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Objective. To evaluate the prognostic value of serum levels of infliximab (IFX) and anti-IFX antibodies for predicting treatment efficacy in children with inflammatory bowel disease (IBD). Patients and methods. This study included 123 children with IBD (65 patients with ulcerative colitis (UC) and 58 patients with Crohn's disease (CD)) aged between 3 and 18 years. Children were examined upon the completion of an induction course of therapy and then after 1 year of therapy and between 1 and 3 years of treatment. The induction course was administered according to the scheme of 0–2–6 weeks; maintenance therapy was administered with 6-week and 8-week intervals. The residual level of IFX and the level of anti-IFX antibodies in serum were measured using enzyme-linked immunosorbent assay. Results. We observed a significant increase in the IFX level during clinical remission of CD (remission – 5.21 [3.32; 7.43] μg/mL; exacerbation – 2.42 [0.42; 4.51] μg/mL, p = 0.001) and UC (remission – 4.57 [3.4; 6.74] μg/mL; exacerbation – 0.63 [0.4; 3.27] μg/mL, p = 0.000). ROC analysis demonstrated high accuracy of our model based on measuring residual IFX level for distinguishing between exacerbation and remission in both CD patients (AUC = 0.812) and UC patients (AUC = 0.851). The optimal minimum level of IFX for maintaining clinical remission was 3.7 μg/mL in children with CD and 3.4 μg/mL in children with UC. Anti-IFX antibodies were detected in 18% of patients; children with exacerbation were 4.7 times more likely to have detectable level of anti-IFX antibodies than those in clinical remission. Conclusion. Monitoring of serum levels of IFX and anti-IFX antibodies is a promising method for predicting treatment efficacy and its optimization in children with IBD. Key words: inflammatory bowel disease, tumor necrosis factor-α, infliximab, residual level of infliximab, anti-infliximab antibodies

Measurements of serum concentrations of therapeutic antibodies and anti-drug antibodies (ADA) can support clinical decisions for the management of non-responders, optimizing the therapy. In the present study we compared the results obtained by classical ELISA and a recently proposed surface plasmon resonance (SPR)-based immunoassay, in 76 patients receiving infliximab for inflammatory bowel diseases. The two methods indicated very similar serum concentrations of the drug, but there were striking differences as regards ADA. All the sera showing ADA by ELISA (14) also showed ADA by SPR, but the absolute amounts were different, being 7–490 times higher with SPR, with no correlation. Eight patients showed ADA only with SPR, and these ADA had significantly faster dissociation rate constants than those detectable by both SPR and ELISA. The underestimation, or the lack of detection, of ADA by ELISA is likely to reflect the long incubation steps which favor dissociation of the patient’s low-affinity ADA, while the commercial, high-affinity anti-infliximab antibodies used for the calibration curve do not dissociate. This problem is less important with SPR, which monitors binding in real time. The possibility offered by SPR to detect ADA in patients otherwise considered ADA-negative by ELISA could have important implications for clinicians.

Background Adalimumab (ADL), infliximab (IFX) and their biosimilars are widely used biological drugs. Some patients, however, generate neutralizing antibodies that hamper the effectiveness of these drugs. Evidence shows therapeutic drug monitoring of serum levels ADL/IFX and anti-drug antibodies (ADA) is useful to improve treatment effectiveness. We evaluated a new rapid quantitative method, Quantum Blue (QB), for determining serum anti-ADL and anti-IFX antibodies (Research Use Only labelling) by comparing it with two established ELISA kits, Promonitor (PM) and Lisa-Tracker (LT). Methods Eighty samples (40 for each drug type) were analysed. Percentage of agreement and kappa statistic were used to compare positive/negative ADA results. Clinical implications for drug treatment in the patients with discordant results were evaluated. The Chi-square test was used to analyze differences for ADA detection in patients with disease flare and without concomitant immunosuppressant treatment. Results Agreement exceeded 80 % among anti-ADL methods. Although LT ELISA showed a lower capacity in detecting anti-ADL antibodies, discrepancies were found for levels close to the cut-off concentration, thus having minimal impact on clinical decisions. Conversely, QB anti-IFX displayed low agreement with PM and LT ELISA kits (67.5 % and 50 %, respectively), and was unable to detect high levels of antibodies, therefore having major clinical implications. Agreement between PM and LT ELISA anti-IFX kits was 82.5 % with all discordant results being undetected for PM and slightly positive for LT. Conclusion QB anti-ADL shows similar performance to ELISA kits while QB anti-IFX needs further improvements to achieve reliable antibody detection.

Many clinical studies in paediatric inflammatory bowel diseases (IBD) use infliximab trough level (IFX-TL) and detection of antibody against infliximab (ATI). Hence, comparison of commercially available assays is needed in paediatric samples to assess their reliability and their comparability. We measured IFX-TL and ATI-TL in sera samples of 53 IBD children using three ELISA kits: Lisa-Tracker® Duo Infliximab (Theradiag®), Ridascreen® IFX monitoring (R-Biopharm®) and Promonitor® IFX (Grifols®). Regarding IFX-TL, median values were comparable (p > 0.05), a good statistical correlation has been observed (0.73 ≤ R2 ≤ 0.85) between tested assays and the Bland-Altman analysis found an excellent agreement with a bias estimated between −0.56 and 0.12 and few values outside the 95% limits of agreement. However, qualitative comparison with therapeutic interval classifications showed some discrepancies (30.2%), mainly due to values near thresholds and more often than not with Theradiag® (22.6%). For ATI, because of non-standardized units, the qualitative comparison found a sensibly good agreement (98.1%). These data show a good agreement of IFX-TL and ATI measurement between three marketed ELISA assays with a small bias obtained. Variations in some results can lead to divergent therapeutic interval classifications and prompt us to be cautious in the interpretation of values near therapeutic thresholds.

Background and aims: Therapeutic drug monitoring (TDM) of infliximab (IFX) and anti-infliximab antibodies (ATIs) is essential for treatment optimisation in inflammatory bowel disease (IBD) patients. The aim of this study was to estimate and compare the agreement and accuracy between a new rapid test and three established enzyme-linked immunosorbent assays (ELISAs) to quantify ATIs levels, and to evaluate the impact of exogenous IFX on the performance of these assays. Methods: We analysed 200 serum samples from 57 IBD outpatients in IFX induction or maintenance therapy at six IBD centres in Portugal. ATI levels were quantified using the rapid test Quantum Blue® (QB) Anti-Infliximab (Bühlmann) and three established ELISAs: In-House, Theradiag (Lisa Tracker Anti-Infliximab), and Immundiagnostik (IDKmonitor Infliximab). ATIs were quantified in patients' serum samples and spiked samples with exogenous IFX, based on analytical and clinical cutoffs. Qualitative agreement and accuracy were estimated by Cohen's kappa (k) with 95% confidence intervals. Results: ATIs quantification with clinical cutoffs showed a slight agreement between QB rapid test and In-House [k = 0.163 (0.051-0.276)] and Immundiagnostik [k = 0.085 (0.000-0.177)]. Regarding IFX/ATIs status, the QB rapid test showed a substantial agreement with Theradiag [k = 0.808 (0.729-0.888)] and a fair agreement with In-House [k = 0.343 (0.254-0.431)] and Immundiagnostik [k = 0.217 (0.138-0.297)]. The QB rapid test could not detect ATI-positive levels in samples with exogenous IFX at 5-300 µg/ml. Interference on ATIs detection was observed at exogenous IFX ⩾30 µg/ml for In-house and Immundiagnostik assays. Conclusion: QB rapid test is only suitable to detect ATI-positive levels in the absence of IFX. The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded in part by the Portuguese IBD Study Group (GEDII), and in part by PDE/BDE/114583/2016, from Portuguese national funds by Fundação para a Ciência e Tecnologia (FCT). info:eu-repo/semantics/publishedVersion

Background: The commercialization of CT-P13, an infliximab (IFX) biosimilar, has the potential to decrease health-related costs and enhance access to biological therapies. This study aimed to address the accuracy and inter-assay agreement of the CT-P13 quantification using four different assays initially developed to assess IFX. Methods: The four different methods, one in-house method and three commercially available kits, were used to quantify exogenously-spiked samples and the sera from 185 inflammatory bowel disease (IBD) patients on CT-P13 therapy. Results: The quantification of the spiked samples unveiled a consistent and accurate behaviour of three of the tested methods, with average percentage recoveries of 90%, 102% and 109%. Results from the clinical samples demonstrated that these three assays were also highly correlated, both concerning Spearman’s rank coefficients (range 0.890–0.947) and intraclass correlation coefficients (range 0.907–0.935). There were a few systematic deviations among them, but their impact in the clinical stratification of the patients using different cut-offs was minimal, particularly when these cut-offs were in the 3–4 µg/ml range, for which the strength of agreement (as assessed by the Kappa statistics that ranged from 0.732 to 0.902) was substantial to almost perfect. Conclusions: Our results indicate that three of the tested IFX quantification methods can be used to accurately quantify CT-P13 without any adjustments.

Three commercially available assays for the measurement of antibodies to infliximab (ATI) are approved for clinical use in Australia: Promonitor anti-infliximab (Grifols), Lisa Tracker anti-infliximab (Theradiag) and Ridascreen anti-IFX (R-Biopharm). All are bridging ELISA assays. Measurement of ATI has been incorporated into treatment algorithms for assessing loss of response to infliximab in patients with inflammatory bowel disease, but results obtained by the three ATI assays have not been systematically compared. We performed a series of experiments to allow comparison of results between the assays. Forty-two patient samples known to be positive for ATI by the Lisa Tracker assay were run on the Promonitor assay in singlicate, of which 26 were run on the Ridascreen assay in duplicate, according to the manufacturers' instructions. The Spearman correlation coefficient for all three pairwise assay comparisons was 0.95. Results were not numerically comparable between the assays. The coefficient of variation (CV) was 2.3% for the Lisa Tracker assay, 7.6% for the Promonitor assay and 7.4% for the Ridascreen assay. The presence of infliximab interfered with all three assays in a dose dependent manner. The cut-point for loss of response to infliximab dose intensification, previously demonstrated to be 200 ng/mL on the Lisa Tracker assay, is equivalent to approximately 60 ng/mL on the Ridascreen assay and between 22.9 and 41 AU/mL on the Promonitor assay. All three assays are suitable for clinical use.

Background Anti-drug-antibodies (ADA) against infliximab are frequently measured in patients receiving infliximab treatment with loss of response and undetectable infliximab concentrations. Different ADA bridging assays (1st generation, 2nd generation and ready-to-use kit) have been developed successively and were applied over the last 10 years, making comparison between ADA concentrations very challenging. A cutoff of 8 μg/ml was established to discriminate low from high ADA concentrations using the 1st generation ADA bridging assay. The objective of this study was to enable comparison of ADA concentrations determined with the different assays that were developed over the years. Methods 166 serum samples were collected from patients with inflammatory bowel disease treated with infliximab. 98 samples were measured simultaneously with the 1st and 2nd generation ADA assay, 67 serum samples were measured with the 2nd generation assay and the ready-to-use kit. Results From our ADA concentration comparison experiments, we deduced that the previously established cutoff of 8 μg/ml with the 1st generation ELISA has a similar impact as the cutoff of 374 ng/ml with the 2nd generation ELISA and a cutoff of 119 ng/ml in the ready-to-use ELISA kit. Conclusion ADA concentrations measured with the different assays were compared and a cutoff concentration was determined for each of them to distinguish between low and high ADA concentrations. These cutoff concentrations may serve as a tool for clinicians to make treatment decisions for patients with a loss of response to infliximab and undetectable infliximab serum concentrations.

OBJECTIVES: The aim of this study was to evaluate the quantitative serum level of infliximab (IFX) as well as the detection of anti-infliximab antibodies (ATIs) in patients with Crohn’s disease (CD). METHOD: Forty patients with CD under treatment at a tertiary center in southeastern Brazil were evaluated. Their use of infliximab was continuous and regular. We analyzed and compared the differences in the IFX and ATI levels between the patients with active CD (CDA) and those with CD in remission (CDR). RESULTS: There was no difference in the IFX level between the CDA and CDR groups (p>0.05). Eighty percent of all patients had IFX levels above the therapeutic concentration (6-10 μg/mL). Two (9%) of the 22 patients with active disease and four (22.2%) of the 18 patients in remission had undetectable levels of IFX. Four (66.6%) of the six patients with undetectable levels of IFX had positive ATI levels; three of these patients were in remission, and one had active disease. In addition, the other two patients with undetectable levels of IFX presented ATI levels close to positivity (2.7 and 2.8 AU/ml). None of the patients with therapeutic or supratherapeutic IFX levels had positive ATI levels. CONCLUSIONS: The undetectable levels of IFX correlated with the detection of ATIs, which was independent of disease activity. Immunogenicity was not the main factor for the loss of response to IFX in our study, and the majority of patients in both groups (CDA and CDR) had supratherapeutic levels of IFX.

Background:Infliximab (Ifx) has proven effective in treating rheumatoid arthritis (RA) and spondyloarthropathies (SpA), although around 40% of cases fails, mainly due to immunogenicity. Formation of immunocomplexes between antibodies to Ifx (ATI) and Ifx can increase drug clearance, leading to treatment failure. Standard ELISA assays which are drug -sensitive are frequently used, being able to detect only free ATI. Interest in drug-tolerant assays to measure total ATI (free and complexed) is increasing.Objectives:To compare the development of ATI using both drug-tolerant and drug-sensitive assays at early stages of Ifx therapy. To analyse the relationship of ATI detected by both assays with the drop-out of treatment.Methods:This is a prospective observational study including 45 patients with RA and 61 with axial-SpA treated with standard doses of Ifx (3mg/kg and 5mg/kg, respectively) enrolled at Biological Therapy Unit of Hospital La Paz. Serum samples were obtained at 2, 6, 12 and 22 weeks (W) after Ifx initiation. The data about discontinuation for inefficacy was obtained from the database. ATI presence was evaluated by a drug-sensitive in-house two-site (bridging) ELISA (bELISA) and a drug-tolerant commercial ELISA assay (Immundiagnostik®,IDK). All comparisons were performed throughout non-parametrical test. In SpA group, due to the low number of ATI+ patients at W12 by bELISA the statistical analysis to compare both assays were not performed.Results:ATI detection by both assays at early stages (≤22 W) of treatment is shown in Table 1a. ATI were always detected earlier by IDK than bELISA and also in RA than in SpA patients probably reflecting the effect of lower Ifx doses. Three out of 106 (3%) vs 0 (0%) patients had ATI at W 2 and 62 (58%) vs 20 (18%) patients at W22, by IDK and bELISA, respectively.Table 1.Patient characteristics of all included patientsW2W6W12W22ARSpAARSpAARSpAARSpAa) ATI+ patients (n, %) at early stagesbELISA003(7%)010(22%)1(2%)13(29%)7(12%)IDK1(2%)2(3%)7(16%)2(3%)16(36%)16(26%)28(62%)34(56%)b) Patients who discontinued (n, %) Ifx therapy considering ATI status at early stagesbELISA+9(90%)*1(100%)*12(92%)4(57%)bELISA-23(66%) 22(37%)20(63%)19(35%)IDK+15(94%)*7(44%)24(86%)13(38%)IDK-17(59%)11(24%)13(77%)10(27%)*pOnce ATIs appeared, regardless both methods, they persisted throughout the follow-up, indicating that immunogenicity was not transient.At W22, only 13/28 (46%) and 7/34 (21%) patients with ATI detected by IDK were also positive by bELISA in RA and SpA, respectively.ATI levels by IDK were higher in ATI+ by bELISA than in ATI- patients at early stages: ATI levels by IDK at W12: 91[74-348] ng/ml ATI+ vs 21.7[15-59.5] ng/ml ATI- (pFree IFX in serum was not detected in bELISA ATI+ patients. In IDK ATI+ patients low circulating Ifx levels were present as compare to ATI- since W6 to the end of follow-up (pMore ATI+ patients dropped out Ifx at W12 and W22 regardless de assay (Table 1.b), being statistically significant for both assays in patients with RA and only for bELISA in patients with SpA.Conclusion:ATI measured by a drug-tolerant assay are always detected earlier than ATI detected by bELISA, indicating that immunogenicity, at least with Ifx, is usually an early event. High levels of ATI by IDK are associated with an earlier detection by bELISA in case of RA patients. ATI detected only by drug tolerant assays are associated with low levels of circulating Ifx but not with a complete drug neutralization and may do not have clinical relevance compared to ATI detected by bELISA. Many patients have low levels of ATI which can only be detected by drug tolerant assays after long-term of follow-up.The reasons why ATI levels rise rapidly in some patients while in others remain low are currently unknown but may be relevant if the clinical effect of immunogenicity is to be minimized.Acknowledgements:We are grateful to all the rheumatologists and nurses of the Daycare Department for Biologics and to the laboratory technicians of the Immunological UnitDisclosure of Interests:ANA MARTÍNEZ-FEITO: None declared, Borja Hernández-Breijo: None declared, Marta Novella-Navarro: None declared, Victoria Navarro-Compán Grant/research support from: AbbVie, Janssen, Lilly, Novartis, Pfizer, and UCB, Cristina Diego: None declared, Irene Monjo: None declared, Laura Nuño: None declared, Alejandro Villalva: None declared, Diana Peiteado: None declared, DORA PASCUAL-SALCEDO: None declared, Pilar Nozal: None declared, Alejandro Balsa Grant/research support from: Abbvie, Pfizer, Novartis, Roche.Amgen, Sandoz, Lilly, UCB. Personal fees and non- financial support from BMS. Grants, personal fees and non- financial support from Nordic., Chamaida Plasencia Grant/research support from: AbbVie, Lilly, Novartis, Pfizer,Sanofi, Biogen and UCB.

Background The goal of this study was to validate the use of capillary blood in a real point-of-care (POC) setting for patients under infliximab treatment by using Promonitor Quick lateral flow (LF) tests. Results were compared to the Promonitor ELISA reference technique in serum samples used by centralised laboratories. Methods A prospective, observational study was designed to evaluate the performance of a rapid LF test (Promonitor Quick IFX, Progenika, Spain). 160 infliximab treated rheumatology consecutive patients (400 samples) were recruited in two hospitals in Galicia, Spain. Prior to the infusion, a finger prick sample was obtained and analysed. Anti-infliximab antibodies were also determined with Promonitor Quick ANTI-IFX1-4. Results were read with the automated portable PQreader instrument. Additionally, a serum sample was collected for subsequent comparative analysis with either LF or ELISA tests. Qualitative (positive (PPA) and negative (NPA) agreements) and quantitative (Pearson correlation and bias) performance of the LF test was compared to ELISA, as well as between different specimens following CLSI EP09-A3. Results Overall agreement between Promonitor Quick IFX finger prick and ELISA test was 91% (88% PPA; 100% NPA). The quantitative comparison showed a good correlation (Pearson correlation coefficient: 0.85 and observed bias: 25%) (Table 1). Similar results were also observed when serum was used with either the LF or the ELISA tests (98% overall agreement, 0.91 correlation coefficient; 6% bias) (Table 1). Overall agreements for visual and automated (PQreader) interpretations with Promonitor Quick ANTI-IFX were 99% and 100% for finger prick and serum specimens, respectively (Table 2). Conclusion Promonitor Quick can be used to reliably quantify infliximab in capillary blood samples and results are comparable to those obtained with the reference ELISA technique. The use of the rapid POC test with finger prick will allow clinicians to monitor their patients in a fully decentralized mode to aid in the decision making process. PQreader is a sensitive portable equipment to report drug as well as antibody levels in the patient samples. References

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.

Anti-drug antibodies can interfere with the activity of anti-tumor necrosis factor (TNF) agents by increasing drug clearance via direct neutralization. The presence of anti-drug antibodies is clinically relevant when trough drug concentrations are undetectable or sub-therapeutic. However, traditional immunoassay is not easily and rapidly accessible, making the translation of the results into treatment adjustment difficult. The availability of a point-of-care (POC) test for therapeutic drug monitoring (TDM) might represent an important step forward for improving the management of inflammatory bowel disease (IBD) patients in clinical practice. In this pilot study, we compared the results obtained with POC tests with those obtained by enzyme-linked immunosorbent assay (ELISA) in a group of IBD patients treated with Infliximab (IFX). We showed that POC test can reliably detect presence of antibody-to-IFX with 100% of specificity and 76% sensitivity, in strong agreement with the ELISA test ( k-coefficient = 0.84).

Background There are many studies presenting data of biologics and several ELISA kits commercially available for monitoring infliximab serum trough levels (s-IFXt) and anti-drug antibodies (ADAb). We propose to compare technical characteristics and results of three different assays on a cohort of 35 patients under infliximab (IFX) and suffering from inflammatory bowel disease (IBD). Patients and methods s-IFXt and ADAb were systematically measured with three ELISA kits: Lisa-Tracker® Duo infliximab (Theradiag®), Ridascreen® IFX Monitoring (R-Biopharm AG®) and Promonitor® IFX (Progenika Biopharma SA®). Results The main technical features that differed between kits for measuring s-IFXt were: (i) TNF coating, (ii) immune complexes revelation strategy and/or (iii) interference with other anti-TNFα agents. For kits measuring ADAb, they were revelation steps and unit of results. There was an excellent mathematical correlation of s-IFXt between assays however Bland-Altman analysis denoted (i) s-IFXt were on average 48 to 69% higher in Ridascreen® than in the other two assays, and (ii) elevated s-IFXt were higher with Promonitor® compared to Lisa-Tracker®. As a consequence, there were some substantial discrepancies between assays for classification of s-IFXt into concentration ranges. Despite unstandardized units, pairwise qualitative comparison showed a perfect agreement between the three pairs of ADAb assays. Conclusion Our data show that the evaluated assays are not quantitatively interchangeable due to substantial variations in some results that could lead, for some patients, to divergent therapeutic decisions. We remind to be cautious when comparing study results issued from different kits and recommend using the same assay for the longitudinal follow-up of IBD patients.

Background Infliximab, a monoclonal antibody directed against tumour necrosis factor, is widely used in the treatment of chronic inflammatory conditions including Crohn’s disease and rheumatoid arthritis. Its use is limited by development of anti-infliximab antibodies, which can lead to loss of therapeutic efficacy. Serum infliximab and anti-infliximab antibody measurements have recently become routinely available in the UK. The study aimed to assess the clinical utility of antibodies as an adjunct to trough infliximab. Methods Serum trough infliximab was measured in 201 samples from 108 gastroenterology and rheumatology patients on maintenance infliximab therapy. Results were correlated with C-reactive protein concentrations. Total anti-infliximab antibodies were measured in 164 samples. Results The median (25th–75th percentile) trough infliximab was 3.7 µg/mL (1.2–5.2 µg/mL) and 23% of samples had a concentration ≤1 µg/mL. A notable proportion had positive anti-infliximab antibodies: 84/164 (51%), which subdivided to 85% and 28% with infliximab ≤1 and >1 µg/mL, respectively. Serum C-reactive protein was found to be significantly higher where infliximab was ≤1 compared to >1 µg/mL (10 mg/L [Conclusion Our findings support measurement of anti-infliximab antibodies only in the context of low infliximab concentrations

Antibodies (Abs) against infliximab (IFX) increase IFX clearance and can result in treatment failure and acute hypersensitivity reactions. However, interpretation of their clinical value is complicated by individual differences in Ab responses and methods used for quantification. The increase in IFX clearance imposed by anti-IFX Abs has generally been evaluated using a binary classification, i.e., positive or negative. This analysis aimed to investigate if anti-IFX Ab concentrations provide a more adequate prediction of alterations in clearance. Data originated from a clinical trial on Crohn's disease patients with IFX treatment failure. The trial was not originally designed for pharmacokinetic analysis. Therefore, published pharmacokinetic models were utilized as priors to enable covariate investigation. The impact of anti-IFX Abs on clearance was assessed using different mathematical relationships and exploiting information from two different quantification assays, measuring semi-quantitative "total" or "unbound neutralizing" concentrations of anti-IFX Ab, respectively. Inclusion of anti-IFX Ab status/concentration improved the model's performance for all investigated relationships. The anti-IFX Ab concentrations were superior to the binary classifications, indicating that the magnitude of increase in IFX clearance imposed by anti-IFX Abs closely relates to their concentration. Furthermore, total anti-IFX Ab concentrations appeared superior to the unbound neutralizing fraction in identifying high clearance individuals. Simulations showed that even at low concentrations, anti-IFX Abs lead to sub-therapeutic IFX concentrations, supporting a need of treatment interventions in all anti-IFX Ab positive patients. The developed model can serve as a basis for further investigations to refine treatment recommendations for patients with anti-IFX Abs.

Background: There is scant information on the accuracy of different assays used to measure anti-infliximab antibodies (ADAs), especially in the presence of detectable infliximab (IFX). We thus aimed to evaluate and compare three different assays for the detection of IFX and ADAs and to clarify the impact of the presence of circulating IFX on the accuracy of the ADA assays. Methods: Blood samples from 79 ulcerative colitis (UC) patients treated with infliximab were assessed for IFX levels and ADAs using three different assays: an in-house assay and two commercial kits, Immundiagnostik and Theradiag. Sera samples with ADAs and undetectable levels of IFX were spiked with exogenous IFX and analyzed for ADAs. Results: The three assays showed 81–96% agreement for the measured IFX level. However, the in-house assay and Immundiagnostik assays detected ADAs in 34 out of 79 samples, whereas Theradiag only detected ADAs in 24 samples. Samples negative for ADAs with Theradiag, but ADA-positive in both the in-house and Immundiagnostik assays, were positive for IFX or IgG4 ADAs. In spiking experiments, a low concentration of exogenous IFX (5 µg/ml) hampered ADA detection with Theradiag in sera samples with ADA levels of between 3 and 10 µg/ml. In the Immundiagnostik assay detection interference was only observed at concentrations of exogenous IFX higher than 30 µg/ml. However, in samples with high levels of ADAs (>25 µg/ml) interference was only observed at IFX concentrations higher than 100 µg/ml in all three assays. Binary (IFX/ADA) stratification of the results showed that IFX+/ADA- and IFX-/ADAs+ were less influenced by the assay results than the double-positive (IFX+/ADAs+) and double-negative (IFX-/ADAs-) combination. Conclusions: All three methodologies are equally suitable for measuring IFX levels. However, erroneous therapeutic decisions may occur when patients show double-negative (IFX-/ADAs-) or double-positive (IFX+/ADAs+) status, since agreement between assays is significantly lower in these circumstances.

*Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands. Laboratory for Pharmaceutical Biology, KU Leuven, Leuven, Belgium. Department of Hospital and Clinical Pharmacy, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands. Division of Gastroenterology, UZ Leuven, Leuven, Belgium. Laboratory for Monoclonal Therapeutics, Sanquin Diagnostics, Amsterdam, the Netherlands. **Department of Immunopathology, Sanquin Research and Landsteiner Laboratory Academic Medical Centre, Amsterdam, the Netherlands. E-mail: d.buurman@umcg.nl Both first authors contributed equally. Both last authors contributed equally.

SummaryBackground Formation of antibodies to infliximab (ATI) inversely correlates with functional drug levels and clinical outcome. Comparison of drug levels and anti-drug antibody monitoring is hampered by lack of standardisation. Aim To determine the correlation between three different assays for measuring infliximab and ATI. Methods Serum samples and spiked controls (total 62) were evaluated in a blinded way in infliximab and ATI assays developed by Sanquin Amsterdam, Netherlands (A), Laboratory for Pharmaceutical Biology, KU Leuven, Belgium (B) and a commercially available kit from Biomedical Diagnostics (BMD), Paris, France (C) performed by the University Medical Center Groningen (UMCG), Netherlands. Results All infliximab assays showed a linear quantitative correlation (Pearson r = 0.91 for A vs. B, 0.83 for A vs. C and 0.73 for B vs. C). Assay C detected infliximab in 11 samples (18%) not detected by A and B, including samples containing only ATI. All ATI assays showed a good linear correlation (Pearson r = 0.95 for A vs. B, 0.99 for A vs. C and 0.97 for B vs. C). Assay A detected ATI in five samples with low ATI that were not detected by assays B and C. Assay B did not detect ATI in three patient samples with low ATI according to assays A and C. Conclusions There is a good correlation of infliximab and antibodies to infliximab measurements between these assays. Nevertheless, the Biomedical Diagnostics kit detected false positive infliximab levels in 18% of the samples.

The formation of antibodies to infliximab (ATIs) is closely associated with the loss of response to infliximab in patients with inflammatory bowel disease (IBD). We evaluated the clinical utility of a novel method to measure serum ATI levels in the presence of infliximab.ATI levels were measured using a novel immunoassay and the conventional method in 58 patients with Crohn's disease (CD) under infliximab maintenance therapy. The serum infliximab trough levels were determined by enzyme-linked immunosorbent assay.ATIs were detected in 16 out of 58 patients (27.6%) by the new method, but the conventional method detected only 2 patients (3.4%) who had the two highest ATI titers assayed by the new method. The presence of ATIs in the samples positive by the new method but negative by the conventional method was confirmed by Western blot analysis. Western blotting analysis also indicated that the new method could restore the binding capacities of the ATIs whose recognition sites were occupied by free infliximab. In the new method, the addition of infliximab to the samples dose-dependently blocked the detection of ATIs. Patients positive for ATIs had significantly lower serum trough levels of infliximab (P0.01) and significantly higher clinical activity scores (P0.001) as compared with patients negative for ATI.The new method makes it possible to measure serum ATI levels in the presence of infliximab. This method is useful for deciding the optimal management strategies for IBD patients with loss of response to infliximab.

Background: Infliximab (IFX) can be immunogenic for humans and lead to the formation of antibodies against IFX (anti-IFX Ab), which could induce acquired IFX resistance. Objective: To test whether the presence of anti-IFX Ab and residual circulating IFX levels are associated with acquired IFX resistance in RA. Methods: A multivariate logistic regression was used to analyze the relationship between anti-IFX Ab, residual IFX concentrations, and acquired IFX resistance in a nested cohort within the Swiss RA registry (SCQM-RA). Results: Sixty-four RA patients on longstanding IFX therapy were included; 24 with an acquired therapeutic resistance to IFX and 40 with continuous good response to IFX. The two groups had similar disease characteristics, but patients with acquired IFX resistance required significantly higher dosage of IFX (5.4 mg/kg versus 4.3 mg/kg, p = 0.02) and shorter infusion intervals (7.1 versus 8.7 weeks, p = 0.01) than long-term good responders. The presence of residual IFX tended to be associated with a decreased risk of acquired therapeutic resistance (OR 0.4 [95% CI: 0.1–1.5]), while the presence of anti-IFX Ab tended to be associated with an increased risk of acquired therapeutic resistance (OR: 1.8 [95% CI: 0.4 – 9.0]). The presence of either high anti-IFX Ab levels or low residual IFX concentrations was strongly associated with acquired therapeutic resistance to IFX (OR 5.9, 95% CI 1.3 – 26.6). However, just 42% of patients with acquired IFX resistance had either low IFX or high anti-IFX Ab levels. Conclusion: These results suggest that the assessment of anti-IFX Ab and residual IFX levels is of limited value for individual patients in routine clinical care.

The development of anti-infliximab antibodies in 8% to 43% of infliximab-treated patients is associated with decreased efficiency and increased risk of adverse effects.1–3 An intervention that can diminish anti-infliximab antibody formation is therefore warranted. Rituximab, a chimeric monoclonal antibody that selectively depletes CD20-positive B lymphocytes, could potentially inhibit the human antibody response against infliximab. Therefore, we assessed the proportion of patients with rheumatoid arthritis (RA) in which treatment with rituximab resulted in the depletion of anti-infliximab antibodies. Consecutive patients with RA with detectable anti-infliximab antibodies, who were initiated on treatment with either rituximab or adalimumab, were included in this prospective controlled cohort study. …

Background Ankylosing spondylitis (AS) is a chronic inflammatory disease which can result in invalidating deformities of the joints and spine at an early age. The introduction of tumor necrosis factor (TNF) blocking agents has changed the treatment options in AS. Nevertheless, the reasons for lack or loss of response to infliximab (IFX) are unclear. So far determinations of both, IFX serum levels and the presence of anti-drug antibodies (ADA) anti-IFX have not been standardized for clinical use. Objectives To assess the correlation between two available versions (V.1 and V.2) of a commercial enzyme-linked immunosorbent assay (ELISA) for serum levels of IFX and IFX-ADA in patients with AS. Methods In this cross sectional study we assessed 40 serum samples taken prior to infusion from patients diagnosed with AS treated with IFX for more than 12 months (1st line of biological treatment). IFX levels and IFX-ADA were measured using two different ELISA assays [Promonitor® IFX R1 and R2 (V.1), Promonitor® IFX and Anti-IFX (V.2) (Progenika Biopharma, Spain)] according to manufacturer9s specifications. The relation comparing V.1 and V.2 for IFX levels and IFX-ADA concentrations was performed using the coefficient of variation (CV), the Cohen9s Kappa coefficient, the Pearson9s r, the intraclass correlation coefficient (ICC) and the Lin9s concordance correlation coefficient (CCC). Bland-Altman plots were drawn to compare both versions of the assays. Results As shown in the table below, we found a greater CV for IFX levels than for IFX-ADA. Regarding the qualitative results the Cohen9s Kappa was from moderate to fair and considering the quantitative results the Pearson9s r was low for IFX levels and high for IFX-ADA, the ICC was questionable for both versions and both determinations. We also determined the CCC and the results showed a poor agreement. Bland-Altman plots showed the difference between both versions for IFX levels (Fig. 1). Conclusions A low reliability for IFX levels and IFX-ADA was obtained for both versions. There is a need to standardize laboratory techniques (variability inter/intra-assay and inter/intra-laboratory) in order to validate this information and its possible clinical application. Disclosure of Interest D. Hernandez Flόrez: None declared, L. Valor: None declared, J. C. Nieto: None declared, L. Martinez: None declared, I. de la Torre: None declared, T. del Rio: None declared, C. Gonzalez: None declared, J. Lopez-Longo: None declared, I. Monteagudo Consultant for: Abbvie, Roche Farma, Bristol-Myers Squibb, Pfizer, UCB, General Electric Healthcare, and Esaote, E. Naredo Grant/research support: UCB and MSD, Consultant for: Abbvie, Roche Farma, Bristol-Myers Squibb, Pfizer, UCB, General Electric Healthcare, and Esaote, M. Montoro: None declared, L. Carreno Perez: None declared DOI 10.1136/annrheumdis-2014-eular.3274

P302 Utility of “trough levels” determination and anti-infliximab antibodies in patients with inflammatory bowel disease. Estimation of individual pharmacokinetic parameters (PK) through population pharmacokinetic model G. Juan1,2 *, A. Alvarino3,4, L. Oltra4, N. Maroto4, N. Cano2, I. Ferrer2, J. Hinojosa2,4. 1Hospital de Manises, Farmacia Hospitalaria, Valencia, Spain, 2Hospital de Manises, Digestivo, Manises, Spain, 3Hospital de Manises, LABCO, Valencia, Spain, 4Hospital de Manises, Gastroenterology, Valencia, Spain

P122 The clinical and immunological significance of low level of infliximab in the absence of anti-infliximab antibodies in patients with IBD B. Ungar1 *, A. Anafy1, M. Yavzori1, O. Picard1, E. Fudim1, U. Kopylov1, Y. Ron2, H. Yanai2, I. Dotan2, Y. Chowers3, R. Eliakim1, S. Ben-Horin1. 1Chaim Sheba Medical Center, Gastroenterology, Ramat Gan, Israel, 2Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, IBD Center, Department of Gastroenterology and Liver Diseases, Tel Aviv, Israel, 3Rambam Health Care Campus & Bruce Rappaport School of Medicine, Technion Institute of Technology, Gastroenterology department, Haifa, Israel

Introduction Commercial assays for monitoring Infliximab (IFX) and anti-drug antibodies (ADAb) are currently available. The heterogeneity of these assays and lack of standardisation of ADAb may cause difficulties in interpretation, in particular comparing bridging ELISA and radioimmunoassay (RIA). Bridging ELISAs have high specificity but are more prone to inhibition by drug hence may underestimate ADAb, whereas false positive results can be seen with RIA. Aims Evaluation of (i) the effect of common interferents in the LISA-Tracker Premium ELISA assay (Theradiag). (ii) the inhibitory effect of IFX on the detection of ADAb. Methods The LISA-Tracker kit was used for the quantitative determination of free drug and ADAb by indirect and bridging assay principles respectively. The measurement range was 0.1 to 5 μg/mL for IFX and 10 to 200 ng/mL for ADAb (> 10 ng/mL considered positive). Interference studies were carried out using samples with known levels of haemolysis, icterus, rheumatoid factor (RF) and paraprotein (IgG kappa = 17 g/L). All patients had no history of exposure to biologics. Samples were run neat and diluted with samples of known IFX and ADAb concentration to assess recovery. Samples with known levels of ADAb were spiked with aqueous solutions of IFX (2.4, 1.2 and 0.3 µg/mL) at a ratio of 1:2. Predicted ADAb concentrations were calculated by sample dilution in drug-free diluent. Results No interference was observed from haemolysis (up to 0.6 g/dL haemoglobin equivalent), icterus (up to 349 μmol/L bilirubin), RF (up to 885 U/mL) and paraprotein as confirmed by recoveries of 93–113% for drug and ADAb. Spiking experiments The inhibitory effect of IFX on ADAb detection was observed in both low and high level ADAb samples with ADAb only detectable in sample 2 i.e. as free, not complex form. Comparing expected with measured concentrations of IFX in the spiked samples indicated a difference in recovery between the low and high ADAb positive samples suggesting decreased availability of free IFX in the presence of ADAb. Conclusion This study shows that the LISA-Tracker kit is not affected by tested interferents. Results from spiking experiments highlight the importance of measuring trough levels and also indicate that the presence of ADAb (detectable or undetectable) can limit the availability of free IFX, leading to sub-therapeutic levels. LISA-Tracker is a simple, robust assay which has been implemented in our hospital and is routinely used as a personalised approach to prescribing and optimising anti-TNFα therapies in IBD. Disclosure of Interest None Declared.

Objectives To analyze association between the presence of antibodies to infliximab (ATI) and the clinical and biological characteristics in rheumatoid arthritis (RA) and spondyloarthritis (SpA) patients Methods Sera from RA (n = 22) or SpA (n = 23) patients treated with infliximab were analyzed. ATI and infliximab trough concentrations were measured using a commercial multiplex enzyme-linked immunosorbent assay kit (LISA-Tracker Infliximab Theradiag®, Marne la Vallee, France). Clinical and biological data were collected retrospectively from each patient’s medical file. Results Infliximab was given in combination with methotrexate (MTX) for 31 (69%) patients (RA patients 90%; SpA patients 43%). Fifteen patients were in remission at the time of analysis. The interval between two consecutive infliximab infusions exceeded 8 weeks for 15 (33%) patients; it was significantly longer for patients who had achieved remission (9.3 weeks) versus the other patients (6.8 weeks; Mann-Whitney U- test, P = 0.0005). Seven (15%) patients were ATI positive (4 RA and 3 SpA), 6 of them were treated in combination with MTX. Duration of infliximab intakedid not differ between ATI positive and negative patients. Their infliximab doses at the time of analysis were comparable, respectively: 4.29mg/kg and 4.13mg/kg (P = 0.74). A longer between-infusions interval was associated with the ATI-positivity (9.6 weeks versus ATI-negativity: 7.3 weeks; P = 0.02). Infliximab trough concentrations were significantly lower in ATI-positive patients (0.18 μ g/ml) vs ATI-negative patients (2.1 μ g/mL, P = 0.0005). A significant inverse correlation was found between ATI titers and infliximab trough concentrations (Spearman’s test, P = 0.0002, R 2 = 0.29). No link was found between the methotrexate intake and ATI-positivity. Conclusions Immunogenicity of infliximab is related with a longer between-infusion interval, which could affect the treatment strategy, because prolonging those intervals for patients in clinical remission might favor ATI production. Disclosure of Interest None Declared

Background Some patients with autoimmune inflammatory diseases and treated with infliximab (an anti-TNF therapy) are suspected of acquired therapeutic resistance and loss of response. Several therapeutic strategies are possible for those patients: increasing infliximab dosage or switching to another TNF-inhibitor or another therapy. However, most of the strategies are based on clinical data. Objectives The aim of this study was to evaluate the presence of anti-infliximab antibodies together with residual infliximab levels in such patients Methods We studied 36 patients presenting rheumatoid arthritis (n=20), psoriasis (n=2), Crohn disease (n=5), ulcerative colitis (n=4), spondylarthritis ankylosis (n=4) treated by infliximab and suspected of therapeutic loss response. For 20 of them, the samples were obtained just before the switch to another biotherapy. Infliximab levels and anti-infliximab antibodies were determined by ELISA (LISA-TRACKER, BMD, France) with a threshold of 10 ng/ml for anti-infliximab antibodies and 0.1 microg/ml for infliximab Results Anti-infliximab antibodies were detected in 21 patients (median: 131 ng/ml, range 10-200 ng/ml). Infliximab levels were weak in 25 patients (median: 0.16 microg/ml, range: Conclusions In conclusion, our preliminary results suggest that in patients suspected of loss of anti-TNF response, the weak residual level of infliximab was mostly related to the presence of anti-infliximab antibodies confirming the necessity in those patients to switch to another TNF-inhibitor instead of increasing infliximab doses. Disclosure of Interest None Declared

P511 Epstein-Barr Virus in inflammatory bowel disease correlation with different therapeutic regimens F. Magro1 *, J. Santos-Antunes1, A. Albuquerque1, F. Vilas-Boas1, G.N. Macedo2, N. Nazareth2, S. Lopes1, J. Sobrinho-Simoes3, S. Teixeira3, C. Camila-Dias4, J. Cabral5, A. Sarmento2, G. Macedo1. 1Centro Hospitalar S. Joao, Gastroenterology, Porto, Portugal, 2CEBIMED Biomedicine Research Center, Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal, 3Centro Hospitalar S. Joao, Clinical Pathology Department, Porto, Portugal, 4Faculty of Medicine, University of Porto, CIDES Department of Health Information and Decision Sciences, Porto, Portugal, 5Faculty of Medicine, University of Porto, Institute of Pharmacology and Therapeutics, Porto, Portugal