Enhanced binding affinity for FcγRIIIa of fucose-negative antibody is sufficient to induce maximal antibody-dependent cellular cytotoxicity
Introduction
Recently, monoclonal antibodies with specific affinity to target antigens have been widely developed and emerged as an important class of therapeutic agent (Janice et al., 2005). Despite this, the therapeutic mechanism of many, if not all, monoclonal antibodies are still controversial. The view is widely held that antibody-dependent cellular cytotoxicity (ADCC) (Reff et al., 1994, Clynes et al., 2005, Manches et al., 2003), complement-dependent cytotoxicity (CDC) (Golay et al., 2000, Di Gaetona et al., 2003, Cragg and Glennie, 2004), apoptosis induction (Shan et al., 2000, Tutt et al., 1998, Hofmeister et al., 2000) may contribute to the therapeutic effects of antibodies, particularly the most studies, the anti-CD20 chimeric antibody rituximab. The clearest mechanistic evidence gleaned from patients highlights the critical role of ADCC (Gennari et al., 2004, Dall’Ozzo et al., 2004, Weng and Levy, 2003, Anolik et al., 2003, Carton et al., 2002) ADCC is triggered following binding of leukocyte receptors (FcγRs) to the antibody constant region (Fc). FcγRs expressed on the leukocyte are composed of three distinct classes: FcγRI (CD64); FcγRII (CD32), including isoforms FcγRIIa, FcγRIIb, and FcγRIIc; FcγRIII (CD16), including isoforms FcγRIIIa and FcγRIIIb (Heijnen and Van de Winkel, 1997, Ravetch, 1997). While FcγRI, FcγRIIa/c and FcγRIIIa are activating receptors, FcγRIIb is an inhibitory receptor. Natural killer (NK) cells, the key effector cells for ADCC, express the activating receptors FcγRIIIa and FcγRIIc. FcγRIIIa is commonly expressed on NK cells but, in contrast, the expression of FcγRIIc on NK cells is dependent on an allelic polymorphism of the gene and is seen in 45% of normal individuals (Ernst et al., 2002). This FcγR expression profile of NK cells leads to the notion that monoclonal antibodies with increased affinity for FcγRIIIa will exhibit improved therapeutic efficacy via enhanced NK cells activation and ADCC.
It has been shown that carbohydrate engineering (Shinkawa et al., 2003, Shileds et al., 2002, Kumpel et al., 1995, Davis et al., 2001, Ripka and Stanley, 1986) and amino acid mutagenesis (Shileds et al., 2001, Greg et al., 2006) increase antibody affinity to the FcγRIIIa and enhance ADCC. The carbohydrates of the Fc region at Asn-297 are crucial to the affinity for FcγRIIIa (Tao and Morrison, 1989, Mimura et al., 2001) and are required to elicit ADCC (Wright and Morrison, 1994, Sarmay et al., 1992). Shinkawa et al. reported that the removal of fucose from the carbohydrates was found to dramatically enhance ADCC of the therapeutic antibody via improved FcγRIIIa binding (Shinkawa et al., 2003). By using anti-CD20 chimeric antibody (rituximab) and an anti-CC chemokine receptor 4 chimeric antibody, antibodies without fucose (fucose-negative) showed almost 100-fold higher ADCC (Niwa et al., 2004a) and significantly higher in vivo anti-tumor activity than fucosylated antibody (Niwa et al., 2004b). Furthermore, fucose-negative antibodies showed potent ADCC at lower antigen density on target cells that conventional, fucosylated antibody could not induce detectable ADCC (Niwa et al., 2005). These results indicate that fucose-negative antibodies with enhanced ADCC are promising candidates as the novel antibody-based therapeutics. Similarly, it has been demonstrated that certain unnatural amino acid mutations in the Fc region also improve binding to the FcγRIIIa and result in higher ADCC without the requirement to remove fucose from the carbohydrate structure (Shileds et al., 2001, Greg et al., 2006).
In the present study, we constructed two types of triple amino acids mutant anti-CD20 chimeric antibodies with fucose attached carbohydrate, and compared their binding affinities to FcγRIIIa and ADCC with fucose-negative wild type antibody. As a previous report indicate that amino acid substitutions and fucose removal in the Fc region may have a synergistic effect on the improvement of FcγRIIIa binding affinity (Shileds et al., 2001), we also constructed an antibody that combined fucose removal and mutagenesis. Here we present the data of in vitro FcγRs binding profiles, ADCC, NK cells binding and activation, and ex vivo ADCC of a series of engineered antibody variants with improved FcγRIIIa affinity.
Section snippets
Cell lines
CHO cell line DG44 was kindly provided by Dr. Lawrence Chasin (Columbia University, New York, NY). FUT 8 KO CHO cell line Ms705 was established from DG44 as previously reported (Yamane-Onuki et al., 2004). CD20-positive human B lymphoma cell line Daudi (ATCC; CCL-213) and Raji (ATCC; CCL-86) were purchased from the American Type Culture Collection. Mouse T-cell lymphoma cell line EL4 (ATCC; TIB-39) was purchased from the American Type Culture Collection, and an EL4 cell line expressing human
Generation and characterization of chimeric anti-CD20 antibodies
To compare the two methods to enhance ADCC, we generated chimeric anti-CD20 antibodies with: (i) two different sets of amino acid mutations in their Fc regions, (ii) without fucose residues in their N-linked oligosaccharides and (iii) combinations of both enhancement methods. The resultant antibodies were designated as follows: WTF, fucosylated antibody with wild type Fc region; WTN, fucose-negative antibody with wild type Fc region; 298F, fucosylated antibody with triple amino acid mutations
Discussion
A recent report revealed that removal of fucose from N-linked oligosaccharides or amino acid mutations in the Fc region of antibody increases FcγRIIIa binding and enhanced ADCC. In the present study, we directly compared in vitro activities of fucose-negative wild type antibody, fucosylated antibodies with amino acid mutations and combinations of the two enhancement methods using a chimeric anti-CD20 antibody as the model. We generated a completely fucose-negative antibody using the FUT8−/− CHO
Acknowledgements
We thank Dr. Philip Wallace and Dr. George Spitalny for helpful suggestions and critical reading of the manuscript.
References (43)
- et al.
Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen
J. Mol. Biol.
(1999) - et al.
The carbohydrate at FcgammaRIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms
J. Biol. Chem.
(2006) - et al.
Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis
Blood
(2000) - et al.
Clustered CD20 induced apoptosis: Src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis
Blood Cells Mol. Dis.
(2000) - et al.
Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype
Blood
(1997) - et al.
In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas
Blood
(2003) - et al.
Role of oligosaccharide residues of IgG1-Fc in Fc gamma RIIb binding
J. Biol. Chem.
(2001) - et al.
Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcgammaRIIIa
J. Mol. Biol.
(2004) Fc receptors
Curr. Opin. Immunol.
(1997)- et al.
Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20
Blood
(1994)
Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc gamma receptor
Mol. Immunol.
The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity
J. Biol. Chem.
The relationship of FcgammaRIIIa genotype to degree of B cell depletion by rituximab in the treatment of systemic lupus erythematosus
Arthritis Rheum.
Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene
Blood
Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets
Nat. Med.
Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents
Blood
Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship
Cancer Res.
Complement activation determines the therapeutic activity of rituximab in vivo
J. Immunol.
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 FC gamma RIII
Biotechnol. Bioeng.
Allelic polymorphisms in the FcgammaRIIC gene can influence its function on normal human natural killer cells
J. Mol. Med.
Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumors overexpressing HER2
Clin. Cancer Res.
Cited by (152)
Neutrophils as potential therapeutic targets for breast cancer
2023, Pharmacological ResearchSerum immunoglobulin and the threshold of Fc receptor-mediated immune activation
2023, Biochimica et Biophysica Acta - General SubjectsIgG glycans in health and disease: Prediction, intervention, prognosis, and therapy
2023, Biotechnology Advances