基于CHO细胞定点整合技术的新型抗体呈现系统的构建

Construction of a new antibody display system based on CHO cell site-specific integration technology

目的建立一个以CHO定点整合系统为基础的高通量抗体筛选平台,筛选获得抗VEGF高亲和力突变体抗体。方法设计并构建了基于CHO细胞定点整合系统的通用minibody展示载体,将3株亲和力已知的抗VEGF抗体(A6,A63,A657)和1个抗VEGF轻链抗体库分别呈现在CHO细胞的表面,通过Western印迹、Bia-core3000、流式细胞分析等对该展示系统进行验证和评价,并通过流式细胞分选技术对抗体库进行了细胞筛选。结果成功实现了抗VEGF抗体在CHO细胞表面的展示,流式细胞分析抗体的亲和力与Biacore测定全抗体亲和力结果一致;通过筛选获得3株抗VEGF高亲和力突变体抗体。结论建立了基于CHO定点整合技术的新型抗体库展示系统,为抗体的亲和力成熟、分子改造等突变库的构建和筛选提供了新的技术支持。

Objective To create a high throughput screening platform for a new antibody display system based on CHO cell site-specific integration technology, on which high affinity anti-VEGF-antibody mutants could be obtained by cell screening. Methods A general plasmid expression vector that could integrate the minibody＇s gene into CHO cell site-spe- cifically was designed and constructed. To evaluate the display system, three anti-VEGF antibodies named A6, A63 and A657 with known affinity were displayed on CHO cell surface respectively. The efficacy was evaluated through Western blot, Biacore 3000 system and flow eytometry analysis. A light chain antibody library against VEGF was constructed and displayed on CHO cell surface,and specific antibodies were selected by FACS. Results Anti-VEGF antibodies and the library were displayed on CHO cell surface successfully. The relative affinity of minibody on CHO cell surface analyzed by FACS was consistent with the affinity of IgG detected by Biacore 3000 system. Three anti-VEGF-antibody mutants with high affinity were selected through the new antibody display system. Conlusion The new antibody display system based on CHO cell site-specific integration technology can provide new technology support for the antibody＇s affinity maturation and other mo- lecular mutations.