HaloCAR - a Universal Chimeric Antigen Receptor (CAR) Programmable by Chloroalkane-Conjugated Adaptors 2241611

Abstract Introduction Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated clinical success against hematologic cancers. However, to date, it has failed against solid tumors due to several issues, including antigen heterogeneity, which leads to incomplete tumor elimination. To address this limitation, we developed a universal CAR T cell system, HaloCAR, that can target multiple tumor antigens via antibody and small molecule “adaptors”. Instead of containing an scFv that binds to an antigen of interest, our CAR contains Halotag, a highly efficient self-labeling haloalkane dehalogenase enzyme. When co-administered with tumor-targeting adaptors containing a chloroalkane (CA) motif, the adaptors covalently attach to the HaloCAR, arming it to attack tumor cells. Methods We generated antibody-based adaptors targeting antigens (CD20, HER2, EGFR, and CAIX). We also synthesized small molecule-based adaptors targeting CAIX and folate receptor-alpha and -beta. In vitro, we assessed the antigen-targeting activity of HaloCAR T cells via dose titration of one or more adaptors on antigen-positive and antigen-negative target cells. We tested the ability of antibody adaptors to conjugate to HaloCAR T cells in vivo in a mouse model. Results Potent and specific T cell activation and tumor cell lysis were observed for both single- and multi-antigen targeting studies. This activity was titratable and dependent on the dose of the adaptor(s). In vivo, we detected robust conjugation of the antibody-adaptor to Halo CAR-T cells, and assembled CAR was detectable for at least 72 hours post-adaptor injection. Conclusion We successfully generated and tested a new covalent universal CAR system targeted by CA-conjugated adaptors. In vitro assays highlight the multi-targeting and tunable capabilities of the platform. In vivo labeling of HaloCAR T cells with antibody adaptors is promising for therapeutic feasibility. We are currently evaluating anti-tumor efficacy in both solid and hematological human tumor xenograft mouse models. Funding Source NIH 1R01CA290866 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)