The Effect of Aglycosylation on the Immunogenicity of a Humanized Therapeutic Cd3 Monoclonal Antibody

The factors affecting the immunogenicity of a humanized γ1 CD3 monoclonal antibody (mAb) were investigated in transgenic mice that express the human CD3 antigen epsilon polypeptide (the mAb target antigen). Two derivatives of the mAb were employed, one with a normal, glycosylated Fc region (γ1 CD3 mAb), and the other with an aglycosylated Fc region (aglycosyl γ1 CD3 mAb). Comparisons of the antiglobulin responses elicited by the two derivatives in transgenic and nontransgenic mice demonstrated that Fab-mediated cell binding activity, dependent on target antigen expression, was a major positive determinant of CD3 mAb immunogenicity. A second positive factor was mAb Fc region glycosylation. At low dose levels the γ1 CD3 mAb consistently produced a higher antiglobulin response than the aglycosyl γ1 CD3 mAb. This was probably a result of the nonspecific, in vivo T cell activating property of the γ1 CD3 mAb, a consequence of its ability to cross-link T cells to Fcγ receptor-bearing cells. (The aglycosyl γ1 CD3 mAb has a reduced Fc binding affinity for Fcγ receptors and so does not activate T cells in vivo.) In support of this hypothesis, the γ1 CD3 mAb was able to nonspecifically enhance humoral immunity to an unrelated, coadministered antigen, whereas the aglycosyl γ1 CD3 mAb was not. The lower immunogenicity of the aglycosyl γ1 CD3 mAb correlated with a longer in vivo half-life and an improved capacity to block the target CD3 antigen. These results suggest that, as well as reducing the cytokine-induced side effects normally associated with CD3 mAb therapy, the nonactivating aglycosyl γ1