Variation in CFHR3 determines susceptibility to meningococcal disease by controlling factor H concentrations.

Neisseria meningitidis protects itself from complement-mediated killing by binding complement factor H (FH). Previous studies associated susceptibility to meningococcal disease (MD) with variation in CFH, but the causal variants and underlying mechanism remained unknown. Here we attempted to define the association more accurately by sequencing the CFH-CFHR locus and imputing missing genotypes in previously obtained GWAS datasets of MD-affected individuals of European ancestry and matched controls. We identified a CFHR3 SNP that provides protection from MD (rs75703017, p value = 1.1 × 10-16) by decreasing the concentration of FH in the blood (p value = 1.4 × 10-11). We subsequently used dual-luciferase studies and CRISPR gene editing to establish that deletion of rs75703017 increased FH expression in hepatocyte by preventing promotor inhibition. Our data suggest that reduced concentrations of FH in the blood confer protection from MD; with reduced access to FH, N. meningitidis is less able to shield itself from complement-mediated killing.

Plasma Exchange Therapy Using Solvent Detergent-Treated Plasma: An Observational Pilot Study on Complement, Neutrophil and Endothelial Cell Activation in a Case Series of Patients Suffering from Atypical Hemolytic Uremic Syndrome.

Introduction: Plasma exchange therapy (PEX) was standard treatment for thrombotic microangiopathy before eculizumab was available and is still widely applied. However, most PEX patients still ultimately progress to end-stage renal disease (ESRD). It has been suggested that infusion of plasma that contains active complement may induce additional complement activation with subsequent activation of neutrophils and endothelial cells, leading to exacerbation of organ damage and deterioration of renal function. Objective: This observational pilot study examines the effect of hemodialysis, eculizumab and PEX before and after treatment in plasma of aHUS patients on complement-, neutrophil and endothelial cell activation. Methods: Eleven patients were included in this pilot study. Six patients were treated with hemodialysis, 2 patients received regular infusions of eculizumab, and 3 patients were on a regular schedule for PEX. Patients were followed during 3 consecutive treatments. Blood samples were taken before and after patients received their treatment. Results: Complement activation products increased in plasma of patients after PEX, as opposed to patients treated with hemodialysis or eculizumab. Increased levels of complement activation products were detected in omniplasma used for PEX. Additionally, activation of neutrophils and endothelial cells was observed in patients after hemodialysis and PEX, but not in patients receiving eculizumab treatment. Conclusion: In this pilot study we observed that PEX induced complement and neutrophil activation, and that omniplasma contains significant amounts of complement activation products. Additionally, we demonstrate that hemodialysis induces activation of neutrophils and endothelial cells. Complement activation with subsequent neutrophil activation may contribute to the deterioration of organ function and may result in ESRD. Further randomized controlled studies are warranted to investigate the effect of PEX on complement- and neutrophil activation in patients with thrombotic microangiopathy.

N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation.

Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N- and O-glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O-glycosylated N-terminal region. Five novel and five known O-glycosylation sites were identified, carrying mainly core1-type O-glycans. In addition, we detected a heavily O-glycosylated portion spanning from Thr82-Ser121 with up to 16 O-glycans attached. Likewise, all known six N-glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N-glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications.

Complement factor H contributes to mortality in humans and mice with bacterial meningitis.

BACKGROUND: The complement system is a vital component of the inflammatory response occurring during bacterial meningitis. Blocking the complement system was shown to improve the outcome of experimental pneumococcal meningitis. Complement factor H (FH) is a complement regulatory protein inhibiting alternative pathway activation but is also exploited by the pneumococcus to prevent complement activation on its surface conferring serum resistance. METHODS: In a nationwide prospective cohort study of 1009 episodes with community-acquired bacterial meningitis, we analyzed whether genetic variations in CFH influenced FH cerebrospinal fluid levels and/or disease severity. Subsequently, we analyzed the role of FH in our pneumococcal meningitis mouse model using FH knock-out (Cfh-/-) mice and wild-type (wt) mice. Finally, we tested whether adjuvant treatment with human FH (hFH) improved outcome in a randomized investigator blinded trial in a pneumococcal meningitis mouse model. RESULTS: We found the major allele (G) of single nucleotide polymorphism in CFH (rs6677604) to be associated with low FH cerebrospinal fluid concentration and increased mortality. In patients and mice with bacterial meningitis, FH concentrations were elevated during disease and Cfh-/- mice with pneumococcal meningitis had increased mortality compared to wild-type mice due to C3 depletion. Adjuvant treatment of wild-type mice with purified human FH led to complement inhibition but also increased bacterial outgrowth which resulted in similar disease outcomes. CONCLUSION: Low FH levels contribute to mortality in pneumococcal meningitis but adjuvant treatment with FH at a clinically relevant time point is not beneficial.

Complement Deposition and IgG Binding on Stored Red Blood Cells Are Independent of Storage Time.

BACKGROUND: In the Netherlands, red blood cells (RBCs) are allowed to be stored up to 35 days at 2-6 °C in saline-adenine-glucose-mannitol (SAGM). During storage, RBCs undergo several changes that are collectively known as storage lesion. We investigated to what extent complement deposition and antibody binding occurred during RBC storage and investigated phagocytic uptake in vitro. METHODS: RBCs were stored for different lengths of time at 2-6 °C in SAGM. Complement deposition and antibody binding were assessed upon storage and after incubation with serum. M1- and M2-type macrophages were generated from blood monocytes to investigate RBC phagocytosis. RESULTS: No complement deposition was directly observed on stored RBCs, while incubation of RBCs with serum resulted in variable donor-dependent C3 deposition and IgG binding, both independent of storage time. Only 1-4% phagocytosis of stored RBCs by macrophages was observed. CONCLUSION: RBCs are susceptible to complement deposition and antibody binding independent of storage time. Limited phagocytic uptake by macrophages was observed in vitro.

Protein array autoantibody profiles to determine diagnostic markers for neuropsychiatric systemic lupus erythematosus.

Objective: The aim was to investigate the association between autoantibodies (autoAbs) and neuropsychiatric (NP) involvement in patients with SLE and to evaluate whether any autoAb or a combination of these autoAbs could indicate the underlying pathogenic process. Methods: Using a multiplexed protein array for 94 antigens, we compared the serum autoAb profiles of 69 NPSLE patients, 203 SLE patients without NP involvement (non-NPSLE) and 51 healthy controls. Furthermore, we compared the profiles of NPSLE patients with clinical inflammatory (n = 38) and ischaemic (n = 31) NP involvement. Results: In total, 75 IgG and 47 IgM autoAbs were associated with SLE patients in comparison with healthy controls. Comparing NPSLE with non-NPSLE and healthy control sera, 9 IgG (amyloid, cardiolipin, glycoprotein 2, glycoprotein 210, heparin, heparan sulphate, histone H2A, prothrombin protein and vimentin) and 12 IgM (amyloid, cardiolipin, centromere protein A, collagen II, histones H2A and H2B, heparan sulphate, heparin, mitochondrial 2, nuclear Mi-2, nucleoporin 62 and vimentin) autoAbs were present at significantly different levels in NPSLE. The combination of IgG autoAbs against heparan sulphate, histone H2B and vimentin could differentiate NPSLE from non-NPSLE (area under the curve 0.845, 99.97% CI: 0.756, 0.933; P < 0.0001). Compared with non-NPSLE, four IgG and seven IgM autoAbs were significantly associated with inflammatory NPSLE. In ischaemic NPSLE, three IgG and three IgM autoAbs were significantly different from non-NPSLE patients. Conclusion: In our cohort, the presence of high levels of anti-heparan sulphate and anti-histone H2B combined with low levels of anti-vimentin IgG autoAbs is highly suggestive of NPSLE. These results need to be validated in external cohorts.

A comparison in therapeutic efficacy of several time points of intravenous StemBell administration in a rat model of acute myocardial infarction.

BACKGROUND: Adipose-derived stromal cells (ASCs) are a promising new therapeutic option for patients with acute myocardial infarction (AMI). Previously, we found that ASCs coupled to antibody-targeted microbubbles (StemBells [StBs]) improved cardiac function when administered intravenously 7 days post-AMI in rats. In this study, we compared the efficacy of intravenous StB administration at different administration time points following AMI in rats. METHODS: AMI, followed by reperfusion, was induced in four groups of male Wistar rats, which subsequently received an intravenous 1 × 106 StB bolus 1 day post-AMI (StB1; n = 8), 7 days post-AMI (StB7; n = 9), at both time points (StB1+7; n = 7) or neither (Control; n = 7). The effect onrdiac function was determined using echocardiography prior to AMI, 7 days post-AMI and 42 days post-AMI. The effect on infarct size and macrophages in the infarct core were determined (immuno)histochemically 42 days post-AMI. RESULTS: At 42 days post-AMI, all three StB groups had a significantly improved fractional shortening compared with the control group. Between the StB-treated groups, the effects did not differ significantly at 42 days post-AMI. At 7 days post-AMI, the StB1 group had a significantly improved fractional shortening compared with the control and StB7 groups. No significant changes in infarct size or macrophage numbers were found compared with the control group for any StB group. CONCLUSIONS: StB administration resulted in long-term improvement of cardiac function, independent of the time point of administration. When administered at 1 day post-AMI, this improvement was already evident at 7 days post-AMI.

TFPI inhibits lectin pathway of complement activation by direct interaction with MASP-2.

The lectin pathway (LP) of complement has a protective function against invading pathogens. Recent studies have also shown that the LP plays an important role in ischemia/reperfusion (I/R)-injury. MBL-associated serine protease (MASP)-2 appears to be crucial in this process. The serpin C1-inhibitor is the major inhibitor of MASP-2. In addition, aprotinin, a Kunitz-type inhibitor, was shown to inhibit MASP-2 activity in vitro. In this study we investigated whether the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI) is also able to inhibit MASP-2. Ex vivo LP was induced and detected by C4-deposition on mannan-coated plates. The MASP-2 activity was measured in a fluid-phase chromogenic assay. rTFPI in the absence or presence of specific monoclonal antibodies was used to investigate which TFPI-domains contribute to MASP-2 inhibition. Here, we identify TFPI as a novel selective inhibitor of MASP-2, without affecting MASP-1 or the classical pathway proteases C1s and C1r. Kunitz-2 domain of TFPI is required for the inhibition of MASP-2. Considering the role of MASP-2 in complement-mediated I/R-injury, the inhibition of this protease by TFPI could be an interesting therapeutic approach to limit the tissue damage in conditions such as cerebral stroke, myocardial infarction or solid organ transplantation.

Blimp-1 homolog Hobit identifies effector-type lymphocytes in humans.

Human cytomegalovirus (CMV) induces the formation of effector CD8(+) T cells that are maintained for decades during the latent stage of infection. Effector CD8(+) T cells appear quiescent, but maintain constitutive cytolytic capacity and can immediately produce inflammatory cytokines such as IFN-γ after stimulation. It is unclear how effector CD8(+) T cells can be constitutively maintained in a terminal stage of effector differentiation in the absence of overt viral replication. We have recently described the zinc finger protein Homolog of Blimp-1 in T cells (Hobit) in murine NKT cells. Here, we show that human Hobit was uniformly expressed in effector-type CD8(+) T cells, but not in naive or in most memory CD8(+) T cells. Human CMV-specific but not influenza-specific CD8(+) T cells expressed high levels of Hobit. Consistent with the high homology between the DNA-binding Zinc Finger domains of Hobit and Blimp-1, Hobit displayed transcriptional activity at Blimp-1 target sites. Expression of Hobit strongly correlated with T-bet and IFN-γ expression within the CD8(+) T-cell population. Furthermore, Hobit was both necessary and sufficient for the production of IFN-γ. These data implicate Hobit as a novel transcriptional regulator in quiescent human effector-type CD8(+) T cells that regulates their immediate effector functions.

HIV Coinfection Enhances Complement Activation During Sepsis.

BACKGROUND: Human immunodeficiency virus (HIV)-induced complement activation may play a role in chronic immune activation in patients with HIV infection and influence the complement system during acute illness. We determined the impact of HIV infection on the complement system in patients with asymptomatic HIV infection and HIV-infected patients with sepsis or malaria. METHODS: We performed a prospective observational study of 268 subjects with or without HIV infection who were asymptomatic, were septic, or had malaria. We measured complement activation products (C3bc and C4bc) and native complement proteins (C3 and C4). levels of mannose-binding lectin and C1q-C4 were measured to examine activation of the lectin and classical pathways, respectively. RESULTS: Asymptomatic HIV infection was associated with increased C4 activation, especially in patients with high HIV loads, and was accompanied by elevated C1q-C4 levels. Similarly, sepsis and malaria resulted in increased C4 activation and elevated C1q-C4 concentrations. HIV coinfection enhanced C4 activation and consumption in patients with sepsis; this effect was not detected in patients with malaria. Mannose-binding lectin deficiency (defined as a mannose-binding lectin level of <500 ng/mL) did not influence complement activation in any group. CONCLUSIONS: HIV activates the complement system, predominantly via the classical pathway, and causes increased C4 activation and consumption during sepsis. HIV-induced complement activation may contribute to tissue injury during chronic infection and acute intercurrent bacterial infections.

Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies.

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 (Kd 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.

Activated protein C inhibits neutrophil migration in allergic asthma: a randomised trial.

Asthma patients show evidence of a procoagulant state in their airways, accompanied by an impaired function of the anticoagulant protein C system. We aimed to study the effect of recombinant human activated protein C (rhAPC) in allergic asthma patients.We conducted a randomised, double-blind, placebo-controlled, proof-of-concept study in house dust mite (HDM) allergic asthma patients. Patients were randomised to receive intravenous rhAPC (24 µg·kg(-1)·h(-1); n=12) or placebo (n=12) for 11 h. 4 h after the start of infusion, a first bronchoscopy was performed to challenge one lung segment with saline (control) and a contralateral segment with a combination of HDM extract and lipopolysaccharide (HDM+LPS), thereby mimicking environmental house dust exposure. A second bronchoscopy was conducted 8 h after intrabronchial challenge to obtain bronchoalveolar lavage fluid (BALF).rhAPC did not influence HDM+LPS induced procoagulant changes in the lung. In contrast, rhAPC reduced BALF leukocyte counts by 43% relative to placebo, caused by an inhibitory effect on neutrophil influx (64% reduction), while leaving eosinophil influx unaltered. rhAPC also reduced neutrophil degranulation products in the airways.Intravenous rhAPC attenuates HDM+LPS-induced neutrophil migration and protein release in allergic asthma patients by an effect that does not rely on coagulation inhibition.

Complement activation by salivary agglutinin is secretor status dependent.

After mucosal damage or gingival inflammation, complement proteins leak into the oral cavity and mix with salivary proteins such as salivary agglutinin (SAG/gp-340/DMBT1). This protein is encoded by the gene Deleted in Malignant Brain Tumors 1 (DMBT1), and it aggregates bacteria, viruses and fungi, and activates the lectin pathway of the complement system. In the lectin pathway, carbohydrate structures on pathogens or altered self cells are recognized. SAG is highly glycosylated, partly on the basis of the donor's blood group status. Whereas secretors express Lewis b, Lewis y, and antigens from the ABO-blood group system on SAG, non-secretors do not. Through mannose-binding lectin (MBL) binding and C4 deposition assays, we aimed to identify the chemical structures on SAG that are responsible for complement activation. The complement-activating properties of SAG were completely abolished by oxidation of its carbohydrate moiety. SAG-mediated activation of complement was also inhibited in the presence of saccharides such as fucose and Lewis b carbohydrates, and also after pretreatment with the fucose-binding lectin, Anguilla anguilla agglutinin. Complement activation was significantly (p<0.01) higher in secretors than in non-secretors. Our results suggest that fucose-rich oligosaccharide sidechains, such as Lewis b antigens, are involved in the activation of complement by SAG.

Complement inhibitors to treat IgM-mediated autoimmune hemolysis.

Complement activation in autoimmune hemolytic anemia may exacerbate extravascular hemolysis and may occasionally result in intravascular hemolysis. IgM autoantibodies as characteristically found in cold autoantibody autoimmune hemolytic anemia, in cold agglutinin disease but also in a considerable percentage of patients with warm autoantibodies are very likely to activate complement in vivo. Therapy of IgM-mediated autoimmune hemolytic anemia mainly aims to decrease autoantibody production. However, most of these treatments require time to become effective and will not stop immediate ongoing complement-mediated hemolysis nor prevent hemolysis of transfused red blood cells. Therefore pharmacological inhibition of the complement system might be a suitable approach to halt or at least attenuate ongoing hemolysis and improve the recovery of red blood cell transfusion in autoimmune hemolytic anemia. In recent years, several complement inhibitors have become available in the clinic, some of them with proven efficacy in autoimmune hemolytic anemia. In the present review, we give a short introduction on the pathogenesis of autoimmune hemolytic anemia, followed by an overview on the complement system with a special focus on its regulation. Finally, we will discuss complement inhibitors with regard to their potential efficacy to halt or attenuate hemolysis in complement-mediated autoimmune hemolytic anemia.

Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies.

In autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal hemolytic anemia. Depending on whether IgG or IgM antibodies are involved, response to therapy is different. Proper identification of the isotype of the anti-erythrocyte autoantibodies is, therefore, crucial. However, detection of IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of complement C3 on a patient's erythrocytes could be used for indirect detection of anti-erythrocyte IgM. To this end, we fractionated patients' sera by size exclusion chromatography and tested which fractions yielded complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an IgM anti-erythrocyte component that could activate complement, even if no such autoantibody had been detected with any other test. This also included all tested patients with only IgG and C3 on their erythrocytes, who would previously have been classified as having an IgG-only mediated autoimmune hemolytic anemia. Depleting patients' sera of either IgG or IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in autoimmune hemolytic anemia is mostly IgM-mediated and the presence of covalent C3 on patients' erythrocytes can be taken as a footprint of the presence of anti-erythrocyte IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy.

IgG antibodies in food allergy influence allergen-antibody complex formation and binding to B cells: a role for complement receptors.

Allergen-IgE complexes are more efficiently internalized and presented by B cells than allergens alone. It has been suggested that IgG Abs induced by immunotherapy inhibit these processes. Food-allergic patients have high allergen-specific IgG levels. However, the role of these Abs in complex formation and binding to B cells is unknown. To investigate this, we incubated sera of peanut- or cow's milk-allergic patients with their major allergens to form complexes and added them to EBV-transformed or peripheral blood B cells (PBBCs). Samples of birch pollen-allergic patients were used as control. Complex binding to B cells in presence or absence of blocking Abs to CD23, CD32, complement receptor 1 (CR1, CD35), and/or CR2 (CD21) was determined by flow cytometry. Furthermore, intact and IgG-depleted sera were compared. These experiments showed that allergen-Ab complexes formed in birch pollen, as well as food allergy, contained IgE, IgG1, and IgG4 Abs and bound to B cells. Binding of these complexes to EBV-transformed B cells was completely mediated by CD23, whereas binding to PBBCs was dependent on both CD23 and CR2. This reflected differential receptor expression. Upon IgG depletion, allergen-Ab complexes bound to PBBCs exclusively via CD23. These data indicated that IgG Abs are involved in complex formation. The presence of IgG in allergen-IgE complexes results in binding to B cells via CR2 in addition to CD23. The binding to both CR2 and CD23 may affect Ag processing and presentation, and (may) thereby influence the allergic response.

Long-term measurement of anti-adalimumab using pH-shift-anti-idiotype antigen binding test shows predictive value and transient antibody formation.

BACKGROUND AND OBJECTIVES: Therapeutic monoclonal antibodies are effective drugs for many different diseases. However, the formation of anti-drug antibodies (ADA) against a biological can result in reduced clinical response in some patients. Measurement of ADA in the presence of (high) drug levels is difficult due to drug interference in most assays, including the commonly used antigen binding test (ABT). METHODS: We recently published a novel method which enables the measurement of complexed antibodies against adalimumab (an anti-TNF antibody) in the presence of drug. Here we use this pH-shift-anti-idiotype ABT (PIA) to measure anti-adalimumab antibodies (AAA) in 99 rheumatoid arthritis (RA) patients treated for up to 3 years with adalimumab. RESULTS: 53 out of 99 RA patients produced AAA. In 50 of these PIA positive patients, AAA could be detected within the first 28 weeks of treatment. Patients in which AAA could be detected in the PIA after 28 weeks of treatment were more prone to declining adalimumab levels (<5 µg/ml) (p<0.01) and high AAA levels which could be detected in the ABT (p<0.05) at later time points. We observed transient AAA formation in 17/53 patients. CONCLUSIONS: Results show that AAA develop early in treatment. However, levels that completely neutralise the drug may be reached much later in treatment. Furthermore, the patients positive for PIA at 28 weeks have an increased chance to develop clinical non-response due to immunogenicity. In some of the patients, AAA formation is transient.

Adalimumab elicits a restricted anti-idiotypic antibody response in autoimmune patients resulting in functional neutralisation.

OBJECTIVES: Millions of patients worldwide are treated with therapeutic monoclonal antibodies. These biological therapeutics can be immunogenic, resulting in anti-drug antibody formation which leads to loss of response. Fully human biological agents, such as the anti-tumour necrosis factor α (anti-TNFα) antibody adalimumab, are considered to be weakly immunogenic, but anti-adalimumab antibodies (AAA) were recently detected in more than half of treated patients with rheumatoid arthritis (RA) within 28 weeks of treatment. A study was undertaken to determine the mechanism by which AAA lead to loss of response. METHODS: The specificity of the repertoire of AAA was investigated in a cohort of 50 AAA-positive RA patients. Inhibition experiments using TNFα and patient-derived anti-adalimumab monoclonal antibodies were performed. 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 serum from 50 AAA-positive patients. The anti-adalimumab response was confined to the TNFα binding region of adalimumab, thereby neutralising its therapeutic efficacy. In line with this restricted specificity, small immune complexes were found in the circulation of AAA-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 neutralisation of the drug, thereby providing a mechanism by which AAA formation leads to clinical non-response.