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Tandem CAR T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

Researchers from Massachusetts General Hospital, the Krantz Family Center for Cancer Research, and Harvard Medical School have demonstrated that a tandem CAR T cell targeting mesothelin and MUC16 ectodomain is able to outmaneuver tumor heterogeneity, outperforming single target CAR-T cells in mixed ovarian and pancreatic models. The team engineered six tandem designs combining SS1 anti mesothelin and 4H11 anti MUC16 ectodomain single-chain variable fragments (scFvs) with one to three G4S linkers to demonstrate that TanCAR1, with SS1 in the distal position and a single G4S linker, bound both soluble antigens without steric interference, showcasing the strongest activation in NFAT reporter Jurkat assays. Patient samples, PDX datasets, and tumor microarrays confirmed the heterogeneous and histology specific expression of the two antigens, motivating dual targeting.

Using acoustic force microscopy, the group quantified avidity between CAR T cells and tumor cells with graded antigen expression. TanCAR1’s avidity matched the stronger corresponding monospecific CAR and scaled with antigen density, indicating one antigen at a time to be binding on double positive cells. In cytotoxicity assays, TanCAR1 lysed both single antigen and dual antigen targets, producing robust Th1 cytokines and surpassing monospecific CARs in two dimensional cocultures that mixed mesothelin only, MUC16ecto only, and dual positive cells. In three dimensional spheroids, TanCAR1 infiltrated deeper, expanded more greatly, and preferentially eliminated the higher density antigen population, consistent with the avidity findings.

In vivo, TanCAR1 equaled monospecific CARs in a uniform OVCAR3 intraperitoneal model while clearly outperforming in a heterogeneous ASPC 1 mixture, ultimately slowing growth, preventing early relapse, and driving higher CD3 infiltration. Flow profiling of residual tumors showcased selective depletion of MUC16ecto high components, mirroring the in vitro density bias. Together, the data position TanCAR1 as a practical design for limiting antigen escape in ovarian and pancreatic cancers and highlights two tunable levers that matter for tandem performance in solid tumors: scFv order and linker length.

Reference:

Salas-Benito D, Birocchi F, Park S, et al. Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time. J Immunother Cancer. 2025;13(9):e012822. Published 2025 Sep 9.

http:/doi.org/10.1136/jitc-2025-012822