Abstract 3992: Tandem CAR-T cells against mesothelin and MUC16ectoto overcome tumor-antigen heterogenicity
Authors: Salas-Benito Diego , Birocchi Filippo , Armstrong Alexander , Bouffard Amanda A. , Kienka Tamina , Korell Felix , Leick Mark B. , Berger Trisha , Maus Marcela V. 
Published: March 2024

Abstract
Introduction: Antigen heterogeneity is a challenge for CAR-T cell therapy in solid tumors due to antigen escape when targeting a single antigen. In ovarian cancer (OC), mesothelin (meso) and Mucin16 (MUC16) are two of the most overexpressed antigens. Moreover, CA125, which is part of MUC16, is lost in some OC patients, but the ectodomain remains on the cell surface. Targeting both antigens (Meso and MUC16ecto) with a tandem-CAR configuration could overcome antigen heterogeneity to improve CAR-T cell efficacy in OC.
Material and Methods: We designed a library of different CARs with tandem Meso and MUC16ecto scFv combinations with varying G4S linker lengths, fused to a CD8 hinge/transmembrane domain and 41BBz intracellular domain. A series of constructs was then tested in an NFAT-GFP reporter system in Jurkat cells to determine which configuration induced maximal T cell activation and antigen-binding capacity via flow cytometry. We identified the top 2 activators and binders, produced primary human tandem CAR-T (TanCAR) cells by LV transduction, and tested their cytotoxicity in vitroagainst dual antigen-expressing or single antigen-expressing tumor cells using luciferase killing assays and Incucyte. We measured the cytokine production with a Luminex® Multiplex Assay and used a z-Movi® Cell Avidity Analyzer to test the tandem scFv avidity towards the antigens. Also, we examined TanCAR activity against a mixed tumor model in NSG mice. Their in vitro and in vivo activity were compared to CAR T cells targeting either single antigen (SS1-anti-Meso or 4H11-anti-MUC16ecto).
Results: We found that TanCAR1 and TanCAR3 as the configurations with the best activation and binding capacity in response to cognate antigen. TanCAR1 and TanCAR3 had superior in vitro cytotoxic capacities compared to SS1 or 4H11 in tumor models expressing both antigens and in mixed cultures of tumor cells expressing one, both, or neither target antigen. From this mixed culture, we analyzed the remaining tumor cell populations by flow cytometry and observed an overgrowth of the population not targeted by CAR. In response to tumor cells, tandem CARs also produced INFg, IL6, IL-18, IL-13, and GM-CSF regardless of whether one or two antigens were expressed. For further experiments, we pursued TanCAR1 due to its slightly better functionality compared to TanCAR3. TanCAR1 had the same avidity for cells expressing the single antigens as SS1 or 4H11 CARs, but an intermediate avidity for the double Ag-expressing cell line compared to the single CARs. Finally, we tested TanCAR1-T cells in an in vivo mixed tumor model, showing that they better controlled and reduced tumor growth compared to either SS1 or 4H11.
Conclusion: Targeting Meso and MUC16ecto through a tandem CAR design allows for a better anti-tumor response compared to CAR T cells targeting a single antigen. This approach may combat antigen heterogeneity by preventing antigen escape.
Citation Format: Diego Salas-Benito, Filippo Birocchi, Alexander Armstrong, Amanda A. Bouffard, Tamina Kienka, Felix Korell, Mark B. Leick, Trisha Berger, Marcela V. Maus. Tandem CAR-T cells against mesothelin and MUC16ectoto overcome tumor-antigen heterogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3992.