Enhancing HIV-1-specific CAR-T cell functional activity by treatment with a novel cytokine-based scaffold with IL-15 superagonist and TGF-β-neutralizing activity

ABSTRACT Chimeric antigen receptor T cell (CAR-T cell) therapy targeting and eliminating HIV-infected cells offers a promising approach to provide people living with HIV (PLWH) with a functional cure by preventing the recurrence of viremia caused by reactivation of latent HIV-1-infected cells. We previously described a bispecific CAR-T cell targeting two highly conserved gp120 epitopes (duoCAR-T cell) with potent anti-HIV-1 activity that is currently in clinical trials. However, elevated levels of transforming growth factor β (TGF-β) present in many PLWH may hinder the activity of both infused HIV-1-specific CAR-T cells, such as duoCAR-T cells and endogenous HIV-1-specific CD8 + T cells, thereby limiting their effectiveness to achieve a functional HIV-1 cure. We hypothesized that HCW9218, a novel bifunctional immunomodulatory protein composed of TGF-βRII and IL-15/IL-15Rα, would enhance anti-HIV-1 immunity by TGF-βRII binding and neutralizing TGF-β, while IL-15/IL-15Rα would stimulate effector cells and reactivate latent HIV-1-infected cells. We used duoCAR-T cells generated from CD4 + and CD8 + T cells from people without HIV (PWoH) and PLWH donors to demonstrate the in vitro capacity of HCW9218 to block TGF-β activity, and enhance duoCAR-T cell proliferation, cytotoxicity, and anti-HIV-1 activity. HCW9218 also functioned as a latency-reversing agent, stimulating HIV-1 production by CD4 + T cells from ART-suppressed PLWH. Production of HIV-1 by HCW9218-treated CD4 + T cells from ART-suppressed PLWH donors was suppressed by co-culture with autologous duoCAR-T cells. Together, these findings highlight the potential of HCW9218 to augment T cell and CAR-T-based therapies and contribute to strategies aimed at achieving a functional cure for HIV-1. IMPORTANCE The persistence of HIV-1 reservoirs remains the primary barrier to an HIV-1 cure because antiretroviral therapy (ART) suppresses viral replication but does not eliminate latent HIV-1-infected cells. Treatment with anti-HIV-1 duoCAR-T cells is a potential strategy to target and eliminate HIV-1-infected cells, but their activity may be impaired by the immunosuppressive environment in lymphoid tissues of people living with HIV (PLWH). Transforming growth factor β (TGF-β), a pleiotropic cytokine elevated in PLWH, is a key mediator of this immunosuppression. Here, we show that HCW9218, a bifunctional fusion protein with TGF-β-neutralizing activity and IL-15 superagonist activity, preserves duoCAR-T cell function in the presence of TGF-β and reactivates HIV-1 production by latent HIV-1-infected cells in ART-suppressed CD4 + T cells from PLWH. These findings highlight HCW9218 as a unique dual-function immunotherapy that may enhance the efficacy of duoCAR-T cells while facilitating clearance of the HIV reservoir.