FROM CODE TO CURE ™

COMPUTATIONAL DISCOVERY OF NOVEL IMMUNE CHECKPOINTSAmit Novik, Itamar Borukhov, Yair Benita, Gady Cojocaru, Assaf Wool, Yossef Kliger, Tomer Zekharya, Zurit Levine, Sergey Nemzer, Eran Ophir, Meir Azulay, Maya Kotturi,Sudipto Ganguly*, Drew M Pardol*, Ofer Levy, Amir Toporik

1

for Cancer Immunotherapy2

* Johns Hopkins University School of Medicine, Baltimore, MD, USA

32461127351118PVRIG

105110348109CTLA4

12912535752CD28

148510733658ICOS

2764714431588BTLA

2403515636076PD1

SignalPeptide Ig Domain TM

ITIM ITSM

ITIM ITSM

ITIM

Extracellular Intracellular Color By Domain

Query

Result

23710733061TIGIT

148510733658ICOSIg Domain

TM

SignalP

Extracellular Intracellular

T GAColor By Intron Phase

T GA T GA

A. The Concept

T GAColor By Intron Phase

T GA T GA

9912933144IL5

10217748135IL4

11114460147IL2

Query

Result

Helix 1 Helix 2+3 Helix 4SPT CellTCRMHC

Co-inhibitoryligand

Co-inhibitoryreceptor

Tumor cell

The immune system constantly identifies and destroys malignant cells. Some tumors evade the immune system by expressing co-inhibitory ligands that inhibit T cells. Blocking the co-inhibitory signal allows the T cells to attack the tumor.Checkpoint blockade is a breakthrough approach to cancer therapy with impressive clinical benefits. However, the majority of cancer patients still do not respond and new targets, target combinations and drug modalities are needed.

Immune genes often have no sequence similarity to any other gene within the organism (have no paralogs). For instance, IL2 and PD-1, do not resemble any other human gene by sequence. If immune related genes, such as cytokines, that have no paralogs by sequence similarity evolved from divergence, there must be a signature in the genome indicative of their related evolutionary origins. We found that gene structure was more conserved than seqeunce for those genes.

Known B7/CD28 proteins have the following typical gene structure:

Based on this gene structure, a BLAST-like algorihm identified BTLA as closest to PD-1 and TIGIT as closest to ICOS. The TIGIT finding was published in 2009 (Proc Natl Acad USA. 2009 Oct 20; 106(42) 7858-7863).

Validation of a Novel Immune Checkpoint for Cancer Immunotherapy3

Introduction B. Discovery of Novel Immune Checkpoints

Normal and Cancer Tissues Highly enriched in normal immune tissues vs normal solid tissues and expressed in multiple solid tumor types

NormalCancer

Immune Cells Tumor CorrelationPVRIG Is expressed and regulated in immune subsets

Expressed In Immune Cells Regulated in Immune Cells

B Cells

T Cells (CD4)

T Cells (CD8)

NK Cells

Monocytes

Neutrophils

PVRIG ExpressionPVRIG Expression

Time

RestimulatedCD3 + CD28

RestimulatedCD3 + CD28

Most correlated PVRIG genes in cancer are T cell genes

PVRIG expression signature in cancer is associated with lymphocytes(colors represent ~30 different tumors )

immune response

Immunological synapse formation

TCR signaling in naïve CD8+ T cells

TCR signaling in naïve CD4+ T cellsImmunoregulatory interactions between

a Lymphoid and a non-Lymphoid cell

T helper cell differentiation

Leucocyte chemotaxis

Signaling in Immune system

Lymphocyte proliferation

TCR signaling

-log(p-value)0 50 100 150 200 250

Top 100 PVRIG correlated genes from each tumor type were tested for gene set enrichment analysis using GeneGo Pathways

T CELL

ANTIGENPRESENTING CELL

TIGITPVRIG DNAM

PVRL2 PVR

IgG4

COM701

Non blocking Ab

Backu

p AbIgG1

0

10

20

30

% P

rolif

erat

ing

(CFS

Elo)

7 10 13 16 20 23 270

1000

2000

3000

4000

5000

Wild-typerIgG2b

Days post-tumor implantation

Tum

or v

olum

e (m

m

7 10 13 16 20 23 270

1000

2000

3000

4000

5000

Wild-typeanti-PDL1

Days post-tumor implantation

Tum

or v

olum

e (m

m

7 10 13 16 20 23 270

1000

2000

3000

4000

5000PVRIG KOrIgG2b

Days post-tumor implantation

Tum

or v

olum

e (m

m

7 10 13 16 20 23 270

1000

2000

3000

4000

5000PVRIG KOanti-PDL1

Days post-tumor implantation

Tum

or v

olum

e (m

m

MC38 Tumor Model

3 )3 )3 ) 3 )

0 5 10 15 20 250

250

500

750

1000

1250

1500

1750

Days

Volu

me

mm

3

PDL-1+rIgG2b

PDL-1+AB 407

***

mIgG1+ rIgG2b

CT26 Tumor Model

Identifying PVRL2 as the PVRIG Counterpart Suggests PVRIG is in the TIGIT Pathway

Antagonist PVRIG Antibodies Increase CD4+ T cell Proliferation in-vitro

PVRIG Knockout Mice Treated with anti-PDL1 Show Tumor Growth Inhibitition Compared to Wild Type

PVRIG Blocking Antibody ReducesTumor Growth in Combination with Anti-PDL1 in Mouse Tumor Model