Use of uteroglobin for the engineering of polyvalent, polyspecific fusion proteins.
We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-alpha, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.
Ventura
Elisa
E
Laboratory of Recombinant Therapeutic Proteins, Advanced Biotechnology Centre, Istituto G Gaslini, Genoa, Italy.
Sassi
Francesca
F
Fossati
Sara
S
Parodi
Arianna
A
Blalock
William
W
Balza
Enrica
E
Castellani
Patrizia
P
Borsi
Laura
L
Carnemolla
Barbara
B
Zardi
Luciano
L
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D008958:Models, Molecular; D009374:Neoplasms, Experimental / Q000276:immunology / Q000473:pathology; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics; D055503:Protein Multimerization; D021382:Protein Sorting Signals / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014598:Uteroglobin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D007376:Interleukin-2; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D014598:Uteroglobin