高亲和力CD34人源化抗体的制备及鉴定

发布时间：2018-01-12 01:05

本文关键词：高亲和力CD34人源化抗体的制备及鉴定　出处：《第二军医大学》2008年硕士论文　论文类型：学位论文

【摘要】： CD34是分子量约为110kDa的穿膜糖蛋白,是目前所知的最早期、最广泛表达于人类HSC上的膜抗原。在临床造血干细胞移植中,CD34单抗已被广泛用于纯化造血干细胞。但现有的抗人CD34单克隆抗体均为鼠源,进入人体后可能诱发人抗鼠抗体免疫应答(HAMA)。因此,为了提高造血干细胞移植的安全性,我们拟制备新型的抗CD34人源化抗体,以降低其免疫原性,提高其在临床使用中的安全性。我们首先采用传统的杂交瘤技术制备了三株抗人CD34单克隆抗体(5812、4C8和2E10),利用试剂盒检测其亚型分别为(IgG2a,κ)、(IgG1,κ)和(IgG3,κ);流式细胞术的结果表明,与商品化的CD34单抗581对照一样,该三株抗体都能与表达CD34抗原的KG-1a细胞特异性结合;Western blotting分析进一步证实它们特异性识别CD34分子;表位鉴定结果表明5B12、4C8和2E10分别识别CD34分子的Ⅰ、Ⅱ、Ⅲ类表位。我们采用5'RACE技术获得了5812、4C8和2E10的可变区全长cDNA,将可变区基因序列输入计算机在GenBank数据库中检索,证明是新克隆的抗体基因。在获得5812、4C8和2E10这三株单抗的可变区基因之后,我们将它们分别与人源抗体的恒定区连接,然后将嵌合抗体轻、重链基因分别克隆到真核表达载体,最后将嵌合抗体轻、重链表达载体共转染CHO-K1细胞,经选择培养基筛选后获得稳定表达抗体蛋白的CHO细胞克隆。获得的CHO细胞培养上清经Protein A亲和分离纯化,获得纯度在90%以上的CD34嵌合抗体c5812、c4C8、c2E10;抗原结合活性结果表明该三株嵌合抗体都能很好地与KG-1a细胞结合;竞争抑制实验结果表明它们都能与各自对应的鼠源抗体竞争,而且它们的IC_(50)值相似,说明三株嵌合抗体都保留了与各自对应的鼠源抗体相似的亲和力和特异性。为了进一步减小嵌合抗体的免疫原性,我们利用计算机辅助设计对Ⅱ类CD34单抗4C8进行人源化改造。在确定了4C8可变区中CDR区和FR区的范围之后,我们将CDR区直接移植到人源抗体模板的FR中,构建CDR移植抗体,并将获得的人源化轻、重链抗体基因克隆到表达载体共转染COS细胞,用流式细胞术测定其抗原结合活性,结果发现这个人源化抗体的亲和力基本完全丧失。为了重建人源化抗体的亲和力,我们利用计算机辅助设计对可能影响抗体亲和力的FR区重要残基进行回复突变分析,分别构建了5条不同的人源化重链和5条不同的人源化轻链,经过活性筛选,我们发现对于人源化重链来说,框架区残基2,46,68,69,82,91的回复突变是必要的,将它们全部突变为鼠源残基后得到了一个活性超过嵌合抗体重链的人源化重链4C8VHb。我们采用同样的方法对人源化轻链基因进行改造,发现将轻链框架区残基3,4,46全部回复突变获得的人源化轻链4C8VLb与4C8VHb组成的人源化抗体h4C8的抗原结合活性超过了嵌合抗体c4C8,在筛选得到高亲和力的人源化抗体h4C8之后,我们又建立了高效表达h4C8的CHO-K1细胞株,为今后的实验和临床研究打下了基础。竞争抑制实验结果表明人源化抗体可竞争抑制原鼠源抗体与CD34阳性细胞的结合并具有与鼠源抗体相似的亲和力,表明h4C8是一个改造成功的人源化抗体综上所述,本课题中构建的抗CD34人源化抗体可能取代现有的鼠源CD34单抗用于临床上造血干细胞的分选,从而提高临床造血干细胞移植的安全性。
[Abstract]:CD34 is a molecular weight of approximately 110kDa transmembrane glycoprotein, is the earliest known at present, the most extensive membrane antigen expression in human HSC. In clinical hematopoietic stem cell transplantation, CD34 monoclonal antibody has been widely used for the purification of hematopoietic stem cells. But the existing anti human CD34 monoclonal antibodies are murine, enter the human body can induce human anti mouse antibody immune response (HAMA). Therefore, in order to improve the safety of hematopoietic stem cell transplantation, we prepared anti CD34 humanized antibody model, in order to reduce its immunogenicity, improve its safety in clinical use.
We used the traditional hybridoma technique was three strains of monoclonal antibodies against human CD34 were prepared (5812,4C8 and 2E10), to detect the subtype using kit respectively (IgG2a, K) (k, IgG1) and (IgG3, K); flow cytometry results showed that, with the commercial CD34 581 single anti control, the three antibodies can be combined with KG-1a cell specific expression of CD34 antigen; Western blotting analysis further confirmed their specific molecular recognition CD34; epitope identification results show that 5B12,4C8 and 2E10 respectively to identify CD34 molecules I, II, III epitopes. We obtained by 5'RACE 5812,4C8 2E10 and the variable region of the full-length cDNA gene sequences of variable regions of input computer retrieval in GenBank database, that is the new antibody gene cloning.
After obtaining the 5812,4C8 and 2E10 variable region genes of the three strains of monoclonal antibodies, we will connect respectively with human antibody constant region, then the chimeric antibody light and heavy chain genes were cloned into the eukaryotic expression vector of the chimeric antibody light and heavy chain expression vector were transfected into CHO-K1 cells, CHO cell clones selection of culture medium were screened with stable expression of antibody protein. The culture supernatant of CHO cells by Protein A affinity purification, the purity of CD34 chimeric antibody c5812, above 90% c4C8, c2E10; antigen binding activity results showed that the three strains of chimeric antibody can combine well with experimental results of competitive inhibition of KG-1a cells; that they can compete with their corresponding monoclonal antibody, and their IC_ (50) value similar to that of three strains of chimeric antibody have retained the mouse antibody with corresponding similar affinity and specificity.
In order to further reduce the immunogenicity of chimeric antibody, we use computer aided design of humanization of class II CD34 mAb 4C8. After determining the scope of the CDR and FR region 4C8 in variable regions, we will CDR transplanted directly to human antibody template FR, construct CDR transplantation and antibody. People will get the source of light, heavy chain antibody gene was cloned into the expression vector were transfected into COS cells. The antigen binding activity was determined by flow cytometry, the results showed that the humanized antibody affinity. In order to complete loss of basic affinity reconstruction of humanized antibody, we use the computer aided design of FR may influence area the important residues of the antibody affinity mutation analysis, we constructed 5 different human heavy chain and 5 different human light chain, through activity screening, we found that the humanized heavy chain, frame area The residue of 2,46,68,69,82,91 mutation is necessary, all of them are mutated into murine residues obtained after a heavy chain activity over the humanized chimeric antibody heavy chain 4C8VHb. we used the same method to transform the human light chain gene, found the residue light chain framework 3,4,46 reply to all human antibody h4C8 mutations of the human source light chain 4C8VLb and 4C8VHb composed of the antigen binding activity than the chimeric antibody c4C8, after screening the humanized antibody with high affinity for h4C8, we established the CHO-K1 cell line expressing h4C8, for future experimental and clinical research. This is the foundation of competitive inhibition the experimental results show that the humanized antibody can competitively inhibit the original mouse antibody and CD34 positive cells in combination with mouse antibody similar affinity, indicating that h4C8 is a successful transformation of the humanized antibody
To sum up, the anti CD34 humanized antibody constructed in this study may replace the existing murine CD34 monoclonal antibody for clinical hematopoietic stem cell sorting, thereby improving the safety of clinical hematopoietic stem cell transplantation.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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