Abstract 4016: mRNA encoding a CAR specific stimulating protein (CSSP) mediates tumor metabolism regulated (TMR) CAR signaling for CAR-T expansion beyond the tumor microenvironment
Authors: Vigant Frederic , Kundu Anirban , Betts Michael , Kerkar Sid P. , Frost Gregory I. 
Published: March 2024

Abstract
Background: Unlike hematologic targets, TMR Chimeric Antigen Receptors with binding restricted to antigen in the tumor microenvironment lack CAR dependent stimulatory signals for proliferation outside the tumor. Increased exposure to CAR-T is associated with improved clinical outcomes and therefore tools to safely and selectively increase proliferation represent potential adjunctive CAR-T therapeutics. Therefore, we developed a surrogate CAR Specific Stimulating Protein (CSSP) approach encoded by synthetic mRNA for expression in antigen presenting cells to boost CAR-T proliferation in vitro and in vivo.
Methods: NFAT-Luc reporter lymphocytes were transduced with lentivectors encoding TMR CARs with or without epitopes reactive to CSSP’s in the CAR. Subsequently, PBMCs were transfected with LNPs containing synthetic mRNAs for either GFP or candidate membrane bound CSSPs. Transfection efficiency was confirmed by microscopy and flow cytometry. The activity of CSSP’s expressed in monocytes cultured with T cells expressing CAR variants with NFAT-luc reporter was measured by luciferase expression and compared to CD3 stimulation. Additionally, the kinetics of CAR stimulation with monocytes expressing CSSPs were determined by serial luciferase measurements. Finally, the proliferation of PBMCs transduced with a TMR-HER2 CAR co-cultured with CSSP treated purified monocytes were evaluated.
Results: Following exposure of PBMCs expressing CSSPs to TMR-CAR-expressing NFAT T cells, CAR specific luciferase signaling was readily detectable compared to non-specific CD3 stimulation, indicating that the CSSPs were capable of CAR specific stimulation through the CD3ζ chain of the CAR. Two of the CSSP variants demonstrated stimulation in a CAR specific fashion at levels similar to or greater than non-specific CD3 stimulation. In kinetic experiments, stimulation of NFAT could be detected over a period of 8 days, indicating persistence of the CSSP similar to GFP in antigen presenting cell controls. Finally, co-culture of CSSP mRNA expressed in purified monocytes were capable of TMR-HER2-CAR T cells stimulation, which do not proliferate when cultured with HER2 antigen expressing cells under physiologic conditions.
Conclusion: TMR-CAR-T cells can achieve proliferative capacity outside the tumor microenvironment using synthetic mRNA encoding CAR Specific Stimulatory Proteins. This novel mRNA approach may provide additional tools to promote the proliferation and/or persistence of solid tumor targeted CAR-T cells outside the tumor environment.
Citation Format: Frederic Vigant, Anirban Kundu, Michael Betts, Sid P. Kerkar, Gregory I. Frost. mRNA encoding a CAR specific stimulating protein (CSSP) mediates tumor metabolism regulated (TMR) CAR signaling for CAR-T expansion beyond the tumor microenvironment [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 4016.