嵌合抗原受体抗肿瘤功能的结构基础

Chimeric antigen receptor (CAR)-engineered T cell (CAR-T) therapy has achieved unprecedented success in the treatment of hematological malignancies. However, CAR-T therapy shows limited effect in treating solid malignancies. In order to design a new generation CAR to improve its clinical applications, it is necessary to understand the molecular mechanism of the antitumor function of CAR, especially the structural basis. In this project, we will first use Nuclear Magnetic Resonance (NMR) Spectroscopy to study the structures of CAR signaling modules and the regulation of CAR structures by lipids. Paramagnetic Relaxation Enhancement (PRE) measurement and other technologies will be used to study the structural dynamics of CAR. To further understand CAR signaling, we will use quantitative Mass Spectrometry (qMS) to study the phosphorylation programs of CAR upon the activation by tumor antigens. Lastly, we will investigate how CAR function is regulated by its structure by tumor immunology methods. The applicant has been working with NMR for 20 years. She is now systematically studying the activation mechanism of key immunoreceptors. The research group that the applicant belongs to has made important original contributions in the T-cell immunology field and has established various technologies of immunology, biochemistry and biophysics (Nature 2013; Nature 2016). The accomplishment of this project will provide new theoretical basis for CAR design, and new insights into the application of CAR-T therapy in treating cancers.

嵌合抗原受体（chimeric antigen receptor，CAR）修饰T细胞（CAR-T）疗法在血液肿瘤治疗中取得了巨大成功，但对实体瘤的疗效却不理想。为设计新一代CAR以提高实体瘤疗效，我们需了解CAR发挥功能的分子机制，尤其是结构基础。本项目将先利用核磁共振研究CAR胞内信号区结构及膜脂环境对结构的调控作用；然后进一步研究其结构动态性；同时利用定量质谱研究CAR被肿瘤抗原活化后的磷酸化模式；最后利用肿瘤免疫学手段验证关键结构位点对CAR抗肿瘤功能的调控机制。申请人从事核磁共振研究已有20年，近来系统性地研究了T细胞关键免疫受体的活化机制；所在研究组在T细胞抗原免疫应答领域做出了原创性贡献（Nature 2013; Nature 2016），建立了多种免疫学、生物化学和生物物理学研究体系。本项目的开展将为CAR的优化设计提供理论依据，为CAR-T疗法在肿瘤治疗中的应用提供新思路。

嵌合抗原受体CAR（chimeric antigen receptor）修饰T细胞（CAR-T）治疗是近年肿瘤免疫治疗的研究热点，在抗肿瘤临床应用中显示出巨大的应用潜力和发展前景。CAR由负责抗原识别的胞外区、跨膜区和负责信号转导的胞内区组成。每一个组成元件都对CAR-T细胞的抗肿瘤效力与安全性、CAR分子的表达量重要相关。T细胞抗原受体TCR（T-cell receptor）的信号转导机制是CAR分子设计的理论基础。本项目以TCR以及CAR的元件CD3ζ和CD28等为研究对象，揭示了一代CAR胞内区（即CD3ζ）3个ITAM信号模体的结构动态异质性；阐明了T细胞共刺激受体CD28的活性调控机制，提出TCR-Ca2+-CD28所组成的正反馈调控环路可以迅速放大T细胞信号强度，从而极大地提高T细胞的抗原响应敏感性。建立了能够同时检测TCR复合物中所有CD3 ITAM的磷酸化模式的绝对定量质谱系统，发现CD3ε呈独特的单磷酸化模式，而单磷酸化的CD3ε可招募抑制型分子Csk，从而为TCR信号转导提供了一种负反馈调节机制，使得TCR分子同时包含活化型和抑制型的功能基序，使之成为可自主控制、自我调节的信号装置。由此获得启示，将CD3ε组装入CD19.28Z CAR分子（即CD19.E28Z CAR）后，发现CD19.E28Z CAR同样可以利用CD3ε ITAM来招募Csk，使CAR分子的磷酸化水平快速衰减，同时CD19.E28Z CAR-T细胞分泌的细胞因子也显著减少，促进细胞生长和存活，整体提高其持续性。在小鼠模型中，相比于“原版”的28Z CAR-T，“升级”后的E28Z CAR-T抗肿瘤活性明显提升。本项研究揭示了CD3ε在TCR信号转导中的多重功能，未来的研究可以充分利用CD3多样性的优势，对CAR分子的信号和功能增加多重调控，为CAR-T细胞治疗提供新思路。