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 PD1/L1, CTLA-4 and IL-2 signaling Antibody-mediated inhibition of PS results in immune activation and durable anti-tumor responses Disclosures of Relevant Financial Relationships I 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 RAB 3 Healthy Cell Plasma Membrane: PS on Interior Leaflet Phosphatidylserine (PS) Exposure of Phosphatidylserine (PS) on the surface of cells Normal Cell under normal conditions Tumor ROS Hypoxia / reoxygenation Tumor cell metabolites Cytokines Tumor cell, endothelial cell, exosome Program cell death Normal dying cell PS and PE PC and SM Brief history of PS targeting VTA Philip E. Thorpe, Ph.D. 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. PS targeting agents Developed in the Thorpe lab and @ Peregrine 3G4 2aG4 Bavituximab PGN635 (1N11) Mouse IgG3 Mouse IgG2a Chimeric human IgG1 Fully human IgG1 β2-Glycoprotein 1-dependent binding of PS targeting agents to exposed PS β2-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) Complex Binding is High Affinity (1 nM) bavitixumab V β2-Glycoprotein 1 I I I I II II II II III III III III IV IV V V 8 IV IV V Combination therapy in rodent tumor models 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% A549 2aG4 + RT Cure (50%) H460 2aG4 + RT 99.9% Rectal SW837 2aG4 + capecitabine + RT 98% Glioma F98 2aG4 + RT 99.9% (15% cures) Lung MECHANISMS Antibody-mediated PS targeting mediates binding of macrophages and destruction of tumor endothelium C 1 day 2 days endothelium monocytes Macrophages 5 days collagen IV A Control IgG endothelial remnants Tumor-associated macrophages (TAMs) FcγR NO Arg-1+ NOS IL-10 TGF-β IL-1, IL-12, TNF-α FcγR FcγR M2 Immunosuppressive Pro-angiogenic Maintain T-regs Do not perform ADCC PREDOMINANT M1 Immunostimulatory Pro-inflammatory Tumoricidal Perform ADCC SCARCE PS blockade increases M1 to M2 ratio of TAMs Arg-1 iNOS Merge 2aG4 C44 F4/80 3 2 1 C44 2aG4 5 10 4 8 M1:M2 ratio 4 0 Yi Y, et al CIR 2013 M1 TAMs (% area) M2 TAMs (% area) 5 3 2 1 0 6 4 2 C44 2aG4 0 C44 2aG4 2aG4 Drives TAMs towards M1 in vivo 10 M1 markers Relative mRNA M2 markers 1 0.1 Yi Y, et al CIR 2013 PS blockade reduces tumor-infiltrating myeloid-derived suppressor cells (MDSCs) 5 tumors pooled 4 10 MDSCs CD 11b /Gr1 /DAPi 72.9 10 6 102 101 2 0 2aG4 2aG4 4 C44 2aG4 CD11b MDSCs (% area) C44 25.4 3 100 100 101 102 103 104 104 103 102 22.2 76 C44 101 Note: Similar data from blood and spleen Yi Y, et al CIR 2013 100 100 101 102 Gr1 103 104 MDSCs treated with 2aG4 differentiate into macrophages and DCs in vitro % total cells MDSCs Macrophages DCs 60 60 40 45 45 30 30 30 20 15 15 10 0 0 0 C44 2aG4 C44 2aG4 C44 2aG4 MDSCs : Gr-1+, CDIIb+ Macrophages: F4/80+ DCs: CD11b+, CDIIchi Yi Y, et al CIR 2013 102 256 103 100 Yi Y, et al CIR 2013 104 100 101 256 101 256 100 MHC II 0 0 CD86 102 103 104 103 104 CD40 0 CD80 0 Events 256 …… and the dendritic cells acquire maturation markers 101 102 103 104 100 101 102 Bavi 2aG4 C44 PS blockade facilitates induction of tumor-specific cytotoxic T-cells MHCII CD40 Antigen presentation Mature DC CD80 CD86 T T Cytotoxic T-cells Tumor blood vessel cell / Tumor cell / Microvesicle Generation of antitumor T-cell immunity in syngeneic F98 glioma in rat2 T Tumor-specific cytotoxicicity Cytotoxic T-cell Generation2 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 Cure of transgenic model of prostate cancer (TRAMP) mice mch1N11 = PS targeting agent 100 Cx + mch1N11 ** Cx + C44 * mch1N11 * C44 Percent survival 80 60 N=23-30 Died of old age 40 Rx start @ week 16 20 0 0 100 200 300 400 Age (days) 1. AACR 2012 Annual Meeting: Yin et al, & Keystone 2014 Huang et al 500 600 700 PS Signaling Suppresses Immune Surveillance Exposed PS in the tumor environment provides a fundamental upstream immunosuppressive signal Normal Cell M1 Macrophage iNOS+ No PS Exposed Why is this important? 1. Prevents auto-immunity 2. Hallmark of cancer PS receptors Fcγ receptor Tumor blood Vessel/ Tumor Cells PS M2 Macrophage, MDSCs PS PS PS • TGF-β • IL-10 Immature DC T cell PS 1. PS Signaling 2. Immune System Signaling 3. Immune Suppression • M2 macrophages engage PS through PS receptors • Signaling initiated • Increased immunosuppressive cytokines • Innate immunity is suppressed • DCs fail to mature • Lack of antigen presentation to T-cells • Impaired cytotoxic T-cell response • M1 macrophages fail to develop Antibody-mediated PS blockade stimulates development of M1 macrophages and T-cell mediated anti-tumor effects Immunosuppressive Signaling Y = Bavituximab CD40 Tumor blood vessel cell / Tumor cell PS Antigen presentation Mature DC Cytotoxic T-cell PS Y Bavituximab Treatment Fcγ receptor Tumor blood vessel cell / Tumor cell PS PS PS PS YY PS receptor PS CD86 PS PS PS CD80 MHCII MDSCs, M2 macs, Immature DCs Tumor-specific cytotoxicicity • IL-12 • TNF-α Tumor blood vessel cell / Tumor cell M1 Mac PS PS iNOS+ Fcγ receptor PS ADCC PS receptor 1. Bavituximab Treatment 2. Immune Signaling Changes • Effector cells engage bavi-coated PS through Fcγ receptors • Inflammatory cytokines • MDSC differentiation • Overrides upstream PS-mediated immunosuppressive signal • M2 to M1 macrophage polarization • DC maturation 3. Immune Activation • Tumor-specific cytotoxic T-cells • ADCC SOURCE: AACR 2013 Annual Meeting: Phosphatidylserine-targeting antibody reactivates tumor immunity and destroys tumor vasculature in mice Yi Yin, Xianming Huang, Gustavo Barbero, Dan Ye, Philip E. Thorpe Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX. BRIEF CLINICAL OVERVIEW >600 patients treated with Bavituximab Second-Line NSCLC Phase III Trial Initiated N = ~ 600 Stage IIIB/IV Randomized 1:1 Placebo-Controlled Bavituximab (3 mg/kg) Weekly Placebo Weekly Docetaxel Docetaxel mg/m2) (75 Day 1, of 21-day cycles up to 6 cycles (75 mg/m2) Day 1 of 21-day cycles for up to 6 cycles or until progression or toxicities • 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: Primary Endpoint: OS Secondary Endpoints: PFS, ORR, Safety • 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 Checkpoint Inhibitor Downstream Immune Treatments Anti-PD-1 Anti-PD-L1 Bavituximab (PS-targeting mAb) Anti-CTLA-4 IL-2 PAP-GMCSF Vaccines 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 UT Southwestern Thorpe/Brekken Labs Philip E. Thorpe, Ph.D. http://www.utsouthwestern.edu/labs/brekken/ 26 Promising Phase II Second-Line NSCLC Data Supports Phase III Development • Consistent, positive trend favoring 3mg/kg bavituximab arm: • • 60% improvement in median overall survival (11.7 months vs. 7.3 months) • Positive safety database 27 Generation of antitumor immunity 4 Anti-CTLA-4 Traffic to tumors Priming and activation 3 5 Infiltrate into tumors 2 6 APC antigen presentation Recognition 1 Release of cancer antigen 7 Effector phase Anti-PD1/PD-L1 Kill cancer cells Chen DS et al, Immunity 2013 PS blockade repolarizes TAMs from M2 TO M1 & induces nitric oxide production IN VITRO M2 % 80 80 60 60 % 40 20 0 0 2aG4 2aG4 F(ab’)2 Relative mRNA 10 1 0.1 Yi Y, et al CIR 2013 M1 markers M2 markers NO 15 40 20 C44 M1 100 NO production (µM) 100 10 5 0 C44 2aG4 2aG4 F(ab’)2 C44 2aG4 2aG4 F(ab’)2 PS blockade (1N11) enhances the efficacy of anti-PD1 in K1735 melanoma model 600 400 200 3 P=0.0211 T u m o r V o lu m e ( m m ) P D -1 3 T u m o r V o lu m e ( m m ) 800 1500 P D -1 + 1 N 1 1 1000 500 0 0 20 30 40 50 60 P D -1 + P 1 N D 1 -1 1 10 D a y s F r o m T u m o r Im p la n t 70 PS blockade enhances the activity of anti-CTLA-4 in K1735 melanoma P e rc e nt su rviva l 100 Control IgG Ch1N11 Anti-CTLA4 Ch1N11 + anti-CTLA-4 80 60 40 20 0 0 20 40 60 80 Days Post Treatment Initiation Confidential PPHM Acknowledgments Brekken Lab Jason Toombs Katie Ludwig Robin Frink Ashley Barraza Kristina Aguilera Mary Topalovski Noah Sorrelle Tori Burton Miao Wang Moriah Hagopian Alan Schroit Xianming Huang Shuzhen Li Lisa Li Gustavo Barbero Dan Ye Olivier Belzile Janie Iglehart Kristi Lynn Andrew (Tom) Ngo Sean Dineen Laura Sullivan Amanda Kirane Collaborators John Minna Joan Schiller Diego Castrilon Tom Wilkie David Gerber Joan Schiller James Lorens – U Bergen Clinical collaborators Peregrine Pharmaceuticals Affitech BerGenBio 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 Brekken lab July 2014 Brekken lab Spring 2014