PSCA靶向的多信号CAR分子修饰T细胞设计及其抗肿瘤效应研究

The therapy of cancer using CAR (chimeric antigen receptor) modified autologous T cells is MHC-independent and is able to artificially control functions of T cells.Therefore,it has been attracting considerable public attention. Clinical trials demonstrated that some patients with cancer could be complete remission. However, not all the clinical trials had achieved the expected aims. The core of the CAR+T cell therapy- - construction of CAR molecules, will directly affect its anti-tumor efficiency. However, there is not a general rule for the design of CAR. In this proposal, we will construct anti-PSCA CARs that can activate multi-signals, and will study the effects of antigen recognition region and intracellular signal domain on anti-tumor efficiency. Furthermore, we will explore the possible mechanism. Previously, we have had some hybridoma cell lines secreting anti-PSCA monoclonal antibodies. The affinity of these antibody-derived scFvs to PSCA will be detected, and the in vitro effects on anti-tumor function of CAR+T cells will be evaluated. Next, we will construct multi-signal CAR molecules based on the highly active scFvs, and will evaluate anti-tumor activity of CAR+T cells by using animal models. Finally, the preliminary mechanism will be investigated. Successful execution of this proposal will answer the key questions for CAR design, and will promote the development of CAR+ T cell therapy in China.

嵌合抗原受体（CAR）修饰的自体T细胞（CAR+T细胞）肿瘤疗法由于打破了MHC分子的限制，并能人为控制T细胞功能而受到极大关注。临床试验结果表明部分肿瘤患者的病情能够达到完全缓解,但并不是所有的试验都达到了预期结果。CAR+T细胞疗法的核心-CAR分子的设计,会直接影响其抗肿瘤效果，但目前没有通用的设计原则。本项目中，我们拟设计针对PSCA抗原的、激活多信号的CAR分子，研究抗原识别区及胞内信号区对其抗肿瘤活性的影响，并探讨其机制。前期研究中我们已获得了多株抗PSCA的单抗。这些抗体来源的scFv与PSCA亲和力将被检测,并研究对CAR+T细胞体外杀伤肿瘤效果的影响；在获得高效scFv基础上，构建能够激活不同的、多信号通路的CAR分子；并在动物模型上评价CAR+T细胞的抗肿瘤效果并初步探讨其机制。本项目的实施，将回答CAR设计中的关键问题，有助于推动CAR+T细胞治疗在我国的发展。

近年来，嵌合抗原受体（Chimeric Antigen Receptor，CAR）修饰的T细胞（CAR-T细胞）治疗在肿瘤免疫治疗领域展现出巨大的应用价值和发展潜力。CAR-T细胞打破了MHC分子的限制，并能人为控制T细胞功能。据估计，全球CAR-T细胞治疗市场在2028年将达到85亿美元。CAR分子的功能主要由胞外的抗原识别区和胞内信号区决定。抗原分子识别区一般有scFv分子构成，但是识别同一抗原的不同scFv是否会影响CAR-T细胞的最终的抗肿瘤效果还并不清楚。.前期研究中获得了多株抗人PSCA (prostate stem cell antigen，前列腺癌干细胞抗原）的单克隆抗体。PSCA在前列腺癌组织中高表达，且表达水平与肿瘤恶性程度正相关，是潜在的免疫治疗靶点。本研究在前期的工作基础上，系统的分析了4株抗PSCA抗体的性质，确定了其各自的与PSCA抗原结合的亲和力水平、特性，以及识别抗原表位的区域。随后，分别构建了基于四株抗体的CAR分子，并利用电转化方法获得了CAR-T细胞。最后，利用表达PSCA抗原的人前列腺癌细胞株评价了四种CAR-T细胞的活性，初步证明了它们均能有效杀伤表达PSCA的肿瘤细胞。.通过本课题的研究，获得了针对PSCA抗原的四株抗体的亲和力及其识别的抗原表位数据，并初步证明了靶向PSCA靶点CAR-T细胞抗肿瘤的活性，具有潜在临床应用价值。另外，本课题的研究还建立了一整套CAR-T细胞制备的方法。包括CAR分子设计、构建、慢病毒和电转方法；T细胞的激活、培养和CAR-T细胞的活性检测方法等。这为以后相关课题的研究奠定了坚实基础。.