Breaking Immune Tolerance by Targeting Key Immunosuppressive Pathways in the Tumor

Microenvironment

Rolf A. Brekken, PhD

Key Messages

Phosphatidylserine (PS) is exposed on the external surface of cells and vesicles in the tumor microenvironment

PS exposure is immunosuppressive

PS signaling is an immune check point that is upstream of PD-1/L1, CTLA-4 and IL-2 signaling

Antibody-mediated inhibition of PS results in immune activation and durable anti-tumor responses

3RAB

Disclosures of Relevant Financial RelationshipsI have the following financial relationships to disclose:• Consultant: Peregrine Pharmaceuticals Inc., and Synergys Biotherapeutics• Grant/Research Support: BerGenBio AS, Imclone Systems/Eli Llly, Affitech AS,

and Peregrine Pharmaceuticals, Inc.• Stock/ Stock Options: Peregrine Pharmaceuticals, Inc. • Co-Founder: Tuevol Therapeutics, Inc

Healthy Cell Plasma Membrane: PS on Interior Leaflet

Phosphatidylserine (PS)

PC and SM

PS and PE

Exposure of Phosphatidylserine (PS) on the surface of cells

Tumor

Hypoxia / reoxygenationTumor cell metabolites

Cytokines

ROS

Normal

Tumor cell, endothelial cell,

exosome

Cell under normal

conditions

Normal dying cell

Program cell death

Brief history of PS targeting

Philip E. Thorpe, Ph.D.

VTA

circa late 90s

In the Thorpe lab we were interested in developing targeting agents that specifically homed to tumor vasculature. PS was a target we recognized.

3G4

Mouse IgG3

2aG4

Mouse IgG2a

Bavituximab

Chimerichuman IgG1

PGN635

Fullyhuman IgG1

PS targeting agentsDeveloped in the Thorpe lab and @ Peregrine

(1N11)

β2-Glycoprotein 1-dependent binding of PS targeting agents to exposed PSβ2-Glycoprotein 1 is an abundant serum protein; antibody induced dimerization of β2GP1 increases affinity for PS by >1000-fold

Monovalent Binding is Low Affinity (1 µM)

V

III

IV

I

II

III

IV

I

II

V

Complex Binding is High Affinity (1

nM)

β2-Glycoprotein 1

V

III

IV

I

II

I

II

III

IVV

bavitixumab

8

Tumor type Name Treatment Growth reduction

Prostate PC3 2aG4 + docetaxel 98%

LNCaP 2aG4 + docetaxel 95%

LNCaP 2aG4 + ADT 99%

Pancreas Pan02 3G4 + gemcitabine 95%

HCC PLC/PRF/5 2aG4 + sorafenib 98%

C3A 2aG4 + sorafenib 75%

Lung A549 2aG4 + RT Cure (50%)

H460 2aG4 + RT 99.9%

Rectal SW837 2aG4 + capecitabine + RT 98%

Glioma F98 2aG4 + RT 99.9% (15% cures)

Combination therapy in rodent tumor models

MECHANISMS

Antibody-mediated PS targeting mediates binding of macrophages and destruction of tumor endothelium

C1 day

A Control IgG

2 days

endothelium

monocytes

endothelial remnants

5 days

collagen IV

Macrophages

Tumor-associated macrophages (TAMs)

M1ImmunosuppressivePro-angiogenicMaintain T-regsDo not perform ADCC

ImmunostimulatoryPro-inflammatoryTumoricidalPerform ADCC

IL-10TGF-β

M2

Arg-1+

FcγR FcγR

FcγR

IL-1, IL-12, TNF-α

NOSNO

PREDOMINANT SCARCE

F4/80 Arg-1 iNOS Merge2a

G4

C44

M1

TAM

s (%

are

a)

2aG4C44

5

3

1

0

4

2

M2

TAM

s (%

are

a)

2aG4C44

5

3

1

0

4

2M

1:M

2 ra

tio

2aG4C44

10

6

2

0

8

4

PS blockade increases M1 to M2 ratio of TAMs

Yi Y, et al CIR 2013

Rela

tive

mRN

A

iNOS

TNF-α IL-12

Ccl5

Cxcl11

CD80CD86

CD40

MHC IIArg-

1

TGF-β IL-

10

CD206Ym

-1Fiz

z-1Ccl2

2Ccl1

7

VEGF-A

VEGF-B

0.1

1

10

M1 markers

M2 markers

2aG4 Drives TAMs towards M1 in vivo

Yi Y, et al CIR 2013

C44

2aG4

CD11b /Gr1 /DAPiMDSCs

0

2

4

6

C44 2aG4

MD

SC

s (%

are

a)

PS blockade reduces tumor-infiltrating myeloid-derived suppressor cells (MDSCs)

100 101 102 103 104100

101

102

103

104

72.9

CD

11b

2aG4

100 101 102 103 104100

101

102

103

104

22.2

Gr1

C44

5 tumors pooled

Note: Similar data from blood and spleen

Yi Y, et al CIR 2013

20

60

45

30

15

0

% to

tal c

ells

MDSCs Macrophages DCs

60

45

30

15

02aG4C44

40

30

10

02aG4C442aG4C44

MDSCs : Gr-1+, CDIIb+ Macrophages: F4/80+ DCs: CD11b+, CDIIchi

MDSCs treated with 2aG4 differentiate into macrophages and DCs in vitro

Yi Y, et al CIR 2013

…… and the dendritic cells acquire maturation markers

100 101 102 103 104

025

6

100 101 102 103 104

025

6

100 101 102 103 104

025

6

100 101 102 103 104

025

6Even

ts

Bavi2aG4C44

CD86 MHC II

CD80 CD40

Yi Y, et al CIR 2013

PS blockade facilitates induction of tumor-specific cytotoxic T-cells

1. He et al, Antiphosphatidylserine Antibody Combined with Irradiation Damages Tumor Blood Vessels and Induces Tumor Immunity in a Rat Model of Glioblastoma. Clin Cancer Res 2009;15:6871-6880

Generation of antitumor T-cell immunity in syngeneic F98 glioma in rat2

Cytotoxic T-cell Generation2

Cytotoxic T-cells

Tumor-specific cytotoxicicity

Antigen presentation

Mature DC

MHCII CD40

CD80 CD86

T

Tumor blood vessel cell /Tumor cell /Microvesicle

T

T

Cure of transgenic model of prostate cancer (TRAMP) mice

Died of old age

0 100 200 300 400 500 600 7000

20

40

60

80

100

Age (days)

Pe

rce

nt s

urvi

val

Cx + C44 *

Cx + mch1N11 **

C44

mch1N11 *

N=23-30

Rx start @ week 16

1. AACR 2012 Annual Meeting: Yin et al, & Keystone 2014 Huang et al

mch1N11 = PS targeting agent

M2 Macrophage,MDSCsTumor blood Vessel/

Tumor Cells

Exposed PS in the tumor environment provides a fundamental upstream immunosuppressive signal

PS

PS receptors

PS

PS

PS

PS

• TGF-β• IL-10

Immature DCT cell

• M2 macrophages engage PS

through PS receptors • Signaling initiated

• Increased immunosuppressive

cytokines• Innate immunity is suppressed• M1 macrophages fail to

develop

• DCs fail to mature • Lack of antigen presentation to T-

cells• Impaired cytotoxic T-cell response

M1 Macrophage

iNOS+

1. PS Signaling 2. Immune System Signaling 3. Immune Suppression

XFcγ receptor

Normal Cell

No PS Exposed

PS Signaling Suppresses Immune Surveillance

Why is this important?1. Prevents auto-immunity2. Hallmark of cancer

Antibody-mediated PS blockade stimulates development of M1 macrophages and T-cell mediated anti-tumor effects

Tumor blood vessel cell /Tumor cell

PS

PS

PS

PS

PS

• IL-12• TNF-α

CD40 CD80

CD86MHCII

Mature DC Cytotoxic T-cell

Tumor-specific cytotoxicicity

Antigen presentation

Tumor blood vessel cell /Tumor cell

PS

PS

PS

M1 Mac

ADCC

• Effector cells engage bavi-coated PS

through Fcγ receptors

• Overrides upstream PS-mediated

immunosuppressive signal

• Inflammatory cytokines

• MDSC differentiation

• M2 to M1 macrophage polarization

• DC maturation

SOURCE: AACR 2013 Annual Meeting: Phosphatidylserine-targeting antibody reactivates tumor immunity and destroys tumor vasculature in miceYi Yin, Xianming Huang, Gustavo Barbero, Dan Ye, Philip E. Thorpe Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX.

Fcγ receptor

MDSCs, M2 macs,

Immature DCs

Y

PS receptor

Fcγ receptor

PS receptor

1. Bavituximab Treatment 2. Immune Signaling Changes 3. Immune Activation

• Tumor-specific cytotoxic

T-cells

• ADCC

Tumor blood vessel cell /Tumor cell

PS

PS

PS

PS

PS

Y Bavituximab Treatment

ImmunosuppressiveSignaling

Y = Bavituximab

Y

iNOS+

YY

BRIEF CLINICAL OVERVIEW

>600 patients treated with Bavituximab

Second-Line NSCLC Phase III Trial Initiated

N = ~ 600Stage IIIB/IVRandomized 1:1Placebo-Controlled

Bavituximab (3 mg/kg) Weekly

Docetaxel(75 mg/m2)

Day 1, of 21-day cycles up to 6

cycles

Primary Endpoint: OS

Secondary Endpoints:PFS, ORR, Safety

Docetaxel(75 mg/m2)

Day 1 of 21-day cycles for up to 6

cycles or until progression or

toxicities

PlaceboWeekly

• Initiated December 2013 • Single Phase III global registration trial• 582 target patient enrollment• >100 sites (U.S, E.U. and Asia Pacific)• Trial is adequately powered to support product

approval

• Patient Criteria:

• Non–squamous NSCLC• Only one prior systemic therapy for advanced

disease

• Unselected for genetic mutations

• Granted Fast Track Designation January 2014

23

MOA supports multiple opportunities with other immunotherapies

Upstream Immune

CheckpointInhibitor

Downstream Immune

Treatments

Bavituximab(PS-targeting

mAb)

Anti-PD-1

Anti-PD-L1

Anti-CTLA-4

IL-2

Vaccines

PAP-GMCSF

Key Messages - Summary

Phosphatidylserine (PS) is externalized in the tumor microenvironment and is a major immunosuppressive signal

PS is a global immune checkpoint

Antibody-mediated blockade of PS signaling breaks immune tolerance reactivating innate and adaptive immunity and results in durable anti-tumor responses in multiple pre-clinical models

Combination pre-clinical studies with other immune checkpoint inhibitors are underway and early phase clinical studies are planned

Acknowledgements and Collaborators

26

UT Southwestern Thorpe/Brekken Labs

http://www.utsouthwestern.edu/labs/brekken/

Philip E. Thorpe, Ph.D.

• Consistent, positive trend favoring 3mg/kg bavituximab arm:

• 60% improvement in median overall survival (11.7 months vs. 7.3 months)

• Positive safety database

Promising Phase II Second-Line NSCLC Data Supports Phase III Development

27

1

2

3

4

5

6

7

Release of cancer antigen

APC antigen presentation

Priming and activation

Effector phase Kill cancer cells

Recognition

Infiltrate into tumors

Traffic to tumors

Chen DS et al, Immunity 2013

Generation of antitumor immunity

Anti-CTLA-4

Anti-PD1/PD-L1

PS blockade repolarizes TAMs from M2 TO M1& induces nitric oxide production IN VITRO

Rela

tive

mRN

A

TNF-αIL-12

CD80CD86

MHC IIiN

OSYm

-1IL-

10Arg-

1

VEGF-A

CD206Fiz

z-1TG

F-β0.1

1

10

M1 markersM2 markers

%

2aG4C44

100

60

20

0

80

40

2aG4F(ab’)2

%

2aG4C44

100

60

20

0

80

40

2aG4F(ab’)2

NO

pro

ducti

on (µ

M)

2aG4C44

15

10

0

5

2aG4F(ab’)2

M2 M1 NO

Yi Y, et al CIR 2013

PS blockade (1N11) enhances the efficacy of anti-PD1 in K1735 melanoma model

Tu

mo

r V

olu

me

(mm

3)

PD-1

PD-1 +

1N11

0

200

400

600

800

P=0.0211

Days From Tumor Implant

Tu

mo

r V

olu

me

(m

m3)

10 20 30 40 50 60 700

500

1000

1500

PD-1

PD-1 + 1N11

PS blockade enhances the activity of anti-CTLA-4 in K1735 melanoma

ConfidentialDays Post Treatment Initiation

Pe

rce

nt

su

rviv

al

0 20 40 60 800

20

40

60

80

100

Control IgG

Ch1N11

Anti-CTLA4Ch1N11 + anti-CTLA-4

PPHM

AcknowledgmentsBrekken LabJason ToombsKatie LudwigRobin FrinkAshley BarrazaKristina AguileraMary TopalovskiNoah SorrelleTori BurtonMiao WangMoriah Hagopian

Sean DineenLaura SullivanAmanda Kirane

CollaboratorsJohn MinnaJoan SchillerDiego Castrilon Tom WilkieDavid GerberJoan Schiller

James Lorens – U BergenClinical collaborators

Peregrine PharmaceuticalsAffitechBerGenBio

Effie Marie Cain Scholarship in Angiogenesis Research, UTSW Department of Surgery, NCI/NIH, Simmons Cancer Center, NSF, Joan’s Legacy Foundation, Mary Kay Foundation, BerGenBio

Alan SchroitXianming HuangShuzhen LiLisa LiGustavo BarberoDan YeOlivier BelzileJanie IglehartKristi Lynn

Andrew (Tom) Ngo

Brekken lab July 2014

Brekken lab Spring 2014