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Antibody engineering is important for many diagnostic and clinical applications of monoclonal antibodies. We recently reported a series of fragment crystallizable (Fc) mutations targeting the neonatal Fc receptor (FcRn) site on a Lewis Y (Le y ) binding IgG1, hu3S193. The hu3S193 variants displayed shortened in vivo half-lives and may have potential for radioimaging or radiotherapy of Le y -positive tumors. Here, we report Fc crystal structures of wild-type hu3S193, seven FcRn-binding site variants, and a variant lacking C1q binding or complement-dependent cytotoxicity (CDC) activity. The Fc conformation of the FcRn-binding sites was similar for wild-type and all mutants of hu3S193 Fc, which suggests that FcRn interactions were directly affected by the amino acid substitutions. The C1q-binding site mutant Fc was nearly identical with the wild-type Fc. Surprisingly, several hu3S193 Fc variants showed large changes in global structure compared with wild-type Fc. All hu3S193 Fc mutants had similar antibody-dependent cellular cytotoxicity, despite some with conformations expected to diminish Fc gamma receptor binding. Several hu3S193 variants displayed altered CDC, but there was no correlation with the different Fc conformations. All versions of hu3S193, except the C1q-binding site mutant, bound C1q, suggesting that the altered CDC of some variants could result from different propensities to form IgG hexamers after engaging Le y on target cells. Overall, our findings support the concept that the antibody Fc is both flexible and mobile in solution. Structure-based design approaches should take into account the conformational plasticity of the Fc when engineering antibodies with optimal effector properties., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In response to the widespread emergence of antibiotic-resistant microbes, new therapeutic agents are required for many human pathogens. A non-mammalian polysaccharide, poly- N -acetyl-d-glucosamine (PNAG), is produced by bacteria, fungi, and protozoan parasites. Antibodies that bind to PNAG and its deacetylated form (dPNAG) exhibit promising in vitro and in vivo activities against many microbes. A human IgG1 mAb (F598) that binds both PNAG and dPNAG has opsonic and protective activities against multiple microbial pathogens and is undergoing preclinical and clinical assessments as a broad-spectrum antimicrobial therapy. Here, to understand how F598 targets PNAG, we determined crystal structures of the unliganded F598 antigen-binding fragment (Fab) and its complexes with N -acetyl-d-glucosamine (GlcNAc) and a PNAG oligosaccharide. We found that F598 recognizes PNAG through a large groove-shaped binding site that traverses the entire light- and heavy-chain interface and accommodates at least five GlcNAc residues. The Fab-GlcNAc complex revealed a deep binding pocket in which the monosaccharide and a core GlcNAc of the oligosaccharide were almost identically positioned, suggesting an anchored binding mechanism of PNAG by F598. The Fab used in our structural analyses retained binding to PNAG on the surface of an antibiotic-resistant, biofilm-forming strain of Staphylococcus aureus Additionally, a model of intact F598 binding to two pentasaccharide epitopes indicates that the Fab arms can span at least 40 GlcNAc residues on an extended PNAG chain. Our findings unravel the structural basis for F598 binding to PNAG on microbial surfaces and biofilms., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.

Background: The targets and mechanisms of human immunity to malaria are poorly understood, which poses a major barrier to malaria vaccine development. Antibodies play a key role in human immunity and may act by inhibiting receptor-binding functions of key merozoite invasion ligands. Antibodies to the major invasion ligand and vaccine candidate, erythrocyte-binding antigen 175 (EBA-175), have been linked with protection, but how these antibodies function has not been established., Methods: We developed 2 new assays that quantify the ability of antibodies to inhibit binding of EBA-175 to its erythrocyte receptor, glycophorin A, using either native or recombinant EBA-175. Binding-inhibitory antibodies were evaluated in a longitudinal cohort study of Papua New Guinean children and related to risk of malaria, age, infection status, and markers of parasite exposure., Results: Binding-inhibition assays (BIAs) were reproducible, and the 2 assays had a high level of agreement. Inhibitory antibodies were common among children, acquired in association with markers of increasing parasite exposure, and high in those children with active infection. Inhibitory antibodies correlated with total immunoglobulin G levels to the EBA-175 binding domain (region II). Importantly, binding-inhibitory antibodies were significantly associated with protection from symptomatic malaria when measured using either BIA., Conclusions: Findings suggest that naturally acquired binding-inhibitory antibodies are an important functional mechanism that contributes to protection against malaria and further supports the potential of EBA-175 as a vaccine candidate. Identifying vaccines and approaches that induce potent binding-inhibitory antibodies may be a valuable strategy in the development of highly efficacious malaria vaccines., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Monoclonal antibodies are being developed as therapeutics to complement drugs and vaccines or to fill the gap where no drugs or vaccines exist. These therapeutic antibodies (ThAb) may be especially important for infectious diseases in which there is antibiotic resistance, toxin-mediated pathogenesis, or for emerging pathogens. The unique structure of antibodies determines the specific nature of the effector function, so when developing ThAb, the desired effector functions need to be considered and integrated into the design and development processes to ensure maximum efficacy and safety. Antibody subclass is a critical consideration, but it is noteworthy that almost all ThAb that are licenced or currently in development utilise an IgG1 backbone. This review outlines the major structural properties that vary across subclasses, how these properties affect functional immunity, and discusses the various approaches used to study subclass responses to infectious diseases. We also review the factors associated with the selection of antibody subclasses when designing ThAb and highlight circumstances where different subclass properties might be beneficial when applied to particular infectious diseases. These approaches are critical to the future design of ThAb and to the study of naturally-acquired and vaccine-induced immunity., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Little is known of the impact of Fc receptor (FcR) polymorphism in macaques on the binding of human (hu)IgG, and nothing is known of this interaction in the pig-tailed macaque (Macaca nemestrina), which is used in preclinical evaluation of vaccines and therapeutic Abs. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcγRIIa (mnFcγRIIa) and inhibitory FcγRIIb (mnFcγRIIb) and predicted their structures using the huIgGFc/huFcγRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcγRIIa and mnFcγRIIb compared with their human FcR counterparts. MnFcγRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcγRIIa (His(131)). In contrast, mnFcγRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcγRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcγRIIa showed that Pro(159) and Tyr(160) impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcγRIIb was shown to be dependent on His(131) and Met(132). Significantly, both His(131) and Met(132) are conserved across FcγRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcγRIIa wherein proline at position 131, also an important polymorphic site in huFcγRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcγRIIa His(131). These marked interspecies differences in IgG binding between human and macaque FcRs and polymorphisms within species have implications for preclinical evaluation of Abs and vaccines in macaques.

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

Bovine herpesvirus 1 (BoHV-1) causes respiratory and genital diseases in cattle for which available vaccines do not confer adequate protection. Since passive immunization with antibodies permits disease prevention, single-chain fragment variable (scFv), originating from a monoclonal bovine IgG1 antibody against BoHV-1, were constructed and expressed in Pichia pastoris in V(lambda)-V(H) orientation via a flexible seven-amino acid linker. Similar to the intact IgG, the purified recombinant scFv neutralized BoHV-1 in vitro and recognized viral antigens in BoHV-1 infected MDBK cells by immunofluorescence. Homology modeling of the Fv predicts two distinct conformations for CDR3H. Firstly, a long protruding CDR3H conformation where no disulfide linkage occurred between two "non-canonical" Cys residues resulted in a large binding cavity between V(lambda) and V(H). Secondly, a smaller potential antigen-binding cavity is predicted with a disulfide linkage between the two Cys residues of CDR3H creating a six-membered loop in the ascending polypeptide, which fitted into the space between V(lambda) and V(H). Despite such potential configurational diversity of the antigen-binding site, the electrostatic surface potentials that would interact with the BoHV-1 epitope are largely similar for both the topographies where salt-bridge type electrostatic interactions likely occur at the edges of the binding site. Given that IgG1 antibody against BoHV-1 is clonally selected, it is likely that disulfide-stabilized broader and flatter surface topography is specifically generated to accommodate the predicted carbohydrate neutralizing B-epitope on BoHV-1. The specificity and neutralizing capacity for BoHV-1 of the scFv should make this bovine antibody fragment a useful diagnostic and potential therapeutic candidate for an important viral pathogen in cattle., ((c) 2009 Elsevier Ltd. All rights reserved.)

A disulphide-constrained peptide that binds to the low affinity Fc receptor, FcgammaRIIa (CD32) has been identified and its structure solved by NMR. Linear (7-mer and 12-mer) and disulphide-constrained (7-mer) phage display peptide libraries were panned on recombinant soluble FcgammaRIIa genetically fused to HSA (HSA-FcgammaRIIa). Peptides were isolated only from the constrained peptide library and these contained the consensus sequence, CWPGWxxC. Phage clones displaying variants of the peptide consensus sequence bound to FcgammaRIIa and the strongest binding clone C7C1 (CWPGWDLNC) competed with IgG for binding to FcgammaRIIa and was inhibited from binding to FcgammaRIIa by the FcgammaRIIa-blocking antibody, IV.3, suggesting that C7C1 and IgG share related binding sites on FcgammaRIIa. A synthetic disulphide-constrained peptide, pep-C7C1 bound to FcgammaRIIa by biosensor analysis, albeit with low affinity (KD approximately 100microM). It was significant that the FcgammaRIIa consensus peptide sequence contained a Proline (Pro3), which when substituted with alanine abrogated FcgammaRIIa binding, consistent with Pro3 contributing to receptor binding. Upon binding of IgG and IgE to their respective Fc receptors (FcgammaRs and FcepsilonRI) Pro329 in the Fc makes a critical interaction with two highly conserved Trp residues (Trp90 and Trp113) of the FcRs. The NMR structure of pep-C7C1 revealed a stabilizing type II beta-turn between Trp2 and Trp5, with Pro3 solvent exposed. Modelling of the pep-C7C1 structure in complex with FcgammaRIIa suggests that Pro3 of C7C1 binds to FcgammaRIIa by inserting between Trp90 and Trp113 of FcgammaRIIa thereby mimicking the molecular interaction made between FcgammaRIIa and IgG.

Analysis of the heavy-chain gene (pTGHC9907) encoding a bovine IgG1 antibody against bovine herpes virus type 1 (BHV-1) isolated from a Holstein cow has led to the identification of a new IgG1 sequence allele. A comparison of nucleotide sequence of pTGHC9907 with the IgG1(a) (clone 2) and IgG1(b) (clone 8.10) sequence variants and unclassified IgG1 cDNA sequence (clone 8.75) has revealed significant differences in the hinge region spanning codons 216-230. The Thr224 and Thr226 of IgG1(a) were replaced with Arg224 and Pro226, while both Thr218 and Pro224 of IgG1(b) were substituted with Arg with deletion of Ser225 in HB9907 antibody. Additional amino acid substitutions were noted in the CH1 (positions 190, 192), CH2 (position 281) and CH3 (position 402) exons. Thus, the polymorphic sites occurred in all constant domains, but were clustered in the hinge region of IgG1. Examination of a three-dimensional model of the HB9907 heavy chain revealed that all sequence variations were on the surface of the IgG and are possible targets for recognition by antisera and effector molecules such as cellular adhesion molecules. The presence in the CH1 domain of a repeating motif of Pro-Ala-Ser-Ser indicated a potential structure-enhancing function and a role in cellular adhesion and migration. Replacement of Thr with Arg residues within the hinge was predicted to have a dual effect of reducing the number of O-linked glycosylation sites and increasing the susceptibility to degradation by protease-secreting bacteria of the hinge region. As unclassified IgG1 cDNA sequence (clone 8.75) is structurally distinct from other variants, it is also classified as IgG1(d). Collectively, these observations support the identification of a new allotypic variant of bovine IgG1, designated as IgG1(c) that is distinct in both sequence and structure from the known sequence variants.

This study explores the structural features of murine monoclonal IgG2a anti-dinitrophenyl (DNP) antibodies that were previously shown to form immune complexes (IC) differing in their capacity to bind complement, their clearance from the circulation and their deposition in the kidney. Interestingly, the sequence of one of these antibodies has a missing stretch of 14 amino acids within FR3. Molecular modeling suggests that this sequence deletion corresponds to the loss of beta-pleated sheet structure for two beta-strands (designated 4-3 and 4-4) on the external surface of the V(H) domain. Despite this sequence and conformational abnormality, the antibody retains affinity for DNP comparable to other IgG2a antibodies. Data presented here identify monoclonal IgG2a antibodies that form IC with varying propensity for both complement binding and renal deposition and yet have similar V(H) domain sequences. In fact, in the case of two IgG2a antibodies that form IC with very different renal tropisms and complement binding capacity, sequence variation within V(H) was observed only at three clustered residues within FR2, a single residue within FR3 and nine clustered residues spanning CDR3 and FR4. Sequence and modeling analysis also yielded the paradoxical finding that an antibody forming IC with a relatively high capacity to serve as a target for complement binding displays a relatively low number of solvent exposed acceptor residues for C4b and C3b. These data underscore the complex relationship between V domain structure, complement activation and renal deposition of model IC.

The VH domains of two human monoclonal antibodies, designated Mcg IgG1(lambda) and Yvo IgM(kappa), were particularly intractable to standard protein sequencing protocols. Peptides liberated from the VH domains of these proteins, using standard enzymatic or chemical cleavages, invariably precipitated during the procedures. Boiling in SDS containing buffers dissolved precipitates and the peptides were separated using SDS-PAGE. Fully overlapped VH sequences were obtained with a series of 'in-gel' cleavages, followed by passive/differential transfers of peptides onto PVDF membranes. Both the in-gel cleavages and passive transfers could be applied to 'wet' or 'dry' gels so that gels could be archived and used at a later date to obtain additional sequence information from a fragment of interest. Repetitive yields of even the most insoluble peptides were such that the sequences of various peptides from relatively complex mixtures of peptides could be assigned with confidence. Despite the overall success of the sequencing, we occasionally referred to electron density maps, calculated for crystals of the Fab of Yvo IgM, to resolve particular sequences and confirm ambiguous amino acid assignments. Methods we describe in this report should be generally useful for obtaining sequences of proteins with intractable cores and may find many applications in the 'post genomic era'., (Copyright 2002 John Wiley & Sons, Ltd.)

A monoclonal immunoglobulin (Ig)M cryoglobulin (Mez) with interesting binding behaviour was isolated from a Waldenström's macroglobulinemia (WM) patient. It demonstrated very strong binding to peptides derived from the sequences of human IgG. However, when tested for binding to intact IgG, this antibody (Ab) did not show any rheumatoid factor (RF) activity. We propose several nonexclusive structural interpretations of the Mez-binding propensities, based on the orientations and solvent accessibilities of ligand residues and the nature of the Ab-binding site. To further characterize the structural features of Mez-peptide binding, IgG-derived octapeptides were docked into the Mez fragment variable (Fv)-binding site, revealing additional reasons for Mez-binding selectivity based on the interactions of the docked peptides with the Mez Fv. The problem was also approached from an immunological perspective. Comparisons of Mez variable region of the light chain (VL)/variable region of the heavy chain (VH) sequences with those of human germlines and known IgM RFs allowed us to provide a possible outline tracing the structural and functional origins of the Mez IgM. Coupled with examinations of interactions in docked complexes, this analysis led us to propose that the potential for RF activity, demonstrated through Mez binding to IgG-derived peptides, was owing to the inherent sequence and structure of the Mez IgM, rather than to somatic mutations. Thus, Mez IgM may occupy an intermediate niche between IgMs with and without RF activity.