Research paper
IgG subclass-independent improvement of antibody-dependent cellular cytotoxicity by fucose removal from Asn297-linked oligosaccharides

https://doi.org/10.1016/j.jim.2005.08.009Get rights and content

Abstract

Fucose depletion from oligosaccharides of human IgG1-type antibodies results in a great enhancement of antibody-dependent cellular cytotoxicity (ADCC). The aim of this study was to clarify the effect of fucose removal on effector functions of all human IgG subclasses. A panel of anti-CD20 chimeric antibodies having a matched set of human heavy chain subclasses with different fucose contents in their oligosaccharides was constructed using wild-type and fucosyltransferase-knockout Chinese hamster ovary cells as host cells. As found previously for IgG1, fucose-negative variant of IgG2, IgG3, and IgG4 exhibited enhanced ADCC and FcγRIIIa binding compared with their highly fucosylated counterparts. In contrast, fucose removal did not affect complement-dependent cytotoxicity (CDC) of any IgGs. Consequently, fucose removal from IgG2 and IgG4 resulted in a unique effector function profile; they had potent ADCC and no CDC. In conclusion fucose depletion can provide a panel of IgGs with enhanced ADCC without an impact on other inherent properties specific for each IgG subclass, such as CDC.

Introduction

The early promise of murine monoclonal antibodies for therapeutic applications was severely impaired by immunogenicity and modest effector functions that limited their clinical utility. To solve these problems, genetic engineering has been used to generate chimeric antibodies and humanized antibodies with human antibody constant region. For therapeutic use the human IgG1 constant region has been employed primarily because of the capacity to induce strong human effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Scheinberg and Chapman, 1995).

ADCC, a lytic attack on cells to which antibodies are bound, is triggered following binding of leukocyte receptors (FcγRs) to the antibody Fc region. Several mouse and clinical studies indicate that ADCC is an important mechanism therapeutically of clinically effective antibodies (Clynes et al., 2000, Cartron et al., 2002, Anolik et al., 2003, Weng and Levy, 2003). Two successful approaches have been reported to improve ADCC by engineering human IgG1 molecule. One is the use of mutated IgG1 Fc sequences with increased affinity for FcγRs obtained by random alanine substitution technique (Shields et al., 2001), and the other is the removal of fucose from IgG1 oligosaccharides (Shields et al., 2002, Shinkawa et al., 2003) which is even more powerful approach than amino acid mutations in Fc region (Shields et al., 2002, Okazaki et al., 2004). A human IgG molecule has two complex-type oligosaccharide chains each linked to Asn297 of heavy chain (Fig. 1) that play a critical role in effector function. In previous studies, we have demonstrated that fucose is the most critical IgG1 oligosaccharide component that affects ADCC, and the removal of fucose from IgG1 oligosaccharides results in a very significant enhancement of both ADCC in vitro (∼ 100 fold) and anti-tumor activity in vivo (Shinkawa et al., 2003, Niwa et al., 2004a). The underlying mechanism by which fucose depletion results in ADCC enhancement is improved binding to FcγRIIIa, the predominant FcγR of NK cells responsible for ADCC mediated by IgG1 (Shields et al., 2002, Niwa et al., 2004a, Niwa et al., 2004b).

However, many therapeutic antibodies currently approved or under clinical development are produced using Chinese hamster ovary (CHO) cells that express high level of α1,6-fucosyltransferase gene (FUT8) and therefore produce IgG1 antibodies with a high fucose content and consequently suboptimal ADCC (Shinkawa et al., 2003). Therefore we generated a fucosyltransferase knockout CHO cell line that can stably produce non-fucosylated antibodies (Fig. 1) with enhanced ADCC that behaves in other respects indistinguishably from the parental line (Yamane-Ohnuki et al., 2004).

FcγRs display IgG subclass specificities despite highly homologous constant region amino acid sequences (93% of sequence except variable hinge region is conserved among four IgGs). The strong binding of IgG1 and IgG3 to FcγRIIIa results in these subclasses being potent ADCC inducer, while IgG2 and IgG4 are weak or inactive subclasses for both ADCC and FcγRIIIa-binding. However, improvement of FcγRIIIa binding and consequent ADCC enhancement by fucose depletion has been verified only for IgG1 subclass (Shields et al., 2002, Shinkawa et al., 2003, Niwa et al., 2004a, Niwa et al., 2004b, Okazaki et al., 2004, Yamane-Ohnuki et al., 2004). In the current study, we constructed human IgG2, IgG3, and IgG4-versions of rituximab, a chimeric IgG1-type antibody targeting the B cell-specific antigen CD20 and is widely used for the treatment for non-Hodgkin's lymphoma (Leget and Czuczman, 1998, Smith, 2003), with different fucose contents in their oligosaccharides. Then we investigated the effect of fucose removal (defucosylation) on the effector functions of different IgG subclasses in addition to IgG1.

Section snippets

Cell lines

CHO cell line DG44 (Urlaub et al., 1986) was kindly provided by Dr. Lawrence Chasin (Columbia University, New York, NY). Ms704, a FUT8 knockout cell line for defucosylated IgG production, has been described previously (Yamane-Ohnuki et al., 2004). Human CD20+ B lymphoma Raji and Daudi were purchased from the American Type Culture Collection (Manassas, VA).

Establishment of cells producing chimeric anti-CD20 IgGs having different IgG heavy chain subclasses

A stable expression vector for chimeric anti-CD20 IgG1 antibody having variable regions identical to those of rituximab (light chain variable

Production and characterization of chimeric anti-CD20 antibodies

In this study the effect of fucose removal on ADCC was determined for a matched set of chimeric anti-CD20 IgGs with different human heavy chain subclasses and the variable regions of rituximab. The human IgG1 heavy chain constant region gene in a chimeric anti-CD20 IgG1 expression vector previously described (Shinkawa et al., 2003) was replaced with human IgG2, IgG3, and IgG4 gene, to construct expression vectors for the production of a matched set of anti-CD20 IgGs. For IgG4, an amino acid

Discussion

Human IgG1-type antibodies have been widely used for therapeutic use, especially in the field of cancer treatment, because of their strong effector functions and long serum half-life (Scheinberg and Chapman, 1995). Other isotypes have been less well studied with IgG2 and IgG4 isotypes being used for blocking antibodies in which effector function was undesirable (Reddy et al., 2000, Yang et al., 2001, Sandborn et al., 2001) or for conjugation to cytotoxic drugs (Carter, 2001).

Recently two

Acknowledgments

We thank Dr. Philip Wallace for helpful suggestions and critical reading of the manuscript.

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