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

• Published in: The Journal of biological chemistry Vol. 282; no. 5; pp. 3196 - 3204
• Main Authors: Grabulovski, Dragan, Kaspar, Manuela, Neri, Dario
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02-02-2007; American Society for Biochemistry and Molecular Biology
• Subjects: Adaptor Proteins, Signal Transducing - chemistry; Adaptor Proteins, Signal Transducing - isolation & purification; Adaptor Proteins, Signal Transducing - metabolism; Animals; Base Sequence; Cloning, Molecular; Dimerization; DNA Primers; Female; Humans; Mice; Models, Molecular; Molecular Sequence Data; Polymerase Chain Reaction; Protein Conformation; Proto-Oncogene Proteins c-fyn - chemistry; Proto-Oncogene Proteins c-fyn - genetics; Proto-Oncogene Proteins c-fyn - isolation & purification; Proto-Oncogene Proteins c-fyn - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; src Homology Domains; Teratoma - metabolism; Teratoma - pathology; Tissue Distribution
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M609211200

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 × 109 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.