Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

Abstract

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-␣, 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.The generation of recombinant polyvalent and/or polyspecific fusion proteins for use as components of novel drugs is still hindered by factors that limit their production, storage, and use, chief of which are issues related to instability and/or inadequate solubility. Here we describe a novel approach based on the use of uteroglobin (UG) 3 as a skeleton for the generation of polyvalent/polyspecific recombinant proteins. Human UG is a small (15.8 kDa) globular, nonglycosylated, and homodimeric secreted protein that was discovered independently by two groups in the 1960s in rabbit uterus (1, 2), and it is the first member of a new superfamily of proteins, the so-called Secretoglobins (Scgb) (3). UG is present in the blood at a concentration of about 15 g/ml and is found in urine and in other body fluids. The UG monomer is composed of about 70 amino acids, depending on the species, and is organized in a four ␣-helix secondary structure; the two subunits are joined in an antiparallel fashion by disulfide bridges established between two highly conserved cysteine residues in amino-and carboxyl-terminal positions (4) (see Fig. 1). The exact functions of UG are not yet clear, but the protein has been reported to have antiinflammatory properties due to its ability to inhibit the soluble phospholipase A 2 . Moreover, UG contains a central hydrophobic cavity able to accommodate hydrophobic molecules such as progesterone, retinol, and prostaglandin D 2 . Theoretically, this cavity could be loaded with different types of therapeutic hydrophobic substances and delivered to targets (for exhaustive reviews on UG, see Refs. 5, 6and references therein).The high solubility and stability of UG to pH and temperature variations, its resistance to proteases, and its homodimeric structure prompted us to consider the protein as a candidate linker for the generation of polyvalent and polyspecific recombinant proteins. We demonstrate here that the use of UG as a linker could provide a general method for...

Use of the Uteroglobin Platform for the Expression of a Bivalent Antibody against Oncofetal Fibronectin in Escherichia coli

“…2B). Identical bands under reducing and non-reducing conditions were observed for L19-UG purified from CHO cells [26]. SEC of the purified and refolded L19-UG shows a single peak with a retention volume of approximately 15 ml, which is in accordance with the molecular mass of dimeric L19-UG and which coincides with the retention volume obtained with the L19-UG purified from CHO cells (Fig.…”

“…We recently described a novel strategy for the generation of divalent and dual-specific tetravalent antibodies based on the use of uteroglobin (UG) [26] , [27] . UG is a seventy-amino acids globular and non-glycosylated homodimeric secreted protein [28] .…”

“…1 ) consists of the fusion of the recombinant antibody sequence at the amino terminal or alternatively at the carboxyl terminal or both the amino and carboxyl terminals of UG; the covalent dimerization of UG allows the dimerization of the fusion proteins and thus the generation of divalent or dual specific-tetravalent molecules, which, when compared with similar fusion proteins without UG, possess enhanced solubility and stability, factors that would improve their storage and clinical use [26] . L19-UG is very soluble and stable and has a better performance with respect to the SIP for in vivo accumulation in neoplastic tissues in tumor-bearing mice [26] . However, until now, both the SIP and UG formats of L19 have been produced in mammalian cells.…”

Selective targeted delivery of the TNF-alpha receptor p75 and uteroglobin to the vasculature of inflamed tissues: a preliminary report

“…The anti-inflammatory potential of the UG component in the fusion protein must also be evaluated, and this should be possible using the fusion protein L19-UG that we have recently described [16]. The therapeutic efficacy of the targeted delivery of TNF-alpha antagonists to the inflamed tissues may thus provide a fertile future research area and yield important contributions for the therapy of degenerative inflammatory diseases.…”

Antibody Vectors for Imaging

“…In vivo , the 99m Tc-labeled tetramer demonstrated the longest serum persistence and highest tumor accumulation (4.3% ID/g at 24 h p.i.). Other multimerization domains that have been used are C4 binding protein to produce scFv octamers 61, and recently dimeric and tetrameric scFv molecules fused to uteroglobin was described 62. However, these have not been evaluated in vivo .…”