Development of PY159, a Monoclonal Antibody That Repolarizes Tumor

Development of PY159, a monoclonal antibody that repolarizes tumor-associated inhibitory myeloid cells, for the treatment of solid tumors 2 Tower Place, Suite 800, Linda Liang, Chris Chan, Venkataraman Sriram, Erick Lu, Joshua L. Pollack, Mikhail Binnewies, Xiaoyan Du, Aritra Pal, Vladi Juric, South San Francisco, CA 94080 Nadine Jahchan, Evan Greger, Kevin P. Baker, Len Reyno, and Michel Streuli

Introduction TREM1 Expression is Higher in Diverse Cancers, and Inversely PY159m Induces Anti-tumor Immunity Correlates With Patient Survival Dosed Dosed Dosed MethodsDosed The major differentially expressed “pathway families” in To improve the proportion of patients who benefit from checkpoint inhibitor (CPI) therapy 1500 PY159m treated MC38 tumors MC38 mono-therapy • Immune pathways

Strata + trem1median=TREM1hi + trem1median=TREM1low ) • Calcium ion transport additional immune pathways likely need to be targeted. Tumor-associated macrophages TREM1 mRNA expression in 3 Negative correlation between TREM1 1000 • Tissue remodeling colorectal cancer • Metabolism and RNA processing 1.00 ++ expression and patient survival also seen in: (TAMs), myeloid-derived suppressor cells (MDSCs), and tumor-associated neutrophils (TANs) ++++++ ++ ++++++ ++ + 500 +++ + exhibit a spectrum of functional phenotypes ranging from immunosuppressive M2-like +++++ • Breast cancer (mm volume Tumor Control The immune pathway families ++ ++ 6 6 ++++++++ +++++++ PY159m + + • Antigen processing and presentation • IFN-g production + +++ • Pancreatic cancer macrophages or N2-like neutrophils that promote tumor growth to pro-inflammatory M1-like 0.75 ++ +++++ 0 • T Cell Receptor signaling pathway • TNF superfamily cytokine production + +++++++++++++ 9 15 18 21 24 27 ++++ +++++++++ • Response to TNF • Regulation of IL6 production ++ +++ Day post implant • NF KB signaling • Regulation of secretion ++++ +++++++++++++ Low • SCC ++++++ +++ TREM1 macrophages and N1-like neutrophils that promote anti-tumor immunity. Therapies that shift ++++ +++++++++++++++ +++++++ +++++++++++ ++++++ + + + 4 4 ++++++++++++ +++ Isolate tumors ++++++++ the balance of inhibitory myeloid cells towards a more pro-inflammatory phenotype are 0.50 + ↓ + + ++++++ High TREM1 mRNA expression seen in:

TREM1 RNAseq of whole tumors ++ +++ • T cell proliferation • Chemokine activity + +++ ++++ ↓ • Adaptive immune response expected to positively impact anti-tumor immune responses and convert CPI-resistant tumors Survival Overall • Chemokine receptor binding • NSCLC (non-SCC) • Lymphocyte mediated immunity Identify pathways upregulated in • Cell chemotaxis 2 2 High • Cell adhesion TREM1 PY159m-treated vs control tumors • Lymphocyte migration

into CPI-sensitive tumors. probability Survival + + • Erk1 & Erk2 cascade 0.25 p=0.008 • HNSCC p = 0.0081 Log2 TREM1 expression TREM1 Log2 The available preclinical and nonclinical data support PY159 immunotherapy, alone or in • Ovarian 0 0 Figure 6. Mice bearing MC38 tumors (n=10 per group) treated with PY159m and isotype antibodies, respectively, were n=566 • Stomach N T nd combination with a CPI, in cancer patients who are resistant or refractory to CPI therapies, to Healthy normColorectal 0.00 harvested on day 19 after implant (48 hours post 2 treatment). RNA was extracted and sequenced. Differentially tissue cancer tissue 0 50 100 150 200 • Bladder induced pathways associated with PY159m treatment were assessed using the Broad Institute’s Gene Set Enrichment improve both the overall response rates as well as the durability of responses. First in human Time (months)Months n=41 n=282 Analysis (GSEA) tool using the c5 collection of gene sets from MSigDB. Significantly upregulated pathways (FDR < 0.1) clinical testing will commence in 2020. were visualized using the Enrichment Map module in Cytoscape using default parameters. Immune families from the Figure 3. RNAseq data from the TCGA colon cohort were downloaded from the Broad Institute (left panel) and RSEM resulting network map are shown with cluster-specific pathways highlighted. values for TREM1 mRNA from tumor and adjacent normal samples were converted to log 2 counts per million. Results TREM1 Background were plotted in R. Normalized TREM1 expression profiles were downloaded from GEO (GSE35982) and divided into two cohorts based on median level of TREM1 (right panel). Kaplan-Meier survival curves were plotted for each cohort PY159 Produces a Pro-inflammatory Response and the associated log-rank test was carried using the survival and survminer packages in R. Breast cancer, pancreatic TREM1: Triggering receptor expressed on myeloid cells 1 cancer and squamous cell carcinoma (SCC) datasets were subjected to similar analysis, also revealing negative correlation between TREM1 mRNA and patient survival (data not shown). Expression: Macrophages, monocyte subsets, neutrophils Upregulated on tumor-associated macrophages PY159 Induces a Highly Selective Set of Anti-tumor Chemokines (TAMs), tumor-associated neutrophils (TANs), and Cell Surface Receptors and myeloid-derived suppressor cells (MDSCs) Function: Activating receptor implicated in innate immunity 20000 Isotype PY159 -/- 40 Genetics: Trem1 mice have a reduced susceptibility to 3540 15000 35 colitis, reduced neutrophil infiltration following 30 10000 2530 Leishmania major infection, increased morbidity 25 5000

20 HLA-DR (gMFI) 20 from Influenza infection, and reduced 15 0 15 0.0 0.5 1.0 1.5 B 15 Antibody concentration (nM) susceptibility to inflammation-induced cancer CD8 CD4 10 NK Plasma Isotype Treg Fold change PY159/Isotype 10 6000 PY159

Ligands: Peptidoglycan recognition protein 1 (PGLYRP1), Fold change (PY159 / Isotype)

TAM pDC Fold change

(PY159 / Isotype) 5 others Monocyte Figure 7. RNA-based fold changes induced by PY159m treatment of MC38 tumors were compared to protein fold (mMDSC) 5 4000 0 changes from PY159-treated RBC-lysed whole blood. Cytokine levels were assessed using the O-link multiplex platform. TREM1 mRNA 2000 0 γ CD40 (gMFI) γ IL-2 IFN- CCL4 CCL2 CCL3 CXCL8 IL-2CCL17 0 CXCL10 IFN- CCL4 CCL2 CCL3 0.0 0.5 1.0 1.5 CXCL8 CCL17 PY159, PIONYR’s Anti-TREM1 mAb, Reprograms CXCL10 Antibody concentration (nM) PY159m has Significant Anti-tumor Activity Dosed Dosed Dosed Suppressive Myeloid Cells 2500 Figure 4. PY159-induced cytokines and activation receptors in human blood. RBC-lysed human whole blood was treated Isotype (20 mpk) Dosed Dosed Dosed Dosed Dosed Dosed Dosed Dosed Dosed Dosed Dosed Dosed Control 1500 2500 600 for 24-hours with PY159 (1 µg/ml). Supernatants were harvested for cytokine and chemokine analysis using MSD and CT26 combo-therapy ControlAnti PD +-1 anti-PD-1 (5 mpk) MC38 mono-therapy ) 2000 Panc-02 mono-therapy

2000 3 )

) Afuc PI-9067 + Control

) PY159m (15 mpk) 3 3 cells were stained with a panel of leukocyte lineage markers and with anti-CD40 and anti-HLA-DR. PY159-induced 3 TREM1 TREM1 TREM1 1000 400 Afuc-PI-9067PY159m (15 mpk+ anti-PD-1) PY159 1500 Reprogramming increase in these activation markers was notable on monocytes. Cytokines shown above were detected >10 pg/ml and + anti PD-1 (5 mpk) Anti-tumor immune cell 1500 towards anti-tumor + + TREM1+ Secretion of pro- recruitment and activation upregulated across experiments from multiple donors. Cytokines that did not meet criteria: IL-10, IL-12p70, IL-13, IL-4, 1000

TREM1 PY159- +/- SD (mm3) 200 myeloid cells anti-tumor inflammatory cytokines • T cells 500 • 10 mice per group pro-tumor mediated (mm volume Tumor Tumor GM-CSF, IL-1b, IL-6, TNF-a, CCL11, CCL26, CCL13, CCL22, IL-12/23p40, IL-15, IL-16, IL-17A. Mean Tumor volume Tumor volume (mm volume Tumor myeloid 500 • Dosing initiated when tumor and induction of • NK cells (mm volume Tumor myeloid TREM1 cross- destruction 1000 volumes reached ~100 mm3 suppressor linking stimulatory co-stimulatory molecules • DC 1 0 cells 0 0 10 15 20 25 30 • MC38: murine colon carcinoma cells • Macrophages 9 15 18 21 24 27 9 12 18 21 24 27 30 Day post implant Day after implant • CT26: murine colon carcinoma Day post implant CR: 20% (range 20-40%) (mm volume Tumor 500 CR: 20% (PY159m + anti-PD-1) • Panc02: murine pancreatic ductal PY159 Induces Signaling Downstream of TREM1 and DAP12 adenocarcinoma ) ) 25002500 )

3 2500 Figure 1. Model of PY159’s mechanism-of-action. Cross-linking of cell surface TREM1 on tumor associated myeloid cell 3 ReimplantReimplantReimplant CT26 CT26CT26 (N=6) 0 3 Figure 8. Eight to ten week old female6 C57BL/610 12 (Jackson14 16 20 Laboratories)22 24 or BALB/c populations by PY159 causes downstream signaling that can induce secretion of a specific set of proinflammatory Neutrophils Monocytes T cells (negative control) 200020002000 HistoricalHistoricalNaive CT26CT26 CT26 (N=6) Human whole (Taconic) mice were implanted with 8x10e5 DayMC38 post (C57BL/6;implant top left), 1x10e6 CT26.WT cytokines and chemokines as well as increase surface expression of HLA-DR and CD40. These immune mediators can blood buffy coats 1500 15001500 (BALB/c; top middle) or 2x10e6 Panc02 (C57BL/6; top right) on right ventral flank. Vertical recruit immune cells including T cells, NK cells, DCs, and macrophages and promote anti-tumor immune cell activation. 1000 RBC lysis 10001000 red lines indicate days when indicated antibodies in the legend were administered ERK

- 500 6 intraperitoneally. BALB/c mice with complete CT26 tumor regression post anti-PY159m +

5 x 10 leukocytes/well (mm volume Tumor 500500 0 0 10 20 30 40 anti-PD-1 treatment were re-challenged with CT26 tumor cells (left panel). Tumor volume (mm volume Tumor + (mm volume Tumor TREM1 is Expressed on Suppressive Myeloid Cells 0 Phospho 0 Day post implant PY159 or 0 0 1010 2020 3030 4040 From Diverse Tumor Types Isotype control DayDay post post implant implant +

1 0.8 0.6 0.4 0.2 PMA/ionomycin High expression PY159 Safety and PK Assessment Summary colon kidney lung ovarian stomach cancer adenocarcinoma cancer adenocarcinoma adenocarcinoma BLAD BREAST CRC CRC CRC CRC CRC CRC CRC CRC CRC CRC CRC CRC CRC CRC CRC CRC KID KID KID KID KID KID KID KID KID KID KID LUAD LUAD LUAD LUAD LUAD LUAD LUSC LUSC LUSC LUSC LUSC LUSC LUSC OV OV OV OV OV OV OV OV OV OV OV OV OV OV OV OV OV OV OV OV PRAD STAD STAD STAD STAD STAD STAD STAD STAD STAD STAD Analysis Summary findings CD3+CD3 cells+ cells 0, 2, 10, 30, 60, and 90 min treatment Rodent PK • PY159m shows dose dependent PK STAT3 Conv.Conv. Monocytes monocytes - Rodent tox • PY159m is well tolerated in mice up to 4 weekly doses of 100 mg/kg TAMsTAMs Phospho-flow Phospho pERK- and pSTAT3- • Terminal half-life (T ) range of 9-11 days between 1 and 10 mg/kg specific mAbs NHP PK 1/2 NeutrophilsNeutrophils • Volume of distribution (Vd) of 70-80 mL/kg, suggesting distribution beyond the vasculature and into tissues No expression detected • Well tolerated up to the top dose tested of 50 mg/kg Single dose NHP pilot • Transient reduction in neutrophils within normal range Figure 2. The indicated human tumors were dissociated and TREM1 expression was assessed on CD45+ leukocyte Figure 5. PY159 cross-linking induces signaling pathways downstream of TREM1 and DAP12. RBC-lysed human • No discernable changes in cytokine and chemokine levels were observed for all groups subsets by flow cytometry. The level of TREM1 expression was measured by mean fluorescence intensity (gMFI) whole blood was stimulated with 5 µg/ml of PY159 or hIgG1 isotype control, or PMA and ionomycin as a positive adjusted to incorporate isotype background levels. These adjusted gMFI values were then normalized across leukocyte • Preliminary findings: PY159 is well tolerated up to 50 mg/kg for 4 weekly doses control for the indicated times. Cells were fixed, permeabilized and stained with a panel of leukocyte lineage Repeat dose NHP pilot • Transient reduction in neutrophils within normal range subsets to highlight those subsets which have high or low TREM1 expression across each indication. markers and with anti-phospho ERK and anti-phospho STAT3 antibodies. PY159 induces phospho- signaling in • No discernable changes in cytokine and chemokine levels were observed for all groups 1 = high expression, 0 = no expression detected. TREM1+ neutrophil and monocyte populations.