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SLT-VEGF is a recombinant cytotoxin comprised of Shiga-like toxin (SLT) subunit A fused to human vascular endothelial growth factor (VEGF). It is highly cytotoxic to tumor endothelial cells overexpressing VEGF receptor-2 (VEGFR-2/KDR/Flk1) and inhibits the growth of primary tumors in subcutaneous models of breast and prostate cancer and inhibits metastatic dissemination in orthotopic models of pancreatic cancer. We examined the efficacy of SLT-VEGF in limiting tumor growth and metastasis in an orthotopic melanoma model, using NCR athymic nude mice inoculated with highly metastatic Line IV Cl 1 cultured human melanoma cells. Twice weekly injections of SLT-VEGF were started when tumors became palpable at one week after intradermal injection of 1 × 106 cells/mouse. Despite selective depletion of VEGFR-2 overexpressing endothelial cells from the tumor vasculature, SLT-VEGF treatment did not affect tumor growth. However, after primary tumors were removed, continued SLT-VEGF treatment led to fewer tumor recurrences (p = 0.007), reduced the incidence of lung metastasis (p = 0.038), and improved survival (p = 0.002). These results suggest that SLT-VEGF is effective at the very early stages of tumor development, when selective killing of VEGFR-2 overexpressing endothelial cells can still prevent further progression. We hypothesize that SLT-VEGF could be a promising adjuvant therapy to inhibit or prevent outgrowth of metastatic foci after excision of aggressive primary melanoma lesions. [ABSTRACT FROM AUTHOR]

Purpose: We recently developed a cysteine-containing peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)-maleimide. We then constructed and expressed C-tagged vascular endothelial growth factor (VEGF) and labeled it with HYNIC. We wished to test 99mTc-HYNIC-C-tagged VEGF (99mTc-HYNIC-VEGF) for the imaging of tumor vasculature before and after antiangiogenic (low continuous dosing, metronomic) and tumoricidal (high-dose) cyclophosphamide treatment. Methods: HYNIC-maleimide was reacted with the two thiol groups of C-tagged VEGF without any effect on biologic activity in vitro. 99mTc-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein (200.300 µCi, 1.2 µg protein) followed by microSPECT imaging 1 h later. Results: Sequencing analysis of HYNIC-containing peptides obtained after digestion confirmed the site-specific labeling of the two accessible thiol groups of C-tagged VEGF. Tumor vascularity was easily visualized with 99mTc/VEGF in Balb/c mice with 4T1 murine mammary carcinoma 10 days after implantation into the left axillary fat pad in controls (12.3±5.0 tumor/bkg, n=27) along with its decrease following treatment with high (150 mg/kg q.o. d. ×4; 1.14±0.48 tumor/bkg, n=9) or low (25 mg/kg q.d. × 7; 1.03±0.18 tumor/bkg, n=9) dose cyclophosphamide. Binding specificity was confirmed by observing a 75% decrease in tumor uptake of 99mTc/biotin-inactivated VEGF, as compared with 99mTc-HYNIC-VEGF. Conclusion: 99mTc can be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. 99mTc-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy. [ABSTRACT FROM AUTHOR]