John-Philip Lawo

Affinity of FVIII-specific antibodies reveals major differences between neutralizing and nonneutralizing antibodies in humans

Recently, we reported that distinct immunoglobulin (Ig) isotypes and IgG subclasses of factor VIII (FVIII)-specific antibodies are found in different cohorts of patients with hemophilia A and in healthy individuals. Prompted by these findings, we further investigated the distinguishing properties among the different populations of FVIII-specific antibodies. We hypothesized that the affinity of antibodies would discriminate between the neutralizing and nonneutralizing antibodies found in different study cohorts. To test this idea, we established a competition-based enzyme-linked immunosorbent assay technology to assess the apparent affinities for each isotype and IgG subclass of FVIII-specific antibodies without the need for antibody purification. We present a unique data set of apparent affinities of FVIII-specific antibodies found in healthy individuals, patients with congenital hemophilia A with and without FVIII inhibitors, and patients with acquired hemophilia A. Our data indicate that FVIII-specific antibodies found in patients with FVIII inhibitors have an up to 100-fold higher apparent affinity than that of antibodies found in patients without inhibitors and in healthy individuals. High-affinity FVIII-specific antibodies could be retrospectively detected in longitudinal samples of an individual patient with FVIII inhibitors 543 days before the first positive Bethesda assay. This finding suggests that these antibodies might serve as potential biomarkers for evolving FVIII inhibitor responses.

A formal comparison of different methods for establishing cut points to distinguish positive and negative samples in immunoassays

Biotechnology derived therapeutics may induce an unwanted immune response leading to the formation of anti-drug antibodies (ADA). As a result the efficacy and safety of the therapeutic protein could be impaired. Neutralizing antibodies may, for example, affect pharmacokinetics of the therapeutic protein or induce autoimmunity. Therefore a drug induced immune response is a major concern and needs to be assessed during drug development. It is therefore crucial to have assays available for the detection and characterization of ADAs. These assays are used to classify samples in positive and negative samples based on a cut point. In this manuscript we investigate the performance of established and newly developed methods to determine a cut point in immunoassays such as ELISA through simulation and analysis of real data. The different methods are found to have different advantages and disadvantages. A robust parametric approach generally resulted in very good results and can be recommended for many situations. The newly introduced method based on mixture models yields similar results to the robust parametric approach but offers some additional flexibility at the expense of higher complexity.

Establishing Bioequivalence in Complete and Incomplete Data Designs Using AUCs

Nonclinical in vivo animal studies have to be completed before starting clinical studies of the pharmacokinetic behavior of a drug in humans. The drug exposure in animal studies is often measured by the area under the concentration versus time curve (AUC). The classical complete data design where each animal is sampled for analysis at every time point is applicable for large animals only. In the case of small animals, where blood sampling is restricted, the batch design or the serial sampling design need to be considered. In batch designs, samples are taken more than once from each animal, but not at all time points. In serial sampling designs, only one sample is taken from each animal. In this article we derive the asymptotic distribution for the ratio of two AUCs and construct different confidence intervals, which are frequently used to assess bioequivalence. The performance of these intervals is then evaluated between the different designs in a simulation study. Additionally, the sample sizes required for the different designs are compared.

A comparison of methods for classifying samples as truly specific with confirmatory immunoassays.

Biotechnology-derived therapeutics may induce an unwanted immune response leading to the formation of anti-drug antibodies (ADAs) which can result in altered efficacy and safety of the therapeutic protein. Anti-drug antibodies may, for example, affect pharmacokinetics of the therapeutic protein or induce autoimmunity. It is therefore crucial to have assays available for the detection and characterization of ADAs. Commonly, a screening assay is initially used to classify samples as either ADA positive or negative. A confirmatory assay, typically based on antigen competition, is subsequently employed to separate false positive samples from truly positive samples. In this manuscript we investigate the performance of different statistical methods classifying samples in competition assays through simulation and analysis of real data. In our evaluations we do not find a uniformly best method although a simple t-test does provide good results throughout. More crucially we find that very large differences between uninhibited and inhibited measurements relative to the assay variability are required in order to obtain useful classification results questioning the usefulness of competition assays with high variability.

Minimum clinically important difference analysis confirms the efficacy of IgPro10 in CIDP: the PRIMA trial

Dear Editor, The PRIMA (PRivigen Impact on Mobility and Autonomy, NCT01184846) trial, a prospective, multi-center, single-arm, open-label, phase III trial, was designed to assess efficacy and safety of IgPro10 (10% liquid IVIG formulated with L-proline, Privigen®, CSL Behring, Berne, Switzerland) in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) (Léger et al., 2013). The primary outcome of the PRIMA study was the responder rate by the 10-point adjusted Inflammatory Neuropathy Cause and Treatment (INCAT) disability score (responders defined as showing an INCAT score improvement ≥1 vs baseline). The success criterion (responder rate ≥35%) was met, making IgPro10 the second IgG product with demonstrated efficacy in CIDP (after IGIV-C) (Hughes et al., 2008; Léger et al., 2013). Here we examine the clinical relevance of the PRIMA study results using the concept of minimal clinically important difference (MCID), which is defined as the smallest difference in clinical score that patients perceive as beneficial and that could lead to a change in the patient’s management (Jaeschke et al., 1989). For this analysis, responder rates for various outcome measures used in the PRIMA trial were recalculated based on MCID cut-off values obtained through selected methods to determine whether the statistically significant results obtained previously also reflect clinically meaningful changes for patients with CIDP. In the PRIMA trial, 28 adult patients with definite or probable CIDP were included. All the enrolled patients first received an IgPro10 induction dose of 2 g/kg body weight in week 1, followed by up to seven infusions of 1 g/kg body weight at 3-week intervals. Outcome measures used in the PRIMA trial were selected based on previous recommendations for assessment in inflammatory neuropathies (Merkies and Lauria, 2006; Lunn et al., 2008). Change in INCAT scores, Medical Research Council (MRC) sum scores,

Subcutaneous immunoglobulin for maintenance treatment in chronic inflammatory demyelinating polyneuropathy (CIDP), a multicenter randomized double-blind placebo-controlled trial: the Path study

Patients with chronic inflammatory demyelinating polyneuropathy (CIDP) often require long-term intravenous immunoglobulin (IVIG) maintenance therapy. Subcutaneous immunoglobulin (SCIG) offers an alternative administration option with anticipated improvements in patient quality of life, convenience, and flexibility. To evaluate IgPro20 (SCIG) as a maintenance treatment in CIDP. A randomized, double-blind trial in CIDP patients (n=172) investigated 0.2 and 0.4 g/kg weekly doses of IgPro20 versus placebo. The primary outcome was percentage of patients with CIDP relapse/withdrawal during 24-weeks of treatment determined by Inflammatory Neuropathy Cause and Treatment score. Secondary endpoints included grip strength and patient satisfaction. Both IgPro20 doses significantly reduced rate of CIDP relapse/withdrawal versus placebo. Grip strength remained stable with Hizentra®, but deteriorated with placebo. Most subjects preferred SCIG over IVIG. Local reactions, reported in 33% of IgPro20-treated patients, were mild or moderate in intensity. IgPro20 is efficacious and well-tolerated as maintenance treatment in CIDP. This article is protected by copyright. All rights reserved