Siglec-15 (S15), a member of sialic acid-binding immunoglobulin- type lectins, is a highly conserved Type I cell surface protein, which was previously reported to play a role in osteoclast differentiation and bone remodeling 1,2 . Here we describe S15 as a novel co-inhibitory ligand expressed on tumors and myeloid cells that suppresses T cell function and promotes cancer growth. Blocking S15 by antibody enhances anti-tumor immunity in preclinical models. 1 Macauley MS et al. Nat Rev Immunol. 2014 Oct;14(10):653-66. 2 Hiruma Y et al. Bone. 2013 Mar;53(1):87-93. Targeting Siglec-15 with NC318, a Novel Therapeutic Antibody to Enhance Anti-Tumor Immunity Linda N. Liu 1 , Jun Wang 2 , Jingwei Sun 2 , Dallas Flies 1 , Chang Song 1 , Melissa Zarr 1 , Kristina Archer 1 , Alison McGuire 1 , Tom O’Neil 1 , Karla Maloveste 1 , Xinxin Nie 2 , Agedi Boto 2,3 , Ron Copeland 1 , Sathya Janardhanan 1 , Tete Obot 1 , Jim Bingham 1 , Kevin N. Heller 1 , Sol Langermann 1 , Lieping Chen 2 1 NextCure Inc., Beltsville, Maryland, USA; 2 Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, USA; 3 Department of Pathology, Yale School of Medicine, New Haven, Connecticut, USA BACKGROUND SIGLEC-15 KNOCK OUT MICE Figure 1: Myeloid-Lineage Cell Expression of S15 Inhibits Antigen-Specific CD8 + OT-I T Cell Responses in Vivo A: Brief outline of the study. B: The kinetics of OT-I T cell expansion and contraction in the blood of WT, S15 KO and LysM-Cre S15 KO mice at indicated time points after OVA/poly (I:C) immunization (n=4/group). Analysis shown is OT-I T cells as a percentage of total CD8 + T cells. C: OT-I T cell proliferation in mouse spleens 5 days after transplantation. Analysis shown is percentage of OT-I of total CD8 + T cells. A B C Figure 2: S15 Suppresses Anti-Tumor Immunity and Promotes GL261 Tumor Growth A) GL261luc tumor growth in wild type or S15 KO mice post intracranial injection. B/C) Flow cytometry analysis of tumor-infiltrating immune cells on Day 14 after GL261 tumor inoculation. CD8 + T cells, CD4 + T cells, CD11b + CD45 high macrophages (MØ), CD11b + CD45low microglia, and CD11c + dendritic cells (DC) in total brain mononuclear cells were quantified. B) Brain mononuclear cells were further re-stimulated with irradiated GL261-luc cells for 5 days. C) Total number of IFN-γ-producing CD8 + T cells and CD4 + T cells was determined based on live cell counting and intracellular cytokine staining. *P < 0.05. Figure 3: S15 Suppresses Anti-Tumor Immunity and Promote B16.GMCSF Tumor Growth A & B) Subcutaneous B16.GMCSF tumor growth in S15 KO or C57Bl/6 wild type mice. Tumor incidence and growth of individual mice (A) as well as percent survival (B). Percentage of immune subsets (C) and T cells with effector phenotype (CD44hiCD62Llo) (D) among total cells in the tumors from wild type or S15 KO mice. E) The % of IFN-γ and TNF- α producing T -cells were shown based on intracellular staining of cells after ex vivo re- stimulation with PMA and ionomycin. SIGLEC-15 SUPRESSES T CELL IN VITRO Figure 4: Siglec-15 Inhibits Antigen-Specific T Cell Responses In Vitro A: Peritoneal macrophages from WT or S15 KO mice were harvested from C57Bl/6 wild type or S15 KO mice peritoneal cavities. B: 293TKbOVA cells were transfected with S15 or control transfection with empty vector. Irradiated 293TKbOVA-S15+ or 293T-KbOVA- control cells were co-cultured with purified OT-I T cells at 1:10 ratio. 3Hthymidine was added to the co-culture two days later. The following day OT-I T cell proliferation was measured by incorporation of 3H-thymidine. C: Supernatants from the cultured cells was harvested prior to addition of 3H-thymidine and assessed for levels of IFN-γ and TNF-α by ELISA. Figure 5: Anti Siglec-15 (S15) Clone 5G12 Reverses S15 Fc Fusion Protein- mediated Suppression on Human T cells. Human PBMCs from healthy donor were labeled with CFSE and added to anti CD3 coated 96-well plate plus S15 Fc fusion protein and indicated S15 mAbs. Three days later, the cells were stained with anti CD4 and anti CD8 followed by FACS analysis of CFSE low cell population. A) Human CD8 T cell proliferation; B) Human CD4 T cell proliferation EFFECTS of 5G12 IN VIVO ➢ S15 immunosuppressive properties in the TME make it a rational target for immunotherapy. ➢ NC318 is a high affinity humanized IgG 1 mAb specific for S15 developed to reverse tumor immune suppression and promote an effective anti-tumor immune response. ➢ NextCure has completed IND-enabling studies and initiated evaluations of NC318 in patients with advanced malignancies: “A Safety and Tolerability Study of NC318 in Subjects With Advanced or Metastatic Solid Tumor” https://clinicaltrials.gov/ct2/show/NCT03665285 A B C ANTIBODY DEVELOPMENT Siglec-15 KO Human Siglec-15 Fc fusion protein immunization Confirmed high serum titer via ELISA Screen clones binding to cells expressing human or mouse Siglec-15 Hybridoma generation Confirm positive clones binding to cells expressing human or mouse Siglec-15 Purified mAbs from positive clones mAb characterization: affinity, binning Test in vitro human PBMC stimulation assay Select top candidate Humanization No CD3 CD3 alone Ctrl Fc S15.Fc 1B2 1C3 1C12 1H3 3H10 5G12 6F8 8C8 8H8 10G9 0 20 40 60 80 % of Divided CD8 + T cells CD3 + 5 g/m L S15.Fc + 12 g/m L m Ab CD3 * No CD3 CD3 alone Ctrl Fc S15.Fc 1B2 1C3 1C12 1H3 3H10 5G12 6F8 8C8 8H8 10G9 0 20 40 60 80 % of Divided CD4 + T cells CD3 + 5 g/m L S15.Fc + 12 g/m L m Ab CD3 * A B Clone K D (nM) (From 3 runs) 1H3 0.11 ± 0.02 5G12 0.30 ± 0.04 6F8 0.44 ± 0.08 10G9 0.55 ± 0.08 1C3 0.58 ± 0.13 3H10 0.60 ± 0.14 8C8 0.89 ± 0.25 8H8 1.30 ± 0.33 1C12 4.01 ± 1.05 1B2 4.33 ± 0.75 Table 1: Average kinetics values Table 2:Summary of Parent 5G12 and NC318 Affinity Binding to Human Siglec-15 K D (nM) R 2 K on (1E+5/Ms) K off (1E-4/s) Parent 5G12 0.37 0.994 3.18 1.19 NC318 0.35 0.999 3.22 1.12 Figure 6: Binding of Anti-S15 Antibodies to Cells Expressing Mouse or Human S15 (A) 293T.hS15 cells (NC318 EC 50 = 2.42 nM) and (B) 293T.mS15 cells (5G12 parent EC 50 = 1.26 nM). A B 0.0001 0.001 0.01 0.1 1 10 100 1000 100 1000 10000 100000 293T-m S15 [A ntibody] (nM ) M FI (A ntibody binding signal) NC318 5G 12 C o n tro l m Ab 0.0001 0.001 0.01 0.1 1 10 100 1000 100 1000 10000 100000 293T-hS15 [A ntibody] (nM ) M FI (A ntibody binding signal) EFFECTS of NC318 IN VITRO CONCLUSION -1 0 1 2 0 500 1000 1500 2000 2500 [NC318] Log g /m L IL-2 (pg/mL) EC 30 = 7 g /m L Donor#1 NC318 Control -1 0 1 2 0 500 1000 1500 2000 2500 3000 [NC318] Log g /m L IL-2 (pg/mL) EC 30 = 15 g /m L Donor#2 NC318 Control -1 0 1 2 0 1000 2000 3000 4000 5000 [NC318] Log g /m L IL-2 (pg/mL) EC 30 = 18 g /m L Donor#3 NC318 Control -1 0 1 2 3000 4000 5000 6000 7000 [NC318] Log g /m L IL-2 (pg/mL) EC 30 = 7 g /m L Donor#4 NC318 Control -1 0 1 2 0 2000 4000 6000 8000 [NC318] Log g /m L IL-2 (pg/mL) EC 30 = 26 g /m L Donor#5 NC318 Control Figure 10: NC318 Dose Dependently Stimulates Production of IL2 in Co- Stimulated PBMC Cultures Human PBMCs from healthy donors were added to anti-CD3 coated 96-well plate plus SEB together with serially diluted NC318 or isotype control mAb. Supernatant was collected three days later for IL-2 analysis. Figure 7: 5G12 Reduces MC38.mS15 Tumor Lung Metastatic Nodules. A: Mice were treated with 5G12 on day 2 at 20 mg/kg, Q4D; B: Mice were treated with 5G12 on Day 3 at 3, 10 or 30 mg/kg, Q7D. **p<0.01 0 10 20 30 0 500 1000 1500 Days post tumor inoculation Tumor Volume (mm 3 ) Control 5G12 a n ti P D -1 Combo * ** **** **** ** **** **** *p<0.05 **p<0.01 ****p<0.0001 A B C Naive Untreated 5G12 PD1 Combo 0 2 4 6 CD8 + IFN + T Cells % of CD8 + C e lls n=3 n=3 n=2 n=2 n=6 Naive Untreated 5G12 PD1 Combo 0.0 0.5 1.0 1.5 2.0 CD8 + TNF + T Cells % of CD8 + C e lls n=3 n=3 n=2 n=2 n=6 0 20 40 60 80 100 120 0 20 40 60 80 100 Days post tumor inoculation Survival (%) Control 5G12 anti PD1 Combo Rechallenge Day 65 Day 100 IL-17 IP-10 MCP-1 0 500 1000 1500 2000 3000 4000 Control (N=10) 5G12 (N=10) PD1 (N=10) 5G12 + PD1 (N=10) Naive (N=8) pg/mL * ** *p<0.05 **p<0.01 ***p<0.001 ** ** * *** vs. Control treated ** * D Figure 8: 5G12 Monotherapy or in Combination with Anti-PD1 In CT26/S15 + BMDM Tumor Model. A: Tumor volume; B: Kaplan-Meier survival plot; C: CT26-specific CD8 + IFN-γ + and CD8 + TNF-α + T cells in mouse spleen collected on Day 107; D: Serum collected on Day 30 and analyzed for 20 mouse cytokines (mouse 20-plex Luminex Kit from ThermoFisher). 2 6 0 2 4 6 8 IL-6 ng/mL Naive LPS+PBS LPS+5G12 Hours 2 6 0.0 0.5 1.0 1.5 TNF- ng/mL Naive LPS+PBS LPS+5G12 Hours 2 6 0 100 200 300 400 500 GM-CSF pg/mL Naive LPS+PBS LPS+5G12 Hours 2 6 0 50 100 150 200 IL-1 pg/mL Naive LPS+PBS LPS+5G12 Hours 2 6 0 200 400 600 800 IL-18 pg/mL Naive LPS+PBS LPS+5G12 Hours Figure 9: 5G12 Significantly Increases LPS-Mediated Immune Activation in Mice. Mice were first injected with PBS or 5G12 (10 mg/kg) and challenged with LPS 1h later. Serum was collected 2h, 6h later and analyzed for pro-inflammatory cytokines.