新单抗3A4的CDR移植抗体及其靶向复发髓系白血病细胞的实验研究

Relapse is still one of the main causes of failure of leukemia treatment. Many studies have showed that leukemia stem cells (LSCs) are the source of disease relapse.Eliminating the LSCs completely is crucial to heal the relapsed acute myeloid leukemia(AML).The monoclonal antibody based targeting therapy is an novel and effective treatment of the relapsed AML. Zhejiang Children's Hospital (ZCH)-6-3A4 or 3A4 was generated in our laboratory by he method described in the literature. It had been identified as a novel mouse anti-human CD45RA monoclonal antibody by the 7th International Workshop and Conference on Human Leucocyte Differentiation Antigens (HLDA7). Our preliminary data have showed the 3A4 can identify the LSCs. 3A4,3A4-NCTD and the chemric antibody scFv-3A4 could block the occurrence of leukemia and prolong the survival of the leukemic animals.We believe 3A4 is probably a new target molecule of leukemia cells and holds a targeting therapeutic potential for relapsed AML. However, as well as the murine antibody 3A4, the scFv3A4-Fc would cause the human anti-mouse antibody response (HAMA) due to the murine origin of its constant regions. Therefore, it is necessary to prepare a completely humanized antibody of 3A4: CDR-grafted antibody. In our project,we will prepare the CDR-grafted antibody through a series of molecular biological technologies such as computer-guided molecular docking, genetic engineering and study its function in eliminating the relapse leukemic cells in vivo and vitro.

彻底消灭白血病干细胞（LSCs）是治愈复发急性髓系白血病 (AML) 的关键，利用单抗靶向杀伤LSCs 可以为治疗复发AML提供新的手段。3A4是本实验研制的鼠抗人CD45亚类新单抗，前期研究表明3A4 能有效靶向LSCs，3A4、免疫毒素3A4-NCTD和融合蛋白ScFv-3A4 均能阻止白血病动物模型发病，延长其生存时间。然而，抗体的鼠源性成份会导致人抗鼠免疫球蛋白的产生。因此，必须对其进行人源化改造，即研制CDR移植抗体。本课题拟采用计算机辅助的分子模拟、基因重组、COS细胞瞬时表达等分子生物学技术获得数株CDR移植抗体，采用固定位置保留结合FR区关键氨基酸回复突变的方法最大程度优化抗体特异性和亲和力，最终通过CHO细胞稳定表达具有天然IgG构象的CDR 移植抗体H3A4，并研究其通过CDC和ADCC效应杀伤复发髓系白血病细胞的作用，为该抗体药物进一步临床靶向复发AML奠定实验基础。

背景.白血病干细胞是白血病耐药和复发的根源。本实验室研制的抗人CD45RA的鼠单抗3A4及其嵌合抗体scFv3A4-Fc能够靶向白血病干细胞，为了降低鼠源抗体的免疫原性，需对其进行人源化改造。本研究的目的是获取高亲和力人源化抗体Hu3A4。.研究内容.1.利用EGF蛋白的稳定性和可视性，建立筛选人源化抗体的新方法：scFv3A4-EGFP蛋白真核表达载体的构建、表达和活性检测.2.高亲和力人源化抗体的筛选：抗体序列设计及真核表达载体的构建、表达与筛选.3.完整IgG形式人源化抗体真核表达载体的构建、表达和功能研究.结 果.1. scFv3A4-EGFP融合蛋白的表达：成功构建pHMCH3/scFv3A4-EGFP质粒，转染CHO细胞，转染后18小时荧光显微镜可观察到EGFP绿色荧光。以转染细胞cDNA为模板，RT-PCR可扩增目的基因；细胞免疫荧光法发现转染细胞胞浆含目的蛋白；流式细胞术确定其存在于培养上清中；SDS-PAGE和Western-Blot鉴定其分子量约57KDa；滴定实验显示0.1µg融合蛋白可饱和1x106KG1a细胞。抗体阻滞实验和细胞表达谱研究证实该抗体与亲本3A4识别相同的抗原表位。.2. 高亲和力人源化抗体的筛选：根据人源化比对结果选择同源性最高的序列为模板，确定关键氨基酸。构建单纯CDR移植pHMCH3/scFv3A4a-Fc和回复突变的人源化抗体pHMCH3/scFv3A4b-Fc表达载体，并分别构建它们和EGFP的融合蛋白用于筛选高亲和力抗体。上述方法明确转染细胞有含目的基因，并表达有活性的目的蛋白。.3. 完整IgG形式人源化抗体Hu3A4的制备及功能研究：成功构建pHMCH3/Hu3A4VH-CH和pHMCH3/Hu3A4VL-CL质粒，转染CHO细胞。RT-PCR可扩增出目的条带；细胞免疫荧光发现转染细胞胞浆含有目的蛋白；SDS-PAGE和Western-Blot鉴定Hu3A4分子量约160 kDa；滴定实验显示约0.5µg Hu3A4可饱和1x106KG1a细胞；其相对亲和力为3.7×1010M-1。Hu3A4 ADCC 作用随效靶比的增高而增强。.科学意义.1 建立通过构建人源化scFv-EGFP融合蛋白以加快人源化筛选的新方法。.2 制备人源化抗体Hu3A4，该抗体具有良好的生物学活性，可进一步用于靶向治疗研究。