Anti-drug Antibody Assay Research Articles

The Immunogenicity of Biologic Therapies.

Virtually all therapeutic proteins (biologics) elicit an immune response with the consequent production of anti-drug antibodies (ADA). The majority of ADA to therapeutic monoclonal antibodies (mAbs) are directed against the antigen-binding site of the therapeutic mAb, and hence are neutralizing. This nature of the ADA response explains why fully human antibodies can still be highly immunogenic. The detection of ADA is technically challenging and all assays have limitations, namely a limited capacity in detecting ADA in the presence of a drug due to immune complex (IC) formation, which may underestimate the ADA incidence. Refined assays, able to disrupt drug-ADA ICs, have revealed the presence of ADA in a higher proportion of patients. The great heterogeneity among ADA assays prevents a direct comparison of immunogenicity between different molecules and across studies. The formation of drug-ADA ICs can significantly alter pharmacokinetics and directly reduce drug efficacy if the ADA titer (i.e., concentration) is sufficiently high and persistent. In patients with low ADA titer, free drug concentrations may remain high enough to be effective, while in patients developing high ADA titer a substantial part of the drug will be neutralized and clinical non-response is likely to occur. ADA can also increase the risk of adverse events, namely hypersensitivity reactions. Several studies have revealed the presence of ADA before a clinically overt adverse reaction, highlighting their predictive value. Algorithms integrating therapeutic drug monitoring and immunogenicity information in the current clinical evaluation of patients receiving biologics are today available to guide therapeutic decisions in clinical practice, helping us to design safer and more cost-effective therapeutic strategies.

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.

Review article: consensus statements on therapeutic drug monitoring of anti-tumour necrosis factor therapy in inflammatory bowel diseases.

Therapeutic drug monitoring (TDM) in inflammatory bowel disease (IBD) patients receiving anti-tumour necrosis factor (TNF) agents can help optimise outcomes. Consensus statements based on current evidence will help the development of treatment guidelines. To develop evidence-based consensus statements for TDM-guided anti-TNF therapy in IBD. A committee of 25 Australian and international experts was assembled. The initial draft statements were produced following a systematic literature search. A modified Delphi technique was used with 3 iterations. Statements were modified according to anonymous voting and feedback at each iteration. Statements with 80% agreement without or with minor reservation were accepted. 22/24 statements met criteria for consensus. For anti-TNF agents, TDM should be performed upon treatment failure, following successful induction, when contemplating a drug holiday and periodically in clinical remission only when results would change management. To achieve clinical remission in luminal IBD, infliximab and adalimumab trough concentrations in the range of 3-8 and 5-12μg/mL, respectively, were deemed appropriate. The range may differ for different disease phenotypes or treatment endpoints-such as fistulising disease or to achieve mucosal healing. In treatment failure, TDM may identify mechanisms to guide subsequent decision-making. In stable clinical response, TDM-guided dosing may avoid future relapse. Data indicate drug-tolerant anti-drug antibody assays do not offer an advantage over drug-sensitive assays. Further data are required prior to recommending TDM for non-anti-TNF biological agents. Consensus statements support the role of TDM in optimising anti-TNF agents to treat IBD, especially insituations of treatment failure.

Generation of assay positive controls for detection of isotype anti-drug antibodies for immunogenicity monitoring.

Development of drug-related surrogate positive controls for isotype anti-drug antibodies assay remains challenging. Efforts on antibody engineering or chemical crosslinking have been made. However, multiple epitope recognition, purity and stability are often of concern. To tackle these challenges, we used LC-SPDP/SMPB crosslinking to conjugate polyclonal anti-drug IgG and human immunoglobulin isotype (hIgE, hIgA, hIgM or hIgG4) with optimized conditions. The final product was a hybrid of anti-drug IgG cross-linked to human isotype immunoglobulin through stable thioether bond. The characteristics of the hybrids and their performance as assay positive controls were further evaluated. The results demonstrate that LC-SPDP/SMPB chemical crosslinking method is suitable for generating stable hybrids to be used as assay positive controls in isotype anti-drug antibodies assay.

Comparison of different immunoassay methods to detect human anti-drug antibody using the WHO erythropoietin antibody reference panel for analytes

Development of an appropriate assay to detect anti-drug antibody (ADA) is important for assessing immunogenicity to therapeutic protein products. However, characterizing ADA assay methods is difficult because human ADA as a reference standard is not available in most cases. We compared the analytical performance of three ligand-binding assay methods for ADA, namely, surface plasmon resonance (SPR), electrochemiluminescence (ECL), and biolayer interferometry (BLI) methods, by using the anti-erythropoietin (EPO) monoclonal antibody reference panel developed by the World Health Organization (WHO) in 2015. Dose-dependent binding responses were observed for all nine anti-EPO antibodies in the anti-EPO panel by the SPR and BLI methods. In contrast, the ECL method did not clearly detect binding of low-affinity anti-EPO antibodies. Regarding IgG2 and IgM antibodies derived from the same clone, IgG2 exhibited a higher binding response in the SPR assay, whereas the IgM binding response was higher than that of IgG2 in the ECL assay. In the case of the BLI method, there was no consistent pattern observed in the binding responses of IgG2 or IgM. Results of the anti-EPO antibody reference panel, which contains a variety of monoclonal antibodies, indicated that the ability to detect ADAs differed among these assay methods. Therefore, with ligand-binding assays, differences in assay platforms can affect the sensitivity and other characteristics of assays to detect ADAs. These results show that understanding the analytical performance of ADA assays is important for an appropriate assessment of immunogenicity. Our study also indicated the benefits of using the established human ADA reference panel to assess the assay methods for ADA detection.

Demonstrating biosimilar and originator antidrug antibody binding comparability in antidrug antibody assays: a practical approach.

Biosimilar drug development has brought new challenges to bioanalytical ligand-binding assays used to determine drug concentration, antidrug antibodies and neutralizing antibodies. One particular challenge is how to demonstrate that the antidrug antibody assay can adequately detect antibodies against both biosimilar and originator. In this paper, we review the current guidelines and literature for practical recommendations and present a gap analysis. Case examples of antibody binding comparability testing are presented, and the challenges and implications are discussed. Based on the lessons learned from our biosimilar assay applications, we recommend a bioanalytical comparability testing approach that is outlined and discussed.

Immunoassays for Measuring Serum Concentrations of Monoclonal Antibodies and Anti-biopharmaceutical Antibodies in Patients.

Monoclonal antibodies (mAbs) may be used as biopharmaceuticals to treat various diseases, ranging from oncology to inflammatory and cardiovascular affections. Trustworthy analytical methods are necessary to study their pharmacokinetics, both during their development and in post-marketing studies. Because biopharmaceuticals are macromolecules, ligand-binding assays (both immunoassays and bioassays) are methods of choice to measure their concentrations. Immunoassays are based on the capture of biopharmaceuticals by their target, which may be a circulating or membrane antigen or by an antibody recognizing their structure. Bioassays measure the activity of the biopharmaceutical in a specific in vitro test. A number of techniques have been reported, but their limits of detection and quantification vary widely. Anti-drug antibodies (ADA) against biopharmaceuticals are often formed and sometimes interfere with clinical efficacy. Accurate and reliable detection of ADA is therefore necessary. Binding of ADA is dependent on affinity and avidity, which makes quantification challenging. In this review, we discuss the benefits and limitations of each method to determine mAb levels and carefully compare ADA assays.

Immunogenicity of Therapeutic Antibodies: Monitoring Antidrug Antibodies in a Clinical Context.

One of the factors that may impact drug levels of therapeutic antibodies in patients is immunogenicity, with potential loss of efficacy. Nowadays, many immunogenicity assays are available for testing antidrug antibodies (ADA). In this article, we discuss different types of immunogenicity assays and their clinical relevance in terms of drug tolerance, relation with pharmacokinetics (PK), neutralizing antibodies, potential adverse events associated with ADA, and prediction of ADA production. Drug-tolerant assays can provide insight into the process of immunogenicity, but for clinical management, these assays do not necessarily outperform drug-sensitive assays. The usefulness of any ADA assay for clinical decision making will be larger when drug concentrations are also measured, and this is true, in particular, for drug-tolerant assays.

A systematic study of the effect of low pH acid treatment on anti-drug antibodies specific for a domain antibody therapeutic: Impact on drug tolerance, assay sensitivity and post-validation method assessment of ADA in clinical serum samples

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and pH also affect drug-specific signal differentially in individual samples. The results presented here represent the most extensive analyses to date on acid treatment of a wide range of ADA affinities to explore sensitivity and drug tolerance issues. They have led to a refinement of our current best practices for ADA method development and provide a depth of data to interrogate low pH mediated immune complex dissociation.

Novel drug and soluble target tolerant antidrug antibody assay for therapeutic antibodies bearing the P329G mutation.

Bridging immunoassays for detection of antidrug antibodies (ADAs) are typically susceptible to high concentrations of residual drug. Sensitive drug-tolerant assays are, therefore, needed. An immune complex assay to detect ADAs against therapeutic antibodies bearing Pro329Gly mutation was established. The assay uses antibodies specific for the Pro329Gly mutation for capture and human soluble Fcγ receptor for detection. When compared with a bridging assay, the new assay showed similar precision, high sensitivity to IgG1 ADA and dramatically improved drug tolerance. However, it was not able to detect early (IgM-based) immune responses. Applied in combination with a bridging assay, the novel assay serves as orthogonal assay for immunogenicity assessment and allows further characterization of ADA responses.

Bridging immunogenicity assays for IgG4 therapeutics: mitigating interference from Fc-Fc interactions.

A bridging immunogenicity assay for a human IgG4 mAb therapeutic was transferred to an automation system to increase throughput. However, background signal increased five- to six-fold during the 6- to 8-h run. Noncovalent Fc contacts formed between labeled IgG4 drugs in reagent solutions stored during the automation run. This generated substantial background signal, reducing assay sensitivity by approximately sixfold. Fc interactions also significantly impacted the confirmation assay. Fc contacts formed between labeled and unlabeled drug, significantly increasing signal inhibition (∼7-70%) in the 6-h run. Storing labeled antibody solutions separately and combining them immediately before adding to samples reduced interference from Fc interactions. Preincubation time for reagent solutions should be strictly controlled for anti-drug antibody assays with IgG4 drugs to avoid false-positive results.

Clinical relevance of detecting anti-infliximab antibodies with a drug-tolerant assay: post hoc analysis of the TAXIT trial

ObjectiveTo 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...

A biotin-drug extraction and acid dissociation (BEAD) procedure to eliminate matrix and drug interference in a protein complex anti-drug antibody (ADA) isotype specific assay

Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors.

P652 Unmet needs of physicians managing inflammatory bowel disease. A Survey of the Italian Group for Inflammatory Bowel Disease (IG-IBD)

Background: It is known that patients affected by Inflammatory Bowel Disease (IBD) present several unmet needs about the management of their illness and its consequences on their Health Related Quality of Life. However, little is known about the unmet needs of physicians caring IBD patients. The aim of the study was to investigate the difficulties and the problems of Italian physicians managing IBD. Methods: A questionnaire was submitted to physicians attending the National Congress and the Residential Courses of IG-IBD. Results: 280 physicians (156 men) completed the questionnaire. Mean age ± SD was 44.4±10.7 years; 95 (33.9%) were working in Academic hospitals. On a 5-point Likert scale, the most problematic issues in managing IBD patients were: increasing bureaucratic charge (3.9±1.2), lacking of extra-gastroenterological IBD expertise (3.4±1.4), lacking of diagnostic techniques (3.1±1.4), budget limitations (2.9±1.3), drugs safety (2.9±1.2), difficulties in guidelines application (2.7±1.2), pharmacy's limitations (2.2±1.1). Ranking from 0 to 9 the most lacking extra-gastroenterological IBD specialist figures led to the following classification: surgeon (2.5±2.5), nobody (3.3±3.4), rheumatologist (3.8±2.3), nutritionist (3.8±2.0), psychologist (3.8±2.2), dermatologist (4.2±2.0), pathologist 4.3±2.5), radiologist (4.8±2.6), stoma therapist (5.4±2.4). Ranking from 0 to 9 the most lacking techniques led to the following classification: nothing (1.6±1.8), anti-drug antibody and trough levels assays (2.7±3.0), enteroscopy (3.1±2.0), exploration under anesthesia of perianal disease (3.2±2.1), entero-MR (3.2±2.4), bowel ultrasonography (3.3±2.8), videocapsule endoscopy (3.9±2.1), entero-CT scan (4.4±2.1), fecal calprotectin (4.7±3.0). Conclusions: In Italy, several situations appear to potentially limit the best management of IBD patients. However, most of them appear to be correctable.

DOP029 Clinical relevance of detecting anti-infliximab antibodies with a drug-tolerant assay: post-hoc analysis of the taxit trial

Background: Anti-drug antibodies (ADA) may develop in up to 51% of patients treated with infliximab maintenance therapy and are associated with infusion reactions and impaired response. Drug-sensitive assays do not detect ADA in the presence of drug and underestimate ADA formation. Drug-tolerant assays have therefore been developed and markedly increased the detection of ADA, although their clinical relevance remains to be shown. Our goal was to evaluate the clinical relevance of anti-drug antibodies (ADA) measured using a drug-tolerant assay in comparison to a drug-sensitive assay in a post-hoc analysis of the Trough Concentration Adapted Infliximab Treatment (TAXIT) randomized controlled trial. Methods: Patients who presented with an infliximab trough concentration (TC) <3 μg/mL at screening (n=76) underwent dose escalation to achieve therapeutic TCs between 3–7 μg/mL prior to randomization. Serum samples were re-analyzed at screening, after optimization and one year after randomization using a drug-tolerant ADA assay. 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 optimization and from 3% to 42% one year after randomization. ADA concentration (median [interquartile range, IQR]) in ADA+ patients grouped into quartiles according to ADA concentration at screening, decreased throughout the study: from 220 [116–737] ng/mL eq at screening to 112 [78–180] ng/mL eq after optimization and to 95 [0–166] ng/mL eq at the end of the study. Patients in ADA Q4 required a higher cumulative infliximab dose (2390 [880–2998] mg) to achieve target TCs (Figure 1) compared to ADA negative patients (450 [365–680] mg, p<0.001) and patients in ADA Q1/Q2 (425 [344–863]/483 [398–719], p<0.001). Figure 1. Cumulative infliximab (IFX) dose required to achieve target concentrations between 3–7 μg/mL A) for ADA+ and ADA- patients, B) across ADA+ quartiles (Q1–Q4) and C) according to the assay (DT, drug-tolerant assay; DS, drug-sensitive assay). However, all but one patient belonging to ADA Q4 were also ADA positive using a drug-sensitive assay. Once dose optimized, patients have similar rates of clinical, biological and endoscopic remission after one year of treatment, regardless of ADA status in a drug-tolerant assay. Conclusions: Upon dose intensification, low concentration ADA, 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 ADA 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.

Inhibition of interleukin-5 induced false positive anti-drug antibody responses against mepolizumab through the use of a competitive blocking antibody

Mepolizumab, a humanized IgG1 monoclonal antibody that blocks native homodimeric interleukin-5 (IL-5) from binding to the IL-5 receptor, has recently been approved for treatment of severe eosinophilic asthma. Our initial immunogenicity assay method for phase I and II studies utilized a bridging electrochemiluminescence format with biotin and ruthenium-labelled mepolizumab linked by anti-drug antibodies (ADA). We discovered that IL-5 significantly increased in dosed subjects from a phase II study and that the increased IL-5 was in the form of a drug-bound complex. We demonstrated that the elevated drug-bound IL-5 produced false-positive response in the in vitro ADA assay, in which drug-bound IL-5 dissociated and then bridged mepolizumab conjugates to yield positive signal. To eliminate the IL-5 interference, we compared two strategies: a solid-phase immunodepletion of IL-5 and an in-solution IL-5 immunocompetition. We identified the best competitive antibody for each purpose. We found both methods demonstrated similar effectiveness in reducing the false positive signal in IL-5 spiked samples; however, the in-solution immunocompetition for IL-5 had fewer false positives in study samples. Additionally, the in-solution immunocompetition method was experimentally simpler to execute. We modified the ADA assay by adding a pre-treatment step with a mepolizumab competitive anti- IL-5 antibody. Using this new method, we retested clinical samples from two phase II studies (MEA112997 and MEA114092). The confirmed ADA positive incidence was reduced from 29% and 61% to 1% and 8% with the modified in-solution immune inhibition method. Target interference is a fairly common problem facing immunogenicity testing, and target-induced false positive cannot be distinguished from true ADA response by the commonly used drug competitive confirmation assay. The approach and method used here for resolving target interference in ADA detection will be useful for differentiating between a true ADA response and target induced false positive as well as similar challenges in other programs.

Mitigation of Pre-existing Antibodies to a Biotherapeutic in Non-clinical Species When Establishing Anti-drug Antibody Assay Cutpoint.

Biotherapeutics are known for their potential to induce drug specific immune responses, which are commonly evaluated by the detection of anti-drug antibodies (ADAs). For some biotherapeutics, pre-existing ADAs against drug have been observed in drug-naïve matrix. The presence of pre-existing drug specific antibodies may significantly complicate assessment of the screening ADA assay cutpoint value, which is usually established based on the statistical analysis of signal distribution from the drug-naïve individuals. A Gaussian mixture model-based approach is presented herein to address high prevalence of pre-existing ADAs to a modified monoclonal antibody-based biotherapeutic (m-mAb). A high prevalence of pre-existing anti-m-mAb antibodies was observed in drug-naïve individual cynomolgus monkey serum samples with signal ranging from 100 to 7000 relative light units (RLU, as determined in an electrochemiluminescence readout-based assay). Application of the industry standard statistical algorithm resulted in a relatively high floating screening assay cutpoint factor (CPF) of 9.80, which potentially would have reported a high percent of false negative samples. An alternative, Gaussian mixture model-based approach was applied to identify the least reactive individual samples in the tested population, which resulted in a floating screening assay CPF of 2.35. The low CPF value significantly reduced the risk of reporting false negative results. The proposed Gaussian mixture model-based approach described herein provides an alternate method for the calculation of biologically relevant screening assay CPF when high prevalence of pre-existing drug specific antibodies is observed.

Validation of a Drug-Resistant Anti-Adalimumab Antibody Assay to Monitor Immunogenicity in the Presence of High Concentrations of Adalimumab.

With respect to patient safety and long-term efficacy, immunogenicity of therapeutic antibodies remains an important issue. Pre-treatment of samples using either higher temperature or acidification in order to separate drug/anti-drug antibody complexes has been implemented in the traditional bridging assay and an in-house-developed affinity capture elution assay but only a limited drug tolerance was obtained. In this study, we aim to apply a drug-resistant anti-drug antibody assay to adalimumab through a combination of adalimumab/anti-adalimumab antibody complex precipitation and acid dissociation. A linear dose-response curve ranging from 3.1 to 200ng/mL was obtained in 1/125 diluted serum, allowing detection of anti-adalimumab antibody concentrations up to 25μg/mL equivalents MA-ADM6A10, a calibrator anti-adalimumab antibody. The cut-off point for detection was determined using 16 samples of adalimumab naïve patients and set at 0.39μg/mL equivalents. Validation of the assay revealed that no detectable anti-adalimumab antibody concentrations were found in samples with either a positive anti-infliximab antibody concentration, a physiologic concentration of TNFα, or a high concentration of rheumatoid factor. Full recoveries were obtained when various concentrations of adalimumab (0, 1, 10, and 50μg/mL) were spiked to 1, 2, and 4μg/mL of MA-ADM6A10. Spiking of 50μg/mL adalimumab to eight individual sera revealed similar anti-adalimumab antibody concentrations as in the absence of adalimumab, with a Pearson r correlation of 0.99 and an interclass correlation of 0.99. The assay allows accurate evaluation of adalimumab immunogenicity during induction or upon dose intensification and in serum samples not taken at trough.

Development, validation, and application of ELISA for detection of anti-HD105 antibodies in pre-clinical safety evaluation using monkeys

Unwanted immunogenicity of protein therapeutics can result in severe side effects and should be assessed in animals before applying the treatment to humans. Monkeys are the most relevant choice for pre-clinical toxicity testing of antibody-based therapeutics. To assess the immunogenicity of HD105, a novel antibody therapeutic that targets both vascular endothelial growth factor and Delta-like-ligand 4, a bridging enzyme-linked immunosorbent assay was developed as an anti-drug antibody (ADA) assay and validated for use in pre-clinical studies using non-human primates. This method was found to have suitable assay sensitivity, intra- and inter-assay precision, confirmation, drug tolerance, recovery, and sample stability for measuring ADA in monkey serum samples. The results showed that ADA elevation occurred following repeated doses of HD105, and that ADA production was negatively associated with serum HD105 concentration. These results suggest that intravenous administration of HD105 induces production of ADA in monkeys and that the detection of ADA may be negatively influenced by free HD105 in serum.

Detection of antidrug antibodies against human therapeutic antibodies lacking Fc-effector functions by usage of soluble Fcγ receptor I.

Bridging immunoassays for the detection of antidrug antibodies (ADAs) are limited to detection of bivalent molecules and are prone to interference by drug and soluble targets. Hence, alternative approaches for ADA detection are desired. Materials& methods: A novel ADA assay with secondary Fc detection using human soluble Fcγ receptor I (hsFcγRI) was established and compared with standard bridging assay. Both assays showed consistent results in human and cynomolgus monkey samples. In contrast to the bridging assay, the hsFcγRI-based assay was insensitive to the presence of oligomeric targets and appeared to have better drug tolerance. The hsFcγRI-based ADA assay can serve as alternative screening assay or as orthogonal confirmation method for preclinical and clinical immunogenicity testing of IgG therapeutics lacking Fc effector functions.