IMGT® Nomenclature of Engineered IGHG Variants Involved in Antibody Effector Properties and Formats

The constant region of the immunoglobulin (IG) or antibody heavy gamma chain is frequently engineered to modify the effector properties of the therapeutic monoclonal antibodies. These variants are classified in regards to their effects on effector functions, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) enhancement or reduction, B cell inhibition by the coengagement of antigen and FcγR on the same cell, on half-life increase, and/or on structure such as prevention of IgG4 half-IG exchange, hexamerisation, knobs-into-holes and the heteropairing H-H of bispecific antibodies, absence of disulfide bridge inter H-L, absence of glycosylation site, and site-specific drug attachment engineered cysteine. The IMGT engineered variant identifier is comprised of the species and gene name (and eventually allele), the letter ‘v’ followed by a number (assigned chronologically), and for each concerned domain (e.g, CH1, h, CH2 and CH3), the novel AA (single letter abbreviation) and IMGT position according to the IMGT unique numbering for the C-domain and between parentheses, the Eu numbering. IMGT engineered variants are described with detailed amino acid changes, visualized in motifs based on the IMGT numbering bridging genes, sequences, and structures for higher order description.


Introduction
The adaptive immune response, acquired by jawed vertebrates (or gnathostomata) more than 450 million years ago and found in all extant jawed vertebrate species from fish to humans, is characterized by a remarkable immune specificity and memory, which are the properties of the B and T cells because of the extreme diversity of their antigen receptors [1]. The antigen receptors of the adaptive immune response [1,2] comprise the immunoglobulins (IG) or antibodies of the B cells and plasmocytes [3,4] and the T cell receptors (TR) of the T cells [5]. The IG recognizes antigens in their native (unprocessed) form, whereas the TR recognizes processed antigens, which are presented as peptides through its highly polymorphic major histocompatibility (MH, in humans HLA for human leucocyte antigens) proteins [6]. Immunoglobulins (IG) or antibodies serve a dual role in immunity. First, they both recognize antigens on the surface of foreign bodies such as bacteria and viruses, and second, they trigger elimination mechanisms such as cell lysis and phagocytosis to rid the body of these invading cells and particles [4]. IMGT ® , the international ImMunoGeneTics information system ® (https://www.imgt.org) (accessed on 11 October 2022) [1], was created in 1989 by Marie-Paule Lefranc in Montpellier, France, Laboratoire d'ImmunoGénétique Moléculaire (LIGM) des Prof G. and M-P. Lefranc (Université de Montpellier and CNRS) to manage the huge diversity of the IG and TR repertoires. For the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J) and constant (C) genes were officially recognized as 'genes' and conventional genes [1,3,5,[7][8][9][10]. Through its creation, IMGT ® marks the advent of a new science, immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics [1]. As an ontology and system, IMGT ® bridges genes, sequences and structures of the antigen receptors to better understand their functions. Focusing on the constant region of the IgG, a standardized definition of engineered variants of therapeutic antibodies is provided based on the IMGT concepts.
Antibodies 2022, 11, x FOR PEER REVIEW 2 of 40 toires. For the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J) and constant (C) genes were officially recognized as 'genes' and conventional genes [1,3,5,[7][8][9][10]. Through its creation, IMGT ® marks the advent of a new science, immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics [1]. As an ontology and system, IMGT ® bridges genes, sequences and structures of the antigen receptors to better understand their functions. Focusing on the constant region of the IgG, a standardized definition of engineered variants of therapeutic antibodies is provided based on the IMGT concepts.

IMGT Databases
IMGT Tools IMGT Web Resources Figure 2. Immunoglobulin IgG1. The structure is that of the antibody b12, an IgG1-kappa, and so far is the only complete human IG crystallized (PDB code: 1hzh, from IMGT ® https://www.imgt.org, IMGT/3Dstructure-DB). H-GAMMA-1 and L-KAPPA (usedfor the chains), VH, CH1, CH2, CH3, V-KAPPA and C-KAPPA (for the domains) are written in capital letters as they are IMGT standardized labels (DESCRIPTION) [1]. This first 3D-structure of a complete Homo sapiens IG shows the expected Y shape with the two Fragment antigen binding (Fab) arms (one L-KAPPA light chain (V-KAPPA-C-KAPPA) paired to the VH-CH1 of each H-GAMMA-1 heavy chain) and the Fragment crystallisable (Fc), made of the paired hinge-CH2-CH3 of the two H-GAMMA-1 heavy chains. The figure also shows the relative position, in space, of the L-KAPPA relative to the VH-CH1 in each Fab (in the front on the left hand side, and the back right hand side). The sequences of the two H-GAMMA1 chains (colored in purple and dark blue for a better visibility) are identical and the sequences of the two L-KAPPA chains (colored in orange and green for a better visibility) are identical (with permission from M-P. Lefranc and G. Lefranc, LIGM, Founders of IMGT ® , the international ImMunoGe-neTics information system ® , https://www.imgt.org). Table 3. The immunoglobulin IgG1 receptor, chain and domain structure labels and correspondence with sequence labels. IMGT standardized labels are in capital letters. They are shown with the example Homo sapiens IgG1-kappa. . Immunoglobulin IgG1. The structure is that of the antibody b12, an IgG1-kappa, and so far is the only complete human IG crystallized (PDB code: 1hzh, from IMGT ® https://www.imgt.org, IMGT/3Dstructure-DB). H-GAMMA-1 and L-KAPPA (usedfor the chains), VH, CH1, CH2, CH3, V-KAPPA and C-KAPPA (for the domains) are written in capital letters as they are IMGT standardized labels (DESCRIPTION) [1]. This first 3D-structure of a complete Homo sapiens IG shows the expected Y shape with the two Fragment antigen binding (Fab) arms (one L-KAPPA light chain (V-KAPPA-C-KAPPA) paired to the VH-CH1 of each H-GAMMA-1 heavy chain) and the Fragment crystallisable (Fc), made of the paired hinge-CH2-CH3 of the two H-GAMMA-1 heavy chains. The figure also shows the relative position, in space, of the L-KAPPA relative to the VH-CH1 in each Fab (in the front on the left hand side, and the back right hand side). The sequences of the two H-GAMMA1 chains (colored in purple and dark blue for a better visibility) are identical and the sequences of the two L-KAPPA chains (colored in orange and green for a better visibility) are identical (with permission from M-P. Lefranc and G. Lefranc, LIGM, Founders of IMGT ® , the international ImMunoGeneTics information system ® , https://www.imgt.org).
In the IMGT system, the C-domain includes the C-DOMAIN of the IG and of the TR [1] and the C-LIKE-DOMAIN of the IgSF other than IG and TR [22]. The C-domain description of any receptor, any chain and any species is based on the IMGT unique numbering for the C-domain (C-DOMAIN and C-LIKE-DOMAIN) [68]. A C-domain ( Figure 3) comprises about 90-100 amino acids and is made up of seven antiparallel beta strands (A, B, C, D, E, F and G), linked by beta turns (AB, DE and EF), a transversal strand (CD) and two loops (BC and FG), and forms a sandwich of two sheets [ABED] [GFC]. A C-domain has a topology and a three-dimensional structure that is similar to that of a V-domain [67], but without the C' and C" strands and the C'C" loop, which is replaced by a transversal CD strand [68]. The lengths of the strands and loops (Table 4) are visualized in the IMGT Colliers de Perles on one layer and two layers ( Figure 3).
for the L chain, or several C-domains (CH) for the H chain. 2 The heavy chain C-REGION also includes the HINGE-REGION, and for membrane IG (mIG), the CONNECTING-REGION (CO), TRANSMEMBRANE-REGION (TM) and CYTOPLASMIC-REGION (CY); for secreted IG (sIG), the C-REGION includes CHS instead of CO, TM and CY. In the IMGT system, the C-domain includes the C-DOMAIN of the IG and of the TR [1] and the C-LIKE-DOMAIN of the IgSF other than IG and TR [22]. The C-domain description of any receptor, any chain and any species is based on the IMGT unique numbering for the C-domain (C-DOMAIN and C-LIKE-DOMAIN) [68]. A C-domain ( Figure  3) comprises about 90-100 amino acids and is made up of seven antiparallel beta strands (A, B, C, D, E, F and G), linked by beta turns (AB, DE and EF), a transversal strand (CD) and two loops (BC and FG), and forms a sandwich of two sheets [ABED] [GFC]. A Cdomain has a topology and a three-dimensional structure that is similar to that of a Vdomain [67], but without the C' and C'' strands and the C'C'' loop, which is replaced by a transversal CD strand [68]. The lengths of the strands and loops (Table 4) are visualized in the IMGT Colliers de Perles on one layer and two layers ( Figure 3). The IMGT Colliers de Perles on two layers show, in the forefront, the GFC strands, and in the back, the ABED strands (located at the interface CH1/CL of the IG), linked by the CD transversal strand. The IMGT Collier de Perles with hydrogen bonds (green lines online, only shown here for the GFC sheet) is generated by the IMGT/Collier de Perles tool [51] integrated in the IMGT/3Dstructure-DB, from experimental 3D structure data. (C) IMGT Collier de Perles on two layers from IMGT/DomainGapAlign [28,49,50]. (D) IMGT Colliers de Perles on one layer. Amino acids are shown in the one-letter abbreviation. All proline (P) are shown online in yellow. IMGT anchors are represented by squares. Hatched circles are IMGT gaps according to the IMGT unique numbering for the C-domain [68]. Positions with bold (online red) letters indicate the four conserved positions that are common to a V-domain and to a C-domain: 23 (1st-CYS), 41 (CONSERVED-TRP), 89 (hydrophobic), 104 (2nd-CYS), and position 118, which is only conserved in V-DOMAIN. The identifier of the chain to which the CH-domain belongs is 1n0x_H (from the Homo sapiens b12 Fab, in IMGT/3Dstructure-DB, https://www.imgt.org) [27][28][29]. The 3D ribbon representation was obtained using PyMOL and "IMGT numbering comparison" of 1n0x_H (CH1) from IMGT/3Dstructure-DB (https://www.imgt.org) [27][28][29]. Table 4. C-domain strands, turns and loops, IMGT positions and lengths, based on the IMGT unique numbering for C-domain (C-DOMAIN and C-LIKE-DOMAIN) [68]. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders of IMGT ® , the international ImMunoGeneTics information system ® , https://www.imgt.org).  [55,56]. b based on the IMGT unique numbering for C-domain (C-DOMAIN and C-LIKE-DOMAIN) [68]. c in number of amino acids (or codons). d IMGT Residue@Position is a given residue (usually an amino acid) or a given conserved property amino acid class, at a given position in a domain, based on the IMGT unique numbering [68].

C Domain Strands, Turns and Loops
There are six IMGT anchors in a C-domain (four of them identical to those of a Vdomain): Positions 26 and 39 (anchors of the BC loop), 45 and 77 (by extension, anchors of the CD strand as there is no C'-C" loop in a C-domain [68]), and 104 and 118 (anchors of the FG loop). A C-domain has five characteristic amino acids at given positions (positions with bold (online red) letters in the IMGT Colliers de Perles). Four of them are highly conserved and hydrophobic [52] and are common to the V-domain: 23 (1st-CYS), 41 (CONSERVED-TRP), 89 (hydrophobic) and 104 (2nd-CYS). These amino acids contribute to the two major features shared by the V and C-domains: The disulfide bridge (between the two cysteines 23 and 104) and the internal hydrophobic core of the domain (with the side chains of tryptophan W41 and amino acid 89). The fifth position, 118, is diverse and is characterized as being an FG loop anchor. In the IMGT system, the C-domains (C-DOMAIN and C- LIKE-DOMAIN) are delimited considering the exon delimitation, whenever appropriate, allowing the integration of strands A and G, which do not have structural alignments.
The 20 usual amino acids (AA) have been classified in eleven IMGT physicochemical classes [52] (IMGT ® https://www.imgt.org, IMGT Education > Aide-mémoire > Amino acids) (Figure 4). is characterized as being an FG loop anchor. In the IMGT system, the C-domains (C-DO-MAIN and C-LIKE-DOMAIN) are delimited considering the exon delimitation, whenever appropriate, allowing the integration of strands A and G, which do not have structural alignments. The 20 usual amino acids (AA) have been classified in eleven IMGT physicochemical classes [52] (IMGT ® https://www.imgt.org, IMGT Education > Aide-mémoire > Amino acids) (Figure 4).

IGHG, IGKC and IGLC2 Engineered Variants
One hundred and fourteen IGHG engineered variants have been defined by their IMGT gene nomenclature, the IMGT unique numbering for C-domain [68] and IMGT motifs in domain strands and/or loops (Table 4, Figure 3), with corresponding Eu positions [97] (IMGT https://www.imgt.org, IMGT Scientific chart > Correspondence between C numberings > Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the EU and Kabat numberings: Human IGHG [97,98] https://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html) (Supplementary Table S1). The IGKC and IGLC2 engineered variants involved in the structure have also been defined similarly by their IMGT gene nomenclature, the IMGT unique numbering for the C-domain [68] and IMGT motifs in the domain strands and/or loops (Table 4), with corresponding Eu positions [97] (IMGT https://www.imgt.org, IMGT Scientific chart > Correspondence between C numberings > Correspondence between the IMGT unique numbering for the C-DOMAIN, the IMGT exon numbering, the EU and Kabat numberings: Human IGKC [97,98]. The correspondence between the IMGT unique numbering and the Eu positions are provided here in a horizontal format for the IGHG1 CH1, hinge, CH2 and CH3-domains ( Figure 5), and hinges of IGHG1, IGHG2, IGHG3 and IGHG4 (Figure 6), and by extension to the alignment of IGKC and IGLC2 with IGHG1 CH1 (Figure 7).

IGHG, IGKC and IGLC2 Engineered Variants
One hundred and fourteen IGHG engineered variants have been defined by their IMGT gene nomenclature, the IMGT unique numbering for C-domain [68] and IMGT motifs in domain strands and/or loops (Table 4, Figure 3), with corresponding Eu positions [97] (IMGT https://www.imgt.org, IMGT Scientific chart > Correspondence between C numberings > Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the EU and Kabat numberings: Human IGHG [97,98] https://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html) (Supplementary Table S1). The IGKC and IGLC2 engineered variants involved in the structure have also been defined similarly by their IMGT gene nomenclature, the IMGT unique numbering for the C-domain [68] and IMGT motifs in the domain strands and/or loops (Table 4), with corresponding Eu positions [97] (IMGT https://www.imgt.org, IMGT Scientific chart > Correspondence between C numberings > Correspondence between the IMGT unique numbering for the C-DOMAIN, the IMGT exon numbering, the EU and Kabat numberings: Human IGKC [97,98]. The correspondence between the IMGT unique numbering and the Eu positions are provided here in a horizontal format for the IGHG1 CH1, hinge, CH2 and CH3-domains ( Figure 5), and hinges of IGHG1, IGHG2, IGHG3 and IGHG4 (Figure 6), and by extension to the alignment of IGKC and IGLC2 with IGHG1 CH1 (Figure 7).

Figure 5.
Correspondence between the Homo sapiens IGHG1 amino acid sequence, based on the IMGT unique numbering for the C-domain [68] and the Eu positions (shown vertically) from 118 to 445 [97]. (A) IGHG1 CH1, CH2 and CH3. The standardized presentation of the IMGT unique numbering on the top two lines [68] can be obtained using IMGT/DomainGapAlign [28,49,50]     Standardized characterization has become a necessity, owing to the increasing number of engineered antibodies of effector properties [99,100] and/or various formats. Based on the IMGT Scientific chart rules, we propose a standardized IMGT nomenclature of engineered variants involved in effector properties (ADCC, ADCP and CDC), half-life and structure of therapeutical monoclonal antibodies. The standardized variant characterization comprises (1) the IMGT engineered Fc variant name (e.g. G1v1), (2) the IMGT variant definition (for each amino acid (AA) change: domain, AA in the one-letter abbreviation [52] and its position in the IMGT unique numbering for C domain [68], e.g. CH2 P1.4, (3) the IMGT amino acid changes on the IGHG CH domain with the Eu numbering between parentheses (e.g., CH2 E1.4 > P (233)), (4) the Eu numbering variant (e.g., E233P), (5) the IMGT motif positions according to the IMGT unique numbering [68], followed between parentheses, by the Eu numbering, motif with AA before and after the AA change in bold (e.g., IGHG1 CH2 1.6-3 (231-239) APELLGGPS > APPLLGGPS; underlined amino acids in the motif correspond to additional positions in the IMGT unique numbering for the C-domain [68,[70][71][72], e.g., APELLG and APPLLG which correspond to 1.6, 1.5, 1.4, 1.3, 1.2 and 1.1), and (6) information from the literature regarding 'property and function'.

Half-life increase
Enhances FCGRT binding at pH 6.0.

Half-life increase
Enhances FCGRT binding at pH 6.0 (1) Ten-fold increase at pH 6.0 [134] and four-fold increases half-life in a cynomolgus pK study [140]. The T18>E amino acid change provides two novel salt bridges between the Fc and BM2 of FCGRT IMGT/3Dstructure-DB: 4n0f, 4n0u [137]. A change of IGHG1 CH2 His H93 (310) into any other amino acid (excluding Cys) leads to an undetectable binding to FCGRT (FcRn) at pH 6.0 [137]. (2) An increased reduction in tumor burden in human FCGRT (FcRn) transgenic tumor-bearing mice treated with an anti-EGFR or an anti-VEGF antibody [135]. From the 3D structure, it is postulated that N114>S (434) allows for additional hydrogen bonds with FCGRT (FcRn) [137] IMGT/3Dstructure-DB: 4n0f, 4n0u.         Reduces acid-induced aggregation [143] Low ADCC Low FcγR binding [143] Low CDC Low C1q binding [143] In the tables, the different columns correspond to the items of the standardized variant characterization detailed above. Engineered amino acid changes are in bold in the IMGT variants (red before the change, green after the change. The motif is in yellow and shown before and after the AA change(s).
The variants involved in B cell inhibition by coengagement of antigen and FcγR on the same cell include one Homo sapiens IGHG1 variant: G1v25 [33,34] (Table 11).
The variants involved in abrogation of binding to Protein A include one Homo sapiens IGHG4 variant: G4v8 (Table 14).
The variants involved in suppression of inter H-L and/or inter H-H disulfide bridges includes three Homo sapiens IGHG1 variants: G1v37, G1v61 and G1v62 (Table 19).
The variants involved in enhancement of hetero pairing H-Linclude two Homo sapiens IGHG1 variants: G1v57 used in association with Homo sapiens IGKC variant: KCv57, and G1v58, used in association with Homo sapiens IGLC2 variant: LC2v58 (Table 21).
The variants involved in control of half-IG exchange of bispecific IgG4 antibodies include one Homo sapiens IGHG4 variant: G4v10 (Table 22).
The variants involved in reducing acid-induced aggregation include one Homo sapiens IGHG2 variant: G2v7 (Table 23).
Two variants have been assigned to two properties belonging to different types and are therefore found in two tables, G1v5 (Tables 5 and 8) and G1v8 (Tables 6 and 9).
Supplementary Table S2 provides the variants of Tables 5-23 in an alphanumeric order of the IMGT engineered variants involved in the effector properties (ADCC, ADCP and CDC), half-life and structure of the therapeutical monoclonal antibodies.

Conclusions
The therapeutic monoclonal antibody engineering field is the most promising in the medical field. A standardized analysis of IG genomic and expressed sequences, structures and interactions is crucial for a better molecular understanding and comparison of the mAb specificity, affinity, half-life, Fc effector properties and potential immunogenicity. IMGT has provided the concepts for the IG loci description of newly sequenced genomes [2], antibody structure/function characterization [4], antibody engineering (single chain Fragment variable (scFv), phage displays, combinatorial libraries) and antibody humanization (chimeric, humanized and human antibodies). IMGT ® standardization allows the repertoire analysis and antibody humanization studies to move to novel, high-throughput methodologies with the same high-quality criteria. The CDR-IMGT lengths are now required for mAb INN applications and are included in the WHO-INN definitions (84)(85)(86). The characterization of the IGHG engineered variants for effector properties, half-life increase, and new structures of bi-and multi-specific antibodies brings a new level of standardized information in the comparative analysis of therapeutic antibodies.