Abstract
The description of hyper-functional factor IX (FIX) Padua triggered the development of BAX 335, an AAV8-based hemophilia B gene therapy vector designed to compensate for low FIX protein expression levels by expressing the FIX Padua variant, thereby reducing the exposure to viral vector. The presence of inactive FIX protein at baseline hindered conventional FIX:Ag ELISA from contributing to a more profound understanding of clinical data from the BAX 335 Phase 1/2 study (ClinicalTrials.gov: NCT01687608). By applying phage display technology, a Fab2 mini-antibody selectively binding to FIX Padua was developed and used to establish a FIX Padua-specific ELISA. The assay adequately performed, utilizing human and monkey plasma samples, and enabled the selective quantification of FIX Padua protein in human plasma samples from the BAX 335 trial. The mini-antibody also allowed the development of a chromogenic FIX Padua-specific activity assay, which adequately performed in human and mouse plasma. Collectively, the isolated FIX Padua-specific mini-antibody enabled the development of transgene-product-specific assays, which should improve the monitoring of hemophilia B gene therapies. The approach applied here for FIX Padua could be leveraged to develop variant-specific activity assays for other therapies where highly active enzymes are instrumental in achieving therapeutic levels of the transgene product.
Highlights
Severe to moderate hemophilia B, an X-linked hereditary deficiency of functionally active human coagulation factor IX (FIX), is characterized by spontaneous or traumatic bleeding episodes.[1]
Three differently modified FIX preparations with prolonged circulatory half-lives have been made available recently for hemophilia B treatment: a recombinant recombinant FIX (rFIX)-immunoglobulin G1 (IgG1) Fc fusion protein, a glycoPEGylated rFIX preparation, and an rFIX preparation linked to human serum albumin.[4,5,6]
The study published by Nathwani et al represented a milestone providing evidence that using an adenovirus-associated virus (AAV) vector-mediated transfer of the FIX gene resulted in the sustained expression of FIX in hemophilia B patients.[12]
Summary
Severe to moderate hemophilia B, an X-linked hereditary deficiency of functionally active human coagulation factor IX (FIX), is characterized by spontaneous or traumatic bleeding episodes.[1]. Data published by Kay et al in 1993 showed sustained partial correction of FIX deficiency in FIX-deficient dogs using a retroviral vector, and since ongoing development of this approach took place.[7,8,9,10,11] The study published by Nathwani et al represented a milestone providing evidence that using an adenovirus-associated virus (AAV) vector-mediated transfer of the FIX gene resulted in the sustained expression of FIX in hemophilia B patients.[12] This basic concept was enriched by using the naturally occurring single amino acid exchange variant FIX Padua, where leucine is substituted for arginine at position 338 (FIX Padua, R338L) as the expression target.[13] This gain-of-function mutation shows specific activity up to 10-fold higher than that of the FIX wild-type.[13] Nonclinical studies, one of which supports an ongoing clinical trial, supported the hypothesis that expression of this FIX variant with higher specific activity will result in adequate FIX activities, despite low protein expression levels.[14,15,16] Current achievements in this experimental field of hemophilia B treatment are reviewed.[17,18,19]. In the background of functionally inactive FIX, as present in cross-reactive-material-positive (CRM+) hemophilia B patients, who are counting for up to 30%–40%.20 we developed a Fab[2] mini-antibody that selectively recognizes the FIX Padua protein harboring a single amino acid exchange and used this antibody to set up FIX Padua-specific methods
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