Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspension-cultured rice cells with coexpression of human [beta]1,4-galactosyltransferase and human CTLA4Ig

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4Ig^sup P^) reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4Ig^sup M^). However, limitations of hCTLA4Ig^sup P^ include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing ([beta]1-2)-xylose and [alpha]1,3-fucose, which cause immunogenic reactions in humans. In the present study, human [beta]1,4-galactose-extended hCTLA4Ig^sup P^ (hCTLA4Ig^sup P^-Gal) was expressed through the coexpression of human [beta]1,4-galactosyltransferase (hGalT) and hCTLA4Ig in an attempt to overcome these unfavorable effects. The results indicated that both encoding hGalT and hCTLA4Ig were successfully coexpressed, and the analysis of N-glycan and its relative abundance in purified hCTLA4Ig^sup P^-Gal indicated that not only were the two glycans containing ([beta]1-4)-galactose newly extended, but also glycans containing both [beta]1,2-xylose and [alpha]1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4Ig^sup P^, respectively. Unlike hCTLA4Ig^sup P^, hCTLA4Ig^sup P^-Gal was effective as an acceptor via ([beta]1-4)-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4Ig^sup P^-Gal in Sprague-Dawley rats was 1.9 times longer than that of hCTLA4Ig^sup P^, and the clearance pattern of hCTLA4Ig^sup P^-Gal was close to that for hCTLA4Ig^sup M^. These results indicate that the coexpression with hGalT and hCTLA4Ig^sup P^ is useful for both reducing glycan immunogens and increasing in vivo stability. This is the first report of hCTLA4Ig as an effective therapeutics candidate in glycoengineered rice cells.