Antigen-boosted CD4 CAR-T cells fail to expand or control viremia in multiple nonhuman primate models of HIV

Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated curative potential in B cell malignancies, yet translating this success to chronic infections like human immunodeficiency virus (HIV) remains a major challenge. In people living with HIV (PLWH) on suppressive antiretroviral therapy (ART), low antigen levels limit CAR-T cell expansion and persistence. We previously reported data from a pilot study which suggested that HIV-targeted CD4CAR-T cells could overcome this barrier through exogenous antigen supplementation, leading to robust in vivo expansion. Here, we sought to comprehensively confirm and expand on those findings. We tested a broad array of strategies to enhance CD4CAR-T cell efficacy, including CRISPR-Cas9-mediated gene editing of immune checkpoint and HIV-associated genes, single and pooled competitive infusions of engineered CAR-T cells, distinct CAR constructs incorporating either CD28 or 4-1BB costimulatory domains, and exogenous antigen boosting. We also developed highly sensitive droplet digital PCR (ddPCR) assays both to quantify CAR-T cell frequency and corroborate flow cytometry-based quantification of CD4CAR T-cell expansion. We evaluated these new approaches across multiple NHP models of HIV, including both simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected, ART-suppressed NHPs. Although CD4CAR-T cell products exhibited antigen-specific proliferation and cytotoxicity ex vivo, they failed to expand, persist, or control viremia in vivo. We were also unable to confirm previously observed CD4CAR T cell expansions from our earlier studies, which will be retracted. Together these data highlight the need for alternative strategies to potentiate anti-HIV CD4CAR-T cells in the immunocompetent setting.