Signal Transduction and Targeted Therapy | Antibody targeting membrane-proximal AXL epitope overcomes resistance to immune checkpoint inhibitors by remodeling the tumor microenvironment

This study reveals the mechanism by which AXL antibodies reprogram the immune-cold tumor microenvironment through activation of innate immune sensing, providing critical experimental evidence for designing combination therapies to overcome resistance to immune checkpoint inhibitors (ICIs), particularly highlighting the therapeutic potential of targeting PD-1hiFoxp3−CD4+ T cells in refractory melanoma.

 

Literature Overview

The article titled 'Reprogramming the tumor microenvironment with antibody against membrane-proximal AXL to overcome immune checkpoint blockade resistance,' published in Signal Transduction and Targeted Therapy, systematically investigates how the monoclonal antibody 6C5, targeting the membrane-proximal epitope of receptor tyrosine kinase AXL, enhances innate immune sensing and remodels the immune-cold tumor microenvironment to overcome resistance to immune checkpoint inhibitors (ICIs). The study not only elucidates the dual regulatory role of AXL in tumor immune escape but also identifies a novel PD-1hiFoxp3−CD4+ T cell subset induced during therapy as a key immunosuppressive factor, offering new insights for future combination strategies.

Background Knowledge

Immune-cold tumors, characterized by a lack of T-cell infiltration, are generally resistant to immune checkpoint inhibitor (ICI) therapy, representing a major clinical challenge in cancer immunotherapy. Although AXL, a member of the TAM receptor family, is highly expressed in various cancers and associated with poor prognosis, it has been considered a potential target for overcoming resistance. However, clinical trials with small-molecule inhibitors and conventional antibodies targeting AXL have shown limited efficacy, suggesting current strategies fail to effectively activate antitumor immunity. The key insight of this study is that conventional antibodies targeting the distal membrane AXL epitopes primarily inhibit tumor growth by blocking the GAS6-AXL signaling pathway but have limited impact on modulating the immune microenvironment. The authors propose that antibodies targeting the membrane-proximal AXL epitope may more effectively activate antigen-presenting cells via FcγR-mediated effector functions (e.g., ADCP), thereby enhancing T-cell responses. This strategy could overcome the current limitations of AXL-targeted therapies, particularly showing translational potential in immune-cold tumors such as melanoma and non-small cell lung cancer.

 

 

Research Methods and Experiments

The research team developed a monoclonal antibody, 6C5, targeting the membrane-proximal fourth fibronectin type III domain (FNIII) of AXL, and compared it with antibodies targeting the distal membrane first Ig-like domain (e.g., 6S2). The antitumor efficacy was evaluated using B16-hAXL melanoma and MC38-hAXL colon cancer models expressing human AXL in C57BL/6 and humanized mice. Transcriptomic changes in tumor-infiltrating immune cells (TIICs) were analyzed by single-cell RNA sequencing, with immune cell subset dynamics validated by flow cytometry. The immune mechanisms dependent on 6C5 were systematically dissected using genetically engineered mouse models including Axl^−/−, Ifnar1^−/−, Myd88^−/−, Batf3^−/−, and CD11c-DTR mice. Furthermore, the hAXL^BAC transgenic mouse model was employed to exclude xenogeneic immune responses, enhancing the translatability of preclinical data.

Key Conclusions and Perspectives

• By binding to the membrane-proximal epitope of AXL, the 6C5 antibody significantly enhances macrophage-mediated ADCP, promoting tumor antigen uptake and presentation, thereby activating the type I interferon pathway — this finding suggests that epitope selection and Fc function optimization should be prioritized in future tumor immunotherapy studies.
• 6C5 therapy activates MyD88-dependent type I interferon signaling, promoting cDC1 maturation and migration, and enhancing CD8+ T-cell infiltration and effector functions — supporting the combination of innate immune-activating antibodies or adjuvants in immune-cold tumor models.
• Although 6C5 monotherapy enhances T-cell responses, it simultaneously induces a PD-1hiFoxp3−CD4+ T-cell subset with Treg-like phenotypes that highly express immune checkpoint molecules such as CTLA-4, LAG3, and TIGIT, and secrete IL-10 and TGF-β — indicating this subset is a novel immunosuppressive target, and future drug development should focus on its specific depletion or functional blockade.
• Combining 6C5 with dual immune checkpoint blockade (anti–PD-1 + anti–CTLA-4) or with a PD-1–targeted IL-2 fusion protein effectively suppresses this inhibitory T-cell subset, leading to robust tumor regression and establishment of durable immune memory — providing clear rationale for clinical translation and combination therapy design.
• 6C5 efficacy depends on macrophages and cDC1s, not NK cells, and its action is independent of AXL signaling inhibition but relies on FcγR-mediated effector functions — emphasizing that when evaluating AXL-targeting antibodies, signal blockade and immune activation functions should be distinguished, with priority given to developing antibodies with strong ADCP capacity.

Research Significance and Prospects

This study redefines the potential of AXL as an immunomodulatory target, indicating that targeting strategies should not be limited to inhibiting tumor cell-autonomous signaling but should focus more on activating innate immunity. It provides a new paradigm for next-generation antibody drug design—selecting specific epitopes to maximize ADCP/ADCC effects. Moreover, the identification of PD-1hiFoxp3−CD4+ T cells as a novel immunosuppressive population suggests their potential as biomarkers to predict ICI response or as therapeutic targets, advancing precision monitoring in clinical settings.

In terms of drug development, this study supports combining AXL antibodies with dual immune checkpoint blockade or targeted cytokine therapies, especially for AXL-high, immune-cold tumors such as melanoma and non–small cell lung cancer. Additionally, the validation of 6C5’s antitumor activity in humanized mouse models strengthens its clinical translatability, and it is recommended to incorporate type I IFN signaling and cDC1 infiltration as pharmacodynamic biomarkers in early clinical trials.

 

 

Conclusion

This study systematically reveals that the anti-AXL antibody 6C5, by targeting the membrane-proximal epitope, activates macrophages and cDC1s to drive type I interferon–dependent T-cell immunity, effectively converting immune-cold tumor microenvironments. Although monotherapy induces a novel PD-1hiFoxp3−CD4+ T cell–mediated immunosuppression, combining 6C5 with dual immune checkpoint blockade or PD-1–targeted IL-2 therapy significantly enhances antitumor efficacy and establishes durable immune memory. These findings not only provide a new mechanistic explanation for overcoming resistance to immune checkpoint inhibitors but also propose clinically viable combination strategies. From bench to bedside, this study delivers critical theoretical support and a translational roadmap for immunotherapy of refractory cancers such as melanoma and non–small cell lung cancer, underscoring the pivotal role of AXL targeting in reshaping the tumor immune landscape and paving the way for the clinical development of next-generation antibody therapies.

 

Zuming Yang, Shuaishuai Cao, Jiaen Zhang, Xuyuan Zhang, and Yong Liang. Reprogramming the tumor microenvironment with antibody against membrane-proximal AXL to overcome immune checkpoint blockade resistance. Signal Transduction and Targeted Therapy.