Isogenica’s next-generationhuman Fab library

Guy Hermans, CSO

Antibody Engineering 2018, San Diego

December 11-15, 2017

Isogenica – corporate overview

Based near Cambridge, UK

Expertise in protein and peptide engineering since 2001

Specialist synthetic antibody library and technologies licensing company

Offering leading edge synthetic library and display technologies

• Colibra™ DNA synthesis

• CIS™ in vitro display

2NON-CONFIDENTIAL3/5/2017

Why choose a synthetic antibody library

Increased diversity

• Million-fold greater sequence space covered

• No immunological response biases or self-tolerance gaps

• Sub-libraries with varying CDR3 length ranges available

Avoiding liabilities

• Potential drug development liabilities or unusual structures excluded(sequence liabilities, free cys, highly extended CDR3 loop-lengths)

• Lack of somatic hypermutation avoids framework region mutations,which can complicate downstream humanization

Time to hit identification

• ‘Single-pot’ library/ies - no lead time for immunization andanimal specific library generation prior to initiation of selections

Greater opportunity to find a developable therapeutic antibody

candidate within short timeframe

33/5/2017

Colibra™ library build technology

• Single or double codon additions per step

• Optimized automation and bioinformatics

• Integrated with NGS for continuous in-process and end product quality control

43/5/2017

Positional Weight Matrix

A G G P

72 possible sequences:NGTYYGDGADDP….

CMC liability motifs:NG, DG, DP

Colibra™ build technology advantages and typical error types

Advantages of ColibraTM:

• Precise control over amino acid composition,no redundant codon usage

• Minimized potential manufacturing liabilitiesby eliminating specific AA motifs, retaining diversity

• Minimal cycle number and in-process QC by NGS strongly reduces compounding of synthesis errors

Synthesis by-products in ColibraTM:

• Most frequent remaining errors (deviations from design) are distinct from those in conventional methods:

• Most frequently -3 or -6 nucleotide in-frame by-products

• Premature stop codons absent

• For antibody loop libraries:

• Shift to shorter products, more pronounced at long loop length syntheses

• Compensated for in library by adjusting mixture of sub-libraries towards design specs

53/5/2017

Avoided: NG, DG, DP

llamdA™: fully synthetic camelid type single domain antibody library

625/04/2017

CIS Display /

Phage Display

Humanized llamdA™FASTtrack

Available, validated offerings:

llamdA™

Design:• Single camelid or humanized framework• All CDRs randomized• 18 CDR3 lengths

• 4 discrete CDR3 length pool sub libraries• 1 ‘single pot’ sub library containing all CDR3 lengths

CDR1FW3 FW4FW1 FW2

CDR2 CDR3

|||

X 18 lengths

and available in:

Alexandria™ specifications

8

Design, build process and QC overview

Design

• Collaboration between Isogenica Ltd. (UK) and Distributed Bio Inc. (US)

• Design based on public and proprietary human immunoglobulin repertoires NGS data sets

• using donors of both sexes

• ages from young adult to elderly

• various ethnicities

• Sequence datasets analysed at Distributed Bio, used to generate library designs (amino acid level positional weight matrices - PWM)

• Isogenica library team designed COLIBRA™ build strategy (nucleotide level)

Build process

• Overall design: split into fixed sequence fragments and library cassettes

• All variable cassettes generated using COLIBRA™ synthesis

93/5/2017

Design: germlines choice

103/5/2017

Germline gene choice rationale

• Regulatory/development familiarity

• Emphasizing human germlines successfully used in therapeutic antibodies

• Aggregation liability

• Emphasis on kappa light chain repertoire – superior protein stability, familiarity and analytics availability

• Avoiding germlines overrepresented in amyloidosis database(Boston University Light Chain Amyloidosis DB - http://albase.bumc.bu.edu/aldb)

• Immunogenicity liabilities

• Avoiding very rare germlines in one or more ethnicities

• Avoiding highly polymorphic germlines

• CDR fold diversity

• Including germlines with diverse CDR1/CDR2/CDRL3 lengths and associated diverse canonical loop structures

• Heavy/Light chain phage expression and pairing efficiencies

Design: germlines choice

Design: 8 VH germlines, 8 VL germlines

113/5/2017

Yellow: Heavy chain

Orange: Light chain

Green: Total of reported heavy or light chain repertoire

Tiller et al.: mAbs 2013 5:3 445

DeKosky et al.: Nature Med 2015 21:1 86

Gene Tiller et al. % DeKosky et al. % Canonical CDR1 Canonical CDR2

VH1-03 NA 1.50 H1-1 H2-2 or H2-3

VH1-46 2.1 2.61 H1-1 H2-3

VH1-69 5.3 4.42 H1-1 H2-2

VH3-07 4.5 4.13 H1-1 H2-3

VH3-15 3.3 2.93 H1-1 H2-4

VH3-23 10.6 9.38 H1-1 H2-3

VH3-30 8.0 5.26 H1-1 H2-3

VH3-48 4.0 3.55 H1-1 H2-3

37.8 33.78

VK1-09 2.4 1.86 L1-2 L2-1

VK1-27 2.2 1.78 L1-2 L2-1

VK1-33 NA 5.07 L1-2 L2-1

VK1-39 14.2 15.20 L1-2 L2-1

VK2-28 NA 4.90 L1-4 L2-1

VK3-11 7.6 7.55 L1-2 L2-1

VK3-15 11.1 8.91 L1-2 L2-1

VK3-20 16.2 17.87 L1-6 L2-1

53.7 63.14

Design: CDR lengths and variability

VL germlines

• Fixed length randomized CDR cassettes, using germline specific dominant lengths

• No ‘universal’ library CDRs: all germlines have specific variable cassette designs

123/5/2017

COLIBRA fragments build

VK2 VK3VK1 VK1FW3 FW4FW1 FW2

VK2 VK3

Fragments assembly

VH germlines

• Fixed length randomized CDR1/2 cassettes, using germline specific dominant lengths

• All germlines have specific variable cassette designs

• 18 different length CDR3 cassettes, maximizing diversity and dispersion

VH2 VH3

||| X 18 lengths

VH1 VH1FW3 FW4FW1 FW2

VH2 VH3

||| X 18 lengths

Design: Heavy chain CDR cassettes

133/5/2017

CDR1, 2 diversitySegment Length Type

CDR1 Fixed - germline specific lengths randomized - germline tailored

CDR2 Fixed - germline specific lengths randomized - germline tailored

CDR3 diversitySegment Length Type

CDR3 5 to 22 (18 lengths) randomized - stems germline tailored

JH region inclusionSegment JH use JH use per length

JH3 Included across all CDR3 lengths Proportions are tailored to CDR3 length

JH4 Included across all CDR3 lengths Proportions are tailored to CDR3 length

JH5 Included across all CDR3 lengths Proportions are tailored to CDR3 length

JH6 Included across all CDR3 lengths Proportions are tailored to CDR3 length

CDR1, 2, 3 lengthsGene CDR1 CDR2 CDR3

VH1-03 9 17 5 to 22

VH1-46 9 17 5 to 22

VH1-69 9 17 5 to 22

VH3-07 9 17 5 to 22

VH3-15 9 19 5 to 22

VH3-23 9 17 5 to 22

VH3-30 9 17 5 to 22

VH3-48 9 17 5 to 22

Design: Light chain CDR cassettes

143/5/2017

CDR definitions following Kabat.

CDR1, 2 diversitySegment Length Type

CDR1 Fixed - germline specific lengths randomized - germline tailored

CDR2 Fixed - germline specific lengths randomized - germline tailored

CDR3 diversitySegment Length Type

CDR3 Fixed - germline specific lengths randomized - germline tailored

CDR1, 2, 3 lengthsGene CDR1 CDR2 CDR3

VK1-09 11 7 11

VK1-27 11 7 11

VK1-33 11 7 11

VK1-39 11 7 11

VK2-28 16 7 11

VK3-11 11 7 11

VK3-15 11 7 11

VK3-20 12 7 11

Design: Alexandria™ Common Light chain variant library

153/5/2017

Alexandria™ core Fab

Alexandria™ CL Common Light chain Fab

• 8 VH x 8 VK germlines• All CDRs randomized• Randomizations to match germline• 18 CDR3 lengths across all VH

• 8 VH x single fixed VK• Fixed VK selected for low immunogenicity, VH pairing• For bispecifics or VK based maturation

Vector and phage display system

Vector system

• Modular new vector system used to clone and park VH and VK repertoires

• VK and VH repertoires combined into functional Fab ready for display and selection

• Combination of VK vector with VH vector yields an M13KO phagemidcompatible Fab phagemid vector

• Modular system allows for full flexibility of final library presentation

1. All VH x All VK, single pot library

2. Individual VH germlines x all 8 VK, yielding 8 VH germline specific sub-libraries

3. Rationally designed combinations of individual VH/VK germlines, based on independently generated VH/VL pairings data: up to 28 optimal VH/VK combinations

• Alexandria™ core library will initially be provided as 8 sub libraries:8 VH germline sub libraries, each combined with all 8 VL germlines

163/5/2017

Vector and phage display system

Vector system advantages

• Future proofing: modular parking system allows for expansion or improvements

• Mass recloning and in-format screening:

• phagemid vector contains heavy and light chains in single plasmid

• ready for mass recloning into IgG format destination vectors

• COLIBRA™ gene synthesis uses ‘compromise’ codon set to allow for shuttling

• small-scale, high throughput transient production system allows for in-format mAb screening

• Binding assays

• Functional (bio)assays

• Initial production yield and stability assessments

• Affinity maturation: light chain shuffling enabled phagemid vector

• primary leads affinity maturation by re-introducing diverse light chain repertoires

173/5/2017

Alexandria™ core library and Alexandria™ CL use patterns

183/5/2017

Alexandria™ core Fab Human IgG

Alexandria™ CL CL Fab Bispecific human IgG

CL Fab

Recombine hits withvariable Light Chain pool,

High stringency re-selection

Outlook

19

Alexandria™ core and CL near term outlook

Alexandria™ core

• Full diversity, functionality analysis (NGS, Sanger)

• Test antigen selection campaigns

• Hit rates

• Sequence diversity analysis

• Epitope diversity analysis

• Affinity range analysis

Alexandria™ Common Light

• Full diversity, functionality analysis (NGS, Sanger)

• Test antigen selection campaigns, as above

• Light Chain Shuffling affinity maturation examples

203/5/2017

3 libraries – functional and structural diversity – endless possibilities

213/5/2017

Humanized llamdA™ VHH Multispecific VHH, other fusion proteins…

Human IgG Bispecific human IgGAlexandria™ coreAlexandria™ CL

Fab

CL Fab

Bispecific human IgG

guy.hermans@isogenica.com sales@isogenica.com