Abstract 2735: Development of a novel, fully human anti-CD19 chimeric antigen receptor for in vivo delivery via CD8-targeted fusosome

Abstract

Abstract Background: We have developed a novel gene therapy platform based on fusogen biology that allows targeted delivery of CAR transgenes to “resting” T cells through systemic administration of a CD8-targeted paramyxovirus-based viral vector (VV). Therapies utilizing autologous CAR T cells have demonstrated success using the murine CD19-specific scFv, FMC63. These murine sequences have the potential to incite T cell and antibody responses against the CAR, which may play a role in CAR T cell elimination. This scenario increases in likelihood in the context of in vivo CAR delivery, where immunosuppression via lymphodepletion is not required. To reduce the potential for CAR-specific immunogenicity, we developed a novel, fully human CD19-specific CAR for use with our in vivo delivery platform. Methods: For efficacy-based screening of human CD19 binders, healthy donor T cells were transduced with VSV-g pseudotyped lentivirus containing second-generation fully human CD19 CARs. FMC63 CARs were produced in a similar fashion and used as benchmark controls for initial characterization studies. CD8-targeted fusogen was generated through targeted mutation to ablate binding to its native receptor. This “blinded” fusogen was subsequently engineered to display a novel scFv that is specific for human CD8α. VV encoding either FMC63 or human CD19 CARs were pseudotyped with CD8-specific fusogen to produce CD8-targeted fusosomes. CD19 CAR efficacy was analyzed in vitro via luciferase assays against NALM-6 or CD19KO NALM-6 tumor lines. Systemic NALM-6 in vivo tumor models (IV injection) were performed in NSG mice. Results: Here we show, our fully human CD19 CAR is comparable to FMC63 CAR in vitro using both short-term luciferase cytotoxicity and long-term Incucyte tumor killing assays. Furthermore, human CD19 CAR T cells demonstrate similar levels of tumor control NALM-6 tumor bearing mice treated with ex vivo generated CD19 CAR+ cells (Mean AUC [n = 3 donors]: FMC63 = 1.26e4, huCD19 = 4.86e4). Our lead human CD19 CAR, like FMC63, lacked killing and cytokine production when cultured with CD19KO NALM-6 tumor cells, demonstrating CD19 antigen specificity. Furthermore, CD8-targeted in vivo delivery of our fully human CD19 CAR showed reduction in NALM-6 tumor load similar to FMC63 CAR (Mean AUC [n = 2 donors]: FMC63 = 1.59e5, huCD19 = 5.06e4). Summary: In vivo delivery of our fully human CD19 CAR with CD8-targeted fusosomes affords tumor clearance comparable to FMC63-CAR in mice bearing NALM-6 tumors. We believe this fully human CD19 CAR has the potential to reduce the risk of immunogenicity compared to a CD19 CAR with the murine scFv FMC63 potentially allowing increased expansion and persistence of CD19 CAR T cells following in vivo delivery in patients. Citation Format: Jeremy M. Kinder, Christie Ciarlo, Chanel Athena Estrada, Neal van Hoeven, Vandana Chaturvedi, Garrett Zipp, Aaron Lampano, Adam J. Johnson, Kutlu Elpek, Terry J. Fry, Aaron E. Foster. Development of a novel, fully human anti-CD19 chimeric antigen receptor for in vivo delivery via CD8-targeted fusosome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2735.