Meena Subramanyam

Risk of Natalizumab-Associated Progressive Multifocal Leukoencephalopathy

BACKGROUND Progressive multifocal leukoencephalopathy (PML) is associated with natalizumab treatment. We quantified the risk of PML in patients with multiple sclerosis, according to the presence or absence of three risk factors: positive status with respect to anti-JC virus antibodies, prior use of immunosuppressants, and increasing duration of natalizumab treatment. METHODS We used data from postmarketing sources, clinical studies, and an independent Swedish registry to estimate the incidence of PML among natalizumab-treated patients with multiple sclerosis, according to positive or negative status with respect to anti-JC virus antibodies, prior or no prior use of immunosuppressants, and duration of treatment (1 to 24 months vs. 25 to 48 months). Blood samples were available for anti-JC virus antibody testing from 5896 patients with multiple sclerosis and from 54 patients with multiple sclerosis who were treated with natalizumab and in whom PML later developed. RESULTS As of February 29, 2012, there were 212 confirmed cases of PML among 99,571 patients treated with natalizumab (2.1 cases per 1000 patients). All 54 patients with PML for whom samples were available before the diagnosis were positive for anti-JC virus antibodies. When the risk of PML was stratified according to three risk factors, the risk of PML was lowest among the patients who were negative for anti-JC virus antibodies, with the incidence estimated to be 0.09 cases or less per 1000 patients (95% confidence interval [CI], 0 to 0.48). Patients who were positive for anti-JC virus antibodies, had taken immunosuppressants before the initiation of natalizumab therapy, and had received 25 to 48 months of natalizumab treatment had the highest estimated risk (incidence, 11.1 cases per 1000 patients [95% CI, 8.3 to 14.5]). CONCLUSIONS Positive status with respect to anti-JC virus antibodies, prior use of immunosuppressants, and increased duration of natalizumab treatment, alone or in combination, were associated with distinct levels of PML risk in natalizumab-treated patients with multiple sclerosis. (Funded by Biogen Idec and Elan Pharmaceuticals.).

Anti-JC Virus Antibodies: Implications for PML Risk Stratification

A study was undertaken to establish an enzyme‐linked immunosorbent assay (ELISA) to detect JC virus (JCV)‐specific antibodies in multiple sclerosis (MS) patients, and to evaluate its potential utility for identifying patients at higher or lower risk (ie, risk stratification) of developing progressive multifocal leukoencephalopathy (PML).

Anti–JC virus antibody levels in serum or plasma further define risk of natalizumab-associated progressive multifocal leukoencephalopathy

The increased risk of progressive multifocal leukoencephalopathy (PML) with natalizumab treatment is associated with the presence of anti–JC virus (JCV) antibodies. We analyzed whether anti‐JCV antibody levels, measured as index, may further define PML risk in seropositive patients.

Anti‐John Cunnigham virus antibody prevalence in multiple sclerosis patients: Baseline results of STRATIFY‐1

A study was undertaken to define the prevalence of anti‐JC virus (JCV) antibodies in multiple sclerosis (MS) patients and to evaluate the analytical false‐negative rate of a 2‐step anti‐JC virus antibody assay.

A second-generation ELISA (STRATIFY JCV™ DxSelect™) for detection of JC virus antibodies in human serum and plasma to support progressive multifocal leukoencephalopathy risk stratification

BACKGROUND JC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). The development and validation of a two-step enzyme-linked immunosorbent assay (ELISA) that detects JCV antibodies in human serum or plasma and its clinical utility for stratification of PML risk have been described. OBJECTIVE To develop a second-generation JCV antibody ELISA kit with improved assay performance characteristics. STUDY DESIGN The assay design was optimized by pre-coating the JC virus-like particles (VLP) on microtiter plates. Assay cut-points were statistically established using sera from >1300 multiple sclerosis patients from natalizumab clinical studies. The assay was analytically validated and then used to determine the presence of JCV antibodies in both treatment-naïve and natalizumab-treated MS patients, as well as in natalizumab-treated PML patients. RESULTS An improved assay for detection of JCV antibodies in human serum and plasma was developed. Key enhancements included improved delineation and reproducibility of low JCV antibody responses and assay ease of use. The assay was validated, demonstrating good agreement with the original two-step JCV antibody ELISA, and similar seroprevalence of 50%-60%. Samples from 63 natalizumab-treated PML patients collected 6-180 months prior to PML diagnosis tested JCV antibody positive. One patient tested JCV antibody negative 15 months prior to PML diagnosis but JCV antibody positive 2 months prior to PML diagnosis. CONCLUSIONS The validated second-generation JCV antibody ELISA offers improved assay design as a kit and enhanced performance characteristics that advance routine clinical use of the assay as a PML risk stratification tool.

Assessment and Reporting of the Clinical Immunogenicity of Therapeutic Proteins and Peptides—Harmonized Terminology and Tactical Recommendations

Immunogenicity is a significant concern for biologic drugs as it can affect both safety and efficacy. To date, the descriptions of product immunogenicity have varied not only due to different degrees of understanding of product immunogenicity at the time of licensing but also due to an evolving lexicon that has generated some confusion in the field. In recent years, there has been growing consensus regarding the data needed to assess product immunogenicity. Harmonization of the strategy for the elucidation of product immunogenicity by drug developers, as well as the use of defined common terminology, can benefit medical practitioners, health regulatory agencies, and ultimately the patients. Clearly, understanding the incidence, kinetics and magnitude of anti-drug antibody (ADA), its neutralizing ability, cross-reactivity with endogenous molecules or other marketed biologic drugs, and related clinical impact may enhance clinical management of patients treated with biologic drugs. To that end, the authors present terms and definitions for describing and analyzing clinical immunogenicity data and suggest approaches to data presentation, emphasizing associations of ADA development with pharmacokinetics, efficacy, and safety that are necessary to assess the clinical relevance of immunogenicity.

Anti-JC virus antibody prevalence in a multinational multiple sclerosis cohort

JC virus (JCV) is an opportunistic virus known to cause progressive multifocal leukoencephalopathy. Anti-JC virus (Anti-JCV) antibody prevalence in a large, geographically diverse, multi-national multiple sclerosis (MS) cohort was compared in a cross-sectional study. Overall, anti-JCV antibody prevalence was 57.6%. Anti-JCV antibody prevalence in MS patients ranged from approximately 47% to 68% across these countries: Norway, 47.4%; Denmark, 52.6%; Israel, 56.6%; France, 57.6%; Italy, 58.3%; Sweden, 59.0%; Germany, 59.1%; Austria, 66.7% and Turkey, 67.7%. Prevalence increased with age (from 49.5% in patients < 30 years of age to 66.5% in patients ≥ 60 years of age; p < 0.0001 comparing all age categories), was lower in females than in males (55.8% versus 61.9%; p < 0.0001) and was not affected by prior immunosuppressant or natalizumab use.

A Novel PEGylated Interferon Beta-1a for Multiple Sclerosis: Safety, Pharmacology, and Biology

This study clinically evaluated a novel PEGylated form of interferon beta‐1a (PEG‐IFN beta‐1a), a potential first‐line treatment for relapsing multiple sclerosis, in healthy volunteers. Two randomized, blinded phase I studies were conducted: a single‐dose study (n = 60) comparing subcutaneous or intramuscular PEG‐IFN beta‐1a (63, 125, or 188 μg) with intramuscular unmodified IFN beta‐1a 30 μg and a multiple‐dose study (n = 69) comparing subcutaneous PEG‐IFN beta‐1a dosed once every 2 or 4 weeks with placebo. Assessments included pharmacokinetic and pharmacodynamic (serum neopterin and 2′,5′‐OAS) measures, exploratory immune assessments, safety, and tolerability. A dose‐proportional increase in PEG‐IFN beta‐1a exposure was observed, with a 4‐fold greater exposure at 63 μg (6 million international units [MIU]) of PEG‐IFN beta‐1a than with 30 μg (6 MIU) intramuscular unmodified IFN beta‐1a. Increases in neopterin and 2′,5′‐OAS levels and changes in T helper cell pathway gene expression and lymphocyte subsets were greater and more sustained with PEG‐IFN beta‐1a than with unmodified IFN beta‐1a. PEG‐IFN beta‐1a was well tolerated, with only transient reductions in absolute neutrophils and some lymphocytes. Flu‐like symptoms were a commonly reported adverse event. These data support the continued clinical development of PEG‐IFN beta‐1a as a potentially effective treatment for patients with relapsing multiple sclerosis.

Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics

The administration of biological therapeutics may result in the development of anti-drug antibodies (ADAs) in treated subjects. In some cases, ADA responses may result in the loss of therapeutic efficacy due to the formation of neutralizing ADAs (NAbs). An important characteristic of anti-drug NAbs is their direct inhibitory effect on the pharmacological activity of the therapeutic. Neutralizing antibody responses are of particular concern for biologic products with an endogenous homolog whose activity can be potentially dampened or completely inhibited by the NAbs leading to an autoimmune-type deficiency syndrome. Therefore, it is important that ADAs are detected and characterized appropriately using sensitive and reliable methods. The design, development and optimization of cell-based assays used for detection of NAbs have been published previously by Gupta et al. 2007 [1]. This paper provides recommendations on best practices for the validation of cell-based NAb assay and suggested validation parameters based on the experience of the authors.

Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development

Humanized monoclonal antibody therapeutics are in many ways indistinguishable from the anti-therapeutic/anti-drug antibodies generated in humans. Therefore, immunogenicity assessments to such therapeutics pose unique challenges in clinical trials especially when significant drug interference is encountered. There are several technology platforms based on the bridging immunogenicity assay format, which have been successfully used for detection and quantification of anti-drug antibodies (ADA) in serum or plasma samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescent (ECL) immunoassay formats are among the most popular technology platforms. Pretreatment of samples with acid can also be used to lower drug interference. While ECL technology platform offered many advantages over traditional solid-phase ELISA methods, reliance on a single (or limited) vendor source became a significant concern within the biopharmaceutical industry especially for immunogenicity assays that need to be implemented over a period of many years in support of a single drug development program. We describe herein a systematic evaluation of solid-phase ELISA, GYROS, AlphaLISA, ECL Immunoassay, and solution ELISA platforms for detection of anti-drug antibodies with the goal of selection and development of a robust technology platform that meets the desired performance characteristics for most immunogenicity assays and can be easily implemented in a typical immunoassay laboratory. As part of this effort the Design of Experiments (DOE) approach was utilized in optimization of sample acid treatment conditions in order to improve drug tolerance in the evaluated assay platforms. After the initial evaluation of various technology platforms, a solution ELISA format was chosen for further development to support clinical trials for a humanized therapeutic antibody. As part of the assay development, flexible use of digoxigenin and 6-(2,4-dinitrophenyl) aminohexanoic acid (DNP) for labeling antibodies was evaluated and is presented in this manuscript. In addition, simple methods for evaluation and qualification of streptavidin-coated plates and overcoming soluble target interference in solution ELISA have also been investigated and highlights of these investigations are discussed. The selection of the solution ELISA format was based on availability of generic reagents, achievement of optimal drug tolerance and robust assay performance on a platform that is readily available in many laboratories. This approach removed the heavy reliance on specialized equipment sourced from a single vendor and assay conditions described here are broadly applicable to other immunogenicity assays across many biologics both during clinical development setting and in the post-marketing arena.