Background ITIL-306 is a genetically engineered autologous TIL cell therapy that amplifies TCR-specific antigen recognition signals (Signal 1) with an FRα-specific CoStimulatory Antigen Receptor (CoStAR; Signal 2).1 Previous work has demonstrated that CoStAR enhances proliferation of T cells in response to target antigen binding, but how this occurs in CD4+ and CD8+ cells, and in particular in response to IL-2 or other common γ-chain cytokines, remains less clear. To explore this effect further we interrogated proliferation dynamics in isolated and mixed CD4+ and CD8+ T cell populations.
Methods T cells from three healthy donors were engineered to express an anti-FRα-specific CoStAR with either a CD28 or CD28.CD40 intracellular signaling domain and subsequently enriched for expression or left non-transduced as control. CD4+ and CD8+ cells were isolated by negative selection and serially stimulated with Ba/F3 cells coexpressing a membrane-anchored OKT3 single chain antibody and FRα in the presence of varying concentrations of IL-2, IL-7/IL-15 or conditioned media. Counts were made every seven days up to day 21.
Results In the presence of high dose exogenous IL-2 or IL-7/IL-15 both CD4+ and CD8+ CoStAR engineered T cells proliferated better than non-transduced T cells. This effect was also observed in the presence of low dose exogenous IL-2 albeit with less robust proliferation of CD8+ T cells. However in the absence of IL-2 only CD4+ and mixed CD4+/CD8+ proliferation was observed with CD28.CD40 CoStAR outperforming a CD28 CoStAR. The effect of high dose IL-2 or IL-7/IL-15 on CD8+ T cells could be recapitulated by the addition of conditioned media from CoStAR-transduced, but not non-transduced, activated T cells. In 1:1 CD4+/CD8+ mixed cultures the ratio was largely maintained over the duration of the restimulations independent of the cytokine milieu.
Conclusions CoStAR provides proliferative benefit to both CD4+ and CD8+ T cells, although the provision of cytokines contributes to the overall magnitude of response. CD28.CD40 provides a substantially more robust signal for cytokine independent expansion of CD4+ and CD4+/CD8+ cultures compared to CD28 CoStAR alone. CoStAR engineered CD4+ T cells support CoStAR engineered CD8+ T cells in a contact independent manner. These data support the ability of CoStAR-transduced T cells to proliferate independent of cytokine support, while implying that potential clinical manipulations known to increase the levels of circulating cytokines, such as increasing intensity of lymphodepleting chemotherapy and addition of exogenous IL-2 infusions, could further augment the activity of CoStAR-TILs in vivo.
Sukumaran S, Kalaitsidou M, Mojadidi M, et al. Costimulatory antigen receptor (CoStAR): a novel platform that enhances the activity of tumor infiltrating lymphocytes (TILs). J Immunother Cancer. 2021;9(Suppl 2):198.
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