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¹ Immunopharmacology and Targeted Therapy Section, Department of Bioimmunotherapy, M. D. Anderson Cancer Center, University of Texas, Houston, TX, ² Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX

Requests for reprints: Michael G. Rosenblum, Immunopharmacology and Targeted Therapy Section, Department of Bioimmunotherapy, M. D. Anderson Cancer Center, University of Texas, 1515 Holcombe Boulevard, Unit 44, Houston, TX 77030. Phone: (713) 792-3554; Fax: (713) 745-3916, E-mail: mrosenbl{at}notes.mdacc.tmc.edu

The serine protease granzyme B (GrB; 25 kDa) is capable of inducing apoptosis through both caspase-dependent and caspase-independent mechanisms. We designed a novel vascular-targeting fusion construct designated as GrB/vascular endothelial growth factor (VEGF)₁₂₁, which is composed of a non-heparin-binding isoform of VEGF and the proapoptotic pathway enzyme GrB fused via a short, flexible tether (G₄S). The chimeric fusion gene was then cloned into a bacterial vector, and the protein was expressed in Escherichia coli and purified by nickel-NTA metal affinity chromatography. Western blotting confirmed incorporation of both VEGF₁₂₁ and GrB proteins into the construct. GrB/VEGF₁₂₁ specifically bound (ELISA) to porcine aortic endothelial (PAE)/FLK-1 cells overexpressing the FLK-1/KDR receptor but not to cells overexpressing the FLT-1 receptor. Immunofluoresence studies showed that the GrB moiety of GrB/VEGF₁₂₁ was delivered efficiently and rapidly into the cytosol of PAE/FLK-1 cells but not into that of PAE/FLT-1 cells after 4 h treatment with GrB/VEGF₁₂₁. Treatment of cells with GrB/VEGF₁₂₁ showed that the IC₅₀ was 10 nM against PAE/FLK-1 cells; however, there were no cytotoxic effects observed on PAE/FLT-1 cells at doses up to 200 nM. GrB/VEGF₁₂₁ induced apoptotic events specifically on PAE/FLK-1 as assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, DNA laddering, and cytochrome c release from mitochondria. In addition, the fusion construct mediated the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase in target endothelial cells within 4 h after treatment. In conclusion, delivery of the human proapoptotic pathway enzyme GrB to tumor vascular endothelial cells or to tumor cells may have significant therapeutic potential and represents a potent new class of targeted therapeutic agents with a unique mechanism of action.

Note: Research conducted, in part, by the Clayton Foundation for Research.

Received 4/17/03; revised 7/ 1/03; accepted 7/31/03.

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