A single chain antibody fragment as therapeutic candidate to neutralize human IFN-alpha subtypes

DEPETRIS, M.; OGGERO EBERHARDT, M.; KRATJE, R. B.; ETCHEVERRIGARAY, M.

Praga. República Checa

Congreso; Protein Expression Europe; 2007

Cambridge Healthtech Institute

Cytokines have a significant role in sustaining normal physiology. However, pathological conditions may arise when they are overproduced. The increased expression of human interferon alpha (hIFN-alfa) is associated with several autoimmune illnesses where the cytokine may be a factor of either initiating or maintaining the disease. Nowadays, several monoclonal antibodies (mAbs) have been approved for human use or are in late stages of clinical trials, representing IFN-alfa an attractive target for antibody based therapy. A panel of 11 murine mAbs was prepared using recombinant hIFN alfa2b as immunogen. All of them 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-alfa 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 alfa family, including the recombinant cytokines hIFN alfa2a and hIFN alfa2b, and a heterogeneous collection of IFN alfa 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, obtaining a new fragment (EP18) with increased affinity and improved neutralizing capacity. Consequently, scFv EP18 becomes an interesting molecule to be used in the treatment of diseases such as systemic lupus erythematosus, multiple sclerosis and rheumatoid arthritis.