Abstract 864: Novel innate immune receptor-based chimeric antigen receptors for in vivo programming of myeloid cells show potent anti-tumor activity in preclinical solid tumor models

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy faces significant challenges in solid tumors due to the immunosuppressive tumor microenvironment (TME). Given their tumor-infiltrating capability, phagocytic activity, and ability to promote adaptive immune responses, CAR-engineered myeloid cells represent a promising therapeutic approach. Here we introduce a novel class of CARs designed for programming myeloid cells in vivo. These CARs comprise antibody binders against tumor antigens fused with truncated innate immune receptors CD89 or NKp44. Like their natural counterparts, activation of CD89 and NKp44-based CARs requires association with their cognate signal adapters FcRγ or DAP12, which are only expressed in myeloid/NK cells. Such segregation of antigen binding from cellular activation ensures immune cell-restricted CAR activity, minimizing off-target effects during in vivo administration. Methods: The CAR constructs comprise a single-chain variable fragments (scFv) against HER2 or gp75, fused to truncated CD89 or NKp44. CAR mRNA was encapsulated in lipid nanoparticles (LNPs) to achieve in vivo programming. Coding sequences and 3’ UTR of the mRNA were optimized for prolonged expression. In vitro, CAR expression and function were evaluated in a THP1 reporter cell line and primary monocytes. Anti-tumor efficacy of the CARs was assessed in a human HER2 (hHER2) transgenic mouse model and a syngeneic B16/F10 melanoma model, in which CAR mRNA/LNPs were administered intravenously. Results: The CD89 or NKp44-based CARs were highly expressed in primary CD14+ monocytes for more than 7 days post electroporation. THP1 reporter cell assays demonstrated CAR-dependent activation of the NFκB pathway. Biochemical studies confirmed specific association and phosphorylation of FcRγ chain or DAP12 with CD89 or NKp44-based CARs, respectively. Transfection of human PBMCs with mRNA/LNP showed preferential CAR expression in myeloid cells. In the hHER2 transgenic mouse model bearing hHER2-MC38 tumors, intravenous administration of mRNA/LNPs encoding HER2-targeting CARs showed potent anti-tumor activity and prolonged survival. In the B16/F10 melanoma model, delivery of gp75-targeting CARs reduced tumor growth and increased CD8+ T cell infiltration into tumors, indicative of TME remodeling toward an immunostimulatory state. Conclusions: We demonstrated that CD89 and NKp44-based CARs achieve robust expression in myeloid cells. In vivo programmed CAR myeloid cells elicit potent anti-tumor activity and promote an T cell-mediated adaptive immune response in syngeneic mouse models. The CARs’ reliance on endogenous signaling adaptors, combined with preferential mRNA/LNP uptake by myeloid cells, ensures immune cell-specific activity and provides an enhanced safety profile for in vivo myeloid cell engineering. Citation Format: Yuxiao Wang, Neha Diwanji, Shannon Argueta, Junming Tong, Ewa Grudzien-Nogalska, Michael Gorgievski, Josephine D'Alessandro, Edward Cochran, Phillip Darcy, Jian Ding, Robert Hofmeister, Daniel Getts. Novel innate immune receptor-based chimeric antigen receptors for in vivo programming of myeloid cells show potent anti-tumor activity in preclinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 864.