Improved effector functions of a therapeutic monoclonal Lewis Y-specific antibody by glycoform engineering

• Published in: Cancer research (Chicago, Ill.) Ill.), 2005-09, Vol.65 (17), p.7934-7941
• Main Authors: SCHUSTER, Manfred, UMANA, Pablo, MUDDE, Geert C, FERRARA, Claudia, BRÜNKER, Peter, GERDES, Christian, WAXENECKER, Gunter, WIEDERKUM, Susanne, SCHWAGER, Cornelia, LOIBNER, Hans, HIMMLER, Gottfried
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Antineoplastic agents; Biological and medical sciences; Cell Line, Tumor; Glycosylation; Humans; Immunoglobulin G - biosynthesis; Immunoglobulin G - genetics; Immunoglobulin G - immunology; Immunotherapy; Lewis Blood-Group System - immunology; Mannosidases - genetics; Mannosidases - metabolism; Medical sciences; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; Neoplasms - immunology; Neoplasms - therapy; Oligosaccharides - immunology; Oligosaccharides - metabolism; Pharmacology. Drug treatments; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transfection; Tumors
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.can-04-4212

The aim of the present study was to produce glycosylation variants of the therapeutic Lewis Y-specific humanized IgG1 antibody IGN311 to enhance cell-killing effector function. This was achieved via genetic engineering of the glycosylation machinery of the antibody-producing host. Antibody genes were transiently cotransfected with acetyl-glycosaminyltransferase-III genes into human embryonic kidney-EBV nuclear antigen cells. A control wild-type antibody, IGN311wt, was expressed in the same host using identical expression vectors, but without cotransfection of genes for acetyl-glycosaminyltransferase-III expression. Both expression products were purified to homogeneity and characterized. The glyco-engineered expression product (IGN312-Glyco-I) showed a remarkably homogenous N-linked glycosylation pattern consisting of one major hybrid-type, non-fucosylated and agalactosylated form carrying a bisecting GlcNAc-group. Wild-type expression product (IGN311wt) on the other hand was glycosylated by a multitude of different core-fucosylated complex-type structures of variable degrees of galactosylation. Target affinity of the glyco-engineered antibody as well as heavy and light chain assembly were not affected by acetyl-glycosaminyltransferase-III expression. In vitro experiments showed a approximately 10-fold increase of antibody-dependent cellular cytotoxicity of the glyco-engineered antibody using different Lewis Y-positive target cancer cell lines (SK-BR-3, SK-BR-5, OVCAR-3, and Kato-III). Complement-mediated cytotoxicity of IGN312-Glyco-I was 0.4-fold reduced using SK-BR-5 as target cell line. The reduction of complement activation could be prevented and even converted into a slight increase of activity by using a different molecular-biological approach directing the glycosylation towards increased levels of complex N-linked oligosaccharides of bisected, non-fucosylated type, as a result of cotransfection of mannosidase II together with acetyl-glycosaminyltransferase-III.