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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: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.

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.

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.

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

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