Thomas Van Stappen

Variability in Golimumab Exposure: A ‘Real-Life’ Observational Study in Active Ulcerative Colitis

BACKGROUND AND AIMS Golimumab has been approved recently to treat refractory moderate-to-severe ulcerative colitis [UC]. To date it is not clear why a considerable fraction of patients do not respond, or lose initial response, to golimumab therapy. Our aim was to investigate whether a low golimumab serum concentration and/or a positive anti-golimumab antibody status reduces the efficacy of this drug in patients with UC. METHODS Serum samples of 21 patients with moderate-to-severe UC were collected during the first 14 weeks of golimumab therapy. For measurement of golimumab serum concentrations, both a tumour necrosis factor [TNF]-coated enzyme-linked immunosorbent assay [ELISA] and a sandwich-type ELISA were developed. Anti-golimumab antibodies were measured using a bridging ELISA and a newly-developed drug-tolerant immunoassay. Clinical response and mucosal healing were assessed 14 weeks after start of treatment. RESULTS Out of 21 patients, 10 [48%] reached partial clinical response at Week 14. Median [interquartile range] serum golimumab concentration was significantly higher in partial clinical responders than in non-responders: 10.0 [7.8-10.5] µg/ml versus 7.4 [4.8-8.3] µg/ml at Week 2 [p = 0.035] and 5.1 [4.0-7.9] µg/ml versus 2.1 [1.8-4.2] µg/ml at week 6 [p = 0.037]. Four out of 21 UC patients developed anti-golimumab antibodies, detectable only using a drug-tolerant immunoassay, and three had a partial clinical response at that time. Clinical non-responders had a significantly more severe colitis, indicated by a higher endoscopic Mayo score at baseline compared with partial clinical responders [p = 0.048]. CONCLUSION Adequate exposure to golimumab drives clinical response. A worse disease at baseline influences clinical response rate negatively.

Generation of a highly specific monoclonal anti-infliximab antibody for harmonization of TNF-coated infliximab assays

Background: Determination of infliximab (IFX) serum concentrations has been used for treatment optimization of patients with inflammatory bowel disease. A wide range of enzyme-linked immunosorbent assays (ELISA) exists to quantitate IFX. Most of these assays lack specificity and cross-react with other anti-tumor necrosis factor (TNF) agents. The ability of these IFX assays to detect IFX in complex with antidrug antibodies is not known. The objective of our study was to develop an IFX-specific immunoassay to monitor IFX serum concentrations and to evaluate the impact of antidrug antibodies on the assay performance. Methods: A panel of monoclonal antibodies toward IFX (MA-IFX) was generated by hybridoma technology and evaluated to replace the polyclonal antibody in a TNF-coated IFX assay. The selected monoclonal antibody–based (MA-based) IFX ELISA was benchmarked to a clinically validated, reference polyclonal antibody–based (pAb-based) IFX ELISA using 209 inflammatory bowel disease serum samples. Results: Fifty-five MA-IFX were generated and grouped into 9 clusters. Of the 22 monoclonal antibodies tested, MA-IFX6B7 was selected for use in the IFX ELISA and the assay was further optimized. MA-IFX6B7 is a high-affinity (KD = 1.40E-09 mol/L), noninhibitory IgG1 antibody that binds to the Fab fragment of IFX and exhibits no cross-reactivity with other anti-TNF drugs. The linearity of an IFX dose–response curve was demonstrated in the range of 1.2–37.5 ng/mL (R2 = 0.988). The MA-based assay showed a good Pearson correlation (R = 0.986) and agreement (intraclass correlation coefficient = 0.985) with the pAb-based assay. The MA-based assay detects IFX in complex with nonneutralizing anti-IFX antibodies but not when complexed with neutralizing anti-IFX antibodies. Conclusions: In this study, a highly specific MA-IFX was developed as detection antibody in an ELISA to quantify IFX serum concentrations. The assay was benchmarked to the clinically validated reference pAb-based IFX ELISA.

Harmonization of Infliximab and Anti-Infliximab Assays Facilitates the Comparison Between Originators and Biosimilars in Clinical Samples.

Background:The availability of an infliximab ELISA for measuring the originator drug Remicade and its biosimilars, such as Remsima and Inflectra (CT-P13), would facilitate the implementation of therapeutic drug monitoring of biosimilars and enhance the extrapolation of treatment stratification algorithms established for Remicade. A universal calibrator for all anti-infliximab antibody bridging assays allows harmonization of anti-drug antibody titers. Methods:A panel of 55 mouse monoclonal antibodies raised against Remicade, including MA-IFX6B7 and MA-IFX10F9, were evaluated for their reactivity toward the biosimilars using a sandwich-type ELISA and surface plasmon resonance. To analyze the similarity of detection of the biosimilars and Remicade in the infliximab ELISA, quality control samples were used. Bridging assays to determine anti-infliximab antibodies were developed according to the bridging ELISA of Remicade using MA-IFX10F9 as calibrator. Serum of 36 patients treated with Remicade was analyzed for anti-infliximab antibodies toward Remicade, Remsima and Inflectra using their respective bridging ELISA. Results:MA-IFX6B7 and MA-IFX10F9 exhibit equal reactivity toward Remicade, Remsima, and Inflectra. The infliximab ELISA quantifies the biosimilars equally well as Remicade. Quantification of anti-infliximab antibodies in the serum of patients treated with Remicade revealed highly correlated titers between biosimilars and Remicade. Conclusions:The assay for therapeutic drug monitoring of Remicade can also be used to determine Remsima and Inflectra concentrations. Anti-drug antibody assays for biosimilars were developed. Anti-Remicade antibodies cross-react with infliximab biosimilars and reveal consistent negative/positive anti-drug antibody responses and highly correlated titers.

An Optimized Anti-infliximab Bridging Enzyme-linked Immunosorbent Assay for Harmonization of Anti-infliximab Antibody Titers in Patients with Inflammatory Bowel Diseases

Background:The formation of anti-infliximab antibodies (ATI) is associated with loss of response and adverse events in patients with inflammatory bowel diseases, leading to the introduction of ATI monitoring for guiding treatment adjustments. However, a lack of standardization among current available assays exists, hampering comparison of results from different studies. This study aimed to improve the harmonization of clinically validated ATI enzyme-linked immunosorbent assays (ELISAs) by introducing a monoclonal anti-infliximab antibody (MA-IFX). Methods:A panel of MA-IFX was evaluated as calibrator in the first generation ATI ELISA. After selection of 1 MA-IFX, assay conditions were optimized and biotin-streptavidin-enhanced detection of bound infliximab was introduced. The novel second generation ELISA was used for reanalysis of 127 serum samples from a cohort of 12 patients with inflammatory bowel disease, previously identified as ATI positive. Results:Of 55 MA-IFX, MA-IFX10F9 was selected as calibrator in the ATI ELISA. After optimization of the assay conditions, a 4-fold improvement in sensitivity was obtained. Reanalysis of 127 serum samples revealed that in 5 of 12 patients (46%), ATI were detected at least 1 time point earlier with the second generation ELISA compared with the first generation ELISA. In 1 patient, the second generation ELISA allowed to detect ATI before the reinitiation of IFX after a drug holiday. Conclusions:In addition to the improved sensitivity and specificity of the second generation ATI ELISA, MA-IFX10F9 can serve as a universal calibrator to achieve assay harmonization. Moreover, the superiority of the second generation assay in analyzing serum of restarters was demonstrated.

Clinical Relevance of Detecting Anti-Infliximab Antibodies with a Drug-Tolerant Assay: Post-HOC Analysis of the Taxit Trial

Objective To evaluate the clinical relevance of antidrug antibodies (ADAs) measured using a drug-tolerant assay in a post hoc analysis of the Trough Concentration (TC) Adapted Infliximab Treatment (TAXIT) randomised controlled trial. Design ADA in serum samples (n=221) of 76 patients enrolled in TAXIT, who presented with an infliximab TC <3 µg/mL at screening, were reanalysed after optimisation and at the end of the study using a drug-tolerant ADA assay. Patients underwent dose escalation to achieve therapeutic TCs between 3 µg/mL and 7 µg/mL prior to randomisation. Patients were grouped into quartiles (Q1–4) according to ADA concentration at screening. Results Using a drug-tolerant assay, the immunogenicity detection rate increased from 21% (drug-sensitive assay) to 63% at screening, from 0% to 51% after optimisation and from 3% to 42% at the end of TAXIT. Patients in ADA Q4 required a higher cumulative infliximab dose (2390 (880–2998) mg) to achieve target TCs, resulting in a higher drug cost (€10 712 (4120–13 596)) compared with ADA-negative patients (€2060 (1648–3296)) and patients in ADA Q1/Q2 (€2060 (1648–4120)/€2060 (1751–3296), p<0.001). However, all but one patient belonging to ADA Q4 were also ADA-positive using a drug-sensitive assay. Conclusions Upon dose intensification, low concentration ADAs, not detectable using a drug-sensitive assay, disappear in more than half of the patients over time and are clinically non-relevant. In contrast, high concentration ADAs which are typically also detected in a drug-sensitive assay, persist over time and necessitate a higher cumulative dose and drug cost. In the latter group, proactive drug switching may be more cost-efficient. Clinical Trials Register 2011-002061-38; Post-results.

Validation of a sample pretreatment protocol to convert a drug-sensitive into a drug-tolerant anti-infliximab antibody immunoassay

A meta-analysis revealed that up to 51% of patients treated with infliximab develop anti-drug Abs (ADA) which are associated with loss of response. Detection of ADA is strongly influenced by assay technique since drug-sensitive ADA assays are not able to detect ADA in the presence of drug and therefore underestimate ADA development. In addition, the lack of a calibrator antibody that can be used in a drug-sensitive and drug-tolerant assay hampers an adequate comparison among different assays. Here we present a sample pretreatment protocol to convert the bridging assay, originally developed as a drug-sensitive assay, into a drug-tolerant assay, allowing use of the same assay and calibrator antibody MA-IFX10F9. Using the sample pretreatment protocol, the bridging assay detects antibodies towards infliximab in samples containing up to 5-fold infliximab over anti-infliximab. Analysis of consecutive serum samples from infliximab treated patients revealed that the drug-tolerant assay detects ADA development up to 40 weeks earlier compared to the drug-sensitive assay. In conclusion, the sample pretreatment protocol can be implemented in various assay formats and allows determination of ADA in the presence of drug, providing the possibility for early treatment optimization. Copyright

Heterobifunctional PEG Ligands for Bioconjugation Reactions on Iron Oxide Nanoparticles

Ever since iron oxide nanoparticles have been recognized as promising scaffolds for biomedical applications, their surface functionalization has become even more important. We report the synthesis of a novel polyethylene glycol-based ligand that combines multiple advantageous properties for these applications. The ligand is covalently bound to the surface via a siloxane group, while its polyethylene glycol backbone significantly improves the colloidal stability of the particle in complex environments. End-capping the molecule with a carboxylic acid introduces a variety of coupling chemistry possibilities. In this study an antibody targeting plasminogen activator inhibitor-1 was coupled to the surface and its presence and binding activity was assessed by enzyme-linked immunosorbent assay and surface plasmon resonance experiments. The results indicate that the ligand has high potential towards biomedical applications where colloidal stability and advanced functionality is crucial.

Current practice for therapeutic drug monitoring of biopharmaceuticals in inflammatory bowel disease

Abstract: Since the late 90s, biopharmaceuticals targeting tumor necrosis factor alpha have revolutionized the treatment of moderately to severely active inflammatory bowel disease. The robust efficacy witnessed in many patients stands in stark contrast with the observation of a proportion of patients who fail to respond or who lose response over time. Therapeutic drug monitoring has been proposed as a means to understand and respond to the variability in clinical response and remission. Various treatment algorithms have been proposed, but optimal use of these measurements in daily practice awaits additional prospective validation trials. This review provides an updated overview on the subject of therapeutic drug monitoring of biopharmaceuticals for the management of inflammatory bowel disease and how we could implement its concepts in a changing landscape.

Generation and characterization of a unique panel of anti-adalimumab specific antibodies and their application in therapeutic drug monitoring assays.

A number of assays are currently available to support therapeutic drug monitoring of adalimumab. A complete characterization of the assays and comparison of different assays has not been performed. The aim of this study, therefore, is to generate and characterize of a panel of monoclonal antibodies towards adalimumab (MA-ADM); to use this panel to develop novel assays to determine adalimumab concentrations; to assess the impact of tumor necrosis factor (TNF) and (non-)neutralizing antibodies on adalimumab detection and to compare the performance of assays. In total, ten specific MA-ADM were generated of which four revealed a neutralizing potency of >78%. At least six different clusters were identified using principal component analysis. MA-ADM40D8 was selected as detecting antibody to determine adalimumab in the TNF-coated ELISA (A) and the MA-ADM28B8/MA-ADM40D8 antibody pair was chosen for use in the MA-coated ELISA (B). The impact of TNF and (non-) neutralizing antibodies was similar in both ELISAs. Finally, serum samples of adalimumab-treated Crohns disease patients were collected and used for an external validation using the assay of Sanquin (C) and the apDia kit (D). All adalimumab assays showed excellent Pearson correlation: r=0.96 for A versus B, 0.96 for A versus C, 0.94 for A versus D, 0.97 for B versus C, 0.95 for B versus D and 0.94 for C and D. The excellent agreement with the two commercially available ELISAs allows harmonization of treatment algorithms in and between different hospitals/infusion centers.

Rapid Test for Infliximab Drug Concentration Allows Immediate Dose Adaptation

OBJECTIVES: Therapeutic drug monitoring of infliximab improves treatment outcomes, but available assays to monitor infliximab lack speed to implement treatment algorithms immediately. Our aim is to validate a rapid, lateral flow‐based assay (LFA) for quantitative determination of infliximab and to assess thresholds associated with mucosal healing in patients with ulcerative colitis. METHODS: Samples (n=190) from 29 anti‐tumor necrosis factor naive patients with ulcerative colitis starting infliximab induction therapy between June 2010 and February 2012 were prospectively collected. All patients had a Mayo endoscopic sub‐score ≥2 at baseline. Mucosal healing (MH), defined as a Mayo endoscopic sub‐score ≤1, was evaluated at week 10–14. Infliximab trough concentrations (TC) were determined with a novel LFA, which was benchmarked with the RIDASCREEN infliximab Monitoring (ELISA). RESULTS: The LFA showed an excellent agreement with enzyme‐linked immunosorbent assay (ELISA) for quantification of infliximab, as observed from Pearson and intraclass correlation coefficients of 0.95 and 0.95 during induction and 0.93 and 0.87 during maintenance therapy, respectively. In total, 45% of patients achieved MH. Using the LFA, week 14 TC ≥2.1 μg/ml (AUROC: 0.819, P=0.008) were associated with MH. After 2 years follow‐up, 77% of patients with MH were still receiving infliximab therapy vs. 25% of patients without MH. CONCLUSIONS: We validated a LFA for quantification of infliximab and identified TC associated with MH. With a time‐to‐result of 20 min, individual sample analysis and user‐friendliness, the LFA outplays ELISA as a rapid, accurate tool to monitor infliximab concentrations.