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Improved Effector Functions of a Therapeutic Monoclonal Lewis Y-Specific Antibody by Glycoform Engineering

¹ igeneon AG, Brunnerstrasse, Vienna, Austria and ² Glycart Biotechnology AG, Wagistrasse, Schlieren, Zurich, Switzerland

Requests for reprints: Manfred Schuster, igeneon AG, Brunnerstrasse 69/3, Vienna A-1230, Austria. Phone: 43-1-90250-208; Fax: 43-1-90250-901; E-mail: manfred.schuster{at}igeneon.com.

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 glycoengineered expression product (IGN312-Glyco-I) showed a remarkably homogenous N-linked glycosylation pattern consisting of one major hybrid-type, nonfucosylated 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 glycoengineered antibody as well as heavy and light chain assembly were not affected by acetyl-glycosaminyltransferase-III expression. In vitro experiments showed a 10-fold increase of antibody-dependent cellular cytotoxicity of the glycoengineered 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, nonfucosylated type, as a result of cotransfection of mannosidase II together with acetyl-glycosaminyltransferase-III.

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