on the route to biobetters: from alternative binding ... · on the route to biobetters: from...
TRANSCRIPT
Prof. Dr. Arne Skerra
Global Engage 2nd Biologics & Biosimilars Congress: Berlin – 2nd Feb. 2016
On the route to Biobetters:
from alternative binding proteins to tuning in vivo half-life!
Prof. Dr. Arne Skerra
The Expanding Universe of Therapeutic Proteins ...
Need for classification: Group I: Protein therapeutics with enzymatic or regulatory activity
Ia: Replacing a protein that is deficient or abnormal (e.g.: insulin, hGH, FVIII) Ib: Augmenting an existing pathway (e.g.: erythropoietin, interferon-α2a/b, tPA, exenatide) Ic: Providing a novel function or activity (e.g.: asparaginase, hirudin, streptokinase)
Group II: Protein therapeutics with special targeting activity IIa: Interfering with a molecule or organism (e.g.: pegvisomant, enfuvirtide, MAbs & Scaffolds ...) IIb: Delivering other compounds or proteins (e.g.: ADCs & Bispecifics ...; Zevalin, Mylotarg ...)
Group III: Protein vaccines IIIa: Protecting against a deleterious foreign agent (e.g.: HBsAg, HPV vaccine) IIIb: Treating an autoimmune disease IIIc: Treating/preventing cancer (e.g.: Cervarix)
Group IV: Protein diagnostics In vivo imaging agents for cancer (e.g.: Arcitumomab)
(Leader et al. 2008 Nat. Rev. Drug Discov.)
The area of Protein- or Peptide-based Biotherapeutics has seen tremendous growth during the last decade: 1982: market approval of the first recombinant protein, insulin (Humulin) 2000: 84 approved biologics in the US + EU 2010: ≥ 200 biopharmaceuticals on the market
(Walsh: Biopharmaceutical Benchmarks – 2003 & 2010) 2013: 907 medicines and vaccines under development in the US,
among those 338 Monoclonal Antibodies (MAbs) (America's Biopharmaceutical Research Companies 2013 Report)
MAb
hGH
Exenatide
Prof. Dr. Arne Skerra
Two Approaches to Biobetters: Anticalins® and PASylation®
The term BIOBETTER refers to a therapeutic protein that is functionally similar (not identical) to an established biopharmaceutical but has improvements over the original in critical attributes and clinical performance. (See, e.g.: Beck, A. (2011) Biosimilar, biobetter and next generation therapeutic antibodies. mAbs 3, 107-110.)
Some examples of Biobetters:
Insulin: Humulin (1982) ⇒ Lantus (2000)
Interferon: Roferon A (1986) ⇒ Pegasys (2002)
G-CSF: Neupogen (1991) ⇒ Neulasta (2002)
Antibody: Herceptin (1998) ⇒ Kadcyla (2013)
Prof. Dr. Arne Skerra
Anticalins® based on the lipocalin scaffold: next generation 'Antibodies'
Advantages:
simpler architecture (one polypeptide chain) - easier manufacturing
and manipulation - facile fusion with
enzymes or toxins (bispecific Duocalins)
- high stability - monovalency - lack of Fc effector module
smaller size - better tissue penetration - tunable plasma half-life - new routes of administration
(inhalation etc.)
Antibody Anticalin (X-ray structure in
complex with CTLA-4)
Target ('Antigen')
Prof. Dr. Arne Skerra
The Lipocalin family of ligand-binding proteins: prominent members
*Crystal structure known (human members of the family)
Prof. Dr. Arne Skerra
The menagery of human lipocalins: a β-barrel fold with 4 variable loops
Schiefner & Skerra, Acc. Chem. Res. 2015
Prof. Dr. Arne Skerra
Lipocalins & Antibodies: structural plasticity in the loop region
10 Fv fragments of different antibodies 6 "prototypic" lipocalin structures: RBP BBP MUP EPA BLG BDA
Prof. Dr. Arne Skerra
Targeted in vitro randomization of a scaffold protein
loop #1 loop #2 loop #3 loop #4
lipocalin cDNA deg. oligo #1 deg. oligo #3
deg. oligo #4 deg. oligo #2
PCR primer
PCR primer
Loop #1 Loop #2 Loop #3 Loop #4
Lipocalin scaffold
combinatorial mutagenesis in vitro
Prof. Dr. Arne Skerra
In vitro evolution of Anticalins: a funnel-like combinatorial design process
Anticalin Library
Target-Specific Clinical Candidate
Phage display selection E. coli surface display High throughput ELISA Secondary ELISA screen
(specificity etc.) Optionally: affinity
maturation Preparative expressions
and biochemical characterization
In vivo activity PK/PD in vivo
Target
Target
Prof. Dr. Arne Skerra
Theoretical aspects of combinatorial library design for a protein scaffold
no. of randomized positions: 12 16 20 24 codons
NNS mutagenesis 32 18 24 30 36 codon mutagenesis 19 15 20 25 30 'informed' mutagenesis ≈10 12 16 20 24
Prof. Dr. Arne Skerra
Evolution of Lcn2-based Anticalin libraries: choice of randomized positions
1 25 50 75 90 . . . . . Lcn2 QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPG Protein ...........................h...........x.x.x.xx.xx...................x.xx...x.x.a.....s... Hapten ...........................h....x..x....x..........x.x.............x.x........x.x.....s... New ...........................h.......x...xx.......x..x...............x.x.xx...x.x.x.....s... ====A=====------#1------===B=== ===C===--#2---===D==== 101 125 150 178 . . . . Lcn2 EFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG Protein ..........xxx.....................x.xx.x.x.a..i.......a................................. Hapten ...........................................x.x.x......a................................. New .....x...x..x..x..................x.x....x.x............................................ ==E=----#3-----===F==== ===G===---#4---===H===
Protein Hapten New
Prof. Dr. Arne Skerra
Tailored Anticalins can tightly bind target molecules of different kinds!
αHepcidin
αCTLA-4
αAβ
αED-B
α
Prof. Dr. Arne Skerra
Fibronectin (FN) was one of the first genes reported to undergo alternative splicing, resulting in 3 isoforms: ED-A, ED-B, IICS.
Isoform B of the extracellular matrix protein FN was found to be associated with neovasculature of solid tumors.
ED-B is identical in several vertebrates, including mice and humans.
ED-B arises exclusively at sites of tissue remodelling (such as during wound healing or in endometrium) but is absent in plasma and normal tissue FN.
ED-B is abundantly expressed around newly formed blood vessels, including neovasculature of various solid tumors, e.g., melanoma, renal cell carcinoma (RCC), breast, colorectal, and non–small cell lung cancer.
Ideal target with broad therapeutic applicability across various cancer types!
Neri & Bicknell 2005 Nat Rev Cancer
Carnemolla et al. 1989 J Cell Biol
Prof. Dr. Arne Skerra
Three Anticalins against ED-B show distinct modes of epitope recognition
Relative orientation of the Anticalins N7A, N7E, and N9B with respect to ED-B:
Relative orientation of ED-B with respect to the Anticalins N7A, N7E, and N9B:
Superposition of ED-B
Superposition of the Anticalins N7A, N7E, and N9B
Gebauer et al., JMB 2013
Prof. Dr. Arne Skerra
Anticalins detect ED-B on primary glioblastoma (WHO°IV GBM) tissue
C
A B
D
E F
G H
Anticalin N7A
Anticalin N7E
ααCD31 (endothelial cell marker)
wtLcn2 (negative control)
wtLcn2 N7A
Immunohistochemical detection of ED-B on human glioblastoma tissue sections: Strep-tag II labeled Anticalin N7A, StrepMAB-Immo, biotinylated α-mouse Ig, streptavidin-HRP / DAB chromogen Cell nuclei were counterstained with hematoxylin.
Albrecht et al., IJC 2016
Prof. Dr. Arne Skerra
Barinka et al., PEDS, published online.
Prof. Dr. Arne Skerra
Prospects for Anticalins in Research, Diagnostics (in vivo) & Therapy
PRS-050, a VEGF antagonist: Safe, well tolerated, biologically active
Safe and well tolerated in a 26-patient Phase I trial (repeated dose escalation study, completed in 2011)
Lack of immunogenicity (no Anti-Drug Antibodies in 17 pts with ≥ 5 doses; one patient was tested for ADA after 17 doses)
Dose proportional pharmacokinetics (6-day half-life for the 40 kDa PEGylated protein)
Durable target engagement
VEGF undetectable in plasma > 21 days following a single i.v. dose
Pro-hypertensive effect consistent with anti-VEGF activity
Sustained inhibition of downstream circulating biomarker
Prof. Dr. Arne Skerra
EUROCALIN Consortium boosts clinical development of αα-Hepcidin project
ααHepcidin
Pieris Pharmaceuticals, Inc.
Human data demonstrating desired drug-like properties - 26 solid tumor patients with VEGF-A antagonist - 36 healthy volunteers with hepcidin antagonist
Several R&D partnerships generating $50+ M in revenue - Potential for future milestone and royalties - Retained commercial rights in major markets
High Caliber Investors - OrbiMed Advisors (~19%), Tekla Capital Management (~10%), Lombard Odier (~6.5%); Ally
Bridge Group, Auriga, Emerald Mutual Fund, Forbion, Gilde, GLSV, Novo Nordisk, Sphera Funds, Zydus
- $110M equity capital raised
Proprietary Next-generation Therapeutic Proteins With Several Degrees of Validation
Anticalins
Non-Confidential 21
22
Anticalin Product Pipeline
Product Target(s) Indication Partner Discovery Preclinical IND-enabling Ph 1 P1b/2a
PRS-080 Hepcidin Anemia
PRS-060 IL4Ra Asthma
PRS-343 CD137/HER2
Immuno-Oncology
PRS-342 CD137/GPC3
PRS300s n.d/n.d.
Roche n.d.
Daiichi Sankyo
n.d. n.d.
n.d. n.d.
Sanofi Pseud. aeuruginosa Inf. Dis.
Zydus cMet Oncology
Stelis n.d. Ophtha
n.d. = not disclosed
PEGylated Anticalin
inhalable Anticalin
mAb-Anticalin fusion
1. Validated Targets
2. IO
3. Non-IO Partnered Programs
Non-Confidential 22
Stelis
Ac/Ac-Fc fusion
multispecifics
February 2016 – Not for public distribution!
Short plasma half-life: the problem of most biopharmaceuticals
kidney: excretion
volume of distribution
Therapeutic Brand Name Size Plasma half-life Sales 2008 Protein e.g. [kDa] [h] (Humans) [Mio. US$]
Epoetin Epogen® 34.0 4-13 5162 G-CSF Neupogen® 18.8 3.5 1341 INFα2b IntronA® 19.3 2-3 234 INFβ1a Avonex® 22.5 10 5351 h-GH Nutropin® 22.1 2.1 3376 IL1-Ra Kineret® 17.3 3.8 75
i.v. injection
0
50
100
0 1 2 3 4
% ID
τ1/2 Size-dependent glomerular filtration
February 2016 – Not for public distribution!
Drug Brand Name Indication PEGylation PK effect Sales 2009
PEG-interferon α2b PEG-Intron® Hepatitis C 12 kDa x 10 844 Mio. US$ PEG-interferon α2a Pegasys® Hepatitis C 40(2) kDa x 12 1539 Mio. US$ PEG-G-CSF Neulasta® Neutropenia 20 kDa x 4 3355 Mio. US$
Drawbacks of PEGylation:
High Cost of Goods
Chemical coupling & processing steps
Often low biological activity
Accumulation in organs (renal tubular cells, macrophages, choroid plexus epithelial cells ...)
Expanded volume
Retarded kidney filtration
Chemical coupling +
PEG Protein drug
PEGylation: an established technology to prolong PK
Ifn
PEG
Clinical development of various PEGylated protein drugs has been terminated or put on hold:
PEGsunercept, PEGylated αIL1β Fab, GlycoPEGylated factor VIIa ...
HO
OX
n
February 2016 – Not for public distribution!
Increased risk awareness of PEGylated drug products
Cellular vacuolation in macrophages and/or histiocytes in various organs and in renal tubular cells
Cases of cellular vacuolation of the choroid plexus epithelial cells (ependymal cells) have been observed in repeat-dose toxicity studies conducted with proteins PEGylated with molecules ≥ 40 kDa
"The ependymal cells of the choroid plexus are the main source of cerebrospinal fluid (CSF) secretion and form the blood-CSF barrier. As such, the choroid plexus is responsible for establishing and maintaining the extracellular milieu throughout the brain and the spinal cord. In addition, the choroid plexus appears to be a source of CSF-borne hormones and growth factors."
"Hence, until more data are available, the risk for ependymal cell vacuolation needs to be addressed before conducting longer term clinical trials in the paediatric population."
February 2016 – Not for public distribution!
PASylation®®: a biological alternative to PEGylation
Computer model of a PASylated Fab fragment
(superposition of 24 PAS conformers)
Pro/Ala-rich Sequences comprising the L-amino acids Proline, Alanine, and/or Serine form a highly soluble biological polymer that can be (i) chemically conjugated or (ii) directly produced as fusion protein with the biopharmaceutical, using various expression systems.
Similar to PEG, PAS polypeptides adopt a stable random coil structure with large apparent volume under physiological conditions, which effectively increases the dimension of the conjugated drug beyond the pore size of the renal glomeruli and, thus, slows down kidney filtration.
PAS sequences are stable in the blood plasma but are efficiently degraded by intracellular enzymes.
PAS sequences are compatible with all biotechnological production systems and allow high secretion titers.
PAS sequences are uncharged and only marginally influence the biological activity of the drug, and they are not immunogenic in animals.
PASylation with 200-1000 residues typically leads to a plasma half-life extension by a factor of 10-100 !
H2N
HN
NH
HN
NH
OH
O
O
O
O
On
R
R
R
R
R
RO
OO
OCH3
n
O
February 2016 – Not for public distribution!
xl020 PAS-hGH Growth Deficiency xl050 ααCD40L Fab-PAS Inflammation xl080 PAS-IFNαα Infection & Inflammation xl081 PAS-IFNantagonist Virology / HIV xl082 PAS-IFNsuperagonist Multiple Sclerosis xl100 PAS-Leptin Obesity / Diabetes xl101 PAS-SLMA Metabolic diseases
xl110 PAS-Exenatide Diabetes xl130 PAS-IL-1Ra Inflammation (RA) xl150 PAS-GMCSF Oncology xl180 PAS-ClottingFactor Hemostasis xl210 ααHer2 Fab-PAS Imaging: Oncology xl220 ααCD20 Fab-PAS Imaging: Oncology xl310 Ex4-PAS-PYY Bispecific: Metab. Disease xl510 PAS substance PEG substitute for coupling
Project status Project Compound Indication
Lead Opt.
In vivo Efficacy
Preclin. GMP
Discovery
IND
XL-protein leverages the versatility of PASylation® as broadly applicable enabling technology!
Clinic
PASylation® platform: Product Development up to in vivo PoC
XL-protein Partnering or Spin-off
February 2016 – Not for public distribution!
Collaboration partners and financiers ...
and several more ...
February 2016 – Not for public distribution!
PASylation®®: a biological alternative to PEGylation
Cheap manufacturing: No in vitro coupling steps
Homogeneous product: No polydispersity of the PAS tag
Biodegradability: Efficient degradation by kidney enzymes
Biological activity: High target affinity is maintained
No immunogenicity: No immunogenicity in mice
Extended half-life: Tunable from 10x to 100x
Expression plasmid
One-step expression
Disordered PAS Chain PAS
Therapeutic protein
gccTCTCCAGCTGCACCTGCTCCAGCAAGCCCTGCTGCACCAGCTCCGTCTGCTCCTGCT ||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AGAGGTCGACGTGGACGAGGTCGTTCGGGACGACGTGGTCGAGGCAGACGAGGACGAcgg AlaSerProAlaAlaProAlaProAlaSerProAlaAlaProAlaProSerAlaProAla
(Receptor)
Therapeutic protein
Pro / Ala / Ser encoding gene cassette:
One polypeptide:
E. coli, P. pastoris, CHO, HEK etc.
February 2016 – Not for public distribution!
Biosynthetic preparation of the monodisperse PA(S) polymer
Carrier Protein: e.g. TrxA
PA(S)
Protease / BrCN
!
!
Reverse phase HPLC:
Analytical SEC:
ESI-MS:
= PAS601
H2N-(Pro/Ala)n-COOH
February 2016 – Not for public distribution!
0
500
1000
1500
2000
0
10
20
30
40
50
60
0 5 10 15 20 25
A225A280
A332
Abs
orpt
ion
at 2
25 n
m /
280
nm [m
AU
]
Absorption at 332 nm
[mA
U]
Elution volume [ml]
Vo Vt
Iodoacetyl-PAS: conjugation to thiol-derivatized peptides or drugs
ESI-MS (deconvoluted):
!
calculated: 48372.23 Da
0
20
40
60
80
100
120
140
160
0 5 10 15 20 25 30
Abs
orp
tion
at 2
25 n
m [m
AU
]
Elution volume [ml]
underivatized PAS600(reference)
PAS600-IA
!
Anion exchange chromatography: Size exclusion chromatography:
0
200
400
600
800
1000
1200
0
10
20
30
40
50
60
0 5 10 15 20 25
A225A280
A332
Abso
rptio
n at
225
nm
/ 28
0 nm
[mAU
]
Absorption at 332 nm [m
AU]
Elution volume [ml]
Coupling with 7-mercapto-4-methylcoumarin:
O OHS
CH3
INH
HN
O
O
(Pro/Ala)n-1-COOH
February 2016 – Not for public distribution!
Site-directed coupling to multiple thiol groups
0
20
40
60
80
100
20
25
30
35
40
11 12 13 14 15 16 17 18 19
A 225 [
% m
ax.] B
uffer B [%
]
Retention time [min]O OHO
HN
OI
OH
O
HS
SH
HS
H2N
H2N COOH SH SH SH
H2N COOH SH
H2N COOH SH SH
Analytical reverse phase HPLC:
Coupling with Fluorescein
iodoacetamide
NHNH
HNNH
NH
O
O
O
O
O
HN NH2
NH HN
HO2C
O
N
O
O
S
S
S
H2N
S
S
S
H2N
Coupling with cyclic RGD peptide
ESI-MS (deconvoluted): Cys-PA200-Cys-PA200-Cys
PA2x200(Cys-Fluorescein)3
calculated: 34881.8 Da
calculated: 32617.4 Da
calculated: 33778.7 Da
Gaertner et al. (2012) Eur. J. Nucl. Med. Mol. Imaging 39, S126-138. Bledzka et al. (2013) Circ. Res. 112, 1189-1200
February 2016 – Not for public distribution!
Comparison of viscosities between PA(S) and PEG polymers
Ranking according to intrinsic viscosity: PA(200) < PEG10kDa < PEG20kDa < PA(600) < PEG30kDa < PEG40kDa
Note that the plasma half-life extending effect of PA(600) lies between PEG(30k) and PEG(40k)!
Viscosity of a PASylated (or PEGylated) drug also strongly depends on fusion/conjugation partner.
PA(200)
PEG(20k)
PA(600)
PEG(40k)
Comparison of viscosities between PA(S) and PEG polymers
0
5
10
15
20
25
30
35
0 20 40 60 80 100
Visc
osity
[mP
a·s]
Concentration [g/L]
(measured on a µVISCTM microviscometer with VROC® chip in PBS)
February 2016 – Not for public distribution!
Leptin: a key regulator of energy homeostasis and fat control
Patients with genetic Leptin deficiency before/after 18 m daily treatment with rec. Leptin:
Friedman&Halaas, Nature 1998
Licinio et al., PNAS 2004
Wabitsch et al. N Engl J Med 2015 Asp79→→Tyr
Metreleptin
Arg84→→Trp * * * * * *
February 2016 – Not for public distribution!
Leptin: a potential biologic to treat obesity and/or diabetes?
Coppari R. & Bjørbæk C. (2012) Leptin revisited: its mechanism of action and potential for treating diabetes. Nat. Rev. Drug Discov. 11, 692-708.
Leptin in clinical settings: new attention as potential drug to treat diabetes
Small peptide hormone (16 kD) ⇒ short plasma half-life (50–150 min) Central role in control of appetite, energy control and metabolism Amgen: initially developed as anti-obesity drug – up to clinical phase II (attempts to develop a PEGylated drug failed) AstraZeneca: US approval of metreleptin (MYALEPT™) to treat congenital or acquired generalised lipodystrophy (2/14) Aptiv Solutions: orphan designation by EC for metreleptin to treat Lawrence syndrome Therapeutic potential for genetic Leptin deficiency, Lipodystrophy, Diabetes (as combination therapy)
Leptin
PAS
February 2016 – Not for public distribution!
Efficient production of PAS-Leptin in the periplasm of E. coli
Periplasmic secretion in E. coli Purification via IMAC, AEX, SEC
February 2016 – Not for public distribution!
Strongly extended half-life owing to expanded molecular volume
Leptin: ττ1/2 = 26 min
PAS600-Leptin: ττ1/2 = 19.6 h
PAS400-Leptin: ττ1/2 = 7.0 h
PAS200-Leptin: ττ1/2 = 3.3 h
PK study in C57BL6/J mice: Proteins were injected i.p. and samples were
taken at time points from 0.17 to 48 h (N=9) Protein concentration in plasma was
determined by sandwich ELISA, followed by fit to the Bateman function
February 2016 – Not for public distribution!
Strong receptor engagement by PAS-Leptin in the hypothalamus
HEK293 cells transfected with murine Leptin receptor and STAT3-regulated luciferase reporter gene
SPR sensor chip charged with murine LepRb-Fc
KD = 4.2 nM
February 2016 – Not for public distribution!
PAS-Leptin: strong decrease in food intake & weight loss in mice
Pharmacodynamics (PD) of Leptin treatment (± PAS) of C57BL/6J mice: male C57BL/6J mice (N=8): measurement of weight and food intake daily i.p. injection of PBS/E, interspersed by single injection of drug on day 0 (0.287 nmol/g b.w.)
Considerably improved efficacy of PASylated Leptin compared to unmodified Leptin!
Superactive Mouse Leptin Antagonist (SMLA) shows the inverse PD effect!
PASylated Leptin efficiently crosses the blood brain barrier (BBB) in the hypothalamus
February 2016 – Not for public distribution!
Repeated PAS#1(600)-Leptin injections in male B6.V-Lepob/J mice
Prof. Dr. Martin Klingenspor Lehrstuhl für Molekulare Ernährungsmedizin N = 4
Collaboration with the laboratory of:
February 2016 – Not for public distribution!
Normalization of body weight, glucose tolerance and insulin level
Insulin levels (not fasted)
Oral Glucose Tolerance Test (day 19)
Glucose Dose = (Lean Mass + 0.2 x Fat Mass) x 2.8 mg/g
ADA assay: No immunogenicity
Day 19 plasma samples, diluted 1:1000, probed with anti-mouse polyvalent Ig-AP, using the following targets:
February 2016 – Not for public distribution!
PASylation®®: boosting the clinical potential of Fab fragments!
Advantages of recombinant Fab fragments:
no dimerization (receptor clustering etc.) [e.g. thrombocytopenia: Avastin; c-Met signalling]
no immunological side effects [e.g. thrombosis: αCD40L Antova]
high yield bacterial production [cf. Cimzia®, MetMAb®]
better stability than scFv fragments
Bottle neck:
Very short circulation in comparison with IgG, unless PEGylated or otherwise modified
PASylation® provides a cheap and effective solution!
Fab fragments in disease therapy:
αGPIIb/IIIa Abciximab (Reopro®) αVEGF Ranibizumab (Lucentis®) αTNF Certolizumab pegol (Cimzia®)
Fab fragments for in vivo imaging:
αCEA 99mTc-Arcitumomab (CEA-Scan®) αCEA/NCA90 99mTc-Sulesomab (Leukoscan®) αCA125 99mTc-Igovomab (Indimacis®)
Fab fragments as antidotes:
αDigitalis DigiFab® αDigitalis Digibind® αSnake venom CroFab®
Fab fragments in clinical development:
αFibrin D-dimer 99mTc ThromboView®
αCD4 99mTc-Fab fragment αMetastasis-ass. 5T4 Naptumomab estafenatox αCD40L CDP7657 (UCP / Biogen Idec) αTNF DLX105 (Delenex) αPcrV KB001 (Kalobios / Sanofi Pasteur) ... etc.
February 2016 – Not for public distribution!
PASylation®® leads to a drastically prolonged plasma half-life
Quantification via sandwich ELISA: 1) wells coated with rec. HER2/ErbB2 2) detection of Fab with αhuCκ-AP conjugate)
Protein sample AUC [h·µg/ml] CL [ml/h/kg] !1/2" PK [h] Fold improvement
Fab 17 293 1.34 1
Fab-PAS#1(100) 123 40.5 2.71 2.0
Fab-PAS#1(200) 254 19.6 5.20 3.9
Fab-PAS#1(400) 870 5.75 14.4 10.7
Fab-PAS#1(600) 1751 2.85 28.2 21.0
Fab-2xPAS#1(200) 2915 1.72 37.2 27.8
Fab-ABD 2376 2.10 28.9 21.6
Fab-PEG(20)2 2726 1.83 35.3 26.4
Pharmacokinetics in Balb/C mice after i.v. injection:
Fab – PAS 200
400 600
February 2016 – Not for public distribution!
Fab PAS-100 PAS-200 PAS-400 albumin binding Rituximab domain
VH VL
CH
1
CL
AB
D
VH VL
CH
1
CL
VH VL
CH
1
CL
VH VL
CH
1
CL
Optimal imaging contrast for PAS-200!
Stronger background with albumin-mediated
plasma half-life extension!
VH VL
CH
1
CL
CH
3 C
H2
PET imaging of CD20+ Granta tumors 24 h p.i. in xenograft mice with PASylated αCD20 124I-Fab
ααCD20 PAS-Fab fragment as optimized in vivo imaging reagent
Prof. Dr. Arne Skerra
Contributors & Collaboration Partners
Pieris: Steffen Schlehuber Gabriele Matschiner Stefan Trentmann Andreas Hohlbaum and others ... IBA: Thomas Schmidt Wacker Biosolutions: Günter Wich Thomas Maier Carsten Bornhövd XL-protein: Uli Binder Lars Friedrich Alexandra Lensch and others ...
TUM lab: Michaela Gebauer Antonia Richter Evelyn Eggenstein Volker Morath Mikhail Barkovskiy Claudia Mendler Sabine Rauth Till Giese Andreas Eichinger Martin Schlapschy and many others ... IBAS Prague: Cyril Barinka Jakub Ptacek Zora Novakova NIH/NIAID: Daniel Douek Krystelle Nganou Makamdop
TUM & MRI: Dirk Haller Martin Klingenspor Markus Schwaiger Hans-Jürgen Wester LMU: Valerie Albrecht Rainer Glaß Jörg-Christian Tonn Transregio 127: Bruno Reichart Eckhard Wolf Elmar Jäckel (MHH) Fatih Noyan (MHH) Weizmann Inst.: Daniel Harari Gideon Schreiber