Targeting signals essential for angiogenesis
Vectors expressing VE-cadherin extracellular domains (EC) 1 and 1+2 were produced to be injected in mice or to be used in the allantois. When these vectors were used to infect human umbilical cord endothelial cells they caused disruption of the endothelial cell monolayer. This indicates that the fragments have the activity of disrupting cell to cell junctions. Therefore it was decided to test these viruses in vivo in a follow-up study to this project. In additional experiments, peptidic sequences of the EC1 domain have been used to further dissect the active region of EC1 domain of VE-cadherin required for its adhesive properties. Different short peptidic sequences spanning the all EC1 domain were tested. Three peptides found to be effective, as inhibitors of angiogenesis have been reformulated in a cyclic conformation.
In order to achieve a specific blockade of VEGF-induced tumour angiogenesis and also to maintain vascular permeability, which would be important for combination therapies with cytostatic compounds, partner 3 and 7 mutated VEGFR1 and targeted the tyrosine residues in the intracellular domain of the VEGFR-2. For a detailed and systematic functional analysis of single tyrosine groups in signalling all tyrosine groups were exchanged against phenylalanine in the murine VEGFR-2 and these VEGFR-2 mutants transfected into human endothelial cells (EC) for Western blot analysis. Publication: Heidenreich R, Machein M, Nicolaus A, Hilbig A, Wild C, Clauss M, Plate KH, Breier G. Inhibition of solid tumor growth by gene transfer of VEGF receptor-1 mutants. Int J Cancer 2004;111:348-57. VEGFR2 data so far unpublished.
The size of a VEGFR-2 promoter/enhancer construct was cut down from 2.3kbp to 510bp without loosing its endothelial specificity and transcriptional strength. This was shown by lacZ reporter analysis of both transient embryonic expression as well as analysis of transgenic mice, in which experimental tumours were analyzed. A patent was applied for and granted for this fragment and its use in gene therapy vectors for specific expression of therapeutic genes in tumor endothelium. Publication: Licht AH, Raab S, Hofmann U, Breier G. Endothelium-specific Cre recombinase activity in flk-1-Cre transgenic mice. Dev Dyn 2004;229:312-8. Patent: Breier G, Kappel A, Risau W, Rönicke V: Regulatory sequences capable of conferring expression of a heterologous DNA sequence in endothelial cells in vivo and uses thereof (European Patent Nr. 1007714, published 14.12.2005 Bulletin 2005/50, International Publication Number WO 1998/055638).
Targeting of adenoviral vector to tumor endothelium has been successfully achieved using different strategies. Adenobodies, obtained by the fusion of a single chain antibody (S11) and a peptide, were able to bind respectively to the adenoviral fiber knob and to selected endothelial receptors. S11 was able to prevent the binding of adenoviral vectors to its natural CAR receptor thereby avoiding uptake of the virus by unselected cells. The peptides on the other hand were able to guide the virus to the receptors VEGFR2, Tie 2 and integrins av(beta)3. (Anna Rita Bellu, Marieke Geel, Anu Kariat, Marianne G.Rots, Hidde J. Haisma. Selective targeting of adenoviral vectors to endothelial receptors avß3 integrins, VEGF and Tie2 by angio-adenobodies. Submitted.) A PEG molecule, bi-functional polyethylene glycol, has been shown to shield the adenovirus so that it was not able to bind to CAR and was not recognized by the immunosystem when injected intravenously in animals thereby incrementing the circulation time. The RGD peptide chemically coupled to the PEG molecule was able both in vitro and in vivo to target the pegylated virus to integrins. Furthermore, the HI loop of the viral fiber knob has been genetically modified for the insertion of peptides such as RGD to target the adenovirus to integrins. This method compared to the non-genetic modifications (adenobodies and pegylation) has the advantage to be a stable modification and is not influenced from external factors. (Anna Rita Bellu, Yunia Sribudiani, Marieke Geel, Ramon Alemany, Gritje Molema, Hidde J. Haisma. Adenoviral targeting of the tumor endothelium using three different approaches. Submitted).
A n important finding during the course of this project was that adenoviral EGR-1 overexpression caused a reduced responsiveness of endothelial cells to growth factors, prevented sprouting and tubule formation in vitro and prolonged expression triggered apoptosis. Furthermore, EGR-1 expressing viruses completely inhibited cell invasion and vessel formation in the murine Matrigel model and resulted in a dramatic blockade of tumor growth in a murine fibrosarcoma model. An analysis of this phenomenon showed that that EGR-1 can actually promote two different activities, one pro-proliferative and pro-angiogenic when expressed at low growth factor-induced level, and an anti-proliferative, anti-angiogenic and pro-apoptotic when overexpressed in a sustained way following infection with recombinant adenoviruses. Obviously, overexpression of EGR-1 causes the preferential induction of potent feed-back inhibitory mechanisms resulting in inhibition of angiogenesis. This could be likely caused by differential modification of EGR-1 when expressed following growth factor stimulation or following adenoviral expression (acetylation versus phosphorylation).
The VEGFR-3 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. Partner 2 has found that homozygous VEGFR-3 targeted mice die around day 10 of embryonic development due to failure of cardiovascular development. He has also purified and cloned the VEGFR-3 ligand, VEGF-C. When overexpressed as a transgene in the RIP-Tag model of pancreatic beta-cell tumors, VEGF-C induced the growth of peritumoral lymphatic vessels and was associated with lymphatic metastasis. VEGF-C overexpression also led to lymphangiogenesis, intralymphatic tumor growth and lymph node metastasis in an orthotopic model of human breast carcinoma in immunoincompetent mice. Furthermore, soluble VEGFR-3, which blocks embryonic lymphangiogenesis, blocked these changes.