Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen

In this work, we have constructed two functional mouse/human chimeric antib odies (IgM kappa and IgG(1)kappa isotypes) by inserting genomic DNA fragmen ts encoding VH and VK variable regions of the murine monoclonal antibody Ig M kappa-83D4 into mammalian expression vectors containing human mu, gamma 1 , and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAc alpha-O-Ser/Thr) express ed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric a ntibodies were purified from the transfected cell line supernatant by affin ity chromatography, and their reactivities against Tn antigen were confirme d by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG(1) kappa and pentamers for IgM kappa, The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface p lasmon resonance showing an affinity constant similar to that of 83D4 nativ e antibody for IgM kappa and a lower affinity constant for IgG(1)kappa chim eric antibody, On the other hand, the replacement of mouse C regions with h uman C regions confers both chimeric antibodies the ability to activate hum an complement. These mouse/human chimeric antibodies should be much less im munogenic and could play an important role in the lysis of tumor cell expre ssing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be con sidered as potential tools for human in vivo studies.