[Show abstract][Hide abstract] ABSTRACT: Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII-activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP-mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal anti-nuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma-purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen crosslinking is involved. In conclusion, FSAP-mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation. This article is protected by copyright. All rights reserved.
Full-text · Article · Dec 2015 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Background Tocilizumab (TCZ) inhibits interleukine-6 (IL-6) receptor resulting in inhibition of C-Reactive Protein (CRP) production, a surrogate marker for IL-6 receptor blockade.1 Evolving evidence showed that pharmacokinetics (PK) of TCZ is probably influenced by target-binding.2,3
Objectives To investigate PK of TCZ and the relationship between TCZ concentrations and disease activity in an observational cohort of rheumatoid arthritis (RA) patients.
Methods This prospective study consisted of 70 consecutive patients (The Netherlands, n=37; Spain, n=33) treated with TCZ 8 mg/ kg intravenously once per 4 weeks, monitored during 48 weeks. Disease activity was measured with CRP (mg/L) and Disease Activity Score of 28 joints (DAS28), using erythrocyte sedimentation rate (ESR)(mm/hr). TCZ serum trough concentrations and anti-drug antibodies were measured using an enzyme linked immunosorbent assay and an antigen binding test, respectively.
Results At baseline, mean DAS28 was 5.4±1.4 and median CRP, 6.9 (2.4-32.5). During 48 weeks, 19 patients discontinued TCZ prematurely due to: inefficacy (n=8), adverse events (n=8) or other reasons (n=3). TCZ concentrations above 1 mg/L were sufficient to normalize CRP production (<10 mg/L)(figure 1A), which was achieved by 69-88% of patients, depending on time point of measurement. Of the 30 patients with increased CRP (>10 mg/L) at baseline, levels were normalized in 17 patients within 4 weeks. In 11 patients normalization of CRP took longer or CRP level was still increased at drop-out. Increased CRP levels in these 11 patients was accompanied by TCZ concentration below 1 mg/L. CRP data of 2 patients was missing. The concentration effect curve at week 24 (last observation carried forward for patients in whom week 12 was the last available visit) showed that the majority was overtreated with TCZ standard dose (figure 1B). At week 24, median TCZ concentration was 11 mg/L (6-19) and mean ΔDAS28 was 2.4±1.6. Mean ΔDAS28 was lower in patients with TCZ concentrations below 1 mg/L vs above, respectively, -0.3±0.8 versus 2.8±1.4 (p<0.001) (independent sample t test). One patient had detectable anti-TCZ antibodies, in combination with low TCZ concentrations, at week 4.
Conclusions Increased CRP levels after baseline were accompanied by TCZ concentrations below 1 mg/L. This suggests that PK of TCZ is influenced substantially by target-binding and only marginally by immunogenicity, since, low TCZ concentration with anti-TCZ antibodies was found in only one patient. Due to the direct relationship between TCZ concentration and CRP inhibition, TCZ might be ideally suited for optimizing treatment via a personalised Therapeutic Drug Monitoring approach, aiming for serum concentrations within the optimal range for target blockade. This creates possibilities for dose reduction, since the majority of patients was overtreated with TCZ standard dose.
Disclosure of Interest E. Kneepkens Speakers bureau: payment for lectures from Pfizer, I. Van Den Oever Speakers bureau: payments for lectures from BMS, C. Plasencia Grant/research support from: Pfizer, paid to institution, Speakers bureau: payment for lectures from Pfizer, D. Pascual-Salcedo Speakers bureau: payment for lectures from Pfizer, D. van der Kleij: None declared, M. Hart: None declared, M. Nurmohamed Consultant for: received consultancy fees from Abbott, Roche, Pfizer, MSD, UCB, SOBI and BMS, Speakers bureau: payment for lectures from Abbott, Roche and Pfizer, A. Balsa Grant/research support from: Pfizer, paid to institution, Consultant for: consultancy fees from Abbvie, Pfizer and MSD, Speakers bureau: payment for lectures from Abbvie, Roche and Pfizer, L. Aarden: None declared, T. Rispens Speakers bureau: received payment for lectures from AbbVie and Pfizer, G. Wolbink Grant/research support from: Pfizer, paid to institution, Speakers bureau: payments for lectures from Pfizer, Amgen, AbbVie, UCB and BMS.
No preview · Article · Jun 2015 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: Background In a subset of patients, anti tumour necrosis factor (TNF) therapeutic antibodies are immunogenic, resulting in the formation of antidrug antibodies (ADAs). Neutralising ADAs compete with TNF for its binding site and reduces the effective serum concentration, causing clinical non-response. It is however unknown to which extent ADAs are neutralising.
Objectives To study which proportion of antibodies to human(ised) anti-TNF (adalimumab, golimumab, certolizumab) as well as chimeric anti-TNF (infliximab) is neutralising.
Methods Neutralising capacity of ADAs was assessed using a TNF competition assay in ADA-positive sera of patients treated with adalimumab (n=21), golimumab (n=4), certolizumab (n=9) or infliximab (n=34) sent in to our diagnostic department.
Results In 34 sera with ADAs to adalimumab, golimumab or certolizumab, >97% of the antibodies were neutralising. In 34 sera with ADAs to infliximab >90% of the antibodies were neutralising. Further characterisation of the broader antibody response to infliximab revealed that non-neutralising antibodies to infliximab do not target murine domains, but may bind infliximab-unique domains not involved in TNF binding (located outside the paratope).
Conclusions Our study shows that ADAs to human(ised) as well as chimeric anti-TNF therapeutic antibodies are largely neutralising. This highly restricted ADA response suggests an immunodominant role for the paratope of anti-TNF therapeutics.
No preview · Article · Oct 2014 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: The production of antibodies to adalimumab in auto-immune 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 sixteen 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 (CDR) was found, indicating an antigen-driven response. We recombinantly expressed eleven 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.
No preview · Article · Oct 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Clinical remission is today the treatment goal for Rheumatoid Arthritis (RA), which requires fast and assertive therapeutic decisions for a tight control of disease activity. Few objective parameters are available to guide clinical decisions, namely in switcher patients. We designed a preliminary algorithm introducing immunogenicity assessment in the current approach to RA patients receiving biotechnologic therapies.
To evaluate the concordance between the new algorithm and current clinical practice, comparing the effectiveness of "immunogenicity-based" versus "empirical-based" switches in a cohort of patients with established RA receiving biologics. Methods: EULAR therapeutic response was evaluated in 105 RA patients (naive or switchers) over one year, through GEE analysis. Serum drug trough levels were assessed by ELISA and anti-drug antibodies (ADAb) by Bridging ELISA. Results During follow-up, 48.6% of patients (Group A) had concordant therapeutic decisions. One year after the therapeutic decision, patients from Group A had a higher probability of achieving response (OR = 7.91, p <0.001, 95% CI = 3.27-19.13) and low disease activity (OR = 9.77, p <0.001, 95% CI = 4.69-20.37). Non-responders to a TNFi in the presence of detectable serum drug trough levels and no detectable ADAb had higher probability of achieving response by switching to a drug with different MOA, rather than another TNFi, even after adjusting for potential confounders, such as DAS28 at the time of switch (OR = 6.76, p = 0.004, 95% CI = 1.82-25.04).
Immunogenicity assessment might help to optimise therapeutic decisions, leading to a better control of disease activity with significant better clinical outcomes in RA patients receiving biotechnologic therapies.
No preview · Article · Mar 2014 · Annals of the rheumatic diseases
[Show abstract][Hide abstract] ABSTRACT: Objective:
Removal of dead cells is essential in the maintenance of tissue homeostasis, and efficient removal prevents exposure of intracellular content to the immune system, which could lead to autoimmunity. The plasma protease factor VII-activating protease (FSAP) can release nucleosomes from late apoptotic cells. FSAP circulates as an inactive single-chain protein, which is activated upon contact with either apoptotic cells or necrotic cells. The purpose of this study was to investigate the role of FSAP in the release of nucleosomes from necrotic cells.
Necrotic Jurkat cells were incubated with serum, purified 2-chain FSAP, and/or DNase I. Nucleosome release was analyzed by flow cytometry, and agarose gel electrophoresis was performed to detect DNA breakdown.
Incubation with serum released nucleosomes from necrotic cells. Incubation with FSAP-deficient serum or serum in which FSAP was inhibited by a blocking antibody was unable to release nucleosomes from necrotic cells, confirming that FSAP is indeed the essential serum factor in this process. Together with serum DNase I, FSAP induced the release of DNA from the cells, the appearance of nucleosomes in the supernatant, and the fragmentation of chromatin into eventually mononucleosomes.
FSAP and DNase I are the essential serum factors that cooperate in necrotic cell DNA degradation and nucleosome release. We propose that this mechanism may be important in the removal of potential autoantigens.
Preview · Article · Mar 2014 · Arthritis & Rheumatology
[Show abstract][Hide abstract] ABSTRACT: Background A substantial part of the rheumatoid arthritis (RA) patients is chronically treated with adalimumab. Some of these patients produce antibodies against adalimumab, which correlate with lower serum drug levels and reduced clinical response. Long term exposure to antigens may result in antigen specific IgG4 production as demonstrated in previous studies on prolonged exposure to antigens such as bee venom, factor VIII and IFN-β.
Objectives Here we investigate whether long term treatment of RA patients with the therapeutic monoclonal antibody adalimumab leads to the production of specific IgG4 antibodies.
Methods We developed radio immunoassays to detect total IgG or IgG4 against adalimumab and applied these in a cohort of 272 RA patients during three years of adalimumab treatment.
Results Of the 272 patients 32% developed antibodies against adalimumab. We observed that a fast majority of the patients also produce IgG4 antibodies against adalimumab (29%). The proportion IgG4 of total IgG against adalimumab varies widely between patients (median; 25-75 percentile 38; 21-67%). In some patients the antibody response even seems to be dominated by IgG4.
Conclusions The role of IgG4 in the immune response against adalimumab is variable in adalimumab treated RA patients. Although IgG4 is often considered to be harmless, because of its lack of effector function, these patients show low adalimumab levels and reduced clinical response.
Disclosure of Interest None Declared
No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: Background In the last years, the “treat to target” strategy revealed the importance of tight control of disease activity in RA patients1. To reach clinical remission or at least low disease activity as soon as possible, this strategy, now part of the EULAR guidelines, requires therapeutic combinations and/or fast switches between different therapies2. Over the last years it became clear that drug immunogenicity is one of the main mechanism behind biologic therapeutic failure3-4. How to integrate the notion of drug immunogenicity in clinical practice remains to be formally established. We conducted a systematic review of the Literature with a meta-analysis evaluating the clinical implications of drug immunogenicity5. Based on the information retrieved we designed a new algorithm which introduces immunogenicity assessment in the EULAR guidelines for the management of RA patients receiving biologic therapy.
Objectives We assessed the influence of adherence to our proposed algorithm on therapeutic responses and low disease activity rates in a prospective cohort of RA patients treated with biologics.
Methods We conducted a prospective cohort study over 2-years evolving 106 consecutive RA patients, 92% female, with a mean age of 55±13 yrs and mean disease duration of 6±4 yrs. Patients had been submitted to biologic therapy by a mean period of 4±3 years. Serum drug trough levels were assessed by ELISA and anti-drug antibodies (ADA) by an optimized Bridging ELISA, which we previously compared with a fluid-phase RIA. Clinicians were blind for the tests results. Therapeutic response and low disease activity were defined according to the EULAR guidelines.
Results At the study onset 22 patients were receiving Infliximab (36.4% anti-infliximab pos), 33 Adalimumab (27.3% anti-adalimumab pos) and 49 Etanercept (0% anti-etanercept pos). Patients with detectable ADAs had undetectable serum drug trough levels and lower therapeutic responses rates (37.5% vs 76.9%, p=0.001 for Infliximab and 33.3 vs 66.7%, p=0.02 for Adalimumab). Not a single patient with detectable ADAs had low disease activity. During follow-up period, therapeutic decision coincident with our proposed algorithm concerned 48.6% of the patients (Group 1), however with a décalage median (IQR) time of 249 days (116-388). Therapeutic decisions were discordant with our algorithm for 51.4% of the patients (Group 2). A significant higher proportion of patients in Group 1, when compared to Group 2, reached therapeutic response at 3 months (97.9% vs 52.2%, p=0.001) and at 12 months (71.4% vs 13% p=p=0.000). Moreover, a significant higher proportion of patients in Group 1 achieved low disease activity at 3 months (71.4% vs 13%, p=0.000) and at 12 months (58.8% vs 3.8%, p=0.000). Considering drug direct costs only, we evaluated that about 1 million euros were spent inefficiently in this cohort over 2 years.
Conclusions Our study strongly suggests that including immunogenicity assessment in EULAR guidelines will allow the design of more cost-effective strategies, tailored to each RA patient receiving biologic therapy.
Disclosure of Interest None Declared
No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: Background Adalimumab is a recombinant human IgG1 monoclonal antibody with high specific binding to human tumor necrosis factor a (TNF). Since 2002 the drug has been approved for the treatment of rheumatoid arthritis (RA) and while several tests have been developed for testing drug levels and immunogenicity, the correlation between serum trough concentrations of this drug and therapeutic effect in clinical practice has not been studied yet. We propose that monitoring drug levels could make this treatment more cost-effective, specially considering the high costs of biologicals.
Objectives To investigate the correlation between clinical improvement and serum trough concentrations of adalimumab in RA patients, 6 months after start of treatment.
Methods Prospective cohort consisting of 201 patients treated with 40mg adalimumab every two weeks. Six months after start of treatment serum trough adalimumab levels were measured by ELISA. Clinical improvement was defined as the difference in Disease Activity Score 28 (DAS28) of the patients before start of the treatment, and after six months. Randomizing data as groups of ten patients gives a median trough and median Delta DAS28 (DDAS28). These findings were studied in a concentration-effect curve.
Results After 6 months of treatment, serum trough concentrations varied between 0 to 33 ug/mL. Patients without detectable adalimumab did not respond clinically. All of these patients had developed anti-adalimumab antibodies. In patients with serum levels ranging from 3 to 12 ug/mL clinical response seemed to be related to serum adalimumab levels. Higher levels of adalimumab did not contribute further to clinical improvement.
Conclusions This study indicates that serum trough levels of adalimumab fluctuate widely. To achieve clinical effect in RA, serum levels of 3 to 12 ug/mL could be sufficient. Hence, this suggests that presently a significant amount of patients is overtreated. Obviously, monitoring of drug levels is needed leading to personalized medication schemes allowing a more rational usage of this drug.
Disclosure of Interest None Declared
No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: Background Over the last years, an increasing body of evidence has highlighted the clinical significance of drug immunogenicity1. Anti-drug antibodies (ADA), by forming immune complexes with the drug, promote its faster clearance from circulation, reducing therapeutic effectiveness2. However, ADA have also been associated with increased incidence of drug-related adverse events (AE), particularly infusion-related AE during infliximab therapy, despite severe thromboembolic phenomena associated with antibodies against adalimumab have also been reported in the literature3,4,5.
Objectives We assessed the association between ADA and infusion-related AE in patients with Rheumatoid Arthritis (RA), Ankylosing Spondylitis (AS), Psoriatic Arthritis (PsA) and Inflammatory Bowel Disease (IBD), receiving infliximab therapy.
Methods We conducted a prospective cohort study over 2-years evolving 84 consecutive patients (22 AR, 33 AS, 9 PsA and 30 IBD) receiving infliximab at 3-5 mg/Kg every 6 or 8 wks, at day care unit of Hospital Garcia de Orta, Almada. Therapeutic response and low disease activity were defined according to the EULAR guidelines (RA and RA-like PsA), ASAS group guidelines (AS and AS-like PsA) and by an expert clinician (IBD patients). ADA were detected by an optimized Bridging ELISA, just prior to next infliximab infusion. Clinicians were blind for the tests results.
Results Seventy-six percent of patients were female, with mean (SD) age of 48 (10.2) years, disease duration of 8 (6.4) years, receiving biologic therapy by 2.9 (2.0) years. All RA patients and 89% of PsA were receiving concomitant MTX. IBD patients were receiving concomitant azathioprine and also hydrocortisone plus anti-histaminic prior to each infliximab infusion. During the follow-up period, a total of 25 patients (30%) had detectable ADA (41% of RA, 33% of PsA, 18% of AS and 23% of IBD patients). Of those, 44% developed an infusion-related AE (4 RA patients, 2 PsA, 2 AS and 4 IBD patients). In all of those cases, ADA detection occurred prior to the AE. All patients with RA, PsA and AS who developed infusion-related AE were unable to maintain therapeutic response over time, while 2 out of 4 patients with IBD were still considered responders during the follow-up period. All the reactions were mild-moderate requiring hydrocortisone and anti-histaminic administration.
Conclusions A significant porpotion of ADA-positives patients develop an infusion-related AE and cannot sustain therapeutic response. The maintenance of therapy in such cases may have serious deleterious effects with no additional therapeutic benefits. Further robust studies are warranted to better evaluate safety aspects related with immunogenicity.
Disclosure of Interest None Declared
No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: To determine a concentration-effect curve of adalimumab in rheumatoid arthritis (RA) patients taking into account the effect of methotrexate (MTX) on concentration and effect and to identify a therapeutic range for adalimumab concentrations.
In a prospective observational cohort study, 221 consecutive patients with RA were treated with 40 mg adalimumab subcutaneously every other week. The relationship between adalimumab trough level and clinical efficacy after 28 weeks of follow-up was determined in a concentration-effect curve. A receiver-operator characteristics (ROC) curve established a therapeutic cut-off concentration. The effect of MTX on adalimumab trough levels was shown by dividing patients that are and are not concomitantly using MTX in the concentration-effect curve and a concentration table.
Clinical efficacy improved with increasing adalimumab concentration and reached a maximum (mean disease activity score in 28 joints improvement of 2) with levels between 5-8 μg/mL. Levels exceeding 8 μg/mL were illustrated to have no additional beneficial effect on disease activity. The ROC curve showed an area under the curve of 0.695 (95% CI 0.626 to 0.764) for European League Against Rheumatism response and adalimumab levels: good responders versus non-responders and moderate responders. A cut-off of 5 μg/mL had a sensitivity of 91% and a specificity of 43%. Adalimumab levels are influenced by concomitant MTX use: patients on adalimumab monotherapy had a median adalimumab level of 4.1 μg/mL (IQR 1.3-7.7), whereas patients concomitantly taking MTX had a median level of 7.4 μg/mL (IQR 5.3-10.6, p<0.001).
Adalimumab trough levels in a range of 5-8 μg/mL are sufficient to reach adequate clinical response. These levels are influenced substantially by concomitant MTX use.
Full-text · Article · Dec 2013 · Annals of the rheumatic diseases
[Show abstract][Hide abstract] ABSTRACT: Abstract Immunogenicity is a major issue of concern for monoclonal antibodies used in human diseases and is by default mainly determined in non-human primates (NHP), as target molecules are considered most similar in NHP compared to human. In this manuscript the predictive value of immunogenicity testing in minipigs for human safety is evaluated, as the immune system of the pig is functionally similar to that in other mammalian species. Adalimumab and infliximab (both monoclonal antibodies blocking TNFα) were used as model substances. Female Göttingen minipigs (4/group) were treated every other week with low (0.1 mg/kg), mid (1.0 mg/kg), or high dose (5 mg/kg) adalimumab or 5 mg/kg infliximab subcutaneous (SC) over a period of 8 weeks. After first and last dosing, pharmacokinetic analysis was performed. Anti-drug antibodies (ADAs) were measured on several time points. Furthermore, hematology, clinical chemistry, body weight, clinical signs, and histopathology of several organs were evaluated. No signs of toxicity of the treatments were observed in the limited organs and tissues collected. Eleven out of 12 minipigs treated with adalimumab elicited a detectable ADA response. Induction of ADA was correlated with decreased plasma levels of adalimumab. Infliximab clearance was comparable after first and last dose. Therefore, the presence of ADA directed to infliximab was considered highly unlikely. It was concluded that the minipig and NHP showed comparable suitability for immunogenicity prediction in humans. More studies with other biopharmaceutical products are needed to strengthen the status of the minipig as an alternative model for immunotoxicity testing including immunogenicity.
Full-text · Article · Jun 2013 · Journal of Immunotoxicology
[Show abstract][Hide abstract] ABSTRACT: Introduction Clinical remission is today the treatment goal for rheumatoid arthritis (RA), which requires fast and assertive therapeutic decisions for a tight control of disease activity. Few objective parameters are available to guide clinical decisions, particularly in switcher patients. We designed a preliminary algorithm introducing immunogenicity assessment in the current approach to patients with RA receiving tumour necrosis factor inhibitors (TNFi).
Objective To evaluate the concordance between the new algorithm and current clinical practice, comparing the effectiveness of ‘immunogenicity-based’ versus ‘empirical-based’ switches in a cohort of patients with established RA receiving biologics.
Methods EULAR therapeutic response was evaluated in 105 patients with RA (naive or switchers) over one year, through generalised estimation equation (GEE) analyses. Serum drug trough levels were assessed by ELISA and antidrug antibodies (ADAb) by Bridging ELISA.
Results During follow-up, 48.6% of patients had therapeutic decisions concordant with the proposed algorithm (Group A), and 51.4% had discordant decisions (Group B). One year after the therapeutic decision, patients from Group A had a higher probability of achieving response (OR=7.91, p<0.001, 95% CI 3.27 to 19.13) and low disease activity (OR=9.77, p<0.001, 95% CI 4.69 to 20.37) than patients in Group B.
Conclusions Immunogenicity assessment might help to optimise therapeutic decisions, leading to a better control of disease activity with significantly better clinical outcomes in patients with RA receiving TNFi.
No preview · Article · May 2013 · Annals of the rheumatic diseases
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jan 2013 · Annals of the rheumatic diseases
[Show abstract][Hide abstract] ABSTRACT: 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
No preview · Article · Jul 2012 · Annals of the rheumatic diseases
[Show abstract][Hide abstract] ABSTRACT: Background The clinical and scientific relevance of monitoring immunogenicity in clinical practice have been recognized and recommended by the European Agency of Medicine (EMA)1. However, its assessment is technically challenging and no consensus exists about the way immunogenicity can be monitored. Newly developed fluid-phase radio-immuno assays (RIA) have proven quite effective in detecting anti-drug antibodies and as been presented as a “gold standard” method to assess immunogenicity2,3. However, RIA requires high doses of radioactivity and special conditions, preventing its use as a routine assay. By contrast, enzyme-linked immunoassay (ELISA) it’s a simple and cheap method, ideal candidate to be used as a high-throughput screening assay. Bridging ELISA is a particular type of ELISA with increased specificity over the conventional ELISAs methods. Several optimizations of this method were made so that it can become a good screeningassay to monitor drug immunogenicity in routine clinical practice.
Objectives We tested, in a same cohort of patients receiving infliximab, this newly optimized bridging ELISA in comparison with a fluid-phase antigen-binding RIA to quantify anti-infliximab antibodies.
Methods A total of 82 consecutive patients were evaluated (38 rheumatoid arthritis patients, 27 ankylosing spondylitis, 9 psoriatic arthritis and 18 patients with inflammatory bowel disease), 61 females, with a mean age of 41 (4.2) years, that were receiving Infliximab in a dosage of 3-5mg/kg every 6 or 8 weeks for a mean period of 3.5 (2.0) years. Blood samples were collected immediately before the next infliximab infusion. Anti-infliximab antibodies were quantified by 1) Bridging ELISA where the antibodies bind to the infliximab coated in a solid phase and revealed by the addition of biotinylated infliximab and by 2) fluid-phase RIA-ABA that uses a sepharose-immobilized protein A, IgG total and IgG4-specific, to bind IgGs in the patient’s serum. Anti-infliximab specific IgGs are revealed by theaddition of 125I-labeled infliximab F(ab’)2. A simple ELISA method was used to quantify serum infliximab levels,based on the principle that infliximab are captured through their ability to bind immobilized TNFα on a solid phase. The binding assessment is revealed by incubation with biotinylated rabbit IgG directed to infliximab idiotype. Therapeutic response was assessed according to validated criteria established for each disease.
Results A total of 22 (27%) were tested positive for the presence of anti-infliximab abs using RIA, coinciding with the samples that were also positives in Bridging ELISA. Bridging ELISA cannot detect monovalent IgG4. No samples testing exclusively IgG4 specific anti-infliximab were detected. All patients (100%) with detectable anti-infliximab antibodies had undetectable serum trough drug levels and were not able to sustain the therapeutic response.
Conclusions By its simplicity, cost and robustness, Bridging ELISA is a suitable test to be implemented in routine clinical practice as a screening assay to monitor drug immunogenicity.
Disclosure of Interest None Declared
No preview · Article · Jun 2012 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: A substantial part of rheumatoid arthritis (RA) patients is chronically treated with adalimumab. Some of these patients produce antibodies against adalimumab, which correlate with lower serum drug levels and reduced clinical response. Long term exposure to antigens may result in antigen specific IgG4 production as was demonstrated in studies on prolonged exposure to antigens such as different allergens, Factor VIII and IFN-β. Here, we investigate whether long term treatment of RA patients with the therapeutic monoclonal antibody adalimumab leads to the production of specific IgG4 antibodies.
We developed radio immunoassays to detect total IgG or IgG4 against adalimumab and applied these in a cohort of 271 consecutive RA patients during 3 years of adalimumab treatment.
In 32 % of the 271 patients antibodies against adalimumab were detectable. IgG4 antibodies were detected in 29 % of the patients. The proportion IgG4 of total IgG against adalimumab varies widely between patients, and IgG4 was found to contribute significantly to the anti drug antibody (ADA) response in some patients.
In the immune response against adalimumab in adalimumab-treated RA patients a considerable part of the ADA is IgG4. Although IgG4 is often considered to be harmless due to its lack of effector function, neutralization of adalimumab by IgG4 antibodies will lead to a reduced clinical response.
No preview · Article · May 2012 · Journal of Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: Factor VII-activating protease (FSAP) is a serine protease that circulates in plasma in its inactive single-chain form and can be activated upon contact with dead cells. When activated by apoptotic cells, FSAP leads to the release of nucleosomes. The serpins C1-inhibitor and α(2) -antiplasmin are reported to be the major inhibitors of FSAP. However, regulation of FSAP activity by Kunitz-type inhibitors is not well studied.
To compare the inhibition of FSAP activity and FSAP-induced nucleosome release from apoptotic cells by tissue factor pathway inhibitor (TFPI) with that of C1-inhibitor and α(2) -antiplasmin.
Apoptotic cells were incubated with plasma or FSAP in presence of the inhibitor, and nucleosome release was analyzed with flow cytometry. Monoclonal antibodies against TFPI and altered forms of TFPI were used to investigate which domains of TFPI contribute to FSAP inhibition.
We show that TFPI abrogates FSAP activity and nucleosome release from apoptotic cells. TFPI is a much more efficient inhibitor than C1-inhibitor or α(2) -antiplasmin. The active site of K2 is required for inhibition of FSAP. A direct binding interaction between FSAP and the C-terminal domain of TFPI is also required for efficient inhibition. Inhibition of FSAP-induced nucleosome release by recombinant TFPI might, in part, explain the anti-inflammatory effects of recombinant TFPI infusion observed in animal and human sepsis.
Preview · Article · Mar 2012 · Journal of Thrombosis and Haemostasis
[Show abstract][Hide abstract] ABSTRACT: Rheumatoid factors are antibodies directed against IgG that may confound immunogenicity testing for therapeutic monoclonal antibodies. We developed antigen-binding assays to monitor anti-drug-antibody (ADA) responses against infliximab and adalimumab using F(ab')2 fragments of the drug. This avoids possible detection of rheumatoid factor activity. During development of these assays, a number of sera from patients before treatment as well as several healthy control sera were tested positive. None of these sera contained antibodies specific for the therapeutic mAb. Instead, they were found to contain anti-hinge antibodies. We demonstrate that this aspecific antibody binding can be inhibited by adding F(ab')2 of intravenous immunoglobulin (IVIG), which consists of pooled polyclonal IgG derived from plasma. Using this protocol, anti-infliximab antibodies can be measured specifically without interference by anti-hinge antibodies.
No preview · Article · Jan 2012 · Journal of immunological methods