K. Fisher1, A.C.N. Brown1. 1PsiOxus Therapeutics Ltd,Abingdon Oxford, United Kingdom

Enadenotucirev (EnAd; formerly called ColoAd1) is a potent, chimericAd11p/Ad3 adenovirus active against a range of epithelial cancer cells,with a shorter time-to-lysis than either wild type Ad11p, Ad3 or Ad5.In normal cells, EnAd is attenuated and shows little or no activity byeither cytotoxicity or by qPCR. In vivo, EnAd shows efficacy in a rangeof xenograft human tumour models following intra-tumoural, intravenousand intra-peritoneal injection, and is currently being evaluated clinicallyfor treatment of several different epithelial cancers. Data from an ongoingclinical mechanism of action study have shown that i.v. dosed EnAd infectsand replicates in tumour cells, producing significant amounts of viral protein(hexon), indicating that transgene encoded proteins will also be made insignificant amounts by tumours following i.v. delivery of an armed EnAdvirus.To develop ‘armed’ variants for delivery of therapeutic agents that enhanceEnAd’s anti-tumour activity, we have developed a system for rapidgeneration of modified viruses that can be dosed systemically to deliverimmunomodulatory antibodies into tumours. We chose to first encode anti-VEGF antibodies since, unlike immunomodulators, they could be readilyevaluated in vivo in immunodeficient mouse human tumour xenograftmodels. We have successfully produced EnAd variants encoding full-length (NG-135) and ScFv (NG-76) forms of anti-human VEGF antibodieswhich have similar virus activity profiles to EnAd in cancer cell linesin vitro (virus replication, gene expression and oncolytic action), butalso express and release the respective anti-VEGF antibody forms intothe culture supernatant. Using either HCT-116 or DLD human coloncarcinoma xenograft models we have shown that the virus infection profilefollowing intra-tumoural injection is similar to the parental EnAd virus (virusreplication and Hexon gene expression). Anti-VEGF antibody expression bythese tumours could be detected in the tissue as both mRNA and functionalantibody. Antibodies were detectable early (within 3 days of infection) andexpression was sustained over several weeks. Furthermore, low levelsof anti-VEGF antibody were detectable in the blood. Production andevaluation of viruses similarly expressing checkpoint inhibitor antibodiesis now in progress, together with evaluation of anti-VEGF armed oncolyticviruses for their impact on the growth and microenvironment of tumourxenografts.

118 POSTERMajor synergy between Coxsackievirus A21 (CAVATAK™) and

radiotherapy or chemotherapy in bladder cancer

G.R. Simpson1, N. Annels1, M. Ajaz1, F. Launchbury1, G. Bolton1,A.A. Melcher2, K.J. Harrington3, G. Au4, D. Shafren4, H. Pandha1. 1TheUniversity of Surrey, Faculty of Health and Medical Sciences, Guildford,United Kingdom; 2St James’s University Hospital Leeds, Instituteof Molecular Medicine, Leeds, United Kingdom; 3Institute of CancerResearch, Targeted Therapy Team, London, United Kingdom; 4ViralyticsLtd & The University of Newcastle, Viralytics Ltd & The University ofNewcastle, Newcastle, Australia

Introduction: There are still no treatments for superficial bladder cancer(SBC) which alter its natural progression, where 20% of patients developmetastatic disease. SBC is often multifocal, has high recurrences aftersurgical resection and recurs after intravesical live BCG.As this is a clinicalsetting in which local live biological therapy is already well established,it presents intriguing opportunities for oncolytic virotherapy. CoxsackievirusA21 (CVA21) has recently been shown to be an efficient oncolytic agent thatspecifically targets and rapidly lyses human malignant melanoma, multiplemyeloma, prostate and breast tumours, which express high levels of theCVA21 cellular uptake receptors both in vitro and in vivo. In addition, aPhase I clinical trial in late stage melanoma patients has recently beencompleted, and has demonstrated that intratumorally administered CVA21is well tolerated in humans, and that 55.55% of patients experiencedstabilization or reduction in injected tumour volumes.Materials and Methods: Infection Radio- & Chemotherapy Synergyassays, Combination index analyses, QPCR and IHC for ICAM-1/DAF,Culture of SBC.Results: Characterization of CVA21 cytotoxicity in a panel of cell linesyielded a range of sensitivities. CVA21 cytotoxicity seems to correlateexpression of viral receptors ICAM-1 & DAF. The addition of radiotherapyor chemotherapy resulted in significantly increased cytotoxicity over CVA21alone. When 5637 or T24 cells were irradiated (4−10Gy) then 24 hours