Selection and characterisation of FVIII-specific single chain variable fragments

Summary

The development of inhibitory anti-FVIII antibodies is currently the most severe complication in the treatment of haemophilia A patients. Inhibitor eradication can be achieved by immune tolerance induction (ITI). Recent findings suggest a correlation between the FVIII-specific IgG subclass distribution and the duration or outcome of ITI. To quantify FVIII-specific IgG subclasses in patients’ plasma FVIII-specific IgG standards are required. Here, the isolation of FVIII-specific single chain variable fragments (scFvs) from synthetic phage display libraries and the characterisation of their FVIII domain specificity are described. The isolated scFv 1G10, which binds to the FVIII A2 domain, was cloned into the context of the four human IgG (hIgG) subclasses and expressed in mammalian cells. Purified 1G10-hIgG1, -hIgG2, -hIgG3 and -hIgG4 are used as standards to determine the absolute amounts and relative contribution of the different FVIII-specific IgG subclasses in future studies. The results from these studies will eventually add to understanding the role of the FVIII-specific IgG subclass distribution as prognostic factor for the outcome of ITI.

Zusammenfassung

In der modernen Therapie von Hämophilie A-Patienten ist die Entwicklung inhibitorischer FVIII-spezifischer Antikörper die schwerwiegendste Komplikation. Toleranz für FVIII kann durch eine Immuntoleranztherapie (ITI) erreicht werden. Aktuelle Untersuchungen deuten auf einen Zusammenhang zwischen der FVIII-spezifischen IgG-Subklassenverteilung und der Dauer sowie dem Erfolg der ITI hin. Um die FVIII-spezifischen IgGSubklassen im Patientenplasma zu quantifizieren, werden FVIII-spezifische IgG-Standards benötigt.

Dafür wurden FVIII-spezifische Antikörperfragmente aus synthetischen Phagen-Bibliotheken isoliert und ihre FVIII Domänen-Spezifität charakterisiert. Der FVIII-spezifische scFv 1G10, der an die FVIII A2-Domäne bindet, wurde in den Kontext der vier humanen IgG (hIgG)-Subklassen kloniert und in Säugerzellen exprimiert. Aufgereinigte 1G10-hIgG1, -hIgG2, -hIgG3 und -hIgG4 werden als Standard verwendet, um in Studien die absoluten und relative Anteile der vier FVIII-spezifischen IgG-Subklassen zu bestimmen. Die Ergebnisse dieser Studien tragen zum weiteren Verständnis über den Zusammenhang von IgG-Subklassenverteilung und ITI-Erfolg bei.

Keywords

Haemophilia A - phage display - FVIII inhibitors - IgG subclasses

Schlüsselwörter

Hämophilie A - Phagen-Display - FVIII-Hemmkörper - IgG-Subklassen

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