A PSMA-targeted CAR-T cell engineered with a CD16A signaling domain mitigates cytokine output while retaining antitumor activity in prostate cancer models

Introduction: Adoptive transfer of Chimeric Antigen Receptor (CAR) T cells has demonstrated limited efficacy in solid tumors, partially attributable to excessive cytokine release driven by synthetic co-stimulatory domains and functional suppression within the tumor microenvironment. The purpose of this study was to engineer a Prostate-Specific Membrane Antigen (PSMA)-targeted CAR incorporating CD16A, an innate immune receptor with a distinct signaling profile to determine if this alternative architecture supports robust antitumor function while attenuating cytokine output. Procedures: A lentiviral vector encoding a humanized J591-derived anti-PSMA single-chain variable fragment (scFv) fused to CD16A transmembrane and intracellular signaling domains (PSMA-CD16A CAR) was constructed, omitting canonical co-stimulatory domains (e.g., 4-1BB, CD28). Primary human T cells were transduced and evaluated for in vitro activity using a luciferase-based cytotoxicity assay against PSMA⁺ LNCaP targets. Cytokine secretion (TNF-alpha, IFN-gamma, IL-2) was quantified by ELISA following co-culture. In vivo antitumor efficacy was assessed in NSG mice bearing subcutaneous LNCaP xenografts (n=5 per group), utilizing untransduced T cells and tumor-only groups as controls. Results: PSMA-CD16A CAR-T cells demonstrated antigen-dependent cytotoxicity in vitro, mediating efficient lysis of LNCaP cells. Compared with untransduced T cells, CAR-T cells exhibited enhanced killing potency while maintaining an attenuated cytokine secretion profile; levels of TNF-alpha, IFN-gamma, and IL-2 were stable and sufficient to support effector function without supraphysiological spikes. In vivo, PSMA-CD16A CAR-T treatment significantly improved tumor control and prolonged survival. Median overall survival (OS) was 97 days in the CAR-T cohort reaching the study endpoint, compared to 71 days (±8) in untransduced T cells, representing a 36.6% survival increase. Three of five CAR-T treated mice survived to the maximal observation period with observed tumor regression with complete response. While survival was ultimately limited by xeno-reactive graft-versus-host disease (GvHD) inherent to the NSG model, tumor burden was significantly reduced compared to controls. Conclusions: We report the successful generation of a PSMA-directed CAR relying exclusively on CD16A signaling. This construct preserves potent antitumor function while exhibiting a moderated cytokine profile. These findings support CD16A-based signaling as a viable alternative to conventional co-stimulatory domains. Ongoing work focuses on expanded functional characterization and evaluation in immunocompetent models to further assess safety, efficacy and persistence.