Research paperFucose removal from complex-type oligosaccharide enhances the antibody-dependent cellular cytotoxicity of single-gene-encoded antibody comprising a single-chain antibody linked the antibody constant region
Introduction
Antibody-dependent cellular cytotoxicity (ADCC) is a lytic attack on antibody-targeted cells that is triggered by the binding of lymphocyte receptors (FcγRs) to the antibody constant region (Fc). ADCC is considered to be one of the major effector functions of therapeutic antibodies.
FcγRIIIa, the FcγR predominantly expressed on natural killer cells and responsible for ADCC activation, has two isoforms, 158Val and 158Phe. The FcγRIIIa/158V allele shows higher binding affinity for IgG1 antibody compared to the FcγRIIIa/158F isoform (Wu et al., 1997, Shields et al., 2001). Importantly, Cartron et al. (2002) have reported recently that the anti-CD20 chimeric IgG1 antibody Rituxan was more effective for follicular non-Hodgkin lymphoma patients with FcγRIIIa/158Val than patients with FcγRIIIa/158Phe. Similar results have been reported by Anolik et al. (2003) for Phase I/II trials of Rituxan treatment of systemic lupus erythematosus. These reports underscore the importance of ADCC of IgG1 in the clinic and highlight the therapeutic value of enhancing ADCC effector function.
Several groups have reported that ADCC enhancement can be achieved by manipulating human IgG1 subclass antibody oligosaccharides. ADCC requires the presence of oligosaccharides in the Fc region and is sensitive to change in the oligosaccharide structure (Nose and Wigzell, 1983, Wright and Morrison, 1997, Jefferis et al., 1998). One IgG molecule contains two asparagine (N)-linked oligosaccharide sites in its Fc region (Rademacher et al., 1986). The general structure of IgG N-linked oligosaccharides is complex-type, characterized by a mannosylchitobiose core with or without bisecting N-acetylglucosamine (GlcNac)/l-fucose and other chain variants including the presence or absence of galactose and sialic acid. Among all of the sugar components in the oligosaccharide, galactose (Kumpel et al., 1994, Kumpel et al., 1995), bisecting-GlcNac (Umana et al., 1999, Davies et al., 2001), and fucose (Shields et al., 2002, Shinkawa et al., 2003) have been reported to affect ADCC.
We have clarified previously the critical importance of fucose among these sugar components. Removal of fucose from humanized anti-interleukin 5 receptor antibody or chimeric anti-CD20 antibody enhanced their ADCC > 50-fold (Shinkawa et al., 2003). Compared with fucose manipulation, the role of bisecting-GlcNac in ADCC was minimal, and galactose modifications did not contribute significantly to ADCC enhancements (Shinkawa et al., 2003). The influence of non-fucosylated oligosaccharide on ADCC has also been reported by Shields et al. (2002) using humanized anti-HER2 IgG1 and humanized anti-IgE IgG1. They showed that the reduction of fucose on IgG1s improved both ADCC and binding to FcγRIIIa. Fucose removal from human IgG1-type antibody is, thus far, one of the most powerful ways to improve antibody effector function. Further, we have demonstrated the superiority of antibodies with enhanced ADCC in vivo. Low fucose chimeric anti-CCR4 IgG1 antibody showed significantly higher anti-tumor activity than the highly fucosylated antibody in a murine xenograft model employing a CC chemokine receptor 4 (CCR4)-positive T-cell lymphoma and human peripheral blood mononuclear cells (PBMCs) (Niwa et al., 2004).
Some cell lines, such as YB2/0 (Shinkawa et al., 2003), had been used to produce IgG molecules with reduced fucose by 30–90%. To generate a new host cell producing a homogeneous product with optimal ADCC enhancement and more appropriate for large-scale manufacture, we disrupted both FUT8 alleles in a Chinese hamster ovary (CHO) cell line by sequential homologous recombination (Yamane-Ohnuki et al., 2004). This cell line is incapable of adding fucose to antibodies as FUT8 encodes an α-1,6-fucosyltransferase that is the only enzyme to catalyze the transfer of fucose from GDP-fucose to N-acetylglucosamine (GlcNac) in an α-1,6 linkage. Importantly, from a manufacturing standpoint, the FUT8−/− cell lines (CHO/FUT8−/−) have morphology and growth kinetics similar to those of the parent, and produce completely fucose-negative recombinant antibodies.
Several groups reported that Fc-fusion molecules, which consist of target binding domains and Fc portion (hinge, CH2 and CH3 domain) of human IgG antibody, have ADCC activity (Shu et al., 1993, Cooper et al., 2003, Heuser et al., 2003, Lorenzo et al., 2004). Single-gene-encoded scFv-Fc is a type of Fc-fusions which contains single-chain Fv (scFv) as a target binding domain (Fig. 1). scFvs are the Fvs designed as single-peptide molecules which consists of the variable region of antibody heavy chain (VH), the variable region of antibody light chain (VL) and peptide-linker. Because of its small size (110 kDa), scFv-Fc molecules are considered to have higher penetration into bulky tumors than whole IgG molecules (150 kDa). Another merit of scFv-Fcs is that they retain immune effector functions mediated by Fc domains. For example, Shu et al. (1993) reported that scFv-Fc which contains scFv from monoclonal antibody CC49, a mouse IgG1, have ADCC activity. CC49 reacts with tumor associated glycoprotein (TAG)-72 expressed on a variety of carcinomas.
Improvement of FcγRIIIa binding and consequent ADCC-enhancement by fucose depletion has been verified for only whole IgG1 molecule. In this study, we produced the fucose-negative anti-TAG-72 scFv-Fc, scFvT-Fc(−), using a CHO/FUT8−/− cell line, and a conventional fucosylated anti-TAG-72 scFv-Fc, scFvT-Fc(+), with a CHO cell line. Then we investigated the effect of the absence of fucose on the effector function of scFv-Fc molecule.
Section snippets
Cell lines
CHO cell line DG44 (Urlaub et al., 1986) was kindly provided by Dr. Lawrence Chasin (Columbia University). CHO/FUT8−/−, a FUT8 knockout cell line for fucose-negative scFv-Fc production, has been described previously (Yamane-Ohnuki et al., 2004). TAG-72-positive human acute T cell leukemia cell line Jurkat [American type Culture Collection (ATCC) TIB-152] was purchased from ATCC. TAG-72-negative human B lymphocytic Burkitt's lymphoma cell line Raji [Japanese Collection of Research Bioresources
Production and characterization of anti-TAG-72 scFv-Fc
We have demonstrated previously that fucose modification is the most critical IgG1 oligosaccharide component for ADCC enhancement, and the removal of fucose from IgG1 oligosaccharides results in a very significant increase of ADCC in vitro (∼ 100-fold) (Shinkawa et al., 2003, Niwa et al., 2004, Okazaki et al., 2004). In this study, the effect of fucose removal on the ADCC of single-gene-encoded scFv-Fc was determined. We generated two chimeric anti-TAG-72 scFv-Fc produced by either CHO/FUT8−/−
Discussion
Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments (McCafferty et al., 1990). For many applications, however, it is useful to restore Fc mediated antibody functions such as avidity, effector functions and a prolonged serum half-life (Shu et al., 1993, Shu-Lian et al., 2000). Powers et al. demonstrated in mice that the increased size of scFv-Fc (approximately 100 kDa) results in a prolonged serum half-life in vivo, with t1/2 of the beta
Acknowledgments
We thank Dr. Philip Wallace for helpful suggestions and critical reading of the manuscript.
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