Instability of Chimaeric Antibody Secretion by Anti-Carcinoembryonic Antigen Producing Hybridoma Cells after Gene Targeting
Objective: To produce a chimaeric version of the 11-285-14 anti-carcinoembryonic antigen (CEA) monoclonal antibody using a gene targeting approach. Materials and Methods: A replacement vector was constructed to insert the human constant γ1 gene within the mouse heavy chain locus of 11-285-14 hybridoma cells. The mouse constant γ1 gene (1.5 kb) and the mouse µ intron fragment (2.2 kb) were amplified by PCR and cloned into a pKO Scrambler vector. The human constant γ1 gene fragment (2.2 kb) was cloned next to the intron fragment. Resistant colonies were screened by ELISA for the presence of the human isotype in their supernatants. Results: Of the 4,370 resistant colonies obtained, 87 colonies showed secretion of the human isotype at levels between 4 and 32 ng/ml. PCR and Southern blot results confirmed the correct integration of the human gene by homologous recombination within the heavy chain locus. Most of the producers ceased to express the human isotype within a few weeks after the initial positive ELISA results. Instability of secretion could not be explained by genetic instability in all the clones, which suggests the presence of other undefined epigenetic or physiologic mechanisms. Conclusion: Gene targeting resulted in transformants with unstable and low production rates of chimaeric anti-CEA antibody.