Anti-drug Antibody Concentrations Research Articles

Impact of immunogenicity on response to anti-TNF therapy in moderate-to-severe plaque psoriasis: results of the PREDIR study

Purpose: This study was conducted to examine the relationship between loss of clinical response to anti-tumor necrosis factor (TNF) therapy and the production of anti-drug antibodies (ADAs) and the potential effects of biologic immunogenicity.Materials and methods: This observational, non-interventional, cross-sectional study included patients with moderate-to-severe plaque psoriasis and secondary failure of adalimumab, etanercept and infliximab who were seen in the clinical practice setting. Clinical data and blood samples were collected after patient enrollment at the time that next doses of anti-TNF therapy were scheduled. ADA and serum drug concentrations were detected at a central reference laboratory using ELISA.Results: Among 137 enrolled patients, ADA were identified in 31/65 (48%), 0/47 and 8/19 (42%) of patients treated with adalimumab, etanercept and infliximab, respectively. The presence of ADA was associated with a slightly worse clinical response in adalimumab-treated patients (Physician Global Assessment score: 3.7 vs. 3.2, ADA-positive vs. ADA-negative patients [p < .05]; correlation between serum ADA titer and body surface area: r = .292 [p = .019]). Concomitant DMARDs were not associated with anti-TNF immunogenicity in any treatment group.Conclusions: Additional evidence is needed from studies of anti-TNF therapy in psoriasis for clinicians to gain a better understanding of the impact of immunogenicity on clinical response.

P637 Anti-infliximab antibody concentrations guide therapeutic decision-making in patients with Crohn's disease losing clinical response

Background: Loss of clinical response (LOR) to infliximab (IFX) maintenance therapy in patients with Crohn's disease (CD) may necessitate treatment intensification. We explored the pharmacokinetics and effectiveness of infusion interval shortening (IS) and dose doubling (DD) and whether IFX and antidrug antibody (ADA) trough concentrations (TC) can guide therapeutic decision-making. Methods: A retrospective cohort study was conducted, including 93 patients with CD who received a double dose IFX (10 mg/kg body weight) and/or a next infusion after a shortened interval following LOR during maintenance therapy. IFX TC and ADA were measured at consecutive time points just before (at T0) and after (at T+1) the treatment intensification. ADA were quantified using an in-house developed drug tolerant assay (1). We compared the short-term evolution of IFX exposure, immunogenicity, clinical (based on physician global assessment) and biological (based on CRP) response during DD, IS and combined DD+IS. Results: Overall, treatment intensification significantly increased the IFX TC from 1.2 μg/mL to 3.6 μg/mL (93 paired samples, p<0.0001) (Table 1). An ADA concentration below 481 ng/mL eq. predicted a therapeutic post-treatment TC (3 μg/mL, 100% specificity, 51% sensitivity, AUROC 0.83, p<0.0001). When ADA were undetectable, all treatment intensification interventions significantly increased the IFX TC and DD+IS resulted in a larger TC increase compared to DD alone (Table 1). When ADA were detectable but below the 481 ng/mL eq. cut-off, only DD significantly increased the IFX TC. When ADA were above the 481 ng/mL eq. cut-off, neither treatment intensification intervention was effective for increasing the IFX TC. A significant TC increase was associated with clinical response in patients undergoing IS, only when they had no detectable ADA (from 2.3 μg/mL [0.7–4.4] to 4.9 μg/mL [1.7–12.1], n=15, p=0.009). Even when ADA were detectable but below the 481 ng/mL eq. cut-off, a significant TC increase was associated with clinical and biological response and remission in patients undergoing DD (biological response: from 1.5 μg/mL [0.4–3.1] to 6.0 μg/mL [3.3–13.5], n=8, p=0.02; biological remission: from 1.7 μg/mL [0.3–3.4] to 4.0 μg/mL [3.2–13.7], n=7, p=0.03; clinical response: from 2.0 μg/mL [1.0–6.2] to 9.2 μg/mL [4.4–13.5], n=8, p=0.008). Table 1. Course of the IFX pharmacokinetics during treatment intensification All values are median [IQR], g/mL. Conclusions: DD is more effective than IS for restoring therapeutic TC and clinical and biological response and remission in patients with low ADA titers. When ADA titers are high, neither treatment intensification strategy is effective.

P226 Comparison of four different immunoassays for measuring golimumab and anti-golimumab antibody concentration in patients with ulcerative colitis

Background: Golimumab is an anti-TNF antibody with higher affinity for TNF compared to infliximab and adalimumab and higher stability compared to infliximab1. The PURSUIT trials showed a significant exposure-response relationship with golimumab in ulcerative colitis (UC). Low drug exposure may be related to primary and secondary non-response as described for infliximab and adalimumab. To assess if assay results obtained with different methods can be considered comparable, we conducted a comparison of different immunoassays measuring levels of golimumab and anti- drug antibodies (ADA). Methods: We included serum samples from 80 patients with UC recruited in an ongoing prospective observational study who started on golimumab treatment. Golimumab was quantified by either an anti-IgG detection antibody (Theradiag) or an antibody directed against the idiotype of golimumab (Sanquin and KU Leuven, Janssen R&D). Bridging drug-sensitive ELISA assays (Theradiag, Janssen R&D, KU Leuven), a bridging drug-tolerant ELISA assay (Janssen R&D), and a radioimmunoassay (Sanquin) were used to quantify ADA. Results: Median (IQR) serum golimumab levels were 4.5 (2.2–6.6), 3.5 (1.7–4.8), 4.9 (2.6–7.0), and 2.4 (1.2–4.0) μg/mL with Theradiag, Sanquin, KU Leuven and Janssen R&D assays, respectively. Median assay values are different between assays in head to head comparisons (p=0.0001), except for comparison of median levels between Theradiag and KU Leuven (p=0.155) and comparison of Sanquin and Janssen R&D assay values (p=0.055). With the Sanquin and Leuven assays, 84% of samples are in the same quartile of distribution of values; this overlap was 71% for Sanquin and Theradiag, 66% for Theradiag and Leuven assays, 68% for Theradiag and Janssen R&D, 81% for Leuven and Janssen R&D and 86% for Sanquin and Janssen R&D. All but not the Theradiag assays showed specificity for golimumab as these make use of an anti-idiotype antibody to golimumab for detection. We were not able to compare ADA concentrations determined by the different assays. However, ADA were detected in the two same patients by Theradiag, Sanquin and KU Leuven assays and in one of those two with the drug-sensitive Janssen R&D assay. ADA were detected in 27.5% of samples (22/80) with the Janssen R&D drug-tolerant assay. Table 1. Correlation between assays to measure Golimumab serum levels Conclusions: There is an acceptable correlation between golimumab serum concentrations measured with Theradiag, Sanquin, KU Leuven and Janssen R&D assays. The low number of ADA positive samples precludes statistical concordance between ADA values measured with the different assays.

Cost-effectiveness of routine measuring of serum drug concentrations and anti-drug antibodies in treatment of rheumatoid arthritis patients with TNF-α blockers.

Monitoring of anti-drug antibodies (ADAbs) or serum concentrations of biologicals in treatment of rheumatoid arthritis could provide an explanation for a loss of efficacy and help in the choice of subsequent medication. Current clinical practices do not generally include such monitoring of tumor necrosis factor (TNF)-α blockers on a routine basis. The main aims of this study were to estimate the probabilities of optimal and nonoptimal treatment decisions if infliximab or adalimumab drug trough level (DL) and ADAbs are tested or not in rheumatoid arthritis, and to model cost-effectiveness of performing such monitoring on a routine basis. Data on DLs and ADAbs concentrations were obtained in Finland from clinically requested monitoring analyses of 486 and 1,137 samples from patients on adalimumab and infliximab, respectively. DL was within the target range in 42% of samples from adalimumab- and 50.4% of infliximab-treated patients. ADAbs were detected in approximately 20% and 13.5% of samples from adalimumab- and infliximab-treated patients, respectively. ADAbs were found in 52.3% and 41.3% of those with low adalimumab or infliximab DLs, respectively. The monitoring data were incorporated into probabilities for making the optimal treatment decision. Economic impact of clinical decision-making was modeled in a short-term (3–6 months) scenario with 100 hypothetical patients. In the model, the combined measurement of DLs and ADAbs was cost-saving compared to the nontesting scenario when the monitoring results affected the treatment decision in at least 2–5 of 100 patients, a proportion which is easily exceeded in real-life clinical practice. This study indicates that routine monitoring of drug level and ADAbs is cost-beneficial in clinical practice, thereby improving the decision-making process in using TNF-α blockers.

The Utility of Infliximab Therapeutic Drug Monitoring among Patients with Inflammatory Bowel Disease and Concerns for Loss of Response: A Retrospective Analysis of a Real-World Experience.

Background. Infliximab (IFX) therapeutic drug monitoring (TDM) allows for objective decision making in patients with inflammatory bowel disease (IBD) and loss of response. Questions remain about whether IFX TDM improves outcomes. Methods. Patients with IBD who had IFX TDM due to concerns for loss of response were considered for inclusion. Serum IFX trough concentration and anti-drug antibody (ADA) concentrations were measured. Patients were grouped by TDM results: group 1, low IFX/high ADA; group 2, low IFX/low ADA; group 3, therapeutic IFX. Changes in management were analyzed according to groupings; remission rates were assessed at 6 months. Results. 71 patients were included of whom 37% underwent an appropriate change in therapy. Groups 1 (67%) and 2 (83%) had high adherence compared to only 9% in group 3. At 6 months, 57% had achieved remission. More patients who underwent an appropriate change in therapy achieved remission, though this did not reach statistical significance (69% versus 49%; P = 0.098). Conclusions. A trend towards increased remission rates was associated with appropriate changes in management following TDM results. Many patients with therapeutic IFX concentrations did not undergo an appropriate change in management, potentially reflecting a lack of available out-of-class options at the time of TDM or due to uncertainty of the meaning of the reported therapeutic range.

Evaluating and Reporting the Immunogenicity Impacts for Biological Products—a Clinical Pharmacology Perspective

Immunogenicity assessment is important for biological products due to potential impacts of immunogenicity on safety and efficacy. We reviewed the prescribing information and the FDA's clinical pharmacology review of 121 approved biological products for evaluating and reporting of immunogenicity data. Of the 121 products, 89% (n = 108) reported the incidence of immunogenicity and 49% (n = 59) reported immunogenicity impact on efficacy. However, only 26% (n = 31) reported whether the immunogenicity affected pharmacokinetics. A subset of 16 products reported effects of anti-drug antibodies (ADA) on both systemic clearance and efficacy; 8 of 16 products had increased systemic clearance coinciding with reduced efficacy, and 6 of 16 products had no changes in either clearance or efficacy. Factors contributing to infrequent reporting of the ADA effect on exposure and methods for determining the effect of ADA on exposure are summarized. Measuring ADA and drug concentrations concurrently over time enables the evaluation of ADA impact on pharmacokinetics. Within-subject comparison of concentration data (before vs. after ADA formation) is a useful alternative to between-subject (ADA+ vs. ADA-) comparison when sample size is limited or when the majority of subjects developed ADA. The biological complexity of immune responses presents challenges to quantifying the ADA impact on pharmacokinetics using model-based methods. Our findings support that pharmacokinetic exposure is more sensitive than efficacy endpoints for evaluating ADA effects. A decrease in drug concentration due to formation of ADA during treatment can serve as an early indicator for potential reduced efficacy occurring at a later time.

Pharmacokinetics of anti-TNF monoclonal antibodies in inflammatory bowel disease: Adding value to current practice.

Since anti-tumor necrosis factor (TNF) antibodies were introduced to treat patients with inflammatory bowel diseases, short- and long-term clinical and endoscopic endpoints can be achieved that were unreachable with conventional anti-inflammatory agents. Although a large proportion of patients (70-90%) initially respond to the treatment, remission rates after induction are still low (20-50%) and patients are at risk to lose response to the drug over time. This inter-individual variability in response is likely to be influenced by the observed inter-individual variability in pharmacokinetics. By extensively reviewing the literature, we evaluated the potential role of therapeutic drug monitoring to optimize dosing of anti-TNF drugs. Thereby we emphasize some of the pharmacokinetic cornerstones that can help to understand the observed concentration-effect relationship. After discussing some of the most commonly used assays to measure anti-TNF drug and anti-drug antibody concentrations, we reviewed the application of those tests and their potential clinical value in retrospective and prospective studies.

Ulcerative Colitis: What is the Optimal Treatment Goal and How Do We Achieve It?

The treatment paradigms and therapeutic options for ulcerative colitis (UC) have rapidly evolved during the past decade. Traditionally, the treatment target has focused on achieving successful induction and maintenance of steroid-free clinical remission. This has been shown to provide a better quality of life and a reduction in complications, hospitalizations, and surgery. Recent studies, however, suggest that achieving "mucosal healing" or endoscopic remission may be the optimal treatment endpoint. In this review, we will examine the treatment goals for UC and the efficacy of each therapy to reach these targets. We will also review the therapeutic options available for UC: mesalamines, steroids, immunomodulators, and biologics, including the first anti-integrin inhibitor, approved in May 2014, for the treatment of UC. Therapeutic drug monitoring, which measures serum drug level and anti-drug antibody concentrations, is emerging as an important clinical decision tool in patients on tumor necrosis factor (TNF)-antagonists. These evolving treatment strategies allow gastroenterologists to optimize control of the disease and offer patients a better quality of life.

Role for therapeutic drug monitoring during induction therapy with TNF antagonists in IBD: evolution in the definition and management of primary nonresponse.

: Primary nonresponse and primary nonremission are important limitations of tumor necrosis factor (TNF) antagonists, occurring in 10% to 40% and 50% to 80% of patients with inflammatory bowel disease, respectively. The magnitude of primary nonresponse differs between phase III clinical trials and cohort studies, indicating differences, e.g., in definition, patient population or blinding. The causes of nonresponse can be attributed to the drug (pharmacokinetics, immunogenicity), the patient (genetics, disease activity), the disease (type, location, severity), and/or the treatment strategy (dosing regimen, combination therapy). Primary nonresponse has been attributed to "non-TNF-driven disease" which is an overly simplified and potentially misleading approach to the problem. Many patients with primary nonresponse could successfully be treated with dose optimization during the induction phase or switching to another TNF antagonist. Therefore, primary nonresponse is frequently not a non-TNF-driven disease. Recent studies from rheumatoid arthritis and preliminary data from inflammatory bowel disease evaluating therapeutic drug monitoring have suggested that early measurement of drug and anti-drug antibody concentrations could help to define primary nonresponse and rationalize patient management of this problem. Moreover, a modeling approach including pharmacological parameters and patient-related covariants could potentially be predictive for response to the treatment. We describe an overview of this evolution in thinking, underpinned by previous findings, and assess the potential role of early measurement of drug and antidrug antibody concentrations in the definition and management of primary nonresponse.

AB0437 Clinical Utility of Measuring Drug and Anti-Drug Antibody Concentration of Biologic Agents in Rheumatoid Arthritis Patients with Moderate and High Disease Activity

BackgroundBiologicals possess a large variation in pharmacokinetics (1). Subtherapeutic drug levels and formation of anti-drug antibodies (ADAb) decrease their efficacy in rheumatoid arthritis (RA) patients (2). Measuring these can become...

Extent and Consequences of Antibody Formation Against Adalimumab in Patients With Psoriasis

In a previously reported cohort of 29 patients with plaque-type psoriasis followed up for 24 weeks, clinically relevant antidrug antibody (ADA) to adalimumab was frequently found. Long-term data were lacking. We now present the extension of this study: 80 patients followed up for 1 year. To assess the extent of ADA and its clinical consequences after 24 weeks of adalimumab treatment for psoriasis in a cohort of 80 patients. A multicenter cohort study, performed in the outpatient dermatology clinic of 2 academic hospitals, included 80 sequential patients receiving adalimumab therapy for plaque-type psoriasis and had a follow-up of 1 year. Outcome assessors were not aware of the presence of antibodies to adalimumab or the adalimumab serum concentration when assessing patients' Psoriasis Area and Severity Index (PASI), and personnel analyzing serum samples were blinded to patients' PASI. For 80 patients treated with adalimumab for psoriasis, disease severity (PASI) was assessed, blood samples were collected, and adalimumab and ADA concentrations was determined at baseline and at weeks 12, 24, and 52. Patient PASI and adalimumab and ADA concentrations. Antidrug antibody formed in 49% of patients, before week 24 in 90% of them. Adalimumab and ADA concentrations, clinical response and ADA concentration, and adalimumab concentration and clinical response had correlations of -0.872, -0.606, and 0.519, respectively. The adalimumab dose interval was shortened because of lack of efficacy in 15 patients, 7 with and 8 without ADA; improvement in responder status occurred in 1 of 7 and 4 of 8, respectively. Patients with no ADA formation in the first 24 weeks of treatment have little chance of it in the following 24 weeks. The presence of ADA is strongly correlated with adalimumab concentration and greatly influences clinical response. If ADA is present, dose interval shortening is less useful.

Current and Future Status of Therapeutic Drug Monitoring in the Treatment of IBD

Although tumor necrosis factor (TNF)-antagonists are highly effective agents for the treatment of inflammatory bowel diseases (IBD), up to 40% of patients ultimately lose response. As a result of the limited availability of treatment options, empiric dose escalation or switching of TNF-antagonists have evolved as strategies to regain response. Recently, therapeutic drug monitoring (TDM) (measurement of serum drug and antidrug antibody concentrations) has emerged to facilitate informed decision-making in patients with secondary loss of response to a TNF-antagonist. The evidence supporting the use of TDM in clinical practice and future applications of this approach will be discussed.

Anti‐adalimumab and anti‐infliximab antibodies developed in psoriasis vulgaris patients reduced the efficacy of biologics: Report of two cases

Biologics such as tumor necrosis factor (TNF) inhibitors are highly effective for treating severe psoriasis, and an increasing number of patients benefit from them. However, subsidiary subjects have arisen in the course of treatment which may directly impact the continued use of these drugs in a given case. In this article, we report two cases of psoriasis vulgaris refractory to treatment with both adalimumab and infliximab. We measured the serum drug concentration and absorbance value of antidrug antibodies using the sandwich enzyme-linked immunoassay method. Antibodies to adalimumab and infliximab were detected in both patients. The low drug concentration and adverse clinical response observed corroborated these findings. Dosage and concomitant medication are considered to be important factors influencing the development of antibody formation. Therapeutic drug monitoring and measurement of antidrug antibodies will assist in maintaining a clinical response and in optimizing the response of patients being treated with biological therapies. The use of concomitant immunosuppressive medication should be considered for patients who will be treated with their second TNF inhibitor after having developed antidrug antibodies against their first TNF inhibitor.

Stratification of Antibody-Positive Subjects by Antibody Level Reveals an Impact of Immunogenicity on Pharmacokinetics

The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic. The antibody responses displayed a wide range of relative concentrations (30 ng/mL to >13 μg/mL) and peaked at various times during the study. To evaluate the impact of immunogenicity on PK, AMG 317 concentration data were analyzed following stratification by dose group, time point, antibody status (positive or negative), and antibody level (relative concentration). With dose group as a stratifying variable, a moderate reduction in AMG 317 levels (<50%) was observed in antibody-positive subjects when compared to antibody-negative subjects, but the difference was not statistically significant in all dose groups. The most significant reduction in AMG 317 levels was revealed when antibody data was stratified by both time point and antibody level. In general, high ADA concentrations (>500 ng/mL) and later time points (week 12) were associated with significantly (up to 97%) lower trough AMG 317 concentrations. The use of quasi-quantitative antibody data and appropriate statistical methods was critical for the most comprehensive evaluation of the impact of immunogenicity on PK.

Therapeutic Drug Monitoring of Tumor Necrosis Factor Antagonists in Inflammatory Bowel Disease

Although tumor necrosis factor (TNF) antagonists have shown clear benefits over conventional treatments for inducing and maintaining clinical remission in both Crohn's disease and ulcerative colitis, a high proportion of patients lose response over time. Given the scarce alternative of treatments when treatment failure occurs, it is highly desirable to optimize both initial response and long-term continuation of TNF antagonists. One of the most well-characterized factors associated with loss of response to these agents is the development of immunogenicity, whereby the production of neutralizing antidrug antibodies accelerates drug clearance, leading to subtherapeutic drug concentrations and, ultimately, to treatment failure. However, other patient-related factors, such as sex and/or body size, and disease severity, including TNF burden and serum albumin concentration among others, also may influence the pharmacokinetics of these agents. Nevertheless, the evidence generated to date about these complex interactions is scarce, and further prospective studies evaluating their influence on the pharmacokinetics of TNF antagonists are needed. Drug adjustment empirically based on clinical symptoms often is inaccurate and may lead to suboptimal outcomes. Recent evidence shows that maintenance of an optimal therapeutic drug concentration is associated with improved clinical outcomes. Therefore, incorporation of therapeutic drug monitoring into clinical practice may allow clinicians to optimize treatment by maintaining effective drug concentrations over time.

Humoral tumor-associated immune responses induced by catumaxomab in patients with malignant ascites.

2575 Background: Catumaxomab is the first EU approved bispecific (anti-EpCAM x anti-CD3) trifunctional antibody for the intraperitoneal (ip) treatment of malignant ascites (MA) in patients (pts) with EpCAM-positive tumors. Methods: In the ongoing phase IIIb study CASIMAS catumaxomab is investigated with/without prednisolone premedication in pts with MA due to epithelial cancer. Pts received four 3-hour infusions of 10, 20, 50 and 150 µg catumaxomab over 11 days. Plasma samples of 15 pts were quantified for autologous anti-EpCAM and anti-HER2 immunoglobulin (Ig) responses by ELISA. Additionally, MA samples were screened for EpCAM-positive tumor cells by immunocytochemistry and anti-drug antibody (ADA) responses were monitored. Results: At screening EpCAM-positive tumor cells were detected in the ascites fluid of all 15 pts. Thereof, 8 individuals (53%) showed a median 3-4 times increase of the initial anti-EpCAM Ig titer after treatment, whereas in 7/15 pts (47%) the titer did not increase. Although anti-EpCAM Ig screening values varied largely (2 -855 ng/ml) no significant distinction between the anti-EpCAM responder group (R) and non-responders (NR) was measured (p = 0.96; Mann Whitney test). However, there was a significant difference in the mean ADA concentrations of available samples determined 7 or 8 d after the last catumaxomab infusion (27,103 [R, n=5] vs. 910 ng/ml [NR, n=5]). Moreover, in 5/11evaluable pts (45%) HER2 -specific Ig could be quantified. In contrast to the augmented anti-EpCAM antibody levels which rapidly appeared (d 10) anti-HER2 responses developed de novo, started delayed (d 18) and peaked at the last sample collection (d 38, median=17 ng/ml). Interestingly, available samples of 2 pts who received a second treatment cycle after recurring MA displayed an anti-HER2 Ig booster reaction with peak levels of 48 and 209 ng/ml. Conclusions: The results indicate an active immunization induced by catumaxomab. Most importantly, the humoral immune response was not restricted to the EpCAM targeted but antigen spreading to the non-targeted HER2 tumor-associated antigen occurred. The impact of this immunization effect for the therapeutic benefit will be further investigated.

Anti-Drug Antibody Responses to Lumiliximab Are Not Detected in Relapsed Refractory CLL Patients Treated with Lumiliximab in Combination with FCR in a Phase 1/II Study

Lumiliximab, an anti-CD23 monoclonal antibody that binds specifically to human CD23, is under investigation for the treatment of patients with relapsed refractory chronic lymphocytic leukemia (CLL). The clinical safety monitoring for Lumiliximab includes testing for the presence of anti-drug antibodies (ADAs) in patient serum samples. In order to determine if patients develop an ADA response to Lumiliximab, serum samples were collected at specific time points throughout a Phase I/II study (152–30) of Lumiliximab in combination with FCR in relapsed CLL. Patient sera from this Phase I/II study were tested in a standard bridging ELISA in which samples were added to a drug coated plate and ADAs were detected using conjugated drug and colorimetric detection. Validation results showed the sensitivity of the assay was 1000ng/ml using a purified polyclonal rhesus anti-Lumiliximab antibody in undiluted human sera. ADAs were not detected in any patients that had evaluable post treatment (Week 13, Week 25, Week 41, and Month 12 post first dose) serum samples in the 152–30 study. Over the last few years, there has been an effort in the immunogenicity field to develop ADA assays with increased sensitivity and to adapt formats that, in general, have increased drug tolerance, i.e. reduction of drug interference in the assay. Therefore, a new ADA assay was developed using Meso Scale Discovery (MSD) technology that has become the reliable and current method for detection of ADA responses and incorporates an acid dissociation step that has been shown in literature to improve drug tolerance. Using the new method the same evaluable samples from the phase I/II study were tested to determine if any previously unseen ADA responses could be detected. The samples were incubated with biotinylated and ruthenylated drug, added to a streptavidin coated plate and ADAs were detected using electrochemiluminescence. Validation results show that the newly developed MSD assay has sensitivity of 250ng/ml using a purified polyclonal chicken anti-Lumiliximab antibody in undiluted human sera. All the samples tested in the MSD assay (n=32) were negative despite greater sensitivity than the ELISA method and an acceptable drug tolerance at the time points samples were drawn. PK concentrations at the specified ADA collection times ranged between 0μg/ml and 114μg/ml whereas drug tolerance ranges from 1μg/ml to 200μg/ml depending on ADA concentrations. Drug interference is a major factor in not being able to detect antidrug responses, yet several of the corresponding post dose PK time points showed low to no drug levels and yet no anti-drug antibodies were detected. These data imply that Lumiliximab when administered in combination with FCR has a low immunogenicity rate.

Detection of High- and Low-Affinity Antibodies Against a Human Monoclonal Antibody Using Various Technology Platforms

The effect of monoclonal antibody (mAb) affinity on the detection limit of enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and electrochemiluminescence (ECL) methods was evaluated using a panel of murine mAbs with affinities ranging from 0.057 to 340 nM. M1 and M7 are anti-idiotypic mAbs against a human mAb, ABX10, with dissociation equilibrium constant (KD) values of 0.057 and 7.2 nM, respectively. HP6030 and HP6002 are anti human IgG mAbs with KD values of 30 and 340 nM, respectively. The limit of detection (LOD) for these mAbs was determined using ELISA, SPR, and ECL technologies and was generally correlated with the rank order of their affinities. The LODs for M1, M7, HP6030, and HP6002 by ELISA were 17 +/- 13, 26,000 +/- 9,020, 344,000 +/- 271,000, and 792,000 +/- 1,050,000 ng/ml, respectively. According to an industry-suggested detection limit of 500 ng/ml, the ELISA was not sensitive enough for detecting M7, HP6030, and HP6002, demonstrating its limitation for detection of low- affinity mAbs. The SPR method lowered the LOD for M7 to 3,900 ng/ml, which was above the industry requirement. The ECL method lowered the LOD for all antibodies tested. Importantly, the ECL method lowered the LOD for M7 to 570 +/- 370 ng/ml, which is close to the industry requirement. Since the ECL method had demonstrated a high serum tolerance, its detection capability may be improved by using a higher percentage of serum in the assay matrix. Although a hook effect was observed with ECL methods, the methods could still detect anti-drug antibody (ADA) concentrations greater than 1 mg/ml, which minimizes concerns that high-titer ADA responses could be missed. The results demonstrated the superiority of an ECL method in detecting high- and low- affinity antibodies when compared to the ELISA and SPR methods.