title: Development of a cell-based assay measuring the activation of FcγRIIa for the characterization of therapeutic monoclonal antibodies.
abstract: Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Tada
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Ishii-Watabe
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Takuo
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
mesh_terms: D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized / Q000627:therapeutic use; D058846:Antibodies, Monoclonal, Murine-Derived / Q000627:therapeutic use; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D000068818:Cetuximab; D005434:Flow Cytometry; D006801:Humans; D019169:Jurkat Cells; D000077544:Panitumumab; D017452:Receptors, IgG / Q000378:metabolism*; D000069283:Rituximab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; C080568:Fc gamma receptor IIA; D017452:Receptors, IgG; D000069283:Rituximab; D000077544:Panitumumab; D000068818:Cetuximab
references.citation: BMC Proc. 2011 Nov 22;5 Suppl 8:P63
references.pmid: 22373329
references.citation: Curr Protoc Immunol. 2001 May;Chapter 7:Unit 7.27
references.pmid: 18432838
references.citation: Nat Rev Immunol. 2008 Jan;8(1):34-47
references.pmid: 18064051
references.citation: Blood. 2002 Feb 1;99(3):754-8
references.pmid: 11806974
references.citation: J Mol Recognit. 1999 Sep-Oct;12(5):279-84
references.pmid: 10556875
references.citation: Am J Health Syst Pharm. 2013 Dec 15;70(24):2162
references.pmid: 24296835
references.citation: Blood. 2011 Oct 13;118(15):4159-68
references.pmid: 21795744
references.citation: MAbs. 2013 Mar-Apr;5(2):229-36
references.pmid: 23406628
references.citation: MAbs. 2012 May-Jun;4(3):310-8
references.pmid: 22531445
references.citation: J Clin Oncol. 2007 Aug 20;25(24):3712-8
references.pmid: 17704420
references.citation: J Immunol. 2010 Jan 1;184(1):512-20
references.pmid: 19949082
references.citation: N Engl J Med. 2006 Sep 7;355(10):1018-28
references.pmid: 16908486
references.citation: MAbs. 2010 May-Jun;2(3):233-55
references.pmid: 20421713
references.citation: Nat Rev Immunol. 2006 May;6(5):343-57
references.pmid: 16622479
references.citation: J Biomed Biotechnol. 2011;2011:379123
references.pmid: 21660134
references.citation: Blood. 2000 Feb 1;95(3):1069-77
references.pmid: 10648424
references.citation: Drugs. 2012 Jun 18;72(9):1293-8
references.pmid: 22686619
references.citation: Mol Cancer Ther. 2008 Aug;7(8):2517-27
references.pmid: 18723496
references.citation: Drugs. 1996 May;51(5):865-94
references.pmid: 8861551
references.citation: J Clin Oncol. 2003 Nov 1;21(21):3940-7
references.pmid: 12975461
references.citation: Crit Rev Ther Drug Carrier Syst. 1993;10(4):307-77
references.pmid: 8124728
references.citation: Cancer Immun. 2012;12:14
references.pmid: 22896759
references.citation: Mol Immunol. 2011 Jul;48(12-13):1512-7
references.pmid: 21570725
references.citation: J Immunol Methods. 2012 Nov 30;385(1-2):45-50
references.pmid: 22914441
references.citation: Biotechnol Bioeng. 2001 Aug 20;74(4):288-94
references.pmid: 11410853
references.citation: Biotechnol Bioeng. 2008 Feb 15;99(3):652-65
references.pmid: 17680659
references.citation: Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4005-10
references.pmid: 16537476
references.citation: Mol Immunol. 2011 Mar;48(6-7):860-6
references.pmid: 21256596
references.citation: MAbs. 2014 Mar-Apr;6(2):409-21
references.pmid: 24492248
references.citation: J Biol Chem. 2003 Jan 31;278(5):3466-73
references.pmid: 12427744
references.citation: Nat Rev Immunol. 2010 May;10(5):317-27
references.pmid: 20414205
references.citation: Transplantation. 1997 Jan 15;63(1):106-12
references.pmid: 9000670
references.citation: J Immunol. 2010 Feb 15;184(4):1968-76
references.pmid: 20083659
references.citation: Expert Rev Clin Immunol. 2009 Sep;5(5):499-521
references.pmid: 20477639
references.citation: Blood. 2012 Mar 15;119(11):2533-44
references.pmid: 22138510
references.citation: Immunogenetics. 2012 Dec;64(12):869-77
references.pmid: 22903236
references.citation: Biotechnol Bioeng. 2006 Apr 5;93(5):851-61
references.pmid: 16435400
references.citation: Drug Discov Today. 2007 Nov;12(21-22):898-910
references.pmid: 17993407
references.citation: Mol Immunol. 2009 May;46(8-9):1878-82
references.pmid: 19269032
references.citation: Protein Sci. 2009 Feb;18(2):424-33
references.pmid: 19165723
references.citation: Expert Opin Biol Ther. 2006 Nov;6(11):1161-73
references.pmid: 17049014
references.citation: J Biol Chem. 2002 Jul 26;277(30):26733-40
references.pmid: 11986321
references.citation: Curr Pharm Biotechnol. 2008 Dec;9(6):468-81
references.pmid: 19075686
references.citation: Cell Immunol. 2000 Feb 25;200(1):16-26
references.pmid: 10716879
references.citation: Blood. 2012 Mar 15;119(11):2432-3
references.pmid: 22422810
references.citation: Curr Opin Biotechnol. 2009 Dec;20(6):685-91
references.pmid: 19896358
references.citation: Immunity. 2008 Jun;28(6):833-46
references.pmid: 18538590
references.citation: Biochemistry. 2008 May 6;47(18):5088-100
references.pmid: 18407665
references.citation: Blood. 2009 Apr 16;113(16):3716-25
references.pmid: 19018092