构建“三元联动”重塑肿瘤免疫微环境的ATP超敏纳米凝胶用于胰腺癌化疗免疫联合治疗研究

Reduced recruitment and inactivation of T cells in the tumor microenvironment are the key factors of immune tolerance. In this project, we intends to explore a novel “ternary synergism” strategy to re-educate the tumor microenvironment for chemo-immunotherapy on pancreatic cancer, based on the combination of inhibiting the tumor-associated macrophages (TAMs), blocking the CXCR4/CXCL-12 (chemotactic factor) axis, and promoting the presenting of tumor cells by dendritic cell (DC). Specifically, a small molecule inhibitor BLZ-945 is used to inhibit the colony stimulating factor 1 receptor (CSF-1R) of the TAMs. The CXCR4 inhibitor AMD3100 is expected to reduce regulatory cells and avail the T cell aggregation in tumors. The drug paclitaxel (PTX) is selected to promote the presenting of tumor cells by DC. Therefore, a multimodal/ATP hypersensitive nanogel (BB@PPC-PTX) is designed to accomplish the novel “ternary synergism” strategy. The BB@PPC-PTX is electrostatic assembled by a negative charged part of BLZ-945 conjugated BSA (BLZ-BSA) and a cationic nanogel PPC-PTX, in which poly-CXCR4 blocker (PAMD) backbone is modified with phenylboronic acid (PBA) to equipped with hypersensitive properties and cholesterol (Chol) to encapsulate PTX. With ATP triggered in tumors, BLZ-BSA firstly releases from the BB@PPC-PTX by the electrostatic repulsion, which could induce TAMs depletion. Subsequently, the exposed PPC-PTX nanogel further penetrates to the deep tumor tissues to initiate the CXCR4 inhibition and tumor impair. The multimodal nanogel integrates the advantages of three drugs co-loading, ordinal drug release and tumor penetration. Taken together, the multimodal/ATP hypersensitive nanogel based on re-educating the tumor-microenvironment with ternary synergism strategy hold tremendous potential to be developed for chemo-immunotherapy on pancreatic cancer.

肿瘤微环境中T细胞钝化、募集抑制是产生免疫耐受的最重要因素。本课题采取抑制肿瘤相关巨噬细胞TAMs活性、阻断趋化因子CXCR4/CXCL-12信号轴及促进DC递呈的三元联动策略，多角度重塑肿瘤免疫微环境，构建ATP超敏纳米凝胶（BB@PPC-PTX）用于胰腺癌化疗免疫联合治疗。本课题避免多药联用载体的复杂性，合成以CXCR4拮抗剂基元构成的“治疗性载体”PBA-PAMD，修饰胆固醇侧基自交联形成可深层渗透的纳米凝胶PPC用于负载化疗药紫杉醇PTX，并以静电吸附表面覆盖BLZ-945-白蛋白小粒（BLZ-BSA）。BLZ-BSA在肿瘤微环境ATP触发下脱落，解除T细胞钝化；暴露出的PPC-PTX深层渗透并阻断CXCR4/CXCL-12信号轴抑制Treg募集；PTX促进T细胞捕获，同时发挥细胞杀伤作用。全面、科学解析纳米制剂构造、解组装与重塑免疫微环境的协同效应，为化疗免疫联合治疗提供新思路。

针对“冷”肿瘤免疫微环境中T细胞钝化、募集浸润抑制所导致的免疫耐受，本课题构建“三元联动”策略用于抑制肿瘤相关巨噬细胞活性、阻断趋化因子CXCR4/CXCL12信号轴及激活免疫循环，多角度重塑乳腺癌免疫抑制微环境。采用CXCR4拮抗剂基元构建的“治疗性载体”PBA-PAMD以避免多药共载的不可控性，其修饰胆固醇侧基自交联用于负载化疗药物紫杉醇PTX实现深层渗透，并利用静电作用力将肿瘤相关巨噬细胞杀伤剂BLZ-945-白蛋白小粒（BLZ-945-BSA）吸附于PPC-PTX表面实现可控释放。所制备的BB@PPC-PTX纳米凝胶具有高载药能力，对于PTX的载药量高达23%；当ATP浓度大于0.4 mM时，BB@PPC-PTX纳米凝胶的表面电位明显下降，可释放93.4%的BLZ945-BSA，证明其具有ATP的超敏感特性可实现精准释药。高转移性乳腺癌小鼠模型中，BB@PPC-PTX纳米凝胶可逐渐蓄积到肿瘤部位且12小时达到最大值，其在肿瘤部位的蓄积量显著高于其他器官组织。经BB@PPC-PTX治疗后，“冷”肿瘤免疫抑制有效解除，具体表现为：肿瘤微环境中肿瘤相关巨噬细胞显著减低（约50.3%），杀伤性T细胞浸润量提高近3倍；PPC-PTX内核深层渗透肿瘤深部，并阻断CXCR4/CXCL12信号轴抑制免疫抑制细胞募集，调节性T细胞显著减少约71%。此外，PPC-PTX纳米凝胶内核缓慢释放PTX，有效杀伤肿瘤细胞并增强其免疫原性，淋巴结内树突状细胞激活量提高近2倍，激活免疫循环。这种解除免疫抑制、阻断趋化因子CXCR4/CXCL12、激活免疫循环的“三元联动”策略可显著抑制肿瘤生长，减少近90%的肿瘤细胞向远端器官的转移，有效延长患癌小鼠中位生存期至40天。体内安全性评价中，BB@PPC-PTX不会导致溶血性贫血、急性感染等并发症，主要脏器未发生病变，证明其具有良好的体内安全性。该课题全面、科学解析纳米制剂构造、解组装与重塑免疫微环境的协同效应，突破了多药共载的固有形式，展现了“治疗性载体”所构建的精准释药体系用于打破肿瘤免疫抑制屏障的优势，临床转化前景良好。本项目超额完成预期研究任务，发表SCI论文28篇（18篇IF＞10），3项专利公开并授权，1项实现高价值转化（1200万）；获得教育部自然科学一等奖等奖励4项。