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The advances in high-throughput DNA sequencing and recombinant antibody technologies has presented new methods for characterizing antibody repertoires and significantly increased our understanding on the functional role of antibodies in immunity and their use in diagnostics, vaccine antigen design and as biological therapeutics. A subset of Bos taurus antibodies possesses unique ultra-long third complementary-determining region of the heavy chain (CDRH3) and are of special interest because they are thought to have unique functional abilities of broadly neutralizing properties - a functional role that has not been fully explored in vaccine development. Next generation sequencing technologies that are widely used to profile immunoglobulin (Ig) repertoires are based on short-read methods such as the Illumina technology. Although this technology has worked well in sequencing Ig V-D-J regions of most jawed vertebrates, it has faced serious technical challenges with sequencing regions in bovine Ig bearing ultra-long CDRH3 sequences, which are longer than 120 bp. To overcome this limitation, we have developed a sequencing strategy based on nested PCR products that allows sequence assembly of full-length bovine Ig heavy-chain (IgH) V-D-J regions. We have used this strategy to sequence IgH V-D-J regions of two Bos indicus breeds, Ankole and Boran. We confirm the presence of ultra-long CDRH3 sequences in IgG transcripts in both African cattle breeds, and provide preliminary evidence for differences and preferences in germline V H , D H and J H allele gene usage as well as differences in the length of the VH region in the two bovine breeds. Our method provides tools that should allow more robust analyses of ultra-long CDRH3 sequences aiding antibody and epitope discovery in different cattle breeds and their role in mediating immunity., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Immunoglobulin G (IgG) antibodies are important for protection against pathogens and exert effector functions through binding to IgG-Fc receptors (FcγRs) on myeloid and natural killer cells, resulting in destruction of opsonized target cells. Despite interspecies differences, IgG subclasses and FcγRs show substantial similarities and functional conservation between mammals. Accordingly, binding of human IgG (hIgG) to mouse FcγRs (mFcγRs) has been utilized to study effector functions of hIgG in mice. In other applications, such as immunostaining with mouse IgG monoclonal antibodies (mAbs), these cross-reactivities are undesired and prone to misinterpretation. Despite this drawback, the binding of mouse IgG (mIgG) subclasses to human FcγR (hFcγR) classes has never been fully documented. Here, we report detailed and quantifiable characterization of binding affinities for all mIgG subclasses to hFcγRs, including functional polymorphic variants. mIgG subclasses show the strongest binding to hFcγRIa, with relative affinities mIgG2a = mIgG2c > mIgG3 >> mIgG2b, and no binding by mIgG1. hFcγRIIa/b showed general low reactivities to all mIgG (mIgG1> mIgG2a/c > mIgG2b), with no reactivity to mIgG3. A particularly high affinity was observed for mIgG1 to the hFcγRIIa-R131 polymorphic variant. hFcγRIIIa showed lower binding (mIgG2a/c > mIgG3), slightly favouring binding to the hFcγRIIIa-V158 over the F158 polymorphic variant. No binding was observed of mIgG to hFcγRIIIb. Deglycosylation of mIgG1 did not abrogate binding to hFcγRIIa-R131, nor did deglycosylation of mIgG2a/c and mIgG3 prevent hFcγRIa binding. Importantly, deglycosylation of the least cross-reactive mIgG subclass, mIgG2b, abrogated reactivity to all hFcγRs. Together, these data document for the first time the full spectrum of cross-reactivities of mouse IgG to human FcγRs., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Preclinical studies require an immune response similar to that of humans in a small animal model that is convenient to operate. Based on genome alignment, tree shrews are small animals considered to be more similar to primates than are rodents, and many human disease models have been established with tree shrews. However, the characteristics of the humoral immune response of tree shrews remain to be elucidated. In this study, the genetic sequence of the heavy chain constant region of tree shrew immunoglobulin (Ig) was complemented, and the results of immunoglobulin domain homology and transcriptome analysis showed that the tree shrew genome encodes only four classes of antibodies and does not encode IgD. The oldest IgM antibody has the highest homology with primates. After the complete sequence of each type of antibody was obtained, the tree shrew antibody protein was further expressed and purified by in vitro recombination, and an IgG quantitative evaluation system was established. The highly effective immuno protective effect induced by HSV-1 infection and the significant bactericidal effect induced by Neisseria meningitidis group C polysaccharide immunization showed that tree shrews exhibited immune responses more similar to humans than to mice. This may provide better predictive value for vaccine preclinical research., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aggregation of therapeutic proteins is a key factor in the generation of unwanted immunogenicity, and can result in reduced serum half-life, neutralization of function and adverse health effects. There is currently little information regarding how aggregates interact with B-cell receptors or cognate antibodies at the protein sequence level, or whether non-native, aggregate-induced epitopes predominate in these interactions. Using an antibody fragment (single chain antibody variable fragment; scFv) that forms aggregates readily at low temperature, anti-scFv IgG antibody responses were generated by intraperitoneal injection of BALB/c strain mice with monomer or aggregate preparations. Aggregate-specific immunosignatures were identified by oligo-peptide microarray fine epitope mapping, using overlapping 15mer peptides based on the linear sequence of scFv, printed onto glass slides. IgG antibodies from mice immunized with aggregated scFv preferentially recognized a patch of overlapping peptides. This region mapped to a β-strand located at the interface between the V H and V L domains. Molecular dynamics simulations indicated that the V L domain is less stable than the V H domain, suggesting the interface region between the two domains becomes exposed during partial unfolding of the scFv during aggregate formation. These data are consistent with the hypothesis that epitopes from partially unfolded states are revealed, or are more fully exposed, in the aggregated state, and that this can augment the IgG antibody response. This observation offers the theoretical possibility that epitopes preferentially associated with aggregates can be identified from the anti-drug antibody serum IgG response which may, in turn, lead to better methods for detection of anti-drug antibody responses, and improved design of therapeutic proteins to control immunogenicity., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders. Here, we show that G1Δnab mutants retain pH-dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0-fold and 1.6-fold for the two antibodies investigated. The transport of the modified G1Δnab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild-type, except that no difference was observed with human umbilical vein endothelial cells. G1Δnab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1Δnab mutations are away from the FcRn-binding site, they have long-distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The binding strength between IgG and FcγR is influenced by the composition of the N-linked glycan at position N297 in the Fc-domain of IgG. Particularly, afucosylation increases the binding affinity of human IgG1 to human FcγRIIIa up to ∼20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up to ∼40 fold. The increase in affinity for afucosylated IgG has previously been shown to depend on direct carbohydrate-carbohydrate interactions between the IgG-Fc glycan with an N-linked glycan at position 162 unique to hFcγRIIIa and hFcγRIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse FcγR, mFcγRIV. The N162-glycan in mFcγRIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFcγRIIIa. However, unlike hFcγRIIIa, mFcγRIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162-glycan in the human FcγRIII family and its orthologous mouse FcγRIV to be functionally conserved., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Immunoglobulin G (IgG) consists of four subclasses in humans: IgG1, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgG1, IgG3 exhibits a similar binding profile to Fcγ receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcγ receptor and C1q complement binding as well as maintaining optimal Fc conformation. We have determined the crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc 2 Man 5 (Man5) high mannose glycoform at 1.8Å resolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcγ receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Human Fc receptor-like 5 (FCRL5) is a novel IgG receptor. We reported that IgG2 samples display a thousand-fold range affinity for FCRL5, indicating that attributes beyond the isotype affect binding. We hypothesized that the complex interaction could be exploited to identify distinct changes in the IgG2 molecule. We investigated using surface plasmon resonance two factors that might affect the interaction between IgG2 and FCRL5; heterogeneity related to disulfide isoforms and charge variants. We found that panitumumab and denosumab samples enriched for the more flexible A disulfide isoform bound FCRL5 with two-fold and 82-fold higher apparent affinity, respectively, than the B isoform. We next assessed whether FCRL5 binding can distinguish panitumumab charge variants which increase during storage, using two approaches. First, samples were stored at 40°C to promote acidic variants. Heat stressed panitumumab had up to four-fold higher apparent affinity for FCRL5. Next, we used conditions that promoted deamidation, a common cause of acidic variants. We found that deamidated panitumumab had up to 14-fold higher apparent affinity for FCRL5, indicating that deamidation promotes the interaction. Statistical analyses of kinetic parameters and similarity scores obtained from sensogram comparisons indicated that IgG2 disulfide isoforms, heat stressed and deamidated samples each bind FCRL5 differently. We conclude that based on FCRL5 binding, we can discern distinct changes in the IgG2 molecule, including the disulfide isoform structure and charge variants related to deamidation. Since both IgG2 deamidation and conversion of disulfide isoforms occur in vivo, these findings elucidate the biological FCRL5 ligand., (Published by Elsevier Ltd.)

Human IgG4 (hIgG4) has weak pro-inflammatory activity. The structural basis for this is still unclear. Here a 3D model of myeloma hIgG4 was created at ∼3nm resolution using electron microscopy (EM) with negative staining and single-particle 3D reconstruction. The hIgG4 model reveals relatively rigid asymmetric Y-like structure. The model shows that one Fab subunit is closer to the upper portion of the Fc subunit (C H 2 domain) than the other Fab. This is in agreement with X-ray crystallography and X-ray/neutron scattering, recently published by others. The same hIgG4 sample was studied with differential scanning calorimetry (DSC) and fluorescence. The thermodynamics and fluorescence observations indicate that one C H 2 domain displays less conformational stability than the other. This finding is consistent with the flipping of one C H 2 domain, observed in pembrolizumab (recombinant hIgG4) by X-ray crystallography. The specific feature of hIgG4 C H 2 domains together with relatively rigid asymmetric Y-like structure, in which one Fab subunit is closer to the upper portion of the Fc subunit (C H 2 domain) than the other Fab, can explain the unique biological properties of hIgG4, such as its weak pro-inflammatory activity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The Fc region of IgG antibodies (Cγ2 and Cγ3 domains) is responsible for effector functions such as antibody-dependent cell-mediated cytotoxicity and phagocytosis, through engagement with Fcγ receptors, although the ability to elicit these functions differs between the four human IgG subclasses. A key determinant of Fcγ receptor interactions is the FG loop in the Cγ2 domain. High resolution cryogenic IgG4-Fc crystal structures have revealed a unique conformation for this loop, which could contribute to the particular biological properties of this subclass. To further explore the conformation of the IgG4 Cγ2 FG loop at near-physiological temperature, we solved a 2.7Å resolution room temperature structure of recombinant human IgG4-Fc from crystals analysed in situ. The Cγ2 FG loop in one chain differs from the cryogenic structure, and adopts the conserved conformation found in IgG1-Fc; however, this conformation participates in extensive crystal packing interactions. On the other hand, at room temperature, and free from any crystal packing interactions, the Cγ2 FG loop in the other chain adopts the conformation previously observed in the cryogenic IgG4-Fc structures, despite both conformations being accessible. The room temperature human IgG4-Fc structure thus provides a more complete and physiologically relevant description of the conformation of this functionally critical Cγ2 FG loop., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The therapeutic activity of monoclonal antibodies can involve immune cell mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC), an activity that is modulated by the structure of Fc-glycans, and in particular the lack of core fucose. The heterogeneity of these glycostructures and the inherent variability of traditional PBMC-based in vitro ADCC assays, have made it challenging to quantitatively assess the impact of other glycostructures on ADCC activity. We applied a quantitative NK cell based assay to generate a database consisting of Fc-glycostructure and ADCC data from 54 manufacturing batches of a CHO-derived monoclonal antibody. Explorative analysis of the data indicated that, apart from afucosylation, galactosylation levels could influence ADCC activity. We confirmed this hypothesis by demonstrating enhanced ADCC upon enzymatic hypergalactosylation of four different monoclonal antibodies derived using standard CHO manufacturing processes. Furthermore we quantitatively compare the effects of galactosylation and afucosylation in the context of glycan heterogeneity and demonstrate that while galactose can influence ADCC activity, afucosylation remains the primary driver of this activity., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.)

In contrast to the glycosylated IgG antibodies secreted by human plasma cells, the aglycosylated IgG antibodies produced by bacteria are unable to bind FcγRs expressed on the surface of immune effector cells and cannot trigger immune effector functions. To avoid glycan heterogeneity problems, elicit novel effector functions, and produce therapeutic antibodies with effector function using a simple bacterial expression system, FcγRI-specific Fc-engineered aglycosylated antibodies, Fc11 (E382V) and Fc (E382V/M428I), containing mutations in the CH3 region, were isolated in a previous study. To elucidate the relationship between FcγRI binding affinity and the structural dynamics of the upper CH2 region of Fc induced by the CH3 mutations, the conformational variation of Fc variants was observed by single-molecule Förster resonance energy transfer (FRET) analysis using alternating-laser excitation (ALEX). In sharp contrast to wild-type Fc, which exhibits a highly dynamic upper CH2 region, the mutations in the CH3 region significantly stabilized the upper CH2 region. The results indicate that conformational plasticity, as well as the openness of the upper CH2 region, is critical for FcγR binding and therapeutic effector functions of IgG antibodies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We determined the X-ray crystal structure of an immunoglobulin fragment crystallizable (Fc) heterodimer, EW-RVT, at a resolution of 2.5Å and found that the designed asymmetric interaction residues located in the heterodimeric CH3 interface favor Fc heterodimer formation. We further generated an inter-CH3 disulfide-bonded heterodimeric Fc variant, EW-RVT(S-S), which exhibited improved heterodimer formation and thermodynamic stability compared with the parent EW-RVT variant. The crystal structure of EW-RVTS-S superimposed very closely with the wild-type Fc structure. Our results provide the detailed structure of heterodimeric Fc scaffolds, which will be useful for the generation of immunoglobulin G (IgG)-like bispecific antibodies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The effects of the Fc silencing mutations such as leucine (L) to alanine (A) substitution at the position 234 and 235 (LALA) and the alanine (A) to asparagine (N) substitution at position 297 (N297A) are well investigated for human IgG. However, the effects of the same two silencing Fc mutations in a mouse IgG backbone are not yet well investigated in respect to binding to mouse Fc gamma receptors (FcγRs), complement and subsequent effector functions. By using a mouse IgG2a tool antibody directed against mouse OX40L, we demonstrate a strongly reduced binding of the two Fc mutants to high and low affinity recombinant and cell expressed mouse FcγRs, when compared to the mouse IgG2a with the wild type (wt) backbone. Reduced FcγR binding by the two investigated Fc mutants could further be confirmed on primary mouse macrophages expressing their native FcγRs. In addition, we reveal that the LALA and N297A mutations in the mIgG2a also slightly reduced binding to C1q of human origin. Thus, here we provide experimental evidence that the two investigated Fc mutations in the mouse IgG backbone lead to similar "silencing" properties as previously demonstrated for the human IgG and thus represent a useful method to alter effector functions in tool antibodies to be used in mouse models., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The Fc region of IgG antibodies, important for effector functions such as antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement activation, contains an oligosaccharide moiety covalently attached to each C(H)2 domain. The oligosaccharide not only orients the C(H)2 domains but plays an important role in influencing IgG effector function, and engineering the IgG-Fc oligosaccharide moiety is an important aspect in the design of therapeutic monoclonal IgG antibodies. Recently we reported the crystal structure of glycosylated IgG4-Fc, revealing structural features that could explain the anti-inflammatory biological properties of IgG4 compared with IgG1. We now report the crystal structure of enzymatically deglycosylated IgG4-Fc, derived from human serum, at 2.7Å resolution. Intermolecular C(H)2-C(H)2 domain interactions partially bury the C(H)2 domain surface that would otherwise be exposed by the absence of oligosaccharide, and two Fc molecules are interlocked in a symmetric, open conformation. The conformation of the C(H)2 domain DE loop, to which oligosaccharide is attached, is altered in the absence of carbohydrate. Furthermore, the C(H)2 domain FG loop, important for Fcγ receptor and C1q binding, adopts two different conformations. One loop conformation is unique to IgG4 and would disrupt binding, consistent with IgG4's anti-inflammatory properties. The second is similar to the conserved conformation found in IgG1, suggesting that in contrast to IgG1, the IgG4 C(H)2 FG loop is dynamic. Finally, crystal packing reveals a hexameric arrangement of IgG4-Fc molecules, providing further clues about the interaction between C1q and IgG., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The deglycosylation of immunoglobulin G (IgG) antibodies leads to a diminished immune response. This reduction in immune response is thought to arise from weakened binding of IgG antibodies to effector molecules as a result of a conformational change in the antibody. The nature of this structural alteration is uncertain due to the conflicting results obtained from different experimental methods. We have examined the impact of deglycosylation by the endoglycosidase PNGase F on the structure of the Fc region of a human IgG antibody using single molecule Förster Resonance Energy Transfer (FRET). The FRET efficiency histograms obtained indicate that the structure of the Fc region becomes more flexible upon deglycosylation. This is demonstrated by a change in the width of the energy transfer efficiency peak, which increases from 0.19 ± 0.02 to 0.6 ± 0.1 upon deglycosylation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Antibody dimers, two self-associated monomers, have been detected on both recombinantly expressed and endogenous human IgG proteins. Nearly 10 years ago, Yoo et al. (2003) described low levels of IgG2 covalent dimer, in human serum, but did not quantify the levels. Here we quantify the total and covalent dimer levels of IgG2 and IgG1 in human blood, and study the origin of covalent dimer formation. Low levels (<1%) of total IgG1 and IgG2 dimers were measured in freshly prepared human plasma. Both IgG1 and IgG2 covalent dimers were also found in plasma. Whereas IgG1 covalent dimer levels were significantly reduced by steps intended to eliminate artifacts during sample preparation, IgG2 covalent dimer levels remain stable in such conditions. About 0.4% of IgG2 in plasma was in a covalent dimer form, yet very little (<0.03%) of IgG1 covalent dimer could be considered naturally occurring. IgG2 dimer also formed in vitro under conditions designed to mimic those in blood, suggesting that formation occurs in vivo during circulation. Thus, small amounts of covalent IgG2 dimer do appear to form naturally., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Until relatively recently the immunoglobulin molecule was viewed as composed of two independent domains comprised of the variable (V) and constant (C) regions. However, recent work has established that the C region mediates allosteric changes in the V region that can influence specificity and affinity. To further explore cross-domain interrelationship in murine IgG structure we carried out solution small angle X-ray scattering (SAXS) measurements for four V region identical IgG isotypes. SAXS analysis revealed elongated Y-shaped structures in solution with significantly different, isotype-dependent domain orientations. To further explore local C region effects on the V region, the IgG₃ Fab crystal structure from the same family was determined to 2.45 Å resolution. The IgG₃ Fab crystal structure differs from a closely related previously solved IgG1 Fab revealing significant structural differences, which may account for isotype-related specificity differences in V region identical Abs. Among the four murine isotypes, IgG₃ was the most different in solution with regards to overall structure as well as aggregate formation in solution suggesting that the greater apparent affinity of this isotype resulted from polyvalent complexes with enhanced avidity. Our results provide additional evidence that Ig V and C domains influence each other structurally and suggest that V region structure can have significant effects on overall Ig structure., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The analyses of two human IgG2 Fc structures, determined in different crystal forms, and the comparison with IgG1 Fc structures reveals molecular features that are involved in accommodating and stabilizing structural conformations. In the IgG2 Fc structures relative positions of the CH2 domains with respect to the CH3 domains vary significantly from those observed for IgG1 Fc structures in similar unit cells. The analysis reveals that the movement of the CH2 domain in all of the Fc structures results from a pivoting around a highly conserved ball-and-socket-like joint in which the CH2 L251 side chain (the ball) interacts with a pocket (the socket) formed by CH3 M428, H429, E430, and H435. Despite the change in positioning of the CH2 and CH3 domains, conserved hydrogen bonds and electrostatic interactions are retained, stabilizing the Fc domain interface. In the high resolution IgG2 and IgG1 Fc structures the position and number of water molecules, and water networks bridging the two domains differ significantly because of the difference in positions of CH2 relative to CH3. At the domain interface, only CH2 T339 in IgG2 differs from alanine found in IgG1 and IgG4. This residue's side chain influences the water structure at the interface by interacting either directly or through a bridging water molecule with D376 in the CH3 BC loop. Thus, the analyses of the IgG2 Fc structures and their comparisons with IgG1 Fc structures reveals similar, but distinctly different dynamic CH2-CH3 interfaces that can accommodate a wide range of CH2-CH3 conformations, that in conjunction with the amino acid residues in the hinge region, may influence FcγR effector function profiles., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Methylene diphenyl diisocyanate (MDI), a low molecular weight chemical important for producing polyurethane foam, coatings, and elastomers is a major cause of occupational asthma, however, mechanisms of disease pathogenesis remain poorly understood. This study characterizes the rearranged germline and hypervariable region cDNA of new anti-MDI secreting hybridomas derived from mice immunized with MDI-conjugated to autologous serum proteins. Six IgG1 secreting clones were identified in initial screening ELISAs, based on differential binding to MDI conjugated human albumin vs. mock exposed albumin. The mAbs secreted by the hybridomas also recognized MDI conjugated to other model proteins (e.g. ovalbumin, transferrin), but did not bind unconjugated proteins, or protein conjugates prepared with other isocyanates (e.g. TDI, HDI). The mAbs displayed MDI-dose dependent binding in ELISA and Western blot, and exhibited varying degrees of cross-competition, suggesting differences in epitope specificity. The cDNA encoding the monoclonal antibodies reveal clonal differences in the CDR3 regions, germline gene usage, and patterns of somatic hypermutation related to epitope specificity. Together, the data provide new insight into the molecular determinants of humoral MDI specificity, and characterize anti-MDI IgG1 mAbs that may be developed into useful diagnostic reagents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human IgG2 antibodies contain three types of disulfide isoforms, classified by the number of Fab arms having disulfide links to the heavy chain hinge region. In the IgG2-B form, both Fab arms have interchain disulfide bonds to the hinge region, and in IgG2-A, neither Fab arm are disulfide linked to the hinge. The IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide linked to the hinge. Changes in the relative levels of these forms over time are observed while IgG2 circulates in humans, suggesting IgG2-A→IgG2-A/B→IgG2-B conversion. Using a flow-through dialysis system, we studied the conversion kinetics of these forms in vitro under physiological conditions. For two IgG2κ antibodies, in vivo results closely matched the kinetics observed in vitro, indicating that the changes observed in vivo were solely conversions between isoforms, not differential clearance of specific forms. Moreover, the combined results validate the accuracy of the physiological model for the study of blood redox reactions. Further exploration of the conversion kinetics using material enriched in the IgG2-A forms revealed that the IgG2-A→IgG2-A/B rate was similar between IgG2κ and IgG2λ antibodies. In IgG2κ antibodies, conversion of IgG2-A/B→IgG2-B was slower than the IgG2-A→IgG2-A/B reaction. However, in IgG2λ antibodies, little IgG2-A/B→IgG2-B conversion was detected under physiological conditions. Thus, small differences in the C-terminus of the light chain sequences affect the disulfide conversion kinetics and impact the IgG2 disulfide isoforms produced in vivo., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Antibodies of the human IgG4 subclass uniquely undergo a process of Fab-arm exchange in which the heavy-chains of antibodies of different specificities can dissociate and then recombine. The mechanism by which the resulting functionally monovalent but bi-specific antibodies are formed is not only key to understanding their biological role, but is also important for the design of therapeutic monoclonal antibodies. Both the hinge region and the C(H)3 domain interface are known to be involved, and of the residues that differ between human IgG1 and IgG4 in C(H)3, residue 409, the only difference at the interface itself, has been implicated. We report the high resolution (1.8Å) structure of the C(H)3 domain dimer of IgG4, and find that Arg409 in IgG4, when compared with Lys409 observed in high resolution IgG1 structures, disrupts a network of water-mediated hydrogen bonding that is conserved in IgG1. Other conformational differences were detected that are a consequence of the presence of Arg409, such as a widening of the separation between residues Asn390 in one domain and Ser 400 in the other, which opens up a groove at the edge of the interface in IgG4 compared with IgG1. The effect of all these differences on the C(H)3 interface, doubled as a result of the interface's two-fold symmetry, is weakening of the inter-domain interaction in IgG4 compared with IgG1. This suggests a mechanism by which Arg409 weakens the C(H)3 interface in IgG4, predisposing this human antibody subclass to Fab-arm exchange., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human IgG4 antibodies are remarkable not only because they can dynamically exchange half-molecules (Fab-arm exchange) but also for their ability to interact with the Fc part of IgG4 and other IgG subclasses. This rheumatoid factor-like binding of IgG4 does not appear to take place spontaneously, because it is only observed to solid-phase or antigen-bound IgG. We hypothesized that Fc-Fc interactions might involve (partial) dissociation of heavy chains. We investigated the molecular basis of these Fc-Fc interactions, and found that the structural features important for the exchange reaction also control the Fc binding activity. In particular, if arginine-409 in the CH(3)-CH(3) interface in IgG4 is mutated to lysine (the equivalent in IgG1), Fc-Fc interactions are formed 3 orders of magnitude less efficiently compared to the wild-type. This mutation was previously found to increase the CH(3)-CH(3) interaction strength in IgG4. Furthermore, of the two hinge isomers of IgG4, the intra-chain (non-covalently linked) form was found to form Fc-Fc interactions, but not the inter-chain form. Together, these results demonstrate that Fc-Fc interactions of IgG4 involve (partial or complete) dissociation of heavy chains. The promiscuity to other IgG subclasses suggests that IgG4 might act as scavenger to IgG molecules with impaired structural integrity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

High resolution glycan mapping was performed on human immunoglobulin G (IgG) obtained from individual healthy subjects and from a combined sample of healthy subjects. In addition to the commonly known complex glycans, a variety of minor glycans are described and quantified, including high mannose forms and several previously unreported hybrid forms. Fc specific glycan analysis was also performed through peptide mapping with LC/MS/MS. Differences in the glycan linked Fc peptide masses allowed glycan profiles to be analyzed and quantified from IgG1 and IgG2 simultaneously for each subject within the same sample. Glycan profiles differed between subtypes, with greater levels of more fully galactosylated species found on IgG1 (e.g. G2F, SG2F) than IgG2. These results also show that Gal attachment on G1F is biased to the Man (alpha1-->6) arm for IgG1 and on the Man (alpha1-->3) arm for IgG2 from individual healthy subjects., ((c) 2010 Elsevier Ltd. All rights reserved.)

In avian species, maternal immunoglobulin (Ig) Y is selectively incorporated into the yolks of maturing oocytes, although the relevance of receptor-mediated uptake is unclear. When administered to birds, several mammalian Igs, including human IgG (hIgG), are also incorporated into the yolks. In the current study, to gain insight into selective Ig transport into yolks, we intended to identify the amino acid residues critical for Ig uptake into egg yolks using alanine and glycine-scanning mutagenesis of 16 residues located along the C(H)2 and C(H)3 domains of hIgG1. Wild-type hIgG1-Fc (WT) and its mutants were synthesized, and their uptakes into the egg yolks of Japanese quail (Coturnix japonica) were determined. The triple mutation of loop MIS252-254 to GGG resulted in a 40% decrease in Fc uptake in comparison to that of the WT. Furthermore, quartet substitution of HEAL429-432 to GGGG located in an exposed loop at the C(H)3 domain completely abolished Fc uptake into egg yolks. Next, the residues HEAL429-432 were individually substituted with either alanine or glycine. Regardless of the glycine and alanine substitution, single mutations of H (429), E (430) and L (432) significantly reduced Fc uptake compared with WT uptake. Notably, the blood clearance rates of these mutants were equivalent to that of the WT. These results suggest that the clustered residues HEAL429-432 in the C(H)3 domain are important for the hIgG1 transport into the egg yolks. The sequence HEAL is conserved in chicken IgY at positions 550-553 within the C(H)4 domain, which might be involved in its uptake into the egg yolks by receptor-mediated endocytosis., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Human IgG4 antibodies are dynamic molecules that in vivo exchange half-molecules to become bispecific antibodies. Here we show that IgG4 antibodies and IgG4 Fc fragments similarly exchange resulting in hybrid antibodies (a single Fab+Fc) with a molecular weight of ca. 100kDa. These antibodies can be separated from IgG4 and IgG4 Fc using size-exclusion chromatography and non-reducing SDS-PAGE. This method does not rely on a cross-linking immunoassay with its potential for false-positive results due to aggregation and it unambiguously demonstrates that the 'Fab-arm' exchange process depends entirely on the Fc part (hinge+CH2+CH3)., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Antibodies (Ab) are bifunctional molecules with two domains, a constant region (C) that confers effector properties and a variable (V) region responsible of antigen (Ag) binding. Historically the C and V regions were considered to be functionally independent, with Ag specificity being solely determined by the V region. However, recent studies suggest that the C region can affect Ab fine specificity. This has led to the proposal that the C(H) domain influences the structure of the V region, thus affecting Ab affinity and fine specificity. An inference from this proposal is that V region identical monoclonal Abs (mAbs) differing in C region (eg isotype) would manifest different secondary structures arising from isotype-induced variation in the V-C regions after Ag binding. We hypothesized that such effects could translate into differences in Circular Dichroism (CD) upon Ag-Ab complexes formation. Consequently we studied the interaction of a set of V region identical IgG(1), IgG(2a), IgG(2b) and IgG(3) mAbs with glucuronoxylomannan (GXM). The native CD spectra of the pairs IgG(1)/IgG(2a) and IgG(3)/IgG(2b) were strikingly similar, implying similar secondary structure content. GXM binding by IgG(1), IgG(2a), IgG(2b) and IgG(3) produced different CD changes, with the pairs IgG(1)/IgG(2a) and IgG(3)/IgG(2b) again manifesting qualitatively similar trends in secondary structure changes. The magnitude of the changes differed among the isotypes with IgG(2a)>IgG(3)>IgG(2b)>IgG(1). These differences in CD changes were interpreted to reflect differences in V-C secondary structures., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Staphylococcal superantigen-like (SSL) proteins are a family of exoproteins that share structural similarity with staphylococcal superantigens but exhibit no superantigenic activity. It was previously reported that two members (SSL5 and SSL7) bound to serum components and cell adhesion molecules involved in host immune response; however, the other family members have not been functionally characterized. In this study, we attempted to isolate SSL10-binding proteins from human serum and found that recombinant His-tagged SSL10 bound two major polypeptides of approximately 50 and approximately 25 kDa after affinity purification and SDS-polyacrylamide gel electrophoresis. These polypeptides were identified as heavy and light chains of human IgG by peptide mass fingerprinting analysis. The specific interaction between recombinant SSL10 and human IgG was confirmed by far Western blot analysis using immobilized SSL10 and pull-down analysis using SSL10-conjugated Sepharose. Surface plasmon resonance analysis revealed that the dissociation equilibrium constant for the interaction between human IgG and recombinant SSL10 was estimated to be 220 nM. We also found that recombinant SSL10 inhibited the binding of complement component C1q to IgG-Sepharose and hemolysis of IgG-sensitized sheep erythrocytes via the classical complement activation pathway. These results suggest that SSL10 may play a role in the evasion of Staphylococcus aureus from the host immune system via interfering complement activation., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Fab arm exchange by a stabilized anti-IL-31 IgG(4)S228P monoclonal antibody (mAb) was studied using physiologically relevant antibody concentrations and thiol exchange conditions, and directly compared to that of matched wild type IgG(4) (IgG(4)wt) and IgG(1) control antibodies. In vitro arm exchange between the test mAbs and a purified IgG(4)wt exchange partner was monitored using capillary isoelectric focusing and a size-exclusion peak shift assay. Arm exchange between the test mAbs and IgG exchange partners with unknown specificity was monitored using only the shift assay. Studies were performed using single isotype human and mouse mAbs, unfractionated human, mouse, and cynomolgus monkey IgG, and human serum as the sources of the exchange partners. In vitro studies using human serum demonstrated that anti-IL-31 IgG(4)S228P did not undergo significant Fab arm exchange with endogenous human IgG(4) whereas anti-IL-31 IgG(4)wt underwent rapid and extensive Fab arm exchange. The in vitro results were corroborated by in vivo studies in which mice were injected with a mixture of either form of the test mAb and an excess of non-specific human IgG(4) exchange partner.

The first three-dimensional structure of a human Fc fragment genetically engineered for improved pharmacokinetics properties is reported. When introduced into the C(H)2 domain of human immunoglobulin G (IgG) molecules, the triple mutation M252Y/S254T/T256E ('YTE') causes an about 10-fold increase in their binding to the human neonatal Fc receptor (FcRn). This translates into an almost 4-fold increase in the serum half-life of YTE-containing human IgGs in cynomolgus monkeys. A recombinantly produced human Fc/YTE fragment was crystallized and its structure solved at a resolution of 2.5A using molecular replacement. This revealed that Fc/YTE three-dimensional structure is very similar to that of other human Fc fragments in the experimentally visible region spanning residues 236-444. We propose that the enhanced interaction between Fc/YTE and human FcRn is likely mediated by local effects at the substitutions sites. Molecular modeling suggested that potential favorable hydrogen bonds along with an increase in the surface of contact between the two partners may account in part for the corresponding increase in affinity.

Monoclonal anti-collagen type II antibody CIIC1 is an arthritogenic autoantibody, which induces arthritis in mice. We crystallized and solved the structure of CIIC1 Fab molecule. Analysis of structure revealed an interaction between the CDR regions of one Fab to the CH1 domain of another Fab, which resembles an antibody-antigen interaction. ELISA experiments confirmed the cross-reactivity of both the full CIIC1 antibody and a single chain Fv fragment to other anti-collagen antibodies which are of different isotypes and epitope specificity. The rheumatoid factor like reactivity of CIIC1 antibody together with its collagen type II specificity may explain the pathogenicity of this antibody.

A comparative in vitro survey of physiologically relevant human and microbial proteinases defined a number of enzymes that induced specific hinge domain cleavage in human IgG1. Several of these proteinases have been associated with tumor growth, inflammation, and infection. A majority of the identified proteinases converted IgG to F(ab')(2), and a consistent feature of their action was a transient accumulation of a single-cleaved intermediate (scIgG). The scIgG resulted from the relatively rapid cleavage of the first hinge domain heavy chain, followed by a slower cleavage of the second chain to separate the Fc domain from F(ab')(2). Major sites of enzymatic cleavage were identified or confirmed from the mass of the F(ab')(2) or Fab fragments and/or the amino-terminal amino acid sequence of the Fc for each enzyme including human matrix metalloproteinases (MMPs) 3 and 12, human cathepsin G, human neutrophil elastase (Fab), staphylococcal glutamyl endopeptidase I and streptococcal immunoglobulin-degrading enzyme (IdeS). The cleavage sites in IgG1 by MMP-3, cathepsin G and IdeS were used to guide the synthesis of peptide analogs containing the corresponding carboxy-termini to be used as immunogens in rabbits. Rabbit antibodies were successfully generated that showed selective binding to different human F(ab')(2)s and other hinge-cleavage fragments, but not to intact IgG. In Western blotting studies of synovial fluids from individuals with rheumatoid arthritis, the rabbit antibodies yielded patterns consistent with the presence of endogenous IgG fragments including F(ab')(2) and the single-cleaved IgG intermediate. The detection in synovial fluid of IgG fragments similar to those observed in the in vitro biochemical studies suggests that proteolysis of IgG may contribute to localized immune dysfunction in inflammatory environments.

We report here the three-dimensional structure of a human Fc fragment engineered for enhanced antibody dependent cell mediated cytotoxicity (ADCC). The triple mutation S239D/A330L/I332E ('3M') was introduced into the C(H)2 portion of a human immunoglobulin G1 (IgG1) Fc. These three substitutions typically result in an about 10-100-fold increase in human IgG1 binding to human Fc gamma RIIIA (CD16). The recombinantly produced Fc/3M fragment was crystallized and its structure solved at a resolution of 2.5A using molecular replacement. No dramatic structural changes were observed in Fc/3M when compared with unmutated human Fc fragments. However, we found that the relative positions of its C(H)2 domains allowed for an unusually 'open' conformation of the entire fragment. Although this particular structural feature could be due to crystallization artifacts or intrinsic variability, we propose that molecular mechanisms at the basis of the enhanced interaction between Fc/3M and CD16 could include enhanced Fc openness as well as the introduction of additional hydrophobic contacts, hydrogen bonds and/or electrostatic interactions at the corresponding interface. The existence of a more pronounced cleft between the two Fc chains as well as of repulsive, electrostatic intra-chain interactions may also account in part for the decreased thermostability of both Fc/3M and a 3M-modified humanized anti-human EphA2 IgG1 when compared with their respective unmutated counterparts.

Recombinant versions of the seven equine IgG subclasses were expressed in CHO cells. All assembled into intact immunoglobulins stabilised by disulphide bridges, although, reminiscent of human IgG4, a small proportion of equine IgG4 and IgG7 were held together by non-covalent bonds alone. All seven IgGs were N-glycosylated. In addition IgG3 appeared to be O-glycosylated and could bind the lectin jacalin. Staphylococcal protein A displayed weak binding for the equine IgGs in the order: IgG1>IgG3>IgG4>IgG7>IgG2=IgG5>IgG6. Streptococcal protein G bound strongly to IgG1, IgG4 and IgG7, moderately to IgG3, weakly to IgG2 and IgG6, and not at all to IgG5. Analysis of antibody effector functions revealed that IgG1, IgG3, IgG4, IgG5 and IgG7, but not IgG2 and IgG6, were able to elicit a strong respiratory burst from equine peripheral blood leukocytes, predicting that the former five IgG subclasses are able to interact with Fc receptors on effector cells. IgG1, IgG3, IgG4 and IgG7, but not IgG2, IgG5 and IgG6, were able to bind complement C1q and activate complement via the classical pathway. The differential effector function capabilities of the subclasses suggest that, for maximum efficacy, equine vaccine strategies should seek to elicit antibody responses of the IgG1, IgG3, IgG4, and IgG7 subclasses.

IgG-dimers in multidonor-derived preparations of IgG antibodies represent not only agents of therapeutic potential, but also molecules of basic immunological interest since their composition mirrors the currently unknown range of clonal concentrations and affinities. To analyze this fundamental type of diversity, a computational model is developed in agreement with a density functional theory and used to simulate the dissociation kinetics of dimers separated from a 5000 donor-derived IgG preparation (protein concentration: 0.74 mg/mL) via superimposition of 8100 arbitrary combinations of off-rates and initial concentrations. The Greedy algorithm-like procedure described requires iterative and consecutive changes of 8 from a total of 11 fitting parameters and allows to approximate the probability density distributions of either quantities within defined limits (apparent off-rates: approximately 4 x 10(-4) to 9 x 10(-17)s(-1); concentrations: approximately 3 x 10(-20) to 1 x 10(-11)M) by lognormal distributions of log-log(10)-type, each of them adapted with four particular parameters, as well as the number of different dimer populations ( approximately 2 x 10(13)). Moreover, reasonably dimensioned equilibrium constants involved in monovalent and bivalent random IgG dimerization are estimated by using a mean on-rate of 2.5 x 10(5)M(-1)s(-1) and interrelationships of molecular parameters derived from known models for antibody-antigen interaction.

Gram-positive bacterial pathogens express immunoglobulin (Ig) binding proteins that perturb Fc-dependent functions such as the interaction with complement or phagocytic Fc-receptors. The possession of such molecules by gram-negative bacteria, including Escherichia coli (E. coli), has also been documented. In many such studies, the detection of Ig binding has relied on the use of polyclonal antibodies as detecting reagents. These are not ideal since such preparations may be contaminated with E. coli specific IgG, allowing for the potential misinterpretation of specific F(ab')(2) binding as non-specific Fc mediated binding. Here we use mono-specific recombinant antibodies to develop a novel assay for Ig binding non reliant on traditional polyclonal antibodies, allowing us to demonstrate the unequivocal absence of Fc binding proteins from E. coli.

Although it is now clear that certain Fc glycan structures on immunoglobulin G (IgG) antibodies (Abs) can have a dramatic influence on binding to selected Fcgamma receptors (FcgammaR) and on Fc-mediated immune functions, the effects of all known Fc glycan structures still have not been exhaustively studied. We report that in vitro analyses of pairs of monoclonal human IgG Abs that differ in the amount of sialic acid in their Fc glycans revealed that, for each of the three Ab pairs we examined, higher levels of sialylation were associated with reduced activity in Ab-dependent cellular cytotoxicity (ADCC) assays. This relationship between sialylation and ADCC activity was observed regardless of whether the differences in the extent of sialylation were derived by different Ab production processes, use of a lectin column to separate monoclonal Ab preparations into differentially sialylated fractions, or use of direct in vitro glycoengineering methods to convert a lesser sialylated Ab into a highly sialylated Ab. Subsequent investigations revealed that, depending on the individual Ab and how the differences in sialylation were derived, the lower ADCC potency of the more sialylated variants was apparently due to lower-affinity binding to FcgammaRIIIa on natural killer (NK) cells and/or, more interestingly, lower-affinity binding to cell-surface antigen. Our data provide the first example of an Fc glycan structure impacting antigen binding and suggest that avoiding Fc glycan sialylation can offer another means of optimizing ADCC activity of Abs.

Many antithrombotic agents have only a small therapeutic window, frequently leading to bleeding problems. However, interfering with platelet adhesion through the collagen-VWF-GPIbalpha axis is expected to cause less bleeding problems. Our group developed a monoclonal antibody, 82D6A3, directed against the von Willebrand factor (VWF) A3-domain, which inhibits the VWF-interaction to fibrillar collagen. 82D6A3 has antithrombotic effects in vivo without bleeding time prolongation. To further investigate the promising features of 82D6A3, we have humanized it by variable domain resurfacing and grafting on the constant regions of a human IgG4. First, the sequence of the variable domains was determined and the murine scFv was constructed. The expressed scFv had a comparable activity as the IgG of 82D6A3, and its DNA was thus used in subsequent humanization procedures. For this, a new approach was introduced to identify non-human like framework surface residues, since the general distribution of accessible residues described for human and murine heavy and light chain variable domains showed several discrepancies with the homology modelled Fv of 82D6A3. Identification of non-human like framework residues and evaluation of their surface accessibility within the context of the homology modelled Fv of 82D6A3, revealed 10 residues that need to be humanized without influencing the conformation of the CDR loops. Indeed, the humanized scFv of 82D6A3, obtained by mutating all 10 residues to their human counterpart, was still binding with high affinity to VWF and retained the inhibitory properties of the murine scFv. Next, in order to increase its half life and to decrease its immunogenicity, the humanized variable domains were grafted on the constant regions of a human IgG4, resulting in h82D6A3 with an in vitro activity comparable to that of the murine IgG.

Analysis of the N-linked oligosaccharides of the murine IgG1 monoclonal antibody (mAb) to Cryptococcus neoformans by LC/MS revealed five different core fucosylated, biantennary complex-type oligosaccharides at Asn-293, with the major species being a mono-galactosylated oligosaccharide with the glycosyl composition of Hex4HexNAc4Fuc (39% of the total glycan pool). The primary sequence predicted from nucleic acid sequencing differed from that measured by mass spectrometry at position 33 (ASN to ASP), a finding that may represent post-translational modification caused by spontaneous ASP deamination. Analysis of mAb 18B7 from three hybridoma clones revealed the same heterogenous N-glycan pattern, indicating that diversity in oligosaccharide structures originated from individual cells. The binding of native and de-glycosylated mAb 18B7 to cryptococcal Ag was comparable but the de-glycosylated 18B7 had shorter serum half-life and did not activate complement (C). De-glycosylated mAb 18B7 was opsonic for C. neoformans with murine macrophages through a mechanism that involved C-independent ingestion through the C receptor. Passive administration of de-glycosylated mAb 18B7 mediated comparable protective efficacy to the native mAb in mice with lethal infection. The results imply that the contribution of N-glycan structure to immunoglobulin function varies depending on the Ag-Ab system.

Ole e 1 is a major allergen from olive pollen with an IgE-binding frequency around 80% among allergic population. Its diagnostic value has been demonstrated, and cross-reactive allergens have been found in ash, lilac and privet. We sought to determine IgE- and IgG-binding regions of Ole e 1. Ole e 1-specific polyclonal antiserum and sera from patients allergic to olive pollen were used to analyze IgG and IgE epitopes, respectively. Short overlapping synthetic peptides covering the complete sequence of Ole e 1 and point mutants of these peptides bound to membranes, as well as long recombinant peptides fused to GST were used in dot blot immunostaining and ELISA. Skin prick tests were performed on 14 allergic patients to assay the response in vivo to the recombinant fusion peptides. Residues at positions 8-11, 29, 32, 33, 55-59, 70, 107-110, 112, 120, 123, 141 of Ole e 1 sequence were found to be antigenically relevant in the IgG-binding. Although amino acids K137, L138, G139, Y141 and P142 were involved in the IgE-recognition of a pool of sera from allergic individuals, the response to the IgEs seemed to be preferentially conformational. IgE-binding capability of recombinant GST-fused peptide T114-M145 was demonstrated by in vivo (prick test) and in vitro (ELISA) experiments. Major IgG and IgE-binding regions of Ole e 1 have been identified being the C-terminal an immunodominant region. These data could help to design hypoallergenic forms of the allergen.

In periodontal disease, IgG1 and IgA1 antibodies produced in situ deposit on antigens in the affected tissues. Thus, there is an interest in the effect of co-deposited IgA1 antibodies on complement activation by IgG1-immune complexes. In the present study, we first analyzed the effect of IgA1-immune complexes on complement using human IgA1 antibodies to dansyl (with dansylated human serum albumin serving as the immobilized antigen). It was observed that these IgA1-immune complexes when incubated for prolonged times with 33% human serum as a source of complement received C4b and C3b deposition. As C4b and C3b deposited on the IgA1 antibodies and on the antigenic surface, the complement-coated IgA1 antibodies departed. These fluid-phase complement-coated IgA1 antibodies were transferred to antigen-coated microtiter-ELISA plates, where they became bound to the antigens. Thus, the complement-coated IgA1 antibodies retained their antigen-binding function, especially as a proportion of their covalently bound C3b progressively degraded to iC3b and C3d. Genetically engineered carbohydrate-deficient mutant human IgA1 antibodies were used to assess the role of carbohydrate in accepting the C4b and C3b depositions, and these studies indicated that the carbohydrate on the Fc-region of IgA1 played a positive role. Another interesting finding generated by this study was that when IgA1 was co-deposited with IgG1 antibodies, and serum complement was added, the IgG1 antibodies tended to remain on the antigenic surface. The co-deposited IgA1 antibodies not only controlled (reduced) the rate of the consumption of the first component of complement (C1) and of classical complement pathway activation by IgG1-immune complexes (and therein reduced the rate of complement-mediated dissolution of the IgG1-immune complexes), but also the co-deposited IgA1 antibodies simultaneously intercepted/accepted C4b and C3b, then departed, as complement began to cover the antigenic surfaces. The process in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.

Immunoglobulins (Ig) require correct folding and assembly of both heavy (H) and light (L) chains to form a functional H2L2 dimer that is secreted from plasma cells. This process is dependent upon the endoplasmic reticulum (ER) chaperone BiP, which targets improperly, folded or assembled Ig molecules for degradation. While investigating the mechanism of low IgG3 secretion, we identified a missense mutation L368P in the Ch3 region of the human gamma3 H-chain that was associated with impaired secretion of intact and functional Ig. The non-secreted H-chains displayed slower electrophoretic migration than secreted H-chains, consistent with them being glycosylated in the ER but not fully processed in the golgi apparatus and secretory pathway. Reversion of the mutated codon to wild type restored secretion of the IgG3, which displayed the same fine specificity for antigen as non-secreted IgG3. However, the non-secreted IgG3 was not opsonic in an in vitro phagocytosis assay. The results indicate that correct IgG3 Ch3 domain folding is essential for secretion and effective function but does not affect specificity for antigen.

While bio-labeled immunoglobulin (IgG) or antibodies are extensively used in imaging studies and therapeutic modality, there have been no reports describing atomic force microscopy (AFM) micrographs of these labeled IgG molecules. Here, AFM studies of phycoerythrin (PE)-conjugated IgG were undertaken to examine whether PE conjugation induced conformational changes or molecular interaction, and subsequently resulted in an alteration in nano-structures of PE-conjugated IgG complex. Once immobilized on mica, single PE-conjugated IgG molecule exhibited globular shape with approximately 60 microns in diameter and 5 microns in height. PE-conjugated IgG were able to form monomers, spindle-like trimers, and hexamers that developed through an end-end connection of two trimers, after they were continuously immobilized on mica. Interestingly, these multimers could aggregate in different directions to form circular monolayers with a highly dense core of PE-conjugated IgG polymers. The formation of these well-organized polymers and aggregates of PE-conjugated IgG may be attributed to the PE conjugation as well as the air-liquid tension on subtract. These findings may help to understand the nano-structures of bio-labeled IgG or antibodies, and facilitate the potential use of PE-conjugated antibodies as markers or immunosensers for AFM bio-analytics of biomolecules in cells and membranes.

Mechanisms of the cryoprecipitation of cryoglobulins have long been studied but not fully understood. In the present study, we characterize a monoclonal immunoglobulin G1 (IgG1) cryoglobulin that forms sharp needle crystals upon cooling and therefore might be an ideal example for the elucidation of a mechanism of cryoprecipitation of cryoglobulins. Limited proteolysis by papain of this cryoglobulin resulted in disappearance of cryocrystallization. Interactions of the proteolytic fragments were examined by surface plasmon resonance measurements. A temperature-dependent isologous Fab-Fab interaction was observed for this cryoglobulin. Glycosylation profiling by use of the HPLC mapping technique revealed that cryoglobulin expresses homogeneous N-glycans in its Fc portion. These results suggest that (1) the temperature-dependent crystallization of the cryoglobulin was ascribed to its specific Fab-Fab interaction and (2) the unusual homogeneity of the Fc glycosylation promote the crystallization of this cryoglobulin.

Variable region domain exchanged IgG, or "inside-out (io)," molecules, were produced to investigate the effects of domain interactions on antibody structure and function. Studies using ultracentrifugation and electron microscopy showed that variable region domain exchange induces non-covalent multimerization through Fab domains. Surprisingly, variable region exchange also affected Fc-associated functions such as serum half-life and binding to protein G and FcgammaRI. These alterations were not merely a consequence of IgG aggregation. Both the extent of multimerization and alterations in Fc-associated properties depended on the IgG isotype.

The 3-D complex structures of extracellular domain of human CD16 (FcgammaRIII) and hIgG1 or B88-9 were obtained by means of computer-guided molecular modeling, respectively. The binding epitopes were predicted and binding energy was calculated theoretically. The epitopes of human CD16 interacted with hIgG1 and B88-9 were different and the binding energy of B88-9 was stronger than hIgG1. The computer competing simulation results showed that B88-9 could bind to CD16 when CD16 was also bound by hIgG1, while IgG1 could not bind to CD16 after CD16 was bound by B88-9. By flow cytometry analysis, the competitive binding activity of B88-9 to CD16 with hIgG1 was evaluated. With the increasing concentrations of hIgG1, the binding activity of B88-9 was not interfered markedly. It suggested that hIgG1 could not inhibit the interaction of CD16 and B88-9 and the binding domain of hIgG1 and B88-9 on CD16 was different.

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.

Immunoglobulins undergoing cold-dependent precipitation are known as cryoglobulins. A type I cryoglobulin after Brouet et al. from serum of a patient with severe cutaneous vasculitis and membranoproliferative glomerulonephritis was purified by reversible temperature-dependent precipitation and analyzed using FPLC, Western blotting and peptide sequencing. The isolated cryoglobulin consisted of a single complex of a molecular weight of above 210kDa observed under non-reducing conditions in SDS-polyacrylamide gel electrophoresis (PAGE). Under reducing conditions, this complex resolved into three bands, two of which were reminiscent of Ig heavy (HC) chains and one of Ig-light chains (LC). The FPLC-purified type I cryoglobulin showed reversible precipitation analyzed by spectrophotometry. Delineation of the peptides involved in complex formation by immunoblot analysis and peptide sequencing revealed IgG3-V(H)4/Igkappa-VkappaIII/JkappaII and IgG1/V(H)3 molecules with evidence of somatic mutation. Coomassie blue-staining suggested that molar amounts of the IgG3-heavy chain were much higher than that of the IgG1-heavy chain. Treatment with SDS and boiling did not disrupt the unusually high molecular weight Ig complex. Pre-treatment of the cryoglobulin in 6M guadinium hydrochloride followed by gel filtration chromatography suggested covalent association of the IgG3, IgG1 and Igkappa molecules. Therefore, it might be that the cryoglobulin was produced by a single plasma B cell clone which passed immunological check-points in terms of B cell selection in the bone marrow in the absence of allelic exclusion, class switching and affinity maturation by somatic mutation.