Frank Horling

Distinct characteristics of antibody responses against factor VIII in healthy individuals and in different cohorts of hemophilia A patients

Neutralizing antibodies against factor VIII (FVIII) remain the major complication in the replacement therapy of hemophilia A patients. To better understand the evolution of these antibodies it is important to generate comprehensive datasets which include both neutralizing and nonneutralizing antibodies, their isotypes, and IgG subclasses. We developed sensitive ELISAs to analyze FVIII-binding antibodies in different cohorts of hemophilia A patients and in healthy individuals. Our data reveal the prevalence of FVIII-binding antibodies among healthy individuals (n = 600) to be as high as 19%, with a prevalence of antibody titers > or =1:80 of 2%. The prevalence of FVIII-binding antibodies was 34% (5% for titers > or =1:80) in patients without FVIII inhibitors (n = 77), 39% (4% for titers > 1:80) in patients after successful immune tolerance induction therapy (n = 23), and 100% (n = 20, all titers > or =1:80) in patients with FVIII inhibitors. We found significant differences for IgG subclasses of FVIII-binding antibodies between the different study cohorts. IgG4 and IgG1 were the most abundant IgG subclasses in patients with FVIII inhibitors. Strikingly, IgG4 was completely absent in patients without FVIII inhibitors and in healthy subjects. These findings point toward a distinct immune regulatory pathway responsible for the development of FVIII-specific IgG4 associated with FVIII inhibitors.

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.

The Mystery of Antibodies Against Polyethylene Glycol (PEG) - What do we Know?

PurposeRecent findings demonstrated anti-PEG antibody formation in some healthy individuals and patients who have not received PEGylated biotherapeutics. Some of these findings evoked criticism because of shortcomings in the antibody assays used. To better understand this topic, we established robust antibody analytics and screened two cohorts of healthy individuals and one cohort of hemophilia patients for the expression of anti-PEG antibodies.MethodsA flow cytometry approach and a fully validated ELISA platform were established to detect specific anti-PEG antibodies. Immunohistochemistry was used to test for potential binding of anti-PEG antibodies to human tissues.ResultsIgM and/or IgG anti-PEG antibodies are expressed by some healthy individuals and by some patients with hemophilia who have not received PEGylated biotherapeutics. These antibodies can be either transient or persistent and recognize PEGs of different sizes with or without terminal methoxy groups. Age and location of healthy individuals influence the prevalence of IgG but not of IgM antibodies. Anti-PEG antibodies do not cross-react with human tissues supporting the safety of the antibodies.ConclusionWe confirm that some healthy individuals and some patients with hemophilia express specific antibodies against PEG which are not associated with any pathology and do not bind to human tissues.

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.

Nonacog gamma, a novel recombinant factor IX with low factor IXa content for treatment and prophylaxis of bleeding episodes

Nonacog gamma is a new recombinant factor IX to treat factor IX deficiency. It is indicated for control of bleeding episodes, perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with hemophilia B. Nonacog gamma was first approved in the USA in June 2013 under the trade name RIXUBIS followed by market approvals in Australia and the EU in 2014, and marketing authorization decision is pending in Japan. Nonacog gamma is derived from a recombinant Chinese hamster ovary cell line using a state of the art biotechnological manufacturing process. Recombinant factor IX is produced by Baxter’s protein-free fermentation technology, which was first developed for ADVATE. The product is purified and formulated in the absence of any human or animal-derived protein. Nonacog gamma was characterized both in comprehensive in vitro and in vivo non-clinical studies as well as in an extensive clinical trial program.

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.

A false sense of security?:can tiered approach be trusted to accurately classify immunogenicity samples?

Detecting and characterizing of anti-drug antibodies (ADA) against a protein therapeutic are crucially important to monitor the unwanted immune response. Usually a multi-tiered approach that initially rapidly screens for positive samples that are subsequently confirmed in a separate assay is employed for testing of patient samples for ADA activity. In this manuscript we evaluate the ability of different methods used to classify subject with screening and competition based confirmatory assays. We find that for the overall performance of the multi-stage process the method used for confirmation is most important where a t-test is best when differences are moderate to large. Moreover we find that, when differences between positive and negative samples are not sufficiently large, using a competition based confirmation step does yield poor classification of positive samples.