A Novel, Non-immunogenic Fyn SH3-derived Binding Protein with Tumor Vascular Targeting Properties

Dario Neri,Manuela Kaspar,Dragan Grabulovski

doi:10.1074/jbc.m609211200

Abstract

The generation of novel binding molecules based on protein frameworks ("scaffolds") represents an emerging field in protein engineering, with the potential to replace antibodies for many research and clinical applications. Here, we describe the design, construction, characterization, and use of a novel human Fyn SH3 phage library, containing 1.2 x 10(9) individual clone members. We also present the isolation and in vitro characterization of Fyn SH3-derived proteins binding to the extra-domain B of fibronectin, a marker of angiogenesis. One specific binding clone, named D3, was further evaluated and showed a remarkable ability to stain vascular structures in tumor sections. Furthermore, quantitative biodistribution studies in tumor-bearing mice revealed the ability of D3 to selectively accumulate in the tumor. In contrast to human scFv antibody fragments administered to mice, neither Fyn SH3 WT nor the D3 mutant was immunogenic in mice after four intravenous injections. The extra-domain B binding D3 protein opens new biomedical opportunities for the in vivo imaging of solid tumors and for the delivery of toxic agents to the tumoral vasculature.

Highlights

Mutated to produce a protein library, which can be screened for binding specificities of interest [1,2,3,4]
In the present article we describe the design, construction, and characterization of a human Fyn Src homology 3 (SH3) phage library containing more than 1 billion individual clones as well as the isolation and in vitro characterization of proteins binding to a pharmaceutically relevant target protein, the extra-domain B (EDB) of fibronectin, a marker of angiogenesis [19]
A total of 10 ␮g of insert was ligated into 75 ␮g of double-digested phagemid vector pHEN1 and electroporated into freshly prepared electrocompetent E. coli TG1 cells, resulting in a library containing a total of 1.2 ϫ 109 individual clones

Summary

Introduction

Mutated to produce a protein library, which can be screened for binding specificities of interest [1,2,3,4]. In the present article we describe the design, construction, and characterization of a human Fyn SH3 phage library containing more than 1 billion individual clones as well as the isolation and in vitro characterization of proteins binding to a pharmaceutically relevant target protein, the extra-domain B (EDB) of fibronectin, a marker of angiogenesis [19].

Results

Conclusion