Preparation And Characterization Of An Anti-human CD3 Humanized Antibody

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 theseresidues 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.