PS - Immune Checkpoint Inhibitors
Expansion and Activation of T-cells via the Targeting of the Immunosuppressive Ligand Phosphatidylserine: Combination Strategy with Other Checkpoint Inhibitors Jeff T. Hutchins, PhD Vice President, Pre-Clinical Research Peregrine Pharmaceuticals, Inc. March 25, 2015 Immune Checkpoint Inhibitors: Validate Novel Pathways, Discover Predictive Biomarkers, Optimize Clinical Strategy Combination Therapy is the Future More Patients Responding to Therapy Combination Treatment * * Hypothetical ± for illustrative purposes only Checkpoint Blockade Yervoy® and Opdivo® are registered trademarks of Bristol Meyers Squibb. Keytruda® is a registered trademark of Merck. Targeted Therapy Conventional Therapy 1. Why do only a sub-set of patients respond to IO Therapy? Role of the immunosuppressive tumor microenvironment Lack of T-cell activation and myeloid cell differentiation 2. How can this patient pool be expanded? Ways a PS blockade changes this environment Consequences of PS targeting -- activation and expansion of T-cells Immune Activity in the Tumor Environment Tumor-Promoting Phenotype: Tumor-Rejecting Phenotype: Patients unable to benefit from checkpoint inhibitors Patients more likely to respond to checkpoint inhibitors MDSC Myeloid-Derived Suppressor Cell Highly immunosuppressive Secrete anti-inflammatory cytokines M2 Mac M2 Macrophage Secrete anti-inflammatory cytokines and growth factors DC Immature Dendritic Cell Incapable of antigen presentation to T-cells T-reg TGF-ǃ IL-10 T-Regulatory Cell Inhibits cytotoxic T-cells Cytokines Inhibit anti-tumor immune responses Keep Immune cells in quiet state MDSC M1 Mac Myeloid-Derived Suppressor Cell Reduction is current goal of immunotherapy M1 Macrophage Secrete inflammatory cytokines Kill tumor cells through ADCC DC Mature Dendritic Cell Present tumor antigens to T-cells Co-stimulatory signaling for T-cells CD8 T-cell Cytotoxic (CD-8+) T-cell Receptor specific tumor cell killing Increased PD-1 expression Cytokines Stimulate anti-tumor immunity TNF-Į IL-12 Immune Suppression in the Tumor Environment Creates a High Hurdle for Immunotherapies MDSCs M2 Macrophages Maintain an immunosuppressive tumor environment TGF-ȕ IL-10 Lack of antigen presentation T-cells do not activate Low levels of PD-1 expression Immature DC Naïve T-cells Ineffective response to tumors M1 Macrophage MDSC TGF-ȕ !" M2 Immature DC IL-10 Naïve T cell Poor Response to Tumor Bavituximab: Immune System Activation with Broad Applicability Immune Signaling Target Monoclonal antibody targeting PS Broad Applicability Activity in multiple cancer types and infectious disease Significant Clinical Experience Immune Activation MOA 19 studies, 500+ patients treated with bavituximab, currently in Phase III Good safety profile alone and in combination with other therapies (no IR safety events) 5HPRYHVWKH³%UDNHV´ and +LWVWKH³$FFHOHUDWRU´ PS Ligand Engages Multiple Receptors BAI1 PS PS PS Expressing Cells Stabilin-2 TIM-1 PS Tumor cell Tumor microvesicle Tumor blood vessel PS TIM-3 PS TIM-4 Immune Cells MDSC M2 Macrophage Immature DC T-Cells Wu, Y., Tibrewal, N., and Birge, R.B. (2006). Phosphatidylserine recognition by phagocytes: a view to a kill. Trends Cell Biol. 16, 189±197Park et al, (2007). BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature 450, 430±434. Hanayama, R., Tanaka, M., Miwa, K., and Nagata, S. (2004). Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells. J. Immunol. 172, 3876±3882 Park et al, (2007). Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor. Cell Death Differ 15, 192±201. ĮȞȕ3 RUĮȞȕ5 PS MFG-E8 PS Tyro-3, Axl, Mer (TAM) Gas6, Protein S Ishimoto Y, Ohashi K, Mizuno K, Nakano T. Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6. J Biochem (Tokyo) 2000;127:411±7. Scott RS, McMahon EJ, Pop SM, et al. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 2001;411:207-11. Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S. Identification of Tim4 as a phosphatidylserine receptor. Nature 2007;450: 435-9. Hanayama R, Miyasaka K, Nakaya M, Nagata S. MFG-E8-dependent clearance of apoptotic cells, and autoimmunity caused by its failure. Curr Dir Autoim- mun 2006;9:162-72. Jinushi M, Nakazaki Y, Dougan M, Carrasco DR, Mihm M, Dranoff G. MFG-E8 mediated uptake of ap- optotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF. J Clin Invest 2007;117: 1902-13 Donna L. Bratton and Peter M. Henson, Apoptotic Cell Recognition: Will the Real Phosphatidylserine Receptor(s) Please Stand up? Current Biology Vol 18 No 2 Mechanisms of PS Immunosuppression Mediated by TAM and TIMs Receptor Signaling TIM Receptors Apoptotic cells and debris with exposed PS TAM Receptors Apoptotic cells polarize to M2 macrophages CD4 Tim-1 Th2 bias CD8 Tim-3 pTh1 response T cell exhaustion Innate immunosuppressive actions stimulated by ligand PS complexes CD8 !I" Tim-4 Tolerance Freeman et al, Immunol Rev. 235: 172±189 (2010) Graham et. al. Nature Reviews 14: 769-785 (2014) TAM signaling inhibits NK antimetastatic effects Activated T cell feedback inhibits innate immunity Strong TAM Kinase Domain Activation Requires PS Courtesy of Lew et. al., and Lemke; eLife 2014;3:e03385 PS Signaling Pathway Placing the ³%UDNHV´ on the Immune System Exposed PS is a checkpoint that places brakes on the immune system PS Signaling Immune System Signaling PS receptor signaling causes increased anti-inflammatory cytokine expression Immune Suppression MDSCs accumulate DCs fail to mature T-cells remain naïve M1 Macrophages fail to develop PS is engaged MDSC By PS receptors PSR Tumor Cell M1 MDSC PS PS PSR = Exposed PS M2 TGF-ȕ 2. Immune System Signaling IL-10 MDSC Naive 3. ImmuneT-cell Suppression Immature DC MDSC Naive T-cell Multiple Pre-&OLQLFDO6WXGLHV6XSSRUW%DYLWX[LPDE¶V02$ 2aG4 treatment of PC3 tumor-bearing mice increases production of immunostimulatory IL-12 and TNF-ĮLQWKHWXPRU microenvironment and decreases the production of immunosuppressive TGF-ȕDQG,/-10. 1 #$%&'(%)'"*+"',(*$-.%&$/)0"(/1%" /)1$%&2%2"(&',$%"342 5" 2aG4 treatment induces expression of T-cell co-stimulatory molecules on CD11b+ cells in PC3 tumors1 Prolonged survival time and cures in rats bearing orthotopic F98 tumors treated with irradiation and 2aG4. Rats bearing orthotopic F98 gliomas were treated with 10 Gy radiation therapy (RT), 2aG4, RT + 2aG4, or RT + C44 5 d after injection of tumor cells as in Fig. 1. The survival in the RT + 2aG4 group was significantly longer than in all other groups (P < 0.001). Thirteen percent of rats treated with RT + 2aG4 were rendered disease free. After 230 d, the disease-free rats were rechallenged by intracerebral injection of 5 î 104 into the contralateral hemisphere. None of the rats developed disease, indicating that their prior treatment had caused them to develop immunity.3 Tumor-specific cytotoxic T-cells 3 Cure of castration resistant prostate cancer in TRAMP mice treated with mchN11 and androgen deprivation therapy. Survival time (until sacrifice) was analyzed by the Log-rank method. Mice treated with Cx+ mch1N11 had longer survival times than mice treated with Sx+ mch1N11, Cx+C44, or Sx+C44. * Significant for mch1N11 or castration alone versus C44 control; ** Significant for Cx+ mch1N11 versus mch1N11 or Cx or alone. Cx, castration. Sx, sham-operated. N = 20-33. 2 ()*+,-,+"./0,0"" 1" #$%&"""""""""""" '&&" 2aG4 directly induces repolarization of TAMs by binding to PS on their cell surface and activating them in an Fc -dependent manner. (a) FACS analyses showing that F4/80+ TAMs from disaggregated PC3 tumors acquire an M1-like phenotype when cultured for 4 days with 2aG4. The M2-like (F4/80+, Arg-1+) population and the M1-like (F4/80+, iNOS+) were quantified. (b) Treatment of F4/80+ TAMs with 2aG4 induces NO production in an Fc-dependent manner. (c) RT-PCR showing that F4/80+ TAMs lose M2 markers and acquire M1 markers when cultured with 2aG4. 1 2aG4 directly induces repolarization of TAMs by binding to PS on their cell surface and activating them in an Fc-dependent manner. (a) FACS analyses showing that F4/80+ TAMs from disaggregated PC3 tumors acquire an M1-like phenotype when cultured for 4 days with 2aG4. The M2-like (F4/80+, Arg-1+) population and the M1-like (F4/80+, iNOS+) were quantified. (b) Treatment of F4/80+ TAMs with 2aG4 induces NO production in an Fc-dependent manner. (c) RT-PCR showing that F4/80+ TAMs lose M2 markers and acquire M1 markers when cultured with 2aG4. 1 Vascular damage is caused by M1-like TAMs generated by 2aG4 treatment. a) Representative frozen sections showing TAM-mediated disintegration of vascular endothelium in PC3 tumors from mice treated for 2 weeks with 2aG4, alone or in combination with docetaxel. b) TAMs congregating around damaged vessels in 2aG4-treated mice costained for iNOS (blue) and F4/80 (green), indicating they were of M1-like phenotype. c) Representative sections showing that 2aG4 treatment causes a shift in the predominant polarity of TAMs from M2-like (F4/80+, green; Arg-1+, red) in C44 mice (upper) to M1-like (F4/80+, green; iNOS+, blue) in 2aG4-treated mice (lower). d) Histograms show the area of PC3 tumor sections occupied by M1 (F4/80+, iNOS+) or M2 (F4/80+, Arg-1+) TAMs. (e) RT-PCR of RNA from PC3 tumor homogenates reveals a general increase in M1 markers and a decrease in M2 markers in tumors from 2aG4-treated mice relative to C44-treated mice. (f) TAMs from 2aG4-treated mice manufacture NO . (g). TAMs from 2aG4-treated mice are tumoricidal. 1 Damage to tumor vasculature induced by treatment with 2aG4. a)Denudation of vascular endothelium in PC3 tumors in mice treated with 2aG4. b) Reduction of tumor vascularity and perfusion in PC3 tumor-bearing mice treated with 2aG4 alone or in combination with docetaxel. c) 3-D power Doppler ultrasound showing a reduction in blood flow in PC3 tumors in mice treated with 2aG4 alone or in combination with docetaxel. 1 1. AACR 2013 Annual Meeting: Phosphatidylserine-Targeting Antibody Induces Differentiation of Myeloid-Derived Suppressor Cells into M1-like Macrophages Yi Yin, Xianming Huang, Dan Ye, Yi Yin, Philip E. Thorpe Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX. 2. AACR 2012 Annual Meeting: Yin et al, Cure of castration-resistant prostate cancer in TRAMP mice by reactivating tumor immunity with a phosphatidylserine-targeting antibody. Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 3. 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 Bavituximab 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 Long-term Immunity Tumor-specific cytotoxicicity Cure of TRAMP Mice by Anti-PS Combination1 Cytotoxic T-cell Generation2 1. AACR 2012 Annual Meeting: Yin et al, Cure of castration-resistant prostate cancer in TRAMP mice by reactivating tumor immunity with a phosphatidylserine-targeting antibody. Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 2. 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 Tumor Response to Bavi Equivalent Involves Increased CD8 T cells and Decreased MDSCs (PS Targeting) EMT-6 breast tumor model in Balb/c mice (N=10/group; mean sem) 200 10 G ro u p M ean C 44 1 .4 7 ch1N 11 4 .1 9 p v a lu e = 0 .0 1 8 5 % M D S C /C D 8 % C D 8 T IL s 15 150 100 G ro u p M ean C 44 7 6 .6 0 ch1N 11 3 0 .8 9 p v a lu e = 0 .0 1 4 50 0 0 0 1000 2000 3000 3 T u m o r V o lu m e ( m m ) 4000 0 1000 2000 3000 3 T u m o r V o lu m e (m m ) 4000 Bavituximab Blocks Immunosuppression (Removes Brakes) Activates Immune Response (Hits Accelerator) Blocks PS Signaling Blocks PS receptor immunosuppressive signal IL-10 and TGF-ȕ levels decrease Activates Immune Response Sustained Immune Response Increased Inflammatory cytokines M1 macrophages kill tumor cells via ADCC Activated T-cells kill tumor cells Fc / )FȖ receptor signaling MDSC differentiation M2 to M1 macrophage polarization DCs mature, present tumor antigens to T-cells Yi Yin, Xianming Huang, Kristi D. Lynn, and Philip E. Thorpe, Phosphatidylserine-Targeting Antibody Induces M1 Macrophage Polarization and Promotes Myeloid-Derived Suppressor Cell Differentiation. Cancer Immunology Research October 2013 1:256-268 Promising and Consistent Pre-Clinical Data Support Phase III Development in Combination with Docetaxel Rationalebased Combination Docetaxel has been shown to increase PS exposure (AACR 2013 Poster #2850) Docetaxel has immunostimulatory effects (Kodumudi et al, Clin Cancer Res 2010;16:4583-4594) Compelling Pre-clinical Data Tumor models show greater than 90% reduction in tumor growth with bavi equivalent plus docetaxel (Cancer Research 2005; 65: (10) 4408-4416) Final Phase II 2nd Line NSCLC Overall Survival Data with Docetaxel 4*(./)%6"4*)'$*7" 4*(./)%6"4*)'$*7" Global Phase III Registration Trial N = 582 Randomized 1:1 Placebo-Controlled Bavituximab (3 mg/kg) Weekly Placebo Weekly Docetaxel Docetaxel Day 1, of 21-day cycles up to 6 cycles Day 1 of 21-day cycles for up to 6 cycles Positive EOP2 discussion with FDA and Ex-US Regulatory Agencies Single Phase III global registration trial > 150 sites (U.S., Europe and Asia Pacific) Est. 24 month enrollment and 12 month followup Powered to show approx. 2 month improvement in median overall survival Enrollment initiated January 2014 Patient Criteria: Primary Endpoint: OS Secondary Endpoints: PFS, ORR, Safety Stage IIIb/IV Non±squamous NSCLC Only one prior systemic therapy for advanced disease Unselected for genetic mutations Granted Fast Track Designation January 2014 Promising ORR, PFS and Survival Data in Breast Cancer Single-Arm Trials n= ORR PFS OS 46 61% 7.4 20.7 29 41% 4.5 11.4 Bavituximab + Carbo/Paclitaxel 46 74% 6.9 23.2 Carboplatin/Paclitaxel 100 62% 4.8 16.0 14 85% 7.3 -- Advanced/MBC ± All Types (1 Prior Chemo) Bavituximab + Docetaxel Docetaxel Advanced/MBC ± All Types (Front-Line) MBC (Her2 neg) Bavituximab + Paclitaxel Breast Docetaxel: ORR: Burstein, H., et al (2000). JCO. PFS/OS: Nabholtz, J., et al (1999). JCO. Peregrine ASCO (2010), 8/11. Breast Paclitaxel/Carboplatin: Loesch D., et al (2002). JCO. Peregrine ASCO (2010). Bavi IST: Alison Stopeck A HCC Responsive Patient Treated with Bavituximab in Combination with Sorafenib Pre Post Pre Post CD68 Pt ID #P2-23 (7.98) IHC score: 3 IHC score: 3 IHC score: 1 IHC score: 0 IHC score: 1 IHC score: 2 FoXP3 H&E CD4 IHC score: 3 CD8 IHC score: 2 IHC score: 3 GranzymeB IHC score: 2 Increase in tumor immune activity: increase CD8+ & CD4+ T cells Hypothesis: Bavituximab Can Potentiate Additional anti-PD-1 Responders Phase II HCC Data: Increased T-cell tumor infiltration Best immune responses observed in patients with greatest % increase in PD-1 and CD8+ cells = poor immune responders PD-L1/PD-1 Signaling = Suppression of T-Cell Function IL-10 APC MDSC PD-L1 + PD-L1 = PD-1 Exhausted or Regulatory Cell T-reg PD-L1 PD-1 PD-1 Endothelial Cell PD-L1 Conversion into T-reg cell PD-1 CD8+ T-cell PD-1 PD-L1 Tumor Cell Adapted from Nguyen et al, Nature Reviews: Immunology 15, 45±56 (2015) and Lee et al, Advances in Bioscience and Biotechnology; 2013, 4, 19-29 PD-L1 PD-1 T-reg PD-1 PD-L1 M1 Mac Conversion into Reg. Mac M2 Mac Anti-inflammatory cytokine production Relationship of PD-L1 Expression and Activity in Tumor Biopsies Evaluable by IHC NSCLC PD-L1 Tumor Expression N= Responses PFS at 6 mo. OS at 6 mo. High 7 4 (57%) 67% 89% Low 21 1 (5%) 11% 33% AACR 2014 Annual Meeting, abstract CT105 MK-3475 (anti-PD-1 monoclonal antibody) for non-small cell lung cancer (NSCLC): Antitumor activity and association with tumor PD-L1 expression Melanoma PD-L1 Tumor Expression N= Responses median PFS OS at 6 mo. Positive 60 29 (48%) 10.6 93% Negative 22 1 (5%) 2.9 75% AACR 2014 Annual Meeting, abstract CT104 Antitumor activity of the anti-PD-1 monoclonal antibody MK-3475 in melanoma (MEL): Correlation of tumor PD-L1 expression with outcome Upstream & Downstream Checkpoint Combinations CTLA-4 = Exposed PS Tumor Cell MDSC TGF-ȕ PSR PS B7-H3L T PD-1 PD-L1 PS B7-H3L PSR IL-10 M2 Macrophage PS / PSR: Upstream ³,PPXQH&KHFNSRLQW´ PS PS Receptors: Axl TIM-1 " " Immunosuppressive Cytokines/ Enzymes TGF-ȕ IL-10 IL-21 TIM-4 Hokkaido University, Japan TIM-3 Immature Dendritic Cell IDO TIM-3 PD-L1 KIR NK B7-H3 Downstream ³,PPXQH&KHFNSRLQWV´ PD-1 PD-L1 CTLA-4 TIM-3 PS Blockade Synergizes with Anti-PD-1 Day 42 Response in Individual Mice 89:;:<55" 3 T u m o r V o lu m e ( m m ) 600 400 200 a n t i- P D - 1 1500 1000 500 0 0 0 10 20 30 40 50 60 70 60 70 PS E x p e r im e n t a l D a y s Mice bearing K1735 melanoma tumors were treated with anti-PD-1 or mch1N11 + anti-PD-1, 2.5 mg/kg 2x per week. " 3 T u m o r V o lu m e ( m m ) an tiP D -1 + an ti- D -1 an tiP Tumor Volume (mm3) 800 1500 a n t i- P D - 1 + a n t i- P S 1000 500 0 0 10 20 30 40 50 E x p e r im e n t a l D a y s Combination Treatment Repolarizes Immune Profile in Spleen Results of PS Blockade + anti-PD-1 Versus anti-PD-1 Alone (K1735 melanoma model) 8"9 CD8+ Cells CD8+ T-cell MDSC CD8 cells 40% Mac DC 0.056 PD-1+ No Change IFN-Ȗ 40% 0.065 IL-2+ 80% 0.034 CD4+ T-cell CD4 cells No Change T-reg No Change PD-1+ No Change IL-2+ 150% 0.018 8"9 MDSC / Macs CD11b+ 8"9 CD4+ Cells No Change MDSC ( via CD11b) 45% PD-L1+ CD11b cells No Change PD-L1+ CD11b/Ly6G- No Change PD-L1+ CD11b/Ly6G- No Change DC (CD11chi/F480lo) 30% Mac (CD11b/F480hi) No Change 0.001 0.020 PS Blockade + Anti-PD-1shifts spleenocytes to immune active phenotype Data support potentiating additional PD-1 responders via PS blockade. PS Targeting Treatment Dramatically Reduces Expression of PDL-1 on Tumor Isolated Leukocytes 4*)'$*7" CD45 30.5% 30.5% &)'/-8=" 7.9% 7.94% DE;<B"*+"83-C5">"4%772" ?@;AB"*+"83-C5">"4%772" &)'/-835" &)'/-835>&)'/-8=" 5.0% 4.97% 31.4% 31.4% ?5;?B"*+"83-C5">"4%772" ?5;FB"*+"83-C5">"4%772" PD-L1 85 to 90% of PD-L1+ cells are CD45+ leukocytes, less than 10-20% PDL-1+ cells are tumor cells in this analysis Combination Treatment Repolarizes Immune Profile in Tumor Results of PS Blockade + Anti-PD-1 Versus anti-PD-1 Alone (K1735 melanoma model) 8"9 CD8+ Cells CD8 cells < 0.01 PD-1+ Lag-3+ 90% < 0.01 IFN-Ȗ 70% 0.017 TNF-Į 70% 0.007 Granzyme-B+ 70% 0.017 8"9 MDSC / Macs Mac MDSC ( via CD11b) 40% 0.02 iNOS+ CD11b cells 90% 0.001 80% <0.01 PD-L1+ TIL CD4 /T-reg Ratio CD4+ T-cell 0.001 MDSC 8"9 CD4+ Cells 0.001 " 100% " 50% CD8 /T-reg Ratio CD8+ T-cell 30% PD-1+ < 0.01 No change CD137+ (41BB) 60% 0.03 IFN-Ȗ 30% < 0.05 TNF-Į 100% < 0.01 60% 0.02 IL-2+ 100% PS Blockade + Anti-PD-1 increases TIL ; shifting TIL and cytokines towards tumorrejecting phenotype. Data support potentiating additional PD-1 responders via PS blockade. Key Takeaway: PS Blockade Optimizes Tumor Environment for Additional Anti-PD-1 Responders Results of PS Blockade + anti-PD-1 versus anti-PD-1 alone (K1735 melanoma model) MDSC CD8+ T-cell PD-1 PD-L1 on TILs 40% reduction* of myeloid-derived suppressor cells 30% increase* in CD8+ T-cells 50% increase* in PD-1 expression 80% decrease* PD-L1 expression on TIL (CD45+) * Statistically significant (p = < 0.02) Positive environment for increasing anti-PD-1 Responders PS Targeted Combination Therapy in PD-1 Resistant Melanoma Increases the Anti-tumor Response Rate C o n tr o l Ig G (C 4 4 ) T u m o r V o lu m e (m m 3500 C o n tro l Ig G 3 3500 3000 2500 2000 1500 1000 500 0 C h1N 11 3000 (Anti-PS) 0 A n t i- P D -1 2500 5 10 15 20 E x p e r im e n t a l D a y s p = 0 .0 0 7 C h 1N 11 4000 T u m o r V o lu m e (m m 3 ) C h 1 N 1 1 + A n ti-P D -1 2000 1500 3500 3000 2500 2000 1500 1000 500 0 0 p = 0 .0 1 8 5 10 15 20 E x p e r im e n t a l D a y s 1000 A n ti-P D -1 T u m o r V o lu m e (m m 3 ) 4000 500 0 0 5 10 15 3500 3000 2500 2000 1500 1000 500 0 20 0 5 10 15 20 E x p e r im e n t a l D a y s E x p e r im e n ta l D a y s A n ti-P D -1 + 1 N 1 1 3 ) 4000 B16F10 Melanoma Tumor Model in C57BL Mice (10/group) T u m o r V o lu m e (m m M e d ia n T u m o r V o lu m e (m m ) 3 ) 4000 3500 3000 2500 2000 1500 1000 500 0 0 5 10 15 20 E x p e r im e n t a l D a y s 28 Immune Cell Changes in the Tumor Microenvironment in the Mouse B-16F10 Model Immune Cell Type (%) Treatment Type (Day 23, after 4 treatments) CD45 CD3* CD8** (Cyto T- CD4** T reg cells) (helper Tcells) Tumor Size (mm3) (WBC) (mature T-cells) Control mAb (2) <:;5! FG;D! AA;E! <:;D! 5G;E! FF<A! ch1N11 (anti- PS) (7) <F;G! EF;G! AF;D! FF;G! 5:;G! <5@A! Anti-CTLA-4 (5) 5@;?! F?;D! EA;5! E:;E! ?;F! <<:E! Anti-PD-1 (6) <G;F! E<;<! E?;E! FF;?! 5?;<! 5G?G! ch1N11 + Anti-PD-1 (5) 2&3&! 4535! 6738! <:;A! 5G;F! 967! "H"8%$1%)'&0%"*+"43EA"I7%,1*1J'%K"L*2/'/M%"1%772" HH"8%$1%)'&0%"*+"43F"I(&',$%"#-1%77K"L*2/'/M%"1%772" (:$:,0:,+$;;/"0,<=,-,+$=:"+>$=<*" Key Observations - 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 in immuno-competent models with PD-1 and CTLA-4: increase CD8 T cells and M1 macrophages decrease MDSCs and M2 macrophages demonstrate statistically significant anti-tumor responses Powerful science. Novel therapies. Dedicated to =L%1/&7"#N&)O2"'*P" patients. Xianming Huang, Ph.D. Rolf Brekken, Ph.D. Dmitry Gabrilovich, M.D., Ph.D. Bruce Freimark, Ph.D. Nikoletta Kallinteris
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