PBS

mO

NCR-1

77PBS

mO

NCR-1

77

0

2000

4000

6000

8000

HS

V g

B M

FI ****

****

PBS

mO

NCR-1

77PBS

mO

NCR-1

77

0

1000

2000

3000

4000A

DP

GK

MF

I

*

PBS

mO

NCR-1

77PBS

mO

NCR-1

77

0

500

1000

1500

2000

2500

RE

PS

MF

I

IFNg

TNFa

IL-2

mONCR-177 oncolytic virotherapy stimulates anti-tumor immune responses

Melissa W. Hayes, Agnieszka Denslow, Jacqueline Gursha, Daniel Wambua, Lingxin Kong, Jacob Spinale, Peter Grzesik, Jennifer S. Lee, Terry Farkaly, Edward M. Kennedy, Lorena Lerner, Christophe Quéva, Brian B. Haines and Sonia Feau. Oncorus, Cambridge, MA, USA

Oncorus is developing ONCR-177, an oncolytic herpes simplex virus engineered for enhanced oncolysis and antitumor immunity.ONCR-177 is replication competent in tumor cells, but its replication and neuropathic activities are attenuated in healthy cellsusing miRNA-dependent degradation of viral transcripts and by mutations in UL37 that prevent retrograde transport in neurons.Efficacy is enhanced by a gain of function in the fusogenic glycoprotein B to enhance viral entry and inactivation of US12 to restoreantigen processing. ONCR-177 is armed with five immunostimulatory transgenes: IL-12, CCL4, FLT3LG, a PD-1 humanizedantagonist VHH nanobody, and the CTLA4 blocking monoclonal antibody ipilimumab. Mechanistic evaluation is performed usingmONCR-177, a mouse surrogate that expresses functionally equivalent immune stimulatory genes and the base vector, ONCR-159,which contains the same enhanced entry and inactivation, and attenuated replication and neuropathic activity, but does not carrythe immunostimulatory payloads.

Introduction

Summary

• mONCR-177 induces potent in situ and abscopalefficacy, inhibits tumor growth and inducescures.

• Treatment with mONCR-177 increased thenumber of infiltrating T cells, as shown withimmunohistochemistry and flow cytometry.Infiltrating cells had a higher activation status inboth injected and non-injected tumor, whilereducing the level of PD-1 expression.

• mONCR-177, compared to PBS control, eliciteda more favorable CD8:Treg ratio.

• Potent viral antigen cytokine responses wereobserved with mONCR-177.

• mONCR-177 treatment resulted in a greaternumber of viral antigen and tumor antigenspecific intratumoral T cells.

• The quality of the T cell response was enhancedin the MC38 models, with an increase inpolyfunctional T cells expressing both IFNɤ andTNFα.

• The transcriptional profiles of treatment withmONCR-177 and combination treatment ofmONCR-177+anti-PD1 resulted in a significantlyimproved abundance of T cells with cytotoxiccapacity. The higher scores were observed inboth injected and non-injected tumors and areindicative of a systemic anti-tumoral response.

• mONCR-177 and anti-PD1 combinationtreatment enhanced the efficacy of eithertreatment alone, improving the T cell andcytotoxicity scores.

mONCR-177 Inhibits Tumor Growth and Induces Cures

ONCR-177 is Uniquely Designed to Activate Multiple Arms of the Immune System

ONCR-177 is delivered by intratumoral administration and is designed to elicit apotent antitumor immune response. As ONCR-177 replicates in cancer cells, itleads to cancer cells oncolysis, tumor antigens release and local production of 5immune stimulatory transgenes. CCL4 and FLT3L promote dendritic cellsrecruitment and differentiation whereas IL-12 is a potent stimulator of NK cells,TH1 and cytolytic T cell responses. Antagonists of the 2 clinically validatedimmune checkpoints CTLA-4 and PD-1 have been added to ONCR-177 in order tofacilitate T cell priming and release from immune suppression within the tumormicroenvironment, respectively. Altogether these mechanisms converge topromote the generation of polyfunctional tumor-antigen specific T cells that caneradicate the injected tumor and patrol of the organism to an abscopal responsein non-injected distant tumors.

C57/Bl6 female mice were inoculated subcutaneously on the upper right flank and the upper left flank. Mice were divided into 3 cohorts of 5 mice per cohort. Treatment was initiated when the right flank tumor reached approximately 100 mm3. Mice received PBS or 6x106 PFU of mONCR-177, injected into a single flank tumor on day 9 and 12. Necropsy was performed on day 14, collecting the injected and non-injected tumors, and spleen from each mouse.

Immunogenicity Study Plan: Bilateral MC38 tumor model, single flank intratumoral injection

CD8 CD4

0

50

100

150

IT C

ells/m

g t

um

or ***

**

CD8 CD4

0

20

40

60

80

No

n-I

T c

ells/m

g t

um

or

*

Injected Tumor

0

5

10

15

20

%C

D8

+ S

LE

C o

f C

D4

5+ *

Injected Tumor

0

5

10

15

20

25

%C

D8

+ S

LE

C o

f C

D4

5+ PBS

mONCR-177

***

mONCR-177 Promotes Accumulation of SLEC CD8+ T cells in Injected and Non-Injected Tumors

Injected Non-Injected Injected Non-Injected

CD8+ (KLRG1+CD127-) were increased in the injected tumor in both tumor models when treated with mONCR-177 as well as CD4+ cells in the MC38model. Treatment with mONCR-177 increased the number T cells in the non-injected tumor in the MC38 model. Infiltrating CD8+ T cells in both injectedand non-injected tumors were predominantly short-lived effector cells.

mONCR-177 Induces a Polyfunctional Viral and Tumor Antigen Response

mONCR-177 Enhances the a Polyfunctional Viral Antigen Response over ONCR-159

Dissociated tumors were stimulated with viral antigen HSV gB, or tumor specific antigens, REPS1 or ADPGK for 5 hours in the presence of a golgi transportinhibitor. Cells were stained for surface phenotyping, fixed and permeabilized, then stained for intracellular cytokines. mONCR-177 induced a significantlyhigher expression of IFNγ in the injected and non-injected tumor in response to viral antigen. mONCR-177 also significantly increased the number of viraland tumor antigen specific IFNγ+CD8+ T cells. In response to viral antigen, mONCR-177 treatment increased the number of polyfunctionalIFNγ+TNFα+CD8+ T cells in the injected tumor. *<0.05; ** <0.01; ***<0.001; ****<0.0001

Injected Non-Injected Injected Non-Injected Injected Non-Injected

mONCR-177 Efficacy is Enhanced by Systemic PD-1 Blockade

MC38 tumor model, mONCR-177: IT, 3x106 PFU, Q3Dx3; anti-PD-1 or isotype: IP, 200 mg, Q3Dx3. Gene expression analysis was performed with the Mouse PanCancer IO 360 Gene Expression Panel (Nanostring), using Day 6 MC38 tumors after 2 rounds of dosing.

MC38 tumor model, mONCR-177: Intratumoral injection once tumors reached ~100mm3 with the indicated PFU, Q3Dx3 in a single flank. Tumor growth was monitored for 20 days. Cell infiltration was evaluated by immunohistochemistry. Infiltrating CD3+ cells were quantified.

The frequency of PD-1+ CD8+ T cells was similar in both tumor models. However, the level of PD-1 expression within the CD8+/PD-1+ population waslowest with mONCR-177, indicative of a less immunosuppressed tumor microenvironment. mONCR-177 improved the CD8:Treg ratio. An increase of DC1in the spleen was observed with mONCR-159 treatment.

mONCR-177 Increases the Frequency of PD-1+ TILS

mONCR-177 Improves the CD8:Treg ratio

mONCR-177 Increases the Number of DC1 in the Spleen

Injected Non-Injected Injected Non-Injected

PBS

mO

NCR-1

77

0

50000

100000

150000

# o

f c

ell

s p

er

sp

lee

n w

ith

in t

he

LIN

-

DC2

DC1

✱✱✱

PBS

mO

NCR-1

77PBS

mO

NCR-1

77

0

2000

4000

6000

8000

0

25

50

75

100

PD

-1 M

FI

% P

D-1

+ C

D8+

T c

ells

%

MFI

*****

PBS

mO

NCR-1

77PBS

mO

NCR-1

77

0

200

400

600

800

CD

8/T

reg

rati

o

**

*

0 4 8 12 16 20 24

19

20

21

22

23

Days on Study

Bo

dy

We

igh

t (g

) PBS

mONCR-177, 3x104 PFU

mONCR-177, 3x105 PFU

mONCR-177, 3x106 PFU

mONCR-177, 3x107 PFU

0 4 8 12 16 20 24

19

20

21

22

23

Days on Study

Bo

dy

We

igh

t (g

) PBS

mONCR-177, 3x104 PFU

mONCR-177, 3x105 PFU

mONCR-177, 3x106 PFU

mONCR-177, 3x107 PFU

C57/Bl6 female mice were inoculated subcutaneously on the upper right flank and the upper left flank. Mice were divided into 4 cohorts of 10 mice per cohort. Treatment was initiated when the right flank tumor reached approximately 100 mm3. Mice received PBS, 200ug/dose of an isotype control, 200ug/dose anti-PD1, 3x106 PFU of mONCR-177, or a combination of anti-PD1 and mONCR-177 on days 1, 4, and 7 of the study. mONCR-177 was delivered intratumorally into a single flank. Anti-PD1 was delivered intraperitoneally. Mice were monitored every 3-4 days.

mONCR-177 PD-1 Combination Treatment Study Plan

PBS

IgG2a

Isoty

pe

anti-P

D-1

mON

CR-171

mON

CR-171

+ a

nti-P

D-1

PBS

IgG2a

Isoty

pe

anti-P

D-1

mON

CR-171

mON

CR-171

+ a

nti-P

D-1

0

2

4

6

8

10

T cell score

Ce

ll t

yp

e s

co

res-

log

2

Injected

Non-Injected

************

************

************

*******

PBS

IgG2a

Isoty

pe

anti-P

D-1

mON

CR-171

mON

CR-171

+ a

nti-P

D-1

PBS

IgG2a

Isoty

pe

anti-P

D-1

mON

CR-171

mON

CR-171

+ a

nti-P

D-1

0

2

4

6

8

10

Cytotoxic score

Ce

ll t

yp

e s

co

res-

log

2

************

***********

*********

**

PBS

IgG2a

Isoty

pe

anti-P

D-1

mON

CR-171

mON

CR-171

+ a

nti-P

D-1

PBS

IgG2a

Isoty

pe

anti-P

D-1

mON

CR-171

mON

CR-171

+ a

nti-P

D-1

-10

-5

0

5

10

Lymphoid compartment

Sig

na

ture

sco

res

************

***********

************

******

PBS

IgG2a

Isoty

pe

anti-P

D-1

mON

CR-171

mON

CR-171

+ a

nti-P

D-1

PBS

IgG2a

Isoty

pe

anti-P

D-1

mON

CR-171

mON

CR-171

+ a

nti-P

D-1

-4

-2

0

2

4

6

Cytotoxicity

Sig

na

ture

sco

res

***********

********* ****

***

***

mONCR-177 and combination treatment elicited a more abundant T cell profile than controls or anti-PD1 alone in both injected and non-injected tumors.

Further, infiltrating cells demonstrated a cytotoxic phenotype. mONCR-177 and combination treatment had the highest cytotoxic scores in both injected and non-injected tumors indicating a greater abundance of these cells.

The lymphoid compartment signature measures the activity of lymphoid cells within the tumor. mONCR-177 and combination treatment demonstrated significantly higher lymphoid compartment score. The improved signature was observed for both the injected and non-injected tumors.

The cytotoxicity signature measures the molecular activity of NK and CD8+ T cells. mONCR-177 and combination treatment demonstrated significantly higher cytotoxicity signature score. The improved signature was observed for both the injected and non-injected tumors.

0 20 40 60 800

25

50

75

100

Days on Study

% S

urv

iva

l

PBS

Anti-mPD-1 mAb

mONCR-171 +

anti-mPD-1 mAb

mONCR-171

mONCR-177 (3x107 PFU, Q3Dx3 Inhibits Bilateral MC38 Tumor Growth and is Curative

**

*<0.05; ** <0.01; ***<0.001; ****<0.0001

*<0.05; ** <0.01; ***<0.001; ****<0.0001

*<0.05; ** <0.01; ***<0.001; ****<0.0001

*<0.05; ** <0.01; ***<0.001; ****<0.0001

*<0.05; ** <0.01; ***<0.001; ****<0.0001 *<0.05; ** <0.01; ***<0.001; ****<0.0001

PBS

mON

CR-177 PBS

mON

CR-177

0.0

0.5

1.0

1.5

2.0

5

10

15

20

HSV gB

# o

f C

D8 T

cells/m

g o

f tu

mo

r

****

*******

PBS

mON

CR-177 PBS

mON

CR-177

0.0

0.1

0.2

0.3

2

4

6

8

10

ADGPK

# o

f C

D8 T

cells/m

g o

f tu

mo

r

****

PBS

mON

CR-177 PBS

mON

CR-177

0

2

4

6

REPS1

# o

f C

D8 T

cells/m

g o

f tu

mo

r

IFN+IL2+TNF+

IFN+IL2+TNF-

IFN+IL2-TNF+

IFN+IL2-TNF-

****