Immunogenicity of biopharmaceuticals in chronic inflammatory diseases

Multiple sclerosis (MS) and rheumatoid arthritis (RA) are chronic inflammatory diseases where genetic and environmental factors influence the pathogenesis. MS is a disease of the central nervous system, while RA primarily affects the joints. Biopharmaceuticals such as interferon beta (IFNβ) and tumor necrosis factor-alpha (TNF-α) inhibitors are widely used treatments to achieve a reduction in disease activity in people with MS and RA respectively. Over time, however, some of the treated patients develop anti-drug antibodies (ADA) or neutralizing ADA (NAb) that can reduce or abrogate the drug efficacy and subsequently lead to loss of clinical response. Five studies are included in this thesis, which assess endogenous immune processes affected and evaluates laboratory methods used for monitoring immunogenicity of IFNβ and TNF-α inhibitors. Collectively, the findings presented in this thesis aim to optimize methods for drug level and ADA screening to allow for easier treatment decisions. Additionally, the thesis highlights the skin site as a potential contributor to ADA development. In study I, we studied the immunomodulatory role of IFNβ and how it was affected by NAb. We found a 3-fold increase of serum IL-7 (genetically associated with MS) in IFNβ treated MS patients and this was related to the lowered IL-7Rα expression on cell surfaces. The presence of high NAb titers to IFNβ resulted in significantly lower serum IL-7 levels compared to NAb negative patients as measured with the myxovirus resistance protein A (MxA) gene expression assay (MGA). Since the MGA method is cumbersome we decided to evaluate a new method, iLite (in study II). We found that the NAb titers had a high degree of correlation between the two assays and that NAb titers of 150 TRU/mL were suggestive of significant neutralization of the drug. However, in the iLite assay (and MGA) NAb titers are calculated using the Kawade principle and this method has statistical limitations. In study III, we therefore continued to validate the iLite assay using a cut-point approach designed to be more sensitive and statistically accurate. By using the cut-point approach, we identified 12% more NAb positive samples compared to using the Kawade method, showing the increased sensitivity achieved with the cut-point design. In study IV, we evaluated different methods for ADA screening of the TNF-α inhibitor infliximab. We showed that ADA could be detected in the majority of samples with low drug levels using ELISA, but that samples with detectable drug levels also tested ADA positive using an acid and dissociation assay (PandA). Thus, the PandA proved useful as a complement to the routinely available ELISA to monitor immunogenicity. Lastly (in study V), to understand the mechanism of ADA induction, we investigated the primary immune response against repetitive injections with biologicals in skin cells. Using a human skin model, we found that the IFNβ injection enhanced dendritic cell maturation and elevated the expression of several inflammatory cytokines, which suggest that the administration triggers an immune response at the injection site

Drug Tolerant Anti-drug Antibody Assay for Infliximab Treatment in Clinical Practice Identifies Positive Cases Earlier

Publisher's version (útgefin grein)A subgroup of patients treated with infliximab lose response to the treatment and one reason for this is the development of anti-drug antibodies (ADA). If used optimally, measuring drug and ADA level could lead to a more personalized and efficient treatment regime, and enable identification of ADA-positive patients before the underlying disease flares or allergic reactions occur. With the use of a drug-tolerant ADA assay which can detect ADA irrespective of drug levels in the sample, we determined the impact of ADA on treatment failure to infliximab. The aims of this study were to estimate the real-life optimal serum infliximab (sIFX) level and set a clinical threshold value for a drug-tolerant ADA assay. Trough levels of sIFX were measured with ELISA. Free ADA was measured with two drug-sensitive methods (ELISA and a bioassay) and one drug-tolerant method (PandA). Two real-life cohorts treated with infliximab were included; a cross-sectional cohort including patients with inflammatory rheumatic diseases (n = 270) and a prospective cohort of rheumatoid arthritis (RA) patients (n = 73) followed for 1 year. Normal range of sIFX was estimated from the prospective cohort and an arbitrary optimal drug level was set to be between 1 and 6 μg/mL. Using this range, optimal sIFX was found in only 60% (163/270) of the patients in the cross-sectional cohort. These patients had significantly better treatment response than those with a drug level under 1 μg/mL, who had an ADA frequency of 34% (19/56) using the drug-tolerant method. In the prospective cohort, the drug-tolerant assay could identify 34% (53/155 samples) as ADA positive in samples with sIFX level >0.2 μg/mL. ADA were seldom detected in patients with >1 μg/mL sIFX, with three interesting exceptions. A clinically relevant ADA threshold was determined to be >3 RECL as measured with the drug-tolerant assay. In a real-life setting, there was a substantial number of patients with suboptimal drug levels and a proportion of these had ADA. Both too low and too high drug levels correlated with worse disease, but for different reasons. Adding a drug-tolerant assay enabled detection of ADA earlier and regardless of drug level at time of sampling.We would like to thank all patients for their participation in this study. We would also like to thank Consuelo Gomez, Pascual Gonzalez, Anna G. Mattsson, Arne St?hl, and Yousra Rehouma for excellent technical assistance. Funding. The research leading to these results has received support from Swelife, Stockholm County Council (ALF project) #20140333 and the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115303, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution. This work was also supported by the grants from Aina (Ann) Wallstr?ms och Mary-Ann Sj?bloms Foundation for Medical Research, Professor Nanna Svartz Foundation, the Gothenburg Medical Society (GLS-889421 to RP), the Swedish Rheumatism Association (R-862061 and R-663511 to RP), Adlerbertska research Foundation and the Regional agreement on medical training and clinical research between the Western G?taland county council and the University of Gothenburg (ALFGBG-926621).Peer Reviewe

Human leukocyte antigen genes and interferon beta preparations influence risk of developing neutralizing anti-drug antibodies in multiple sclerosis.

A significant proportion of patients with multiple sclerosis who receive interferon beta (IFNβ) therapy develop neutralizing antibodies (NAbs) that reduce drug efficacy. To investigate if HLA class I and II alleles are associated with development of NAbs against IFNβ we analyzed whether NAb status and development of NAb titers high enough to be biologically relevant (>150 tenfold reduction units/ml) correlated with the HLA allele group carriage in a cohort of 903 Swedish patients with multiple sclerosis treated with either intramuscular IFNβ-1a, subcutaneous IFNβ-1a or subcutaneous IFNβ-1b. Carriage of HLA-DRB1*15 was associated with increased risk of developing NAbs and high NAb titers. After stratification based on type of IFNβ preparation, HLA-DRB1*15 carriage was observed to increase the risk of developing NAbs as well as high NAb titers against both subcutaneous and intramuscular IFNβ-1a. Furthermore, in patients receiving subcutaneous IFNβ-1a carriage of HLA-DQA1*05 decreased the risk for high NAb titers. In IFNβ-1b treated patients, HLA-DRB1*04 increased the risk of developing high NAb titers, and in a subgroup analysis of DRB1*04 alleles the risk for NAbs was increased in DRB1*04:01 carriers. In conclusion, there is a preparation-specific genetically determined risk to develop NAbs against IFNβ high enough to be clinically relevant in treatment decisions for patients with multiple sclerosis if confirmed in future studies. However, choice of IFNβ preparation still remains the single most significant determinant for the risk of developing NAbs

HLA association to development of NAbs and biologically relevant titers.

a<p>Major HLA alleles (>5% allele frequency) nominally associated or previously reported to be associated with development of NAbs and biologically relevant titers are presented. All HLA alleles can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090479#pone.0090479.s002" target="_blank">Table S2</a></b> and <b>S3</b>.</p>b<p>nominal <i>P</i>-values assessed by Chi square test,</p>c<p>Bonferroni corrected <i>P</i>-values (76 allele groups tested).</p><p>Abbreviations: BRT = biologically relevant titers, C.I. = confidence interval, NAb = neutralizing antibodies, OR = odds ratio.</p

Carrier frequency and absolute risk for HLA allele groups associated to NAb development and biologically relevant titers^a.

a<p>All HLA allele groups can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090479#pone.0090479.s005" target="_blank">Table S5</a></b>.</p>b<p>Treatment only is the frequency of NAb development and biologically relevant titers for each treatment regardless of genotype.</p>c<p>The absolute risk for each HLA allele group is calculated with Bayes' theorem and assessed based on frequency and impact on NAb development.</p>d<p>Nominal <i>P</i>-values from Fishers exact test.</p>e<p>Bonferroni corrected <i>P</i>-values (allele groups tested: 12 tests for i.m. IFNβ-1a, 19 tests for IFNβ-1b, 38 for s.c. IFNβ-1a).</p><p>Abbreviations: AR = absolute risk, C.I. = confidence interval, OR = odds ratio, IFNβ = interferon beta, n/a = not applicable, NAb = neutralizing antibodies.</p

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

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

Characteristics of the cohort.

a<p>IFNβ preparation could be assigned to 358 of the 364 NAb positive patients, while the type of preparation used was unspecified for 6 NAb positive patients.</p><p>Abbreviations: IQR = interquartile range, i.m. = intramuscular, s.c. = subcutaneous, TRU/ml = tenfold reduction units per milliliter, IFNβ = interferon beta, NAb = neutralizing antibodies</p