A scFv antibody fragment as a therapeutic candidate to neutralize a broad diversity of human IFN-alpha subtypes

Abstract

Despite their significant role in maintaining the normal physiology, cytokines may cause pathological conditions when they are overproduced. In this way, the increased expression of human interferon alpha (hIFN-α) is associated with acute viral infections, inflammatory disorders and several autoimmune illnesses, where the cytokine may be a factor in either initiating or maintaining the disease. Currently, there are several mAbs marketed for a variety of indications and many more in clinical trials, in which IFN-α represents a potential target for antibody-based therapy. A panel of 11 murine mAbs was prepared using recombinant hIFN-α2b as immunogen, all of which bound to the native form of the cytokine with affinity constants ranging from 1.7 × 107 M- 1 to 1.4 × 1010 M- 1. An epitope mapping protocol demonstrated four spatially distinct areas of the protein recognized by the mAbs. Taking into account the characterization of the antibodies and their ability to inhibit the IFN-α biological activity, four mAbs were selected to produce scFv fragments. One of these fragments (CA5E6) was able to neutralize a wide spectrum of subtypes of the IFN-α family, including the recombinant cytokines hIFN-α2a and hIFN-α2b and a heterogeneous collection of IFN-α produced by activated leukocytes and Namalwa cells. With the aim of improving the affinity of the selected fragment, a standard error-prone PCR method was carried out. By using this strategy, it was possible to generate a new fragment (EP18) with increased affinity and ability to neutralize a broad diversity of IFN-α subtypes. Consequently, the scFv EP18 represents a potential therapeutic agent for those immune and inflammatory diseases which are associated with an increased IFN-α expression.