Multimerization is required for antigen binding activity of an engineered IgM/IgG chimeric antibody recognizing a skin-related antigen.

Monoclonal antibodies offer great tools for research. We encountered a potentially useful mouse IgM monoclonal antibody whose antigen is expressed in normal skin but lost in human skin cancer. Because IgM is difficult to work with and the antigen was unknown, we decided to convert the IgM (µ) to IgG (γ) version. After cDNA for the antibody was obtained by RACE PCR, we made a series of molecules with different combinations of IgM and IgG domains. Whereas V-Cµ1-Cµ2-Cγ3 and V-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 functionally bound to the antigen, V-Cγ1-Hinge-Cγ2-Cγ3, V-Cµ1-Hinge-Cγ2-Cγ3, and V-Cµ1-Cµ2-Cγ2-Cγ3 did not. Gel filtration analyses revealed that the functional molecules tend to form multimers and the multimeric forms retained antigen binding activity. Furthermore, the mutation of amino acid residue p.309Q > C of mouse IgG and addition of IgM tailpiece to the C-terminus of the molecules induced multimer formation, dramatically enhanced antibody functionality and all non-functional molecules became strongly functional. The functional molecules could be bound by protein A/protein G and other IgG specific reagents and therefore should be useful for further characterization of the antigen. Our study revealed that multimerization of converted IgM is functionally important for antigen binding activity of engineered IgM/IgG chimeric antibodies.