Construction and Expression of Two Mouse—Human Chimeric Antibodies with High Specificity and Affinity for Carcinoembryonic Antigen

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Abstract
We have previously reported that a group of monoclonal antibodies (MAbs) to carcinoembryonic antigen (CEA), designated Group F MAbs, are able to discriminate CEA in tumor tissues from the CEA-related normal antigens and that CEA assay systems utilizing at least one Group F MAb show the improved cancer diagnosis. In this study, we cloned the genes coding for two Group F MAbs (Fl1-35 and Fl1-39) and deduced the amino acid sequences of the variable regions for their heavy and light chains. The variable region for the heavy chain of Fl1-35 contained a possible N-glycosylation site (Asn/Asp/Thr) at amino acid positions 89-91. Then, we constructed two mouse-human chimeric antibodies by using the Fl1-35 and Fl1-39 variable region genes of heavy and light chains (VH and Vκ) and human heavy and light chain constant region genes (γ1 and κ) derived from a human plasma cell leukemia line (ARH77). The chimeric gene constructs were sequentially co-transfected into murine non-Ig-producing myeloma (P3-U1) or hybridoma (Sp2/0) cells by electroporation. The resulting chimeric heavy chain of Fl1-35 showed a slightly but significantly higher molecular weight than that of Fl1-39, but the molecular weights of their unglycosylated peptides synthesized in the presence of tunicamycin were similar, indicating the glycosylation at the possible N-glycosylation site in the variable region of the Ch Fl1-35 heavy chain. Both chimeric antibodies exhibited the same specificity and affinity for CEA as those of the parental murine hybridoma antibodies, respectively. Ascites production of Sp2/0 transfectomas is sufficiently high (600-900 μg/ml) for initial clinical studies with the chimeric antibodies.

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