抗体库优化策略对特异性抗肝癌单链抗体的人源化  

Optimized humanization of a single chain variable fragment against hepatocellular carcinoma using a combinatorial library

作　　者：叶刚[1] 杨冬华[1] 汤绍辉[1] 黄卫[1] 丁世华[1] 罗静兰[1] 

机构地区：[1]暨南大学附属第一医院内科,广东省广州市510632

出　　处：《世界华人消化杂志》2008年第11期1144-1150,共7页World Chinese Journal of Digestology

基　　金：广东省科技计划基金资助项目;No.2006B19901014;广东省名医工程研究基金资助项目;No.2004-199;广州市科委领域专项创新药物基金资助项目;No.2004Z3-E4l21;暨南大学博士创新基金资助项目;暨南大学附属第一临床医学院科研培育专项基金资助项目~~

摘　　要：目的:对特异性抗肝癌单链抗体(single chain variable fragment,scFv)DM同步进行人源化和优化,以获得亲和力高和免疫原性低的人源化抗肝癌单链抗体.方法:通过分子结构和序列分析,确定对抗原结合位点的构象有重要影响的骨架区(FR)残基,把难以判断回复突变残基的人、鼠源副本同时编入人源化抗体序列中;通过重叠延伸PCR技术合成人源化抗体全基因,利用噬菌体展示技术构建人源化组合抗体库;通过肝癌细胞筛选阳性克隆,ELISA方法测定各个克隆抗原结合活性;用硫氰酸盐洗脱法测定并比较亲本抗体和人源化scFv的相对亲和力.结果:成功构建人源化组合抗体库,实际库容4.77×105,包含由25不同重链和22不同轻链组成的128种人源化DM;经过筛淘及ELISA鉴定,获得HDM1、HDM2和HDM33个阳性克隆,相对亲和力指数分别为HDM11.6mol/L、HDM21.2mol/L和HDM32.2mol/L.结论:抗体库优化法是一个高效、简便的人源化方法;获得的3株阳性克隆HDM1、HDM2、HDM3与亲本抗体DM同样具有良好的抗原结合特异性和较高的亲和力.AIM： To humanize and optimize the single chain variable fragment （scFv） DM specific against hepatocellular carcinoma （HCC）, and simultaneously to get humanized scFvs with high affinity and low immuogenicity. METHODS： The amino acid sequence and themodel structure of scFv DM were investigated to identify murine framework residues that most likely contribute to the integrity of binding site. All the residues that were difficult to decide were included as human and murine alternatives in the combinatorial library. The full-length humanized scFv gene fragments were synthesized with splicing overlap extension-polymerase chain reaction （SOE-PCR）. Phage display technique was used to construct a humanized combinatorial library. Hepatocarcinoma cell-specific scFvs were selected by panning with HCC cell line, and the specificity of those selected scFvs for HCC was detected by enzyme linked immunosorbent assay （ELISA）. The relative affinities of phage antibodies of the positive clones and the primitive clone DM were measured by ELISA using thiocyanate elution. RESULTS： A humanized combinatorial library was constructed successfully, and the real content of the library was 4.77 × 10^5 and contained a total of 128 variant humanized DM consisting of 2^5 different heavy chains and 2^2 different light chains. Three selected positive clones HDM1, HDM2, and HDM3 were obtained after two rounds of panning and ELISA detection, and their relative affinities were 1.6, 1.2 and 2.2 mol/ L, respectively. CONCLUSION： Combinatorial library strategy is an effective and simple method for humanization of antibodies. All of the selected positive clones, HDM1, HDM2 and HDM3, have high specificity and affinity against HCC as the primitive clone DM.

关 键 词：人源化 单链抗体 组合抗体库 肝癌 重叠延伸PCR 硫氰酸盐洗脱法 酶联免疫吸附试验 噬菌体展示技术 

分 类 号：R735.7[医药卫生—肿瘤] R512.62[医药卫生—临床医学]