Four-year review of New Zealand laboratory infliximab and adalimumab concentration results indicating potential for improved dosing.

A review of laboratory results across New Zealand for therapeutic drug monitoring (TDM) of infliximab and adalimumab concentrations and antidrug antibodies (ADAs) over 4 years was completed. Of 6591 results, the median serum concentration for infliximab was 5.7 mg/L and for adalimumab was 5.5 mg/L. Subtherapeutic drug concentrations (<7 mg/L) were measured in 54% of samples. Drug concentrations <2 mg/L were measured in 23% of samples, with ADAs detected in 51% of these. The high number of samples with subtherapeutic drug concentrations and common ADA detection is consistent with failing therapy but could also suggest that standard dosing is frequently too low for patients. These results reinforce the value of antitumour necrosis factor drug TDM in making decisions to adjust dosing or switch agents in patients taking infliximab and adalimumab.

Development of a competitive binding homogeneous mobility shift assay for the quantification of adalimumab levels in patient serum.

Adalimumab is a TNF specific monoclonal widely used therapeutically. Monitoring adalimumab levels is important for guiding treatment strategies and is predominantly performed using an ELISA. The homogeneous mobility shift assay (HMSA) has many advantages over an ELISA for adalimumab monitoring but current HMSA methodologies do not discriminate between adalimumab and other TNF specific monoclonals such as infliximab. The development and validation of a competitive binding HMSA (cHMSA) specific for adalimumab is reported here. The cHMSA had a lower limit of quantitation of 1.25 µg/ml and the intra-assay and inter-assay coefficents of variation (CV) were <20%. No signal was detected in adalimumab naïve control serum including those containing rheumatoid factor or infliximab. The majority (14/20) of adalimumab patient samples containing anti-adalimumab antibodies gave a cHMSA signal >3 standard deviations lower than the controls. The performance of the cHMSA and an ELISA was compared using adalimumab patient samples (n = 82). There was a strong correlation between the assays (r = 0.91) and the intra-class correlation coefficient (0.88) was indicative of good-excellent inter-assay reliability. Bland-Altman plots showed little overall bias and comparison of the sub-groups defined using cut-points (1.25 or 7.3 µg/ml) gave percent agreement (>90%) and Cohens kappa (95% CI: 0.61-0.93) values indicative of substantial-almost perfect agreement. These results demonstrate that cHMSA provides an accurate and specific method for monitoring adalimumab levels and can additionally provide an initial screen for the presence of anti-adalimumab antibodies.

Infliximab and adalimumab concentrations and anti-drug antibodies in inflammatory bowel disease control using New Zealand assays.

BACKGROUND: Therapeutic drug monitoring of anti-tumour necrosis factor (TNF) drugs and anti-drug antibodies (ADA) is now recommended in the treatment of inflammatory bowel disease. However, assay types and drug concentration thresholds are still debated. AIM: To correlate inflammatory bowel disease activity in a New Zealand cohort with trough concentrations of infliximab and adalimumab, and ADA using locally developed competitive-binding enzyme-linked immunosorbent assays (ELISA) to establish threshold concentrations. METHODS: Patients with ulcerative colitis (UC) and Crohn disease (CD) from Christchurch and Dunedin on anti-TNF drugs >12 weeks were enrolled. Trough blood samples were assayed for drug and ADA concentrations. Other data included quality of life, blood count, C-reactive protein, albumin, renal function and disease activity indices. RESULTS: Of 103 patients, 53 were on infliximab (36 CD, 15 UC and 2 unclassified) and 50 adalimumab (48 CD and 2 UC). Median (range) infliximab and adalimumab concentrations were 10.5 (0-41) and 9.61 mg/L (0-30). CD remission, Crohn Disease Activity Index <150, correlated with infliximab and adalimumab concentration in CD (infliximab, P = 0.03; adalimumab, P = 0.04), with too few UC patients for analysis. Receiver operator curve analysis suggested a threshold value of 5.1 mg/L for distinguishing active disease from remission for infliximab and 7.3 mg/L for adalimumab in CD. Of 13 patients with infliximab <2 mg/L, 10 were ADA positive by homogeneous mobility shift assay (HMSA), including five with neutralising antibodies using ELISA. Of six with adalimumab <2 mg/L, three were ADA positive using HMSA, including one with neutralising antibodies. CONCLUSION: Using the New Zealand ELISA assay, threshold concentrations of 5 mg/L for infliximab and 7 mg/L for adalimumab are suggested to aid dosing decisions, consistent with results internationally. Both neutralising (ELISA) and non-neutralising ADA (HMSA) are associated with low drug concentrations.

Discrimination of Anti-drug Antibodies With Neutralizing Capacity in Infliximab- and Adalimumab-Treated Patients: Comparison of the Homogeneous Mobility Shift Assay and the Affinity Capture and Elution Assay.

BACKGROUND: The measurement of anti-drug antibody (ADA) levels in adalimumab (ADAL)-treated and infliximab (IFX)-treated patients is critical for guiding therapeutic strategies. The homogeneous mobility shift assay (HMSA) and affinity capture elution (ACE) assay provide effective, drug-tolerant formats for measuring total ADA levels. However, their ability to discriminate between ADA from samples with or without neutralizing capacity is unclear and therefore was analyzed in this study. METHODS: Sera from ADAL and IFX patients with low drug levels (<1 mcg/mL) were analyzed by ACE, HMSA, and bridging assay. Neutralizing capacity was determined by competitive ligand-binding assay. RESULTS: HMSA and ACE detected high ADA levels in all ADAL (19/42) and IFX (27/64) samples with neutralizing capacity. ADA was also detected in most of the samples without neutralizing capacity, but levels were significantly lower (P < 0.0001). Receiver operator characteristic curve analysis demonstrated that for both assays, ADA levels were a strong discriminatory marker of neutralizing ADA (area under the curve > 0.9, P < 0.0001). Using a signal >8× background as a cut-point, neutralizing ADA could be identified with high specificity (HMSA > 95%, ACE > 85%) and sensitivity (HMSA > 70%, ACE > 80%). The detection of multimeric drug-ADA complexes after HMSA was also a highly specific marker (specificity > 95%) of neutralizing ADA in both ADAL and IFX patients. Results using ACE and HMSA were highly correlated. CONCLUSIONS: Results obtained after HMSA and ACE analysis are strongly correlated, and in both assays, high ADA levels are a specific marker of neutralizing capacity. The detection of multimeric complexes by HMSA also selectively identifies sera with neutralizing capacity. These data support the use of these assays as quantitative rather than simple qualitative measures of ADA.

Development of an ELISA-Based Competitive Binding Assay for the Analysis of Drug Concentration and Antidrug Antibody Levels in Patients Receiving Adalimumab or Infliximab.

BACKGROUND: There is increasing interest in measuring both drug and antidrug antibody (ADA) levels in patients receiving anti-tumor necrosis factor treatment as part of algorithms for guiding therapeutic strategies. Many of the current assays for ADA detection have limitations with respect to specificity, sensitivity, and/or laboratory requirements. Specific identification of ADA based on their inhibitory activity in a simple competitive binding assay remains problematic. The development of an enzyme-linked immunosorbent assay (ELISA)-based method for detection of both drug and ADA in patients receiving either adalimumab or infliximab would widen availability of monitoring for these patients. METHODS: An ELISA for the specific detection of adalimumab and infliximab using widely available reagents was developed. A simple modification for the detection of ADA capable of competitively inhibiting the in vitro binding of drug to solid phase tumor necrosis factor was also developed. Drug and ADA levels were analyzed in patients with rheumatoid arthritis and inflammatory bowel disease. RESULTS: The ELISA specifically detected drug concentrations in patient sera with no evidence of positive or negative interference by rheumatoid factor positive control sera. A subset of those patients with low drug concentrations had detectable levels of ADA with inhibitory activity in a competitive binding assay. Spiking with both drugs confirmed the specificity of the ADA detected. CONCLUSIONS: A modified ELISA protocol can be used to for the detection of both drug concentrations and ADA in patients receiving either adalimumab or infliximab. The ELISA incorporates those features identified in the literature as important for the accurate analysis of these antibodies and uses laboratory facilities and reagents that are widely available. It therefore provides a relatively simple and low cost assay for therapeutic drug monitoring of inpatients receiving adalimumab or infliximab.