抗CD3人源化抗体的制备及生物学特性研究
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摘要
为了克服鼠源性单克隆抗体应用于人体时所出现的严重过敏反应，人源化抗体应运而生，并很快成为十余年来国内外生物医学研究的重大热点之一。12F6是我所自制的抗人CD3的小鼠单克隆抗体，实验发现它竞争抑制OKT3、SK7及UCHT1等CD3单抗与抗原的结合并具有与OKT3相似的激活及抑制T细胞的功能，提示12F6有可能作为免疫抑制剂应用于急性移植排斥反应的治疗。但作为小鼠杂交瘤技术生产的抗体，12F6同OKT3一样存在鼠源抗体应用于人体时可能产生的一系列问题。因此，我们计划对12F6进行人源化改造，期望获得一个即保留高亲和力，免疫原性又大大下降的人源化抗体。
首先采用5'RACE技术获得了12F6的可变区全长序列，在确定了12F6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，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。经过活性筛选，我们发现对于人源化重链来说，框架区残基6，23，24，27，28，30， 48，49，71，73， 76，78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RHj。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21，46，47，73全部回复突变获得的人源化轻链RLi与RHj组成人源化抗体hu12F6的抗原结合活性超过了嵌合抗体。在筛选得到高亲和力的人源化抗体hu12F6之后，我们采用DHFR扩增系统建立了高效表达hu12F6的CHO细胞株，为人源化抗体今后的动物实验及临床研究打下了基础。
竞争抑制实验结果表明人源化抗体与原鼠源抗体存在竞争抑制关系并具有相似的亲和力；生物学功能实验表明hu12F6有效刺激T细胞增殖、释放细胞因子和表达活化标记抗原，并能有效调变TCR/CD3复合体，提示它与鼠源12F6一样具有激活和抑制T细胞的双向功能，是一个改造成功的人源化抗体。
人源化抗体解决了抗体鼠源性的问题，但CD3单抗激活T细胞引起首剂效应的问题仍然存在。为此我们构建了恒定区特定位点突变的人源化抗体
hu12F6m。它对T细胞的激活作用明显减弱而保留了与hu12F6相似的亲和力和调变功能，可望成为一种抗排异能力强、免疫原性和毒副作用小的有效免疫抑制剂用于急性移植排斥反应的治疗。
To reduce the immunogenicity of murine monoclonal antibodies in humans, humanized antibodies have been developed during the past ten years. 12F6 is a murine anti-human CD3 mAb produced in our lab. It competes with other several anti-CD3 mAbs (OKT3, UCHT1 or Leu4) for binding to human T cells and possesses potent T cell activation and suppression properties in vitro similar to OKT3. This indicates 12F6 may be used as an immunosuppressive agent for treatment of acute allograft rejection. However, 12F6 is a murine antibody and is highly immunogenic in humans. Therefore, we plan to develop a humanized version of 12F6, which has the same binding affinity and less immunogenicity compared to the parental 12F6 antibody.
The variable region cDNAs for the light and heavy chains of 12F6 were cloned from the hybridoma cells by 5'RACE. The obtained variable region genes were sequenced and analyzed. The three Complementarity-determining regions from 12F6 light chain or heavy chain were grafted into human antibody light chain or heavy chain frameworks to generate humanized antibody genes. Then the humanized light chain and heavy chain genes were inserted into expression vectors respectively and were co-expressed transiently in COS cells, yielding humanized antibody (RLa,RHa). FCM was performed to determine the binding of humanized antibody (RLa,RHa) to Peripheral blood mononuclear cells (PBMCs). However, it was demonstrated that this antibody almost lost all the binding activity. Basing on the theory that some changes in the human FRs are essential to reconstitute full binding activity of the humanized antibody, important residues that may have influences on binding activity in the human FRs were analysis and backmutation assay was carried out. A number of light and heavy chain versions were produced to evaluate the contribution of FR residues to antigen binding activity. It was demonstrated that residues 27，28，30，71，6，23，24，48，49，73，76 ,78 on the heavy chain FRs or residues 21，46，47，73 on the light chain FRS were important to the binding activity of heavy chain or light chain. Backmutation of all these
residues generated humanized antibody hu12F6 (RLi,RHj). This humanized antibody completely restore the binding activity of murine 12F6. In order to meet the need of further experimental studies and clinical applications, we expressed humanized antibodies in CHO cells using DHFR amplification system and obtained CHO cell lines that produced antibodies at high levels.
Competitive binding assay demonstrated hu12F6 compete with 12F6 for binding to HPBMCs, with similar binding affinity to 12F6. T cell activation potency of hu12F6 was evaluated by proliferation, cytokine release and early activation marker expression. The suppressive property of hu12F6 was evaluated by modulation of the TCR/CD3 complex. These experimental results indicate that hu12F6 possesses both T cell activation and suppression properties similar to 12F6. So we thought hu12F6 was a very successful humanized antibody.
The immunogenicity of 12F6 in humans has been reduced though humanization. But the first-dose reaction associated with T cell activation by anti-CD3 Abs still exists. So humanized antibody containing mutated Fc region(hu12F6m) was also constructed. Hu12F6m was shown to be significantly less mitogenic to T cells but had binding affinity and suppressive property similar to hu12F6. In conclusion, Hu12F6m may be an effective immunosuppressive agent with less immunogenicity and toxicity for acute solid organ rejection.
首先采用5'RACE技术获得了12F6的可变区全长序列，在确定了12F6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，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。经过活性筛选，我们发现对于人源化重链来说，框架区残基6，23，24，27，28，30， 48，49，71，73， 76，78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RHj。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21，46，47，73全部回复突变获得的人源化轻链RLi与RHj组成人源化抗体hu12F6的抗原结合活性超过了嵌合抗体。在筛选得到高亲和力的人源化抗体hu12F6之后，我们采用DHFR扩增系统建立了高效表达hu12F6的CHO细胞株，为人源化抗体今后的动物实验及临床研究打下了基础。
竞争抑制实验结果表明人源化抗体与原鼠源抗体存在竞争抑制关系并具有相似的亲和力；生物学功能实验表明hu12F6有效刺激T细胞增殖、释放细胞因子和表达活化标记抗原，并能有效调变TCR/CD3复合体，提示它与鼠源12F6一样具有激活和抑制T细胞的双向功能，是一个改造成功的人源化抗体。
人源化抗体解决了抗体鼠源性的问题，但CD3单抗激活T细胞引起首剂效应的问题仍然存在。为此我们构建了恒定区特定位点突变的人源化抗体
hu12F6m。它对T细胞的激活作用明显减弱而保留了与hu12F6相似的亲和力和调变功能，可望成为一种抗排异能力强、免疫原性和毒副作用小的有效免疫抑制剂用于急性移植排斥反应的治疗。
To reduce the immunogenicity of murine monoclonal antibodies in humans, humanized antibodies have been developed during the past ten years. 12F6 is a murine anti-human CD3 mAb produced in our lab. It competes with other several anti-CD3 mAbs (OKT3, UCHT1 or Leu4) for binding to human T cells and possesses potent T cell activation and suppression properties in vitro similar to OKT3. This indicates 12F6 may be used as an immunosuppressive agent for treatment of acute allograft rejection. However, 12F6 is a murine antibody and is highly immunogenic in humans. Therefore, we plan to develop a humanized version of 12F6, which has the same binding affinity and less immunogenicity compared to the parental 12F6 antibody.
The variable region cDNAs for the light and heavy chains of 12F6 were cloned from the hybridoma cells by 5'RACE. The obtained variable region genes were sequenced and analyzed. The three Complementarity-determining regions from 12F6 light chain or heavy chain were grafted into human antibody light chain or heavy chain frameworks to generate humanized antibody genes. Then the humanized light chain and heavy chain genes were inserted into expression vectors respectively and were co-expressed transiently in COS cells, yielding humanized antibody (RLa,RHa). FCM was performed to determine the binding of humanized antibody (RLa,RHa) to Peripheral blood mononuclear cells (PBMCs). However, it was demonstrated that this antibody almost lost all the binding activity. Basing on the theory that some changes in the human FRs are essential to reconstitute full binding activity of the humanized antibody, important residues that may have influences on binding activity in the human FRs were analysis and backmutation assay was carried out. A number of light and heavy chain versions were produced to evaluate the contribution of FR residues to antigen binding activity. It was demonstrated that residues 27，28，30，71，6，23，24，48，49，73，76 ,78 on the heavy chain FRs or residues 21，46，47，73 on the light chain FRS were important to the binding activity of heavy chain or light chain. Backmutation of all these
residues generated humanized antibody hu12F6 (RLi,RHj). This humanized antibody completely restore the binding activity of murine 12F6. In order to meet the need of further experimental studies and clinical applications, we expressed humanized antibodies in CHO cells using DHFR amplification system and obtained CHO cell lines that produced antibodies at high levels.
Competitive binding assay demonstrated hu12F6 compete with 12F6 for binding to HPBMCs, with similar binding affinity to 12F6. T cell activation potency of hu12F6 was evaluated by proliferation, cytokine release and early activation marker expression. The suppressive property of hu12F6 was evaluated by modulation of the TCR/CD3 complex. These experimental results indicate that hu12F6 possesses both T cell activation and suppression properties similar to 12F6. So we thought hu12F6 was a very successful humanized antibody.
The immunogenicity of 12F6 in humans has been reduced though humanization. But the first-dose reaction associated with T cell activation by anti-CD3 Abs still exists. So humanized antibody containing mutated Fc region(hu12F6m) was also constructed. Hu12F6m was shown to be significantly less mitogenic to T cells but had binding affinity and suppressive property similar to hu12F6. In conclusion, Hu12F6m may be an effective immunosuppressive agent with less immunogenicity and toxicity for acute solid organ rejection.
引文
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2 Abramowicz D, Norman DJ, Vereerstraeten P, Goldman M, De Pauw L, Vanherweghem JL, Kinnaert P, Kahana L, Stuart FP Jr, Thistlethwaite JR Jr, Shield CF, Monaco A, Wu SC, Haverty TP. OKT3 prophylaxis in renal grafts with prolonged cold ischemia times: association with improvement in long-term survival. Kidney Int, 1996,49(3):768-72.
3 Jaffers GJ, Fuller TC, Cosimi AB, Russell PS, Winn HJ, Colvin RB. Monoclonal antibody therapy. Anti-idiotypic and non-anti-idiotypic antibodies to OKT3 arising despite intense immunosuppression. Transplantation,1986, 41(5):572-8
4 Norman DJ, Shield CF 3rd, Henell KR, Kimball J, Barry JM, Bennett WM, Leone M. Effectiveness of a second course of OKT3 monoclonal anti-T cell antibody for treatment of renal allograft rejection. Transplantation,1988,46(4):523-9.
5 Adair JR. Engineering antibodies for therapy. Immunolog Rev,1992,130: 5-40
6 Kettleborough CA, Saldanha J, Heath VJ, Morrison CJ, Bendig MM. Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation. Protein Eng, 1991,4(7):773-83
7 Kolbinger F, Saldanha J, Hardman N, Bendig MM. Humanization of a mouse anti-human IgE antibody: a potential therapeutic for IgE-mediated allergies. Protein Eng, 1993, 6(8):971-80.
8 Abramowicz D, Schandene L, Goldman M, Crusiaux A, Vereerstraeten P, De Pauw L, Wybran J, Kinnaert P, Dupont E, Toussaint C. Release of tumor necrosis factor, interleukin-2, and gamma-interferon in serum after injection of OKT3 monoclonal antibody in kidney transplant recipients. Transplantation,1989, 47(4):606-8.
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