1

Discovery of Potent, Functional Anti-TIGIT Antagonists from Three Different Phage Display Platforms

Aaron K. Sato, Ph.D.CSO, Head of Antibody Center

2Passion for antibody discovery …

3Multiple shots on goal for antibody discovery and engineering …

4

ØDistributed Bio’s SuperHuman 2.0 scFv synthetic phage library • Hits against any epitope and fully natural CDR diversity • >70 billion antibody diversity• 100% germline frameworks and improved thermostability

ØXOMA’s 031 human Fab naïve phage library • Routinely selected pM affinity antibodies • Fully human, very large (>1011), multiple open reading frames• Leads in multiple clinical programs

Ø Immune custom phage libraries: Mouse, Rat, Rabbit, Chicken, Llama

ØAffinity Maturation

ØHigh throughput reformatting into IgG isotypes from different species

ØHigh throughput 96-well transfections and high-yield purifications from proprietary CHO cell lines

Ø IgG specificity, KD assessments using Octet and Carterra Array SPR

LakePharma Antibody Discovery – Phage Display

5Project: Phage Display Target POC with Multiple Libraries

§ Goal: Highlight the strengths of the Antibody Center

§ Target– Single pass transmembrane receptor with recombinant protein available– Known receptor – ligand interaction that upon blocking results in therapeutic benefit– Low human/cyno homology– Regarded as difficult (subjective)

§ Libraries– Naïve phage display library: Xoma– Mouse immune library: Co-immunized with both human/cyno

§ Panning– Select only against human antigen to find the very best human antibodies

§ Screening– Conduct large HT screen to capture all target binders– Assess both human and cyno cross-reactivity– Check for affinity, binning, receptor-ligand blocking, polyspecificity, and function

6TIGIT: MOA – Similar to B7/CTLA4/CD28 Pathway

§ Coinhibitory receptors can suppress immune responses by direct signaling in cis, by inducing ligand signaling in trans, and by competition with costimulatory receptors.

§ Competition: TIGIT Impairs CD226 Function by Directly Disrupting CD226 Homodimerization

§ TRANS: Ligation of its ligand PVR (CD155) on dendritic cells, resulting in the conversion of those dendritic cells to a tolerogenic phenotype characterized by increased IL-10 and decreased IL-12 production

77TPP: TIGIT

Receptor Family/Superfamily Ig Superfamily

Expression Profile – cell types some T cells and NK cells

Known Ligand(s) CD155 (PVR) on DCs/macrophages with high affinity, CD112

(PVRL2) with lower affinity

Human Homology (AA) 58.6% (mouse), 87.8% (cyno)

Specificity Requirement/Epitope Block CD155 binding (trans) and inhibition CD226

costimulation signal by preventing dimerization (cis)

Mechanism of Antibody Action Antagonist, Block TIGIT/CD155-mediated suppression of T-cell

function à promote CD155/CD226 signaling

Affinity Requirement <1 nM

Monotherapy vs. Combo Therapy? Monotherapy and/or Combo therapy with anti-PD1, anti-PDL1,

or anti- CTLA4

ADCC/CDC Requirement? Recommended Fc? No, recommend hIgG4 (PE) or hIgG2

Toxicity associated with anti-target antibody therapy

Not available

Most advanced competitor antibody & company RG6058, Genentech, Phase I (BENCHMARK)

88

Ø 4 rounds of selections using XOMA Library– Subsequent rounds of selections performed with decreasing Biotin-human TIGIT coupled to Streptavidin-coated magnetic beads– Stringency of selections increased with number and duration of washes in successive selection rounds – Bacterial periplasmic extracts (PPEs) screened for human and cyno-TIGIT binding by ELISA (HighRes automation deck)

Ø 3 rounds of selections with LakePharma immune library, following human and cyno-TIGIT mouse immunizations: – 3 Rds of selections with Biotin-human TIGIT on Streptavidin-coated ELISA plate wells

Phage Panning and Screening Workflow

Infect E. coli

amplify phage

C. SCREENING PPEs by ELISA

Recover bound phage

Wash off unbound

phage

A. Phage panning selections with XOMA 031 Fab & LakePharmaimmune libraries

SA-SA-

B. ENRICHMENT OF PHAGE using biotinylated human-TIGIT coupled to streptavidin-coated magnetic beads

B- Biotin-human-TIGIT

Magnetic beads coated with streptavidin (SA)

BSA-

B

B

D. IgG4 reformatting

SA-

99

Ø XOMA Fab naïve Library: Sequenced 1116 HuTIGIT binders from 12 x 96-well plates of clones

– 61 sequence-unique binders identified– One clone highly enriched – Clones with identical VH sequences were coupled to multiple VL sequences – 30 unique human-TIGIT binders, including all VH-unique sequences, selected for human IgG4 reformatting

and characterization:• All clones with unique VH • Clones with most frequent L3CDR• All clones were only able to recognize human TIGIT

Ø LakePharma scFv Immune Library: Sequenced 1116 HuTIGIT binders from 12 x 96-well plates of clones

– 67 sequence-unique binders identified– 8 clones with highly enriched H3CDR and 9 clones with highly enriched L3CDR – 56 sequence-unique human-TIGIT binders, including all VH unique sequences, selected for human IgG4 reformatting:

• All clones with unique VH • Clones with most frequent L3CDR

Sequences of TIGIT-binding Abs from library selection campaigns

10ELISA binding of TIGIT-specific Fab clones in bacterial PPEs following panning with the XOMA phage library

Ø Four rounds of phage selections performed against biotinylated human TIGIT antigen

Ø 1116 individual clones of bacterial Fab PPEs screened by ELISA against cynomolgus and human TIGIT protein, using LakePharma’s HighResautomation deck

Ø No cynomolgus TIGIT binders were identified from the XOMA Fab library selections

Sequencing analysis allowed the selection of 30 unique human TIGIT Fab binders which were subsequently reformatted into human IgG4 for further kinetic and functional characterizations

0

0.2

0.4

0.6

0.8

1

1.2

1.4

XO_C1_P1

XO_C3_P1

XO_D4_P2

XO_G1_P2

XO_H8_P2

XO_B1_P3

XO_B8_P3

XO_D3_P3

XO_A10_P5

XO_A5_P6

XO_A9_P6

XO_D9_P6

XO_A5_P7

XO_A7_P7

XO_C12_P7

XO_D6_P7

XO_D8_P7

XO_D2_P8

XO_F4_P8

XO_F10_P8

XO_A8_P9

XO_C5_P10

XO_C8_P10

XO_E8_P10

XO_C7_P11

XO_D9_P11

XO_D12_P11

XO_F6_P11

XO_C8_P12

XO_D12_P12

A(49

0nm

)

biotin-huTIGIT biotin-cyTIGIT

11ELISA binding of TIGIT-specific scFv clones in bacterial PPEs following panning with immune phage library

Sequencing analysis allowed the selection of 56 unique TIGIT scFv binders that were subsequently reformatted into a chimeric IgG4 isotype for further kinetic and functional characterizations

Ø Following co-immunization of mice with human and cynomolgus TIGIT-Fc antigen using LakePharma’s optimized hybridoma protocols, a diverse scFv immune phage library was generated with cDNA template from murine spleen mRNA

Ø Three rounds of phage panning selections were performed using biotinylated human TIGIT

Ø 1116 individual clones of bacterial scFv PPEs screened by ELISA against cynomolgus and human TIGIT protein, using LakePharma’s HighResautomation deck

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

H10_P1_Imm

C10_P2_Imm

G7_P2_Imm

H1_P2_Imm

A3_P3_Imm

B5_P3_Imm

C4_P3_Imm

C6_P3_Imm

E10_P3_Imm

F8_P3_Imm

H11_P3_Imm

A8_P4_Imm

B7_P4_Imm

B8_P4_Imm

C6_P4_Imm

C8_P4_Imm

D6_P4_Imm

F7_P4_Imm

F12_P4_Imm

G9_P4_Imm

A6_P5_Imm

B5_P5_Imm

B7_P5_Imm

C12_P5_Imm

F8_P5_Imm

F11_P5_Imm

A11_P6_Imm

D10_P6_Imm

E3_P6_Imm

E12_P6_Imm

F10_P6_Imm

A5_P7_Imm

A6_P7_Imm

E3_P7_Imm

F3_P7_Imm

F8_P7_Imm

P8_A4_P8_Imm

P8_A7_P8_Imm

P8_C9_P8_Imm

P8_F1_P8_Imm

B6_P9_Imm

D12_P9_Imm

A6_P10_Imm

A8_P10_Imm

A12_P10_Imm

B6_P10_Imm

D9_P10_Imm

D10_P10_Imm

D11_P10_Imm

H4_P10_Imm

H9_P10_Imm

A6_P11_Imm

B1_P11_Imm

D2_P11_Imm

E4_P11_Imm

H2_P11_Imm

A9_P12_Imm

C7_P12_Imm

D8_P12_Imm

biotin-huTIGIT biotin-cyTIGIT

12HT IgG4 Transient Production using LakePharma’s TunaCHOTM cell line

Ø TunaCHOTM is LakePharma’s proprietary cell line (same parental cell line as CHO-GSN)

Ø Consistent anti-TIGIT IgG4 production in 10ml scale transient transfections

Ø More cost-effective than other premium CHO transient cell line production systems

XOMA

Immune

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Yie

ld (

mg

)

Number of Antibodies Reformatted

Anti-TIGIT IgG4 Production Yields

XOMA Immune

Superb expression of 86 IgG4-reformatted anti-TIGIT Abs from XOMA and immune phage

display libraries (average yield ~4.6mg from 10ml transfections)

13Octet analysis (sensogram example shown) demonstrates CD155 (PVR) blocking of TIGIT-binding antibodies

Ø Streptavidin Octet biosensor loaded with Biotin-human TIGIT (3 min)

Ø TIGIT-specific IgG4 Abs added at saturating concentration (8 min)

Ø Human CD155 ligand allowed to bind (1 min)

Capturing biotin-TIGIT

Binding Anti-TIGIT Abs CD155 Binding

XOMA blocking IgG4XO_A5_P6 XO_C5_P10

XO_A5_P7 XO_C8_P12

XO_A7_P7 XO_D3_P3

XO_A8_P9 XO_D4_P2

XO_A9_P6 XO_D9_P11

XO_B1_P3 XO_D9_P6

XO_B8_P3 XO_F10_P8

XO_C1_P1 XO_F6_P11

XO_C12_P7 XO_G1_P2

XO_C3_P1

CD155-blocking antibodies were discovered following XOMA andin-house immune phage display library selections

Immune blocking IgG4Imm_A12P10 Imm_D9P10

Imm_A3P3 Imm_E10P3

Imm_A6P10 Imm_E3P6

Imm_A6P5 Imm_E3P7

Imm_A7P8 Imm_E4P11

Imm_B1P11 Imm_E9P12

Imm_B5P3 Imm_F10P6

Imm_B6P10 Imm_F12P4

Imm_B7P5 Imm_F3P7

Imm_B8P4 Imm_F8P7

Imm_C4P3 Imm_H11P3

Imm_C6P3 Imm_H4P10

Imm_D10P10 Imm_H9P10

Imm_D2P11

Imm_D6P4

Imm_D8P12

14

0.02000.04000.06000.08000.0

10000.012000.014000.016000.018000.0

TIGIT

benc

hmark

antib

ody

Antib

ody f

ree co

ntrol

XO_G

1_P2

XO_D

2_P8

XO_F

4_P8

XO_D

9_P6

XO_D

8_P7

XO_F

10_P

8

XO_C

5_P1

0

XO_C

8_P1

2

XO_A

10_P

5

XO_D

12_P

11

XO_A

5_P6

XO_A

8_P9

XO_B

8_P3

XO_C

12_P

7

XO_A

7_P7

XO_D

12_P

12

XO_C

7_P1

1

XO_E

8_P1

0

XO_A

5_P7

XO_A

9_P6

XO_D

6_P7

XO_D

9_P1

1

XO_D

3_P3

XO_H

8_P2

XO_C

1_P1

XO_B

1_P3

XO_F

6_P1

1

XO_C

8_P1

0

XO_C

3_P1

XO_D

4_P2

Alph

a Co

unts

XOMA library IgGs

AlphaLISA ligand CD155 blockade assay for Immune and XOMA library reformatted clones

ØTIGIT:CD155 Homogeneous Assay Kit in AlphaLISA format (BPS Bioscience) using biotin-huTIGIT and human CD155 ligand

ØAnti-TIGIT IgGs (40-160 µg/ml) incubated with human TIGIT before adding CD155

ØAcceptor and donor beads added before monitoring Alpha Counts

ØLower Alpha Count values indicate CD155 blockade

ØFull titrations conducted with functional leads (see later)

0100002000030000400005000060000700008000090000

TIGI

T be

nchm

ark

antib

ody

Antib

ody

free

cont

rol

Imm

_B7P

4Im

m_F

8P7

Imm

_A6P

7Im

m_F

9P7

Imm

_C9P

8Im

m_F

11P5

Imm

_H11

P3Im

m_G

9P4

Imm

_B6P

9Im

m_D

9P10

Imm

_E9P

12Im

m_B

8P4

Imm

_C6P

3Im

m_F

7P4

Imm

_H2P

11Im

m_B

7P5

Imm

_A5P

7Im

m_C

6P4

Imm

_D8P

12Im

m_H

1P2

Imm

_C7P

12Im

m_E

3P7

Imm

_H4P

10Im

m_C

4P3

Imm

_B5P

3Im

m_A

4P8

Imm

_A7P

8Im

m_C

10P2

Imm

_C8P

4Im

m_F

10P6

Imm

_C12

P5Im

m_D

6P4

Imm

_H10

P1Im

m_A

8P10

Imm

_B6P

10Im

m_D

10P1

0Im

m_F

1P8

Imm

_A12

P10

Imm

_A3P

3Im

m_F

8P5

Imm

_D12

P9Im

m_H

9P10

Imm

_F3P

7Im

m_F

12P4

Imm

_E12

P6Im

m_A

6P10

Imm

_A8P

4Im

m_D

11P1

0Im

m_D

2P11

Imm

_F8P

3Im

m_A

6P5

Imm

_E4P

11Im

m_B

1P11

Imm

_E10

P3Im

m_E

3P6

Imm

_G7P

2

Alph

a Co

unts

Immune Library IgGs

15KD distribution of IgG4-reformatted TIGIT-binding antibodies by Carterra

X OM

A li

b rary

Imm

u n e lib ra

ry

Co n tr

o l Ab

-1 0

-9

-8

-7

-6

L o g [K D ] d is tr ib u tio n fo r tw o lib ra r ie s a g a in s t h u T IG IT -H is

Lo

g[K

D]

X OM

A li

b rary

Imm

u n e lib ra

ry

Co n tr

o l Ab

-1 0

-9

-8

-7

-6

L o g [K D ] d is tr ib u tio n fo r tw o lib ra rie s a g a in s t c y T IG IT -H is

Lo

g[K

D]

Ø Binding assays performed using Array SPR

Ø Cyno and Human TIGIT were injected as monovalent analytes as a 3-fold dilution series (0.4-300nM) over IgGs captured onto discrete spots to create a 192-spotted array via anti-human IgG Fc-coated CMD50M chip

Ø Kinetic rate constants were calculated using a monovalent (1:1) binding model

Ø No cynomolgus TIGIT IgGs from XOMA library

Spot 135, Imm_B7P5, KD =563pM

Human Cyno

16Human TIGIT (affinity ranked from high to low): Representative Data from 96 spots within a 192 spot array

17Ligand blockadeKD (nM) hTIGITKD (nM) cyTIGITBin

18Color by ligand blockade Color by bin

19

0.0200.0400.0600.0800.0

1000.01200.01400.01600.01800.02000.0

TIGIT

benc

hmark

antib

ody

Antib

ody f

ree co

ntrol

XO_G

1_P2

XO_D

2_P8

XO_F

4_P8

XO_D

9_P6

XO_D

8_P7

XO_F

10_P

8

XO_C

5_P1

0

XO_C

8_P1

2

XO_A

10_P

5

XO_D

12_P

11

XO_A

5_P6

XO_A

8_P9

XO_B

8_P3

XO_C

12_P

7

XO_A

7_P7

XO_D

12_P

12

XO_C

7_P1

1

XO_E

8_P1

0

XO_A

5_P7

XO_A

9_P6

XO_D

6_P7

XO_D

9_P1

1

XO_D

3_P3

XO_H

8_P2

XO_C

1_P1

XO_B

1_P3

XO_F

6_P1

1

XO_C

8_P1

0

XO_C

3_P1

XO_D

4_P2

RLUs

XOMA library IgGs

Cell-Based Immunoblockade Assay (Promega) for Immune (A) and XOMA (B) library reformatted clones

Ø CD226-induced luminescence is inhibited when Jurkat effector T cells expressing human TIGIT with luciferase reporter are co-cultured with CHO-K1 cells expressing CD155

Ø Anti-TIGIT IgGs (33-132 µg/ml) block interaction of TIGIT/CD155, resulting in CD226-activated luminescence (Higher RLUs indicate CD155 blockade)

Ø Full titrations conducted with functional leads (see later)

0.0200.0400.0600.0800.0

1000.01200.01400.01600.01800.02000.0

TIGI

T be

nchm

ark

antib

ody

Antib

ody

free

cont

rol

Imm

_B7P

4Im

m_F

8P7

Imm

_A6P

7Im

m_F

9P7

Imm

_C9P

8Im

m_F

11P5

Imm

_H11

P3Im

m_G

9P4

Imm

_B6P

9Im

m_D

9P10

Imm

_E9P

12Im

m_B

8P4

Imm

_C6P

3Im

m_F

7P4

Imm

_H2P

11Im

m_B

7P5

Imm

_A5P

7Im

m_C

6P4

Imm

_D8P

12Im

m_H

1P2

Imm

_C7P

12Im

m_E

3P7

Imm

_H4P

10Im

m_C

4P3

Imm

_B5P

3Im

m_A

4P8

Imm

_A7P

8Im

m_C

10P2

Imm

_C8P

4Im

m_F

10P6

Imm

_C12

P5Im

m_D

6P4

Imm

_H10

P1Im

m_A

8P10

Imm

_B6P

10Im

m_D

10P1

0Im

m_F

1P8

Imm

_A12

P10

Imm

_A3P

3Im

m_F

8P5

Imm

_D12

P9Im

m_H

9P10

Imm

_F3P

7Im

m_F

12P4

Imm

_E12

P6Im

m_A

6P10

Imm

_A8P

4Im

m_D

11P1

0Im

m_D

2P11

Imm

_F8P

3Im

m_A

6P5

Imm

_E4P

11Im

m_B

1P11

Imm

_E10

P3Im

m_E

3P6

Imm

_G7P

2

RLUs

Immune Library IgGs

20Polyspecificity ELISA of selected TIGIT-blocking antibodies

Polyspecificity ELISA experimental design[1]: Ø Coating with Baculovirus particle (BVP)

Ø Loading anti-TIGIT Abs (150, 50, 16.7 µg/ml)

Ø Detection with HRP-conjugated anti-human Fc (2nd Ab)

Ø Positive control (PC) and negative control (NC) Abs

Ø RG6058 benchmark anti-TIGIT IgG4

Ø Background BVP wells coated only with 2nd Ab

Ø Cut-off: 5x coated background signal

BVP Score Calculation [1]: Ø BVP score is determined by normalizing

absorbance on control wells with 2nd Ab onlyØ BVP score = OD450 average of antibody

(150 µg/mL) /OD450 average of 2nd Ab only

XOMA blocking Abs

Immune Library blocking Abs

[1] Jain et al. Biophysical properties of the clinical-stage antibody landscape. PNAS, 2017, 114(5), 944-949.

P C N C

R G6 0 5 8

2 n d Ab O

n ly

X O_G

1 _P 2

X O_B 8 _P 3

X O_C 1 _P 1

X O_F 1 0 _ P 8

X O_C 1 2 _ P 7

X O_D 9 _P 1 1

X O_B 1 _P 3

X O_C 3 _P 1

X O_C 5 _P 1 0

X O_A 5 _P 6

X O_A 7 _P 7

X O_A 5 _P 7

X O_D 3 _P 3

X O_F 6 _ P 1 1

X O_D 4 _P 2

X O_D 9 _P 6

X O_C 8 _P 1 2

X O_A 8 _P 9

X O_A 9 _P 6

Imm

_B 7P 5

Imm

_C 4P 3

Imm

_A 1 2P 1 0

Imm

_F 1 2P 4

Imm

_A 6P 5

Imm

_B 5P 3

Imm

_D 6P 4

Imm

_A 3P 3

Imm

_E 4P 1 1

Imm

_F 8P 7

Imm

_D 9P 1 0

Imm

_A 6P 1 0

Imm

_E 9P 1 2

Imm

_D 8P 1 2

Imm

_A 7P 8

Imm

_B 1P 1 1

Imm

_B 8P 4

Imm

_B 6P 1 0

Imm

_H 9P 1 0

Imm

_E 1 0P 3

Imm

_C 6P 3

Imm

_D 1 0P 1 0

Imm

_D 2P 1 1

Imm

_E 3P 6

Imm

_E 3P 7

Imm

_F 10P 6

Imm

_F 3P 7

Imm

_H 1 1P 3

Imm

_ H4P 1 0

0

1 0

2 0

3 0

4 0

B V P S c o re

BV

P S

co

re

21Lead human TIGIT-blocking reformatted antibodies from immune library phage library selection campaigns (3 best candidates highlighted in green color)

Immune Library clones

Multiple TIGIT-blocking antibodies from our immune library phage display selections (12) were able to meet most ligand blocking, affinity, productivity and polyspecificity criteria

Blocking huTIGITAbs (Octet)

Yield in CHOTunaTM (mg)

KD (human TIGIT) (nM)

KD (cyno TIGIT) (nM)

Blocking huTIGIT Abs

AlphaLISA (<3000 counts)

AlphaLISA (IC50)

IO assay (>500 RLUs)

IO Assay (IC50)

Polyspecificity(BVP score < 5)

Imm_A6P5 6.61 46 177 ü 5.46E-09 ü 3.85E-07 ü

Imm_A6P10 5.87 15.3 351 ü 3.93E-09 ü 1.46E-07 ü

Imm_A7P8 5.27 15.4 759 ü 2.94E-09 ü 3.02E-08 ü

Imm_A12P10 8.28 69 276 ü 6.11E-09 ü 4.48E-08 ü

Imm_B5P3 5.96 12.3 226 ü 2.99E-09 ü 1.72E-08 ü

Imm_C4P3 7.13 2.2 4 ü 2.75E-09 ü 5.01E-09 ü

Imm_C6P3 7.67 42 311 ü 3.60E-09 ü 4.88E-08 ü

Imm_D2P11 6.25 50 186 ü 4.36E-09 ü 1.52E-01 ü

Imm_D8P12 5.13 1.8 4 ü 6.07E-09 ü 3.94E-09 ü

Imm_E3P6 3.01 14.4 476 ü 6.45E-09 ü 1.20E-08 ü

Imm_E3P7 4.5 17.1 1609 ü 3.07E-09 ü 5.70E-07 ü

Imm_F3P7 5.06 0.46 0.1 ü 3.61E-09 ü 3.90E-09 ü

Benchmark 0.13 21 ü 4.12E-09 ü 6.12E-09 ü

22CONCLUSIONS

Ø We were able to discover multiple TIGIT-specific antibodies following selections with the XOMA 031 Fab and LakePharma scFv immune libraries

Ø There was a very high sequence diversity of TIGIT binders derived from the XOMA and LakePharma scFvphage libraries

Ø A high number of cynomolgus and human TIGIT cross-reactive binders was discovered following panning with the LakePharma immune libraries

Ø Co-immunization of mice with human and cynomolgus TIGIT resulted in higher affinity CD155 blocking antibodies, as shown by both AlphaLISA and cell-based immuno oncology assays

Ø Most TIGIT Abs originating from the LakePharma immune library did not bind non-specifically to baculovirusparticles (low BVP score), possibly predicting fewer developability issues