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