CD4‐Based Chimeric Antigen Receptor (CAR)‐T Cells With Resistance to HIV‐1 Infection and Enhanced Anti‐HIV Efficacy: Covalent Interaction Between CD4‐CAR and HIV‐1 Envelope Glycoprotein

The current highly active antiretroviral therapy (HAART or ART) effectively suppresses de novo HIV-1 infection but fails to eliminate HIV reservoir cells, which leads to rapid viral rebound upon ART cessation. Chimeric antigen receptor (CAR) T cells engineered to target HIV-1 Env⁺ cells offer a promising strategy to eliminate or control these persistent reservoirs and achieve durable control of HIV-1 infection. However, a major challenge is the susceptibility of such CAR-T cells to HIV infection, especially those soluble CD4 (sCD4)-based CAR-T cells. In this study, an sCD4-based CAR incorporating the S85C mutation in the CD4 Ig-like domain 1 (termed D1C) was engineered to enable disulfide bond formation with the HIV-1 envelope glycoprotein (Env), thereby reducing viral entry and conferring protection against HIV infection. D1C/sCD4 CAR-T cells exhibited enhanced T-cell activation and cytotoxicity in response to Env stimulation while demonstrating resistance HIV-1 infection in vitro and in vivo. Furthermore, the herpesvirus entry mediator (HVEM) intracellular domain was identified as an optimal costimulatory domain, enhancing cytokine induction, cytotoxicity, and promoting a favorable central memory phenotype and persistence of CAR-T cells. In humanized mouse models, D1C/sCD4 CAR-T cells demonstrated superior persistence and improved control of HIV rebound following ART interruption compared to wild-type (WT)/sCD4 CAR-T cells. These findings highlight a novel strategy to enhance the efficacy and durability of HIV-targeted CAR-T cell therapy by combining HIV resistance and optimized co-stimulation.