Development and application of methods for the selective measurement of the human single amino acid exchange variant factor IX Padua

A. Weber1, A. Engelmaier1, D. Voelkel2, R. Pachlinger2, F. Scheiflinger1, H. Rottensteiner1 (1Vienna, Austria, 2Orth, Austria)

Clinical Science
Date: 17.02.2017,
Time: 11:00 - 12:00

Objective: The naturally occurring single amino exchange (R338L) variant factor IX (FIX) Padua is used in the BAX 335 gene therapy Phase 1/2 clinical trial since this gain-of-function mutation shows a 5 – 10 fold higher activity than that of FIX wild type. Assessment of the gene therapy’s success would benefit from a method that enables specific detection of the transgene product, particularly in the presence of inactive FIX as in cross-reactive material positive patients. Therefore, a Fab2 mini antibody, selectively binding to FIX Padua, was developed and applied for the development of FIX Padua-specific methods.

Methods: A Fab2 mini antibody, specifically binding to FIX Padua and isolated by phage display, was applied to capture FIX Padua. This Fab2 mini antibody was used in combination with a polyclonal anti FIX antibody to establish a specific ELISA for analysis of subject samples in clinical trials. The performance of this ELISA was checked in terms of accuracy, precision and parallelism. In addition, this Fab2 mini antibody could also be used to set up a FIX Padua-specific chromogenic FIX activity assay.

Results: The Fab2 mini antibody selected showed exclusive binding to FIX Padua with no cross-reactivity to wild-type FIX. The ELISA, using this Fab2 mini antibody to selectively capture FIX Padua in combination with a biotinylated polyclonal anti-FIX antibody and streptavidin peroxidase, covered a FIX Padua protein concentration range of 0.9 to 27.1 ng/mL. Spike-recovery carried out with representative patients’ samples showed acceptable recoveries and there was no influence of the citrated plasma matrix on the assay performance. Furthermore, there was clear correlation between FIX Padua protein concentration and FIX activity, while the presence of functionally inactive FIX had no impact on the assay. The calibration curve of the FIX Padua-specific chromogenic activity assay, carried out after selective capture of FIX Padua with the Fab2 mini antibody, ranged from 0.1 to 3.3 mU FIX Padua/mL, while a normal reference plasma pool showed no response.

Conclusion: The FIX Padua ELISA can be used in clinical settings to selectively quantify FIX Padua antigen levels. In addition, the Fab2 mini antibody offers the possibility to set up a FIX Padua-specific chromogenic activity assay.