Reprogramming the tumor microenvironment with antibody against membrane-proximal AXL to overcome immune checkpoint blockade resistance.

Immune-cold tumors fail to respond to immunotherapy due to insufficient lymphocyte infiltration within the tumor tissue. Increasing the objective response rate remains an urgent challenge. Here, we report the development of a monoclonal antibody (6C5) that specifically targets the membrane-proximal epitope of the receptor tyrosine kinase AXL and modulates antitumor immunity. Unlike traditional membrane-distal AXL antibodies, which suppress tumor growth primarily by blocking AXL signaling, 6C5 significantly enhanced innate immune sensing by promoting macrophage-mediated antigen uptake and type I interferon production. These effects further activated dendritic cells and increased CD8(+) T-cell infiltration and effector function, thereby reshaping the tumor microenvironment. However, AXL antibody treatment concurrently induced a suppressive subset of PD-1(hi)Foxp3(-)CD4(+) T cells. These cells displayed a transcriptomic profile resembling that of Tregs, characterized by elevated expression of immune checkpoint molecules (including PD-1 and CTLA4), reduced IL-2 production, and increased IL-2 receptor expression, thereby attenuating antitumor immune responses. The combination of 6C5 with dual immune checkpoint blockade (anti-PD-1 plus anti-CTLA-4) or a PD-1-targeted IL-2 fusion protein therapy mitigated this immunosuppression, resulting in potent tumor regression and durable immune memory. Our findings demonstrate that a membrane-proximal AXL-targeting antibody effectively converts the immune-cold tumor microenvironment, overcoming resistance to both conventional and next-generation immune checkpoint inhibitors.