naturally optimized human antibodies® roland buelow, ph.d. · • 20 ighv, 11 igkv, and 12 iglv...
TRANSCRIPT
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Roland Buelow, Ph.D.
Naturally optimized human antibodies®
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Four animal platforms & three species createthe broadest antibody repertoires available
An industry-leading patented, validatedhuman antibody rat
Added species yields additional antibodiesand increased epitope
coverage
Rat with singlecommon light chain,
designed for bispecifichuman antibodies
3rd species with unique epitope coverage
“Three Species – One License”
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• 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
OmniAb: A Best-in-Class Technology
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OmniAb® PartnersA Growing and Diverse List
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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
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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 2 152 880 B1
— EP 2 336 329 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
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Platform Development
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OmniAb® Platform Development
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• 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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Inactivation of Endogenous Rat Ab Expression
• Zn-finger technology– Exclusive license for rat Ig knockout– One cut per genome– Double strand break repair– Mutation
Target sequence (exon of coding gene)
Non-homologous end-joining (NHEJ)
deletion insertion
Gene disruption
Double strand break
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Targeted Mutagenesis in Rats Using ZFNs
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X
One-cell embryo
Extract DNA to look for ZFN activity
ZFN1/ZFN2Transfer to pseudo-pregnant females
Newborns
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OmniAb® Platform Development
• Inactivation of endogenous rat Ig genes– Heavy chain J-locus– Light chain Cκ– Light chain Cλ
100% rat Ig expression inactivated
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Geurts et al. Science (2009)Menoret et al. Eur J Immunol (2010)
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Science-First Rat Ig Gene Knock-Out
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Geurts et al. Science2009, July 24, 325: 433-
Menoret et al. Eur. J. Immunology2010, 40: 2932–2941
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Transgenic Immunoglobulin LociRepresenting the Human V(D)J Repertoire
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mRNA
Human Antibody
Human LC locusVL…………..VL1 J CL +
VJ C
VH…..VH1 D J EµCµCd Cγ2b E A
Easy conversion
Heavy chain locus
VDJ C
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Heavy Chain: Complete VH Repertoire
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HC30
HC14
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Light Chain: Kappa and Lambda Loci
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4.4kbmodified E24 ~157 kb PmeI-AsiSI
3.5kbG22 ~153 kb SacII
modified D9 ~158 kb NruI
UR
A
LC
KC
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Normal Human J- and D-Genes Usage
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Osborn et al. J. Immunol. 2013
44 functional VH genes (100% of HC)
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Normal CDR3 Length Distribution in OmniRat
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Normal Expression of Human Kappa Chain
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Osborn et al. J. Immunol. 2013
20 functional Vκ genes
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Normal Expression of Human Lambda Chain
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15 functional Vλ genes
Osborn et al. J. Immunol. 2013
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Immune Development Is Fully Restored
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SD wt
JKO/JKO
JKOJKO/LKOLKO/HuL
Bone marrow SpleenIgMµ FITC
CD45R PEOsborn et al. J. Immunol. 2013
Human Igκ titer
Human Igλ titer
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OmniRat and OmniMouse
• Functional recombinant immunoglobulin loci– Productive rearrangement of all functional human VH, DH, JH; Vκ, Jκ and Vλ, Jλ– 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
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Journal of Immunology 2013
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OmniFlic® Rats For Bi-Specific Antibody Development
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• Productive rearrangement of all functional human VH, DH, JH• Somatic mutations and high affinities (single digit to sub-nanomolar)• Functional activity
Rearranged human κ L-chain expressed with any (human) IgH locus
Monospecific IgG
Bispecific IgG
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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
• Different immunogenicity in different species– Human antigen : rat immunogen ≠ mouse immunogen– Functional antibodies: OmniMouse® vs OmniRat®
RatMouse
Epitope coverage
Antigen Human/Mouse Human/Rat Mouse/Rat
CD30 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-1β 64.7% 63.8% 86.9%
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Antigen sequence homology
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OmniAb: A Best-in-Class TechnologyOmniChicken™: Expanded Epitope Coverage
Addition of OmniChicken offers partnersunparalleled epitope coverage
Mouse Response Rat Response
Chicken Response
Overlap area represents novel and cross-reactive epitopesNon-overlap area
represents novelepitopes
• Because of evolutionary distance between birds and mammals, chickens enable the generation of novel antibodies against targets that are not immunogenic in rodents
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Antibody Discovery
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Immunization Protocols
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• 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
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Maximum Success Strategy
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• 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)
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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)
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Good Diversity Against Conserved Targets
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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
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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
1 62 463
2 74 80
3 97 285
4 99 199
5 95 225
6 87 264
7 92 470
8 71 282
9 68 282
10 46 395
11 63 484
12 67 1089
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OmniRat vs Phage Display Antibody Binding
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Target 1
* Estimated affinities
Target 2
Target 3
KD*= 1.9 nM
OMT-A1
KD= 2.3 nM
KD= 15 nM KD= 0.01 nM
OMT-C2
KD*= 0.001 nM KD*= 0.02 nM
OMT-B1
OMT-C3
KD= 7 nM
OMT-C4
KD*= 4 nM
OMT-C1
KD= 0.07 nM
KD*= 0.13 nM
OMT-B2
KD= 0.33 nM
OMT-B3
OMT-A2
KD= 219 nM
Phage display-derived IgG
OmniRat-derived IgG
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Targeting a GPCR
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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
• 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%)
Parallel immunization with KO mice was unsuccessful
Human target (transient)Mouse target (transient)
Control target (transient)Human target (stable)
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Sequence-Based Discovery at TeneoBio
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• Platform is a unique combination of– Antibody repertoire deep sequencing– Custom bioinformatics analysis– High-throughput vector assembly– Recombinant expression and screening
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Screening Strategy
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• Primary screen: All prominent CDR3 sequence families (ELISA, affinity, functional)• Secondary screen: Complete lineages of primary hits (affinity, functional)
Primary Screen:100-400 diverse CDR3 sequences
Guided by lineage rank analysis
SecondaryScreen:50-300 unique sequences per lineage
Includes rare sequences in lineages of interest
hit
hit
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PD-L1 Antibody Discovery in OmniFlic
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• 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
6 OmniFlic rats,injected with PD-L1
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HCAb Repertoire Rank Analysis
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1 2 3 4 5Adj only rats Adj+antigen rats
LN samples used for hybrid/yeast display
Blue= hybridoma/yeast display sequences from rat #6
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
Antigen-selected samples
high freq seqs found in antigen rats and adj-only rats
1 2 3 4 5 6 7
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Selection of Antibodies For Primary Screen
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• 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
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PD-L1 Binding and Affinity 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 bindingBlue= negative binding
Primary Screening
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PD-L1 Ligand-Blocking Sequence Families
• Identified 2 antigen-specific antibody families with ligand blocking activity
• Identify family members with higher affinity and fewer sequence liabilities
Primary Screen Secondary Screen
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VD Recombination in OmniFlic Rats
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Ig-Seq Analysis of Single B Cells
# o
f V g
ene
retr
ieve
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Heavy chain germline families (IGHV)
D genes
N= 1874 sequenced B cells
• Excellent diversity and recombination
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CDRs Diversity Within a Clonal Cluster
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• Evidence of in vivo somatic hypermutations
IGVH4-39 & CDR3 with 15 aa (N=504)
HCDR1 HCDR2 HCDR3
Amin
o ac
id fr
eque
ncy
(%) 100
75
50
25
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OmniFlic Generate High Affinity IgG
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• Similar affinities to benchmark antibody
#6448A01 #6450G07 #6474B02
Benchmark
KD= 4.2 nM KD=3.7 nM KD=18.2 nM KD=6.6 nM
IgG isolated from OmniFlic rats
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OmniAb Antibody Platform
Three Species – One License
Platform also includes OmniFlic rat designed for bispecific antibodies
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Slide Number 1Slide Number 2Slide Number 3OmniAb® Partners�A Growing and Diverse List�Strategic Service Partners�OmniAb®�Intellectual Property and Freedom-to-OperateSlide Number 7OmniAb® Platform DevelopmentInactivation of Endogenous Rat Ab ExpressionTargeted Mutagenesis in Rats Using ZFNsOmniAb® Platform DevelopmentScience-First Rat Ig Gene Knock-OutTransgenic Immunoglobulin Loci�Representing the Human V(D)J RepertoireHeavy Chain: Complete VH RepertoireLight Chain: Kappa and Lambda LociNormal Human J- and D-Genes UsageNormal CDR3 Length Distribution in OmniRatNormal Expression of Human Kappa ChainNormal Expression of Human Lambda ChainImmune Development Is Fully RestoredOmniRat and OmniMouseJournal of Immunology 2013OmniFlic® Rats For Bi-Specific Antibody DevelopmentTwo Species For Higher Diversity and Broader Epitope Coverage�Slide Number 25Slide Number 26Immunization ProtocolsMaximum Success StrategyOmniAb HybridomasGood Diversity Against Conserved TargetsAntigen-Specific B Cell Cloning Is an Efficient Method �Analysis of Screening Campaigns with Single Ag+ B Cell Cloning��OmniRat vs Phage Display Antibody BindingTargeting a GPCRSequence-Based Discovery at TeneoBioScreening StrategyPD-L1 Antibody Discovery in OmniFlicHCAb Repertoire Rank AnalysisSelection of Antibodies For Primary ScreenPD-L1 Binding and Affinity ScreeningPD-L1 Ligand-Blocking Sequence FamiliesVD Recombination in OmniFlic RatsCDRs Diversity Within a Clonal ClusterOmniFlic Generate High Affinity IgGOmniAb Antibody PlatformSlide Number 45