Accelerated formation of fibrin clots in a tumor microenvironment can be used for targeted delivery of interferon gamma (IFN gamma) to tumor cells. Here, we selected cysteine-arginine-glutamic acid-lysine-alanine (CREKA) as the fibrin clot-binding peptide and designed 2 types of fusion proteins for tumor targeting. The CREKA peptide was fused to IFN gamma's C-terminus, with or without a matrix metalloproteinase-2 (MMP2)-cleavable linker (IFN gamma-mmp-CREKA or IFN gamma-CREKA, respectively). The former was designed to release IFN gamma from IFN gamma-mmp-CREKA bound to fibrin clots, to ensure IFN gamma's function in the tumor milieu. IFN gamma-activated sequence-dependent reporter gene expression in B16-BL6 cells revealed that the biological activities of IFNg-CREKA and IFN gamma were comparable, whereas that of IFN gamma-mmp-CREKA was approximately 60% that of IFNg. Plasma concentrations of IFN gamma-CREKA and IFN gamma-mmp-CREKA remained at effective levels for at least 4 weeks after gene transfer into mice. After gene transfer to tumor-bearing mice, intratumoral concentration of IFN gamma in pCpG-IFN gamma-mmp-CREKA group was tended to be higher than those of the other groups. Inhibition of colon-26 tumor growth was significantly more with gene transfer of IFN gamma-mmp-CREKA than with IFNg or IFN gamma-CREKA. These results indicate that targeted delivery of IFN gamma to fibrin clots through IFN gamma-mmp-CREKA fusion can enhance the therapeutic efficacy of IFN gamma in cancer gene therapy. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.