Antibodies • Anti-human KIT mAb (KTN0158, Kolltan Pharmaceuticals) • Anti-mouse KIT mAb (ACK2, Biolegend) • Anti-CTLA-4 (UC10-4F10-11, BioXCell) • Anti-PD-1 (RMP1-14, BioXCell) • Anti-mast cell tryptase (AA1, Dako) Immune Cell Flow Cytometry T-Cell Panel: CD45, CD3, CD4, CD8, CD25, FoxP3. Myeloid Cell Panel: CD45, CD3, CD11b, Ly6G (g-MDSC), Ly6C (m-MDSC). Dosing and Sampling Schedules (Flow Cytometry Pharmacodynamic Studies) Tumors were staged to 150-250 mm 3 , dosed q3d x2, then sampled for flow cytometry at day 7. Dosing Schedules (Efficacy Studies) ACK2: 15 or 3 mg/kg, biweekly x2 (from day 3). Anti-CTLA-4: 5 mg/kg x1 (day 8), 2.5 mg/kg (days 11, 14). Anti-PD-1: 5 mg/kg biweekly x2 (from day 3). Colon26 (cell line) and Pan02 (tumor fragments) dosed from day 3 of implant. CloudmanS91 (cell line) dosed from day 3 after growth to 140-160 mm 3 . CR indicates number of animals exhibiting complete responses (no measurable tumor mass) within a treatment group. • KTN0158 is a potent humanized anti-KIT mAb in clinical development. Tumor-infiltrating mast cells represent a potential target for anti-KIT antibodies within the tumor microenvironment. • The combination of an anti-KIT antibody with immune checkpoint inhibitors showed enhanced anti-tumor activity in the Colon26, Pan02 and CloudmanS91 models. • High pre-treatment m-MDSC counts are associated with reduced survival in melanoma patients treated with checkpoint inhibitors. • Anti-KIT treatment in vivo reduced m-MDSC numbers, which may result in reduced suppression of anti-tumor immunity. • The data support clinical evaluation of KTN0158 in combination with anti-PD-(L)1 and/or anti-CTLA-4 for the treatment of cancer. • KTN0158 is a humanized anti-KIT IgG1 monoclonal antibody that binds to the extracellular domain of KIT, and is being developed as a potential therapy for cancer (Phase 1 study: NCT02642016) and other mast cell-related diseases such as neurofibromatosis type 1. • Expression of KIT in immune cell types, including mast cells, suggests the potential for additional roles of KIT in indirect modulation of tumor progression. • KIT may be involved in modulating the activity of mast cells and MDSC’s in tumors ( Danelli et al, Cancer Immunol Res. 2015; Saleem et al, J Immunol. 2012; Pan et al, Blood 2008). • In melanoma patients, prolonged overall survival is associated with lower numbers of monocytic myeloid-derived supressor cells in peripheral blood prior to treatment with ipilimumab or nivolumab (Kitano et al, Cancer Immunol Res. 2014; Weber et al, Cancer Immunol Res. 2016). • The ability of anti-KIT mAb treatment to relieve immune suppression and enhance anti-tumor activity of immune checkpoint inhibitors was evaluated in a panel of preclinical tumor models. Inhibition of KIT In Vivo Modifies Immune Cell Populations to Improve The Efficacy of Checkpoint Inhibitors in Syngeneic Mouse Tumor Models Anti-KIT Treatment Enhances the Anti-Tumor Activity of Anti-PD-1 in the CloudmanS91 Mouse Melanoma Tumor Model Immune Cell Profiling of Colon26 Tumors Following Dosing with Antibodies Targeting KIT, PD-1 and CTLA-4 Anti-KIT Treatment Enhances the Anti-Tumor Activity of Immune Checkpoint Inhibitors in the Colon26 Tumor Model Anti-KIT Treatment Reduces Monocytic Myeloid-Derived Suppressor Cell Counts In Multiple Mouse Tumor Models Andrew J. Garton, Lori Lopresti-Morrow, Scott Seibel, Theresa LaVallee, Richard Gedrich Kolltan Pharmaceuticals, Inc., New Haven, CT Introduction Results Conclusions 4020 0 20 40 60 0 800 1600 2400 Study Day Tumor Volume (mm 3 ) CTLA-4 CTLA-4 + KIT PD-1 PD-1 + KIT 0 CR 2 CR Control IgG KIT Control IgG CTLA-4 + PD-1 Control IgG CTLA-4 Control IgG PD-1 0 20 40 60 0 800 1600 2400 Study Day Tumor Volume (mm 3 ) 0 20 40 60 0 800 1600 2400 Study Day Tumor Volume (mm 3 ) 0 20 40 60 0 800 1600 2400 Study Day Tumor Volume (mm 3 ) 0 20 40 60 0 800 1600 2400 Study Day Tumor Volume (mm 3 ) 0 20 40 60 0 800 1600 2400 Study Day Tumor Volume (mm 3 ) Dosing Dosing Dosing Dosing Dosing Dosing CD4 T-Cells CD8 T-Cells Treg g-MDSC m-MDSC Control IgG CTLA-4 PD-1 CTLA-4 + PD-1 KIT CTLA-4 + KIT PD-1 + KIT Antibody Treatments Treatment Group Treatment Group 0 10 20 30 40 50 Tumor Cell Counts (% of Live Cells) 0 10 20 30 0 600 1200 1800 Study Day Tumor Volume (mm 3 ) 0 10 20 30 0 600 1200 1800 Study Day Tumor Volume (mm 3 ) 0 10 20 30 0 600 1200 1800 Study Day Tumor Volume (mm 3 ) Dosing Dosing Dosing CTLA-4 CTLA-4 + KIT (3 mg/kg) Control IgG CTLA-4 CTLA-4 CTLA-4 + KIT (15 mg/kg) -14 -12 -10 -8 -6 0 10000 20000 30000 0 2000 4000 6000 log [Ab] (M) Mouse mast cell p-KIT (RLU) Human mast cell p-KIT (RLU) KTN0158 (human KIT) ACK2 (mouse KIT) IC 50 285 pM IC 50 418 pM Mast Cell Staining of Colon26 Tumors Toluidine Blue Mast Cell Tryptase Inhibition of Human and Mouse KIT in SCF-Treated Mast Cells • The potency of KIT inhibition in mouse mast cells by ACK2 was comparable to KTN0158 inhibition of KIT in human mast cells. • The immune cell content of Colon26 tumors included mast cells identified by ex vivo staining of tumor tissue with toluidine blue and an anti-mast cell tryptase antibody. • Anti-CTLA-4 and anti-PD-1 were both efficacious as single agents in the Colon26 model. • Anti-KIT enhanced activity of both anti-CTLA-4 and anti-PD-1, but had no activity as a single agent in the Colon26 model. • Combination of anti-KIT and anti-PD-1, or anti-CTLA-4 and anti-PD-1, yielded additional anti-tumor activity in the CloudmanS91 melanoma model compared to single agent treatments. The Combination of Anti-KIT and Anti-CTLA-4 Exhibits Anti-Tumor Activity in the Pan02 Mouse Pancreatic Tumor Model • The combination of anti-KIT and anti-CTLA-4 treatment yielded anti- tumor activity in the Pan02 pancreatic tumor model. • Anti-CTLA-4 and anti-PD-1 did not exhibit strong anti-tumor activity in the Pan02 model when dosed as single agents or in combination. • Anti-KIT treatment reduced monocytic myeloid-derived suppressor cell (m-MDSC) counts in Colon26 tumors. • Anti-KIT treatment did not further enhance CD8+ T-cell infiltrates induced by anti-CTLA-4 or anti-PD-1 treatments. • m-MDSC numbers were reduced in the spleens of tumor-bearing mice following dosing with an anti-KIT mAb, both in the absence and presence of anti-CTLA-4 or anti-PD-1. Potential Mechanism for Enhanced Efficacy of Immune Checkpoint Inhibitors in Combination with a KIT mAb KIT mAb reduced m-MDSC KIT and SCF Expression in Mouse Tumor Cell Lines Methods Colon26 4T1 Cloudman S91 MC38 mKIT-CHO KIT Protein Expression in Mouse Cell Lines Secretion of SCF by Mouse Cell Lines Colon26 4T1 Cloudman S91 MC38 0 250 500 750 1000 [SCF](pg/ml) Colon26 4T1 CloudmanS91 MC38 Lack of Effect of SCF and KIT Inhibition on Cell Growth In Vitro 0.00 0.25 0.50 0.75 1.00 1.25 Relative Cell Growth (vs. Medium Control) Low Serum - SCF Low Serum + SCF Low Serum + SCF + KIT mAb Growth Medium Growth Medium + KIT mAb • No evidence for KIT-dependent proliferation in mouse tumor cell lines in vitro , regardless of KIT or SCF expression levels Growth Medium Conditioned Medium Anti-KIT Treatment Inhibits KIT Phosphorylation in Mast Cells and Mast Cells are Present in the Tumor Microenvironment Colon26 (Tumor Fragments) Pan02 (Tumor Fragments) Colon26 (Cell line) Antibody Treatments Control IgG CTLA-4 + PD-1 PD-1 CTLA-4 ▲ Control mice (No-tumors) KIT ▼ PD-1 + KIT CTLA-4 + KIT 0 5 10 15 Spleen m-MDSC Counts (% of Live Cells) 0 CR 2 CR 1 CR 2 CR 0 CR 0 CR 0 CR 0 CR 0 CR 1 CR 0 10 20 30 0 250 500 750 1000 Median Tumor Volume (mm 3 ) Control IgG Anti-PD-1 Anti-CTLA-4 Anti-CTLA-4 + Anti-PD-1 Anti-KIT 3 mg/kg Anti-KIT 15 mg/kg Anti-CTLA-4 + Anti-KIT 3 mg/kg Anti-CTLA-4 + Anti-KIT 15 mg/kg Anti-PD-1 + Anti-KIT 15 mg/kg Study Day Dosing