Anti-drug Antibodies Testing Research Articles

Quantification of ADA of Humanized CD19 CAR-T Cell Therapy in R/R LBCL

Purpose: Humanized CD19 chimeric antigen receptor (CAR) T-cell therapy results in a high response rate in patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL). And many clinical trials have discovered the impact of anti-drug antibody (ADA) on CAR-T efficacy. But the ADA has yet been quantified. And its influence in patients with CAR-T infusion is still unknown. Patients and methods: We enrolled 30 patients with R/R LBCL treated with humanized CD19 CAR-T cells between April, 2022 and July, 2023, and collected their plasma at pre-FC, day7, 14, and follow up months for ADA testing using flow cytometry. Results: The detection of ADA within plasma of humanized CAR-T was performed using flow cytometry, and the relative quantification of ADA was achieved by setting up a calibration curve with CD19 protein as a standard. R 2 of the calibration curve can be 0.999 (Figure 1a). The physiologic threshold was determined as 0.1nM (95%CI) by using plasma from 10 normal subjects. 27/30 patients who experienced only one infusion and did not relapse had mostly low ADA (<3nM). Only one exception having a maximum value higher than 5nM with PFS maintained 1 year. All of the 27 patients had ADA presence before FC, but it did not affect CAR-T amplification. 3/30 patients who received second CAR-T cell infusion, 2 of which had 2 infusions of the same product, and 1 of which relapsed after relma-cel infusion and followed by the humanized product. 2 of the 3 patients (P08 and P09) who had a high ADA before the second infusion (of 3.52 and 3.27) had extremely limited CAR-T expansion after the second infusion (maximum CAR copies of 470 and 3950, respectively), and both relapsed 1 month after the second infusion (Figure 1b-1c). The other one patient (P18) had a low ADA both before and after the second infusion (maximum is before infusion, 0.65 nM), and had normal subsequent CAR copies amplification (maximum 60800 and minimum 10850), and did not relapse during the follow-up period (Figure 1d). We consider that ADA >3nM suggests poor CAR-T amplification and poor prognosis, but further validation is needed due to the small number of patients. Conclusions: The quantification of ADA is essential and credible, which contributes to the unification of standard. Pre-existing ADA does not affect CAR-T amplification for the first infusion but possibly suggests a negative impact on CAR-T efficacy and amplification of multiple-infusion. However, further research is needed.

B-095 Determination of Error Rates with Duplicate Analysis in Anti-Drug Antibody Testing

Abstract Background Duplicate measurements can be used to characterize assay imprecision, identify outliers, and estimate error rates. The benefits of duplicate analysis, however, need to be balanced against the increased financial cost. The objective of this study was to evaluate the need for continued replicate measurement in a laboratory developed assay measuring anti-drug antibodies to infliximab (ATI). To this end, duplicate patient sample measurements from the ATI assay over a two-year period were analyzed to calculate outlier and error frequencies. Methods ATI were measured by electrochemiluminescence (ECL) on the MesoScale Discovery (MSD) platform, with a standard curve from 20–1250 U/mL using commercially available monoclonal ATIs. All patient samples tested for ATI over a 27-month period of time (n = 9582) were subject to inclusion in this study. Only patient samples with ATI levels within the analytical measuring range (AMR) of the assay, however, were selected for further analysis. Samples were divided equally into 7 bins across the measuring range based on mean replicate concentration. Within-run imprecision was determined for each bin using the standard deviation (combined from all replicates using sum of squares) and mean concentration derived from all replicates. Outliers were defined as replicate measurements with a percent difference greater than ±3 SD of the calculated assay imprecision. Errors were defined as samples which had replicate values on opposite sides of the clinical cutoff of positive ATI (≥50 U/mL) and a percent difference greater than 3SD of the assay imprecision. Results Of the 9582 total patient samples analyzed, 2467 had ATI values within the AMR and 1655 had levels over the clinical cut-off of 50 U/mL (17.3% positivity rate). Within-run imprecision determined from the mean and combined SD of the duplicate measurements ranged from 15.7% at the lowest concentration bin (20–27.5 U/mL) to 10.0% at the highest concentration bin (358–1243 U/mL). The mean percentage of outliers in each bin ranged from 0.8%–3.4% resulting in an overall outlier frequency of 2.1% when considering all samples within the AMR. There were 66 samples within the AMR which demonstrated qualitative discrepancies between the replicates. However, only 21 (31.8%) of these samples, had a percent agreement greater than 3SD of the assay imprecision resulting in an overall error rate of 0.2% when considering all 9582 samples. Conclusion In this study, the analysis of replicate patient samples enabled the establishment of a precision profile and estimation of error rates for a laboratory developed test measuring ATI. The calculated error rate of 0.2%, however, is well within the reported range of most clinical assays indicating that the continued practice of duplicate measurement may not be necessary. Additionally, updating the laboratory protocol to eliminate replicate testing would have positive impact in laboratory workflow, cost, and turn-around-time for results.

Measuring Serum Vedolizumab and Vedolizumab Antibodies: Comparison of Commercial Assays with the Vedolizumab Clinical Development Assay.

Vedolizumab (VDZ) is an anti-α 4 β 7 integrin monoclonal antibody approved for inflammatory bowel disease treatment. VDZ serum and antidrug antibody (ADA) concentrations may be used for treatment optimization. In this article, the results of 5 commercial assays (Grifols, Immundiagnostik, Progenika, Sanquin, and Theradiag) measuring VDZ concentration and ADA were compared with those of the reference assays used in VDZ clinical studies. Our findings will assist clinicians in interpreting commercial assay results in the context of VDZ clinical trial data. VDZ-treated patient samples were used to evaluate the agreement between commercial assays and the reference VDZ serum concentration assay, based on linear regression, Bland-Altman, and qualitative agreement analyses. VDZ ADAs were detected using qualitative assays. Specificity, selectivity, accuracy, and precision were assessed using serum samples from healthy donors or patients with IBD (VDZ serum concentration <0.5 mcg/mL) spiked with VDZ, with/without other biologics (identical sample sets per assay). All assays were specific and selective for VDZ. Overall, the commercial assay results for VDZ-spiked samples correlated well with those of the reference serum concentration assay (R 2 ≥ 0.98). Compared with the Immundiagnostik and Theradiag assays, the Grifols, Sanquin, and Progenika assays had the best reference assay agreement (based on regression analysis, Bland-Altman plots, and qualitative agreement [Cohen's kappa ≥0.92]). All immunogenicity assays detected VDZ ADAs; only the reference assay detected VDZ ADAs in the presence of 15 mcg/mL VDZ, advising caution with commercial ADA assays if VDZ is present. All 5 commercial assays are suitable for VDZ therapeutic monitoring and ADA testing. However, the absolute values from the reference assays and the different commercial assays were not comparable, indicating that the same assay must be used for repeated monitoring of VDZ serum concentrations.

Development, Validation and Application of a Bridging ELISA for Detection of Antibodies against GQ1001 in Cynomolgus Monkey Serum.

Immunogenicity is a major issue associated with the PK, efficacy, and safety evaluation of therapeutic protein products during pre-clinical and clinical studies. A multi-tiered approach consisting of screening, confirmatory, and titration assays has been widely adopted for anti-drug antibody testing. GQ1001, a recombinant humanized anti-human epidermal growth factor receptor 2 monoclonal antibody covalently linked to a cytotoxin of DM1, possesses a novel format of antibody-drug conjugates. In this study, we reported the development, validation, and application of an acid-dissociation bridging enzyme-linked immunosorbent assay for the detection of antibodies against GQ1001 in cynomolgus monkey serum. The sensitivity of the screening assay was 126.141 ng/mL in undiluted serum. The screening assay and confirmatory assay were neither affected by the naïve monkey serum nor by 2% and 5% (v/v) erythrocyte hemolysates. Moreover, the assay was not subject to interference by 2500 ng/mL of human IgG1 in the samples. Drug interference at low positive control (150 ng/mL) and high positive control (8000 ng/mL) of anti-GQ1001 antibodies was not observed when GQ1001 concentrations were below 3.125 μg/mL and 100 μg/mL, respectively. Furthermore, no hook effect was observed for the positive antibodies in the concentration range of 8 to 64 μg/mL. The validated assay was, thereafter, successfully applied to a single-dose toxicity study of GQ1001. Anti-drug antibody positive rates among dosing animals and testing samples were reported, and no significant impact was found on toxicokinetic outcomes.

Antidrug Antibodies to Tumor Necrosis Factor α Inhibitors in Patients With Noninfectious Uveitis

Tumor necrosis factor inhibitors (TNFis) can induce antidrug antibody (ADA) formation and loss of therapeutic response. However, the utility of ADA testing and the association between ADAs and treatment response in patients with noninfectious uveitis (NIU) is not well understood. To assess the frequency of ADAs and their association with drug levels and clinical response in patients with NIU treated with adalimumab or infliximab. This retrospective cross-sectional study included patients diagnosed with NIU who received adalimumab or infliximab and underwent testing for serum drug level and ADAs at the National Eye Institute from September 2017 to July 2021. Serum drug level testing with reflex testing for ADA levels was performed. The main outcome was the association between drug levels and ADAs, clinical response, and concurrent antimetabolite use in patients treated with TNFis for NIU. Of 54 patients included in the study, 42 received adalimumab (mean [SD] age, 43.6 [19.6] years; 25 [59.5%] female) and 12 received infliximab (mean [SD] age, 42.7 [20.4] years; 7 [58.3%] male). In the adalimumab group, mean (SD) drug level was 9.72 (6.82) μg/mL, mean (SD) ADA level was 84.2 (172.9) arbitrary units/mL, and ADA frequency was 35.7% (15 of 42 patients). Mean drug level was lower in those with ADAs compared with those without ADAs (mean [SD], 2.8 [2.6] μg/mL vs 13.6 [5.2] μg/mL; difference: 10.8 μg/mL; 95% CI, 8.3-13.2 μg/mL; P < .001). There was a higher mean drug level with concurrent antimetabolite use compared with monotherapy (mean [SD], 11.0 [7.3] μg/mL vs 6.8 [4.5] μg/mL; difference: -4.2 μg/mL; 95% CI, -8.7 to 0.2 μg/mL; P = .06). Multivariable modeling showed that a 1-arbitrary unit increase in ADAs was associated with a -0.02 μg/mL (95% CI, -0.01 to -0.34 μg/mL) difference in mean drug level (P < .001). Favorable clinical response was associated with a threshold drug level above 2.7 μg/mL or an antibody level below 15.2 μg/mL. The mean (SD) drug level in the infliximab group was 27.02 (18.15) μg/mL, and no ADAs were detected. In this study, 35.7% of adalimumab-treated patients with NIU had ADAs. The presence of ADAs was associated with lower drug levels, and higher ADA levels were associated with increased risk of TNFi treatment failure. Although limited by the retrospective design, our results suggest that therapeutic drug monitoring may be considered among patients experiencing therapy failure to help exclude ADAs as a potential cause of treatment failure.

P001 What is the role of antidrug antibody and drug level testing in patients treated with infliximab and adalimumab?

Abstract Background/Aims Approximately 30-40% of rheumatology patients fail to respond to first-line biologics. Secondary inefficacy is mediated by immune complex formation between biologic agents and anti-drug antibodies. Anti-drug antibody testing has been undertaken at Sheffield Teaching Hospitals since October 2015. However, there are currently no national guidelines or consensus on what levels of anti-drug antibodies are clinically significant or what changes to therapy are suggested as a consequence of these tests. We aimed to review the reasons for and outcomes of anti-drug antibody levels tested at STH in patients on Adalimumab or Infliximab. Methods Retrospective review of records of all Rheumatology patients having antidrug antibody levels tested October 2015 - April 2019. Results 237 patients were included in this analysis. The mean age of patients was 48 years. 43% were male. The most common reasons for testing antibody levels were clinical evidence of a flare in disease (n = 92) and patient reported worsening of symptoms (n = 88). 66% (n = 157) of antibody levels tested were negative, 21% (n = 49) of tests were strongly positive (antibody titre &amp;gt;50). Serum drug concentrations were subtherapeutic in 20 % (n = 47), therapeutic in 22% (n = 51) and supratherapeutic in 38% (n = 91). In 51% of patients (n = 119) the current treatment regime was continued. However, 38% (n = 90) changed biologics, and dosing schedule was changed in 2% (n = 6). Antibody titres were more likely to be strongly positive in patients who had clinically active disease compared to those who had symptoms but no clinical evidence of disease (30% vs 10% p = 0.009). Those with strongly positive antibodies were more likely to switch biologics than those with normal antibodies (84% vs 28%, p = 0.01). Patients with clinically active disease but normal antibodies and drug levels were more likely to switch biologics than patients with no evidence of active disease but positive antibodies (p = 0.03). Underlying diagnosis (p = 0.23) or concomitant DMARD use (p = 0.92) were not associated with positive autoantibodies. Of the 47 patients with subtherapeutic drug levels, 61% (n = 29) had strongly positive anti-drug antibodies and 73% (n = 34) subsequently switched biologics. 49% (n = 111) of patients had both therapeutic drug levels and normal antibodies. Of these, 22% (n = 25) switched biologics. Of the 25 patients that switched biologics 24% (n = 6) did not have evidence of active disease and 76% (n = 19) had active disease. Conclusion 33% of patients had positive autoantibodies. 39% of patients switched biologics following testing. There was no protective effect of DMARDs identified. Patients with active disease were more likely to have positive antibodies and to switch biologics than those with no clinical evidence of disease. 25% of patients had subtherapeutic drug levels. However, only 2% of patients had a dose schedule adjustment. Therefore, dosing schedule alterations could be considered in these patients prior to escalating to a more expensive biologic. Disclosure M. Cox: None. R. Smith: None. G. Wild: None. L. Dunkley: None.

Immunogenicity of Biologic and Biosimilar Therapies for Psoriasis and Impact of Novel Immunoassays for Immunogenicity Detection.

Anti-drug antibodies (ADAs) may develop against originator biologic and biosimilar therapies used for the treatment of psoriasis and may be the cause of initial therapeutic non-response or diminished therapeutic response over time. Comparing immunogenicity between therapeutic agents is challenging owing to the variation in assays used for detection, among other reasons. Using the results of a PubMed search for psoriasis clinical trials disclosing the rates of ADAs for originator biologic and biosimilar therapies approved for the treatment of psoriasis within the last 5 years, this review discusses the rates and potential clinical impact of ADA formation in patients with psoriasis managed with originator biologic and biosimilar therapies, along with novel methods of ADA testing. Anti-drug antibodies are detectable in all biologic and biosimilar therapies approved for the treatment of psoriasis in the last 5 years, and the effect of ADAs on clinical response varies by agent. Novel immunoassays used for the detection of ADAs may have increased sensitivity compared with traditional assays, although the increased rate of detection may not correlate with decreased clinical response and the decision to test for the presence of ADAs may vary from patient to patient. Though ADA formation seems ubiquitous with the use of biologic agents for the treatment of psoriasis, the increased rates of ADAs detected by novel immunoassays may not necessarily correlate with decreased treatment efficacy.

Implementation of a three-tiered approach to identify and characterize anti-drug antibodies raised against HIV-specific broadly neutralizing antibodies

The ability to detect, quantify, and interrogate the properties of immune responses raised against biological therapeutics is not only important to our understanding of these molecules, but also to their success in the clinic. A tiered assay approach to identify the presence, specificity, and titer of anti-drug antibody (ADA) responses has been adopted as a gold standard by industry leaders, the FDA, and the EMA. In order to support pre-clinical and clinical trials, these assays must be standardized, and their performance sufficiently characterized to ensure the accuracy and reproducibility of results under relevant testing conditions. Here we present implementation of electrochemiluminiscence assays that fit into the tiered paradigm of ADA testing for five HIV broadly neutralizing antibodies (3BNC117, 3BNC117-LS, 10–1074, PGT121, and PGDM1400) in compliance with Good Clinical Laboratory practices. Assay sensitivities and matrix effects were evaluated and used to inform the development of positivity cut points. Once cut points were established, assay precision, specificity, free-drug tolerance, and robustness were defined. In all cases, assay characteristics met or surpassed recommendations set forth by the FDA. To further evaluate the performance of these assays and the tiered approach, samples from non-human primates that had received a subset of the five therapeutics were evaluated. In sum, this study reports qualification of a set of ADA assays available to the scientific community as pre-clinical and clinical trials of broadly HIV-neutralizing antibodies proceed, and a framework that is easily adapted as new drug products are advanced in the clinic.

Optimization and qualification of a functional anti-drug antibody assay for HIV-1 bnAbs

The recent identification of human monoclonal antibodies with broad and potent neutralizing activity against HIV-1 (bnAbs) has resulted in substantial efforts to develop these molecules for clinical use in the prevention and treatment of HIV-1 infection. As with any protein therapeutic drug product, it is imperative to have qualified assays that can accurately detect and quantify anti-drug antibodies (ADA) that may develop in patients receiving passive administration of HIV-1 bnAbs. Here, we have optimized and qualified a functional assay to assess the potential of ADA to inhibit the neutralizing function of HIV-1 bnAbs. Using a modified version of the validated TZM-bl HIV-1 neutralization assay, murine anti-idiotype antibodies were utilized to optimize and evaluate parameters of linearity, range, limit of detection, specificity, and precision for measuring inhibitory ADA activity against multiple HIV-1 bnAbs that are in clinical development. We further demonstrate the utility of this assay for detecting naturally occurring ADA responses in non-human primates receiving passive administration of human bnAbs. This functional assay format complements binding-antibody ADA strategies being developed for HIV-1 bnAbs, and when utilized together, will support a multi-tiered approach for ADA testing that is compliant with Good Clinical Laboratory Practice (GCLP) procedures and FDA guidance.

Rheumatology, the multitude of options

The number of therapeutic antibodies available in rheumatology increases every year, mainly indicated in chronic inflammatory rheumatisms and other immune-mediated inflammatory diseases. The choice between all these monoclonal antibodies depends on their specificities, patients and diseases characteristics. Until now, there is no reliable biomarker, predictive of response for specialized medicine purpose. Today, therapeutic drug monitoring and anti-drug antibodies testing can help to better adapt the dose of the drug and to help in the decision to switch to another biological drug according to the activity of the inflammatory disease.

Analysis of regulatory guidance on antidrug antibody testing for therapeutic protein products.

Therapeutic proteins have the potential to induce unwanted immune responses. The potential impact of immunogenicity on pharmacokinetics, pharmacodynamics, safety and efficacy are well established. Here, we analyze key aspects of current US FDA and EMA guidelines on the development and validation of antidrug antibody assays. Although FDA and EMA guidance documents are in harmony on most points, EMA allows greater leeway for scientific judgement, while FDA recommends specific approaches that may not be appropriate in some situations. Many white papers suggest approaches different from the guidance documents, however, these can conflict with each other and are themselves only scientifically valid in certain situations. Here, we indicate when alternatives to guidance may be needed and what those approaches might be.

Modeling Immunization To Infliximab in Children With Crohn's Disease Using Population Pharmacokinetics: A Pilot Study.

Antidrug antibodies (ADAs) dramatically increase infliximab clearance and are responsible for underexposure to the drug, leading to treatment failure. This pilot study aimed at developing a population pharmacokinetic model to detect and describe an early increase in infliximab clearance due to ADA. Twenty children with Crohn's disease (CD) were followed for 1 year or until treatment failure. Infliximab trough concentration, ADA, C-reactive protein (CRP), and Paediatric Crohn's Disease Activity Index (PCDAI) were recorded at each visit. A time-varying clearance population pharmacokinetic model was built to detect and describe an increase in infliximab clearance, independent from ADA testing. Factors associated with clearance variation and the relationships between infliximab concentrations, clearance variation, and clinical response were investigated. The model detected important increases in clearance in 4 patients. These patients had suboptimal early response, with higher mean PCDAI (P = 0.0086) and CRP (P = 0.028) compared with other patients. Two of them had detectable ADA. Clearance increase as described by the model and lower infliximab trough concentration at week 2 were associated with poorer outcomes in a multivariate Cox model (P = 0.001 and P = 0.0048, respectively). Being able to detect an increase in infliximab clearance, this model could allow the early detection of immunization to infliximab and therefore could help with dose adjustment in patients with CD. Moreover, the results suggest that clearance variations could be used as a predictive marker of clinical response. These findings need to be confirmed in a larger cohort, however, and predictive factors of clearance increase have to be investigated.

Drug Target Interference in Immunogenicity Assays: Recommendations and Mitigation Strategies

Sensitive and specific methodology is required for the detection and characterization of anti-drug antibodies (ADAs). High-quality ADA data enables the evaluation of potential impact of ADAs on the drug pharmacokinetic profile, patient safety, and efficacious response to the drug. Immunogenicity assessments are typically initiated at early stages in preclinical studies and continue throughout the drug development program. One of the potential bioanalytical challenges encountered with ADA testing is the need to identify and mitigate the interference mediated by the presence of soluble drug target. A drug target, when present at sufficiently high circulating concentrations, can potentially interfere with the performance of ADA and neutralizing antibody (NAb) assays, leading to either false-positive or, in some cases, false-negative ADA and NAb assay results. This publication describes various mechanisms of assay interference by soluble drug target, as well as strategies to recognize and mitigate such target interference. Pertinent examples are presented to illustrate the impact of target interference on ADA and NAb assays as well as several mitigation strategies, including the use of anti-target antibodies, soluble versions of the receptors, target-binding proteins, lectins, and solid-phase removal of targets. Furthermore, recommendations for detection and mitigation of such interference in different formats of ADA and NAb assays are provided.

Recommendations for Systematic Statistical Computation of Immunogenicity Cut Points.

Today, the assessment of immunogenicity is integral in nonclinical and clinical testing of new biotherapeutics and biosimilars. A key component in the risk-based evaluation of immunogenicity involves the detection and characterization of anti-drug antibodies (ADA). Over the past couple of decades, much progress has been made in standardizing the generalized approach for ADA testing with a three-tiered testing paradigm involving screening, confirmation, and quasi-quantitative titer assessment representing the typical harmonized scheme. Depending on a biotherapeutic's structural attributes, more characterization and testing may be appropriate. Unlike bioanalytical assays used to support the evaluation of pharmacokinetics or toxicokinetics, an important component in immunogenicity testing is the calculation of cut points for the identification (screening), confirmation (specificity), and titer assessment responses in animals and humans. Several key publications have laid an excellent foundation for statistical design and data analysis to determine immunogenicity cut points. Yet, the process for statistical determination of cut points remains a topic of active discussion by investigators who conduct immunogenicity assessments to support biotherapeutic drug development. In recent years, we have refined our statistical approach to address the challenges that have arisen due to the evolution in biotherapeutics and the analytical technologies used for quasi-quantitative detection. Based on this collective experience, we offer a simplified statistical analysis process and flow-scheme for cut point evaluations that should work in a large majority of projects to provide reliable estimates for the screening, confirmatory, and titering cut points.

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk. A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen "putative positive" samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β) and percentage of inhibition is calculated. The assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control. An ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

A Proposal to Redefine Clinical Immunogenicity Assessment.

With more than 100 therapeutic proteins (TP) approved since the first EMA guidance on immunogenicity in 2007, a vast amount of clinical experience with a variety of therapeutic proteins has been gained. This has provided data on anti-drug antibodies (ADA) and their observed clinical impact, or lack thereof. It has become evident that not all ADA responses are clinically relevant. The current "standard practice" is to test for ADA in all patients on every study. It is essential that we acknowledge the immunogenicity data gained from marketed TPs and that options for immunogenicity testing reflect this information. Improvements in bioanalytical support throughout the drug development process will eliminate extraneous, non-impactful practices. We propose that low-risk therapeutic proteins could be supported with an event-driven ("collect-and-hold") immunogenicity testing strategy throughout early phases of the clinical program. In the absence of an event, only pivotal studies (where ADA incidence and impact can be decisively assessed) would include default ADA testing. In keeping with the "standard practice," immunogenicity risk assessment must be an on-going and real-time evaluation. This approach has the potential to deliver meaningful, clinically relevant immunogenicity results while maintaining an emphasis on patient safety.

A microcosting study of immunogenicity and tumour necrosis factor alpha inhibitor drug level tests for therapeutic drug monitoring in clinical practice.

Objectives. To identify and quantify resource required and associated costs for implementing TNF-α inhibitor (TNFi) drug level and anti-drug antibody (ADAb) tests in UK rheumatology practice. Methods. A microcosting study, assuming the UK National Health Service perspective, identified the direct medical costs associated with providing TNFi drug level and ADAb testing in clinical practice. Resource use and costs per patient were identified via four stages: identification of a patient pathway with resource implications; estimation of the resources required; identification of the cost per unit of resource (2015 prices); and calculation of the total costs per patient. Univariate and multiway sensitivity analyses were performed using the variation in resource use and unit costs.Results. Total costs for TNFi drug level and concurrent ADAb testing, assessed using ELISAs on trough serum levels, were £152.52/patient (range: £147.68–159.24) if 40 patient samples were tested simultaneously. For the base–case analysis, the pre-testing phase incurred the highest costs, which included booking an additional appointment to acquire trough blood samples. The additional appointment was the key driver of costs per patient (67% of the total cost), and labour accounted for 10% and consumables 23% of the total costs. Performing ELISAs once per patient (rather than in duplicate) reduced the total costs to £133.78/patient.Conclusion. This microcosting study is the first assessing the cost of TNFi drug level and ADAb testing. The results could be used in subsequent cost-effectiveness analyses of TNFi pharmacological tests to target treatments and inform future policy recommendations.

Sa1972 Factors Influencing Infliximab and Adalimumab Antibody Formation and 6 Month Retrospective Follow up in a Tertiary IBD Centre

Introduction Infliximab(IFX) and adalimumab(Ada) are established treatments for IBD.However, immunogenicity can lead to anti-drug antibodies(ADAb) which is associated with a poor clinical response.Little is known regarding the factors which influence ADAb formation or the long term outcomes. Methods Patients with detectable ADAb between May 2012 and October 2014 were identified.Assays had been performed using LISA-Tracker DUO Kits which detects free drug levels(DLs) and ADAb only.Patients were analysed for disease phenotype, prior anti-TNF treatment and ADAb formation,and the use of concomitant immunomodulation(IM). Results 330 IFX DLs and ADAb levels from 199 patients and 143 Ada DLs and ADAb levels from 105 patients were analysed.21 positive ADAb, 19 IFX(9.5%) and 2 Ada(1.9%),were detected with a median ADAb of 141.8 U/ml(range 11–200),all DLs were >1.0μg/ml. 47.6% were male, age range 21–84,median 33.85.7% had Crohn’s Disease.In the majority of patients DL and ADAb were measured due to loss of response(66.7%). 6/19 patients who had IFX ADAb had prior exposure to IFX and 1 had prior exposure to Ada.Time to antibody detection ranged from 2–140 weeks(median 14).Of the 6 patients who had previous exposure to IFX,median time to ADAb detection was 6 weeks.Both patients who developed Ada ADAb had prior exposure to Ada,one also had prior exposure to IFX. 18/21 patients were prescribed concomitant IM(2 methotrexate, 16 thiopurines).8/16 on thiopurines had subtherapeutic TGNs( IFX ADAb group(n = 19):11 patients were switched to Ada, all of whom had a clinical response(mean HBI at week 26 = 2.1, no ADAb detected). 1 was switched out of class to vedolizumab and responded well. 3 stopped biologic treatment,1 patient continued on IFX at their own request and 1 patient was dose escalated but failed to respond. 2 patients had concomitant IM optimised,1 of whom on subsequent ADAb testing showed loss of ADAb. Ada ADAb group(n = 2):1 patient stopped anti-TNF due to recurrent infections and the other was lost to follow up. Conclusion 42.9% of patients with ADAb had been exposed to anti-TNF previously,all with a gap of >6 months prior to repeat exposure suggesting this is a risk factor for earlier ADAb formation. All patients switched to Ada from IFX responded well without developing ADAb,highlighting that switching to another anti-TNF is an acceptable option before switching out of class. Our use of concomitant IM is high but 50% of this cohort had subtherapeutic TGNs.The 2 patients in whom a thiopurine was optimised achieved clinical remission while remaining on IFX. However the level at which TGNs should be targeted in this context is still unclear Disclosure of Interest B. Warner: None Declared, E. Johnston: None Declared, J. Digby-Bell: None Declared, Z. Arkir: None Declared, N. Unsworth: None Declared, S. Anderson: None Declared, J. Sanderson: None Declared, P. Irving Grant/research support from: PI has been advisor to, in receipt of educational or research grants from, or invited lecturer for Abbvie, Falk, Ferring, Genetech, Hospira, Merck, Pharmacosmos, Shire, Takeda, Tillotts, Topivert, Vifor and Warner Chilcott, Paid instructor for: as above

Using monoclonal antibodies as an international standard for the measurement of anti-adalimumab antibodies

Comparing studies investigating anti-drug antibody (ADA) formation is hampered by the lack of comparability between study protocols, assay formats, and standardized reference materials. In this respect, the use of an international standard would mean a major step forward. Here we compared 11 fully human monoclonal antibodies against adalimumab in two assays commonly used for ADA measurement; the bridging ELISA and the antigen binding test (ABT). Our results show non-parallel titration of the monoclonal antibodies in both assays, which we also find for polyclonal ADA sources. Moreover, we observed that the output of the bridging ELISA depends to a large degree on the affinity of the monoclonal antibody. For the ABT, results reflect a combination of affinity and avidity. This suggests that rather than reporting ADA values in nanogram per milliliter, arbitrary units may be more appropriate. Together our data highlight the difficulty of ADA standardization by identifying several pitfalls that should be taken into account when selecting a standard for ADA testing.

Development of a Method That Eliminates False-Positive Results due to Nerve Growth Factor Interference in the Assessment of Fulranumab Immunogenicity

Fulranumab, a human IgG2 monoclonal antibody that neutralizes nerve growth factor (NGF), is currently in development for the treatment of pain. Our initial immunogenicity test method was found to be prone to NGF interference, leading to a high apparent incidence of anti-drug antibody (ADA) in phase 1 studies. The ADA immunoassay comprised a homogeneous bridging electrochemiluminescence (ECL) format with biotin and ruthenium-labeled fulranumab bound together ("bridged") by ADA in test samples for detection. In this assay, NGF produced a false-positive signal due to its ability to bridge fulranumab molecules. Thus, we developed a specificity assay to eliminate the NGF false-positive results. We encountered the challenge of eliminating drug interference as well as drug target interference, and discovered that the acid-dissociation-based pretreatment of samples used for mitigating drug interference dramatically increased drug target interference. Several strategies were investigated to eliminate the NGF interference; yet only one strategy specifically removed NGF and produced true fulranumab-specific ADA results by using competitive inhibition with fulranumab and utilizing an alternative NGF binding antibody to eliminate NGF interference. Using this new method, we confirmed that the high apparent anti-fulranumab antibody incidence (>60%) in clinical study samples was in fact due to fulranumab-bound NGF released during the acid-dissociation step of the ADA testing method. We conclude that our revised method accurately identifies anti-fulranumab antibodies by incorporating steps to eliminate fulranumab and NGF interference. We advise that acid-dissociation pretreatment must not be universally applied to improve ADA assays without investigating its bioanalytical risks versus benefits.