Fcγr Binding and ADCC Activity of Human Igg Allotypes
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fimmu-11-00740 May 4, 2020 Time: 17:25 # 1 ORIGINAL RESEARCH published: 06 May 2020 doi: 10.3389/fimmu.2020.00740 FcgR Binding and ADCC Activity of Human IgG Allotypes Steven W. de Taeye1,2*†, Arthur E. H. Bentlage2, Mirjam M. Mebius3, Joyce I. Meesters3, Suzanne Lissenberg-Thunnissen2, David Falck4, Thomas Sénard4, Nima Salehi1, Manfred Wuhrer4, Janine Schuurman3, Aran F. Labrijn3, Theo Rispens1‡ and Gestur Vidarsson2‡ 1 Sanquin Research and Landsteiner Laboratory, Department of Immunopathology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands, 2 Sanquin Research and Landsteiner Laboratory, Department of Experimental Edited by: Immunohematology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands, 3 Genmab, Utrecht, Eric O. Long, Netherlands, 4 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands National Institute of Allergy and Infectious Diseases (NIAID), United States Antibody dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function Reviewed by: of IgG important for anti-viral immunity and anti-tumor therapies. NK-cell mediated Amy W. Chung, ADCC is mainly triggered by IgG-subclasses IgG1 and IgG3 through the IgG-Fc- The University of Melbourne, Australia receptor (FcgR) IIIa. Polymorphisms in the immunoglobulin gamma heavy chain gene Geoffrey Thomas Hart, University of Minnesota Twin Cities, likely form a layer of variation in the strength of the ADCC-response, but this has United States never been studied in detail. We produced all 27 known IgG allotypes and assessed *Correspondence: FcgRIIIa binding and ADCC activity. While all IgG1, IgG2, and IgG4 allotypes behaved Steven W. de Taeye [email protected]; similarly within subclass, large allotype-specific variation was found for IgG3. ADCC [email protected]; capacity was affected by residues 291, 292, and 296 in the CH2 domain through altered [email protected] affinity or avidity for FcgRIIIa. Furthermore, allotypic variation in hinge length affected † Present address: ADCC, likely through altered proximity at the immunological synapse. Thus, these Steven W. de Taeye, Department of Medical Microbiology, functional differences between IgG allotypes have important implications for therapeutic Amsterdam UMC, University of applications and susceptibility to infectious-, allo- or auto-immune diseases. Amsterdam, Amsterdam, Netherlands ‡These authors have contributed Keywords: antibodies, IgG polymorphism, Fc gamma receptor, antibody dependent cellular cytotoxicity, glycosylation equally to this work Specialty section: This article was submitted to INTRODUCTION NK and Innate Lymphoid Cell Biology, a section of the journal In the defense against invading pathogens, Immunoglobulins are formed by B cells. These bind the Frontiers in Immunology pathogen via their Fab domains and subsequently activate both complement but also immune cells Received: 17 January 2020 by immunoglobulin Fc-receptors (1). The largest portion are Immunoglobulin g (IgG) antibodies Accepted: 01 April 2020 that mediate their effector functions through Fc gamma receptors (FcgR) on myeloid and Natural Published: 06 May 2020 Killer (NK) cells (2–4). Antibody dependent cellular cytotoxicity (ADCC) is one of the major Citation: Fc-dependent effector functions of IgG, that is particularly important in the clearance of viral de Taeye SW, Bentlage AEH, infections and is mostly mediated by NK cells (4–6). NK cells mediate ADCC through binding Mebius MM, Meesters JI, of antibody opsonized target cells by membrane expressed FcgRIIIa and induce cytotoxicity by Lissenberg-Thunnissen S, Falck D, releasing granzymes and perforins stored in intracellular granules (7,8). This is an important Sénard T, Salehi N, Wuhrer M, effector mechanism that contributes to the killing of tumor cells upon immunotherapy (9, 10). Schuurman J, Labrijn AF, Rispens T and Vidarsson G (2020) Fcγ R Binding The four IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are defined by unique structural and and ADCC Activity of Human IgG functional characteristics. Besides variation in terms of half-life, antigen binding and Fab-arm Allotypes. Front. Immunol. 11:740. exchange, the subclasses differ in their capacity to activate the complement system or bind FcgR doi: 10.3389/fimmu.2020.00740 on immune effector cells (2, 11). This variation in responses allows for the generation of tailored Frontiers in Immunology| www.frontiersin.org 1 May 2020| Volume 11| Article 740 fimmu-11-00740 May 4, 2020 Time: 17:25 # 2 de Taeye et al. IgG Polymorphisms Influence ADCC Activity antigen-specific responses with optimal effector functions (2, 12– specific IgG3 antibodies crossing the placental membrane more 15). The strength of ADCC activity is not only influenced by efficiently as a result of increased binding to FcRn (41, 42). the IgG subclass, but also by N297-linked glycosylation of the To capture the structural and functional diversity between antibody, and FcgR polymorphisms (16–20). IgG allotypes, we generated the complete set of IgG allotypes IgG-heavy chain polymorphisms, IgG allotypes, form that have been described to date and compared their binding to another layer of variation. Although it is likely that these FcgR and capacity to induce ADCC in vitro. Whereas allotypic polymorphisms influence functional and structural features variants within the IgG1, IgG2 and IgG4 subclass did not change of the IgG subclasses, this has never been systematically binding to FcgR or ADCC, the differences in hinge length and studied (2). IgG polymorphic variants were first detected with CH2 domain between IgG3 allotypes affected FcgRIIIa binding serological tests, classically by hemagglutination inhibition and ADCC substantially. Understanding the functional diversity (21–23). The identification of IgG polymorphisms with within IgG subclasses may shed light on associations found serological tests was a powerful tool to study population with infectious diseases or auto-immune diseases and potentially variations (24). Later, sequencing of the immunoglobulin initiate new strategies to improve therapeutic antibodies. genes of different ethnic groups revealed that even more polymorphic variants are present that could not be detected with serological testing, in particular for IgG3 (2, 22, 25). MATERIALS AND METHODS IgG polymorphisms were found to be associated with infectious-and auto-immune diseases in multiple studies Cell Lines (26–32), suggesting that IgG allotypes may affect the humoral FreeStyle Expi293F cells were cultured in FreeStyle 293 antibody response. However, the structural or functional expression medium according to the manufacturer’s instructions characteristics of IgG allotypes underlying these associations (Invitrogen). Additional cell lines were obtained from the remain to be elucidated. American Type Culture Collection (ATCC). Raji (human CD20- The fact that serological tests are used to determine IgG positive Burkitt’s lymphoma) cells were cultured in RPMI allotypes, indicates that the amino acid variation in the 1640 medium (Lonza), supplemented with 10% heat-inactivated antibody constant domain is an immunogenic determinant Donor Bovine Serum with Iron (DBSI; Life Technologies), 4 mM and that therapeutic antibodies potentially induce an anti- L-glutamine, 25 mM Hepes and 1 mM Sodium Pyruvate (Lonza). allotype response, in the case of an allotype mismatch (33). Wien-133 (human CD52-positive Burkitt’s lymphoma) cells were However, patients treated with IgG1 allotype mismatched anti- cultured in Iscove’s Modified Dulbeco’s Medium (IMDM) with TNF therapeutic antibodies were not found to raise anti-allotype HEPES and L-Glutamine (Lonza), supplemented with 10% heat- ◦ antibodies, suggesting that allotypic variation is only a minor inactivated DBSI. All cell lines were maintained at 37 C in a 5% immunogenic determinant (34, 35). IgG3 allotypic variants might CO2 humidified incubator. be more immunogenic, as more amino acid variation is present between allotypes. Cloning and Production of Anti-RhD and IgG allotypes were found to correlate with plasma IgG Anti-TNP Antibodies level, most likely as a result of variation in the non-coding All IgG polymorphic variants known at the initiation of the switch regions or unfavorable RNA transcripts. In addition, study, deposited in the IMGT-web site, and described previously IgG polymorphisms may also be associated with class-switching by Vidarsson et al. (2) were cloned into a pEE6.4 expression efficiency and thereby serum concentrations, through variations vector containing an anti-Rhesus D (anti-RhD) heavy chain within the non-coding switch regions inherited haplotype of a previously described clone (43). Restriction sites flanking (36, 37). Recently IgG1 allotypic variants were found to be all individual IgG constant domains (CH1, hinge, CH2 and associated with the subclass distribution (IgG1/IgG2) of an HIV- CH3) were introduced to allow the exchange of allotype specific specific antibody response, illustrating the association of IgG gene fragments (Integrated DNA technologies) and generate all polymorphisms with IgG class switching (38). the different polymorphic variants. From the anti-RhD allotype In addition to the association of IgG allotypes with antibody constructs, all 27 IgG allotype constant domains were also cloned expression and IgG class switching in B-cells, the Fc-mediated in a pcDNA3.1 vector containing the anti-TNP heavy chain effector functions are also likely to be different between IgG by swapping all polymorphic variant constant