Steven Pirie-Shepherd

The Hemostatic System as a Regulator of Angiogenesis

Angiogenesis is the process of sprouting and configuring new blood vessels from pre-existing blood vessels, whereas the hemostatic system maintains the liquid flow of blood by regulating platelet adherence and fibrin deposition. Both systems normally appear quiescent, yet both systems remain poised for repair of injury. With vessel injury, a rapid sequence of reactions must occur to occlude the vessel wall defect and prevent hemorrhage. Activated platelets link the margins of the defect and form a provisional barrier that is quickly enmeshed with polymerized fibrin. This clot structure initially requires immobilized vascular endothelial cells to anchor the clot and prevent further bleeding. Thereafter, endothelial cells at the clot margins become mobile, dismantling and invading the cross-linked fibrin structure to rebuild a new vessel wall. Although the positive and negative regulators that control the delicate balance of platelet reactivity and fibrin deposition have been elucidated over the past four decades, analogous proteins that control endothelial cell growth and inhibition have only been discovered within the past decade. Hemostasis and angiogenesis are becoming increasingly inter-related. Proteins generated by the hemostatic system coordinate the spatial localization and temporal sequence of clot/endothelial cell stabilization followed by endothelial cell growth and repair of a damaged blood vessel. We focus here on the regulation of angiogenesis during vessel repair mediated by proteins secreted by platelets and derived as cryptic fragments from the coagulation cascade and fibrinolytic system.

Specifically Targeting Angiopoietin-2 Inhibits Angiogenesis, Tie2-Expressing Monocyte Infiltration, and Tumor Growth

Purpose: Angiopoietin-1 (Ang1) plays a key role in maintaining stable vasculature, whereas in a tumor Ang2 antagonizes Ang1s function and promotes the initiation of the angiogenic switch. Specifically targeting Ang2 is a promising anticancer strategy. Here we describe the development and characterization of a new class of biotherapeutics referred to as CovX-Bodies, which are created by chemical fusion of a peptide and a carrier antibody scaffold. Experimental Design: Various linker tethering sites on peptides were examined for their effect on CovX-Body in vitro potency and pharmacokinetics. Ang2 CovX-Bodies with low nmol/L IC50s and significantly improved pharmacokinetics were tested in tumor xenograft studies alone or in combination with standard of care agents. Tumor samples were analyzed for target engagement, via Ang2 protein level, CD31-positive tumor vasculature, and Tie2 expressing monocyte penetration. Results: Bivalent Ang2 CovX-Bodies selectively block the Ang2–Tie2 interaction (IC50 < 1 nmol/L) with dramatically improved pharmacokinetics (T½ > 100 hours). Using a staged Colo-205 xenograft model, significant tumor growth inhibition (TGI) was observed (40%–63%, P < 0.01). Ang2 protein levels were reduced by approximately 50% inside tumors (P < 0.01), whereas tumor microvessel density (P < 0.01) and intratumor proangiogenic Tie2+CD11b+ cells (P < 0.05) were significantly reduced. When combined with sunitinib, sorafenib, bevacizumab, irinotecan, or docetaxel, Ang2 CovX-Bodies produced even greater efficacy (∼80% TGI, P < 0.01). Conclusion: CovX-Bodies provide an elegant solution to overcome the pharmacokinetic–pharmacodynamic problems of peptides. Long-acting Ang2 specific CovX-Bodies will be useful as single agents and in combination with standard-of-care agents. Clin Cancer Res; 17(5); 1001–11. ©2011 AACR.

Disruption of the KEX1 gene in Pichia pastoris allows expression of full-length murine and human endostatin.

Endostatin is a potent angiogenesis inhibitor. In order to isolate sufficient quantities of soluble protein for in vivo studies in mice, we expressed murine endostatin in Pichia pastoris. Analysis of the expressed protein by mass spectrometry indicated that the protein was truncated. N‐terminal sequence analysis determined that the N‐terminus was intact, suggesting that the C‐terminal lysine was missing. In Saccharomyces cerevisiae, Kex1p can cleave lysine and arginine residues from the C‐terminus of peptides and proteins. We hypothesized that the KEX1 homologue in P. pastoris is responsible for the loss of the C‐terminal lysine of endostatin. To test this hypothesis, we cloned and disrupted the P. pastoris KEX1 gene. Although the overall amino acid identity between the P. pastoris and the S. cerevisae Kex1p is only 36%, the amino acid residues involved in the catalytic activity or close to the active residues are highly conserved. Disruption of the KEX1 reading frame allowed expression of murine and human endostatin with the C‐terminal lysine. The KEX1 disruption strain may be a useful tool for the expression of other proteins with a C‐terminal basic amino acid. Addition of a lysine to the C‐terminus of recombinant proteins may protect the C‐terminus from degradation by other carboxypeptidases. 3·5 kb of the P. pastoris KEX1 gene locus have been deposited in the GeneBank database and are available under Accession No. AF095574. Copyright

Evolution of potent and stable placental-growth-factor-1-targeting CovX-bodies from phage display peptide discovery.

Novel phage-derived peptides are the first reported molecules specifically targeting human placental growth factor 1 (PlGF-1). Phage data enabled peptide modifications that decreased IC(50) values in PlGF-1/VEGFR-1 competition ELISA from 100 to 1 μM. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold 1 (CVX-2000), generating bivalent CovX-Bodies with 2 nM K(D) against PlGF-1. In vitro and in vivo peptide cleavage mapping studies enabled the identification of proteolytic hotspots that were subsequently chemically modified. These changes decreased IC(50) to 0.4 nM and increased compound stability from 5% remaining at 6 h after injection to 35% remaining at 24 h with a β phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h β half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored.

Abstract LB-396: FGFR4-antagonistic CovX-Body™ inhibits tumor growth

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL FGFR4 is the receptor for FGF19. Published data has shown FGF19 transgenic animals develop hepatocellular carcinoma (HCC), suggesting that the FGFR4:FGF19 axis may play a role in HCC. FGFR4 upregulation has been shown to be a negative prognostic indicator in multiple tumor types. CVX-63, an FGFR4-specific CovX-Body , was produced by fusing a chemically modified peptide to the specific Fab binding site of a specially designed antibody. CVX-63 was shown to inhibit FGF19 binding to FGFR4 in a competition ELISA with an IC50 of 10 nM, and had no cross reactivity with FGFR1–3. CVX-63 has a beta half life of 55 hrs in the mouse. The anti-tumor activity of CVX-63 was demonstrated in a staged Colo-205 xenograft model, with CVX-63 administered i.v. weekly. Maximal activity of 30% tumor growth inhibition was seen at dosing 10 mg/kg. Immunohistochemistry analysis of treated tumors shows a >90% reduction in phospho-ERK and 50% downregulation of FGFR4 on tumor cells. Ki67, a marker of proliferation was reduced, but active caspase 3 was not found in treated tumors. A patient-derived HCC xenograft was identified which expresses both FGFR4 and FGF19. When tested in this HCC model, CVX-63 did not show any effect inhibiting tumor growth. These data demonstrate that CVX-63 effectively inhibits FGFR4 activity in xenografts. FGFR4 inhibition has a modest effect on human tumor progression and FGFR4 does not seem to be a critical driver of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-396. doi:10.1158/1538-7445.AM2011-LB-396

Ein neuer Angiogenese-Inhibitor (DBP-maf) inhibiert die Endothelzell-Biologie und das Tumorwachstum im Maus-Model

Background: Tumor growth is dependent upon the balance of positive and negative regulators of angiogenesis. At least three antiangiogenic molecules have been previously isolated from conditioned media of tumors where these tumors have shown antiangiogenic potential in a mouse model. Methods and Results: BxPc-3 conditioned media (containing 5% fetal calf serum) was applied to a heparin Sepharose column and eluted with a 0.5 M NaCl steps. Fractions that inhibited capillary endothelial cell proliferation were further purified using Q-Sepharose and monoQ anion exchange chromatography. The inhibitory activity was associated with a protein of 53 kD. Sequence analysis revealed identity to Vitamin D binding protein (DBP). Purified DBP itself had no significant effect on endothelial cell proliferation. Therefore we tested the ability of serum free conditioned media from BxPC3 cells to process DBP by incubating DBP in conditioned media. The converted DBP specifically inhibited endothelial proliferation and was able to stimulate macrophages. Vitamin D binding protein-macrophage activating factor (DBP-maf) was able to suppress tumor growth in a dose dependent manner significantly. Immunhistological analysis showed a significantly reduced micovessel density and a elevated apoptotic rate and an unchanged proliferation rate. Conclusions: Human pancreatic cancer cell line express enzymatic activity which converts DPB to DBP-maf. This molecule inhibits endothelial cell biology in vitro and angiogenesis in vivo and is therefore a new candidate for antiangiogenic tumor therapy.