Linda Ohrmund

The relationship between infliximab concentrations, antibodies to infliximab and disease activity in Crohn's disease

Objective Although low infliximab trough concentrations and antibodies to infliximab (ATI) are associated with poor outcomes in patients with Crohns disease (CD), the clinical relevance of ATI in patients with adequate infliximab concentrations is uncertain. We evaluated this question using an assay sensitive for identification of ATI in the presence of infliximab. Design In an observational study, 1487 trough serum samples from 483 patients with CD who participated in four clinical studies of maintenance infliximab therapy were analysed using a fluid phase mobility shift assay. Infliximab and ATI concentrations most discriminant for remission, defined as a C-reactive protein concentration of ≤5 mg/L, were determined by receiver operating characteristic curves. A multivariable regression model evaluated these factors as independent predictors of remission. Results Based upon analysis of 1487 samples, 77.1% of patients had detectable and 22.9% had undetectable infliximab concentrations, of which 9.5% and 71.8%, respectively, were positive for ATI. An infliximab concentration of >2.79 μg/mL (area under the curve (AUC)=0.681; 95% CI 0.632 to 0.731) and ATI concentration of <3.15 U/mL (AUC=0.632; 95% CI 0.589 to 0.676) were associated with remission. Multivariable analysis showed that concentrations of both infliximab trough (OR 1.8; 95% CI 1.3 to 2.5; p<0.001) and ATI (OR 0.57; 95% CI 0.39 to 0.81; p=0.002) were independent predictors of remission. Conclusions The development of ATI increases the probability of active disease even at low concentrations and in the presence of a therapeutic concentration of drug during infliximab maintenance therapy. Evaluation of strategies to prevent ATI formation, including therapeutic drug monitoring with selective infliximab dose intensification, is needed.

Development and validation of a homogeneous mobility shift assay for the measurement of infliximab and antibodies-to-infliximab levels in patient serum

Antibody-based drugs such as infliximab (IFX) are effective for the treatment of inflammatory bowel disease (IBD) and other immune-mediated disorders. The development of antibodies against these drugs may result in unfavorable consequences, including the loss of drug efficacy, hypersensitivity reactions, and other adverse events. Therefore, accurate monitoring of serum drug and anti-drug antibody levels should be an important part of therapy for patients being treated with an antibody-based drug. Current methods for the assessment of anti-drug antibodies and drug levels, involving various bridging ELISA and radioimmunoassay techniques, are limited by their sensitivity, interference, and/or complexity. To overcome these limitations, we have developed a non-radiolabeled homogeneous mobility shift assay (HMSA) to measure the antibodies-to-infliximab (ATI) and IFX levels in serum samples. Full method validation was performed on both the ATI- and IFX-HMSA, and the clinical sample test results were also compared with those obtained from a bridging ELISA method to evaluate the difference in performance between the two assays. Validation of the ATI-HMSA revealed a lower limit of quantitation of 0.012 μg/mL in serum. The linear range of quantitation was 0.029-0.54 μg/mL. The intra- and inter-assay precision was less than 20% of coefficient of variation (CV), and the accuracy (% error) of the assay was less than 20%. In serum samples, ATI as low as 0.036 μg/mL can be measured, even in the presence of 60 μg/mL of IFX in the serum. Sera from 100 healthy subjects were tested to determine the cut point of the assay. ATI-positive samples that had been previously analyzed by using a bridging ELISA from 100 patients were also measured by the new method. There was a high correlation between the two methods for ATI levels (p<0.001). Significantly, the new method identified five false-positive samples from the bridging ELISA method. Validation of the mobility shift IFX assay also showed high assay sensitivity, precision and accuracy. The HMSA method may also be applied to other protein-based drugs to accurately detect serum drug and anti-drug antibody levels.

A prospective cohort study to determine the relationship between serum infliximab concentration and efficacy in patients with luminal Crohn's disease

Patients with Crohns disease (CD) may experience disease relapse on maintenance infliximab. Anti‐drug antibodies likely contribute to loss of response, and serum infliximab levels likely correlate with efficacy.

Monitoring of adalimumab and antibodies-to-adalimumab levels in patient serum by the homogeneous mobility shift assay

This report describes the analytical validation and application of the homogeneous mobility shift assay (HMSA) method for the measurement of adalimumab and human antibodies-to-adalimumab (ATA) in serum samples from patients who have lost response to adalimumab treatment. Validation of the ATA- and the adalimumab-HMSA revealed a lower limit of detection to be 0.026 U/mL for ATA and 0.018 μg/mL for adalimumab in serum samples. Intra-assay and inter-assay precision determination yielded a coefficient of variation of less than 15%, and the accuracy of both assays was within 20%. Adalimumab drug tolerance in the ATA-HMSA was up to 20 μg/mL in the test serum. Serum samples from 100 drug-naïve healthy subjects were tested to set-up the cut point of 0.55 U/mL for ATA and 0.68 μg/mL for adalimumab. Analysis of 100 serum samples from patients who were losing response to adalimumab showed that 26% had an adalimumab level below the cut point, of these 68% were ATA positive. Overall, 44% of the patients (44/100) were positive for ATA. This study presents evidence that drug and anti-drug antibody levels are important determinants of patient response to therapy.

565 Novel Infliximab (IFX) and Antibody-to-Infliximab (ATI) Assays are Predictive of Disease Activity in Patients With Crohn's Disease (CD)

Background: Infliximab (IFX), a chimeric mouse/human IgG1 monoclonal antibody against tumor necrosis factor alpha is successfully used in the treatment of Crohns disease and ulcerative colitis. Despite its beneficial effect, IFX can provoke an immunogenic response which can lead to loss of response to the drug. It is generally believed that, once elicited, an antibody response to a certain protein cannot be overcome. We hypothesised that in patients who elicit only low levels of antibodies to infliximab (ATI), these may disappear with dose optimisation and that patients may recapture response. Methods: In this retrospective analysis in 52 IFX-treated patients a total of 693 serum samples (average of 13.3 samples per patient) were analysed. Patients were selected based on ATIs detected on at least one time point during follow up with an in-house developed ELISA. All consecutive samples were then analysed for IFX trough levels (TLI; expressed in μg/ml; cut-off value: 0.91 μg/ ml) and ATIs (expressed in U/ml; cut-off value 7.95 U/ml) using a fluid phase mobility shift assay (Prometheus Laboratories, San Diego US). Treatment decisions to optimise and stop therapy were made on clinical grounds and CRP, without knowledge of ATI or TLI. Results: Patients developed ATIs after a median of 16 weeks (IQR 8-39) following initiation of IFX and after a median of 5 (IQR 3-7) infusions. In 14 (27%) patients ATIs disappeared over time whereas in 38 (73%) patients ATIs persisted. There was no difference in time to ATI onset between patients with transient or sustained ATIs. Patients with transient ATIs had significantly lower ATI levels (median 18.7 U/ml; IQR 10.6-31.5) compared to patients with sustained ATIs (median 22.1 U/ml; IQR 14.0-45.7; p<0.01; Mann-Whitney U test). Patients with sustained ATIs had a two-fold higher risk (RR 2.1; 95%CI 0.9-4.6; p<0.05) to suffer an acute infusion reaction compared to patients with transient ATIs. Concomitant immunomodulator (IMM) use was associated with lower ATI levels (median ATI level with IMM: 8.8 U/ml versus 15.5 U/ml without IMM; p<0.01; Mann-Whitney U test). In 8/14 patients (57%) ATIs disappeared following dose optimisation and in the other 6 patients (43%), ATIs disappeared spontaneously. Patients with sustained ATIs more often discontinued IFX treatment due to loss of response and/or hypersensitivity reactions compared to patients with transient ATIs (68% versus 14 % respectively; p<0.001; Fishers Exact test). Conclusion: Antibodies to infliximabmay be transient and can disappear after dose optimisation. Sustained high levels of ATIs lead however to permanent loss of response. When low or undetectable TLI are detected, knowledge of ATIs is therefore important as low titre ATIs can be overcome mostly with dose optimisation and high titre ATIs lead to a higher risk of adverse events and necessitate treatment stop.

P253 Transient versus sustained antibodies to infliximab: Possibility to overcome low titer antibody responses by dose optimisation

have shown that ustekinumab induces and maintains clinical response in Crohn’s disease (CD). Data of the effectiveness of ustekinumab in CD in clinical practice are lacked. The aim of this study was to assess the effectiveness and safety of ustekinumab in CD after multi-drug failure, in a compassionate program. Methods: Consecutive patients with CD in whom ustekinumab was administrated under compassionate use until October 2011, in 15 Spanish centers were retrospectively included. Demographic characteristics, medical and surgical history, dosage and schedule of ustekinumab administration were registered. The clinical response and remission to induction and maintenance treatment were evaluated based on the Harvey Bradshaw index (HBI), and by medical judgment. Side effects were also recorded. Results: Thirty CD patients (18 female, median age 35 years, range 17 80) followed up for 189±148 days were included. Most of them had ileocolonic involvement (60%), non-stenosant non-penetrating behavior (60%), and longstanding disease (median 10 years, range 3 34). Sixteen patients had at least one previous intestinal resection (range 1 7). Twentyfour (67%) patients had previously failed to at least two immunosuppressants and 80% to at least two anti-TNFs agents. Twenty-eight (93.3%) patients had received at least 4 doses of ustekinumab. Twenty-four patients received ustekinumab induction and the most frequent (71%) schedule was 90mg weekly during 4 weeks subcutaneously. Nineteen (82.6%) patients received 90mg of ustekinumab every eight weeks subcutaneously as maintenance schedule therapy. Clinical remission, response and failure rates after induction and at the end of follow-up are shown in the table.

W1256 Measurement of Human Anti-Chimeric Antibodies (Haca) and Infliximab Levels in Patient Serum Using a Novel Homogeneous Assay

G A A b st ra ct s DNMT -1, -3A and -3B were un-methylated in CD affected, non affected and controls. DNMT3L was differentially methylated between DNA from matched affected and nonaffected tissue in 17 confirmed CD cases and 11 control samples(CD Affected average=0.55 CD non-affected average=0.33, p<0.0002. Control average=0.39, p<0.006, Students t-test. Level of methylation of DNMT3L for each CpG site in CD affected and non-affected CD and controls revealed that DNMT3L is hypermethylated in affected CD (Fig 1). Relative gene expression of DNMT3L to GAPDH revealed that DNMT3L expression was less in Crohns affected tissue samples compared with matched normal tissue controls. Conclusion: DNMT3L is important for normal gut function and inflammation in CD affects its gene expression.

Abstract 3179: Functional profiling of multiple signal pathway proteins in breast cancer patients

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The COllaborative Proximity ImmunoAssay (COPIA) is a multiplexed protein microarray platform that utilizes the formation of a unique immuno-complex requiring co-localization of two detector-antibodies. The detector-antibodies are conjugated with corresponding channeling-enzymes, glucose oxidase (GO) and horseradish peroxidase (HRP). Once target proteins are bound by the capture antibodies, the channeling events between GO and HRP in proximity enables the profiling of the target proteins with extreme sensitivity. COPIA delivers extremely high analytical specificity as it requires multiple entities within target specific proximity for the signal generation/amplification. COPIA can also be configured for each specific target protein to allow differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., full length-HER2). We applied COPIA to investigate the levels of expression and activation of HER1, HER2, p95HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, panCK, and other targets in signal transduction pathways. Here, we report the functional pathway signatures for multiple proteins in 250 frozen tissues obtained from BCA patients with various primary histology and from 50 fine needle aspirate (FNA) samples collected from metastatic sites (mFNA) in advanced BCA patients with various ER/PR/HER2 status. There was a high concordance between primary HER2-IHC status and COPIA-HER2 expression analysis. Significant levels of p95HER2 were observed in over 40% of HER2-positive (HER2: 3+ and 2+ with FISH+) patients, and low but detectable levels in some sample tissues with IHC-HER2 negative (2+ with FISH - / 1+ /0) were also observed. Over 50% of p95HER2-expressors had activated p95HER2, and over 25% of HER2-positive samples also had HER1, HER3, IGF1-R and other RTKs and transduction protein expression and/or activation. As the disease profile often shifts in recurrent breast cancer, our unique assay format can be utilized to provide valuable clinical information on limited samples obtained from evolving disease to help oncologists adjust their disease treatment options for each patient according to their ‘personal’ cancer profile-shift. Having the ability to profile tumors at different metastatic sites with an expanded pathway panel could provide information on their differential metastatic potentials; hence minimally invasive single-passage-mFNA samples may be utilized to tailor therapy options accordingly. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3179.