新型生物支架介导的恶性黑素瘤肿瘤免疫综合治疗方法研究

Adoptive immunotherapy for malignant melanoma (MM) is challenged by generating sufficient number of activated CD8(+) cytoxic T lymphocytes (CTL) to kill tumor cells. Furthermore, the activity of effector CTLs is largely suppressed and their killing tumor effect is not sustained due to immune escape mechanism. In addition, as adoptive immunotherapy usually contains administration of cytokines, it's challenging to regulate cytokines release and optimize their therapeutic effect. Thus the outcome of this type of immunotherapy is unsatisfactory. Based on our previous work on tissue engineering scaffold, we propose to use scaffold as a delivering vehicle for cells and cytokines in this application for the treatment of malignant melanoma. We propose to use synthetic antigen presenting cell（aAPC) to induce enough quantity of melanoma antigen specific CTLs and then transfer CTLs and DCs to the scaffold. In the meantime, we propose to encapsulate cytokines such as granulocyte macrophage colony stimulating factor (GM-CSF)in the matrix of the scaffold. When used in combination with tumor lysate, GM-CSF could enhance DC antigen presenting effect and stimulating DC recruitment and proliferation, which in turn activates CTLs; in addition, GM-CSF is capable of down-regulating inhibitor B7-H4 expression to tilt the balance towards protective anti-tumor immunity. .The scaffold will be implanted at the tumor site and the outcome of the immunotherapy and the anti-tumor mechanism will be studied. Our strategy may provide theoretical and experimental proof that may contibute to future clinical treatment of malignant melanoma.

黑素瘤免疫过继治疗通常因难以诱导机体产生足够数量的能杀灭肿瘤的CD8(+)细胞毒性T淋巴细胞(CTL)、到达病灶部位的CTL活性受抑制、免疫应答持续时间短、难以控制辅助性细胞因子释放等原因导致抗瘤疗效有限。在前期工作基础上，我们拟利用新型生物组织工程支架作为CTL和辅助性细胞因子的载体来治疗黑素瘤：利用aAPC在体外扩增获得足够数量的黑素瘤抗原特异性CTL，然后将CTL和树突状细胞（DC）移植到生物支架上，同时在支架中包埋可活化DC的细胞因子GM-CSF以及肿瘤细胞裂解物，以促进CTL存活、增强其抗瘤作用、并降低负调控因子B7-H4的免疫抑制作用来减少肿瘤免疫逃逸效应；最后将生物支架植入黑素瘤病灶部位，研究其抗瘤效果及机制，为黑素瘤的临床免疫治疗进步，提供实验和理论依据。

我们团队开创性地利用蚕茧中的天然成分丝胶研制出具有优越的生物学活性的水凝胶，并且研发出海藻酸盐和丝胶水凝胶混合支架，以及可用于搭载恶性黑色素瘤治疗药物的氧化葡聚糖丝胶复合水凝胶支架。这些生物支架具有天然的自发荧光活性可用于体内示踪，良好的生物相容性性和细胞粘附性,使其可以作为细胞3D生物支架，可用于体外3D培养和治疗性细胞的体内移植。此外，我们利用丝胶经过化合反应与肿瘤化疗药物阿霉素偶联形成嵌合体分子，通过聚合得到丝胶-阿霉素偶联纳米颗粒，并将该纳米颗粒与叶酸偶联，使其具有肿瘤细胞靶向性，在小鼠肿瘤模型实验中此纳米颗粒的抗肿瘤效果优越。我们还通过化学键连接首次将丝胶蛋白包裹于介孔二氧化硅表面，制备了具有pH/蛋白酶双敏感的智能纳米控释体系。搭载了抗肿瘤药物阿霉素的丝胶/介孔硅纳米粒能够高效地被肿瘤细胞吞噬并进入溶酶体，通过溶酶体内环境触发快速释放药物，成功地克服了肿瘤的多重耐药性，且具有良好的生物安全性。本课题组研发出以海藻酸盐水凝胶作为生物支架的肿瘤免疫治疗制剂，联合特异性非甾体抗炎药塞来昔布和免疫抑制阻断抗体αPD-1作为配方，有望成为人类肿瘤治疗的可用处方。该支架在发挥抗肿瘤效果中起到了关键作用，塞来昔布和PD-1阻断抗体在该生物支架的给药体系中，通过抑制调节性T淋巴细胞和髓性抑制性细胞，降低肿瘤微环境的血管生成作用和炎症反应，协同性地提高了细胞毒性T淋巴细胞的活性，从而有效抑制黑色素瘤的生长和转移。本项目研发的这些新型的生物支架和治疗技术有望转化为临床肿瘤治疗的新制剂。