Identification of Clinically and Pathophysiologically Relevant Rheumatoid Factor Epitopes by Engineered IgG Targets

Objective: Rheumatoid factors (RFs), which are anti-IgG autoantibodies strongly associated with rheumatoid arthritis (RA), are also found in other diseases and in healthy individuals. RFs bind to various epitopes in the constant (Fc-) domain of IgG. Therefore, disease-specific reactivity patterns may exist. This study was undertaken in order to develop a new approach to dissecting RF epitope binding patterns across different diseases. Methods: We analyzed RF reactivity patterns in serum from patients with seropositive arthralgia, patients with RA, and patients with primary Sjögren’s syndrome (SS) using bioengineered, natively folded IgG-Fc targets that demonstrated selective RF binding toward several distinct regions of the IgG-Fc domain. Results: Rheumatoid factor responses primarily bound the Fc Elbow region, with a smaller number of RFs binding the Fc Tail region, while the Fc receptor binding region was hardly targeted. A restricted reactivity against the IgG-Fc Tail region was associated with less positivity for anti–citrullinated protein antibodies (ACPAs) and less arthritis development in arthralgia, whereas combined reactivity toward IgG-Fc Tail and Elbow regions was associated with more arthritis development. Reactivity toward the IgG-Fc Tail region was observed far more frequently in RA than in primary SS. Conclusion: Bioengineered IgG targets enable serologic characterization of RF reactivity patterns, and use of this approach appears to reveal patterns associated with ACPA detection and arthritis development in patients with arthralgia. These patterns are able to distinguish RA patients from primary SS patients. This new methodology improves the clinical value of RFs and our understanding of their pathophysiologic processes

Dynamics of circulating TNF during adalimumab treatment using a drug-tolerant TNF assay

Patients with rheumatoid arthritis (RA) can be successfully treated with tumor necrosis factor (TNF) inhibitors, including the monoclonal antibody adalimumab. Once in remission, a proportion of patients can successfully discontinue treatment, indicating that blocking TNF is no longer required for disease control. To explore the dynamics of circulating TNF during adalimumab treatment, we developed a competition enzyme-linked immunosorbent assay that can quantify TNF in the presence of large amounts of TNF inhibitor, i.e., a “drug-tolerant” assay. In 193 consecutive adalimumab-treated patients with RA, we demonstrated that circulating TNF increased in average of &gt;50-fold upon treatment and reached a stable concentration in time for most patients. A similar increase in TNF was found in 30 healthy volunteers after one dose of adalimumab. This implies that TNF in circulation during anti-TNF treatment is not primarily associated with disease activity. During treatment, TNF was in complex with adalimumab and could be recovered as inactive 3:1 adalimumab-TNF complexes. No quantitative association was found between TNF and adalimumab concentrations. Low TNF concentrations at week 4 were associated with a higher frequency of antidrug antibodies (ADAs) at subsequent time points, less frequent methotrexate use at baseline, and less frequent remission after 52 weeks. Also in healthy volunteers, early low TNF concentrations are associated with ADAs. In conclusion, longitudinal TNF concentrations are mostly stable during adalimumab treatment and may therefore not predict successful treatment discontinuation. However, early low TNF is strongly associated with ADA formation and may be used as timely predictor of nonresponse toward adalimumab treatment.</p

Factors affecting IgG4-mediated complement activation

Of the four human immunoglobulin G (IgG) subclasses, IgG4 is considered the least inflammatory, in part because it poorly activates the complement system. Regardless, in IgG4 related disease (IgG4-RD) and in autoimmune disorders with high levels of IgG4 autoantibodies, the presence of these antibodies has been linked to consumption and deposition of complement components. This apparent paradox suggests that conditions may exist, potentially reminiscent of in vivo deposits, that allow for complement activation by IgG4. Furthermore, it is currently unclear how variable glycosylation and Fab arm exchange may influence the ability of IgG4 to activate complement. Here, we used well-defined, glyco-engineered monoclonal preparations of IgG4 and determined their ability to activate complement in a controlled system. We show that IgG4 can activate complement only at high antigen and antibody concentrations, via the classical pathway. Moreover, elevated or reduced Fc galactosylation enhanced or diminished complement activation, respectively, with no apparent contribution from the lectin pathway. Fab glycans slightly reduced complement activation. Lastly, we show that bispecific, monovalent IgG4 resulting from Fab arm exchange is a less potent activator of complement than monospecific IgG4. Taken together, these results imply that involvement of IgG4-mediated complement activation in pathology is possible but unlikely

Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA

Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1 μg of biotinylated protei

Room temperature structure of human IgG4-Fc from crystals analysed in situ

The Fc region of IgG antibodies (C gamma 2 and C gamma 3 domains) is responsible for effector functions such as antibody-dependent cell-mediated cytotoxicity and phagocytosis, through engagement with Fey receptors, although the ability to elicit these functions differs between the four human IgG subclasses. A key determinant of Fey receptor interactions is the FG loop in the C gamma 2 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 C gamma 2 FG loop at near-physiological temperature, we solved a 2.7 angstrom resolution room temperature structure of recombinant human IgG4-Fc from crystals analysed in situ. The C gamma 2 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 C gamma 2 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 C gamma 2 FG loop. (C) 2016 The Authors. Published by Elsevier Lt

Mechanism of immunoglobulin G4 Fab-arm exchange

Immunoglobulin G (IgG) antibodies are symmetrical molecules that may be regarded as covalent dimers of 2 half-molecules, each consisting of a light chain and a heavy chain. Human IgG4 is an unusually dynamic antibody, with half-molecule exchange ("Fab-arm exchange") resulting in asymmetrical, bispecific antibodies with two different antigen binding sites, which contributes to its anti-inflammatory activity. The mechanism of this process is unknown. To elucidate the elementary steps of this intermolecular antibody rearrangement, we developed a quantitative real-time FRET assay to monitor the kinetics of this process. We found that an intrinsic barrier is the relatively slow dissociation of the CH3 domains that noncovalently connect the heavy chains, which becomes rate determining in case disulfide bonds between the heavy chains are reduced or absent. Under redox conditions that mimic the previously estimated in vivo reaction rate, i.e., 1 mM of reduced glutathione, the overall rate is ca. 20 times lower because only a fraction of noncovalent isomers is present (with intra- rather than interheavy chain disulfide bonds), formed in a relatively fast pre-equilibrium from covalent isomers. Interestingly, Fab arms stabilize the covalent isomer: the amount of noncovalent isomers is ca. 3 times higher for Fc fragments of IgG4 (lacking Fab domains) compared to intact IgG4, and the observed rate of exchange is 3 times higher accordingly. Thus, kinetic data obtained from a sensitive and quantitative real-time FRET assay as described here yield accurate data about interdomain interactions such as those between Fab and/or Fc domains. The results imply that in vivo, the reaction is under control of local redox condition

The enzymatic removal of immunoglobulin variable domain glycans by different glycosidases

About 15% of immunoglobulin G (IgG) molecules contain glycans linked to the antigen-binding fragments (Fab arms) in addition to the glycans linked to the crystallizable fragment (Fc tail) of all IgGs. Fab glycosylation appears to be an important feature of antibodies, for example by influencing antigen binding and antibody stability. The reliable generation of antibodies that either have or lack Fab glycans would be very helpful to study the role of Fab glycans in more detail. In this study, we set out to remove Fab glycans by treating polyclonal and monoclonal human IgG antibodies with two commonly used glycosidases and an improved version of one of the two (Endo F3, PNGase F, and Rapid™ PNGase F). Fc glycans can be removed using PNGase F and Rapid™ PNGase F, but not with Endo F3. For most antibody clones, Endo F3 partially cleaved off the Fab glycans. In contrast, PNGase F left the Fab glycans of most clones unaffected, but could remove glycans of some clones. Rapid™ PNGase F showed a higher glycosidase efficacy than PNGase F, and more clones could be deglycosylated using this enzyme. In summary, not all Fab glycans can be cleaved off by the tested glycosidases (under non-denaturing conditions), suggesting that Fab glycans are exposed to different degrees

IgG Subclass Specificity Discriminates Restricted IgM Rheumatoid Factor Responses From More Mature Anti-Citrullinated Protein Antibody-Associated or Isotype-Switched IgA Responses

To investigate the presence and patterns of specific IgG subclass recognition by IgM rheumatoid factor (IgM-RF) and IgA-RF with a newly developed enzyme-linked immunosorbent assay (ELISA), which can discriminate between polyspecific and restricted RF responses. Polyspecific and restricted RF responses were determined with our ELISA, which uses individually coated recombinant IgG subclasses instead of polyclonal IgG as target antibodies. Fine specificity was determined using target antibodies with single amino acid mutations in the Fc region. In a screening panel of 93 sera that were previously found to be IgM-RF positive in a conventional RF assay, we were able to discriminate between sera with polyspecific IgM-RF responses (i.e., RF responses directed against all 4 IgG subclasses) and those with restricted IgM-RF responses, with low or absent relative reactivity against IgG2, IgG3, or IgG4. We found the largest variation for anti-IgG3 reactivity. Samples without detectable anti-IgG4 reactivity formed an independent group from the other restricted RF responses and the polyspecific RF responses. The specificity of these anti-IgG4-negative sera could be pinpointed to single amino acid differences between IgG1 and IgG4. Polyspecific RF responses more often showed signs of RF response maturation, with more isotype switching to IgA-RF, as compared to restricted RF responses. In a cohort of IgM-RF+ and/or anti-citrullinated protein antibody (ACPA)-positive arthralgia patients, we found restricted RF responses in 35% (49 of 140) of RF+/ACPA- patients, while RF+/ACPA+ patients, who have a much higher risk of developing rheumatoid arthritis, virtually always (123 of 128 [96%]) showed a polyspecific RF response. IgG subclass-specific RF distinguishes between immature restricted RF responses and potentially more pathogenic, ACPA-associated polyspecific response

Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange

Tumor necrosis factor (TNF) is a homotrimeric cytokine that is a key mediator of inflammation. It is unstable at physiological concentrations and slowly converts into an inactive form. Here, we investigated the mechanism of this process by using a Forster resonance energy transfer (FRET) assay that allowed monitoring of monomeric subunit exchange in time. We observed continuous exchange of monomeric subunits even at concentrations of TNF high enough to maintain its bioactivity. The kinetics of this process closely corresponds with the appearance of monomeric subunits and disappearance of trimeric TNF in time at ng/ml concentrations as monitored by high-performance size-exclusion chromatography (HP-SEC). Furthermore, of the five therapeutic TNF inhibitors that are currently used in the clinic, three (adalimumab, infliximab, etanercept) were found to completely inhibit the monomer exchange reaction and stabilize TNF trimers, whereas golimumab and certolizumab could not prevent monomer exchange, but did slow down the exchange process. These differences were not correlated with the affinities of the TNF inhibitors, measured with both surface plasmon resonance (SPR) and in fluid phase using fluorescence-assisted HP-SEC. The stabilizing effect of these TNF inhibitors might result in prolonged residual TNF bioactivity under conditions of incomplete blocking, as observed in vitro for adalimuma

Biased N-Glycosylation Site Distribution and Acquisition across the Antibody V Region during B Cell Maturation

Abs can acquire N-linked glycans in their V regions during Ag-specific B cell responses. Among others, these N-linked glycans can affect Ag binding and Ab stability. Elevated N-linked glycosylation has furthermore been associated with several B cell-associated pathologies. Basic knowledge about patterns of V region glycosylation at different stages of B cell development is scarce. The aim of the current study is to establish patterns of N-glycosylation sites in Ab V regions of naive and memory B cell subsets. We analyzed the distribution and acquisition of N-glycosylation sites within Ab V regions of peripheral blood and bone marrow B cells of 12 healthy individuals, eight myasthenia gravis patients, and six systemic lupus erythematosus patients, obtained by next-generation sequencing. N-glycosylation sites are clustered around CDRs and the DE loop for both H and L chains, with similar frequencies for healthy donors and patients. No evidence was found for an overall selection bias against acquiring an N-glycosylation site, except for the CDR3 of the H chain. Interestingly, both IgE and IgG4 subsets have a 2-fold higher propensity to acquire Fab glycans compared with IgG1 or IgA. When expressed as rmAb, 35 out of 38 (92%) nongermline N-glycosylation sites became occupied. These results point toward a differential selection pressure of N-glycosylation site acquisition during affinity maturation of B cells, which depends on the location within the V region and is isotype and subclass dependent. Elevated Fab glycosylation represents an additional hallmark of TH2-like IgG4/IgE responses