CT26 syngeneic model

ANTI-PVRIG BLOCKING ANTIBODIES ENHANCE TIL ACTIVATION

Introduction

• Background: While antibody blockade of the CTLA4 and PD1 pathways has emerged as an effective treatment modality for cancer, the majority of patients do not derive long term benefit, suggesting a need for targeting of additional immune checkpoints. Employing our unique computational algorithms to define new members of the B7/CD28 family we identified PVRIG, which is expressed by multiple subsets of T and NK cells. We report here its expression pa�ern, functional characterization, and an�-tumor activity of blocking antibodies targeting this molecule.

• Materials and Methods: Utilizing Compugen’s Predictive Discovery platform we identified PVRIG as a potential novel immune checkpoint, after which a retroviral cell screening library was used to identify its cognate binding counterpart. Target effects on T-cell modulation were assessed with primary and tumor-derived T-cell assays, taking advantage of target overexpression, knockdown, and antagonist antibody approaches. Antibodies against the human protein were screened for their ability to enhance T-cell activation in vitro, while antibodies targeting the mouse orthologue were assessed in vivo for effects on tumor growth inhibition in syngeneic models.

Use of ‘Functional Homology’ in absence of sequence similarity based on exon size, phase, and functional elements within exons

PVRIG-Fc (20ug/ml)

Retrogenix Cell Microarray

PVRL2

PVRIG-Fc binding to PVRL2expressing HEK293 cells

PVRIG-Fc bindingto PVRL2 (ELISA)

PVRPVRL3

DNAM

PVRL2

PVRL2 IS A LIGAND IN THE DNAM-1/TIGIT IMMUNECHECKPOINT AXIS

Martinet & Smyth, 2015 (modified)ϲ

BINDING INTERACTIONS IN THE PVRIG/TIGIT/DNAM AXIS

7

PVRIG (co-inhibitory): Binds to PVRL2

DNAM-1 (co-stimulatory):Binds to PVR and PVRL2

TIGIT (co-inhibitory):Binds to PVR

Anti-PVRL2 blocks PVRIG binding to Expi293 cells

Anti-PVR blocks TIGIT bindingto Expi293 cells

PVRL2 binding (ELISA)

PVR binding (ELISA)

PVRIG EXHIBITS TUMOR EXPRESSION CHARACTERISTICSCONSISTENT WITH T-CELL RECEPTOR CHECKPOINTS

PVRIG Correlation with CD8 positive T cells and PD-1(Kidney Clear Cell Cancer)

CD8 vs PVRIG PD-1 vs PVRIG

IDENTIFICATION OF PVRL2AS THE LIGAND FOR PVRIG CONFIRMATION OF PVRIG BINDING

TO PVRL2

PVRIG FUNCTIONAL GENE STRUCTURE MATCHES KNOWNIMMUNE CHECKPOINT RECEPTORS

DEVELOPMENT OF COM701: A HIGH AFFINITY PVRIG ANTAGONIST

Human phage display and standard hybridoma

An�bodies screened for:

COM701 selected as therapeu�c leadIND an�cipated in 2017

High affinity (KD < 1nM)Ability to block PVRIG/PVRL2 bindingIn vitro enhancement of T-cell ac�va�on

B cells

PVRIG

CD56 bright NK cells CD56 dim NK cells

monocytes CD4 T cells CD8 T cells

Red= isotypeBlue= PVRIG

PVRIG EXPRESSION ON NAÏVE PBMC SUBSETSγδ/NKT enriched

PVRIG

CMV ACTIVATED CD8+ CELLS CD4+ CD8+

CHO/PVRL2/OKT3

PVRIG PVRL2

ANTAGONIST PVRIG ANTIBODIES INCREASE CD4+ T-CELL PROLIFERATION

ϭϭ

gp100 pulsed CHO-HLA-A2 MART-1/624-mel

624-mel orpeptide-pulsed CHO-HLA-A2

PVRIG PVRL2

gp100/MART-1specific TILs

COMBINING PVRIG AND TIGIT BLOCKADE INCREASESTIL ACTIVATION

Compugen In house

624 Mel orpeptide-pulsed

CHO-HLA-A2

PVRIG/TIGIT PVRL2 /PVR

MART-1 or gp100 specific

TILs

gp100 pulsed CHO-HLA-A2 MART-1/624-mel

PVRIG EXPRESSION IS INDUCED FOLLOWING T-CELL ACTIVATION AND ELEVATED ON TEMRA AND TEM CELLS

PVRIG BLOCKING ANTIBODIES REDUCE TUMOR GROWTH AND INCREASE SURVIVAL IN COMBINATION WITH PD1 PATHWAY BLOCKADE

COMBINING PVRIG AND TIGIT BLOCKADE RESULTS IN ENHANCED ACTIVATION OF CMV REACTIVE CD8+ CELLS

ϭϭ

SUMMARY

Tumor growth Survival

mIgG1+ rIgG2b

αPDL-1+rIgG2b

αPDL-1+AB 407

PD-1

PVRIG/TIGIT PVRL2/PVR

Peptide pulsed 624-mel (blue) or

624-mel/PVR (green)

CMV reactiveCD8+ T-cells

• Results: A PVRIG-Fc-fusion protein was found to bind PVRL2, with binding specificity confirmed both by ELISA and flow cytometry analysis. PVRIG demonstrated unique expression kinetics upon T-cell activation, with detection of the target on memory T-cells, as well as on NK cells and γδ T-cells. A panel of high affinity human antibodies with the ability to block interaction of PVRIG with PVRL2 were generated, which when tested in vitro were shown to enhance activation of both primary CD4+ and tumor-derived CD8+ T-cells through a PVRL2-dependent mechanism. The lead antibody, COM-701, is currently in preclinical development.

• Since COM-701 is not mouse cross-reactive, in vivo studies were conducted with a surrogate blocking an�-mouse PVRIG antibody. When combined with an�-PDL1 blockade, an�-mouse PVRIG inhibits growth of established tumors in both the CT26 and MC38 colorectal cancer models. Combination testing with additional immune checkpoint inhibitors, as well as in PVRIG knockout mice, is ongoing.

• Conclusion: We describe the identification of PVRIG as a novel immune checkpoint on T cells, as well the development of a high affinity antagonistic antibody, COM-701, that is currently in preclinical development. COM-701 is able to enhance human T-cell activation, and a surrogate antibody with similar characteristics shows synergy with PD-L1 in vivo in multiple syngeneic models. Overall, our data demonstrates the utility of targeting PVRIG in addition to other B7 family checkpoints for the treatment of cancer.

IFNγ IL-2

FROM CODE TO CURE: Computa�onal Iden�fica�on, Func�onal Characteriza�on and An�body Blockade of a New Immune Checkpoint in the TIGIT Family of Interac�ng Molecules

Ofer Levy (1), Chris Chan (2), Gady Cojocaru (1), Spencer Liang (2), Eran Ophir (1), Sudipto Ganguly (3), Maya Ko�uri (2), Tal Friedman (1),Benjamin Murter (3), Liat Dassa (1), Ling Leung (2), Shirley Greenwald(1), Meir Azulay (1), Sandeep Kumar(2), Zoya Gluzman (1),Xiaoyu Pan (3), Arthur Machlenkin (1), Andy Drake (2), Ran Salomon (1), John Hunter (2), Zurit Levine (1), Drew Pardoll (3), and Mark White (2)

1. Compugen Ltd, Holon Israel | 2. Compugen USA, Inc, South San Francisco CA | 3. Bloomberg~Kimmel Ins�tute for Cancer Immunotherapy, Johns Hopkins University, Bal�more MD

PVRIG Expression

NormalCancer