Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII.

The gene encoding the rat glycosylation enzyme beta1-4-N-acetylglucosaminyltransferase III (GnTIII) was cloned and coexpressed in a recombinant production Chinese hamster ovary (CHO) cell line expressing a chimeric mouse/human anti-CD20 IgG1 antibody. The new cell lines expressed high levels of antibody and have growth kinetics similar to that of the parent. Relative QPCR showed the cell lines to express varying levels of mRNA. High-performance liquid chromatography (HPLC) analysis showed the enzyme to have added bisecting N-acetylglucosamine (GlcNAc) residues in most (48% to 71%) of the N-linked oligosaccharides isolated from antibody preparations purified from the cell lines. In an ADCC assay the new antibody preparations promoted killing of CD20-positive target cells at approximately 10- to 20-fold lower concentrations than the parent. This activity was blocked using an anti-Fc gamma RIII antibody, supporting the role of Fc gamma RIII binding in this increase. In addition, cell binding assays showed the modified antibody bound better to Fc gamma RIII-expressing cells. The increase in ADCC activity is therefore likely due to an increased affinity of the modified antibody for the Fc gamma RIII receptor.

[1]  J. Ledbetter,et al.  Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. , 1998, Blood.

[2]  S. Narasimhan.  Control of glycoprotein synthesis. UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III, an enzyme in hen oviduct which adds GlcNAc in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-glycosyl oligosaccharides. , 1982, The Journal of biological chemistry.

[3]  James E. Bailey,et al.  Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity , 1999, Nature Biotechnology.

[4]  K. Welte,et al.  IgM and IgG secretion in human B-cell lines regulated by B-cell-inducing factors (BIF) and phorbol ester. , 1983, Immunology letters.

[5]  G. Distefano,et al.  A new Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins. , 2000, Biochimica et biophysica acta.

[6]  P. Chinn,et al.  Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. , 1994, Blood.

[7]  J. Bailey,et al.  Tetracycline-regulated overexpression of glycosyltransferases in Chinese hamster ovary cells. , 1999, Biotechnology and bioengineering.

[8]  Metabolic Engineering of N-Linked Glycoform Synthesis Systems in Chinese Hamster Ovary (CHO) Cells , 1997 .

[9]  M J Keen,et al.  Glycosylation and biological activity of CAMPATH-1H expressed in different cell lines and grown under different culture conditions. , 1995, Glycobiology.

[10]  J W Larrick,et al.  Antibody engineering at the millennium. , 2000, BioTechniques.

[11]  I Royston,et al.  IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma. , 1997, Blood.

[12]  E. Clark,et al.  Phosphorylation of the CD20 phosphoprotein in resting B lymphocytes. Regulation by protein kinase C. , 1989, The Journal of biological chemistry.