Within the first four years of its existence (established May 2006) the Angio Core has accomplished all of the above sited goals and is now expanding their efforts in these individual areas, involving: 1) Collaboration interactions with Dr. David Salomon (NCI/CCR/Mammary Biology and Tumorigenesis Laboratory), who has been working with cripto/nodal/apelin/apj involvement in EMT/Breast CA development, has resulted in the identification of a second angiogenic factor encoded in the apelin gene. This new growth factor is generated by the enzymatic processing of apelin-36 giving rise to two distinct peptides;the previously know split-product form the carboxy-terminal region (apelin-13) and a second entity from the amino-terminal region denoted as Selective Apelin-36 Cutting and Amidation peptide (Salcut-NH2). Salcut-NH2 was shown to be a growth stimulator of both blood vessel and lymphatic endothelial cells, mast cells and several anatomically different solid tumor cells at subnanomolar concentrations. Rabbit antisera to Salcut-NH2 easily identified peptide expression in paraffin section of normal and diseased tissue. A quantitative ELISA was developed that selectively discriminated Salcut-NH2 from apelin-36, having a sensitivity in the sub nanogram/milliliter range. EIR filed with NCI/TTC 2/27/08, TRG Approval 4/9/09, Provisional Patent filed 2/27/09 (61/156, 351), Posted on Federal Register 5/1/09, Possible CRADA interest response from Vivo Biosciences, Inc. 6/11/09, actively involved in Scope-of-Work negotiations. Subsequent studies with Salcut-NH2 demonstrated that only the amide derivative was biologically active and neither its free-acid (Salcut-OH) nor its glycine-extended intermediate form (Salcut-Gly) induced cell proliferation. In addition, Salcut-NH2 mediated blood vessel and lymphatic endothelial cell tube formation over a dose range of [0.1nM to 100nM], with maximum tube density observed at [10nM]. Finally, using Salcut-NH2 induced tube formation assay, it was shown that although the apelin antagonist (apelin-13[F13A]) could block apelin-13 induced tube formation, it did not inhibit the Salcut-NH2 mediated event demonstrating the Salcut-NH2 activity was not dependent on the apelin-13 receptor (APJ). A non-provisional US patent encompassing all of the above claims was filed 3/1/2010 (Pat. Application No. 12/715,338 The identification of a novel amidated peptide derived from the N-terminus of apelin-36 that stimulates endothelial cell and tumor growth at nanomolar concentrations). 2) Collaborating with Dr. William Douglas Figg (NCI/CCR/Clinical Pharmacology Program) assessing his thalidomide small molecule derivatives (Gu compounds) as anti-angiogenic drugs in both the established in vitro assay systems of the Angio Core and using nude mouse xenograft model of low vascular density human tumors (lung CA, A549) and high vascular density tumors (leiomyosarcoma, SK-LMS-1). Initial animal studies were looking for toxicity effects and determining the maximum tolerable dose of Gu compounds. Follow-up analysis examined a variety of Gu derivative at [100uM] using the following protocol;100ul Rx injected 3X/wk/mouse for 4wks (10mice/Rx grp). Genentechs Avastin (bevacizumab) was used as our positive control (1mg/ml) and when dealing with a GRP-dependent tumor cell line (ie A549) our peptide neutralizing monoclonal antibody (MoAb2A11, 1mg/ml)) was also used. Preliminary results with Gu992 and Gu998 demonstrated that these compounds were equally effective as Avastin at suppressing the xenograft growth of A549. However, parallel Rx challenges in SK-LMS-1 xenografts prove Gu992 to be uneffective, Gu998 to be partially suppressive and Avastin to dramatically inhibit tumor growth. 3) Development of high throughput assays for the identification of anti-antigenic drugs. Dr. Enrique Zudaire, the groups staff scientist, took the lead on this project and created a fluorescent based, 96-well assay system for detecting drug candidates that blocked endothelial cell proliferation, tube formation or both. Dr. Zudaire used this analytical technology to evaluate the DTP diversity set of small molecule as a Proof-of-Principle approach. Over the past 2.5 yrs and through multiple repeat testing, he has identified several small molecule compounds having anti-angiogenic activity. Recently Dr. Zudaire submitted his finding to the NCI Technology Transfer Center (NCI/TTC) as an EIR, circumventing TRG review via Dr. Robert Wiltrouts signed approval, and resulting in an NCI provisional patent (61/230,667) being filed 7/31/09 thus giving Dr. Zudaire the ability to present his science at the Angiogenesis Gordon Research Conference in Newport, RI (8/2/09 to 8/7/09) without jeopardizing IP status. Dr. Zudaire has chosen seven of his best candidate drugs (that include NSC numbers;19630, 292222, 259969, 122657, 150117, 48300 and 292596 representing 0.4% of the total number of small molecule drugs screened) to do further in vivo characterization studies via DIVAA and nude mouse xenograft testing on low angiogenic and high angiogenic human tumors (50mg drug aliquots received from DTP/Fisher BioServices 8/21/09). Dr. Zudaire has now expanded his analytical strategy to evaluate initial selected small molecule compounds that make it through his 3 step assay screen for the follwing: (a) Structural activity relationship with prior FDA approved anti-angiogenic Rxs, (b) Determine it they are tubulin binders, (c) Assess for tyrosine kinase inhibition, (d) determine gene profile using 170+ array that covers gene marker either up-regulating angiogenesis or suppressing this process, thus eliminating ant compounds that are similar to previously established anti-angiogenic Rxs. A new US provisional patent was file 7/30/2010 entitled Antiangiogenic Small Molecule Signatures (Pat. Application No. PCT/US2010/043998), 4) Developing an in vitro 3-dimentional co-culture assay platform that mimics the in vivo microenvironment around tumors and apply this strategy to determine drug sensitivity responses for a given tumor. Dr. Changge Fang has designed a 3D co-culture assay that utilizes stably transfected fluorescent protein immortalized endothelial cells (GFP and YFP) evenly dispersed in a gel matrix to which a single cloned spheroid colony of human tumor (CFP) has been suspended. Within 5 days the endothelial cells migrate towards the tumor colony and begin to form tubular structures. In the case of highly vascularized human tumors, endothelial cell migrate to the tumor spheroid, encapsulating it and form sprouts that similar to what is observed in vivo. In addition, for highly metastatic tumors (ocular melanoma, leiomyosarcoma, neuroblastoma) migration of tumor cells along vascular highways is clearly seen, a form of metastatic spread used by tumor cells know as angiotrophism. Hence, Dr. Fangs newly developed 3D co-culture assay had the potential to not only screen for drugs capable of inhibiting tumor induced angiogenesis or lymphangiogenesis but also suppress perivascular spread of tumor cells. A new US provisional patent (Continuation-In-Part, CIP) was filed 6/10/2010 entitled Monitoring Angiogenesis and Metastasis in Three Dimensional Co-Cultures (Pat Application No. 12/802,666), and 5) The Angio Core has now signed a contract (approved by NCI/TTC) with Springer Scientific (Netherlands) to edit a textbook on in vitro/in vivo methodologies and applications of angiogenesis and lymphangiogenesis. The chapter contributors have been recruited from world recognized experts in the field that have made paradigm shifts in assay development. The manual, entitled A Textbook of Angiogenesis and Lymphangiogenesis: Methods and Applications is due out in March of 2011.
