Background
We previously described a bispecific CAR-T cell targeting two conserved gp120 epitopes (duoCAR-Ts) with potent anti-HIV activity, now in clinical trials. However, despite ART, many people living with HIV (PLWH) develop systemic inflammation associated with increased levels of TGF-β which may suppress the activity of infused duoCAR-Ts. We hypothesized that inhibiting TGF-β activity may unleash a more potent anti-HIV immune response mediated by our duoCAR-Ts, further amplified by treatment with an IL-15 based fusion molecule.
Methods
HCW Biologics developed bifunctional immunomodulatory proteins including HCW9218, composed of TGF-βRII and IL-15/IL-15Rα. TGF-βRII acts as a TGF-β sink, effectively neutralizing its activity and the linked IL-15/IL-15Rα activates NK and CD8+ T cells. As controls, we used HCW9228, composed of TGF-βRII with IL-15/IL-15Rα mutated to reduce activity and HCW9238 with a functional IL-15/IL-15Rα but inactivated TGF-βRII. TGF-β neutralizing activity of HCW proteins were evaluated by treating duoCAR-Ts with HCW proteins and TGF-β, followed by quantifying TGF-β induced-phosphorylation of SMAD2/3. IL-15 function was assessed by staining for IL-15 induced STAT5 phosphorylation in HCW protein treated T cells and measuring cell growth of duoCAR-Ts stimulated with HCWs and TGF-β after 5 days (Fig. 1A). Anti-HIV activity of duo-CAR-Ts in the presence of TGF-β and the HCW proteins was evaluated by cytotoxicity assays targeting 293T-gp120-Luc cells and HIV viral suppression assays using autologous HIV-LucR-infected CD4+ T cells (Fig 1B).
Results
TGF-β induced SMAD2/3 phosphorylation was inhibited by HCW9218 but not HCW9238 which lacks functional TGF-βRII. HCW9218 displayed robust IL-15 activity by phosphorylating STAT5 and stimulating proliferation of an IL-15 dependent cell line. HCW9218, but not HCW9228 treatment prevented TGF-β suppression of duoCAR-T cell proliferation (n=4 donors) and HCW9218 IL-15 activity stimulated significantly greater cell growth of duoCAR-Ts compared to HCW9228 treatment (Fig. 1A). duoCAR-Ts treated with TGF-β maintained cytotoxicity and HIV suppression (n=3 donors) in the presence of HCW9218 but not HCW9238 (Fig. 1B).
Conclusions
HCW9218 neutralizes TGF-β activity and thereby prevents TGF-β-mediated inhibition of duoCAR-T cell proliferation, cytotoxicity, and HIV suppression. HCW9218 may serve as an adjunct therapy to increase the anti-HIV activity of duoCAR-Ts in PLWH with elevated levels of TGF-β to enable sustained anti-HIV immune responses.