建立一种基于抗原抗体共展示技术的膜蛋白抗体文库筛查系统

Multi-pass membrane proteins (MMPs) are important molecular targets of monoclonal antibody drugs, however it is hard to develop their antibodies because of the difficulty of MMPs purification. Current methods include improving antigen purification methods or the cell-based membrane proteins immunization. These methods are not only difficult to completely solve the problem of antigen preparation, but also have to use hybridoma technology to select antibodies, which wastes time and energy and cannot directly obtain human antibody. In order to improve the efficiency of the development of MMPs antibody, this study aims to develop an antibody library screening system based on Antigen Antibody Co-Display (AACD) technology. The system displays both the MMPs and the antibody library on the surface of yeast cell membrane, and reports the interactions between them by the coupled detection system. In AACD system, the antibody was displayed on the surface of yeast cell membrane through a transmembrane peptide fused at the N-terminus of antibody. This strategy enables MMPs and antibodies to directly interact on the surface of cell membrane, which not only ensures the natural conformation of antigen, but also realizes the screening of antibody library. At the same time, the affinity of antigen-antibody can be observed directly by the growth rate of clones, so as to avoid the pointless affinity assay of the plenty of candidate antibodies. The establishment of this system will greatly improve the efficiency of the development of MMPs antibody, and it is expected to become the mainstream technology in MMPs antibody development.

多次跨膜蛋白（MMPs）是单抗药物的重要分子靶点，但因为天然MMPs纯化困难导致其抗体研发缓慢。目前的解决手段包括改良抗原纯化方法或者用细胞荷载膜蛋白免疫动物。这些方法不但难以彻底解决抗原制备的问题，而且需要通过杂交瘤技术筛选抗体，费时费力，且无法直接获得全人抗体。为了提高MMPs抗体研发效率，本课题拟开发一种基于抗原抗体共展示技术的抗体文库筛查系统。该系统在酵母细胞膜表面同时展示MMPs抗原和抗体文库，通过所偶联的检测系统报告两者的相互作用。本系统通过融合跨膜肽将抗体展示在酵母细胞膜表面，替代了传统的细胞壁展示技术。这一策略使得MMPs得以在细胞膜表面与抗体文库直接对话，既保证了抗原的天然构象，又实现了抗体的文库筛查。同时，抗原抗体的亲和力通过克隆的生长速度得以直接观察，避免了盲目地对候选抗体亲和力的测定。该系统的建立将大大提升膜蛋白抗体开发的效率，有望成为MMPs抗体开发的主流技术。

GPCR是一类重要的药物靶点。本课题成功了一种基于抗原抗体共展示技术的抗体文库筛查系统。该系统在酵母细胞膜表面同时展示MMPs抗原和抗体文库，通过所偶联的检测系统报告两者的相互作用。本系统通过融合跨膜肽将抗体展示在酵母细胞膜表面，实现了GPCR抗体的高通量筛选。.本课题通过以下几个方面的研究建立了一套以抗原-抗体共展示策略的GPCR单克隆抗体筛选方法：（1）抗原抗体模块结构的优化。选用CXCR4作为抗原，与其有亲和力的抗体At1-scFv作为模型，优化抗体模块的拓扑结构、跨膜肽、柔性肽以及抗原模块的信号肽。（2）抗体的特异性检测。使用AACD检测CXCR4和CXCR5与6个scFv的相互作用，证明该方法具有较强的特异性与普遍适用性。（3）抗体与抗原表位的检测。分别将CXCR4抗原的4个胞外区与柔性肽GSSSS进行替换，使用AACD检测其与At1-scFv的相互作用，结果显示At1-scFv作用于CXCR4的表位为ECL1和ECL2区域。（4）抗体文库的筛选。建立酵母scFv文库，并从中筛选出CXCR4特异性抗体6株，通过流式细胞术验证其确实为CXCR4的特异性抗体。..通过所建立的AACD平台，我们开展了以下应用：（1）对3个重要的GPCR受体CXCR3、CXCR4和CXCR5进行了抗体筛选，获得了多株单克隆抗体，亲和力范围在10-7~10-9M范围内。（2）研究了抗体亲和力（Kd）与筛选指标β-半乳糖苷酶活性的相关性。证明了该平台可以有效筛选GPCR单克隆抗体，并且通过报告基因半乳糖苷酶活性对亲和力进行早期评估，提高筛选效率。..重要技术参数：所建立的AACD系统包括以下重要参数：（1）所构建的抗体Fab文库为轻链和重链的杂交文库，两个独立文库库容均在106~107范围。（2）两个文库通过杂交获得Fab文库，每杂交100mL的轻、重链文库可获得5x109的Fab抗体文库库容。（3）使用5x109文库库容的Fab文库可筛选出抗体5~20株高亲和力的单克隆抗体。..该技术的建立为GPCR这类多次跨膜蛋白的抗体筛选提供了简便、高效的方法。

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