短肽水凝胶负载可分泌PD-1阻断scFv的CAR-T细胞的抗乳腺癌研究

On the basis of chimeric antigen receptor (CAR) T cell therapy is a promising strategy to combat cancer. But for most kinds of solid tumor, such as breast cancer, the current therapy just showed the potential due to the self-formed tumor microenvironments including the release of inhibitory cytokines and metabolites and the presence of immune checkpoint receptors on effector T-cells. Besides, immunotherapy based on the injection of CAR-T suffers from low cell viability upon injection, short duration in vivo and poor homing efficiency, which all restrict the therapy outcomes. In this study, the CAR targeting human epidermal growth factor receptor 2 (HER2) was constructed for breast cancer treatment. Alternatively, the CAR construct has been engineered to coexpress factors that boost CAR-T cell function in the tumor microenvironment. We modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells acted in both a autocrine and paracrine manner to improve the anti-tumor activity. On the basis of previous work, we constructed short peptide or covalently conjugating epitope on peptide-based hydrogel by self-assembly technique, as well as to play a role as a scaffold and effective carrier of CAR-T cells. Thus, in this project, we will focus on 1) clarifying the influence of the local injection of a PD-1-blocking scFv by CAR-T cells-loaded hydrogel on the duration in vivo and homing efficiency, and 2) elucidating the antitumor efficiency and mechanism by which covalently conjugating epitope on peptide-based hydrogel activates the endogenous immune cells and combination of CAR-T cells. This project will provide theoretical basis and clinical strategy for the use of tissue-engineering approaches in the field of immunotherapy of breast cancer.

嵌合性抗原受体（CAR）为基础的T细胞疗法是一种很有前景的手段。但对于乳腺癌等实体瘤，由于抑制性微环境等因素的影响，只显示出了一定清除肿瘤的潜力。如何打破免疫耐受，提高抗肿瘤效应，同时解决CAR-T细胞体内作用时间短、归巢效率低等缺陷是提高治疗效果的重要策略。我们首先以HER2为乳腺癌治疗靶点，制备了可以自身分泌抗PD-1单链抗体（scFv）的CAR-T细胞，研究CAR激活与PD-1阻断是否对T细胞杀伤乳腺肿瘤有协同作用。进一步，在前期工作的基础上，通过自组装技术制备短肽及键合表位的纳米结构水凝胶，作为CAR-T细胞的支架材料和递送载体，重点阐述：1）水凝胶负载CAR-T细胞瘤旁局部注射对细胞体内存活时间及归巢效率的影响，2）键合表位的水凝胶激活内源性免疫细胞并联合促进CAR-T细胞抗乳腺肿瘤的作用及机制，以期为组织工程学手段治疗乳腺癌提供重要的理论基础和临床策略。

嵌合性抗原受体（CAR）为基础的T细胞疗法是一种很有前景的手段。但对于乳腺癌等实体瘤，由于抑制性微环境等因素的影响，只显示出了一定清除肿瘤的潜力。如何打破免疫耐受，提高抗肿瘤效应，同时解决CAR-T细胞体内存活时间短、归巢效率低等缺陷是提高治疗效果的重要策略。我们首先以HER2为乳腺癌治疗靶点，制备了可以自身分泌抗PD-1单链抗体（scFv）的CAR-T细胞，阐明了PD-1阻断scFv通过自分泌和旁分泌的方式发挥抗乳腺癌作用的机制；进一步，在前期工作的基础上，筛选并制备了键合T细胞特异性扩增功能序列的自组装短肽水凝胶材料，通过调整刚度和配体密度来模拟体内淋巴组织微环境，促进了CAR-T细胞的快速增殖，并保持了高效的杀伤能力；体内通过局部注射的方式增强CAR-T细胞的体内存活时间和肿瘤归巢效率，且没有出现系统的全身性毒性，明确了联合治疗的抗肿瘤效应及机制，为乳腺癌免疫治疗提供新型方式和临床策略。

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