naturally optimized human antibodiescontent.stockpr.com/omniab/db/252/746/file/omniab.pdf• sd vs...
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Industry‐leading platforms for hmAb discovery
– Patented technology with freedom‐to‐operate– Engineering of custom animals
VH
CH3
CH2
hinge
VL
VH C
CH1
CH2
CH3
hinge
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Partners23 partners– 17 with unlimited access for milestones and/or royalties– Three fully paid up LLC-style– One academic, anti-PD-1 for China and OmniFlic for CART
Strategic partners– Three animal breeders and knock-out providers– Four CROs - US, Europe and Japan
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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
Improved genetic engineering based on proven technology
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Novel and proprietary knockout technology
Sangamo Zn‐finger technology– Exclusive license for Rat Ig knockout
Zn‐finger nuclease– One cut per genome– DSB repair– Mutation
Inactivation of rat antibody expression
Target sequence (exon of coding gene)
Non Homologous End‐Joining(NHEJ)
deletion insertion
Gene Disruption
DSB
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Targeted mutagenesis in rats using ZFNs
X
One‐cell embryo
Extract DNA to look for ZFN activity
ZFN1/ZFN2
Transfer to pseudopregnant females
Newborns
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OmniRat/OmniFlic/UniRat
New KO Technology–Heavy chain J‐locus deletion–Kappa light chain mutation–Lambda constant region deletion
Rat antibody expression 100% inactivated
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Improved genetic engineering
AbgenixMedarexHematechKirinTherapeutic Human Polyclonals (THP)
Old, suboptimal New, improved
Suboptimal BCR signaling Normal BCR signaling
RegeneronKyMabOMT
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Transgenic immunoglobulin lociRepresenting the human V(D)J repertoire
m‐RNA Human Antibody
Human LC locus
VL…………… VL1 J Ck +
VJ C
VH………VH1 D J E Cm Cd C2b E A
Easy
Conversion
Heavy chain locus
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Rat and mouse transgenesis
X
One‐cell embryo
Microinject DNA construct
Implant in hormone‐treated female
Transgenic offspring
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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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ImmunizationStandard (every 3‐4 weeks +/‐ adjuvant)Rapid (2x/week for 4 weeks)Rapid (1x at base of the tail)DNA prime/protein boostCell prime/DNA boostAddition of T‐cell epitope
• Sprague Dawley• Brown Norway• Lewis
• B6/SJL
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Two species = more antibodies = better epitope coverage
Different antibody titers– Different immune response genes
• SD vs BN vs LEW vs Mouse Bl6/SJL
– Human an gen ≠ rat an gen ≠ mouse an gen– Different V‐genes (human vs rat vs mouse)
• Blocking Antibodies: OmniMouse vs OmniRat vs Mouse vs Rat
– Isotype Switching• Increased IgM+/decreased IgG+ B‐cells in OmniRat• Mouse vs Rat Fc
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%
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Hybridoma generationOMT rats make antibodies as well as normal rats
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
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High number of unique binders
Target Homology human/rat
# screened clones
# binders # unique binders
1 99% 7896 511 1752 98% 6768 337 1303 67% 2880 297 49
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Good diversity against conserved targets
20 IGHV, 11 IGKV, and 12 IGLV different germ lines isolated from 3 screening campaigns
Some V genes are specific to one target selection
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OmniRat vs phage display antibody binding
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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DNA immunization
MONOCLONALPOLYCLONAL
Polyclonal sera (8 weeks) Monoclonal antibodies(4 months)
No need for isolated protein or peptide during the process or for screening
Start from electronic cDNA sequences
Cloning of human CEACAM5 cDNA into our specially designed
vectors
cDNA immunisation (GENOVAC Antibody Technology®) of OmniRats™ and
Wistar Rats. Immune response against the
native antigen
Fusion and hybridoma generation
Screening and subcloning
Serum and supernatant test on native protein. Cross‐reactivity tests.
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Receptor protein complex
Immune serum (1:1000 dilution) of a representatative animal is tested on
mammalian cells transfected with the cDNA encoding for the target antigen
(green curves) or with an irrelevant construct (red curves)
Three fusions with 7 immunized animals– 505 positive hits out of 960 tested samples (53%)– 1122 positive hits out of 1632 tested samples (69%)– 792 positive hits out of 864 tested samples (69%)
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GPCR
Immune serum (1:1000 dilution) of a representatative 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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Transgenic animals for hmAb discovery
Fixed L‐chain IgG antibodies
OmniFlic with rearranged human L‐chain expressed with any (human) IgH locus
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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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OmniDeep™ sequence‐based discovery
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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OmniDeep screening strategy– 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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6 OmniFlic rats,injected with PDL1
6 OmniFlic rats,injected with PDL1
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
Example: PDL1 antibody discovery in OmniFlic
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1 2 3 4 5 6
PDL1 OmniFlic Rats
Eac
h ro
w is
a u
niqu
e C
DR
3 fa
mily
Each column is an independent sample
Heat map key:Red= highest frequency sequenceBlue= lowest frequency sequence
Antibody repertoire lineage rank analysis
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Selection of antibodies for primary screenHigh‐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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Primary screen: PDL1 ELISA binding & affinity
– 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
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PDL1 ligand‐blocking sequence families
Primary Screen– Identified 2 antigen‐specific antibody families with ligand blocking activity
Secondary Screen– Identify family members with higher affinity and fewer sequence liabilities
Primary Screen Secondary Screen
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affinity (nM) CDR seq liability1.31.32.2 met,nglyc2.7 met4.0 met5.0 nglyc6.36.87.88.08.28.4 nglyc9.0 nglyc
10.0 deam,met13.7 met,nglyc13.714.4 met from primary screen16.118.421.923.225.4 deam29.038.548.249.0 from primary screen49.553.7 nglyc79.5
PDL1 ligand‐blocking family #1
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PDL1 ligand‐blocking family #2affinity (nM) CDR seq liability
4.4 isom,cys4.9 isom from primary screen5.9 isom6.1 isom6.3 isom6.9 isom6.9 isom7.8 isom,cys8.0 isom
10.1 isom11.3 isom11.8 isom,cys12.3 isom17.5 nglyc20.6 deam,isom23.5 isom61.4 isom62.8 isom67.0 deam,isom82.0 deam,isom92.0 isom
870.0 deam,isom980.0 isom
1665.0 deam,isom1750.0 deam,isom1920.0 deam,isom2480.0 deam,isom2670.0 deam,isom2710.0 deam,isom