Antibodies To Infliximab Levels Research Articles

Clinical Experience with Measurement of Serum Infliximab and Antibodies to Infliximab Using a New Homogenous Mobility Shift Assay: Results of a Multi-center Observational Study

Purpose: To characterize utilization of a new test for monitoring serum infliximab (IFX) and antibodies to infliximab (ATI) in Inflammatory Bowel Disease (IBD) patients on IFX therapy. The main advantage of this new test, using a liquid phase homogeneous assay, over the existing ELISA is that serum IFX does not interfere with quantification of ATI. Therefore, physicians can measure serum levels of IFX and ATI at any time during IFX therapy. Methods: IBD patients undergoing IFX therapy were enrolled in the study if their treating physician determined a need to measure IFX and ATI levels at any time during the course of therapy. Indications for the test, clinical status, and dosing information were collected at the time of blood draw. IFX and ATI levels were communicated to the physicians within 7 days and actions taken in response to the test results were documented. Results: As of this analysis, baseline data were available for 48 patients (28 CD, 19 UC and 1 indeterminate colitis). Mean age was 48y, mean BMI was 26.4 kg/m2 and mean duration of disease was 9.3 years. Forty-two percent of physicians ordered the test because their patients had an inadequate response or loss of response whereas 58% ordered the test in the absence of a lack or loss of response. Median IFX concentration was 9.45 μg/mL and 14 of 48 patients (29%) were positive for ATI. Among ATI-positive patients, 86% had IFX concentrations < 3 μg/mL, whereas only 9% of ATI-negative patients had IFX concentrations < 3 μg/mL (Odds Ratio = 0.02; p <<0.0001, Fisher's Exact test). Median IFX concentrations were 1 μg/mL and 20 μg/mL in ATI-positive and ATI-negative patients, respectively. Patients with lower disease scores had higher median IFX concentrations (median IFX = 9.3 μg/mL for HBI <7 vs. 5.2 μg/mL for HBI ≥7 in CD and median IFX = 34.00 μg/mL for partial Mayo score < 6 vs. 14.25 μg/mL for partial Mayo score ≥ 6 in UC). Changes made to the treatment regimen in response to knowing results of IFX/ATI testing included increasing the dose of IFX, changing the dosing interval, switching to another biologic, or performing additional work-up. Follow-up data for these patients will assess the clinical impact of adjusting therapy based on IFX / ATI levels. Conclusion: Results of this study are consistent with previous reports in the literature demonstrating that ATI development is negatively associated with serum IFX concentrations and clinical status. Physicians appear to find the measurement of serum IFX and ATI levels useful in multiple clinical scenarios including inadequate response or loss of response. Disclosure: Dr. Wolf: None Dr. Shringarpure: Employee of Prometheus Laboratories Inc. Dr. Lockton: Employee of Prometheus Laboratories Inc. Dr. Corey: None. Dr. Woods: None. Dr. Aguilar: None. Dr. Chuang: Employee of Prometheus Laboratories Inc. This research was supported by an industry grant from Prometheus Laboratories Inc.

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 0.012μg/mL in serum. The linear range of quantitation was 0.029–0.54μg/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 0.036μg/mL can be measured, even in the presence of 60μg/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.

566 Infliximab Concentration and Clinical Outcome in Patients With Ulcerative Colitis

Background: Infliximab (IFX), a chimeric mouse/human IgG1 monoclonal antibody against tumor necrosis factor alpha is successfully used in the treatment of Crohn's 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; Fisher's 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.

Development of a new Immunoassay for the accurate determination of anti-infliximab antibodies in Crohnʼs Disease

The formation of antibodies to infliximab (ATIs) is closely associated with the loss of a response to infliximab in patients with Crohn's disease (CD). We evaluated the clinical utility of a novel method to measure serum ATI levels in the presence of infliximab. ATI levels were measured by a novel method of immunoassay with protein A and the conventional method in a cohort of 58 patients with 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 2 highest ATI titers assayed by the new method. The presence of ATIs in those samples positive according to the new method but negative by the conventional method was confirmed by Western blotting analysis. It also indicated that the new method could restore the binding capacities of the ATIs to infliximab, which were occupied by free infliximab. In the current method, the addition of infliximab to the samples dosedependently blocked the detection of ATIs. Patients positive for ATIs had significantly lower serum trough levels of infliximab (p<0.01), and significantly higher clinical activity scores (p<0.001) and higher laboratory markers for inflammation (CRP and ESR) (p<0.01) as compared to patients negative for ATI. The current method makes it possible to measure serum ATI levels in the presence of infliximab. This new method seems to be useful for deciding the optimal management strategies for CD patients with a loss of response to infliximab.

Development of a new immunoassay for the accurate determination of anti-infliximab antibodies in inflammatory bowel disease

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 (P < 0.01) and significantly higher clinical activity scores (P < 0.001) 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.

Oral ulcerations are associated with the loss of response to infliximab in Crohn's disease

We describe a 25-year-old Caucasian man with a 13-year history of inflammatory Crohn's disease (CD) who was suffering recurrent severe oral and esophageal ulcerations for the past 3 years. His CD had been treated with infliximab infusions among other medications. The loss of efficacy was confirmed by antibodies to infliximab (ATI) and serum infliximab tests that showed high levels of ATIs and undetectable levels of infliximab respectively. These findings were consistent with significant immunogenic response to infliximab leading to loss of effect. Infliximab infusions and prednisone were discontinued and treatment of the CD was instituted with adalimumab, a human anti-tumor necrosis factor (TNF)-alpha biologic agent, to control the inflammatory small intestinal disease and dapsone for the oral and esophageal CD ulcerations. The patient's oral and esophageal lesions as well as the enteric CD are under control after 5 months of therapy.

Intravenous hydrocortisone premedication reduces antibodies to infliximab in Crohn's disease: A randomized controlled trial

Background & Aims: We assessed the relationship between antibodies to infliximab (ATI) and the loss of response postinfliximab, infusion reactions and, in a randomized trial, investigated whether intravenous hydrocortisone premedication can reduce ATI. Methods: Initially, we prospectively evaluated clinical response, adverse events, and ATI levels in 53 consecutive patients with Crohn's disease who received 199 infliximab (5 mg/kg) infusions. Subsequently, 80 patients with Crohn's disease were randomized to intravenous hydrocortisone 200 mg or placebo immediately before their first and subsequent infliximab infusions. The primary endpoint was reduction in median ATI levels at week 16. Analysis was by intention to treat. Results: Nineteen of our initial 53 patients (36%) developed ATI, including all 7 patients with serious infusion reactions (median ATI level, 19.6 μg/mL). Eleven of 15 patients (73%) who lost their initial response were ATI positive compared with none of 21 continuous responders, (8.9 vs. 0.7 μg/mL, P < 0.0001). Administering a second infusion within 8 weeks of the first (OR, 0.13; 95% CI, 0.03-0.5; P = 0.0007) or concurrent immunosuppressants (OR, 0.19; 95% CI, 0.04-1.03; P = 0.007) significantly reduced ATI formation. In the placebo-controlled trial, ATI levels were lower at week 16 among hydrocortisone-treated patients (1.6 vs. 3.4 μg/mL, P = 0.02), and 26% of hydrocortisone-treated patients developed ATI compared with 42% of placebo-treated patients, P = 0.06. Conclusions: Loss of initial response and infusion reactions post-infliximab is strongly related to ATI formation and level. Administering a second infusion within 8 weeks of the first and concurrent immunosuppressant therapy significantly reduce ATI formation. Intravenous hydrocortisone premedication significantly reduces ATI levels but does not eliminate ATI formation or infusion reactions.