title: Improved effector functions of a therapeutic monoclonal Lewis Y-specific antibody by glycoform engineering.
abstract: 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.
authors.affiliation: igeneon AG, Brunnerstrasse, Vienna, Austria. manfred.schuster@igeneon.com
authors.forename: Manfred
authors.initials: M
authors.lastname: Schuster
authors.forename: Pablo
authors.initials: P
authors.lastname: Umana
authors.forename: Claudia
authors.initials: C
authors.lastname: Ferrara
authors.forename: Peter
authors.initials: P
authors.lastname: Brünker
authors.forename: Christian
authors.initials: C
authors.lastname: Gerdes
authors.forename: Günter
authors.initials: G
authors.lastname: Waxenecker
authors.forename: Susanne
authors.initials: S
authors.lastname: Wiederkum
authors.forename: Cornelia
authors.initials: C
authors.lastname: Schwager
authors.forename: Hans
authors.initials: H
authors.lastname: Loibner
authors.forename: Gottfried
authors.initials: G
authors.lastname: Himmler
authors.forename: Geert C
authors.initials: GC
authors.lastname: Mudde
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D000920:Antibody-Dependent Cell Cytotoxicity; D045744:Cell Line, Tumor; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007983:Lewis Blood Group Antigens / Q000276:immunology*; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000276:immunology / Q000628:therapy; D009844:Oligosaccharides / Q000276:immunology / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007983:Lewis Blood Group Antigens; C094162:Lewis Y antigen; D009844:Oligosaccharides; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase