抗人CD30单克隆抗体的制备和活性研究

Preparation and activity study of an anti-CD30 monoclonal antibody

目的制备抗人CD30单克隆抗体并进行活性研究,为其作为抗体偶联药物的靶向运输载体提供研究依据。方法通过基因工程技术构建真核表达载体pIZDHL-CD30-Ig G,将质粒稳定转染CHO/dh Fr^-细胞并筛选,获得稳定表达anti-CD30-IgG抗体的细胞株并纯化抗体,通过HPLC检测其纯度。ELISA、Biacore和流式细胞术实验检测anti-CD30-IgG的亲和活性;免疫荧光共聚焦实验观察其在肿瘤细胞的内吞情况;利用小动物活体成像技术研究抗体在NOD/SCID鼠移植瘤模型中的靶向性。结果成功构建了anti-CD30-IgG抗体的表达载体,并筛选出稳定表达的单克隆细胞株CHO-CD30-IgG,纯化后的抗体anti-CD30-IgG纯度达到98%以上。Anti-CD30-IgG与重组人CD30抗原具有很好的亲和活性,亲和常数为4.15×10~8 L/mol,其可通过肿瘤细胞表面CD30受体介导的内吞进入细胞。在NOD/SCID小鼠L540和Karpas299移植瘤模型中,anti-CD30-IgG可以选择性地富集并滞留在肿瘤部位。结论 Anti-CD30-IgG对抗原和肿瘤细胞具有高亲和性和特异性,并且可被内吞进入肿瘤细胞内部,在小鼠体内可选择性靶向并且长时间滞留在肿瘤部位,可作为抗体偶联药物中"弹头"部分的靶向输送载体。

Objective The aim of this study is to prepare an anti-CD30 monoclonal antibody and investigate its activity, by which we provide an evaluation for its use as a drug career in antibody-drug conjugates. Methods The eukaryotic expression vector of pIZDHL-CD30-IgG was constructed by genetic engineering, and transfected into CHO/dhFr- cells to obtain file single cell clones which stably expressed the antibody of anti-CD30-IgG Anti-CD30-IgG was purified using affinity chromatography and its purity was detected by HPLC. The antigen-binding activity of anti-CD30-IgG was analyzed by ELISA, Biacore and flow cytometry and its internalization in tumor cells was observed by immunofluorescence confocal microscopy. The in vivo tumor-targeting ability of anti-CD30-IgG was monitored by living images. Results In this study, we constructed the expression vector of anti-CD30-IgG and selected the CHO single cell clone which successfully expressed high level of anti-CD30-IgG After purification, we obtained file antibody of anti-CD30-IgG with purity of more than 98%. Anti-CD30-IgG showed specific and high affinity binding to recombinant CD30 with the affinity constant of 4.15×10^8 L/mol. Besides, it could bind to CD30^＋ cancer cells and be internalized into target cells through the antigen-mediated endocytosis Anti-CD30-IgG also exhibited excellent tumor-targeting capability in L540 and Kappas299 xenograft models. Conclusion Anti-CD30-IgG shows attractive specificity and affinity to CD30 antigen and it can be internalized into CD30^＋ tumor cells. Anti-CD30-IgG selectively targets tumor tissues in NOD/SCID mice xenograft models. Taken together, anti-CD30-IgG is an appropriate targeted vehicle for the delivery of cytotoxic payloads in antibody-drug conjugates.