Axl-RTK Inhibition Modulates T Cell Functions and Synergizes with Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies

Despite the remarkable results of CD19 directed chimeric antigen receptor T cell (CART19) therapy, the durable responses in B cell lymphoma and chronic lymphocytic leukemia (CLL) is limited. This may be due to the loss of CART persistence, poor trafficking and inhibition by the tumor microenvironment. Strategies to enhance CART function are thus needed. Recent data have shown that abnormal expression of receptor tyrosine kinase (RTK) AXL portends poor prognosis. Inhibition of AXL with TP0903, a high affinity AXL inhibitor, has been found to induce robust apoptosis of CLL B cells. We therefore examined the role of AXL inhibition with TP0903 on T cell and CART19 function. First, we investigated the effect of AXL inhibition on naive T cells. Cells stimulated with PMA/Ionomycin and cultured with TP0903 showed significant reduction of Th2 cytokines and inhibitory receptors (Fig 1a). Effector and regulatory T cells (Treg) treated with TP0903 showed a preferential reduction of Tregs (Fig 1b). Next, we investigated the influence of TP0903 on 41BB costimulated CART19. TP0903 led to polarization of CARTs into a Th1 phenotype when T cells were stimulated with the CD19 + mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP0903 treatment also significantly downregulated inhibitory receptors on activated CARTs, including cytokine release syndrome (CRS) related cytokines (Fig 1c). The combination of CART19 and TP0903 yielded a synergistic cytotoxicity against JeKo in vitro (Fig 1d). We compared the transcriptome of activated CARTs treated with TP0903, and noted >100 genes that were differentially expressed compared to non-treated cells. Among these, cell junction, cell migration, and immune synapse related genes were significantly increased in activated CARTs treated with TP0903. To investigate the effect of AXL inhibition of CARTs with TP0903 in vivo , we established MCL xenografts via the injection of JeKo into NSG mice. The mice were then treated with vehicle alone, TP0903 alone, CART19 alone, or TP0903 + CART19. Three weeks after the treatment, mice were rechallenged with JeKo. Mice treated with CART19 and TP0903 rejected the JeKo challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART persistence (Fig 1e). Finally, we validated our findings in a phase I clinical trial of TP0903 (NCT02729298). T cells isolated from 3 cancer patients showed significant reduction of Tregs. This will be further investigated in a planned phase I clinical trial of TP0903 in relapsed/refractory CLL (NCT03572634). In summary, we showed for the first time that AXL inhibitior polarizes T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes CARTs in B cell malignancies. These findings encourage further study of TP0903 as an enhancer of T cell immunotherapies.