RnDSy-lu-2945 VSIG8 is a Co-inhibitory and an Molecule for Human T Cells Jinghua Wang, Brian Manick, Mark Renelt, Louis Hansen, Guoping Wu, and Vassilios Kalabokis | R&D Systems, 614 McKinley Place NE, Minneapolis, MN 55413

Abstract Results Cell surface-localized and secreted immunoglobulin superfamily IL-6 IFN-γ IGFBP3 IP-10 IL-2 2.5 (IgSF) play central roles in regulating adaptive and CD8 0.13 CD3 CD3 0.12 CD4 2.0 innate immune responses, and are the primary targets for the ) ) CD4 0.11 CD8 development of new immunotherapeutics. In this work, we 450 450 1.5 0.10 IgG1-Fc provide biologic and functional insight on VSIG8, one member of 0.09

1.0 (OD IL-2 this important class of proteins. VSIG8 inhibits the production of IFN- γ (OD 0.08 0.07 0.5 cytokines (IL-2, IFN-g, IL-17, IL-6, and IL-19), chemokines (MCP-1, 0.06

MCP-3, and IP-10), and other proteins (IGFBP3 and RBP4) on IL-19 MCP-1 MCP-3 RBP4 IL-17 10-2 10-1 100 101 10-2 10-1 100 101 anti-CD3 activated human CD3+ T cells. Furthermore, VSIG8 IL-6 IFN-γ IGFBP3 IP-10 IL-2 VSIG8-Fc (µg/mL) VSIG8-Fc (µg/mL) significantly reduces the production of IFN-g and IL-2 from both Figure 2. VSIG8 inhibits anti-CD3-induced IL-2 and IFN-g production by human + + + + + + + CD3 , CD4 , or CD8 T cells in a dose-dependent manner. Human CD3 , CD4 , CD4 and CD8 T cells in the presence of or CD8+ T cells were isolated from PBMCs using the MagCellect™ Human CD3+, signaling. In addition, VSIG8 markedly suppresses anti-CD3- VSIG8-Fc CD4+, or CD8+ T Cell Isolation Kits (Catalog # MAGH101, # MAGH102, or induced human T cell proliferation and profoundly decreases the # MAGH112, respectively). Human CD3+, CD4+, or CD8+ T cells were then differentiation of naïve CD4+ T cells into Th1 cells. Thus, we have incubated with an immobilized Mouse Anti-Human CD3 Monoclonal (1 μg/mL) and the indicated concentrations of Recombinant Human VSIG8-Fc or identified VSIG8 as a new immune checkpoint molecule that is g Recombinant Human IgG1-Fc for 24 hours. The levels of IL-2 and IFN- in the cell able to inhibit human T cell activation. This novel human T cell IL-19 MCP-1 MCP-3 RBP4 IL-17 culture supernatants were measured using the Human IL-2 or IFN-g Quantikine® Figure 1. VSIG8 inhibits the production of cytokines (IL-2, IL-17, IL-6, IL-19, and ELISA Kits (Catalog # D2050 or # DIF50, respectively). IgG -Fc controls did not co-inhibitory ligand may be a unique target for developing new 1 IFN-γ), chemokines (MCP-1, MCP-3, and IP-10), and other proteins (IGFBP-3 and alter anti-CD3 induced IL-2 or IFN-g secretion by CD3+, CD4+, or CD8+ T cells immunotherapy strategies for the treatment of human , RBP4) by anti-CD3-activated human CD3+ T cells. Human CD3+ T cells were (data not shown). autoimmune disorders, and infectious diseases. isolated from PBMCs using the MagCellect™ Human CD3+ T Cell Isolation Kit IgG1-Fc VSIG8-Fc (Catalog # MAGH101). The isolated cells were then treated with a combination 4 4 IFN-γ 10 10 of plate-bound Mouse Anti-Human CD3 Monoclonal Antibody (1 μg/mL; 23.8% 15.1% Catalog # MAB100) and either plate-bound Recombinant Human VSIG8-Fc Introduction 103 103 (10 μg/mL; Catalog # 9200-VS) or Recombinant Human IgG -Fc (10 μg/mL; VSIG8 (V­-set and immunoglobulin domain containing 8), also 1 Catalog # 110-HG) for 24 hours. Cytokine levels in the supernatants were 102 102 known as C1orf204, is an approximately 45 kDa type I measured using the Proteome Profiler™ Human XL Cytokine Array Kit

(Catalog # ARY022B). 1 1 transmembrane belonging to the immunoglobulin 10 10 superfamily (IgSF). Mature human VSIG8 consists of a 242 76.2% 84.9% A 2400 0 0 amino acid (aa) extracellular domain (ECD) containing two V-type­ 100 101 102 103 104 100 101 102 103 104 2200 CD4

Ig-­like domains, a 21 aa transmembrane domain, and a 130 aa IgG1-Fc + cytoplasmic domain. Within the ECD, human VSIG8 shares 88% 2000 VSIG8-Fc Figure 4. VSIG8 inhibits the differentiation of naïve CD4 cells to Th1 cells. + ™ and 89% aa identity with mouse and rat VSIG8, respectively. Human naïve CD4 T cells were isolated from PBMCs using the MagCellect 1800 Human Naive CD4+ T Cell Isolation Kit (Catalog # MAGH115). To induce Th1 cell Alternative splicing generates a long isoform of human VSIG8 (RFI) Proliferation differentiation, naïve CD4+ cells were treated for 5 days with immobilized Mouse 1600 with a substitution in the cytoplasmic juxtamembrane region Anti-human CD3 Monoclonal Antibody (1 µg/mL) in 5% FBS-RPMI1640 medium 100 101 and a 124 aa extension at the C-terminus.­ VSIG8 was identified µg/mL supplemented with Recombinant Human IL-2 (10 ng/mL; Catalog # 202-IL) and 1,2 B 60 60 Recombinant Human IL-12 (10 ng/mL; Catalog # 219-IL). To determine the effect IgG -Fc VSIG8-Fc + by proteomic analysis of human hair shafts, and was found to 1 of VSIG8 on Th1 cell differentiation, naïve CD4 cells were treated with be expressed in the hair follicle and shaft, and superficial layers 50 50 immobilized Recombinant Human VSIG8-Fc (10 µg/mL), or Recombinant Human 3 40 85% 40 63% IgG -Fc (10 µg/mL). On day 5, the cells were fixed, permeabilized, and stained of the nail matrix and oral epithelium. A published US patent 1 30 30 using an APC-conjugated Mouse Anti-Human IFN-g Monoclonal Antibody (Catalog

(WO2016090347 A1) has reported that VSIG8 is a receptor for Counts Counts # IC285A) and a Fluorescein-conjugated Mouse Anti-Human CD4 Monoclonal 20 20 VISTA/-H5 and can mediate the suppressive effects of VISTA/ Antibody (Catalog # FAB3791F). Quadrants were set based on staining with the M1 M1 B7-H5 on T cell immunity. Furthermore, VSIG8 has been 10 10 appropriate isotype controls. 0 0 reported to be the counterpart of CXAR, serving the analogous 100 101 102 103 104 100 101 102 103 104 function of maintaining tight junctions in stratified epithelia CFSE + Summary through homophilic trans-dimerization.4 However, the role of Figure 3. VSIG8 inhibits anti-CD3 induced human CD3 T cell proliferation in a dose-dependent manner. (A) Human T cells were incubated with an immobilized • VSIG8 inhibits cytokine and chemokine production by VSIG8 as a co-inhibitory ligand capable of modulating T cell Mouse Anti-Human CD3 Monoclonal Antibody (1 μg/mL) and the indicated immunity, has not been previously described. concentrations of Recombinant Human VSIG8-Fc or Recombinant Human human T cells. IgG -Fc for 3 days. Cell proliferation was assessed by a fluorometric assay using 1 • VSIG8 suppresses human T cell proliferation. the redox-sensitive dye, Alamar Blue (Resazurin). IgG1-Fc controls did not alter In this study, VSIG8-Fc fusion proteins were produced by cloning anti-CD3-induced cell proliferation of CD3+ T cells. (B) CFSE-labeled T cells were + the extracellular domain of human VSIG8 (aa 1—263) fused to treated with a combination of plate-bound Mouse Anti-Human CD3 Monoclonal • VSIG8 decreases the differentiation of naïve CD4 T cells to the Fc region of human IgG in mammalian expression vectors. Antibody (1 μg/mL) and either plate-bound Recombinant Human VSIG8-Fc Th1 cells. 1 (10 μg/mL) or Recombinant Human IgG -Fc (10 μg/mL) for 5 days. T cells were This VSIG8-Fc fusion protein showed negative regulation of 1 analyzed by flow cytometry. In conclusion, VSIG8 is a novel negative regulator of T cell human T cells in various in vitro experimental systems, responses, with potential roles in the modulation of immune suggesting that VSIG8 is a co-inhibitory ligand involved in References responses. regulating T cell-mediated immunity. As an IgSF protein with 1. Rice, R.H. et al. (2010) J. Proteome Res. 9:6752. robust T cell inhibitory activity, VSIG8 represents a novel B7-like ligand that exerts negative immune modulation via interaction 2. Lee, Y.J. et al. (2006) Mol. Cell. Proteomics 5:789. Acknowledgments with a receptor expressed on activated T cells, thereby defining it We thank personnel of the Molecular Biology, Cell Culture, Protein as a novel immune checkpoint molecule. 3. Rice, R.H. et al. (2011) J. Invest. Dermatol. 131:1936. Development, and Antibody Development Departments for their 4. Eng-Hui Y. et al. (2014) J. Mol Biol. 426:945. support and production of the recombinant proteins and .