The Design Of A Novel CD3-specific Antibody And Its Function

The anti-CD3 antibody OK.T3 was approved for the reversal of acute kidney transplant rejection in 1986. Since then, the CD3-specific antibody has been widely used in solid organ and non-solid organ transplantation, such as kidney, heart and bone marrow. Previous studies have shown that anti-CD3 antibodies are also effective in treatment of some autoimmune diseases such as type I diabetes and some other auto-immune diseases. However, the acute side effects have been observed in clinical trials, resulted from the immunogenicity of mouse antibody and the mitogenicity, which induced by the interaction of the antibody and Fc receptors. Through humanization and the mutation at CH₂ region, which binds to the FcR, several research groups have reduced the mitogenicity and immunogenicity successfully. These corresponding products have proceeded into clinical phases and achieved the efficacy. For both academic and applied purposes, it is significant to construct a novel antibody of our own intellectual property.In the present study, based on yCD3, a CD3-specific mouse monoclonal antibody maintained in our laboratory, and implemented on the established platform of constructing antibody molecules with new structure using the information of antigen-antibody interacting conformation, a novel CD3-specific small-molecule antibody with non-mitogenicity and humanization was designed. Furthermore, the preliminary study on its function mechanism was performed. The research results were shown as follows: 1. The heavy/light chain variable domain genes of the CD3-specific mouse monoclonal antibody yCD3 was cloned from the hybridization. The CD3-specific chimeric antibody was constructed and expressed. And then, the functions were preliminarily investigated. It was observed that the chimeric antibody could specifically bind to CD3⁺ cell line Jurkat and peripheral blood mononuclear cells(PBMC). In the mixed lymphocyte culture, the antibody exhibited desirable immunosuppressive activity.2. Using the platform of constructing novel antibody molecules based on the information of antigen-antibody interacting conformation, a novel small molecule antibody named as mini-yCD3 was designed. In this molecule, VH—Linker—VL—hinge—Human IgG1 CH₃ domain forms the single chain. The hinge region was improved to facilitate the two single chains to form disulfide bond, hence to a novel bivalent antibody. In theory, this bivalent antibody could interact with two CD3 molecules, and possessed the same affinity with the parent antibody.3. The mini-yCD3 was consistently expressed in CHO cell. Western Blot showed that the antibody existed as dimmers under non-reductive conditions. In competitive binding experiments, the affinity of mini-yCD3 was consistent with its parent antibody. Mini-yCD3 also showed the potent immunorepression in mixed lymphocyte culture, whereas no ability of activationg T cell. The dramatic release of neither IL-2 nor other inflammatory factors was observed. In other words, the mini-yCD3 presented a novel bivalent construction and inherited the affinity of its parent antibody, but the mitogenicity is significantly reduced.4. Chosing some key processes in the activation of T cell (such as the phosphorelation of theζchain in CD3 molecule, the recruitment of Zap70, the change of Ca²⁺ in cytoplasm and the translocation of NFAT into nuclear), the difference between the novel antibody and the parent antibody was analyzed. The yCD3 could induce the complete phosphorelation of theζchain, while the mini-yCD3 just maintained that in a low level. So, yCD3 could induce efficiently the activation of Zap70, inflow of Ca²⁺, translocation of NFAT into nuclear and the expression of IL-2, then it caused the activation of T cell. Nevertheless, the mini-yCD3 increased the concentration of Ca²⁺ in a low level only in its higher dose, and it was not enough to turn on the follow signal passway and active the T cell.