Abstract 4098: Chimeric antigen receptor (CAR) T cells overexpressing Bcl-xL increase proliferation and antitumor activity alone and in combination with BH3 mimetics
Authors: Korell Felix , Olson Michael , Salas-Benito Diego , Leick Mark B. , Larson Rebecca C. , Silva Harrison , Gasparetto Alessandro , Berger Trisha R. , Bouffard Amanda , Kann Michael C. , Mergen Markus , Kienka Tamina , Wehrli Marc , Bailey Stefanie R. , Letai Anthony , Maus Marcela V. 
Published: April 2023

Abstract
Background: Chimeric antigen receptor (CAR) T cells have become a well-established treatment option for patients, with six products approved for different hematologic diseases and new approvals allowing for their therapeutic use as early as second line. However, relapse rates of around 50% have been observed in all patient subsets, with one major mechanism associated with CAR T failure being cancer cell resistance to apoptosis. A form of cancer therapeutic named BH3-mimetics has been designed to inhibit members of the anti-apoptotic B cell lymphoma-2 (Bcl-2) family and, therefore, directly activate the apoptotic machinery in malignant cells. We hypothesized that integration of these anti-apoptotic molecules into CAR T cells would induce resistance towards the BH3 mimetics and allow combinational therapeutic approaches.
Methods: 4-1BB and CD28 CAR constructs were designed to overexpress one of four anti-apoptotic proteins: wildtype Bcl-2, a Venetoclax-resistant Bcl-2 variant (G101V), B cell extra-large (Bcl-xL), or myeloid cell leukemia-1 (Mcl-1). CAR T cells made from these constructs were tested against leukemia (Nalm6) and lymphoma (JeKo-1) cell lines in combination with three different BH-3 mimetics: Venetoclax (ABT-199, an FDA-approved Bcl-2 inhibitor), Navitoclax (ABT-263, a Bcl-2/Bcl-xL inhibitor), and AZD5991 (an Mcl-1 inhibitor).
Results: CAR T cells with a 4-1BB costimulatory domain tended to have increased killing over CARs with CD28 and were less susceptible to apoptosis; therefore, 4-1BB CARs were used for all further testing. Integration of Bcl-2 family proteins into CAR T cells didn’t impair or even increase tumor cell clearance in vitro; however, in combination with Venetoclax, Navitoclax, or AZD5991, killing capacity significantly increased compared to control CAR T cells. Even without combination with drugs, CAR T cells overexpressing Bcl-xL and Bcl-2 (both wildtype and mutant) provided higher anti-tumor activity and prolonged survival against JeKo-1 cells in vivo, whereas only Bcl-xL overexpression showed increased tumor control compared to regular 4-1BB CARs against Nalm6 cells.
Conclusion: Of the tested antiapoptotic proteins, Bcl-xL overexpressing CAR T cells proved superior, having higher proliferation and increased anti-tumor activity in combination with or without BH3 mimetics, providing a new strategy to optimize CAR T cell function for the treatment of leukemia and lymphoma.
Citation Format: Felix Korell, Michael Olson, Diego Salas-Benito, Mark B. Leick, Rebecca C. Larson, Harrison Silva, Alessandro Gasparetto, Trisha R. Berger, Amanda Bouffard, Michael C. Kann, Markus Mergen, Tamina Kienka, Marc Wehrli, Stefanie R. Bailey, Anthony Letai, Marcela V. Maus. Chimeric antigen receptor (CAR) T cells overexpressing Bcl-xL increase proliferation and antitumor activity alone and in combination with BH3 mimetics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4098.