Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspension-cultured rice cells with coexpression of human β1,4-galactosyltransferase and human CTLA4Ig

Abstract

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4Ig(P)) reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4Ig(M)). However, limitations of hCTLA4Ig(P) include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing (β1-2)-xylose and α1,3-fucose, which cause immunogenic reactions in humans. In the present study, human β1,4-galactose-extended hCTLA4Ig(P) (hCTLA4Ig(P)-Gal) was expressed through the coexpression of human β1,4-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(P)-Gal indicated that not only were the two glycans containing (β1-4)-galactose newly extended, but also glycans containing both β1,2-xylose and α1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4Ig(P), respectively. Unlike hCTLA4Ig(P), hCTLA4Ig(P)-Gal was effective as an acceptor via (β1-4)-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4Ig(P)-Gal in Sprague-Dawley rats was 1.9 times longer than that of hCTLA4Ig(P), and the clearance pattern of hCTLA4Ig(P)-Gal was close to that for hCTLA4Ig(M). These results indicate that the coexpression with hGalT and hCTLA4Ig(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.

Purification, characterization, and N‐glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures

“…The presence of these structures suggests that, despite low detection of rrBChE in the culture medium, the molecule is indeed being processed through the secretory pathway as intended. The structure, relative abundance, and heterogeneity of these glycans across the sites match what has been previously reported for recombinant proteins secreted from rice cell suspension cultures (Jung, Kim, Jang, Shin, & Yang, ; Kang et al, ), and are similar to those secreted from whole Nicotiana benthamiana plants (Alkanaimsh et al, ; Schneider, Marillonnet et al, ). Glycans terminating in galactose residues are likely a result of incomplete formation of the LewisA structure, which is commonly found in the rice secretome (Jung et al, ), though not always detected in high abundance (Kang et al, ; Shin, Chong, Yang, & Kwon, ).…”

Exploring the N-Glycosylation Profile of Glycoprotein B from Human Cytomegalovirus Expressed in CHO and Nicotiana tabacum BY-2 Cells

“…Similarly, N-glycans released with Peptide-N-Glycosidase PNGase A from recombinant human DNaseI (two N-sites) secreted in the BY-2 culture medium correspond mainly to GnGnXF (51%) and GnMXF (45%) [14]. Similar relative proportions were reported for N-glycans released from human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig; three N-sites) secreted in the culture medium of rice suspension cells: GnGnXF (49%) and GnMXF (44%) [15]. Analysis of N-glycans released from human acid alpha-glucosidase secreted from rice suspension cells also showed the presence of a large amount of complex bi-antennary structures like GnGnXF (49%) and GnGnX (12%), some paucimannosidic structures (e.g., GnMX (14%), GnMXF (8%)), as well as a small proportion of Lewis A-containing structures (e.g., Le a GnXF (7.5%)) [16].…”