Background Despite the success of CAR-T therapy in treating relapsed/refractory B-cell malignancies, >40% of patients experience relapse in part due to reduction of CAR-T function and poor persistence. Traditional αCD3/αCD28-based activation of T-cells prior to CAR transduction is associated with production of more differentiated CAR-Ts, corresponding to reduced functional persistence. This may be overcome by targeting early memory T-cells. TSCM-cells are the least differentiated T-cell population with self-renewal capacity and a crucial role in the diversification of immune memory.1 We hypothesize that treatment of T-cells with specific cytokines will drive cell-cycle transitioning to enable lentiviral transduction and expansion of CAR-Ts displaying the TSCM phenotype. Using cytokine/protein scaffolds, we evaluated the impact that IL-7, IL-15, IL-21 and the TGF-β sink (TGFβRII) had on the generation of TSCM-like CAR-Ts.
Methods HCW Biologics has developed a bifunctional fusion molecule (HCW9218), which incorporates the extracellular domain of TGFβRII and an IL-15/IL-15 receptor α complex into a tissue factor-based scaffold.2 A similar scaffold comprised of IL-7, IL-15 and IL-21 (HCW9206) was also produced (figure 1). To generate CAR-Ts, primary T-cells isolated from donor-derived PBMCs are treated with HCW scaffolds prior to transduction with a lentivirus expressing an αCD19-CAR and a GFP reporter. 6-days post transduction we compared T-memory differentiation of HCW-treated CAR-T cells to αCD3/αCD28-activated cells by flow cytometry (figure 2). The functional activity of HCW-treated CAR-Ts was determined by a B-cell leukemia cytotoxicity assay.
Results After stimulation with HCW scaffolds for 3-days prior to transduction, the population of T-cells were enriched with a TSCM-like phenotype, which mediated the generation of CAR-TSCMs (figure 3). HCW-stimulated CAR-Ts also displayed more potent dose-responsive cytotoxic function towards a B-cell leukemia cell line than CAR-T cells generated using traditional aCD3/aCD28 activation (figure 4). Finally, these TSCM-like CAR-Ts will be assessed in future experiments for their longevity in vivo and their functional persistence to a relapse event in a humanized mouse model.
Conclusions HCW scaffolds are a novel class of immunotherapeutic that are currently being evaluated in Phase I clinical trials. We have demonstrated that activation with cytokine scaffolds significantly increases the number of TSCM-like CAR-Ts, while also retaining cytotoxic function. Our results indicated that treatment with cytokine scaffolds generate a large population of CD19 CAR-Ts with an early memory T-cell phenotype which should enhance the persistence of CAR-Ts in patients. This strategy will likely enhance long-term in vivo survival of CAR-T therapy and enable sustained suppression of relapsed/refractory B-cell malignancies.
Arcangeli S, et al. CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome. JCI. 2022;132(12).
Liu B, et al. Bifunctional TGF-β trap/IL-15 protein complex elicits potent NK cell and CD8+ T cell immunity against solid tumors. Molecular Therapy. 2021;29(10):2949–2962.
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