Abstract 2878: Mediating NK cell function against solid tumors, via targeting of tumor stroma, using a TEM8-targeting Tri-specific Killer Engager (TriKE®)
Authors: Felices Martin , Kaminski Michael , Hinderlie Peter , Hopps Rachel , Bendzick Laura , Geller Melissa , Miller Jeffrey S. 
Published: June 2022

Abstract
Immunotherapies for solid tumors need to overcome barriers within the solid tumor microenvironment (TME), including stromal cells, cancer-associated fibroblasts, endothelial cells, pericytes, and immune cells. These can comprise a significant portion of the tumor mass in many common carcinomas and contribute to immune cell dysfunction within the tumor. Stromal cells support cancer cells through several mechanisms and can promote metastasis. Tumor vasculature supplies cancer cells with nutrients and oxygen necessary for tumor growth and dissemination. Thus, targeting these components of cancer can enhance tumor rejection directly and increase sensitization of surviving tumors to immune infiltration and chemotherapeutic treatments through weakening of the tumor architecture. While there are current antiangiogenic therapies being tested clinically, they have associated toxicities in normal angiogenic processes, limiting clinical efficacy. Tumor Endothelial Marker 8 (TEM8, encoded by the ANTXR1 gene) is a highly conserved integrin-like adhesion molecule that was discovered on the endothelium of colorectal cancer, and subsequent studies identified its expression on multiple stromal cells—endothelial cells, fibroblasts, and pericytes—in the tumor microenvironment of diverse human cancer types. Little to no expression has been demonstrated in normal human stroma, indicating that this would be a good target for immunotherapy. Using a mammalian expression system, we designed and expressed a TEM8 targeting TriKE (cam1615TEM8) consisting of a humanized anti-CD16 single domain antibody, a wild-type IL-15 moiety, and an anti-TEM8 scFv. cam1615TEM8 induces NK cell degranulation and cytokine production against TEM8+ tumor and stromal cell lines (endothelial cells and fibroblasts). Using TEM8- cancer lines and a TEM8-CRISPR knockout version of A549 lung cancer lines, we show that cam1615TEM8 only activates NK cells in the presence of tumor antigen. cam1615TEM8 also preferentially stimulates TEM8+ tumor spheroid killing. Moreover, the camelid anti-CD16 nanobody selectively enhances signaling by the IL-15 moiety to NK cells, specifically promoting NK cell survival and proliferation in vitro and in vivo. In an in vivo xenogeneic mouse model, containing human NK cells and tumor, cam1615TEM8-treated mice had enhanced NK cell tumor infiltration, significantly decreased tumor growth, and enhanced survival when compared to IL-15 treated mice. Since TEM8 is highly conserved, and the scFv within the TriKE is reactive to mouse TEM8, we were able to show reduced tumor endothelial density in the tumors of mice treated with cam1615TEM8. Given the findings of this study cam1615TEM8 could be leveraged in several clinical settings by inducing NK cell specific targeting of the tumor itself and/or the tumor endothelium and stroma.
Citation Format: Martin Felices, Michael Kaminski, Peter Hinderlie, Rachel Hopps, Laura Bendzick, Melissa Geller, Jeffrey S. Miller. Mediating NK cell function against solid tumors, via targeting of tumor stroma, using a TEM8-targeting Tri-specific Killer Engager (TriKE®) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2878.