Reprogramming the Tumor Microenvironment with a Membrane-Proximal AXL Antibody to Overcome Immune Checkpoint Blockade Resistance

Immune-cold tumors failed to respond to immunotherapy due to insufficient lymphocytes infiltration within tumor tissue. How to increase the objective response rate is an urgent challenge. Here, we report the development of a monoclonal antibody (6C5) that specifically targets the membrane-proximal epitope of receptor tyrosine kinase AXL, which modulates the anti-tumor immunity. Unlike traditional membrane-distal AXL antibodies, 6C5 significantly enhanced innate immune sensing by promoting macrophage-mediated antigen uptake and type I interferon production via the MyD88 pathway. This reshaped the tumor microenvironment , further boosting CD8+ T cell infiltration and effector function. However, AXL antibody treatment concurrently induced a suppressive subset of PD-1hiFoxp3-CD4+ T cell, attenuating antitumor responses. The combination of AXL Ab with dual immune checkpoint blockade (anti-PD-1 plus anti-CTLA-4) or PD-1-targeted IL-2 fusion protein therapy mitigated this immunosuppression, achieving potent tumor regression and durable immune memory. Our findings demonstrate that membrane-proximal AXL targeting antibody effectively converts immune-cold tumor microenvironment, overcoming resistance to both conventional and next-generation immune checkpoint inhibitors. Overall design: Tumor-bearing mice treated with an anti-AXL antibody. After treatment, tumors were collected and enzymatically dissociated into single-cell suspensions. CD4+T subsets were subsequently isolated by fluorescence-activated cell sorting (FACS) and subjected to RNA sequencing.

免疫冷肿瘤（immune-cold tumors）因肿瘤组织内淋巴细胞浸润不足而无法对免疫治疗产生应答，提高客观缓解率（objective response rate）是当前亟待解决的挑战。本研究开发了一种特异性靶向受体酪氨酸激酶AXL（receptor tyrosine kinase AXL）膜近端表位（membrane-proximal epitope）的单克隆抗体（monoclonal antibody）6C5，其可调控抗肿瘤免疫。与传统靶向AXL膜远端表位的抗体相比，6C5可通过MyD88通路（MyD88 pathway）促进巨噬细胞介导的抗原摄取（macrophage-mediated antigen uptake）与I型干扰素（type I interferon）产生，显著增强固有免疫感知（innate immune sensing）能力。该过程重塑了肿瘤微环境（tumor microenvironment），进一步促进CD8+T细胞（CD8+ T cell）浸润及其效应功能的发挥。但AXL抗体治疗同时会诱导产生PD-1hiFoxp3-CD4+ T细胞抑制性亚群，进而削弱抗肿瘤免疫应答。将AXL抗体与双重免疫检查点阻断（immune checkpoint blockade，即抗PD-1联合抗CTLA-4治疗）或靶向PD-1的IL-2融合蛋白（IL-2 fusion protein）疗法联合使用，可缓解该免疫抑制效应，实现强效的肿瘤消退（tumor regression）与持久的免疫记忆（durable immune memory）。本研究结果表明，靶向AXL膜近端表位的抗体可有效将免疫冷肿瘤微环境重塑为免疫激活状态，克服对传统及新一代免疫检查点抑制剂（next-generation immune checkpoint inhibitors）的耐药性。
实验整体设计：将抗AXL抗体应用于荷瘤小鼠。治疗后收集肿瘤组织，通过酶解法制备单细胞悬液；随后采用荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离CD4+T细胞亚群，并进行RNA测序（RNA sequencing）。