Background AXL receptor tyrosine kinase (AXL) is a transmembrane protein that is over-expressed in a variety of cancer and immune cells. AXL signaling has been implicated in creating an immunosuppressive tumor microenvironment (TME) through both tumor-intrinsic and immunomodulatory mechanisms1,2,3,4,5 promoting resistance to various therapies.6,7,8,9
Methods Compound inhibition potency against the kinase activity of AXL and other kinases was determined by detecting phosphorylated substrate using homogeneous time-resolved fluorescence (HTRF). Binding affinity of inhibitor to intracellular AXL kinase was determined by monitoring displacement of a competitive fluorescent tracer using an AXL NanoBRET assay. Recombinant Gas6, cancer cell lines, whole blood or isolated cells from healthy donors were used to determine the reduction in AXL-mediated signaling in-vitro. PK/PD and anti-tumor effects of selected AXL inhibitors were evaluated in murine models.
Results AXL is highly expressed on a subset of immune cells, including DC’s, NK cells and M2 macrophages as well as fibroblasts, which contribute to a blunted anti-tumor response. Consistent with these observations, AXL is strongly associated with increased infiltration of macrophages, exhausted NK and T-cells, as well as significantly increased CD73 expression in multiple cancer types in TCGA. Additionally, AXL expression is strongly and significantly correlated with epithelial-mesenchymal transition (EMT), which further generates an immunosuppressive TME and promotes resistance to immune, targeted and chemotherapies. High expression of AXL is also strongly associated with poor survival in NSCLC, pancreatic, breast, head & neck, stomach, colorectal, ovarian & prostate adenocarcinomas, especially in the metastatic setting. AXL inhibitors that exhibit high potency in both biochemical (IC <5nM) and cell-based (IC <25nM) assays in addition to good selectivity against closely related kinases MER and TYRO3 (>90x and >25x fold selectivity, respectively) as well as other kinases involved in downstream signaling such as PI3K have been developed. Initial studies in animal models indicate a favorable pharmacokinetic profile and anti-tumor efficacy.
Conclusions AXL is a promising therapeutic target involving both immunomodulatory and tumor-intrinsic mechanisms. AXL inhibition reduces the immunosuppressive TME, enables activation of an anti-tumor immune response and renders tumors more susceptible to previously resistant therapies. Highly potent and selective AXL inhibitors have been designed, displaying biological profiles superior to those of less-selective molecules currently advancing through clinical development.
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