Antigen-boosted CD4CAR 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 such as HIV remains a major challenge. In people living with HIV who are receiving suppressive antiretroviral therapy (ART), low-antigen levels limit CAR T-cell expansion and persistence. We previously reported data from a pilot study suggesting 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 polymerase chain reaction assays to quantify CAR T-cell frequency and to corroborate the flow cytometry-based quantification of CD4CAR T-cell expansion. We evaluated these new approaches across multiple nonhuman primate (NHP) models of HIV, including both simian immunodeficiency virus- and simian-human immunodeficiency virus-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 We were also unable to confirm the previously observed CD4CAR T-cell expansions from our earlier studies, which have been retracted. Together, these data highlight the need for alternative strategies to potentiate anti-HIV CD4CAR T cells in the immunocompetent setting.