一种高选择性ITK抑制剂的发现：其在体外和体内均能促进Th1分化并缓解T细胞耗竭

ITK is a member of the TEC family of kinases, expressed in T, NK, and mast cells. ITK plays important roles in TCR signaling and T cell differentiation. Following TCR stimulation, ITK is recruited to the cell membrane and then leads to phosphorylation and activation of PLCgamma1. ITK knockout mice show defects in Th2 differentiation, while retaining the ability to differentiate into Th1 cells, a phenomenon known as Th1 skewing. It is generally accepted that Th1 cells are the primary Th cell subtype associated with tumor elimination. ITK and RLK double knockout in T cells has a more substantial signaling defect, resulting in a profound loss of normal T cell function. Thus, it holds great therapeutic promise to treat cancer with selective ITK inhibitors while sparing RLK.Here, we report that ATH-409 is a highly selective, covalent ITK inhibitor with an IC50 of < 5 nM and more than 300-fold selectivity against RLK and at least 100-fold selectivity against the remaining 10 cysteine-containing kinases. In the kinase panel assay involving 430 kinases, ATH-409 exhibits inhibitory activity against less than 5 of these kinases, with an IC₅₀ selectivity of at least 60-fold for all of the non-target kinases. ATH-409 potently suppresses ITK-mediated phosphorylation of PLCgamma1 and TCR activation-induced IL2 release in PBMCs. At high concentrations, ATH-409 exerts an anti-proliferation effect on human primary T cells, while showing no impact on T cell viability. Under both polarizing and non-polarizing conditions, ATH-409 does not affect the production of Th1 cytokines, but decreases the release of Th2 cytokines. Additionally, ATH-409 attenuates the occurrence of T cell exhaustion. ATH-409 alone does not suppress the proliferation of CT26 cells in vitro , but does inhibit CT26 tumor growth in vivo , indicating that the anti-tumor effect of ATH-409 is achieved through regulating the immune system. In in vivo model, the ITK occupancy increases with the elevation of ATH-409. Furthermore, when ATH-409 is combined with PD-1 antibody and CTLA-4 antibody, the inhibitory effect on CT26 tumors is further enhanced. Analysis of tumor-infiltrating lymphocytes (TILs) reveals that under the combination treatment, the proportion of Th1 cells in tumors increases and T cell exhaustion is alleviated, which is consistent with the phenomena observed in in vitro experiments.