Research paper
Fucose 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

https://doi.org/10.1016/j.jim.2005.07.025Get rights and content

Abstract

Fucose removal from complex-type oligosaccharide of human IgG1-type antibody 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 a single-gene-encoded antibody with an scFv used as the binding domain. We generated both a fucose-negative anti-tumor associated glycoprotein (TAG)-72 scFv-Fc using α-1,6-fucosyltransferase knock-out CHO cells and a highly fucosylated scFv-Fc from parental CHO cells. Expression, assembly and antigen binding activity of the scFv-Fcs were not influenced by fucose removal. The scFv-Fc lacking fucose exhibited significantly more potent FcγRIIIa binding and ADCC compared to highly fucosylated scFv-Fc. These results prove that ADCC enhancement by fucose-removal is effective in not only whole IgG1, but also scFv-Fc, and thus increases the potential of Fc-fusion proteins as therapeutic candidates.

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.

References (29)

  • J.C. Cooper et al.

    Alefacept selectively promotes NK cellular deletion of CD45R0+ human T cells

    Eur. J. Immunol.

    (2003)
  • J. Davies et al.

    Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FcγRIII

    Biotechnol. Bioeng.

    (2001)
  • S.R. Hamilton et al.

    Production of complex human glycoproteins in yeast

    Science

    (2003)
  • C. Heuser et al.

    An anti-MUC1-antibody-interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells

    Br. J. Cancer

    (2003)
  • Cited by (53)

    • Human IgG Glycosylation in Inflammation and Inflammatory Disease

      2021, Comprehensive Glycoscience: Second Edition
    • Engineered Sialylation of Pathogenic Antibodies In Vivo Attenuates Autoimmune Disease

      2018, Cell
      Citation Excerpt :

      Studies over the last decade have demonstrated the composition of the Fc glycan exerts profound influence over IgG effector functions (Jefferis, 2005, 2009a, 2009b). IgG with afucosylated Fc glycans have 50-fold enhanced affinity to the activating FcγR, FcγRIIIA, compared to fucosylated IgG and exhibit markedly enhanced ADCC in vivo (Ferrara et al., 2011; Natsume et al., 2005; Okazaki et al., 2004; Shields et al., 2002). Recent studies have linked dengue-specific IgG with afucosylated Fc glycans with Dengue hemorrhagic fever (Wang et al., 2017) and tuberculosis (TB)-specific afucosylated IgG in controlling latent TB infections (Lu et al., 2016).

    • Biosimilars from a practicing rheumatologist perspective: An overview

      2016, Autoimmunity Reviews
      Citation Excerpt :

      See Figs. 2–4.) Glycans can be responsible for the stability of the immunoglobulin structure [7,8], can have profound influence on binding to Fc receptors on effector cells such as antibody-dependent cellular cytotoxicity (ADCC) [9,10] and on immune mediators such as complement-dependent cytotoxicity (CDC). Binding of the C1q to the Fc domain requires the presence of at least two N-acetylglucosamines with multiple galactose and sialic acids and mutated aglycosylated IgG molecules have markedly reduced binding of FcγR [11].

    • In vivo anti-tumor efficacy of afucosylated anti-CS1 monoclonal antibody produced in glycoengineered Pichia pastoris

      2015, Journal of Biotechnology
      Citation Excerpt :

      Currently, strategies involving modification of antibodies to enhance FcγRIIIA binding either by selective mutations or afucosylation are in progress. The enhancement of ADCC due to afucosylation has been shown to be relevant for all human IgG subclasses (Niwa et al., 2004), scFv-Fc proteins (Natsume et al., 2005, 2006) and Fc-fusion proteins (Shoji-Hosaka et al., 2006). Afucosylated antibodies can be produced by inactivation of the fucosylation pathway, which has been demonstrated by fucosyltransferase (FUT8) knockout in Chinese Hamster Ovary (CHO) (Yamane-Ohnuki et al., 2004; Mori et al., 2004).

    View all citing articles on Scopus
    View full text