Anti-drug Antibody Assay Research Articles

P066 The relationship between serum adalimumab and corresponding anti-adalimumab antibody levels: analysis of over 20,000 patient results

BACKGROUND: Adalimumab (ADL) is a TNF inhibitor administered by injection. Serum assays of ADL and its anti-drug antibodies (ADAb) are utilized to manage failure to respond and loss of response. These monitoring assays may also be used to proactively titrate doses and dosing intervals (1). Here, 22,012 adalimumab (ADL) patient results were analyzed. METHODS: Measurements of drug and ADAb levels were performed by lab developed chemiluminescent immunoassays (2). The lower limits of quantitation are 0.6 μg/mL for adalimumab and 25 ng/mL for anti-adalimumab antibodies. Drug assays measure free (ADAb-unbound) drug while ADAb assays detect total antibodies (including IgM and IgG subtypes) and are drug- tolerant to the presence of up to >20 μg/mL of drug. Clinical histories and blood collection timing are unknown. RESULTS: Of 22,012 measured adalimumab samples, 60% (13,176) were ADAb-free and 40% (8,836) had measurable anti-adalimumab antibodies. Of all ADAb-free samples, the majority (10,171 or 77%) had ADL concentrations between 3 and 30 μg/mL while drug levels were <3 μg/mL in 2,495 (19%). Of ADAb-positive samples, most were positive in the low to intermediate titer range (25 to 200 ng/mL) Furthermore, as ADAb increase, their corresponding mean drug concentrations diminish. Low concentrations of ADAb (<100 ng/mL) appear to have minimal impact on mean drug levels where the baseline mean adalimumab (6.1 μg/mL) was calculated from 11,085 ADAb-free samples with adalimumab from 0.6 to 15 μg/mL. ADAb titers between 100 and 300 ng/mL are associated with a moderately diminished mean adalimumab (3.4–5.6 μg/mL) while high ADAb titers (>300 ng/mL) are invariably associated with diminished free ADL concentrations <30% of the mean. CONCLUSION(S): Of 22,012 samples, 60% were ADAb-free. Of those, about one third (37%) were within the target range of 5–10 μg/mL for adalimumab while 36% of samples were subtherapeutic at <5.0 μg/mL. Anti-adalimumab antibodies occurred in 40% of samples, and ADL drug concentrations were inversely related to anti-adalimumab antibody titers where low titer ADAb may have little or no impact on free drug levels while high titer ADAbs are associated with very diminished or absent drug. These findings are consistent with American Gastroenterological Association Critical Care Pathways for Crohn's Disease and Ulcerative Colitis where low and high antibody scenarios are managed very differently. It may be possible to treat away low titer ADAb by increasing drug or adding an immunomodulator (3). Therefore, drug-tolerant anti-drug antibody assays that have been designed both (1) to detect low level ADAb and (2) to distinguish low to intermediate to high titers with high resolution (from 25 to 2,000+ ng/mL) may be most helpful in preventing, managing and treating immunogenicity.

Anti-drug Antibody Assay Validation: Improved Reporting of the Assay Selectivity via Simpler Positive Control Recovery Data Analysis.

Anti-drug antibody (ADA) assay selectivity is evaluated during assay validation to assess the potential for individual matrices to interfere with detection of ADA. While current EMA and FDA guideline documents suggest comparative analysis with and without matrix, they do not provide specific recommendations on the acceptance criteria such as an acceptable percent positive control (PC) recovery range or positive rate. Industry has adopted an approach where recovery of PC spiked sample is expected to fall within ± 20% (80 to 120%) vs. that for the PC material spiked in negative control matrix or assay buffer. Here, it is proposed that ADA assay selectivity evaluated using a qualitative assessment of PC recovery vs. a PK-like quantitative method may be more appropriate. The PC recovery test should focus on the reliability of the method to detect the low PC level in individual samples and avoid false-negative ADA reporting. Therefore, it is proposed that assessment of high PC level as well as the assessment of quantitative percent recovery (within ± 20%) should not be included in the test. The recovery test may be viewed as acceptable should a pre-selected number of individual samples (for example at least 8 or 9 out of 10) prepared at the low PC concentration of the assay score as ADA positive.

Anti-drug Antibody Assay Conditions Significantly Impact Assay Screen and Confirmatory Cut-Points.

Assays for the detection and confirmation of anti-drug antibodies (ADA) are commonly used tools for assessing the immunogenicity of drug candidates in both clinical and nonclinical studies. During the development of such assays, it is typical to optimize the assay conditions based on factors such as sensitivity or signal/noise ratio (S/N) and is commonly done using an assay positive control (PC). However, even carefully optimized methods often suffer with problems due to low cut-point factors and failure to distinguish assay "noise" from a true biological response. In this paper, we describe an approach to assay development in which the impacts of assay conditions on the response and variability, both analytical and biological, of drug-naïve samples are tested by way of PC-independent assay condition optimization. Using two ADA methods as model systems, we examine the impact of minimum required dilution, assay reagent (labeled drug) concentrations, incubation time, assay, and wash buffer composition. We find that the choice of assay conditions, particularly the labeled drug concentration, can greatly affect the distribution of naïve sample responses and thus impact screening and confirmatory assay cut-points. In two case studies presented, screening assay cut-point (SCP) varied from 1.38 to 2.20 and 1.04 to 1.20 while the confirmatory assay cut-point (CCP) varied from 58.5 to 95.6% and 26.2 to 16.2% depending on the conditions tested. Some of the conditions produced unacceptably high CCP values. It is proposed that the degree of the observed impact of the assay conditions on SCP and CCP values depends on the compound nature and assay matrix composition and is likely connected with the diversity of interactions between drug protein and matrix components. Because it was also observed that higher assay SCP can associate with a loss of the PC-based assay sensitivity, additional assessment of the assay conditions would be required to determine an overall assay performance acceptability, including assay PC-based sensitivity, drug, and target tolerance characteristics. In conclusion, it is suggested that by assessing performance of treatment-naïve samples at various assay conditions, one can identify potential assay protocols that allow to avoid undesirably low screening (e.g., < 1.2) and confirmatory (e.g., < 25%) cut-points.

Evaluation of potential biotin interference in immunogenicity testing.

Aim: One-step bridging assays typically used for immunogenicity testing may be challenged by biotin interference (BI) caused by widely available dietary supplementation or medically prescribed high-dose therapies. We investigated BI in two one-step antidrug antibody assays. Results: Both assays showed biotin-related interference, with the peptide-based assay being less affected than the antibody-based assay. BI was reduced by minimum required dilution adaption from 10 to 1% and eliminated by a depletion-based sample pretreatment. Conclusion: Increased biotin levels have the potential to interfere with immunogenicity testing methods that use biotin technology. Since the extent of interference differs from assay to assay, assessment during development phase is recommended. Minimum required dilution adjustment or sample pretreatment are options to reduce or eliminate BI.

Report on the AAPS Immunogenicity Guidance Forum.

In September 2018, the American Association of Pharmaceutical Scientists (AAPS) conducted an Annual Guidance Forum on the considerations related to immunogenicity testing for therapeutic protein products. In addition to a broad representation by the pharmaceutical industry, the event included strong representation by leading scientists from the US Food and Drug Administration (FDA). The agency and industry perspectives and updates to the guidance were presented. Specific topics that were discussed included the strategies of anti-drug antibody (ADA) assay cut-point assessments, the selection of ADA-positive controls (PCs), and the evaluation of PC performance. Assessment strategies and relevance of ADA assay attributes were also discussed, including assay drug tolerance and ADA assay sensitivity. The following is a summary of the discussion.

New insights on critical reagent optimization for antidrug antibody assays.

Aim: Conjugated critical reagents are a pillar of ligand binding analysis. Although good practices for characterization have already been discussed, little is known about how assays are affected by specific conjugation parameters. Results: Here we developed, characterized and screened a toolset of bioconjugates that provided new insights about the optimization of conjugated critical reagent attributes. Biotinylated and sulfo-tagged trastuzumab were utilized as capture and detection antibodies, respectively, in an antidrug antibody (ADA) assay. The optimal conjugation window was identified by functional assessment. Excess of unlabeled biotin, but not sulfo-tag, affected the assay performance. An increase in the assay baseline was observed when sulfo-tagged trastuzumab underwent increasing freeze-thaw cycles. Conclusion: Upfront systematic screening and characterization of conjugated critical reagent attributes benefit assay robustness.

Update on immunogenicity in severe asthma: Experience with mepolizumab

Update on immunogenicity in severe asthma: Experience with mepolizumab

Neutralizing Antibody Assay Development with High Drug and Target Tolerance to Support Clinical Development of an Anti-TFPI Therapeutic Monoclonal Antibody.

Immunogenicity is a major challenge for protein therapeutics which can potentially reduce drug efficacy and safety and is often being monitored by anti-drug antibody (ADA) and neutralizing antibody (NAb) assays. Circulating targets and residual drugs in matrices can have significant impacts on accuracy of results from ADA and NAb assays, and sufficient drug and target tolerance for these assays are necessary. Here, we report the development of a competitive ligand binding (CLB) NAb assay for an anti-TFPI (tissue factor pathway inhibitor) monoclonal antibody (PF-06741086) with high drug and target tolerance to support ongoing clinical studies. A double acid affinity capture elution approach was used to mitigate drug interference, and a robust target removal strategy was employed to enhance target tolerance. The validated NAb assay has sensitivity of 313ng/mL, drug tolerance of 50μg/mL, and target tolerance of 1200ng/mL. A step-by-step tutorial of assay development is described in this manuscript along with the rationale for using a high drug/target tolerant NAb assay. The NAb assay cut point factor obtained was 0.78. Other assay performance characteristics, e.g., precision and selectivity, are also discussed. This validated method demonstrated a superior drug and target tolerance to warrant specific and precise characterization of the NAb responses in support of ongoing clinical studies.

Elucidation of the Statistical Factors that Influence Anti-Drug Antibody Cut Point Setting Through a Multi-Laboratory Study

Aim: Appropriateness of anti-drug antibody (ADA) assay is critical for immunogenicity assessment of biopharmaceuticals. Although cut point setting in ADA assay has a large impact on the results, a standard statistical approach for its setting has not been well established. Methodology: In this multi-laboratory study, to elucidate factors influencing the cut point setting, we compared the statistical approaches and calculated cut points for multiple datasets of ADA assays using the individual procedure employed at each laboratory. Conclusion: We showed that outlier exclusion, false-positive rate and investigating data distribution have the greatest impact on both screening and confirmatory cut points. Our results would be useful for industry researchers and regulators engaged in immunogenicity assessment of biopharmaceuticals.

Compatibility of immunogenicity guidance by the EMA and the US FDA.

The guidelines for immunogenicity studies by the European Medicines Agency and the US FDA are based on different legislations and regulatory philosophies. In spite of the different background, the main guidelines are compatible on the scientific level, especially for new innovative therapeutic protein products. The importance of sensitive and drug-tolerant antidrug antibody assays and multidisciplinary approach to development and assessment are highlighted by both agencies. The main differences are in the field of biosimilars. The nonclinical in vivo immunogenicity studies are seen more useful by the FDA than by the European Medicines Agency. The draft FDA guidance on interchangeability will complicate global biosimilar development by requiring clinical switch studies with US sourced reference product.

Immunogenicity is not the driving force of treatment failure in vedolizumab-treated inflammatory bowel disease patients.

The pivotal GEMINI trials reported low immunogenicity of vedolizumab. However, anti-vedolizumab antibodies (AVA) are frequently underestimated because most assays are not drug-tolerant and unable to detect antidrug antibodies while there is drug in the circulation. This study aimed to explore which antidrug antibody assay is best suited to detect AVA and investigated immunogenicity of vedolizumab in inflammatory bowel disease (IBD) patients discontinuing vedolizumab therapy. A drug-tolerant affinity capture elution (ACE) assay was developed for the measurement of AVA in the presence of vedolizumab and compared with the previously established drug-resistant and drug-sensitive assays. Vedolizumab and AVA were measured at week 6, at the last infusion, and 12-20weeks after treatment discontinuation in a cohort of 40 vedolizumab-treated IBD patients who stopped treatment due to primary non-response, loss of response, or adverse events. The drug-tolerant ACE assay could detect AVA in samples that the drug-resistant and drug-sensitive assays were unable to. Using the drug-tolerant ACE assay, 3 (8%) out of 40 vedolizumab-treated IBD patients who discontinued therapy were AVA positive at week 6, whereas no AVA were detected at the last infusion nor after treatment discontinuation. Primary non-responders had numerically lower median vedolizumab concentrations at week 6 compared with patients with loss of response (20.3 vs 30.7μg/mL, respectively, P=0.0570). Immunogenicity of vedolizumab is not the driving force of treatment failure, and AVA do not increase upon treatment discontinuation in vedolizumab-treated IBD patients. Underexposure during induction might partially be responsible for primary non-response.

Wide variation in the use and understanding of therapeutic drug monitoring for anti-TNF agents in inflammatory bowel disease: an inexact science?

ABSTRACTBackground: We aimed to understand the way in which therapeutic drug monitoring (TDM) is used, understood and interpreted for anti-TNF agents in IBD.Research design and methods: We designed an 18-question survey that included 5 TDM-based clinical scenarios, for which the ‘most appropriate’ responses were based on the BRIDGe groups ‘Anti-TNF Optimizer’. This resource combines TDM evidence with expert consensus.Results: We received 110 complete responses: 50 (45%) consultants, 30 (27%) trainees, 25 (23%) IBD nurse specialists and 5 (5%) gastroenterology pharmacists. Over half (61, 55%) only carry out TDM in non-response. The remainder use TDM routinely, including during stable maintenance therapy for patients in remission. Lower therapeutic thresholds used were variable. Most (82, 75%) were unsure whether their laboratory uses a drug-tolerant or drug-sensitive antidrug antibody assay and few (15, 14%) understand the difference. Consultants, high-frequency users (> 3requests/month) and clinicians with larger anti-TNF cohorts (> 100) were significantly more likely to select the ‘most appropriate’ answer to at least 1 of the 5 TDM-based clinical scenarios.Conclusions: There exists marked heterogeneity in the practical use, understanding and interpretation of biologic TDM. Biologic decision-making, informed by TDM, should involve consultation with experienced clinicians who are frequent TDM users, ideally, as part of a multidisciplinary, biologics-focused IBD meeting.Abbreviations: TDM: therapeutic drug monitoring; CNS: clinical nurse specialist; ELISA: enzyme-linked immunosorbent assay; RIA: radioimmunoassays; HMSA: homogenous mobility shift assays; BSG: British Society of Gastroenterology.

Unmet needs of Italian physicians managing patients with inflammatory bowel disease

BackgroundLittle is known about the unmet needs of physicians caring for patients with inflammatory bowel disease (IBD). AimsThis study explored the practical difficulties and needs for professional updating of Italian IBD physicians. MethodsA questionnaire was distributed to 600 physicians attending IG-IBD meetings. Results280 physicians completed the questionnaire (46.7%). On a 5-point Likert scale (from 1, strongly disagree to 5, strongly agree), they identified the most problematic issues in managing IBD patients as increasing bureaucracy (3.9), lack of extra-gastroenterological IBD expertise (3.4), lack of diagnostic techniques (3.1) and budget limitations (2.9). The most lacking techniques, ranked from 1 (greatest need) to 9 (lowest need), were: anti-drug antibody and trough level assays (2.7), device-assisted enteroscopy (3.1), exploration under anaesthesia (3.2), MR enterography (3.2), and bowel ultrasonography (3.3). About professional updating, respondents indicated (on a 5-point Likert scale) that helpful topics were practical medicine (4.3), managing difficult patients (4.1), and guidelines (4.0). The most desired modality for updating was residential courses on clinical practice (4.3). ConclusionSeveral factors potentially limit the best management of IBD patients in Italy. Satisfying these unmet needs could improve care for IBD patients.

Sensitivity and drug tolerance of antidrug antibody assays in relation to positive control characteristics.

Detection and characterization of antidrug antibodies (ADA) play a key role in understanding the relation of ADA with safety and efficacy. Positive controls (PCs) are used to estimate the sensitivity and assay sensitivity in the presence of drug (drug tolerance) of assays to detect ADA. We investigated a number of factors that may drive sensitivity and drug tolerance. We found no correlation between affinity and sensitivity and sensitivity in the presence of drug in multiple assays with two antibody-drug conjugates. Multiple factors that influenced sensitivity and drug tolerance were observed, yet these had limited overall predictive value for all investigated assay formats, PCs and compounds. Assay sensitivity and drug tolerance as studied here, are likely driven by multiple factors such as cut point confidence level, stoichiometry of PC-drug complexes and presentation of epitopes.

Immunogenicity testing of therapeutic antibodies in ocular fluids after intravitreal injection.

High drug concentrations in ocular fluids after intravitreal administration preclude the use of drug-sensitive immunoassays. A drug-tolerant immunoassay is therefore desirable for immunogenicity testing in ophthalmology. Immune complex (IC) antidrug antibody (ADA) assays were established for two species. The assays were compared with the bridging assay in ocular and plasma samples from two preclinical studies. The IC assays showed high drug tolerance, which enabled a reliable ADA detection in ocular fluids after intravitreal administration. The IC assays were superior to the bridging assay in the analysis of ocular fluids with high drug concentrations. The IC assay allows a reliable ADA detection in matrices with high drug concentrations, such as ocular fluids.

Detection of Memory B Activity Against a Therapeutic Protein in Treatment-Naïve Subjects.

Bridging immunoassays commonly used to detect and characterize immunogenicity during biologic development do not provide direct information on the presence or development of a memory anti-drug antibody (ADA) response. In this study, a B cell ELISPOT assay method was used to evaluate pre-existing ADA for anti-TNFR1 domain antibody, GSK1995057, an experimental biologic in treatment naive subjects. This assay utilized a 7-day activation of PBMCs by a combination of GSK1995057 (antigen) and polyclonal stimulator followed by GSK1995057-specific ELISPOT for the enumeration of memory B cells that have differentiated into antibody secreting cells (ASC) in vitro. We demonstrated that GSK1995057-specific ASC were detectable in treatment-naïve subjects with pre-existing ADA; the frequency of drug-specific ASC was low and ranged from 1 to 10 spot forming units (SFU) per million cells. Interestingly, the frequency of drug-specific ASC correlated with the ADA level measured using an in vitro ADA assay. We further confirmed that the ASC originated from CD27+ memory B cells, not from CD27--naïve B cells. Our data demonstrated the utility of the B cell ELISPOT method in therapeutic protein immunogenicity evaluation, providing a novel way to confirm and characterize the cell population producing pre-existing ADA. This novel application of a B cell ELISPOT assay informs and characterizes immune memory activity regarding incidence and magnitude associated with a pre-existing ADA response.

Collaborative study using common samples to evaluate the performance of anti-drug antibody assays constructed by different companies

This study was undertaken to evaluate the performance of anti-drug antibody (ADA) assays constructed by each participating company using common samples including ADA, drug and human serum. The ADA assays constructed by each company showed good sensitivity and precision for evaluation of ADA. Cut points for screening and confirmatory assays and assay selectivity were determined by various calculation methods. In evaluations of blind ADA samples, nearly similar results were obtained by the study companies in determinations of whether samples were positive or negative except at the lowest sample concentration (5 ng/mL). In measurement of drug tolerance, for almost samples containing ADA and drugs, more positive results were obtained in assays using acid dissociation compared to those without acid dissociation. Overall, the performance of ADA assays constructed by the 10 companies participating in this study was acceptable in terms of sensitivity and reproducibility for detection and evaluation of immunogenicity in both patients and healthy subjects. On the other hand, based on results for samples containing ADA and drugs, validity of results for ADA assays conducted without acid dissociation was less meaningful and more difficult to evaluate. Thus, acid dissociation was confirmed to be useful for improving drug tolerance.

Therapeutic Drug Monitoring of the Biosimilar SB2 (RENFLEXIS™, Infliximab-abda) Using LabCorp Infliximab Assays for Drug Level and Anti-drug Antibodies

BACKGROUND: Therapeutic drug monitoring (TDM) of infliximab (IFX) concentration and anti-infliximab antibody (ADA) titer has helped physicians make informed clinical decisions in the management of inflammatory bowel disease (IBD). A TDM-based approach to treating IBD patients has been used to improve clinical efficacy of anti-TNF agents. The LabCorp IFX TDM test (drug and ADA levels) has been validated in peer-reviewed literature and is widely used by clinicians to help maximize treatment response. Recently approved by the FDA for treatment of IBD, RENFLEXIS (SB2, infliximab-abda) is a biosimilar of the originator infliximab (TNF alpha inhibitor). The aim of this study was to validate the LabCorp IFX TDM test for the quantification of serum SB2 concentrations and ADA. METHODS: To demonstrate ability to measure RENFLEXIS using the LabCorp IFX TDM test, SB2-specific immunoassays were developed. SB2 calibrators and SB2-conjugated key reagents were used to compare with IFX TDM kits. Analytical performance including accuracy, precision, spike and recovery, specificity and selectivity was assessed. Exogenous SB2 was spiked into donor serum to assess recovery using the IFX drug assay. 124 IFX-treated IBD patients were split-tested using SB2 and IFX drug and ADA assays to assess equivalency and interchangeability between the two methods. RESULTS: Both SB2 drug level and ADA showed excellent agreement to IFX drug level and ADA respectively. SB2 assay accuracy (<11% bias) and precision (<7% CV) were equivalent to IFX accuracy (<5% bias) and precision (<10% CV) across the measuring range of the assay. More importantly, spike and recovery of RENFLEXIS using the IFX drug assay was equivalent to the recovery of reference product IFX using the IFX drug assay. SB2 was spiked into the donor serum pool to generate 0.25, 0.5, 1.0, 2.0, 4.0, 5.0, 6.0, & 8.0 ug/mL targets. The same target concentrations were generated for IFX. Both sets of targets were read using the IFX drug assay. Percent recovery of SB2 ranged from 87% to 114%; IFX recovery was 83% to 113%. Average spike and recovery was 99% for both SB2 and IFX using the IFX drug assay. Both the SB2 drug assay and IFX drug assay were used to analyze 59 serum samples of IBD patients treated with reference product IFX. The calculated linear regression between the two methods was Y=1.11x-0.13 and r2=1.0. The SB2-specific anti-drug antibody assay performed essentially identically to the reference product IFX ADA assay in terms of precision (<10% CV), accuracy (<10% bias), linearity, drug tolerance and sensitivity. 65 ADA positive serum samples from IBD patients were tested using the SB2 ADA assay. Antibody titer values were compared. Linear regressions of Y=1.09x-8.7, r2=0.99 were obtained. CONCLUSION(S): This study demonstrates complete cross-reactivity of RENFLEXIS (infliximab-abda) in LabCorp IFX TDM assay. Healthcare providers can confidently use LabCorp IFX TDM test to monitor RENFLEXIS (infliximab-abda) serum drug levels and ADA titers in their patients.

Immunogenicity of therapeutic protein products: current considerations for anti-drug antibody assay in Japan.

Immunogenicity assessment is an important issue for ensuring the safety and efficacy of therapeutic protein products. Although the reliability of the anti-drug antibody (ADA) assay is one of the key points, there are some difficulties in assessing its validity because the analytes are polyclonal antibodies with variable and unknown characteristics. To elucidate the points to consider for the ADA assay, a Japanese research group was established that discusses the issues raised on the immunogenicity assessment. In this review, we first introduce the current situation regarding the development and immunogenicity assessment of therapeutic protein products in Japan. We then present our current view and recommendations on the ADA assay by considering its unique features.

Immunogenicity considerations for antibody-drug conjugates: a focus on neutralizing antibody assays.

Immunogenicity considerations for antibody-drug conjugates: a focus on neutralizing antibody assays.