Naturally optimized human antibodies. Roland Buelow, Ph.D.

Transcription

1 Naturally optimized human antibodies Roland Buelow, Ph.D. 1

2 Three Species One License An industry-leading patented, validated human antibody rat Added species yields additional antibodies and increased epitope coverage Rat with single common light chain, designed for bispecific human antibodies 3 rd species with unique epitope coverage Four animal platforms & three species create the broadest antibody repertoires available 2

3 OmniAb: A Best-in-Class Technology 31 partners >300 antibody projects >20,000 unique fully human binders Good manufacturability, high affinity, expected PK 5 INDs/Phase I Genentech, Janssen, Gloria, Aptevo, HanAll First Phase I trial in 2016

4 4 OmniAb Partners A Growing and Diverse List

5 Strategic Service Partners Breeding All rats at Charles River Laboratories (US) All mice at Taconic (Denmark) Downstream antibody discovery services Abcellera (Canada) Aldevron (Germany & US) Antibody Solutions (US) ImmunoPrecise (Canada) Syngene International (India) Teneobio (US) WuXi AppTec (China) Knock-out animal generation Horizon Discovery (SAGE Labs) Taconic 5

6 OmniAb Intellectual Property and Freedom-to-Operate Broad protection exists under issued OmniAb patents US 8,703,485 B2 US 8,907,157 FB2 EP B1 EP B1 US 9,475,859 (issued October 2016) New OmniRat2 patent application filed in January 2017 Freedom-to-Operate for all indications worldwide Other antibody discovery technologies have been subject of significant complexities relating to Freedom-to-Operate 6

7 Platform Development

8 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 8

9 Inactivation of Endogenous Rat Ab Expression Target sequence (exon of coding gene) Zn-finger technology Exclusive license for rat Ig knockout One cut per genome Double strand break repair Mutation Double strand break Non-homologous end-joining (NHEJ) deletion insertion Gene disruption 9

10 Targeted Mutagenesis in Rats Using ZFNs X Transfer to pseudopregnant females ZFN1/ZFN2 One-cell embryo Newborns 10 Extract DNA to look for ZFN activity

11 OmniAb Platform Development Inactivation of endogenous rat Ig genes Heavy chain J-locus Light chain Cκ Light chain Cλ 100% rat Ig expression inactivated Geurts et al. Science (2009) Menoret et al. Eur J Immunol (2010) 11

12 Science-First Rat Ig Gene Knock-Out Geurts et al. Science 2009, July 24, 325: 433- Menoret et al. Eur. J. Immunology 2010, 40:

13 Transgenic Immunoglobulin Loci Representing the Human V(D)J Repertoire Human LC locus VL..VL 1 J CL + Heavy chain locus VH..VH 1 D J EµCµCd Cγ2b E A VJ C VDJ C mrna Human Antibody Easy conversion 13

14 Heavy Chain: Complete VH Repertoire HC14 HC30 14

15 Light Chain: Kappa and Lambda Loci LC G22 ~153 kb SacII 3.5kb modified D9 ~158 kb NruI 4.4kb modified E24 ~157 kb PmeI-AsiSI URA KC 15

16 Normal Human J- and D-Genes Usage 44 functional VH genes (100% of HC) Osborn et al. J. Immunol

17 17 Normal CDR3 Length Distribution in OmniRat

18 Normal Expression of Human Kappa Chain 20 functional Vκ genes Osborn et al. J. Immunol

19 Normal Expression of Human Lambda Chain 15 functional Vλ genes Osborn et al. J. Immunol

20 Immune Development Is Fully Restored IgMµ FITC Bone marrow Spleen Human Igκ titer SD wt JKO/JKO Human Igλ titer JKOJKO/ LKOLKO/HuL CD45R PE Osborn et al. J. Immunol

21 OmniRat and OmniMouse Functional recombinant immunoglobulin loci Productive rearrangement of all functional human V H, D H, J H ; V κ, J κ and V λ, J λ Normal human frequencies of V-, D-, J-gene usage Normal human CDR3 length distribution Normal B cell development High expression of human antibodies Normal hypermutation and affinity maturation Sprague Dawley/ Brown Norway/ Lewis B6.SJL 21

22 Journal of Immunology

23 OmniFlic Rats For Bi-Specific Antibody Development Productive rearrangement of all functional human V H, D H, J H Somatic mutations and high affinities (single digit to sub-nanomolar) Functional activity Monospecific IgG Rearranged human κ L-chain expressed with any (human) IgH locus Bispecific IgG 23

24 Two Species For Higher Diversity and Broader Epitope Coverage Different immune response genes Rat SD vs BN vs LEW vs mouse B6.SJL Different V-genes (human vs rat vs mouse) Isotype switching Mouse Fc vs rat Fc Mouse Epitope coverage Rat Different immunogenicity in different species Human antigen : rat immunogen mouse immunogen Functional antibodies: OmniMouse vs OmniRat Antigen sequence homology Antigen Human/Mouse Human/Rat Mouse/Rat CD % 50.1% 83.4% CD % 56.9% 77.7% CD % 61.3% 80.9% CD % 43.4% 63.4% CD % 41.0% 64.9% IL-1β 64.7% 63.8% 86.9% 24

25 OmniAb: A Best-in-Class Technology OmniChicken : Expanded Epitope Coverage Because of evolutionary distance between birds and mammals, chickens enable the generation of novel antibodies against targets that are not immunogenic in rodents Addition of OmniChicken offers partners unparalleled epitope coverage Mouse Response Rat Response Non-overlap area represents novel epitopes Overlap area represents novel and cross-reactive epitopes Chicken Response 25

26 Antibody Discovery

27 Immunization Protocols Standard (every 3-4 weeks +/- adjuvant) Rapid (2x/week for 4 weeks) Rapid (1x at base of the tail) DNA prime/protein boost Cell prime/dna boost Addition of T-cell epitope Sprague Dawley/ Brown Norway/ Lewis B6.SJL 27

28 Maximum Success Strategy Immunization of many animals 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 (FACS, microfluidics) NGS repertoire analysis Display technologies (phage or yeast) 28

29 OmniAb Hybridomas >250 antibody discovery projects >100 human antigens >1,300 animals (6-20) >250 fusions (1-5) >15,000 unique antibodies ~10% hit rate (Ag-specific antibody producing hybridomas) Low immortalization frequency ~20% failure (= no hybridomas) 29

30 Good Diversity Against Conserved Targets Germline Usage from Sequenced Hybridomas 20 IGHV, 11 IGKV, and 12 IGLV different germlines isolated from 3 screening campaigns Some V genes are specific to one target selection 30

31 Antigen-Specific B Cell Cloning Is an Efficient Method Analysis of Screening Campaigns with Single Ag + B Cell Cloning > 25 target antigens 2% ±1% of cloned/expressed antibodies are unique and Ag-specific >4500 unique Ag-specific antibodies 100% project success rate Antigen % aa homology # of unique mabs

32 OmniRat vs Phage Display Antibody Binding Phage displayderived IgG OmniRat-derived IgG OMT-A1 OMT-A2 Target 1 K D *= 1.9 nm K D = 2.3 nm K D = 219 nm OMT-B1 OMT-B2 OMT-B3 Target 2 K D *= nm K D *= 0.02 nm K D *= 0.13 nm K D = 0.33 nm OMT-C1 OMT-C2 OMT-C3 OMT-C4 Target 3 K D = 15 nm K D = 0.07 nm K D = 0.01 nm K D = 7 nm K D *= 4 nm * Estimated affinities 32

33 Targeting a GPCR Hybridoma Screening by FACS Immune serum (1:1000 dilution) of a representative animal tested on mammalian cells transfected with the cdna encoding for the target antigen Human target (transient) Mouse target (transient) Human target (stable) Control target (transient) Three fusions with 10 immunized animals 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%) Parallel immunization with KO mice was unsuccessful

34 Sequence-Based Discovery at TeneoBio Platform is a unique combination of Antibody repertoire deep sequencing Custom bioinformatics analysis High-throughput vector assembly Recombinant expression and screening 34

35 Screening Strategy Primary screen: All prominent CDR3 sequence families (ELISA, affinity, functional) Secondary screen: Complete lineages of primary hits (affinity, functional) hit Primary Screen: diverse CDR3 sequences Guided by lineage rank analysis Secondary Screen: unique sequences per lineage Includes rare sequences in lineages of interest hit 35

36 PD-L1 Antibody Discovery in OmniFlic 6 OmniFlic rats, injected with PD-L1 LN tissue from 2 legs X 2 tech reps of each = 4 total samples per rat Deep sequencing and analysis done on each sample Candidates expressed as fully human IgG with fixed light chain 36

37 HCAb Repertoire Rank Analysis Adj only rats Adj+antigen rats LN samples used for hybrid/yeast display Antigen-selected samples Blue= hybridoma /yeast display sequences from rat #6 37 Blocks of red= high freq seqs unique to one antigen rat high freq seqs found in multiple antigen rats but not in adj-only rats high freq seqs found in antigen rats and adj-only rats

38 Selection of Antibodies For Primary Screen High-frequency sequences chosen from 2 categories CDR3 families found in multiple rats CDR3 families found in individual rats 234 total heavy chain sequences 106 unique CDR3 sequence families Expressed as fully human IgG with fixed LC in HEK cells ELISA screen 38

39 PD-L1 Binding and Affinity Screening Primary Screening Each row is a unique VH sequence expressed as IgG with fixed LC 127 of 234 total abs positive by ELISA 62 of 106 unique CDR3 families positive by ELISA 1.3 nm highest affinity 2 different CDR3 sequences with ligand-blocking activity in cell-based assay Red= strong binding Blue= negative binding

40 PD-L1 Ligand-Blocking Sequence Families Primary Screen Secondary Screen Identified 2 antigen-specific antibody families with ligand blocking activity Identify family members with higher affinity and fewer sequence liabilities

41 VD Recombination in OmniFlic Rats Ig-Seq Analysis of Single B Cells N= 1874 sequenced B cells # of V gene retrieved D genes Heavy chain germline families (IGHV) Excellent diversity and recombination 41

42 CDRs Diversity Within a Clonal Cluster IGVH4-39 & CDR3 with 15 aa (N=504) Amino acid frequency (%) HCDR1 HCDR2 HCDR3 Evidence of in vivo somatic hypermutations 42

43 OmniFlic Generate High Affinity IgG IgG isolated from OmniFlic rats Benchmark #6448A01 #6450G07 #6474B02 K D = 4.2 nm K D =3.7 nm K D =18.2 nm K D =6.6 nm Similar affinities to benchmark antibody 43

44 OmniAb Antibody Platform Three Species One License Platform also includes OmniFlic rat designed for bispecific antibodies

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