LAUSR

The survival benefit of checkpoint immunotherapies is currently limited, with de novo and acquired resistance to therapy occurring in over 75% of patients. A major mechanism of resistance accounting for these poor responses is a lack of tumor immunogenicity, resulting from loss of function mutations in antigen presentation pathways (e.g., B2M, MHC1), or a lack of tumor expressed neoantigens. Dual-antibody T cell engagers (DATE) represent a promising immunotherapeutic approach for poorly immunogenic tumors as these agents engage CD3 on T cells while binding a tumor-associated antigen, resulting in intratumoral T-cell activation, independent of antigen recognition or co-stimulation. To harness the therapeutic potential of this approach for treatment of clear cell renal cell carcinoma (ccRCC), a carbonic anhydrase 9 (CA9) targeting DATE was developed, given the near ubiquitous and selective expression of this antigen on ccRCC tumors. To engineer the CA9-DATE, a panel of CA9 antigen binding fragments (Fabs) was developed through phage display and characterized for CA9-membrane fragment esterase inhibition. A CA9-inhibiting Fab was subsequently cloned into a mammalian expression system containing the anti-CD3 single chain variable fragment. CA9-DATE binding specificity was measured by flow cytometry utilizing a panel of CA9-expressing and -knockout cell lines. Functionally, T-cell activation was assessed through CD25 levels using flow cytometry and IFN-γ secretion using ELISA. In vitro T-cell mediated cytotoxicity was tested utilizing luciferase expressing ccRCC cells and LDH release assays. Finally, humanized NOG mice bearing patient-derived RCC tumors were employed for in vivo efficacy studies. CA9-DATE showed antigen-specific binding towards both CD3- and CA9 in the low-nanomolar ranges. CA9-DATE treatment of patient-derived ccRCC cell lines co-cultured with healthy isolated CD3+ lymphocytes or peripheral blood mononuclear cells resulted in rapid CD4+ and CD8+ lymphocyte activation characterized by a proliferative response, increased CD25 expression, and IFN-γ production. In addition, CA9-DATE resulted in CA9 specific-lysis across 5 patient-derived ccRCC cell lines in in vitro cytotoxicity assays. Preliminary in vivo results demonstrate significant reductions in tumor burden following CA9-DATE therapy relative to vehicle control-treated mice. Collectively, these data provide proof of principle to support the clinical study of CA9-DATE targeting strategies in clear cell RCC. Citation Format: Jason Moffat, Xiaoyu Zhang, Keith Lawson, Sunandan Banerjee, Xiaowei Wang, Jarrett Adams, James Pan, Laurie Ailles, Antonio Finelli, Sachdev Sidhu. Development and characterization of a novel CA9 targeting dual-antibody T-cell engager for renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B49.