BackgroundThe tumor microenvironment contains stromal cells, including endothelial cells and fibroblasts, that aid tumor growth and impair immune cell function. Many solid tumors remain difficult to cure because of tumor-promoting stromal cells, but current therapies targeting tumor stromal cells are constrained by modest efficacy and toxicities. TEM8 is a surface antigen selectively upregulated on tumor and tumor stromal cells, endothelial cells and fibroblasts that may be targeted with specific natural killer (NK) cell engagement.MethodsA Tri-specific Killer Engager (TriKE) against TEM8—‘cam1615TEM8’—was generated using a mammalian expression system. Its function on NK cells was assessed by evaluation of degranulation, inflammatory cytokine production, and killing against tumor and stroma cell lines in standard co-culture and spheroid assays. cam1615TEM8-mediated proliferation and STAT5 phosphorylation in NK cells was tested and compared with T cells by flow cytometry. NK cell proliferation, tumor infiltration, and tumor and tumor-endothelium killing by cam1615TEM8 and interleukin-15 (IL-15) were assessed in NOD scid gamma (NSG) mice.Resultscam1615TEM8 selectively stimulates NK cell degranulation and inflammatory cytokine production against TEM8-expressing tumor and stromal cell lines. The increased activation translated to superior NK cell killing of TEM8-expressing tumor spheroids. cam1615TEM8 selectively stimulated NK cell but not T cell proliferation in vitro and enhanced NK cell proliferation, survival, and tumor infiltration in vivo. Finally, cam1615TEM8 stimulated NK cell killing of tumor and tumor endothelial cells in vivo.ConclusionsOur findings indicate that the cam1615TEM8 TriKE is a novel anti-tumor, anti-stroma, and anti-angiogenic cancer therapy for patients with solid tumors. This multifunctional molecule works by selectively targeting and activating NK cells by costimulation with IL-15, and then targeting that activity to TEM8+ tumor cells and TEM8+ tumor stroma.

1202 Figure 1 Structure of a PSMA TriKE molecule. A PSMA TriKE molecule consists of a humanized anti-CD16 single domain antibody (sdAb) derived from camelid, an anti-PSMA single chain variable fragment (scFv) and an IL-15 molecule. IL-15, interleukin-15; mCRPC, metastatic castration-resistant prostate cancer; PSMA, prostatespecific membrane antigen; TriKE, tri-specific killer engager Abstract 1202 Figure 2 PSMA TriKE relieves suppressed NK cell cytotoxicity induced by hypoxia. NK cells were incubated with 0.06nM IL-15 or 3nM PSMA TriKE (at equifunctional concentrations) in normoxia (20% oxygen) or hypoxia (1% oxygen) for 7 days then harvested and plated at effectors to targets (E:T) ratio of 2:1 for 48 hours in normoxia to assess NK cell cytotoxicity against C4-2 using IncuCyte live cell imaging. Representative data of N=5.