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Because of the ongoing increase in antibiotic-resistant microbes, new strategies such as therapeutic antibodies and effective vaccines are required. Bacterial carbohydrates are known to be particularly antigenic, and several monoclonal antibodies that target bacterial polysaccharides have been generated, with more in current development. This review examines the known 3D crystal structures of anti-bacterial antibodies and the structural basis for carbohydrate recognition and explores the potential mechanisms for antibody-dependent bacterial cell death. Understanding the key interactions between an antibody and its polysaccharide target on the surface of bacteria or in biofilms can provide essential information for the development of more specific and effective antibody therapeutics as well as carbohydrate-based vaccines., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

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.)

Structure-guided approaches to HIV-1 vaccine design depend on knowledge of the presentation of neutralizing epitopes on gp120, such as the epitope for the broadly neutralizing mAb b12. Here, we characterized predicted three-dimensional structures of functionally diverse gp120 proteins in their b12-bound conformation, to better understand the gp120 determinants that expose or occlude the b12 epitope. Mapping the gp120-b12 binding interface identified amino acid polymorphisms within the C2, C3, C4 and V5 regions of gp120 associated with augmented b12 binding, and importantly, identified residues in the b12-exclusive binding domain of gp120 that are important for b12 neutralization resistance. Structural studies suggest that these b12 resistance variants promote reduced conformational flexibility in the b12 recognition site, which we show involves structural alterations within the gp120 CD4 binding loop and the V4 loop. Together, our studies provide new mechanistic insights into the gp120 determinants influencing sensitivity and resistance to HIV-1 neutralization by b12., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The murine mAb, K-1-21, recognizes a conformational epitope expressed on free Ig kappa light chains (FκLCs) and also on cell membrane-associated FκLCs found on kappa myeloma cells. This has led to the development of a chimeric version of K-1-21, MDX-1097, which is being assessed in a Phase II clinical trial for the treatment of multiple myeloma. The epitope recognized by K-1-21 is of particular interest, especially in the context that it is not expressed on heavy chain-associated light chains such as in an intact Ig molecule. Using epitope excision techniques we have localized the K-1-21 epitope to a region spanning residues 104-110 of FκLC. This short strand of residues links the variable and constant domains, and is a flexible region that adopts different conformations in FκLC and heavy chain-associated light chain. We tested this region using site-directed mutations and found that the reactivity of K-1-21 for FκLC was markedly reduced. Finally, we applied in silico molecular docking to generate a model that satisfied the experimental data. Given the clinical potential of the Ag, this study may aid the development of next generation compounds that target the membrane form of FκLC expressed on the surface of myeloma plasma cells., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Carbohydrates are notoriously flexible molecules. However, they have an important role in many biochemical processes as specific ligands. Understanding how carbohydrates are recognized by other biological macromolecules (usually proteins) is therefore of considerable scientific value. Interfering with carbohydrate-protein interactions is a potentially useful strategy in combating a range of disease states, as well as being of critical importance in facilitating allo- and xenotransplantation. We have devised an in silico protocol for analyzing carbohydrate-protein interactions. In this study, we have applied the protocol to determine the structures of alphaGal-terminating carbohydrate antigens in complex with a panel of xenoreactive antibodies. The most important feature of the binding modes is the fixed conformation of the Galbeta(1,4)Glc/GlcNAc linkage across all of the binding modes. The preferred conformation of the terminal Galalpha(1,3)Gal linkage varies depending on the antibody binding site topography, although it is possible that some of the antibodies studied recognize more than one Galalpha(1,3)Gal conformation. The binding modes obtained indicate that each antibody uses distinct mechanisms in recognizing the target antigens.

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.)

Antibody-carbohydrate interactions play central roles in stimulating adverse immune reactions. The most familiar example of such a process is the reaction observed in ABO-incompatible blood transfusion and organ transplantation. The ABO blood groups are defined by the presence of specific carbohydrates expressed on the surface of red blood cells. Preformed antibodies in the incompatible recipient (i.e., different blood groups) recognize cells exhibiting host-incompatible ABO system antigens and proceed to initiate lysis of the incompatible cells. Pig-to-human xenotransplantation presents a similar immunological barrier. Antibodies present in humans recognize carbohydrate antigens on the surface of pig organs as foreign and proceed to initiate hyperacute xenograft rejection. The major carbohydrate xenoantigens all bear terminal Gal alpha(1,3)Gal epitopes (or alphaGal). In this study, we have developed and validated a site mapping technique to investigate protein-ligand recognition and applied it to antibody-carbohydrate systems. This site mapping technique involves the use of molecular docking to generate a series of antibody-carbohydrate complexes, followed by analysis of the hydrogen bonding and van der Waals interactions occurring in each complex. The technique was validated by application to a series of antibody-carbohydrate crystal structures. In each case, the majority of interactions made in the crystal structure complex were able to be reproduced. The technique was then applied to investigate xenoantigen recognition by a panel of monoclonal anti-alphaGal antibodies. The results indicate that there is a significant overlap of the antibody regions engaging the xenoantigens across the panel. Likewise, similar regions of the xenoantigens interact with the antibodies.

We describe IgM class human autoantibodies that hydrolyze amyloid beta peptide 1-40 (Abeta40). A monoclonal IgM from a patient with Waldenström's macroglobulinemia hydrolyzed Abeta40 at the Lys-28-Gly-29 bond and Lys-16-Ala-17 bonds. The catalytic activity was inhibited stoichiometrically by an electrophilic serine protease inhibitor. Treatment with the catalytic IgM blocked the aggregation and toxicity of Abeta40 in neuronal cell cultures. IgMs purified from the sera of patients with Alzheimer disease (AD) hydrolyzed Abeta40 at rates superior to IgMs from age-matched humans without dementia. IgMs from non-elderly humans expressed the least catalytic activity. The reaction rate was sufficient to afford appreciable degradation at physiological Abeta and IgM concentrations found in peripheral circulation. Increased Abeta concentrations in the AD brain are thought to induce neurodegenerative effects. Peripheral administration of Abeta binding antibodies has been suggested as a potential treatment of AD. Our results suggest that catalytic IgM autoantibodies can help clear Abeta, and they open the possibility of using catalytic Abs for AD immunotherapy.

Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.

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.