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

Elisa Ventura,Francesca Sassi,Sara Fossati,Arianna Parodi,William Blalock,Enrica Balza,Patrizia Castellani,Laura Borsi,Barbara Carnemolla,Luciano Zardi

doi:10.1074/jbc.m109.025924

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-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.

Highlights

Member of a new superfamily of proteins, the so-called Secretoglobins (Scgb) [3]
We demonstrate here that the use of UG as a linker could provide a general method for the generation of covalently linked bivalent and tetravalent antibodies, either monospecific or bispecific, as well as of different kinds of fusion proteins, which, compared with similar fusion proteins without UG, possess generally enhanced properties of solubility and stability, factors that expedite their storage and clinical use
We describe the use of UG for the production of a bivalent and tetravalent format of L19, an scFv specific for the angiogenesis-associated extra domain B (ED-B) of fibronectin (FN) [7], of an immunocytokine composed of IL2 and L19, and of a tetravalent dual specificity antibody composed of L19 and the scFv D2E7, a human antibody able to neutralize TNF-␣ activity [8]

Summary

EXPERIMENTAL PROCEDURES

Human and Mouse UG cDNAs—Human UG cDNA was obtained by reverse transcription-PCR from normal human lung RNA using the Titan One-Step reverse transcription-PCR system (Roche Diagnostics), with primers TI-36 (see Table 1 for primers sequences) and TI-18, containing the EcoRI and NotI restriction sites, respectively. The resulting product was digested EcoRI/NotI and was ligated into EcoRI/NotI-digested

Sequences of the primers used

RESULTS

SvHsd mice bearing the syngeneic

DISCUSSION