Clinicogenomic factors of biotherapy immunogenicity in autoimmune disease: A prospective multicohort study of the ABIRISK consortium.

BACKGROUND: Biopharmaceutical products (BPs) are widely used to treat autoimmune diseases, but immunogenicity limits their efficacy for an important proportion of patients. Our knowledge of patient-related factors influencing the occurrence of antidrug antibodies (ADAs) is still limited. METHODS AND FINDINGS: The European consortium ABIRISK (Anti-Biopharmaceutical Immunization: prediction and analysis of clinical relevance to minimize the RISK) conducted a clinical and genomic multicohort prospective study of 560 patients with multiple sclerosis (MS, n = 147), rheumatoid arthritis (RA, n = 229), Crohn's disease (n = 148), or ulcerative colitis (n = 36) treated with 8 different biopharmaceuticals (etanercept, n = 84; infliximab, n = 101; adalimumab, n = 153; interferon [IFN]-beta-1a intramuscularly [IM], n = 38; IFN-beta-1a subcutaneously [SC], n = 68; IFN-beta-1b SC, n = 41; rituximab, n = 31; tocilizumab, n = 44) and followed during the first 12 months of therapy for time to ADA development. From the bioclinical data collected, we explored the relationships between patient-related factors and the occurrence of ADAs. Both baseline and time-dependent factors such as concomitant medications were analyzed using Cox proportional hazard regression models. Mean age and disease duration were 35.1 and 0.85 years, respectively, for MS; 54.2 and 3.17 years for RA; and 36.9 and 3.69 years for inflammatory bowel diseases (IBDs). In a multivariate Cox regression model including each of the clinical and genetic factors mentioned hereafter, among the clinical factors, immunosuppressants (adjusted hazard ratio [aHR] = 0.408 [95% confidence interval (CI) 0.253-0.657], p < 0.001) and antibiotics (aHR = 0.121 [0.0437-0.333], p < 0.0001) were independently negatively associated with time to ADA development, whereas infections during the study (aHR = 2.757 [1.616-4.704], p < 0.001) and tobacco smoking (aHR = 2.150 [1.319-3.503], p < 0.01) were positively associated. 351,824 Single-Nucleotide Polymorphisms (SNPs) and 38 imputed Human Leukocyte Antigen (HLA) alleles were analyzed through a genome-wide association study. We found that the HLA-DQA1*05 allele significantly increased the rate of immunogenicity (aHR = 3.9 [1.923-5.976], p < 0.0001 for the homozygotes). Among the 6 genetic variants selected at a 20% false discovery rate (FDR) threshold, the minor allele of rs10508884, which is situated in an intron of the CXCL12 gene, increased the rate of immunogenicity (aHR = 3.804 [2.139-6.764], p < 1 × 10-5 for patients homozygous for the minor allele) and was chosen for validation through a CXCL12 protein enzyme-linked immunosorbent assay (ELISA) on patient serum at baseline before therapy start. CXCL12 protein levels were higher for patients homozygous for the minor allele carrying higher ADA risk (mean: 2,693 pg/ml) than for the other genotypes (mean: 2,317 pg/ml; p = 0.014), and patients with CXCL12 levels above the median in serum were more prone to develop ADAs (aHR = 2.329 [1.106-4.90], p = 0.026). A limitation of the study is the lack of replication; therefore, other studies are required to confirm our findings. CONCLUSION: In our study, we found that immunosuppressants and antibiotics were associated with decreased risk of ADA development, whereas tobacco smoking and infections during the study were associated with increased risk. We found that the HLA-DQA1*05 allele was associated with an increased rate of immunogenicity. Moreover, our results suggest a relationship between CXCL12 production and ADA development independent of the disease, which is consistent with its known function in affinity maturation of antibodies and plasma cell survival. Our findings may help physicians in the management of patients receiving biotherapies.

Clinical practice of analysis of anti-drug antibodies against interferon beta and natalizumab in multiple sclerosis patients in Europe: A descriptive study of test results.

Antibodies against biopharmaceuticals (anti-drug antibodies, ADA) have been a well-integrated part of the clinical care of multiple sclerosis (MS) in several European countries. ADA data generated in Europe during the more than 10 years of ADA monitoring in MS patients treated with interferon beta (IFNß) and natalizumab have been pooled and characterized through collaboration within a European consortium. The aim of this study was to report on the clinical practice of ADA testing in Europe, considering the number of ADA tests performed and type of ADA assays used, and to determine the frequency of ADA testing against the different drug preparations in different countries. A common database platform (tranSMART) for querying, analyzing and storing retrospective data of MS cohorts was set up to harmonize the data and compare results of ADA tests between different countries. Retrospective data from six countries (Sweden, Austria, Spain, Switzerland, Germany and Denmark) on 20,695 patients and on 42,555 samples were loaded into tranSMART including data points of age, gender, treatment, samples, and ADA results. The previously observed immunogenic difference among the four IFNß preparations was confirmed in this large dataset. Decreased usage of the more immunogenic preparations IFNß-1a subcutaneous (s.c.) and IFNß-1b s.c. in favor of the least immunogenic preparation IFNß-1a intramuscular (i.m.) was observed. The median time from treatment start to first ADA test correlated with time to first positive test. Shorter times were observed for IFNß-1b-Extavia s.c. (0.99 and 0.94 years) and natalizumab (0.25 and 0.23 years), which were introduced on the market when ADA testing was already available, as compared to IFNß-1a i.m. (1.41 and 2.27 years), IFNß-1b-Betaferon s.c. (2.51 and 1.96 years) and IFNß-1a s.c. (2.11 and 2.09 years) which were available years before routine testing began. A higher rate of anti-IFNß ADA was observed in test samples taken from older patients. Testing for ADA varies between different European countries and is highly dependent on the policy within each country. For drugs where routine monitoring of ADA is not in place, there is a risk that some patients remain on treatment for several years despite ADA positivity. For drugs where a strategy of ADA testing is introduced with the release of the drug, there is a reduced risk of having ADA positive patients and thus of less efficient treatment. This indicates that potential savings in health cost might be achieved by routine analysis of ADA.

Occurrence of Anti-Drug Antibodies against Interferon-Beta and Natalizumab in Multiple Sclerosis: A Collaborative Cohort Analysis.

Immunogenicity of biopharmaceutical products in multiple sclerosis is a frequent side effect which has a multifactorial etiology. Here we study associations between anti-drug antibody (ADA) occurrence and demographic and clinical factors. Retrospective data from routine ADA test laboratories in Sweden, Denmark, Austria and Germany (Dusseldorf group) and from one research study in Germany (Munich group) were gathered to build a collaborative multi-cohort dataset within the framework of the ABIRISK project. A subset of 5638 interferon-beta (IFNß)-treated and 3440 natalizumab-treated patients having data on at least the first two years of treatment were eligible for interval-censored time-to-event analysis. In multivariate Cox regression, IFNß-1a subcutaneous and IFNß-1b subcutaneous treated patients were at higher risk of ADA occurrence compared to IFNß-1a intramuscular-treated patients (pooled HR = 6.4, 95% CI 4.9-8.4 and pooled HR = 8.7, 95% CI 6.6-11.4 respectively). Patients older than 50 years at start of IFNß therapy developed ADA more frequently than adult patients younger than 30 (pooled HR = 1.8, 95% CI 1.4-2.3). Men developed ADA more frequently than women (pooled HR = 1.3, 95% CI 1.1-1.6). Interestingly we observed that in Sweden and Germany, patients who started IFNß in April were at higher risk of developing ADA (HR = 1.6, 95% CI 1.1-2.4 and HR = 2.4, 95% CI 1.5-3.9 respectively). This result is not confirmed in the other cohorts and warrants further investigations. Concerning natalizumab, patients older than 45 years had a higher ADA rate (pooled HR = 1.4, 95% CI 1.0-1.8) and women developed ADA more frequently than men (pooled HR = 1.4, 95% CI 1.0-2.0). We confirmed previously reported differences in immunogenicity of the different types of IFNß. Differences in ADA occurrence by sex and age are reported here for the first time. These findings should be further investigated taking into account other exposures and biomarkers.

Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta.

Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNß) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low positive titers needs to be further evaluated.

Development of an ultra-sensitive single molecule counting assay for the detection of interleukin-13 as a marker for asthmatic severity.

OBJECTIVE: Interleukin-13 (IL-13) has been difficult to quantify within human serum due to low abundance. Available assays have not been sensitive enough to detect IL-13 at the femtogram level. Thus, there are inconsistencies within the published literature as to IL-13 concentrations in normal or disease populations. To better understand IL-13 serum concentrations, a highly sensitive immunoassay was developed and used to determine concentrations from asthmatics with varying clinical severities. METHODS: A single molecule counting (SMC) method was used to analyze serum samples from a total of 145 individuals (60 severe asthma, 60 moderate asthma, 60 mild asthma and 23 healthy donors). RESULTS: IL-13 concentrations correlated with severity of asthma, with overlapping ranges. Mean IL-13 levels were highest in severe asthma. Mean IL-13 levels in moderate asthma population were second highest followed by mild asthma with the lowest IL-13 concentration. IL-13 concentrations in healthy donors were similar to the mild asthmatic population. The average concentrations of IL-13 in severe, moderate, mild and healthy donors were 1.286pg/mL, 0.672pg/mL, 0.508pg/mL and 0.155pg/mL respectively. CONCLUSION: Severe asthma patients have elevated levels of IL-13.

Development and validation of a cell-based SEAP reporter assay for the detection of neutralizing antibodies against an anti-IL-13 therapeutic antibody.

A cell-based bioassay capable of detecting neutralizing antibodies (NAb) specific to a therapeutic anti-IL-13 monoclonal antibody was developed, validated and used to analyze normal human and asthma serum samples. At the time of this study, a neutralizing assay was unavailable for anti-IL-13 antibody therapeutics with sufficient rigor for validation. Thus, we describe here a method and considerations for validation. The assay used IL-13 responsive HEK293 cells transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cells were plated at 5.4×10(4) per assay well due to 90% confluence on the subsequent day. Optimal IL-13 and anti-IL-13 concentrations were determined to be 600 pg/mL and 900 ng/mL respectively. We demonstrated the assay's cut point, sensitivity, specificity/cross reactivity, selectivity/matrix interference, and precision. Also, we demonstrated how the drug inhibitory concentration (IC(50), IC(75), and IC(90)) can affect sensitivity and dynamic range/assay window. We characterized the differences in assay response between serum samples of normal population and asthma population. Asthma samples demonstrated an elevated OD ratio in average compared to normal samples. Thus, separate cut points were needed and calculated to be 1.78 and 2.43 for normal and asthma serum, respectively. The assay sensitivity was 670 ng/mL with the positive control (affinity purified rabbit anti-drug polyclonal antibodies). Potential false positives resulting from endogenous serum cytokines including IL-13, IL-4, and Interferon alpha (INF-α) were evaluated and the results indicated that the interfering concentrations for these cytokines are much higher than the respective physiological concentrations. Based on these data, the risk of false positive by endogenous cytokines was considered to be low. In addition, irrelevant anti-drug positive control antibodies were evaluated for assay specificity and did not demonstrate neutralizing capability. Further, no matrix interference in the intended patient population was found when using a final assay serum concentration of 16.7%. The validated assay had acceptable intra- and inter- assay precision in that all %CVs were ≤25%. Overall, this assay successfully proceeded through validation and was used to determine NAb responses within serum samples.