利用肿瘤穿透肽瘤内靶向递送TNFa增强抗肿瘤作用及机制研究

Recently, series of progresses made immunotherapy as a breakthrough in clinic cancer therapy. According to the growing knowledge of the immunosuppressive tumor microenvironment, harnessing the immune system to attack tumors became a hot topic in cancer therapy. Allergy of endothelium of tumor vessels is an important mechanism for keeping immunosuppressive tumor microenvironment and induction of drug resistance of tumor. It is known that TNFa exerts superior antitumor activity through intratumoral immune responses triggered by injury or activation of tumor endothelial cells. However, systemic toxicity of natural TNFa is obvious. The premise for application of TNFa in clinic cancer therapy is intratumoral delivery. In our previous works, we have identified a tumor-penetrating peptide which can specifically bind tumor endothelial cells and penetrate into the solid tumor. It was found that fusion to the tumor-penetrating peptide significantly enhanced the antitumor effect, but simultaneously reduced the systemic toxicity of TNFa, which strongly triggered our interesting to deliver TNFa using more tumor-penetrating peptides. In this project, we will extend the screening of tumor-penetrating peptide candidates and produce series of fusion proteins containing tumor-penetrating peptide and TNFa. And we will determine the antitumor effect of these fusion proteins as a monotherapy or in combination with other drugs, followed by investigation of their mechanisms. The systemic toxicity of fusion protein will be evaluated and compared with that of TNFa. In addition, the potential of these fusion proteins as novel agents for cancer immunotherapy will be suggested. Tumor-penetrating peptides used in this project are tumor-targeting as well as tumor-penetrating, which is helpful to deliver the big protein deeper into the solid tumor thus might exert better antitumor effect. By using these peptides as carrier of TNFa, it is possible to develop several fusion proteins with improved antitumor effect but lower toxicity. And these proteins might be novel alternations for immunotherapy of solid tumors.

免疫疗法极有可能开创临床肿瘤治疗新局面。针对肿瘤免疫抑制微环境，发展肿瘤免疫增强型药物是热点问题。血管内皮惰性化是肿瘤造就免疫抑制微环境并产生药物抗性的重要机制。TNFa通过活化血管内皮细胞触发瘤内免疫反应而显示超强抗肿瘤活性。但天然TNFa系统毒性强。因此，靶向运输是TNFa广泛临床应用的前提和保障。课题组前期利用一种肿瘤穿透肽与TNFa融合，显著增强其抗肿瘤作用并切实降低了系统毒性。本项目拟进一步筛选更多肿瘤穿透肽，组建系列靶向性TNFa融合蛋白，确定其单独或联合用药的抗肿瘤效果及机制，评价将其发展为新型肿瘤免疫治疗药物的可能性。项目选择的肿瘤穿透肽既有肿瘤靶向性，又有组织穿透性，可将大分子蛋白运输至瘤体深部，显示更强大的抗肿瘤效果。将其与TNFa融合，可望推出系列自主知识产权、靶向性好、机制独特的高效、低毒抗肿瘤蛋白，为实体瘤免疫治疗提供新选择。

TNFα可损伤血管活化免疫系统而显示强烈的抗肿瘤作用，但系统毒性限制了其作为抗肿瘤药物的应用。将TNFα靶向递送至肿瘤内部极有可能减小其副作用，扩大其在抗肿瘤中的应用。已有研究主要将TNFα靶向递送至肿瘤血管内皮细胞。但选择的分子靶标也在正常血管内皮细胞上表达。因此，以内皮细胞为靶细胞递送TNFα仍会对正常血管造成伤害。除内皮细胞外，周细胞也是主要的血管壁细胞。肿瘤血管内皮不完整，其血管周细胞通常暴露。而正常血管周细胞被完整的血管内皮覆盖，不容易接触到血液中的药物。若将TNFα递送至肿瘤血管周细胞，不容易伤害正常血管。因此，本项目设计以周细胞为靶细胞，选择特异识别周细胞的导向分子，与TNFα融合从而将其靶向递送到瘤内肿瘤血管周细胞上，以期能激活瘤内免疫反应，改善肿瘤血管结构和功能从而降低药物抗性。.根据这一想法，课题组首先调查了血管周细胞在瘤体中的分布。结果发现，多数类型肿瘤血管均富含周细胞。这些细胞高表达PDGFRβ。因此，课题组发现，能特异识别PDGFRβ的亲合体ZPDGFRβ可通过结合周细胞而富集于肿瘤部位。将其与TNFα融合组建的Z-TNFα也因具有周细胞结合能力而富集于肿瘤内部。Z-TNFα的安全性显著优于TNFα。高剂量Z-TNFα（10μg/鼠）可破坏肿瘤血管，通过出血性坏死而显示抗肿瘤作用。低剂量Z-TNFα（1μg/鼠）无直接抗肿瘤作用，但其可轻微损伤血管周细胞，激活瘤内免疫系统，诱导巨噬细胞富集于血管部位。Z-TNFα可抑制周细胞分泌VEGF并减少瘤内新生血管数量，增加肿瘤血管平滑肌细胞，使肿瘤血管壁结构更完整，分支减少，长度增加，对氧气和化学药物的运输能力增强。将低剂量Z-TNFα与阿霉素联用，显著提高了阿霉素的抗肿瘤作用。这些结果说明，将TNFα靶向递送至肿瘤血管周细胞，可促进肿瘤血管正常化而提高化疗药物抗肿瘤效果。低剂量Z-TNFα与化疗药物联用，可能成为新的抗肿瘤策略。