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Construction and characterization of a humanized anti‐human CD3 monoclonal antibody 12F6 with effective immunoregulation functions

12F6 is a murine anti‐human CD3 monoclonal antibody, which competes with OKT3 for binding to human T cells and possesses more effective T‐cell suppression and activation properties compared to OKT3. It thus exhibits the potential to be developed as an immunoregulation agent for manipulating T‐cell functions and preventing acute allograft rejection. In an attempt to minimize the immunogenicity of murine 12F6 (m12F6) for potential clinical application, a humanized version of 12F6, denoted as hu12F6, was successfully constructed by complementary determining region (CDR) grafting and shown to maintain both T‐cell activation and suppression activities similar to m12F6. Furthermore, in order to reduce the first dose reaction syndrome caused by T‐cell activation following the first administration of anti‐CD3 antibodies, two amino acid mutations were introduced into the Fc region of hu12F6, resulting in the Fc‐mutated 12F6 humanized antibody (hu12F6mu). This Fc‐mutated version displayed a similar antigen‐binding affinity and specificity compared with hu12F6 and m12F6 but with much weaker FcR binding activity. hu12F6mu was shown to be much less potent in the induction of T‐cell proliferation, cytokine release (tumour necrosis factor‐α, interferon‐γ and interleukin‐10) and early activation marker expression on the cell surface (CD69 and CD25) than parental 12F6 and OKT3 did. In contrast, hu12F6mu was effective in modulating T‐cell receptor/CD3 and inhibiting mixed lymphocyte reaction with a similarity as compared to m12F6 and OKT3. In conclusion, the resultant hu12F6mu was much less mitogenic to T cells but retained potent immunosuppression, suggesting it might be an alternative to OKT3 as an immunosuppressive drug with less immunogenicity and toxicity for clinical application.