Document resource
Background Chimeric Antigen Receptor (CAR) T cell therapy is an adoptive cell therapy in which cells are genetically modified to express a receptor that binds and kills tumor cells via target antigen recognition. This approach 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 universal CAR T cells that target multiple tumor antigens via antibody and small molecule adaptors. Instead of directly binding to the antigen of interest, we developed our universal CAR, HaloCAR, a self-labeling system based on Halotag, a mutated haloalkane dehalogenase. When co-administered with one or more 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 several antigens (CD20, HER2, EGFR, and CAIX). We also synthesized several molecule-based adaptors targeting CAIX and folate receptor-alpha (FOLR1) and -beta (FOLR2). 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 for 24 or 48 hours. Finally, 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 dual 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 that remained on the cell surface for 1-2 days. All adaptors were extensively characterized, and structure-activity relationship data based on the number of ligands and spacer length were generated.Conclusions We successfully generated and tested a new covalent universal CAR system targeted by CA-conjugated antigen-binding adaptors. In vitro assays highlight the multi-targeting and tunable capabilities of the platform with minimal off-target activity. In vivo labeling of HaloCAR T cells with antibody adaptors is promising for therapeutic feasibility, and anti-tumor activity testing in human tumor xenograft mouse models is ongoing.