Identification of novel immune regulators of tumor growth using highthroughput screening in vivo Tom Brennan 1
Five Prime s Unique Platform Tests Nearly Every Extracellular Protein to Identify Protein Drugs and Antibody Targets Library of > 5700 Extracellular Proteins Soluble Extracellular Domains Secreted Factors Cell Surface Proteins 2
Five Prime s Unique Platform: Screening a Library of the Extracellular Proteome to Identify Novel Targets and Therapeutics Library of > 5700 Extracellular Proteins Proprietary Screens Protein Therapeutics Secreted Factors Cell-based Screens Antibodies Cell Surface Receptors/Ligands In Vivo Screens Soluble Extracellular Domains Receptor-Ligand Matching Soluble Extracellular Domains 3
in vivo Screening Platform: Rapid in vivo Protein Production System (RIPPS ) DNA RIPPS Tumor Monitor Efficacy Inject DNA encoding protein of interest DNA instructs liver to make protein Protein distributed in circulation The RIPPS system bypasses the need for protein production to obtain rapid in vivo proof of concept. ECD-Fc, secreted factors, and even antibodies can be RIPPS ed Sustainable levels of protein in circulation for weeks after a single injection. Ideal for screening novel genes and hypothesis-driven targets, as well as early validation of hits from parallel in vitro screens Typically screen 10 mice per DNA construct 4
RIPPS Screening has Been Highly Successful for Discovering Targets In Multiple Indications Partner Indication Results Fibrosis Related Conditions 4 claimed targets Muscle atrophy 1 claimed target FPT only Oncology 2 targets validated, IND s on schedule for 2017, 2018 5
The FivePrime Immunome Library is a Subset of the Total Library and Includes Immune Regulatory Proteins FivePrime Immunome : ~700 potential immune-regulatory proteins based on structure, phylogeny and/or expression Ig domain containing proteins ITIM/ITAM/ITSM domain containing TNF family members Expression on immune cells Prioritized by cell type (CD8, CD4, monocytes, NK, macrophages) Prioritized by narrowness of expression VISTA TIM3 B7H3 B7H4 PDL1 PDL2 PD1 LAG3 Discovery Strategy: Identify therapeutic targets by screening the immunome library in vivo in mouse tumor models Screen across multiple tumor models Screen ligands and extracellular domain of receptors (Fused to Fc of IgG) Rank and prioritize hits for follow-up with recombinant protein or antibodies 6
Multiple Tumor Models Were Chosen for Screening to Capture the Diversity Associated with Human Cancer RNAseq profiling and gene signature CD3 IHC 4T1 CT26 MC38 The immune cell repertoire and immunohistochemistry varies among mouse tumor types 7
A Multi-Pronged Approach for Identifying Novel Immuno-Oncology Targets CT26 and MC38 Model subcutaneous colon cancer models Sensitive to traditional checkpoint inhibitors (low bar models) Can we reprogram the immunosuppressive environment? Orthotopic 4T1 model in combination with anti-pd-1 Insensitive to traditional IO therapeutics, including anti-pd-1 (high bar model) High MDSC populations, limited TILs - Ability to impact multiple IO mechanisms Can we make a resistant model sensitive to anti-pd-1 therapy? TC-1 in combination with vaccine based approach Limited TILs Vaccine activates tumor antigen-specific T cells Vaccination sensitizes model to co-stimulatory & co-inhibitory molecules Can we identify targets that combine with tumor antigen-specific therapy? 8
Monotherapy Screen in MC38 and CT26 Subcutaneous Syngeneic Tumor Models Screen Design Subcutaneous Models C57Bl/6 BALB/c MC38 CT26 Implant tumor Day 0 80-100 mm 3 RIPPS Day 8 Tumor Measurement Evaluate Efficacy Day 21 MC38 and CT26 are sensitive to T cell modulation via traditional checkpoint inhibitors and immunostimulatory molecules Distinct TIL profiles compared to other syngeneic models, including prominent TAM/Myeloid population in MC38 RIPPS is performed in the absence of combination with other IO therapy 9
T u m o r v o lu m e (M e a n m m 3 S D ) Screening in Combination with anti-pd-1 in Orthotopic 4T1 Breast Tumor Model 4T1 has a highly immunosuppressive tumor microenvironment Large number of MDSC, limited T, NK cells. Limited response to checkpoint inhibitors including anti-pd-1 monotherapy PD-L1 is induced after treatment with FGFR2 targeted therapy RIPPS is performed in combination with anti-pd-1 therapy in each treated group F P A 1 4 4 S h o w s A d d itiv e E ffe c t in R e d u c in g 4 T 1 T u m o r w ith A n ti-p D -1 5 0 0 C o n tr o l Ig G 1 a n ti-f G F R 2 4 0 0 a n ti-p D -1 a n ti F G F R 2 + a n ti-p D -1 3 0 0 2 0 0 1 0 0 1 0 1 2 1 4 1 6 1 8 2 0 D a y s P o s t In o c u la tio n Orthotopic 4T1 Model Screen Design Implant 4T1 tumor cells 40~50 mm 3 RIPPS a-pd-1 Tumor Measurement Evaluate Efficacy BALB/c mice Day 0 Day 8 Day 9 Day 23 10
T u m o r v o lu m e (M e a n m m 3 S D ) Screening in Combination with Peptide Vaccine in TC-1 Subcutaneous HPV+ Tumor Model Screen Design Subcutaneous TC-1 Model Implantation of E7/PADRE TC-1 tumor cells RIPPS 60~75 mm 3 E7/PADRE Tumor Measurement Evaluate Efficacy C57Bl/6 mice Day 0 Day 11 Day 12 Day 17 Day 28 TC-1 lung epithelial cell line engineered to express HPV-associated antigens E6/E7 E7/PADRE peptide vaccine stimulates an antigen-specific T-cell response Model is sensitive to co-stimulatory & coinhibitory molecules RIPPS performed in combination with two doses of vaccine (E7/PADRE) 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0 K n o w n Im m u n s tim u la to ry M o le c u le in T C -1 M o d e l S a lin e 4-1 B B L V a c V a c + 4-1 B B L 0 0 8 1 6 2 4 3 2 D a y a fte r In o c u la tio n 11
Tumor Growth Volume % The In Vivo Screen of the Immunome Identified Both Immune Activators and Inhibitors CT26 Screen Data 400 300 200 Potential immune stimulator hits CD80 GITRL 41BBL OX40L Potential checkpoint hits CTLA4 100 0 Individual Library Proteins Several targets were chosen for advanced validation CD80 and GITR have advanced to therapeutic campaigns Additional targets are continuing to be evaluated and validated 12
% G r o w th % G r o w th % G r o w th % G r o w th Each Model Screened With the Immunome Library Produced A Broad Spectrum Of Hits CT26 4T1 + anti-pd-1 2 5 0 2 0 0 3.4% hit rate 2 5 0 2 0 0 1.4 % hit rate 1 5 0 1 5 0 1 0 0 1 0 0 5 0 5 0 0 0 MC38 TC-1 + vaccine 2 5 0 2 0 0 1 5 0 1 0 0 5 0 0 2.6 % hit rate 1.0 % hit rate 2 5 0 2 0 0 1 5 0 1 0 0 5 0 0 13
Summary Of RIPPS Screens The immunome library of ~700 immune related proteins was screened in 4 models Inhibitors and stimulators of tumor growth were identified Many hits were common across multiple models Suggests possible broad therapeutic application Magnitude of efficacy correlated with immune profile If high suppressive signature in model (e.g. 4T1) low efficacy If high TIL signature in model high efficacy Unique hits were observed in each model May reflect distinct therapeutic applications for subtype indications Multiple hits have moved into validation and development programs CD80-Fc Anti-GITR agonist Ab 14
FPT155 A Soluble CD80-Fc That Modulates Multiple Signaling Pathways On T Cells
FPT155 is One of Most Potent Tumor Inhibitors Identified in Our In Vivo Screens of More Than 700 Immunome Proteins CD80 FPT155 (CD80-Fc) Activates T Cells Through Three Pathways Co-stimulatory molecule expressed on antigen presenting cells Binds to the T cell activating receptor CD28, the T cell inhibitory receptor CTLA4, and PD-L1 CTLA4 Engagement (activates T cells) CD28 Activation (activates T cells without superagonism) PD-L1 Blockade (prevents checkpoint inhibition) 16
The Murine Surrogate mfpt155 Has Potent Anti-tumor Activity in Multiple Tumor Models 17
FPT155 Induces a Favorable Environment for Anti-Tumor Immune Response CT26 model: tumor IF & flow cytometry after 2 doses mfpt155 increases the ratio between effector T cells and T reg mfpt155 control NKp46 T cell infiltration into tumor mass 1 2 0 * R a tio to T r e g 8 0 4 0 **** 0 C D 4 T e ff C D 8 T CD3 CD8 control mfpt155 (0.3 mg/kg) 18
2016 Five Prime Therapeutics, Inc. All Rights Reserved mfpt155 Treatment Induces Long Term Anti-Tumor Immunity Mice re-challenged with tumors 8 weeks after clearance of initial primary tumor were protected from tumor growth 19
IF N (p g /m l) C e ll C o u n t FPT155 Induces T cell Activation and proliferation in Vitro C y to k in e R e le a s e P r o life r a tio n 2 5 0 0 2 0 0 0 0 2 0 0 0 1 5 0 0 0 1 5 0 0 1 0 0 0 1 0 0 0 0 5 0 0 5 0 0 0 0 C D 2 8 S u p e r a g o n is t A b F P T 1 5 5 O K T 3 F P T 1 5 5 + O K T 3 0 C D 2 8 S u p e r a g o n is t A b F P T 1 5 5 O K T 3 F P T 1 5 5 + O K T 3 Active on purified T cells leading to cytokine release and proliferation Not active as a superagonist because it depends on TCR costimulation Not active in cytokine release assays designed to predict CRS infusion reactions 20
2016 Five Prime Therapeutics, Inc. All Rights Reserved FPT155 Is On Track for IND Filing in Mid 2018 FPT155 ECD-Fc lead has been selected Induces strong anti-tumor responses Induces TIL profiles favorable for anti-tumor immunity Does not display superagonist activity Non-human primate studies are underway Manufacturing activities have been initiated IND filing planned in mid 2018 21
Acknowledgements RIPPS David Bellovin Justin Chou Janine Powers Nebiyu Wondyfraw Tonirose Hidalgo Marina Jaquez Jennifer Stokes David Yang Pharmacology / IHC Barbara Sennino Jackie De La Torre Quinn Walker Marc Jobon Cell based / in vitro Susannah Barbee David Busha Maggie Best Monica Macal Bioinformatics Ernestine Lee Kevin Hestir Molecular Biology Elizabeth Bosch Katrina LeGris Frank Connolly Erika Smith Sheila Lou 22
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