Background and Purpose: Millions of patients with inflammatory diseases are treated with tumour necrosis factor (TNF) inhibitors (TNFi). Individual treatment response varies, in part related to variable drug clearance. The role of TNF-TNFi complexes in clearance of the different TNFi is controversial. Moreover, mechanistic insight into the structural aspects and biological significance of TNF-TNFi complexes is lacking. We hypothesized a role for Fc-mediated clearance of TNF-TNFi immune complexes. Therefore, we investigated circulating TNF-TNFi complexes upon treatment with certolizumab-lacking Fc tails-in comparison with adalimumab, golimumab, infliximab and etanercept.Experimental Approach: Drug-tolerant ELISAs were developed and used to quantify TNF during adalimumab, golimumab, etanercept, certolizumab and infliximab treatment in patients with inflammatory arthritis or ulcerative colitis for a maximum follow-up of 1 year. Effects on in vitro TNF production and Fc-mediated uptake of TNF-TNFi complexes were investigated for all five TNFi.Key Results: Circulating TNF concentrations were >20-fold higher during certolizumab treatment compared with adalimumab, reaching up to 23.1 ngml(-1). Internalization of TNF-TNFi complexes by macrophages depended on Fc valency, with efficient uptake for the full antibody TNFi (three Fc tails), but little or no uptake for etanercept and certolizumab (one and zero Fc tail, respectively). TNF production was not affected by TNFi. Total TNF load did not affect clearance rate of total TNFi.Conclusions and Implications: Differences in TNFi structure profoundly affect clearance of TNF, while it is unlikely that TNF itself significantly contributes to target-mediated drug disposition of TNFi.

IgM is secreted as a pentameric polymer containing a peptide called the joining chain (J chain). However, integration of the J chain is not required for IgM assembly and in its absence IgM predominantly forms hexamers. The conformations of pentameric and hexameric IgM are remarkably similar with a hexagonal arrangement in solution. Despite these similarities, hexameric IgM has been reported to be a more potent complement activator than pentameric IgM, but reported relative potencies vary across different studies. Because of these discrepancies, we systematically investigated human IgM-mediated complement activation. We recombinantly generated pentameric and hexameric human IgM (IgM+J and IgM-J, respectively) mAbs and measured their ability to induce complement deposition and complement-dependent cytotoxicity when bound to several Ags at varying densities. At high Ag densities, hexameric and pentameric IgM activate complement to a similar extent as IgG1. However, at low densities, hexameric IgM outcompeted pentameric IgM and even more so IgG1. These differences became progressively more pronounced as antigenic density became critically low. Our findings highlight that the differential potency of hexameric and pentameric IgM for complement activation is profoundly dependent on the nature of its interactions with Ag. Furthermore, it underscores the importance of IgM in immunity because it is a more potent complement activator than IgG1 at low Ag densities.

Abstract Objectives Characterisation of the human antibody response to SARS‐CoV‐2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT‐PCR‐positive SARS‐CoV‐2 convalescent adults during the first 250 days after onset of symptoms. Methods We measured antibody responses to the receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT‐PCR‐positive SARS‐CoV‐2 convalescent adults. With a median of 5 (range 2–18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed‐effects modelling. Results All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow‐up analysis. Anti‐RBD IgG and anti‐nucleocapsid IgG levels declined with median half‐lives of 62 and 59 days, respectively, 2–5 months after symptom onset, and several‐fold variation in half‐lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow‐up, which points towards long‐term immunological memory. The magnitude of the anti‐RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in‐house developed competitive assay. Conclusion The result of this study gives valuable insight into the long‐term longitudinal response of antibodies to SARS‐CoV‐2.

AbstractThe novel SARS-CoV-2 virus emerged in late 2019 and has caused a global health and economic crisis. The characterization of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes as well for treatment options such as transfusion with convalescent plasma or immunoglobin products derived from convalescent plasma. In this study, we measured antibody responses in 844 longitudinal samples from 151 RT-PCR positive SARS-CoV-2 convalescent adults during the first 34 weeks after onset of symptoms. All donors were seropositive at the first sampling moment and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels slowly declined with median half-life’s of 62 and 59 days during 2-5 months after symptom onset, respectively. The rate of decline of antibody levels diminished during extended follow-up. In addition, the magnitude of the IgG response correlated with neutralization capacity measured in a classic plaque reduction assay as well in our in-house developed competition assay. The result of this study gives valuable insight into the longitudinal response of antibodies to SARS-CoV-2.

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of B cells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 B cells, circulating IgG4 B cells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive B cells with an IgG4-inducing combination of T-FH/T-H2 cytokines. Consistent with these observations, we found a low abundance of IgG4 B cells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20(+) B cell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 B cells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections often cause only mild disease that may evoke relatively low Ab titers compared with patients admitted to hospitals. Generally, total Ab bridging assays combine good sensitivity with high specificity. Therefore, we developed sensitive total Ab bridging assays for detection of SARS-CoV-2 Abs to the receptor-binding domain (RBD) and nucleocapsid protein in addition to conventional isotype-specific assays. Ab kinetics was assessed in PCR-confirmed, hospitalized coronavirus disease 2019 (COVID-19) patients (n = 41) and three populations of patients with COVID-19 symptoms not requiring hospital admission: PCR-confirmed convalescent plasmapheresis donors (n = 182), PCR-confirmed hospital care workers (n = 47), and a group of longitudinally sampled symptomatic individuals highly suspect of COVID-19 (n = 14). In nonhospitalized patients, the Ab response to RBD is weaker but follows similar kinetics, as has been observed in hospitalized patients. Across populations, the RBD bridging assay identified most patients correctly as seropositive. In 11/14 of the COVID-19-suspect cases, seroconversion in the RBD bridging assay could be demonstrated before day 12; nucleocapsid protein Abs emerged less consistently. Furthermore, we demonstrated the feasibility of finger-prick sampling for Ab detection against SARS-CoV-2 using these assays. In conclusion, the developed bridging assays reliably detect SARS-CoV-2 Abs in hospitalized and nonhospitalized patients and are therefore well suited to conduct seroprevalence studies.

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N-linked glycans, a process conditional on the introduction of consensus amino acid motifs (N-glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

PURPOSE:Intravenous immunoglobulin (IVIg) products contain various amounts of dimeric IgG complexes. Current insights into the possible biological activities of these dimers remain controversial, and both immunemodulating and immune-activating effects have been reported. Here, we analyzed the putative immune-activating effects of dimers isolated from IVIg.METHODS:Dimers isolated from IVIg were purified by high-performance size-exclusion chromatography (HP-SEC) and tested for the ability to induce neutrophil degranulation in vitro.RESULTS:Dimers isolated from IVIg were found to be incapable of inducing in vitro neutrophil degranulation or complement activation, even at concentrations exceeding those expected to be reached upon administration in patients. These results depend on the removal of artefactual activation by using 0.1 micron filtration and the use of poloxamer to prevent adsorption of IgG onto the solid phase.CONCLUSIONS:The data suggest dimeric IgG found in IVIg may bind to Fc-receptors without causing activation.

Complement is activated as part of the innate immune defense against invading pathogens. Also, it helps to remove apoptotic debris and immune complexes from the circulation. Impaired complement function due to aberrant plasma levels of complement proteins may be indicative for complement-mediated diseases or can be involved in susceptibility for infections. To determine whether plasma levels are abnormal, reference intervals (RIs) are used from adult healthy donors. Since many complement-mediated diseases have an onset during childhood, it is important to know whether these RIs can be extrapolated to children. RIs of Factor H (FH), the crucial fluid-phase regulator, and the FH-related proteins (FHRs), its homologous counterparts, are unknown in healthy children. While FH is measured to diagnose and monitor therapy of patients with atypical hemolytic uremic syndrome, recent studies also implicated increased plasma levels of FHRs in disease. Here, we investigated the levels of FH and FHRs in healthy children using recently developed specific ELISAs. We found that levels of FH, FHR-2, and FHR-3 were equal to those found in healthy adults. Levels of FHR-4A and FHR-5 were lower in children than in adults. However, only the FHR-5 levels associated with age. The RIs of these FH family proteins now serve to support the interpretation of plasma levels in prospective and retrospective studies that can be used for routine diagnostic and monitoring purposes including pediatric patient samples.

Objectives Therapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these 'anti-idiotype' complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies. Methods Size distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA. Results Size and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation. No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions. Conclusions Anti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.

OBJECTIVES:Controversy exists on the role of IgE antidrug antibodies (IgE-ADA) in infusion reactions (IR) on infliximab treatment, partly due to the lack of a positive control used for assay validation. We sought to (1) develop a robust assay to measure IgE-ADA, including a positive control, (2) determine the association between IgE-ADA and IR and (3) determine the incidence of IgE-ADA in infliximab treated patients. METHODS:A recombinant human IgE anti-infliximab monoclonal antibody was developed as standard and positive control. With this antibody, we set up a novel robust assay to measure IgE-ADA. IgE-ADA was determined in three retrospective cohorts (n=159) containing IR+ (n=37) and IR- (n=39), and longitudinal sera of 83 spondyloarthritis. RESULTS:IgE-ADA was found in 0/39 IR-, whereas 4/37 (11%) IR+ showed low levels (0.1-0.3 IU/mL, below the 0.35 IU/mL threshold associated with elevated risk of allergic symptoms). All patients who were IgE-ADA positive also had (very) high IgG-ADA levels. The incidence of IgE-ADA in patients with infliximab-treated spondyloarthritis was estimated at less than approximately 1%. CONCLUSIONS:IgE-ADA is rarely detected in infliximab-treated patients. Moreover, the absence of IgE-ADA in the majority of IR+ patients suggests that IgE-ADA is not associated with infusion reactions.

Many therapeutic mAbs have been found to elicit an immune response to various degrees. Antidrug antibodies (ADAs) can diminish efficacy by neutralization or enhanced clearance of the therapeutics, and occasionally cause hypersensitivity reactions or other adverse events.1Vennegoor A. Rispens T. Strijbis E.M.M. Seewann A. Uitdehaag B.M.J. Balk L.J. et al.Clinical relevance of serum natalizumab concentration and anti-natalizumab antibodies in multiple sclerosis.Mult Scler. 2013; 19: 593-600Crossref PubMed Scopus (47) Google Scholar Therapeutic antibodies are classified as chimeric, humanized, or "fully human," in order of increasing homology of the variable domains with human germline sequences, and thereby their expected decrease in immunogenicity. Recently, our group investigated the antibody repertoire in patients with an immune response against any of 4 different anti-TNF therapeutic antibodies.2van Schie K.A. Hart M.H. de Groot E.R. Kruithof S. Aarden L.A. Wolbink G.J. et al.The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region.Ann Rheum Dis. 2015; 74: 311-314Crossref PubMed Scopus (91) Google Scholar, 3van Schouwenburg P.A. Kruithof S. Votsmeier C. van Schie K. Hart M.H. de Jong R.N. et al.Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies.J Biol Chem. 2014; 289: 34482-34488Crossref PubMed Scopus (21) Google Scholar For the human(ized) adalimumab, certolizumab, and golimumab, more than 94% were found to bind the TNF-binding site, demonstrating that these ADAs are essentially all neutralizing. Strikingly, the antibody response to the chimeric infliximab, of which the entire variable domains are of mouse origin, was also more than 90% directed to the TNF-binding site. These results prompted the question whether this restricted response is due to specific immunogenic properties of the anti–TNF-binding site, or that the antigen-binding site (or paratope) is an inherently immunodominant part of the antibody, irrespective of its target. If so, antidrug antibodies to any therapeutic antibody might be expected to be predominantly neutralizing. To shed light on this question, we investigated the antibody response to natalizumab, a humanized antibody that differs in target (α4-integrin), subclass (IgG4), and patient group (relapsing-remitting multiple sclerosis [RRMS]) from anti-TNF therapeutics. Natalizumab contains several nonhuman germline determinants outside of its paratope (Fig 1, A) and can thus theoretically elicit a broad nonneutralizing antibody response. In this study, we examined the neutralizing capacity of the anti-natalizumab response of patients with RRMS. We therefore first developed human monoclonal anti-natalizumab antibodies. Variable domain sequences of in total 3 natalizumab-specific peripheral blood B cells were obtained from 2 patients (see Table E1 in this article's Online Repository at www.jacionline.org). These sequences were recombinantly expressed in HEK293F cells, resulting in 3 human monoclonal anti-natalizumab antibodies. Analysis of the clones using ImMunoGeneTics/V-QUEry and STandardization (IMGT/V-Quest)5Brochet X. Lefranc M.P. Giudicelli V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis.Nucleic Acids Res. 2008; 36: W503-W508Crossref PubMed Scopus (0) Google Scholar revealed a distinct V(D)J gene usage for each clone. Using surface plasmon resonance, the affinity of anti-natalizumab 1.1 and 2.1 was found to be moderate, whereas anti-natalizumab 2.2 had a high affinity (1 × 10−11 mol/L) (see Fig E1 and Table E2 in this article's Online Repository at www.jacionline.org). To investigate the natalizumab-binding site for each clone relative to the others, the antibodies were tested in a competitive ELISA. In this assay, the binding of a labeled clone was assessed in the presence of a competing nonlabeled clone (Fig 1, B). The results showed that the mAbs all competed with each other for binding to natalizumab (Fig 1, C), indicating that all 3 mAbs bind to a restricted site on natalizumab. We further examined whether the mAbs could block the binding of natalizumab to its target α4-integrin. This was tested using fluorescently labeled natalizumab that binds α4-integrin expressed on Jurkat cells. Preincubation of natalizumab with any of the 3 mAbs abrogated the binding of natalizumab to α4-integrin (Fig 1, D and E). Similar results were found for the binding of exchanged (ie, monovalent; see "IgG4 half-molecule exchange" section in this article's Online Repository at www.jacionline.org) natalizumab to α4-integrin (see Fig E2 in this article's Online Repository at www.jacionline.org). This indicates that all 3 monoclonal anti-natalizumab antibodies compete with α4-integrin for binding to natalizumab, and are thus all neutralizing. Next, we investigated the neutralizing capacity of polyclonal anti-natalizumab antibodies from 15 ADA-positive patients. A competitive ELISA was set up in which α4-integrin blocks the paratope of natalizumab (Fig 2, A). For all patients, the binding of ADA to natalizumab was reduced with increasing concentrations of α4-integrin (Fig 2, B). With the highest amount of α4-integrin tested, the signal was reduced by at least 91% compared with the uninhibited signal (Fig 2, C). This indicates that for all patients tested, at least 91% of all ADAs to natalizumab are neutralizing. We further analyzed which parts of natalizumab are targeted by the anti-natalizumab response using a similar competitive assay, but now using neutralizing, but less bulky, Fab fragments of monoclonal anti-natalizumab 2.2 to block the binding site of natalizumab (Fig 2, D). Again, the binding of patient ADAs to natalizumab could be reduced in a concentration-dependent manner, ultimately preventing at least 92% of ADAs from binding natalizumab using this single mAb (Fig 2, E and F). This implies that most ADAs from patients with RRMS bind determinants of natalizumab located in or in proximity to the paratope. The results from this present study on the anti-natalizumab response, in combination with our previous findings on the neutralizing capacity of ADAs to anti-TNF therapeutics,2van Schie K.A. Hart M.H. de Groot E.R. Kruithof S. Aarden L.A. Wolbink G.J. et al.The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region.Ann Rheum Dis. 2015; 74: 311-314Crossref PubMed Scopus (91) Google Scholar, 3van Schouwenburg P.A. Kruithof S. Votsmeier C. van Schie K. Hart M.H. de Jong R.N. et al.Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies.J Biol Chem. 2014; 289: 34482-34488Crossref PubMed Scopus (21) Google Scholar indicate that the immune response preferentially targets the paratope of at least several classes of therapeutic antibodies. The antigen specificity of the drug seems irrelevant for this response. In addition, the nonhuman germline determinants outside of the paratope found in natalizumab, as well as those in the chimeric infliximab (and even in the other humanized and fully human anti-TNFs), apparently are less immunogenic. This observation is also consistent with the lack of immune response toward mismatched allotypes of therapeutic antibodies.6van Schie K.A. Wolbink G.J. Rispens T. Cross-reactive and pre-existing antibodies to therapeutic antibodies–effects on treatment and immunogenicity.MAbs. 2015; 7: 662-671Crossref PubMed Scopus (72) Google Scholar The paratope of therapeutic antibodies, but perhaps of any antibody, might be intrinsically more immunogenic than other determinants outside of the paratope. It is however unknown whether the paratope displays specific structural features that facilitate its potential role as epitope. For infliximab, attempts were made to more precisely locate immunogenic amino acid sequences using synthetic peptides. Homann et al7Homann A. Röckendorf N. Kromminga A. Frey A. Jappe U. B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera.J Transl Med. 2015; 13: 339Crossref PubMed Scopus (17) Google Scholar found reactivity of ADA-positive patients to 4 peptide sequences located in or in proximity to the complementarity-determining regions, although 2 of these peptides were also recognized by healthy controls.7Homann A. Röckendorf N. Kromminga A. Frey A. Jappe U. B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera.J Transl Med. 2015; 13: 339Crossref PubMed Scopus (17) Google Scholar In contrast, a study by Kosmač et al8Kosmač M. Avčin T. Toplak N. Simonini G. Cimaz R. Šerbec V.C. Exploring the binding sites of anti-infliximab antibodies in pediatric patients with rheumatic diseases treated with infliximab.Pediatr Res. 2011; 69: 243-248Crossref PubMed Scopus (32) Google Scholar showed no reactivity to synthetic peptides nor to denatured infliximab Fab, suggesting that anti-infliximab antibodies target conformational rather that linear epitopes. It therefore remains unclear which precise determinants on infliximab are targeted. For adalimumab, we demonstrated that anti-adalimumab antibodies bind to multiple overlapping, but distinct epitopes in the paratope.3van Schouwenburg P.A. Kruithof S. Votsmeier C. van Schie K. Hart M.H. de Jong R.N. et al.Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies.J Biol Chem. 2014; 289: 34482-34488Crossref PubMed Scopus (21) Google Scholar Human(ized) and chimeric therapeutic antibodies all have fully human constant domains. Although there is little evidence for drug-induced antibody responses toward these regions, preexisting antibodies such as rheumatoid factors (low-affinity IgM antibodies binding human IgG Fc) can be present. The available literature suggests that the presence of such antibodies generally has little consequences.6van Schie K.A. Wolbink G.J. Rispens T. Cross-reactive and pre-existing antibodies to therapeutic antibodies–effects on treatment and immunogenicity.MAbs. 2015; 7: 662-671Crossref PubMed Scopus (72) Google Scholar Our findings on the neutralizing capacity of ADAs to natalizumab are in line with a study of Calabresi et al9Calabresi P.A. Giovannoni G. Confavreux C. Galetta S.L. Havrdova E. Hutchinson M. et al.The incidence and significance of anti-natalizumab antibodies: results from AFFIRM and SENTINEL.Neurology. 2007; 69: 1391-1403Crossref PubMed Scopus (282) Google Scholar in which ADAs to natalizumab were analyzed using an ELISA that measures all ADAs and a cell-blocking assay that measures only neutralizing ADAs. The strong correlation between the 2 assays suggested that most or all ADAs to natalizumab were neutralizing. In conclusion, this study shows that most of the polyclonal anti-natalizumab response in patients with RRMS specifically targets the antigen-binding site of natalizumab, and thereby extends the results previously obtained for anti-TNF antibodies.2van Schie K.A. Hart M.H. de Groot E.R. Kruithof S. Aarden L.A. Wolbink G.J. et al.The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region.Ann Rheum Dis. 2015; 74: 311-314Crossref PubMed Scopus (91) Google Scholar, 3van Schouwenburg P.A. Kruithof S. Votsmeier C. van Schie K. Hart M.H. de Jong R.N. et al.Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies.J Biol Chem. 2014; 289: 34482-34488Crossref PubMed Scopus (21) Google Scholar Although exceptions cannot be ruled out, these results suggest that the immune response toward antibody therapeutics may preferentially be directed toward the paratope of the drug, and will thus inherently neutralize the therapeutics. We thank Laura Lighaam, Ellen Vermeulen, and Hong Dai for their contribution to the B-cell screening. For the development of human monoclonal anti-natalizumab antibodies, EDTA blood was collected from 2 ADA-positive patients with RRMS. For competitive assays, serum samples of 7 patients with anti-natalizumab antibodies were selected from a prospective observational cohort previously described by Vennegoor et al.E1Vennegoor A. Rispens T. Strijbis E.M.M. Seewann A. Uitdehaag B.M.J. Balk L.J. et al.Clinical relevance of serum natalizumab concentration and anti-natalizumab antibodies in multiple sclerosis.Mult Scler. 2013; 19: 593-600Crossref PubMed Scopus (97) Google Scholar After obtaining informed consent, blood was taken from these patients. The study was approved by the ethics committee of the VU Medical Center, Amsterdam. In addition, anti-natalizumab antibody positive sera were obtained from our diagnostic department (n = 8). Materials were used anonymously without any connection to clinical data. Anti-natalizumab positivity was measured using a previously described antigen-binding test.E2Rispens T. Hart M.H. Ooijevaar-de Heer P. van Leeuwen A. Vennegoor A. Killestein J. et al.Drug interference in immunogenicity assays depends on valency.J Pharm Biomed Anal. 2013; 85: 179-185Crossref PubMed Scopus (17) Google Scholar Natalizumab-specific B cells were isolated essentially as described before,E3van Schouwenburg P.A. van de Stadt L.A. de Jong R.N. van Buren E.E.L. Kruithof S. de Groot E. et al.Adalimumab elicits a restricted anti-idiotypic antibody response in autoimmune patients resulting in functional neutralisation.Ann Rheum Dis. 2013; 72: 104-109Crossref PubMed Scopus (159) Google Scholar with the modification that 4′-6-diamidino-2-phenylindole, dihydrochloride–negative, anti-CD20-PerCP-Cy5.5–positive, anti-IgD-fluorescein isothiocyanate–negative (all from BD Biosciences, San Jose, Calif) memory B cells were selected, after which cells double positive for natalizumab F(ab)2-AF647 (Invitrogen, Paisley, United Kingdom, labeled according to the manufacturer's instructions) and biotinylated natalizumab F(ab)2 combined with streptavidin-phycoerythrin (BD Biosciences) were placed 1 cell per well in 96-well flat-bottom plates. Determining the type of subclass and light chain of monoclonal anti-natalizumab antibodies, and production of the recombinant antibodies, was done as described before.E4van Schouwenburg P.A. Kruithof S. Votsmeier C. van Schie K. Hart M.H. de Jong R.N. et al.Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies.J Biol Chem. 2014; 289: 34482-34488Crossref PubMed Scopus (48) Google Scholar B-cell supernatants and recombinant monoclonal anti-natalizumab antibodies were tested for anti-natalizumab specificity using a bridging ELISA as described before, with slight adjustments.E3van Schouwenburg P.A. van de Stadt L.A. de Jong R.N. van Buren E.E.L. Kruithof S. de Groot E. et al.Adalimumab elicits a restricted anti-idiotypic antibody response in autoimmune patients resulting in functional neutralisation.Ann Rheum Dis. 2013; 72: 104-109Crossref PubMed Scopus (159) Google Scholar In short, samples (diluted appropriately in PBS containing 0.2% gelatin (PTG) [Merck, Darmstadt, Germany] and 0.1% Tween-20) were added to a natalizumab-coated plate (0.25 μg/mL in PBS), followed by detection with biotinylated natalizumab F(ab)2 (50 ng/mL). F(ab′)2 and Fab′ fragments were generated using pepsin, and subsequent reduction and alkylation with dithiothreitol and iodoacetamide, respectively, essentially as described.E5van Schouwenburg P.A. Bartelds G.M. Hart M.H. Aarden L. Wolbink G.J. Wouters D. A novel method for the detection of antibodies to adalimumab in the presence of drug reveals "hidden" immunogenicity in rheumatoid arthritis patients.J Immunol Methods. 2010; 362: 82-88Crossref PubMed Scopus (143) Google Scholar Fab fragments were generated by papain digestion as described,E6Guhr T. Bloem J. Derksen N.I.L. Wuhrer M. Koenderman A.H.L. Aalberse R.C. et al.Enrichment of sialylated IgG by lectin fractionation does not enhance the efficacy of immunoglobulin G in a murine model of immune thrombocytopenia.PLoS One. 2011; 6: e21246Crossref PubMed Scopus (95) Google Scholar with the exception that the enzyme:antibody ratio was 1:10. Surface plasmon resonance measurements were performed using a Biacore T200 instrument (GE Healthcare, Uppsala, Sweden) at 25°C. Mouse anti-human IgG (MH16-1, Sanquin) was immobilized at a concentration of 5 μg/mL in 10 mM sodium acetate, pH 5.0, at a flow rate of 10 μL/min on a CM5 sensor chip using N-hydrosuccinimide/1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride. Anti-natalizumab mAbs were dissolved at 5 μg/mL in PBS, pH 7.4, containing 0.05% Tween-20 (PBS-T), and passed through the cells at 15 μL/min yielding approximately 100 to 300 response units of bound antibody. Subsequently, binding of natalizumab Fab′ (0.125-1.25 μg/mL) dissolved in PBS-T was measured at a flow rate of 15 μL/min. After each run, immobilized ligand was regenerated by removing the bound analyte with 5 μL of 0.1 mol/L phosphoric acid. Adsorptions obtained in the reference channel without bound anti-natalizumab antibody were subtracted from adsorptions in the other cells. Association and dissociation kinetics were fitted using the models provided with the Biacore analysis software (GE Healthcare, Uppsala, Sweden). A 96-well flat-bottom MaxiSorp plate was coated with 0.25 μg/mL natalizumab in PBS and incubated overnight. Plates were washed with PBS-T. Biotinylated anti-natalizumab was preincubated at 0.15 μg/mL in PTG together with unlabeled anti-natalizumab at a final concentration of 1 or 5 μg/mL and subsequently added to the plate. After 1-hour incubation, plates were washed and incubated for 25 minutes with streptavidin-poly horseradish peroxidase (Sanquin, Amsterdam, The Netherlands) diluted in PTG, after which plates were washed again and developed using substrate solution. Samples were measured at 450 nm and 540 nm. IgG4 antibodies have the property to exchange half-molecules (ie, 1 heavy chain and its attached light chain) with other IgG4 molecules. This half-molecule exchange occurs under physiological conditions in patients and healthy people. Natalizumab is an IgG4 antibody and is able to exchange with autologous IgG4, thus becoming monovalent.E7Aalberse R.C. Stapel S.O. Schuurman J. Rispens T. Immunoglobulin G4: an odd antibody.Clin Exp Allergy. 2009; 39: 469-477Crossref PubMed Scopus (606) Google Scholar To create monovalent fluorescently labeled natalizumab, natalizumab labeled with DyLight488 (natalizumab-AF488, Invitrogen, labeled according to the manufacturer's instructions) was incubated with a 100-fold excess of anti-adalimumab IgG4 in the presence of 1 mM GSH (reduced glutathione) at 37°C as described previously.E2Rispens T. Hart M.H. Ooijevaar-de Heer P. van Leeuwen A. Vennegoor A. Killestein J. et al.Drug interference in immunogenicity assays depends on valency.J Pharm Biomed Anal. 2013; 85: 179-185Crossref PubMed Scopus (17) Google Scholar After 24 hours, the reaction was stopped by adding iodoacetamide to a concentration of 1 mM. A bridging ELISA was used to check the exchange of natalizumab: exchanged natalizumab is unable to form a bridge between coat and detection antibody. Jurkat cells (a kind gift of M. Schreier [Sandoz, Basel, Switzerland]) were washed with PBS + 0.1% BSA (Sigma-Aldrich, Steinheim, Germany). Natalizumab-AF488 was preincubated for 15 minutes with anti-natalizumab in a ratio of 1:1, 1:5, or 1:25 (natalizumab-AF488 [1 μg/mL]:anti-natalizumab) and subsequently added to the cells in a 20× dilution. In addition, natalizumab-AF488 without inhibiting anti-natalizumab was added to the cells in a 20× dilution. A similar setup was performed with exchanged (ie, monovalent) natalizumab-AF488. Binding of natalizumab-AF488 to Jurkat cells was determined using the Beckton Dickinson LSR Fortessa (BD Biosciences) and analyzed with FACS DIVA software (BD Biosciences) and FlowJo software (v7.6.2; Treestar, Ashland, Ore). First, patient sera were titrated to determine the optimal serum dilution (out of plateau) for every patient. Next, 96-well flat-bottom MaxiSorp plates were coated with 31 ng/mL natalizumab in PBS and incubated overnight. For each sample, 12 wells were incubated with the optimal serum dilution (diluted in PTG). The standard curve consisted of a titration of monoclonal anti-natalizumab 2.2. Samples were incubated for 1 hour. Biotinylated natalizumab (5 ng/mL) was preincubated with a titration of the inhibitor (anti-natalizumab 2.2 Fab [generated using papain] or α4β1 [R&D Systems, Minneapolis, Minn], both ranging from 0 to 15 μg/mL) and subsequently added to each patient serum. For the standard curve, biotinylated natalizumab without inhibitor was used. After 1-hour incubation, plates were washed and incubated with streptavidin-poly horseradish peroxidase diluted in PTG for 25 minutes. Plates were washed again and developed using substrate solution as described above. Samples were measured at 450 nm and 540 nm. Optical density was converted into arbitrary units (AUs) using the standard curve. Percentage of inhibition was calculated by dividing the AUs of inhibited samples by the AUs of noninhibited samples.Fig E2Monoclonal anti-natalizumab antibodies compete with α4-integrin for binding natalizumab. A, Schematic representation of the competition FACS experiment. Exchanged natalizumab binds α4-integrin expressed on Jurkat cells. Neutralizing antibodies inhibit this binding. B, Inhibition of exchanged natalizumab binding to cell surface α4-integrin by all 3 mAbs determined by FACS. FACS, Fluorescence-activated cell sorting; MFI, mean fluorescence intensity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Characteristics of human monoclonal anti-natalizumab antibodiesCloneHeavy chainLight chainIsotypeV geneD geneJ geneRSκ/λV geneJ geneRS1.1IgG138-25-053105λ1-472412.1IgG13-113-10463κ3-112002.2IgG11-036-13594κ1-334137For each clone, the isotype, V(D)J usage, number of mutations leading to amino acid replacements (R), or silent mutations (S) and κ/λ is described. Sequences were aligned using the ImMunoGeneTics/V-QUEry and STandardization (IMGT/V-Quest) database.E8Brochet X. Lefranc M.P. Giudicelli V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis.Nucleic Acids Res. 2008; 36: W503-W508Crossref PubMed Scopus (834) Google Scholar Open table in a new tab Table E2Estimated affinities of anti-natalizumab clones for natalizumab Fab′Cloneka (M−1 s−1)kd (s−1)Kd (M)1.11.01 × 1064.7 × 10−34.7 × 10−92.16.1 × 1051.5 × 10−32.4 × 10−92.22.1 × 1062 × 10−51 × 10−11Association and dissociation rates and Kd of anti-natalizumab to natalizumab Fab′ are calculated from surface plasmon resonance measurements. Open table in a new tab For each clone, the isotype, V(D)J usage, number of mutations leading to amino acid replacements (R), or silent mutations (S) and κ/λ is described. Sequences were aligned using the ImMunoGeneTics/V-QUEry and STandardization (IMGT/V-Quest) database.E8Brochet X. Lefranc M.P. Giudicelli V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis.Nucleic Acids Res. 2008; 36: W503-W508Crossref PubMed Scopus (834) Google Scholar Association and dissociation rates and Kd of anti-natalizumab to natalizumab Fab′ are calculated from surface plasmon resonance measurements.

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 Förster 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 adalimumab.

We read with a great interest the report by Van Schie et al 1 which highlighted that the antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region. The results described in this report confirm very clearly the results that we obtained recently. This study adds to the one we performed, which shows the neutralising capacity of antidrug antibodies (ADAs) against infliximab (IFX) in patients with inflammatory bowel disease (IBD) treated by IFX. We assessed ADAs’ biological activity by a functional assay using a reporter HEK-Dual TNF cell line developed for TNF detection (InvivoGen). Briefly, sera of patients with IBD were first incubated …

Comparing studies investigating anti-drug antibody (ADA) formation is hampered by the lack of comparability between study protocols, assay formats, and standardized reference materials. In this respect, the use of an international standard would mean a major step forward. Here we compared 11 fully human monoclonal antibodies against adalimumab in two assays commonly used for ADA measurement; the bridging ELISA and the antigen binding test (ABT). Our results show non-parallel titration of the monoclonal antibodies in both assays, which we also find for polyclonal ADA sources. Moreover, we observed that the output of the bridging ELISA depends to a large degree on the affinity of the monoclonal antibody. For the ABT, results reflect a combination of affinity and avidity. This suggests that rather than reporting ADA values in nanogram per milliliter, arbitrary units may be more appropriate. Together our data highlight the difficulty of ADA standardization by identifying several pitfalls that should be taken into account when selecting a standard for ADA testing.

Background: Therapeutic antibodies such as adalimumab can elicit anti-drug antibodies. Results: Monoclonal patient-derived antibodies were generated and found to compete for binding to adalimumab and are neutralizing, but they show markedly different fine-specificity. Conclusion: Anti-adalimumab antibodies bind to overlapping but distinct epitopes on adalimumab. Significance: Even for a fully human therapeutic antibody, there may be multiple determinants that contribute to immunogenicity.The production of antibodies to adalimumab in autoimmune patients treated with adalimumab is shown to diminish treatment efficacy. We previously showed that these antibodies are almost exclusively neutralizing, indicating a restricted response. Here, we investigated the characteristics of a panel of patient-derived monoclonal antibodies for binding to adalimumab. Single B-cells were isolated from two patients, cultured, and screened for adalimumab specificity. Analysis of variable region sequences of 16 clones suggests that the immune response against adalimumab is broad, involving multiple B-cell clones each using different combinations of V(D)J segments. A strong bias for replacement mutations in the complementarity determining regions was found, indicating an antigen-driven response. We recombinantly expressed 11 different monoclonal antibodies and investigated their affinity and specificity. All clones except one are of high affinity (K-d between 0.6 and 233 pm) and compete with TNF as well as each other for binding to adalimumab. However, binding to a panel of single-point mutants of adalimumab indicates markedly different fine specificities that also result in a differential tendency of each clone to form dimeric and multimeric immune complexes. We conclude that although all anti-adalimumab antibodies compete for binding to TNF, the response is clonally diverse and involves multiple epitopes on adalimumab. These results are important for understanding the relationship between self and non-self or idiotypic determinants on therapeutic antibodies and their potential immunogenicity.

Background Worldwide millions of patients are treated with monoclonal antibodies. These biological therapeutics can be immunogenic, resulting in antibody formation which lead to loss of response. Fully human biologics such as the anti-TNFα antibody adalimumab are considered to be weakly immunogenic, but we found that the majority of Rheumatoid Arthritis patients develop anti-drug antibodies (ADA) within 28 weeks of treatment. Objectives Here we unraveled the mechanism by which ADA lead to loss of response by investigating the full specificity of the repertoire of anti-adalimumab antibodies. Methods The specificity of the repertoire of anti-adalimumab antibodies in a cohort of 50 ADA-positive RA patients was elucidated. In inhibition experiments using TNFa and patient derived anti-adalimumab monoclonal antibodies that were recombinantly generated after isolation of adalimumab-specific B cells. Results The antibody response against adalimumab is highly restricted: Fab fragments of a single, monoclonal antibody specific for the idiotype of adalimumab inhibited 98.65% (25th-75th percentiles: 98.25- 99.90) of the total anti-adalimumab reactivity in sera of 50 different ADA-positive RA patients. Furthermore, we found that the anti-adalimumab response is confined to the TNFα binding region of adalimumab, thereby neutralizing its therapeutic efficacy. In line with this restricted specificity, we observed small immune complexes in the circulation of ADA-forming patients. Conclusions The humoral immune response against adalimumab is highly restricted and limited to the idiotype of the therapeutic antibody. All antibodies result in functional neutralization of the drug, thereby providing a mechanism in which ADA formation leads to clinical non-response. Disclosure of Interest None Declared

Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1 pM. During the analysis of several human-derived monoclonal antibodies to adalimumab, we found a clone with a very high affinity that could not be measured using conventional surface plasmon resonance assays. We developed a straightforward and robust method to measure affinities in the nanomolar to sub-picomolar range. The assay is based on separation of bound and free fluorescently labeled antigen using size exclusion chromatography and quantification by in-line fluorescence detection. We describe optimal conditions and procedures that result in a very sensitive assay that can be used to reliably determine ultra-high affinities. Using the method described in this article, a dissociation constant of 0.78 pM could be determined for the anti-adalimumab antibody.

OBJECTIVEACPAs are thought to play a pathogenic role in RA. Because of their polyclonal nature it is difficult to study characteristics of ACPAs such as cross-reactivity or affinity. This study aimed to analyse the ACPA response at clonal level.METHODSCitrullinated fibrinogen-specific B cells were isolated from blood derived from an RA patient by fluorescent automated cell sorting (FACS). Antigen specificity was verified by ELISA of culture supernatant. RNA of antigen-specific B cells was isolated and VH and VL chains were cloned and subsequently expressed as IgG1 antibodies.RESULTSTwo human recombinant antibodies were obtained that bind to citrullinated fibrinogen peptide (cFib). Both monoclonal antibodies originate from different naive B cells, undergo extensive somatic hypermutation and bind to cFib (but not to Fib) with moderate avidity. Furthermore, they show distinct cross-reactivity patterns towards other citrullinated peptides, suggesting that both antibodies have different primary targets.CONCLUSIONTogether these data suggest that ACPAs are formed by antigen-driven maturation, and that multiple citrullinated antigens are involved in activating the B-cell response.