Antiangiogenic treatment with the three thrombospondin-1 type 1 repeats recombinant protein in an orthotopic human pancreatic cancer model.

PURPOSE: This study investigates the antiangiogenesis and antitumor efficacy of a recombinant protein composed of the three type 1 repeats (3TSR) of thrombospondin-1 in an orthotopic human pancreatic cancer model and provides useful preclinical data for pancreatic cancer treatment. EXPERIMENTAL DESIGN: Human pancreatic cancer cells (AsPC-1) were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR (3 mg per kg per day) or PBS for 3 weeks. Subsequently, the effects of 3TSR on tumor growth, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. The in vitro effects of 3TSR on human pancreatic cancer cells were also studied. RESULTS: 3TSR treatment significantly reduced angiogenesis and tumor growth of orthotopic pancreatic cancer. 3TSR-treated mice had a 69% reduction in tumor volume (316.6 +/- 79.3 versus 1,012.2 +/- 364.5 mm(3); P = 0.0001), and a significant increase in tumor necrotic area. After 3TSR treatment, both the vessel number and average microvessel size were significantly decreased, and microvessel density was decreased from 8.0% to 3.7% (P < 0.0001). The apoptotic rate of tumoral endothelial cells in 3TSR-treated tumors increased to 14.7% comparing to 4.2% in control tumors (P < 0.0001). 3TSR showed no direct effects on pancreatic cancer cell proliferation or apoptosis either in vivo or in vitro. CONCLUSION: 3TSR, a domain of a natural occurring angiogenesis inhibitor, showed potent therapeutic effect in pancreatic cancer by inhibiting tumor angiogenesis and may prove to be a promising agent for clinical pancreatic cancer treatment.

Discovery of high-affinity peptide binders to BLyS by phage display.

B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) family member and a key regulator of B cell responses. We employed a phage display-based approach to identify peptides that bind BLyS with high selectivity and affinity. Sequence analysis of first-generation BLyS-binding peptides revealed two dominant peptide motifs, including one containing a conserved DxLT sequence. Selected linear peptides with this motif were found to bind BLyS with K(D) values of 1-3 microM. In order to improve the binding affinity for BLyS, consensus residues flanking the DxLT sequence were seeded into a second-generation, BLyS affinity maturation library (BAML). BAML phage were subjected to stringent binding competition conditions to select for isolates expressing high-affinity peptide ligands for BLyS. Post-selection analysis of BAML peptide sequences resulted in the identification of a core decapeptide motif (WYDPLTKLWL). Peptides containing this core motif exhibited K(D) values as low as 26 nM, approximately 100-fold lower than that of first-generation peptides. A fluorescence anisotropy assay was developed to monitor the protein-protein interaction between BLyS labeled with a ruthenium chelate, and TACI-Fc, a soluble form of a BLyS receptor. Using this assay it was found that a BAML peptide disrupts this high-affinity protein-protein interaction. This demonstrates the potential of short peptides for disruption of high affinity cytokine-receptor interactions.