New Discoveries in Regulation and Inhibition VEGF Vascular Endothelial Growth Factors

Agenda

Monday, October 23, 2000

Angiogenesis Regulating Factors

9:05 Oncogene-Mediated Regulation of VEGF Through The ERK and P38 Signaling Pathways
Unnur P. Thorgeirsson, M.D., Section Head, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH
We showed that different MAPK pathways are involved in up-regulation of VEGF mRNA expression in v-Ha-ras, v-raf, and v-myc transformed rat epithelial cell lines. VEGF was up-regulated through the ERK pathway and suppressed through the p38 pathway in the v-Ha-ras transformed and human breast carcinoma cell lines, whereas the p38 pathway was responsible for VEGF up-regulation in the v-raf and v-myc transformants. These findings may have important implications in the design of anti-angiogenic agents that target VEGF signaling pathways.

9:40 VEGFs, Angiopoietins and Ephrins: Emerging Views on the Individual and Collaborative Actions of These Angiogenic Factors
George D. Yancopoulos, M.D., Ph.D., Chief Scientific Officer, Sr. VP of Research, Regeneron Pharmaceuticals, Inc.
The recently discovered Angiopoietins join the vascular endothelial growth factor (VEGF) family as the only known growth factor families largely specific for vascular endothelial cells. A single member of the very large Ephrin family of growth factors, EphrinB2, also appears to have selective actions on blood vessels.
Emerging data indicates that VEGF and the Angiopoietins work in complementary and coordinated fashion during normal vascular development and remodeling, as well as in pathologic angiogenesis as occurs in tumors. Angiopoietin-1 seems to serve an important role during later stages of vessel maturation and stabilization, by optimizing formation of the vessel wall. While the hypervascularity formed in the presence of excess VEGF is leaky and fragile (and associated with tissue edema and hemorrhage), vessels made in the presence of excess Angiopoietin-1 are actually resistant to vascular leak induced by VEGF or inflammatory mediators. The ability to decrease plasma leakage and the resulting edema could have important clinical benefit in numerous disease settings, including diabetic retinopathy, tumor-associated ascites, brain edema associated with tumors or ischemic stroke, as well as in arthritis and other inflammatory conditions.
Recent gene knockout studies suggest that Angiopoietin-2 also plays a key role within the vessel wall, in regulating vessel de-stabilization and vessel regressions, and in regulating development of lymphatics. Interestingly, recent work with EphrinB2 also suggest a key role in vessel wall formation, particularly for arterial vessels.
The discovery of multiple new angiogenesis regulatory factors, together with novel characterization approaches made possible by the use of knockout and transgenic technologies, is clearly leading to a new understanding of the molecular basis of blood vessel development that seems likely to have important therapeutic implications.

10:15 Role of Integrins, Extracellular Matrix &
Matrix Metalloproteinases In The Modulation of VEGF
Shaker A. Mousa, Ph.D., MBA, FACC, FACB, Fellow & Professor of Pharmacology, DuPont Pharmaceuticals Co.
Angiogenesis depends on the cooperation of growth factors, Integrins, cell adhesion proteins & Matrix Metalloproteinases (MMP). While integrins avb3 and avb5 have been shown to play critical roles in two distinct pathways of angiogenesis, integrins are also involved in the modulation of MPP and VEGF. The interactions and the regulations among those different angiogenic components will be discussed.

10:50 Refreshment Break and Poster/Exhibit Viewing

Clinical trials and animal models

11:50 Gene Therapy for Therapeutic Angiogenesis
Ronald G. Crystal, M.D., Director, Institute of Genetic Medicine; Director, The Arthur and Rochelle Belfer Gene Therapy Core Facility; Bruce Webster Professor of Medicine, Joan and Sanford I. Weill Medical College of Cornell University
Therapeutic angiogenesis describes a strategy where blood vessel formation is induced for the purposes of treating and/or preventing ischemic disease. Gene therapy with an adenovirus expressing a gene coding for a protein capable of inducing angigogensis has several advantages over protein therapy in that it is local, and is capable of expressing the relevant gene for 1-2 weeks, an ideal time to elicit new blood vessel growth. Adenovirus mediated angiogenesis with the gene coding for human vascular endothelial growth factor 121 has been successful in experimental models of cardiac and peripheral vascular disease, as well as in plastic surgery and wound healing applications, and preliminary data in human studies of cardiovascular disease are encouraging.

12:25 Lunch, sponsored by The Knowledge Foundation

Clinical Trials And Animal Models II

2:20 Efficacy of a Recombinant Adenovirus Expressing FGF and VEGF in an Animal Model of Myocardial Ischemia
H. Kirk Hammond, M.D., Associate Professor of Medicine, University of California, San Diego and VA Medical Center, San Diego; Scientific Founder, Collateral Therapeutics*
We have previously demonstrated in an ameroid pig model of myocardial ischemia that regional heart function and perfusion are improved after intracoronary delivery of an adenovirus (Ad5) expressing FGF. We have now constructed a recombinant Ad5 expressing variants of both FGF-4 and VEGF-145, and tested this construct (Ad5-dual) at various doses in the ameroid model of myocardial ischemia. The results indicate that an Ad5 expressing both FGF and VEGF genes is associated with improved regional function and perfusion in myocardial ischemia. The data indicate that the efficacy of the Ad5-dual is increased compared to Ad5 expressing single growth factors. *Collaborating authors: Gao M.H., Lai N.C., Bell D.J., Dalton N, McKirnan M.D., Roth D.A.

2:55 Refreshment Break and Poster/Exhibit Viewing

VEGF Receptors

3:50 A unique signal transduction of VEGF-VEGF receptors in angiogenesis.
Masabumi Shibuya, Professor, University of Tokyo, Japan
I will discuss (1) induction of VEGF gene by non-TPA type tumor promoter, Okadaic acid, (2) a unique KDR (VEGFR-2) signaling for endothelial cell proliferation which mostly utilizes PLCgamma-PKC-MEK-MAP kinase pathway but not Ras-MAP kinase pathway, (3) dual function of Flt-1 (VEGFR-1), positive and negative role in angiogenesis in vivo.

4:25 Novel VEGF Receptors on Tumor Cells and Their Role in VEGF-Deriven Angiogenesis
Shay Soker Ph.D., Research Associate, Dept. of Urology, ChildrenŐs Hospital and Harvard Medical School
VEGF is a potent angiogenic factor that has been implicated in physiological and pathological neovascularization. We have recently purified and cloned a new VEGF receptor, neuropilin-1 (NRP-1), from breast carcinoma-derived cells. Conditional over-expression of NRP-1 by rat prostate tumor cells significantly enhanced tumor growth and its vascularization. On the other hand, co-expression of NRP-1 with KDR/Flk-1 on EC increased VEGF165 activities. Accordingly, we have developed anti-neuropilin approaches to inhibit VEGF-mediated endothelial cell proliferation.

5:00 VEGFR-2 and Neuropilin-1 (NPN-1) Form A Co-Receptor Complex That Is Responsible For The Enhanced Signaling Potency of VEGF165 vs. VEGF121
Jan S. Rosenbaum, Ph.D., Principal Scientist, Cardiovascular Research, Procter & Gamble Pharmaceuticals
Despite similar receptor binding affinity, VEGF165 is considerably more potent at stimulating VEGFR-2/KDR autophosphorylation in HUVEC than is the VEGF121 isoform. This discrepancy can be explained by formation of a VEGFR-2 + NPN-1 heteromeric receptor complex to which only VEGF165 can bind appreciably. Antagonism of binding to NPN-1 reduces the potency of VEGF165 but not VEGF121, implicating NPN-1 as a potentiator of signaling through VEGFR-2.

5:30 End of Day One

Tuesday, October 24, 2000

8:45 Poster/Exhibit Viewing & Coffee and Danish

Anti-Angiogenesis

9:30 Tumor Angiogenesis: Molecular Facts and Therapeutic Opportunities
Prof. Dr. Dieter Marme, Tumor Biology Center, Freiburg, Germany
Tumor angiogenesis depends on the co-operation of two ligand/receptor systems: VEGFŐs/VEGF-receptors and angiopoietins/TIE-2. Interruption of both systems by experimental techniques leads to inhibition of tumor growth and decreased incidence of metastases formation. Therapeutic strategies including the use of recombinant soluble receptors for angiogenic ligands and VEGF-receptor tyrosine kinases have been developed and are currently under preclinical and clinical investigation.

10:05 Anti-Angiogenesis Strategies Using
Monoclonal Antibodies
Peter Bohlen, Ph.D., Vice President, Research, ImClone Systems Inc.
ImClone is developing two monoclonal antibodies (mAb IMC-1C11, in phase 1 trials, and VE-Cadherin mAb). Both block angiogenesis and tumor growth by targeting cell surface molecules essential for angiogenesis. IMC-1C11 binds to VEGF receptor KDR and blocks its activation by the angiogenic growth factor. VE-Cadherin mAb interferes with the ability of endothelial cells to adhere to each other, thus preventing their assembly into tubes.

10:40 Essential Role of VEGF/VEGFR-2 Autocrine and Paracrine Signaling Pathways and Bone Marrow-Derived Endothelial Precursor Cells in the Proliferation and Invasion of Human Leukemias.
Shahin Rafii M.D., Associate Professor of Medicine, Division of Hematology-Oncology Laboratory of Vascular Hematology, Cornell University Medical College
We have shown that a subset of human leukemias express functional vascular endothelial growth factor-2 (VEGFR-2, also known as KDR, Flk-1). Since leukemic cells also express VEGF, this results in the generation of VEGF/VEGFR2 autocrine loop that support the proliferation and invasiveness of leukemias. Similar to solid tumors, the growth of leukemias may also be angiogenesis dependent. In this regard, we have shown that paracrine factors released by VEGFR2+ mature endothelium and endothelial precursor cells support the proliferation of leukemic cells. VEGF released by leukemic cells support recruitment of endothelial precursors cells to the leukemic vascular beds.
We have developed an in vivo xenotransplant model where the effect of anti-VEGFR2 MoAbs in inhibiting the growth of leukemic cells could be evaluated in immunocompromised NOD-SCID mice. Injection of human primary lymphoid or myeloid leukemic cells into the NOD-SCID mice results in the engraftment of the leukemic cells in the marrow of the NOD-SCID mice. Within two weeks, the inoculated leukemic cells invade the peripheral circulation, spleen and liver, and if untreated will result in the demise of the inoculated mice. Injection of the MoAb selective for the inhibition of murine VEGFR2 (Flk-1, paracrine loop) resulted in increased survival of the treated mice for three weeks. However, injection of anti-VEGFR2 MoAb that selectively blocks the human VEGFR2 (KDR, autocrine loop) resulted in increased survival of the treated NOD-SCID mice as long as the mice are treated with MoAb, suggesting that the VEGF/VEGFR2 autocrine loop plays a more critical role in the proliferation of leukemic cells. Moreover, MoAb to both the murine and human VEGFR-2 (blocking both autocrine and paracrine loops), resulted in complete remission and long-term survival of human leukemias xenotransplanted onto NOD-SCID mice. In addition, anti-VEGFR2 MoAb inhibited the recruitment of endothelial precursors into leukemic vascular beds. These results suggest that subsets of VEGFR2+ leukemias may be amenable to anti-angiogenic therapy, and lay the foundation for designing clinical trials where the efficacy of anti-angiogenic therapy could directly be examined in patients with leukemias and lymphomas.* Collaborating authors: S. Dias, Z. Zhu, D. Hicklin, L. Witte, P. Bohlen, Cornell University Medical College, and ImClone Systems

11:15 Refreshment Break and Poster/Exhibit Viewing

11:45 Immunotoxins targeted on VEGF-R2 selectively destroy tumor vessels.
Jean Plou‘t, M.D., Ph.D.; Institut de Pharmacologie et Biologie Structurale, CNRS; AbTech, Toulouse, France
The recent enthusiasm for the discovery of new anti-cancer therapies targeted on endothelial cells rather than on cancer cells is very promising. A large body of evidence has acknowledged the VEGF system as a major regulator of angiogenesis. We prepared selective agonists of VEGF-R2 by the anti-idiotypic antibody strategy. These antibodies linked to a toxin can destroy tumor angiogenic endothelial cells, thus leading to tumor shrinkage, without affecting resting endothelial cells of the normal vasculature. Preclinical data obtained with monoclonal anti-idiotypic antibodies will be presented.

12:20 Tumor regression by SU6668, an angiogenesis inhibitor which blocks signalling via the VEGF, PDGF and FGF receptors.
A. Douglas Laird, Ph.D., Associate Group Leader, Oncology Development, SUGEN, Inc.
SU6668, a synthetic small molecule angiogenesis inhibitor currently in Phase I clinical trials, competitively inhibits (with respect to ATP) the kinase activities of the receptor tyrosine kinases Flk-1/KDR, PDGFR and FGFR. SU6668 treatment causes regression or growth arrest of large established human tumors grown in athymic mice. Molecular and histological analyses yielding insights into the mechanisms underlying these responses will be presented.

1:00 End of Conference

Call for Posters

Call for Posters
Industry, government and academic researchers are encouraged to submit poster abstracts for this event. One-page abstracts (8 1/2” x 11” with 1-inch margins) must be submitted no later than September 1, 2000 for inclusion in the program course book. Additional poster submissions will be accepted until September 15, 2000 but may not be included in the course book. Note: The poster board reservation fee is $35. If you are submitting a poster, you MUST be registered and paid in advance to ensure that a poster board is reserved for you.

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