Functional consequences of allotypic polymorphisms in human immunoglobulin G subclasses.

Heritable polymorphisms within the human IgG locus, collectively termed allotypes, have often been linked by statistical associations, but rarely mechanistically, to a wide range of disease states. One potential explanation for these associations is that IgG allotype alters host cell receptors' affinity for IgG, dampening or enhancing an immune response depending on the nature of the change and the receptors. In this work, a panel of allotypic antibody variants were evaluated using multiplexed, label-free biophysical methods and cell-based functional assays to determine what effect, if any, human IgG polymorphisms have on antibody function. While we observed several differences in FcγR affinity among allotypes, there was little evidence of dramatically altered FcγR-based effector function or antigen recognition activity associated with this aspect of genetic variability.

Systematic analysis of the varied designs of 819 therapeutic antibodies and Fc fusion proteins assigned international nonproprietary names.

By combining and cross-checking data from the World Health Organization's lists of international nonproprietary names with three other databases, we assembled a dataset of amino acid sequences of 819 antibody-related therapeutics. This enabled the systematic tabulation of 57 different molecular formats and about 90 different constant-region variants (47 for silencing, 14 for enhancement, 8 for modifying binding to FcRn, 13 for heterodimerization, 4 for site-specific modification and 4 for stabilization), as well as the frequencies of different targets and immunoglobulin allotypes. The curated dataset provides a resource for researchers, giving insights into trends in antibody engineering and a guide to the most frequently tested designs.

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein.

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both severe acute respiratory syndrome coronavirus 2 infection severity and long-term immunity, after either recovery or vaccination. A hallmark of coronavirus disease 2019 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon, we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides across ten common allotypes of endoplasmic reticulum aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for major histocompatibility complex class I molecules, including known severe acute respiratory syndrome coronavirus 2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses in coronavirus disease 2019.

Two Human Monoclonal HLA-Reactive Antibodies Cross-React with Mamu-B*008, a Rhesus Macaque MHC Allotype Associated with Control of Simian Immunodeficiency Virus Replication.

MHC class I molecules play an important role in adaptive immune responses against intracellular pathogens. These molecules are highly polymorphic, and many allotypes have been characterized. In a transplantation setting, a mismatch between MHC allotypes may initiate an alloimmune response. Rhesus macaques (Macaca mulatta, Mamu) are valuable as a preclinical model species in transplantation research as well as to evaluate the safety and efficacy of vaccine candidates. In both lines of research, the availability of nonhuman primate MHC-reactive mAbs may enable in vitro monitoring and detection of presence of particular Mamu molecules. In this study, we screened a collection of thoroughly characterized HLA class I-specific human mAbs for cross-reactivity with rhesus macaque MHC class I allotypes. Two mAbs, OK4F9 and OK4F10, recognize an epitope that is defined by isoleucine (I) at amino acid position 142 that is present on the Indian rhesus macaque Mamu-B*008:01 allotype, which is an allotype known to be associated with elite control of SIV replication. The reactive pattern of a third mAb, MUS4H4, is more complex and includes an epitope shared on Mamu-A2*05:01 and -B*001:01-encoded Ags. This is the first description, to our knowledge, of human HLA-reactive mAbs that can recognize Mamu allotypes, and these can be useful tools for in vitro monitoring the presence of the relevant allelic products. Moreover, OK4F9 and OK4F10 can be powerful mAbs for application in SIV-related research.

Comprehensive Analysis of CD4+ T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II.

Common human coronaviruses have been circulating undiagnosed worldwide. These common human coronaviruses share partial sequence homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therefore, T cells specific to human coronaviruses are also cross-reactive with SARS-CoV-2 antigens. Herein, we defined CD4+ T cell responses that were cross-reactive with SARS-CoV-2 antigens in blood collected in 2016-2018 from healthy donors at the single allele level using artificial antigen-presenting cells (aAPC) expressing a single HLA class II allotype. We assessed the allotype-restricted responses in the 42 individuals using the aAPCs matched 22 HLA-DR alleles, 19 HLA-DQ alleles, and 13 HLA-DP alleles. The response restricted by the HLA-DR locus showed the highest magnitude, and that by HLA-DP locus was higher than that by HLA-DQ locus. Since two alleles of HLA-DR, -DQ, and -DP loci are expressed co-dominantly in an individual, six different HLA class II allotypes can be used to the cross-reactive T cell response. Of the 16 individuals who showed a dominant T cell response, five, one, and ten showed a dominant response by a single allotype of HLA-DR, -DQ, and -DP, respectively. The single allotype-restricted T cells responded to only one antigen in the five individuals and all the spike, membrane, and nucleocapsid proteins in the six individuals. In individuals heterozygous for the HLA-DPA and HLA-DPB loci, four combinations of HLA-DP can be expressed, but only one combination showed a dominant response. These findings demonstrate that cross-reactive T cells to SARS-CoV-2 respond with single-allotype dominance.

Beyond Allotypes: The Influence of Allelic Diversity in Antibody Constant Domains.

Polymorphic diversity in antibody constant domains has long been defined by allotypic motifs that cross react with the sera of other individuals. Improvements in sequencing technologies have led to the discovery of a large number of new allelic sequences that underlie this diversity. Many of the point mutations lie outside traditional allotypic motifs suggesting they do not elicit immunogenic responses. As antibodies play an important role in immune defense and biotechnology, understanding how this newly resolved diversity influences the function of antibodies is important. This review investigates the current known diversity of antibody alleles at a protein level for each antibody isotype as well as the kappa and lambda light chains. We focus on evidence emerging for how these mutations perturb antibody interactions with antigens and Fc receptors that are critical for function, as well as the influence this might have on the use of antibodies as therapeutics and reagents.

MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation.

Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging. We propose here a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of the N-glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fc-specific beads followed by an on-bead C H 2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards. While the glycosylation patterns were similar within allotypes of the same subclass, clear differences were observed between IgG subclasses and donors, highlighting the relevance of the proposed approach. In a single analysis, glycosylation levels specific for each allotype, relative abundances of subclasses and information on co-occurring modifications are obtained. This middle-up method represents an important step toward a comprehensive analysis of immunoglobulin G-Fc variants.

FcγR Binding and ADCC Activity of Human IgG Allotypes.

Antibody dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. NK-cell mediated ADCC is mainly triggered by IgG-subclasses IgG1 and IgG3 through the IgG-Fc-receptor (FcγR) IIIa. Polymorphisms in the immunoglobulin gamma heavy chain gene likely form a layer of variation in the strength of the ADCC-response, but this has never been studied in detail. We produced all 27 known IgG allotypes and assessed FcγRIIIa binding and ADCC activity. While all IgG1, IgG2, and IgG4 allotypes behaved similarly within subclass, large allotype-specific variation was found for IgG3. ADCC capacity was affected by residues 291, 292, and 296 in the CH2 domain through altered affinity or avidity for FcγRIIIa. Furthermore, allotypic variation in hinge length affected ADCC, likely through altered proximity at the immunological synapse. Thus, these functional differences between IgG allotypes have important implications for therapeutic applications and susceptibility to infectious-, allo- or auto-immune diseases.

IgG3 anti-Kell allotypic variation results in differential antigen binding and phagocytosis.

BACKGROUND: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT). STUDY DESIGN AND METHODS: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages. RESULTS: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients. CONCLUSION: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn.

Distinct Polymorphisms in HLA Class I Molecules Govern Their Susceptibility to Peptide Editing by TAPBPR.

Understanding how peptide selection is controlled on different major histocompatibility complex class I (MHC I) molecules is pivotal for determining how variations in these proteins influence our predisposition to infectious diseases, cancer, and autoinflammatory conditions. Although the intracellular chaperone TAPBPR edits MHC I peptides, it is unclear which allotypes are subjected to TAPBPR-mediated peptide editing. Here, we examine the ability of 97 different human leukocyte antigen (HLA) class I allotypes to interact with TAPBPR. We reveal a striking preference of TAPBPR for HLA-A, particularly for supertypes A2 and A24, over HLA-B and -C molecules. We demonstrate that the increased propensity of these HLA-A molecules to undergo TAPBPR-mediated peptide editing is determined by molecular features of the HLA-A F pocket, specifically residues H114 and Y116. This work reveals that specific polymorphisms in MHC I strongly influence their susceptibility to chaperone-mediated peptide editing, which may play a significant role in disease predisposition.

Novel Approach to Cell Surface Discrimination Between KIR2DL1 Subtypes and KIR2DS1 Identifies Hierarchies in NK Repertoire, Education, and Tolerance.

Cumulative activating and inhibitory receptor signaling controls the functional output of individual natural killer (NK) cells. Investigation of how competing signals impact response, however, has been hampered by the lack of available antibodies capable of distinguishing inhibitory and activating receptors with highly homologous ectodomains. Utilizing a novel combination of monoclonal antibodies with specificity for discrete inhibitory KIR2DL1 and activating KIR2DS1 allotypes found among 230 healthy donors, we investigated allele-specific receptor expression and function driven by KIR2DL1 and KIR2DS1 alleles. We found that co-expression of the HLA-C2 ligand diminishes KIR2DL1, but not KIR2DS1, cell surface staining, but does not impact the respective frequencies of KIR2DL1- and KIR2DS1-expressing cells within the NK repertoire. We can distinguish by flow cytometry NK cell populations expressing the most common KIR2DL1-C245 allotypes from those expressing the most common KIR2DL1-R245 allotypes, and we show that the informative differential binding anti-KIR2DL1/S1 clone 1127B is determined by amino acid residue T154. Although both KIR2DL1-C245 and KIR2DL1-R245 subtypes can be co-expressed in the same cell, NK cells preferentially express one or the other. Cells expressing KIR2DL1-C245 exhibited a lower KIR2DL1 cell surface density and lower missing-self reactivity in comparison to cells expressing KIR2DL1-R245. We found no difference, however, in sensitivity to inhibition or cell surface stability between the two KIR2DL1 isoforms, and both demonstrated similar expansion among NKG2C+ KIR2DL1+ NK cells in HCMV-seropositive healthy individuals. In addition to cell surface density of KIR2DL1, copy number of cognate HLA-C2 hierarchically impacted the effector capacity of both KIR2DL1+ cells and the tolerization of KIR2DS1+ NK cells. HLA-C2 tolerization of KIR2DS1+ NK cells could be overridden, however, by education via co-expressed self-specific inhibitory receptors, such as the heterodimer CD94/NKG2A. Our results demonstrate that effector function of NK cells expressing KIR2DL1 or KIR2DS1 is highly influenced by genetic variability and is calibrated by co-expression of additional NK receptors and cognate HLA-C2 ligands. These findings define the molecular conditions under which NK cells are activated or inhibited, potentially informing selection of donors for adoptive NK therapies.

Identification of a new European rabbit IgA with a serine-rich hinge region.

In mammals, the most striking IgA system belongs to Lagomorpha. Indeed, 14 IgA subclasses have been identified in European rabbits, 11 of which are expressed. In contrast, most other mammals have only one IgA, or in the case of hominoids, two IgA subclasses. Characteristic features of the mammalian IgA subclasses are the length and amino acid sequence of their hinge regions, which are often rich in Pro, Ser and Thr residues and may also carry Cys residues. Here, we describe a new IgA that was expressed in New Zealand White domestic rabbits of IGHVa1 allotype. This IgA has an extended hinge region containing an intriguing stretch of nine consecutive Ser residues and no Pro or Thr residues, a motif exclusive to this new rabbit IgA. Considering the amino acid properties, this hinge motif may present some advantage over the common IgA hinge by affording novel functional capabilities. We also sequenced for the first time the IgA14 CH2 and CH3 domains and showed that IgA14 and IgA3 are expressed.

Identification of high-affinity anti-CD16A allotype-independent human antibody domains.

CD16A (FcγRIIIA) is an activating receptor mostly expressed on natural killer (NK) cells and monocytes/macrophages. It can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) through low-affinity interaction with human immunoglobulin G (IgG) Fc. It can also mediate cell lysis if NK cells are guided by bispecific killer cells engagers (BiKEs). BiKEs showed some success in clinical trials of cancer and are promising candidate therapeutics. However, currently reported BiKEs are based on antibody fragments (scFvs) of relatively large size. The CD16A-specific antibodies are also typically from animal origin. Decreasing the BiKE size could result in enhanced penetration into solid tumor and normal tissues, and using fully human antibodies could decrease the likelihood of immunogenicity. Here we report the identification and characterization of two antibody domains, D6 and E11, isolated from a very large human VH antibody domain library displayed on phage. D6 and E11 bound CD16A with EC50 of 4nM and 8nM, respectively, but not other Fc gamma receptors (FcγRs) such as CD64 (FcγRI), CD32 (FcγRII) and CD16B (FcγRIIIB). They bound to both CD16A allotypes (158F,V) with equal affinity and competed with each other as well as with human IgG1 and the mouse anti-CD16A antibody 3G8. These and other results were used to build a molecular docking model predicting that D6 and E11 may bind to the CD16A membrane proximal D2 domain by interacting with its BC, C'E and EF loops. Importantly, cross-linked (bivalent) D6 and E11 induced secretion of IL-2 after binding to CD16A-expressing Jurkat T cells. The small size of these antibody domains combined with their high-affinity, specific, allotype-independent, activating interactions with CD16A could allow generation of novel highly effective BiKEs and other candidate protein therapeutics.

Serological blind spots for variants of human IgG3 and IgG4 by a commonly used anti-immunoglobulin reagent.

BACKGROUND: Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty-nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized. STUDY DESIGN AND METHODS: A novel monoclonal anti-K antibody (PugetSound Monoclonal Antibody 1 [PUMA1]) was isolated and sequenced, and a panel of PUMA1 variants was expressed, consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was preincubated with K-positive red blood cells (RBCs) and then subjected to testing with currently approved anti-IgG by flow cytometry, solid phase systems, gel cards, and tube testing. RESULTS: A US Food and Drug Administration (FDA)-approved monoclonal anti-IgG (gamma-clone) failed to recognize 2 of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 of 3 IgG4 isoallotypes (IgG4-01, IgG4-02, and IgG4-03). In contrast, an FDA-approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes. CONCLUSION: These findings demonstrate "blind spots" in isoalloantibody detection by a monoclonal anti-IgG. If a patient has anti-RBC antibodies predominantly of an IgG3 subtype (the IgG3-03 and/or IgG3-13 variety), then it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 have an estimated frequency of 1% to 3% in Caucasian populations and 20% to 30% in certain African populations. Nonreactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported.

IgG1 Allotypes Influence the Pharmacokinetics of Therapeutic Monoclonal Antibodies through FcRn Binding.

Because IgG1 allotypes might have different half-lives, their influence on infliximab (G1m17,1 allotype) pharmacokinetics was investigated in a group of spondyloarthritis patients. Infliximab was found to have a shorter half-life in patients homozygous for the G1m17,1 allotypes than in those carrying the G1m3 with no G1m1 (G1m3,-1) allotype. Because the neonatal FcR (FcRn) is involved in the pharmacokinetics of mAbs, the interaction of different IgG1 allotypes with FcRn was examined using cellular assays and surface plasmon resonance. G1m17,1 mAbs, such as infliximab and rituximab, were shown to bind more efficiently to FcRn and to be transcytosed better than the G1m3,-1 mAb cetuximab, which explains why infliximab is a better competitor for endogenous IgG1 in G1m3,-1 allotype-bearing patients. A set of four allotype variants of adalimumab (G1m17,1; G1m17,-1; G1m3,1; and G1m3,-1) was also tested for its binding to FcRn, revealing that the G1m3,1 variant, not present in commercial mAbs, binds more efficiently to FcRn and is transcytosed better than the other three variants, all of which are found in therapeutic mAbs.

Association of complement factor B allotypes and serum biomarkers in rheumatoid arthritis patients and their relatives.

The aim of the study was to investigate the allotypic variability of complement factor B (BF) in patients and relatives with rheumatoid arthritis (RA) and its association with serological biomarkers and clinical features of the disease. BF allotypes were determined by high-voltage agarose gel electrophoresis in serum samples of 180 patients with RA, 198 relatives and 98 controls from Southern Brazil. Anticyclic citrullinated peptide (anti-CCP), antimutated citrullinated vimentin (anti-MCV) and IgA-rheumatoid factor (RF) were determined by ELISA and IgM-RF by latex agglutination in all samples. No significant differences were found in the allotypic variants of BF between patients with RA, relatives and controls, nor associations with gender and age of RA onset. BF*S07 allotype was significantly associated with extra-articular manifestations (EAMs; Secondary Sjögren Syndrome, pneumonitis, rheumatoid nodules) in patients with RA (P = 0.02; OR = 6.62). Patients with phenotype BF F had lower positivity for anti-MCV biomarker (P = 0.02; OR = 0.22) and those with allotype BF*S had higher prevalence of this autoantibody (P = 0.02; OR = 3.77). An increased frequency of RF-IgA was detected in relatives of patients with RA with BF FS07 phenotype (P = 0.02; OR = 7.78). Complement BF variability did not influence the development of RA in the studied patients, but BF variants may act as markers of disease prognosis, such as development of EAMs, corroborating with the role of the alternative pathway in the pathogenesis of RA.

Equine immunoglobulins and organization of immunoglobulin genes.

Our understanding of how equine immunoglobulin genes are organized has increased significantly in recent years. For equine heavy chains, 52 IGHV, 40 IGHD, 8 IGHJ and 11 IGHC are present. Seven of these IGHCs are gamma chain genes. Sequence diversity is increasing between fetal, neonatal, foal and adult age. The kappa light chain contains 60 IGKV, 5 IGKJ and 1 IGKC, whereas there are 144 IGLV, 7 IGLJ, and 7 IGLC for the lambda light chain, which is expressed predominantly in horses. Significant transcriptional differences for IGLV and IGLC are identified in different breeds. Allotypic and allelic variants are observed for IGLC1, IGLC5, and IGLC6/7, and two IGLV pseudogenes are also transcribed. During age development, a decrease in IGLVs is noted, although nucleotide diversity and significant differences in gene usage increased. The following paper suggests a standardization of the existing nomenclature of immunoglobulin genes.

Rheumatoid arthritis response to treatment across IgG1 allotype - anti-TNF incompatibility: a case-only study.

INTRODUCTION: We have hypothesized that incompatibility between the G1m genotype of the patient and the G1m1 and G1m17 allotypes carried by infliximab (INX) and adalimumab (ADM) could decrease the efficacy of these anti-tumor necrosis factor (anti-TNF) antibodies in the treatment of rheumatoid arthritis (RA). METHODS: The G1m genotypes were analyzed in three collections of patients with RA totaling 1037 subjects. The first, used for discovery, comprised 215 Spanish patients. The second and third were successively used for replication. They included 429 British and Greek patients and 393 Spanish and British patients, respectively. Two outcomes were considered: change in the Disease Activity Score in 28 joint (ΔDAS28) and the European League Against Rheumatism (EULAR) response criteria. RESULTS: An association between less response to INX and incompatibility of the G1m1,17 allotype was found in the discovery collection at 6 months of treatment (P = 0.03). This association was confirmed in the replications (P = 0.02 and 0.08, respectively) leading to a global association (P = 0.001) that involved a mean difference in ΔDAS28 of 0.4 units between compatible and incompatible patients (2.3 ± 1.5 in compatible patients vs. 1.9 ± 1.5 in incompatible patients) and an increase in responders and decrease in non-responders according to the EULAR criteria (P = 0.03). A similar association was suggested for patients treated with ADM in the discovery collection, but it was not supported by replication. CONCLUSIONS: Our results suggest that G1m1,17 allotypes are associated with response to INX and could aid improved therapeutic targeting in RA.