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OmniAb®Naturally optimized human antibodies®
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Transgenic animals for hmAb discovery
Only company to offer three platformsPatented technology with freedom‐to‐operate
VL
VH C
CH1
CH2
CH3
hinge
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28 current OmniAb users
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Strategic partners
OmniAb Animal Breeding– All rats at CRL– All mice at Taconic
Antibody Discovery– Aldevron (Germany & US)– Antibody Solutions (US)– Teneobio (US)– WuXi (China)– Syngene International (India)
Knock‐outs– Horizon Discovery (SAGE Labs)– Taconic
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OmniAb status
28 partners– 14 partners < 18 months– 14 partners > 18 months
>300 antibody projects– >20,000 unique fully human binders
Good manufacturability, high affinity, expected PK3 INDs in 20165‐10 INDs in 2017
First Phase I trial in 2016
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Why?
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Broad protection under issued patents– US 8,703,485 B2, US 8,907,157 FB2, US 9,475,859 (October 25, 2016)– EP 2 152 880 B1, EP 2 336 329 B1
FTO for all indications worldwide
Freedom‐to‐operate (FTO)
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Litigation Update: Homologous Recombination in the MouseRegeneron vs Merus, re U.S. Patent No 8,502,018– 018 found invalid by the District Court in New York– Notice of appeal to the Federal Circuit filed December 17, 2015– Motion granted to file amicus curiae in support of Regeneron (Mach 25, 2016)– More than a dozen patent applications still pending in the same Velocimmune
patent family before the USTPO
EPO Board of Appeal and UK High Court re EP 1360287– EPO Board of Appeal and UK High Court have issued conflicting decisions on the
validity of Regeneron’s main European patent for the Velocimmune platform– EPO revoked EP 136027, but the patent was subsequently maintained on appeal
in view of claim amendments– By contrast, during infringement proceedings in the UK brought by Regeneron
against KyMab and Novo Nordisk, it was found that KyMab was infringing the patent but that the patent was invalid
– Regeneron still has the opportunity to appeal the UK High Court decision
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Three platforms
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Bioengineering: 6‐12 monthsBioengineering: 6‐12 months
Immune system: 7‐14 daysImmune system: 7‐14 days
Immune system faster than engineering
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Approved/pending therapeutic antibodies
2011 ‐ 33 antibodies0 homo sapiens‐derived
31 animal‐derived2 phage‐derived
6 transgenic animal‐derived
2016 ‐ 65 antibodies1 homo sapiens‐derived
59 animal‐derived5 phage‐derived
19 transgenic animal‐derived
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OmniAb platform development
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OmniAb platform development
Inactivation of endogenous rat Ig genes– Heavy chain J‐locus– Light chain Cκ– Light chain Cλ
Recombinant immunoglobulin loci– Kappa light chain– Lambda light chain– Heavy chain
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Science‐first rat Ig gene knock‐out
Science2009, July 24, 325: 433‐
European Journal of Immunology2010, 40: 2932–2941
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OmniRat and OmniMouseFunctional recombinant immunoglobulin loci– Productive rearrangement of all functional human VH, DH, JH and VL, JL– Normal human frequencies of V‐, D‐, J‐gene usage– Normal human CDR3 length
Normal B‐cell developmentHigh expression of human antibodiesNormal hypermutation and affinity maturation
• Sprague Dawley• Brown Norway• Lewis
• B6/SJL
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Journal of Immunology 2013
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Antibodies
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Immunize animals• More animals = more unique antibodies (6‐20)• Protein, cells, DNA, etc.
Adjuvant systems• CFA/IFA, Ribi, etc. (>1)
Hybridoma technology• Inexpensive, but inefficient and variable• More fusions = more antibodies (> 2 fusions using 4‐10 animals)
Isolation of B‐cells + cDNA cloning + antibody expression• Technically challenging, but efficient
NGS repertoire analysis + HT gene assembly + expression• Technically challenging, but efficient
Antibody discovery
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OmniAb Hybridomas
>250 antibody discovery projects>100 human antigens>1300 animals (6‐20)>250 fusions (1‐5)>15000 unique antibodies̴̴10% hit rate (antigen‐specific antibody producing hybridomas)Low immortalization frequency̴̴20% failure (= no hybridomas)
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9% 63% 41% 23% 10% 20%
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Animal Antigen Cells* fusions titer hybrids IgGs** Kd***
SD PG LN 1 38400 3520 38 0.3‐1.0 nM
OmniRat PG LN 1 12800 1600 148 0.7‐2.4 nM
OmniRat hGHR LN 3 4800 704‐1024 18, 3, 2 ND
SD TAU/KLH LN 1 20000 1728 99 0.6‐2.4 nM
OmniRat TAU/KLH LN 1 4800 1880 118 0.5‐3.2 nM
SD HEL LN 1 12800 1564 26 0.02‐0.1 nM
OmniRat HEL LN 3 25600 288‐640 0, 2, 7 0.6‐1.5 nM
SD OVA LN 1 9600 1488 10 1.1‐4.8 nM
OmniRat OVA LN 4 8000 512‐2240 0, 30, 0, 1 0.7‐1.5 nM
5 different antigens
Single immunization on day 0Lymph node fusion on day 21
16 fusions
Similar titers
Similar # of hybridomas
502 mAbs confirmed by Biacore
5 highest affinity Abs
OmniRat makes Abs like normal rats
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FACS Sorting of IgG+ B‐cells• > 25 target antigens• 2% +/‐ 1% of cloned/expressed antibodies are unique and antigen‐specific• >4500 unique antigen‐specific antibodies
100% project success rate
Antigen % aa homology # of unique mAbs1 62% 4632 74% 803 97% 2854 99% 1995 95% 2256 87% 2647 92% 4708 71% 2829 68% 28210 46% 39511 63% 48412 67% 1089
B cell isolation, cDNA/cloning, mAb expression
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Summary of discovery output: Total
Total number of discovery projects 44
Total number of animals 520
Total number of targets 27
Total number of NGS sequence reads generated 584,251,034
Total number of unique antibodies screened 11,631
Total number of antigen‐specific antibodies 2,905
100% project success rate
Teneobio
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Potential failure and possible improvements
Potential Failure• No immune response• No epitope coverage• Low affinity binders
Possible Improvements• Use many animals from different strains• Use different animal species
Sometimes Chicken is better than Beef!
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OmniFlic animals bred on 3 genetic backgrounds:
Lewis, Sprague Dawley, Brown Norway
Does the genetic background of Flic animals have an effect on antibody production?
– Serum titer
– V‐gene usage
– Antibody sequence diversity
OmniFlic strain comparison
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Brown Norway showed significantly higher IgM and IgG serum levels
OmniFlic total Ig serum levels
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Brown Norway showed significantly higher antigen‐specific IgG
OmniFlic antigen‐specific serum levels
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Immune response – cellular perspectiveMultiple injections, 5‐6 week immunization time course Harvest cells from lymph nodes
Millions of naïveB cells in circulation
Affinity maturation in GCs of LNs• ~2M total B cells per LN• ~20K ag‐specific B cells per LN (1%)• ~200 ag‐specific CDR3 families per animal
(based on GC model of normal rodent)
Plasma or memory cell differentiation
Our analysis is focused onLN‐derived B‐cells
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Primary Screen:300‐400 diverse CDR3 sequences
Guided by lineage rank analysis
SecondaryScreen:50‐300 unique sequences per lineage
Includes rare sequences in lineages of interest
hit
hit
Primary screen: Diverse CDR3 sequence families (ELISA, affinity, functional)
Secondary screen: Complete lineages of primary hits (affinity, functional)
Sequence‐based antibody discovery
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1 2 3 4 5 6
Immunized Rats
Each ro
w is a uniqu
e he
avy chain CD
R3 fa
mily
Each column is an independent sample
Heat map key:Red= expanded familiesBlue= non‐expanded families
Sequence‐based antibody discoveryAntibody repertoire lineage rank analysis
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Sequence‐based antibody discoveryPrimary Screen Secondary Screen
Each row is a unique antibody
Primary screen• Apply high‐throughput primary
screen assays (expression, binding, function)
• Identify families with preferred functional activity
Secondary screen• Identify family members with
higher affinity and fewer sequence liabilities
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V‐gene usage – OmniFlic strain comparision
Red= 60% Blue= 0%
Lewis
Sprague Dawley
Brown Norway
IGHV
4‐39
IGHV
3‐38
IGHV
3‐35
IGHV
4‐34
IGHV
3‐33
IGHV
4‐31
IGHV
3‐30
IGHV
4‐28
IGHV
2‐26
IGHV
1‐24
IGHV
3‐23
IGHV
3‐11
IGHV
3‐9
IGHV
1‐8
IGHV
3‐7
IGHV
2‐5
IGHV
7‐4
IGHV
4‐4
IGHV
1‐3
IGHV
1‐2
IGHV
6‐1
Sig diff (p<0.05)
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Seq analysis – OmniFlic strain comparision
Num
ber o
f uniqu
e CD
R3s
0
20
40
60
80
100
Unique CDR3 seqs with ≥10 counts
Spr. Daw. Lewis Brown Norway
T‐test p‐valueSprague Dawley Brown Norway 1.3E‐02Sprague Dawley Lewis 8.5E‐05Brown Norway Lewis 8.7E‐01
Sig diffSig diff
Individual animals
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Analysis of convergent sequences
Each ro
w is a uniqu
e sequ
ence
Red= high freq seq Blue= absent seq
>99.9% of sequences are unique to a single animal– 61% of convergent sequences within a strain are unique
to that strain
Differences in genetic background drive sequence convergence within strain but not between strains
Sprague Dawley Brown Norway
Example of convergent sequences in 2 strains
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Two species = more antibodies = better epitope coverage
RatMouse
Epitope coverage
Gene Human/Mouse Human/Rat Mouse/RatCD30* 54.0% 50.1% 83.4%CD22* 58.7% 56.9% 77.7%CD14 63.7% 61.3% 80.9%CD80 39.2% 43.4% 63.4%CD52 36.1% 41.0% 64.9%
IL‐1 beta 64.7% 63.8% 86.9%
Different immune response genes• SD vs BN vs LEW vs Mouse Bl6/SJL
Human antigen ≠ rat antigen ≠ mouse antigen
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Kinetics and epitope binning of anti‐PG mAbs
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GPCR
Immune serum (1:1000 dilution) of a representative animal is tested on
mammalian cells transfected with the cDNA encoding for the target antigen
(human = green curves, mouse = blue), on a stable cell line, or with an irrelevant
construct (red curves)
Parallel immunization with KO mice unsuccessful!
Three fusions with 10 immunized animals– 11 positive hits out of 1824 tested samples (0.6%)– 34 positive hits out of 1920 tested samples (1.8%)– 2 positive hits out of 1920 tested samples (0.1%)
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Conclusions
>99.9% of antibody sequences in an individual animal are unique• More animals = more diverse antibodies
Different genetic background result in different antibodies• Immunize various different animal strains• Immunize mice and rats
Use of many genetically diverse animals increases epitope coverage
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Maximum success strategyImmunize many animals• Use different animal strains• Use mice and rats• Use at least 2 adjuvant systems• Include OmniFlic animals
Hybridoma technology• More animals + more fusions = higher success rate• Retain B cell RNA for NGS or display technology
Alternative technologies• B cell isolation + cDNA cloning + antibody expression• NGS repertoire analysis + high‐throughput gene assembly and expression
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OmniAb
Best Antibodies for the Most Difficult Targets