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Heme (Fe protoporphyrin IX) serves as a prosthetic group of numerous proteins implicated in oxidative metabolism. This molecule is abundantly present in the red blood cells where it serves as a cofactor of hemoglobin. As consequence of various pathological conditions, the membrane of red blood cells can be damaged and therefore large quantities of hemoglobin and subsequently heme released in the extracellular space. Since heme is a highly reactive compound, when released extracelluarly it can influence the functional activity of different plasma components. Thus, previous investigations have demonstrated that heme can interact with components of complement system and immunoglobulins, profoundly affecting their functions. Here we propose two basic protocols that can be used for characterization of interaction of free heme with complement proteins and immunoglobulins. The first technique is based on UV-Vis absorbance spectroscopy. It allows general characterization of the heme binding to the protein and estimation of the number of heme binding sites. The second protocol consists in the use of biosensor assay based on surface plasmon resonance. This protocol would be useful for evaluation of heme binding kinetics and equilibrium affinity. Besides for complement components and immunoglobulins, the presented protocols can be utilized for characterization of the interaction of heme with other proteins.

Hemolytic diseases such as sickle-cell disease, β-thalassemia, malaria, and autoimmune hemolytic anemia continue to present serious clinical hurdles. In these diseases, lysis of erythrocytes causes the release of hemoglobin and heme into plasma. Extracellular heme has strong proinflammatory potential and activates immune cells and endothelium, thus contributing to disease pathogenesis. Recent studies have revealed that heme can interfere with the function of plasma effector systems such as the coagulation and complement cascades, in addition to the activity of immunoglobulins. Any perturbation in such functions may have severe pathological consequences. In this review we analyze heme interactions with coagulation, complement, and immunoglobulins. Deciphering such interactions to better understand the complex pathogenesis of hemolytic diseases is pivotal., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Antigen-binding specificity of immunoglobulins is important for their function in immune defense. However, immune repertoires contain a considerable fraction of immunoglobulins with promiscuous binding behavior, the physicochemical basis of which is not well understood. Evolution of immunoglobulin specificity occurs through iterative processes of mutation and selection, referred to as affinity maturation. Recent studies reveal that some somatic mutations could compromise the thermodynamic stability of the variable regions of immunoglobulins. By integrating this observation with the wealth of data on the evolution of novel enzyme activities, we propose that antibody specificity is linked to the thermodynamic stability of the antigen-binding regions, which provides a quantitative distinction between highly specific and promiscuous antibodies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients., (© 2013 APMIS. Published by John Wiley & Sons Ltd.)

An Ab molecule or a BCR that is able to bind multiple structurally unrelated Ags is defined as polyreactive. Polyreactive Abs and BCRs constitute an important part of immune repertoires under physiological conditions and may play essential roles in immune defense and in the maintenance of immune homeostasis. In this review, we integrate and discuss different findings that reveal the indispensable role of Ag-binding polyreactivity in the immune system. First, we describe the functional and molecular characteristics of polyreactive Abs. The following part of the review concentrates on the biological roles attributed to polyreactive Abs and to polyreactive BCRs. Finally, we discuss recent studies that link Ig polyreactivity with distinct pathological conditions.

An antibody molecule that can bind to multiple distinct antigens is defined as polyreactive. In the present study, we performed statistical analyses to assess sequence correlates of polyreactivity of >600 antibodies cloned from different Bcell types of healthy humans. The data revealed several sequence patterns of variable regions of heavy and light immunoglobulin chains that determine polyreactivity. The most prominent identified patterns were increased number of basic amino acid residues, reduced frequency of acidic residues, increased number of aromatic and hydrophobic residues, and longer length of CDR L1. Importantly, our study revealed that antibodies isolated from different B-cell populations used distinct sequence patterns (or combinations of them) for polyreactive antigen binding. Furthermore, we combined the data from sequence analyses with molecular modeling of selected polyreactive antibodies and demonstrated that human antibodies can use multiple pathways for achieving antigen-binding promiscuity. These data reconcile some contradictions in the literature regarding the determinants of antibody polyreactivity. Moreover, our study demonstrates that the mechanism of polyreactivity of antibodies evolves during immune response and might be tailored to specific functional properties of different B-cell compartments. Finally, these data can be of use for efforts in the development and engineering of therapeutic antibodies. [ABSTRACT FROM AUTHOR]

The interaction of some human antibodies with heme results in posttranslational acquisition of binding to various self- and pathogen-derived antigens. The previous studies on this phenomenon were performed with oxidized heme (Fe3+). In the present study, we elucidated the effect of other pathologically relevant species of heme, i.e., species that were formed after contact of heme with oxidizing agents such as hydrogen peroxide, situations in which heme's iron could acquire higher oxidation states. Our data reveal that hyperoxidized species of heme have a superior capacity to heme (Fe3+) in triggering the autoreactivity of human IgG. Mechanistic studies demonstrated that oxidation status of iron was of critical importance for the heme's effect on antibodies. We also demonstrated that hyperoxidized heme species interacted at higher affinities with IgG and that this binding occurred through a different mechanism as compared to heme (Fe3+). Regardless of their profound functional impact on the antigen-binding properties of antibodies, hyperoxidized species of heme did not affect Fc-mediated functions of IgG, such as binding to the neonatal Fc receptor. The obtained data contribute to a better understanding of the pathophysiological mechanism of hemolytic diseases and of the origin of elevated antibody autoreactivity in patients with some hemolytic disorders. [ABSTRACT FROM AUTHOR]

Intravascular hemolysis occurs in diverse pathological conditions. Extracellular hemoglobin and heme have strong pro-oxidative and pro-inflammatory potentials that can contribute to the pathology of hemolytic diseases. However, many of the effects of extracellular hemoglobin and heme in hemolytic diseases are still not well understood. Here we demonstrate that oxidized hemoglobin (methemoglobin) can modify the antigen-binding characteristics of human immunoglobulins. Thus, incubation of polyclonal or some monoclonal human IgG in the presence of methemoglobin results in an appearance of binding reactivities towards distinct unrelated self-proteins, including the protein constituent of hemoglobin i.e., globin. We demonstrate that a transfer of heme from methemoglobin to IgG is indispensable for this acquisition of antibody polyreactivity. Our data also show that only oxidized form of hemoglobin have the capacity to induce polyreactivity of antibodies. Site-directed mutagenesis of a heme-sensitive human monoclonal IgG1 reveals details about the mechanism of methemoglobin-induced antigen-binding polyreactivity. Further here we assess the kinetics and thermodynamics of interaction of a heme-induced polyreactive human antibody with hemoglobin and myoglobin. Taken together presented data contribute to a better understanding of the functions of extracellular hemoglobin in the context of hemolytic diseases. Oxidized hemoglobin induces polyreactivity and autoreactivity of human IgG through direct transfer and binding of heme to the variable region of IgG, which contributes to a better understanding of the physiopathology of hemolytic diseases. [ABSTRACT FROM AUTHOR]

Broadly neutralizing antibodies against rapidly evolving viruses (e.g., HIV-1 and influenza virus), often manifest antigen-binding promiscuity. Based on a recent study, we hypothesize on the significance of antibody polyreactivity in neutralization of rapidly evolving viruses. We propose that polyreactivity contributes to toleration of viral variants and shortens the time for generating neutralizing antibodies. [ABSTRACT FROM AUTHOR]

Therapeutic antibodies have revolutionized modern medicine. At present, antibodies are successfully used for treatment of diverse human diseases, ranging from cancer to viral infections. All clinically approved antibodies rely on highly specific recognition of their target antigen. Antigen-binding promiscuity, binding to autoantigens, and propensity for self-binding (homophilic interaction) are highly undesirable characteristics of antibody drug candidates. Nevertheless, the immune system of all healthy individuals constantly produces and uses large quantities of antibodies that can be classified as inappropriate for development as drugs. Here, I provide arguments that antibodies with 'aberrant' properties have therapeutic potential. They could be useful in certain complex pathological conditions, thus enriching our armamentarium for treatment of human diseases. Therapeutic antibodies have been successfully used for treatment of diverse pathological conditions. Antibodies exert their therapeutic effects through different mechanisms – inhibition of receptor–ligand interactions or induction of innate immune cytotoxic reactions via their constant fragments. Drug development programs exclusively select highly specific antibodies, that is, antibodies that recognize a single disease-associated target. Antibodies with antigen-binding promiscuity, tendency for self-association, or off-target autoreactivity are usually rejected in the early phases of development. The immune system constantly produces promiscuous antibodies, self-binding antibodies, and autoantibodies. These antibodies play important roles in immune defense and in immune regulation. Antibodies with 'aberrant' behavior could offer unique opportunities for therapeutic innovations. [ABSTRACT FROM AUTHOR]

The typical functions of antibodies are based on linking the process of antigen recognition with initiation of innate immune reactions. With the introduction of modern research technologies and the use of sophisticated model systems, recent years have witnessed the discovery of a number of noncanonical functions of antibodies. These functions encompass either untypical strategies for neutralization of pathogens or exertion of activities that are characteristic for other proteins (cytokines, chaperones, or enzymes). Here, we provide an overview of the noncanonical functions of antibodies and discuss their mechanisms and implications in immune regulation and defense. A better comprehension of these functions will enrich our knowledge of the adaptive immune response and shall inspire the development of novel therapeutics. Antibodies are major players in adaptive immunity. They participate in immune defense and have potent immunomodulatory potential. Antibody molecules have two functional centers: they bridge specific antigen recognition through the antigen-binding site with the interaction and activation of innate immune receptors via their constant regions. These integrated interactions are essential for classical antibody functions. Antibodies can utilize alternative or noncanonical strategies for broadening their functional competency. Thus, antibodies perform atypical activities by acquiring the typical functions of cytokines, chaperones, transporters, enzymes, etc. The noncanonical functions of antibodies contribute to immune defense and the maintenance of physiological homeostasis. Many of these activities are operational in healthy individuals. Others appear only as a result of immunopathology. The noncanonical functions of antibodies arise as a result of the enormous diversity of adaptive immune repertoires and microevolutionary selection of B cells. They provide a rich source of potential therapeutic modalities. [ABSTRACT FROM AUTHOR]

The development of antibodies against therapeutic factor VIII (FVIII) represents the major complication of replacement therapy in patients with severe hemophilia A. Amongst the environmental risk factors that influence the anti-FVIII immune response, the presence of active bleeding or hemarthrosis has been evoked. Endothelium damage is typically associated with the release of oxidative compounds. Here, we addressed whether oxidation contributes to FVIII immunogenicity. The control with N -acetyl cysteine of the oxidative status in FVIII-deficient mice, a model of severe hemophilia A, reduced the immune response to exogenous FVIII. Ex vivo exposure of therapeutic FVIII to HOCl induced a mild oxidation of the molecule as evidenced by the loss of free amines and resulted in increased FVIII immunogenicity in vivo when compared to native FVIII. The increased immunogenicity of oxidized FVIII was not reverted by treatment of mice with N -acetyl cysteine, and did not implicate an increased maturation of professional antigen-presenting cells. Our data document that oxidation influences the immunogenicity of therapeutic FVIII. [ABSTRACT FROM AUTHOR]

Polyreactive antibodies play an important role for neutralization of human immunodeficiency virus (HIV). In addition to intrinsic polyreactive antibodies, the immune system of healthy individuals contains antibodies with cryptic polyreactivity. These antibodies acquire promiscuous antigen binding potential post-translationally, after exposure to various redox-active substances such as reactive oxygen species, iron ions, and heme. Here, we characterized the interaction of a prototypic human antibody that acquires binding potential to glycoprotein (gp) 120 after exposure to heme. The kinetic and thermodynamic analyses of interaction of the polyreactive antibody with distinct clades of gp120 demonstrated that the antigen-binding promiscuity of the antibody compensates for the molecular heterogeneity of the target antigen. Thus, the polyreactive antibody recognized divergent gp120 clades with similar values of the binding kinetics and quantitatively identical changes in the activation thermodynamic parameters. Moreover, this antibody utilized the same type of noncovalent forces for formation of complexes with gp120. In contrast, HIV-1-neutralizing antibodies isolated from HIV-1-infected individuals, F425 B4a1 and b12, demonstrated different binding behavior upon interaction with distinct variants of gp120. This study contributes to a better understanding of the physiological role and binding mechanism of antibodies with cryptic polyreactivity. Moreover, this study might be of relevance for understanding the basic aspects of HIV-1 interaction with human antibodies. [ABSTRACT FROM AUTHOR]

Therapeutic monoclonal antibodies have exerted a transformative impact on clinical practice in last two decades. However, development of a therapeutic antibody remains a complex process. Various physiochemical and functional liabilities can compromise the production or the therapeutic efficacy of antibodies. One of these liabilities is the susceptibility to oxidation. In the present study, we portrayed an oxidation-dependent vulnerability of immunoglobulins that can be of concern for therapeutic antibodies. By using a library of 119 monoclonal IgG1 molecules, containing variable domain matching clinical-stage antibodies, we demonstrated that a substantial number of these molecules acquired antigen-binding polyreactivity upon exposure to ferrous ions. Statistical analyses revealed that the potential for induction of polyreactivity by the redox-active metal ions correlated with a higher number of somatic mutations in V genes encoding variable domains of heavy and light immunoglobulin chains. Moreover, the sensitive antibodies used with biased frequencies particular V gene families encoding variable domains of their light chains. Besides the exposure to ferrous ions the induction of polyreactivity of therapeutic antibodies occurred after contact with an unrelated pro-oxidative substance—hypochlorite ions. Our data also revealed that induction of polyreactivity by pro-oxidative agents did not impact the binding of antibodies to their cognate antigens. The results from this study may contribute for better selection of antibody therapeutics with suitable developability profiles. [ABSTRACT FROM AUTHOR]

Abstract: Polyspecific antibodies represent a significant fraction of the antibody repertoires in healthy animals and humans. Interestingly, certain antibodies only acquire a polyspecific antigen-binding behavior after exposure to protein-modifying conditions, such as those found at inflammation sites, or used in small- and large-scale immunoglobulin purification. This phenomenon is referred to as “criptic polyspecificity”. In the present study, we compare the potential of different chemical agents to induce IgG polyspecificity. Depending on the treatment used, quantitative and qualitative differences in the recognition of individual antigens from a standard panel were observed. Antibodies with cryptic polyspecificity utilized common mechanisms for the recognition of structurally unrelated antigens when exposed to a particular inductor of polyspecificity. Our study contributes to the understanding of the mechanisms underlying the cryptic polyspecificity. [Copyright &y& Elsevier]

IgG molecules are exposed on a regular basis to acidic conditions during immunoaffinity purification procedures, as well as during the production of some therapeutic immunoglobulin preparations. This exposure is known to induce in them an antigen-binding polyreactivity. The molecular mechanisms and the possible biological significance of this phenomenon remain, however, poorly understood. In addition to the previously reported ability of these modified IgG antibodies to interact with a large panel of self-antigens, enhanced binding to non-self-antigens (bacterial), an increased ability to engage in F(ab′)2/F(ab′)2 (idiotype/anti-idiotype) interactions and an increased functional antigen-binding affinity are reported here. The newly acquired ‘induced polyreactivity’ of low-pH buffer-exposed IgG is related to structural changes in the immunoglobulin molecules, and is at least partly attributable to the enhanced role of the hydrophobic effect in their interactions with antigen. Our results suggest that data from many previous studies on monoclonal and polyclonal IgG antibodies purified by low-pH buffer elution from protein A or protein G immunoaffinity columns should be reconsidered, as the procedure itself may have dramatically affected their antigen-binding behavior and biological activity. Low-pH buffer-treated pooled therapeutic immunoglobulins acquire novel beneficial properties, as passive immunotherapy with the pH 4.0 buffer-exposed, but not with the native therapeutic intravenous immunoglobulin preparation, improves the survival of mice with bacterial lipopolysaccharide-induced septic shock. [ABSTRACT FROM AUTHOR]

Abstract: Reactive oxygen species (ROS) released from activated phagocytes are involved in the innate immune defense against pathogens. However, when released in excess and when the antioxidant systems are impaired, ROS may induce cellular and tissue damage and dissociation of iron ions or iron containing compounds (heme) from protein-bound state. Free iron ions and free heme are prooxidative. Immunoglobulins usually perform their biological functions at sites of inflammation, where they may encounter reactive oxygen species and/or redox active compounds. It has been demonstrated that the exposure of some antibodies to heme, to transition metal ions or to reactive oxygen species induces an appearance of new binding specificities for various autoantigens. This review article is devoted to the interplay between redox active agents and antibodies. The biological significance of the appearance of new antigen binding specificities on antibodies after exposure to redox-active agents is discussed. [Copyright &y& Elsevier]

Abstract: A fraction of antibodies present in all healthy individuals begins to recognize large number of self-antigens only after a transient exposure to certain protein-destabilizing conditions, including low or high pH, high salt concentration, chaotropic factors and redox-active agents. Recent findings on the molecular mechanisms of the inducible antibody auto-reactivity, obtained by using kinetic and thermodynamic analysis on the interactions of single monoclonal antibody with its cognate antigen are discussed in the review. [Copyright &y& Elsevier]

Various pathological processes are accompanied by release of high amounts of free heme into the circulation. We demonstrated by kinetic, thermodynamic, and spectroscopic analyses that antibodies have an intrinsic ability to bind heme. This binding resulted in a decrease in the conformational freedom of the antibody paratopes and in a change in the nature of the noncovalent forces responsible for the antigen binding. The antibodies use the molecular imprint of the heme molecule to interact with an enlarged panel of structurally unrelated epitopes. Upon heme binding, monoclonal as well as pooled immunoglobulin G gained an ability to interact with previously unrecognized bacterial antigens and intact bacteria. IgG-heme complexes had an enhanced ability to trigger complement-mediated bacterial killing. It was also shown that heme, bound to immunoglobulins, acted as a cofactor in redox reactions. The potentiation of the antibacterial activity of IgG after contact with heme may represent a novel and inducible innate-type defense mechanism against invading pathogens. [ABSTRACT FROM AUTHOR]

Abstract: Polyspecificity is defined as the ability of a given antibody molecule to bind a large panel of structurally diverse antigens. A fraction of circulating IgG in all healthy individuals acquires promiscuous antigen-binding activity only after a transient exposure to certain protein destabilizing factors. The molecular mechanisms of this phenomenon are not well understood. Exposures to protein destabilizing agents are common steps in immunoglobulin isolation and purification processes. We performed kinetic and thermodynamic analyses using surface plasmon resonance-based technique in order to characterize the interactions of a single mouse monoclonal antibody to its cognate antigen before and after induction of promiscuous antigen-binding activity. The obtained results, suggest that enhanced antigen binding activity induced by exposure to mild denaturing condition resulted from an increase in the structural flexibility of the antigen-binding site. Further pH and ionic strength-dependence analyses of the antibody/antigen interactions demonstrated that the transition to promiscuous antigen-binding was accompanied by a change in the type of non-covalent forces involved in the complex formation. Moreover, from this study, it is evident that an antibody molecule could use two distinct thermodynamic pathways for binding to the same antigen while retaining the same value of the binding affinity. The obtained results may contribute to the understanding of the molecular mechanisms that lay behind natural antibody polyspecificity. [Copyright &y& Elsevier]

Polyspecific antibodies represent a first line of defense against infection and regulate inflammation, properties hypothesized to rely on their ability to interact with multiple antigens. We demonstrated that IgG exposure to pro-oxidative ferrous ions or to reactive oxygen species enhances paratope flexibility and hydrophobicity, leading to expansion of the spectrum of recognized antigens, regulation of cell proliferation, and protection in experimental sepsis. We propose that ferrous ions, released from transferrin and ferritin at sites of inflammation, synergize with reactive oxygen species to modify the immunoglobulins present in the surrounding microenvironment, thus quenching pro-inflammatory signals, while facilitating neutralization of pathogens. [ABSTRACT FROM AUTHOR]

Abstract: The evaluation of antibody avidity by elution with chaotropic agents is a frequently used approach in research and diagnostics. It provides important information on the functional relevance of antibodies. However, in the literature, there is a large heterogeneity in the experimental settings for the determination of this important parameter. Here, we demonstrate that antibody concentration and the choice of chaotropic agent are critical for the reliable estimation of antibody avidity. [Copyright &y& Elsevier]

Abstract: Two recent articles in the Journal of Molecular Biology, provide strong evidence that certain intracellular proteins, mostly involved in the cellular signalling, acquire functional activity only after partial denaturation. Thus, non-native forms of such proteins acquire functional activity, while the native forms of the proteins are devoid of these activities. As described by Korzhniev (2013), the transition to non-native (non-ground state) conformer of a given protein can be induced by changes in the environment, post-translational modification, cofactor binding or spontaneous unfolding of the protein due to intrinsic thermal fluctuations. In this correspondence we would like to discuss another case of gain of functional activity of proteins after a shift from the native conformation to non-native conformation. Here we provide arguments that similar structure-function transitions may be typical for one of the most abounded plasma proteins – immunoglobulins. [Copyright &y& Elsevier]

Geographical expansion and re-emerging new genotypes of the Japanese encephalitis virus (JEV) require the development of novel therapeutic approaches. Here, we studied a non-conventional approach for antibody therapy and show that, upon exposure to heme, a fraction of natural human immunoglobulins acquires high-affinity reactivity with the antigenic domain-III of JEV E glycoprotein. These JEV-reactive antibodies exhibited neutralizing activity against recently dominant JEV genotypes. This study opens new therapeutic options for Japanese encephalitis. [ABSTRACT FROM AUTHOR]

Abstract: Intravenous immunoglobulins (IVIG) are known to have a therapeutic effect in some autoimmune diseases. We examined the effect of IVIG and heme-exposed IVIG on the development of immune mediated diabetes induced in C57BL/6 mice by multiple low doses of streptozotocin. IVIG were used in a dose of 200mg/kg daily for 15days. Treatment with IVIG resulted in significant attenuation of diabetes induction as evaluated by glycemia, glycosuria and HbA1c level. Interestingly, heme-exposed IVIG had a still stronger antidiabetogenic effect. Serum levels of proinflammatory cytokines TNF-α, IFN-γ and IL17 were lower in IVIG treated animals when compared with controls, while IL10 level was higher. The number of CD4+Foxp3+ cells was higher in pancreatic lymph nodes of heme-exposed IVIG treated mice. Our results show that IVIG may downregulate diabetes induction possibly by favouring induction of T regulatory cells and suggest enhanced effect upon heme-binding to IVIG. [Copyright &y& Elsevier]

• Human immunoglobulin repertoires contain antibodies that bind to dinitrophenol. • Among 112 clinical-stage therapeutic IgG1 antibodies about 20 % bind dinitrophenol considerably. • Interaction of antibodies with dinitrophenol correlated with important developability parameters. Therapeutic antibodies should cover particular physicochemical and functional requirements for successful entry into clinical practice. Numerous experimental and computational approaches have been developed for early identification of different unfavourable features of antibodies. Immune repertoires of healthy humans contain a fraction of antibodies that recognize nitroarenes. These antibodies have been demonstrated to manifest antigen-binding polyreactivity. Here we observed that >20 % of 112 clinical stage therapeutic antibodies show pronounced binding to 2,4-dinitrophenol conjugated to albumin. This interaction predicts a number of unfavourable functional and physicochemical features of antibodies such as polyreactivity, tendency for self-association, stability and expression yields. Based on these findings we proposed a simple approach that may add to the armamentarium of assays for early identification of developability liabilities of antibodies intended for therapeutic use. [ABSTRACT FROM AUTHOR]

• The process of development of therapeutic antibodies is complex. • Many antibodies fail to reach clinic because of inappropriate physicochemical behaviour. • Assessment of physicochemical parameters of antibodies and identification of problematic molecules is of great importance. • Physicochemical properties of antibodies relevant for development depend of the sequence features of variable region. • Discovered sequence correlates may contribute for early identification of antibodies with developability issues. Therapeutic antibodies have transformed the clinical practice. Not surprisingly, development of antibody therapeutics is currently the main focus of the biotechnology industry. Nonetheless, the development process is complex, and many antibodies do not reach the clinic. Reasons for the failures include, undesired binding behavior (polyreactivity), low stability, poor expression yields, unfavorable pharmacokinetics etc. Numerous studies have proposed different analytical methods for assessment of physicochemical parameters of antibodies and identification of problematic molecules at early stages of the development process. These studies, however, have not addressed the basic mechanistic question of how sequence features of variable regions determinate the different biophysical characteristics and the binding behavior of the antibodies. In a recent study, Jain et al assessed 12 biophysical qualities of 137 monoclonal therapeutic antibodies. We used the raw data from this comprehensive study to perform correlation analyses of different biophysical measurables with various sequence features of variable regions of the antibodies - number of mutations, length of hypervariable loops, and frequency of amino acid residue types. The obtained data reveled significant relationships between the sequence characteristics of the variable domains and different physiochemical properties of antibodies. The data from this study can assist in design of a set of criteria for early identification of antibodies with developability issues. Moreover, our findings provide novel fundamental insights into the sequence-function relationship of antibodies. [ABSTRACT FROM AUTHOR]

Therapeutic intravenous immunoglobulin preparations (IVIg) are used for treatment of wide range of autoimmune and inflammatory diseases. Versatile mechanisms have been reported to contribute to the immunomodulatory effects of IVIg. Here we demonstrate that IVIg has a strong potential to inhibit pro-inflammatory effect of extracellular heme. Indeed, the presence of immunoglobulins reduced the potential of heme to activate the complement system on the surface of human endothelial cells. Since extracellular heme is considered as one of the principal pathogenic factors in hemolytic disorders, its therapeutic scavenging by IVIg may have significant clinical repercussions. [ABSTRACT FROM AUTHOR]

Recently, numerous studies report bats as reservoirs of emerging pathogens with little to no signs of infections. This is thought to be connected to the unique immune system of bats, which remains poorly characterized. Despite the physiological importance of the Neonatal Fc receptor (FcRn) in the homeostasis of IgG antibodies, it is unclear how its functional activity is evolutionary conservative among mammals, and so is the case for bats. Using surface plasmon resonance-based technology, we tested the interactions of IgG antibodies isolated from three bat species with recombinant human and mouse FcRn. Our data show that IgG from the studied bat species binds to both human and mouse FcRn, albeit with distinct affinities. Importantly, the binding pattern of bat IgG is similar to human IgG. This confirms the conservative nature of IgG-FcRn interaction and highlights the importance of FcRn IgG salvaging system in bats. • Neonatal Fc receptor (FcRn) plays important role of homeostasis of IgG in different species. • Recombinant FcRn binds IgG from three different bat species. • Human FcRn binds bat IgG with similar quantitative parameters as human IgG. • Bat IgG might be recycled and transferred from mother to foetus in a same way as other species IgG. [ABSTRACT FROM AUTHOR]

Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. [ABSTRACT FROM AUTHOR]

The healthy immune repertoire contains a fraction of antibodies that bind to various biologically relevant cofactors, including heme. Interaction of heme with some antibodies results in induction of new antigen binding specificities and acquisition of binding polyreactivity. In vivo, extracellular heme is released as a result of hemolysis or tissue damage; hence the post-translational acquisition of novel antigen specificities might play an important role in the diversification of the immunoglobulin repertoire and host defense. Here, we demonstrate that seronegative immune repertoires contain antibodies that gain reactivity to HIV-1 gp120 upon exposure to heme. Furthermore, a panel of human recombinant antibodies was cloned from different B cell subpopulations, and the prevalence of antibodies with cofactor-induced specificity for gp120 was determined. Our data reveal that upon exposure to heme, ~24% of antibodies acquired binding specificity for divergent strains of HIV-1 gp120. Sequence analyses reveal that heme-sensitive antibodies do not differ in their repertoire of variable region genes and in most of the molecular features of their antigen-binding sites from antibodies that do not change their antigen binding specificity. However, antibodies with cofactor-induced gp120 specificity possess significantly lower numbers of somatic mutations in their variable region genes. This study contributes to the understanding of the significance of cofactor-binding antibodies in immunoglobulin repertoires and of the influence that the tissue microenvironment might have in shaping adaptive immune responses. [ABSTRACT FROM AUTHOR]

Highlights: [•] Heme induces polyreactivity of IgG cloned from splenic marginal zone B-cell lymphoma. [•] Heme-induced polyreactive antibody binds various autoantigens. [•] Induction of polyreactivity does not affect Fc-fragment dependant binding of IgG. [•] Heme-induced polyreactivity of BCR might have repercussions for the pathology. [ABSTRACT FROM AUTHOR]

Abstract: Abzymes are immunoglobulins endowed with enzymatic activities. The catalytic activity of an abzyme resides in the variable domain of the antibody, which is constituted by the close spatial arrangement of amino acid residues involved in catalysis. The origin of abzymes is conferred by the innate diversity of the immunoglobulin gene repertoire. Under deregulated immune conditions, as in autoimmune diseases, the generation of abzymes to self-antigens could be deleterious. Technical advancement in the ability to generate monoclonal antibodies has been exploited in the generation of abzymes with defined specificities and activities. Therapeutic applications of abzymes are being investigated with the generation of monoclonal abzymes against several pathogenesis-associated antigens. Here, we review the different contexts in which abzymes are generated, and we discuss the relevance of monoclonal abzymes for the treatment of human diseases. [Copyright &y& Elsevier]

Abstract: Common variable immunodeficiency (CVID) is associated with low serum immunoglobulin concentrations and an increased susceptibility to infections and autoimmune diseases. The treatment of choice for CVID patients is replacement intravenous immunoglobulin (IVIg) therapy. IVIg has been beneficial in preventing or alleviating the severity of infections and autoimmune and inflammatory process in majority of CVID patients. Although the mechanisms of action of IVIg given as ‘therapeutic high dose’ in patients with autoimmune diseases are well studied, the underlying mechanisms of beneficial effects of IVIg in primary immunodeficiencies are not completely understood. Therefore we investigated the effect of ‘replacement dose’ of IVIg by probing its action on B cells from CVID patients. We demonstrate that IVIg at low doses induces proliferation and immunoglobulin synthesis from B cells of CVID patients. Interestingly, B cell stimulation by IVIg is not associated with induction of B cell effector cytokine IFN-γ and of transcription factor T-bet. Together, our results indicate that in some CVID patients, IVIg rectifies the defective signaling of B cells normally provided by T cells and delivers T-independent signaling for B cells to proliferate. IVIg ‘replacement therapy’ in primary immunodeficiencies is therefore not a merepassive transfer of antibodies to prevent exclusively the recurrent infections; rather it has an active role in regulating autoimmune and inflammatory responses through modulating B cell functions and thus imposing dynamic equilibrium of the immune system. [Copyright &y& Elsevier]