title: 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-alpha) 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-alpha represents a potential target for antibody-based therapy. A panel of 11 murine mAbs was prepared using recombinant hIFN-alpha2b as immunogen, all of which bound to the native form of the cytokine with affinity constants ranging from 1.7x10(7) M(-1) to 1.4x10(10) 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-alpha 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-alpha family, including the recombinant cytokines hIFN-alpha2a and hIFN-alpha2b and a heterogeneous collection of IFN-alpha 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-alpha subtypes. Consequently, the scFv EP18 represents a potential therapeutic agent for those immune and inflammatory diseases which are associated with an increased IFN-alpha expression.
authors.affiliation: Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje El Pozo-C.C. 242, S3000ZAA, Santa Fe, Argentina. mdepetri@fbcb.unl.edu.ar
authors.forename: M
authors.initials: M
authors.lastname: Depetris
authors.forename: P
authors.initials: P
authors.lastname: Casalis
authors.forename: R
authors.initials: R
authors.lastname: Kratje
authors.forename: M
authors.initials: M
authors.lastname: Etcheverrigaray
authors.forename: M
authors.initials: M
authors.lastname: Oggero
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D003001:Cloning, Molecular; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000276:immunology* / Q000302:isolation & purification; D007370:Interferon Type I / Q000276:immunology*; D016898:Interferon-alpha / Q000037:antagonists & inhibitors / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins
chemical_list: D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D007370:Interferon Type I; D016898:Interferon-alpha; D011994:Recombinant Proteins