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Patent Issued for Compositions and Methods Related to anti-FGF Agents

August 18, 2014



By a News Reporter-Staff News Editor at Cancer Vaccine Week -- From Alexandria, Virginia, NewsRx journalists report that a patent by the inventors Takada, Yoshikazu (Davis, CA); Mori, Seiji (Houston, TX), filed on March 30, 2012, was published online on August 5, 2014 (see also The Regents of the University of California).

The patent's assignee for patent number 8796209 is The Regents of the University of California (Oakland, CA).

News editors obtained the following quote from the background information supplied by the inventors: "The present invention relates to FGF-1 and mutants of FGF-1 affecting the signaling of cellular growth, differentiation, and angiogenesis.

"Various publications are referred to in parentheses throughout this application. Each of these publications is incorporated by reference herein. Complete citations of scientific publications are set forth below, or in the text of the specification. Assoian, R. K. (1997) Anchorage-dependent cell cycle progression. J Cell Biol, 136, 1-4. Belford, D. A., Hendry, I. A. and Parish, C. R. (1992) Ability of different chemically modified heparins to potentiate the biological activity of heparin-binding growth factor 1: lack of correlation with growth factor binding. Biochemistry, 31, 6498-6503. Brooks, P., Clark, R. and Cheresh, D. (1994a) Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science, 264, 569-571. Brooks, P., Montgomery, A., Rosenfeld, M., Reisfeld, R., Hu, T., Klier, G. and Cheresh, D. (1994b) Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell, 79, 1157-1164. Brooks, P. C., Stromblad, S., Klemke, R., Visscher, D., Sarkar, F. H. and Cheresh, D. A. (1995) Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin. J Clin Invest, 96, 1815-1822. Brown, K. J., Hendry, I. A. and Parish, C. R. (1995) Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: implications for potentiation of growth factor action by heparin. J Cell Biochem, 58, 6-14. Burgess, W. H., Shaheen, A. M., Ravera, M., Jaye, M., Donohue, P. J. and Winkles, J. A. (1990) Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue. J Cell Biol, 111, 2129-2138. Comoglio, P. M., Boccaccio, C. and Trusolino, L. (2003) Interactions between growth factor receptors and adhesion molecules: breaking the rules. Curr Opin Cell Biol, 15, 565-571. DiGabriele, A. D., Lax, I., Chen, D. I., Svahn, C. M., Jaye, M., Schlessinger, J. and Hendrickson, W. A. (1998) Structure of a heparin-linked biologically active dimer of fibroblast growth factor. Nature, 393, 812-817. Eliceiri, B. P. (2001) Integrin and growth factor receptor crosstalk. Circ Res, 89, 1104-1110. Friedlander, M., Brooks, P. C., Shaffer, R. W., Kincaid, C. M., Varner, J. A. and Cheresh, D. A. (1995) Definition of two angiogenic pathways by distinct alpha v integrins. Science, 270, 1500-1502. Friesel, R. E. and Maciag, T. (1995) Molecular mechanisms of angiogenesis: fibroblast growth factor signal transduction. Faseb J, 9, 919-925. Frisch, S. M. and Screaton, R. A. (2001) Anoikis mechanisms. Curr Opin Cell Biol, 13, 555-562. Fromm, J. R., Hileman, R. E., Weiler, J. M. and Linhardt, R. J. (1997) Interaction of fibroblast growth factor-1 and related peptides with heparan sulfate and its oligosaccharides. Arch Biochem Biophys, 346, 252-262. Goodsell, D. S. and Olson, A. J. (1990) Automated docking of substrates to proteins by simulated annealing. Proteins, 8, 195-202. Hynes, R. O. (2002) Integrins: bidirectional, allosteric signaling machines. Cell, 110, 673-687. Kaplow, J. M., Bellot, F., Crumley, G., Dionne, C. A. and Jaye, M. (1990) Effect of heparin on the binding affinity of acidic FGF for the cloned human FGF receptors, flg and bek. Biochem Biophys Res Commun, 172, 107-112. Klint, P. and Claesson-Welsh, L. (1999) Signal transduction by fibroblast growth factor receptors. Front Biosci, 4, D165-177. Kwabi-Addo, B., Ozen, M. & Ittmann, M. The role of fibroblast growth factors and their receptors in prostate cancer. Endocr Relat Cancer 11, 709-24 (2004). LaVallee, T. M., Prudovsky, I. A., McMahon, G. A., Hu, X. and Maciag, T. (1998) Activation of the MAP kinase pathway by FGF-1 correlates with cell proliferation induction while activation of the Src pathway correlates with migration. J Cell Biol, 141, 1647-1658. Lishko, V. K., Kudryk, B., Yakubenko, V. P., Yee, V. C. and Ugarova, T. P. (2002) Regulated unmasking of the cryptic binding site for integrin alpha M beta 2 in the gamma C-domain of fibrinogen. Biochemistry, 41, 12942-12951. Liu, J., Huang, C. and Zhan, X. (1999) Src is required for cell migration and shape changes induced by fibroblast growth factor 1. Oncogene, 18, 6700-6706. Morris, G. M., Goodsell, D. S., Halliday, R. S., Fig Huey, R., Hart, W. E., Belew, R. K. and Olson, A. J. (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comp. Chem., 19, 1639-1662. Morris, G. M., Goodsell, D. S., Huey, R. and Olson, A. J. (1996) Distributed automated docking of flexible ligands to proteins: parallel applications of AutoDock 2.4. J Comput Aided Mol Des, 10, 293-304. Pages, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, S. and Pouyssegur, J. (1993) Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA, 90, 8319-8323. Pellegrini, L., Burke, D. F., von Delft, F., Mulloy, B. and Blundell, T. L. (2000) Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature, 407, 1029-1034. Plotnikov, A. N., Hubbard, S. R., Schlessinger, J. and Mohammadi, M. (2000) Crystal structures of two FGF-FGFR complexes reveal the determinants of ligandreceptor specificity. Cell, 101, 413-424. Powers, C. J., McLeskey, S. W. and Wellstein, A. (2000) Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer, 7, 165-197. Prater, C. A., Plotkin, J., Jaye, D. and Frazier, W. A. (1991) The properdin-like type I repeats of human thrombospondin contain a cell attachment site. J. Cell Biol., 112, 1031-1040. Presta, M., Dell'Era, P., Mitola, S., Moroni, E., Ronca, R. and Rusnati, M. (2005) Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev, 16, 159-178. Rusnati, M., Tanghetti, E., Dell'Era, P., Gualandris, A. and Presta, M. (1997) alphavbeta3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells. Molecular Biology of the Cell., 8, 2449-2461. Sahni, A. and Francis, C. W. (2004) Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires alphavbeta3. Blood, 104, 3635-3641. Saphire, E. O., Parren, P. W., Pantophlet, R., Zwick, M. B., Morris, G. M., Rudd, P. M., Dwek, R. A., Stanfield, R. L., Burton, D. R. and Wilson, I. A. (2001) Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science, 293, 1155-1159. Schlessinger, J. (2000) Cell signaling by receptor tyrosine kinases. Cell, 103, 211-225. Schwartz, M. A. and Assoian, R. K. (2001) Integrins and cell proliferation: regulation of cyclin-dependent kinases via cytoplasmic signaling pathways. J Cell Sci, 114, 2553-2560. Schwartz, M. A. and Ginsberg, M. H. (2002) Networks and crosstalk: integrin signalling spreads. Nat Cell Biol, 4, E65-68. Shimaoka, M. and Springer, T. A. (2003) Therapeutic antagonists and conformational regulation of integrin function. Nat Rev Drug Discov, 2, 703-716. Takagi, J., Erickson, H. P. and Springer, T. A. (2001) C-terminal opening mimics `inside-out` activation of integrin alpha5beta1. Nat Struct Biol, 8, 412-416. Tanghetti, E., Ria, R., Dell'Era, P., Urbinati, C., Rusnati, M., Ennas, M. G. and Presta, M. (2002) Biological activity of substrate-bound basic fibroblast growth factor (FGF2): recruitment of FGF receptor-1 in endothelial cell adhesion contacts. Oncogene, 21, 3889-3897. Thornton, S. C., Mueller, S. N. and Levine, E. M. (1983) Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science, 222, 623-625. Ullrich, A. and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell, 61, 203-212. Wang, W. and Malcolm, B. A. (1999) Two-Stage PCR Protocol Allowing Introduction of Multiple Mutations, Deletions and Insertions Using QuikChange.TM. Site-Directed Mutagenesis. BioTechniques, 26, 680-682. Yokoyama, K., Erickson, H. P., Ikeda, Y. and Takada, Y. (2000) Identification of amino acid sequences in fibrinogen y-chain and tenascin C C-terminal domains critical for binding to integrin .alpha.v.beta.3. J. Biol. Chem., 275, 16891-16898. Yokoyama, K., Zhang, X. P., Medved, L. and Takada, Y. (1999) Specific binding of integrin .alpha.V.beta.3 to the fibrinogen .gamma. and .alpha.E chain C-terminal domains. Biochemistry, 38, 5872-5877. Zhu, H., Anchin, J., Ramnarayan, K., Zheng, J., Kawai, T., Mong, S. and Wolff, M. E. (1997) Analysis of high-affinity binding determinants in the receptor binding epitope of basic fibroblast growth factor. Protein Eng, 10, 417-421.

"Fibroblast growth factors (FGFs) constitute a family of heparin-binding polypeptides involved in the regulation of biological responses such as growth, differentiation, and angiogenesis. They are also implicated in inflammation, excess wound healing, and resistance of tumor cells to chemotherapeutic agents (chemoresistance).

"The FGF family currently consists of 24 members, with FGF-1 (acidic FGF) and FGF-2 (basic FGF) the most extensively studied. The biological effects of FGFs are mediated by four structurally related receptor tyrosine kinases, denoted FGFR1, FGFR2, FGFR3, and FGFR4. The binding of FGF to its receptor results in receptor dimerization and subsequent autophosphorylation on specific tyrosine residues within the intracellular domain (Klint and Claesson-Welsh, 1999; Powers et al., 2000; Presta et al., 2005; Ullrich and Schlessinger, 1990)

"Integrins are a family of cell adhesion receptors that recognize extracellular matrix ligands and cell surface ligands (Hynes, 2002). Integrins are transmembrane .alpha.-.beta. heterodimers, and at least 18 .alpha. and .beta. subunits are known (Shimaoka and Springer, 2003). Integrins transduce signals to the cell upon ligand binding, and their functions are in turn regulated by the signals from within the cell (Hynes, 2002). Ligation of integrins triggers a large variety of signal transduction events that serve to modulate cell behavior including proliferation, survival/apoptosis, shape, polarity, motility, gene expression, and differentiation. Integrin-stimulated pathways are very similar to those triggered by growth factor receptors and are intimately coupled with them. It has been proposed that many cellular responses to soluble growth factors, such as epidermal growth factor, platelet-derived growth factor, and thrombin, are dependent upon the cell's adherence to extracellular matrix ligands via integrins. Integrins lie at the basis of such anchorage-dependent cell survival and proliferation (Assoian, 1997; Frisch and Screaton, 2001; Schwartz and Assoian, 2001).

"It has been proposed that FGF-2-induced angiogenesis requires integrin signaling from the extracellular matrix (crosstalk between integrins and FGF receptors). Indeed antibody against integrin .alpha.v.beta.3 blocks FGF-2-induced angiogenesis (Brooks et al., 1994a; Brooks et al., 1994b). It has been reported that FGF-2 enhances .alpha.v.beta.3 expression during angiogenesis (Brooks et al., 1994a). Antibody or cyclic peptide antagonist of .alpha.v.beta.3 inhibits this .alpha.v.beta.3 upregulation (Brooks et al., 1994a; Brooks et al., 1995; Friedlander et al., 1995). It has been shown that integrin and growth factors are colocalized under certain condition. For example coimmunoprecipitation studies revealed direct biochemical interaction between .alpha.v.beta.3 and FGFR1 in the presence of both FGF-2 and fibrinogen (Sahni and Francis, 2004). These findings suggest integrin and FGFR are colocalized on the membrane in the presence of FGF-2. It has not been established how integrins and FGFR crosstalk in FGF-2 signaling.

"It has been reported that substrate-bound FGF-2 promotes endothelial cell adhesion by interacting with integrin .alpha.v.beta.3 (Rusnati et al., 1997) and induces endothelial cell proliferation, motility, and the recruitment of FGFR1 in cell substrate contact (Tanghetti et al., 2002). Anti-.alpha.v.beta.3 antibodies block cell proliferation on immobilized FGF-2, but deletion of the tyrosine kinase portion of FGFR blocks cell proliferation induced by immobilized FGF-2. Thus it has been proposed that .alpha.v.beta.3 is required but not sufficient to transduce mitogenic signals of FGF-2 (Tanghetti et al., 2002). It is unclear how integrins interact with FGF-2 or whether this interaction is biologically relevant since heat-denatured FGF-2 still supports integrin binding (Tanghetti et al., 2002)."

As a supplement to the background information on this patent, NewsRx correspondents also obtained the inventors' summary information for this patent: "The invention relates to an isolated amino acid comprising at least a portion of the FGF protein amino acid sequence, and including a mutation in the integrin .alpha.v.beta.3 binding region of FGF-1.

"In one preferred embodiment, the isolated amino acid comprises a mutation in the region Asn-33, GIy-34, GIy-35. In another preferred embodiment, the isolated amino acid comprises a mutation in the region His-36, Arg-39, Leu-41. In a further preferred embodiment the isolated amino acid comprises a mutation in the region Asp-43, Thr-45, Val-46. In a different preferred embodiment, the isolated amino acid comprises a mutation in the region Asp-47, GIy-48, Thr-49.

"In a further preferred aspect of this embodiment, the isolated amino acid comprises a mutation in the region Arg-50, Asp-51, Arg-52. In a further preferred aspect of this embodiment, the isolated amino acid comprises a glutamine substitution for Arg-50. In a still further preferred aspect, the isolated amino acid is the protein R50E.

"In a further preferred embodiment, the isolated amino acid comprises a mutation in the region Ser-53, Asp-54.

"In a different preferred embodiment, the isolated amino acid comprises a mutation in the region Lys-127, Lys-128, Asn-129. In a further preferred aspect of this embodiment, the isolated amino acid comprises a glutamine substitution for Lys-127 and Lys-128. In a still further preferred aspect, the isolated amino acid is the mutant 4xE.

"In a different preferred embodiment, the isolated amino acid comprises a mutation in the region Gly-130, Ser-131, Cys-132. In a different preferred embodiment, the isolated amino acid comprises a mutation in the region Lys-133, Arg-134, Arg-137. In a further preferred aspect of this embodiment, the isolated amino acid comprises a glutamine substitution for Lys-133 and Arg-134. In a still further preferred aspect, the isolated amino acid is the mutant 4xE.

"In a different preferred embodiment, the isolated amino acid comprises a mutation in the region 138, GIy-141, GIn-142.

"In a different preferred embodiment, the isolated amino acid binds to FGFR, and preferably acts as a dominant negative mutant against FGF inducing activity.

"In another aspect of the invention, the isolated amino acid comprises a mutation in the region where the amino acid blocks the angiogenesis inducing activity of FGF-1. In a further aspect of this preferred embodiment, the isolated amino acid blocks the tumor growth inducing activity of FGF. In a different aspect of the invention, the isolated amino acid blocks the inflammation inducing activity of FGF. In still different aspect of the invention, the isolated amino acid blocks the excess wound healing inducing activity of FGF. In a further aspect of the invention, the isolated amino acid blocks the resistance of tumor cells to chemotherapeutic agents.

"The isolated amino acid preferably acts as an antagonist to FGF signaling.

"In a different embodiment, the isolated amino acid comprises at least a portion of the FGF protein amino acid sequence, and includes a mutation in the FGFR binding region of FGF-1. The isolated amino acid preferably binds to integrin .alpha.v.beta.3. In one preferred embodiment, the isolated amino acid comprises a mutation in the region of amino acids 100 to 110 of FGF-1. In a further such embodiment, the isolated amino acid is the mutant 3xA. The isolated amino acid preferably acts as a dominant negative mutant against FGF inducing activity.

"The invention further comprises a pharmaceutical composition containing the amino acid. The pharmaceutical composition preferably is selected from the group of pharmaceutical compositions consisting of a diagnostic agent, a preventive agent, and a therapeutic agent for disease condition involving accelerated or abnormal cell growth.

"In a different aspect of the embodiment, the accelerated or abnormal cell growth is selected from the group of conditions consisting of angiogenesis, cancer growth, inflammation and excess wound healing.

"In a further aspect of the embodiment, the cancer growth condition is selected from the group of conditions consisting of acute myelogenous leukemia, breast cancer, prostate cancer, colon cancer, hepatic, cancer, myeloma, uterine leiomyoma, malignant tumor, or solid tumor.

"In a still different aspect of the embodiment, the inflammation condition is selected from the group of conditions consisting of rheumatoid arthritis (RA), lupus (SLE), inflammatory bowel diseases (IBD), experimental allergic encephalomyelitis (EAE), multiple sclerosis (MS), and diabetic retinopathy.

"In another aspect of the embodiment, the angiogenesis condition is selected from the group of conditions consisting of cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis and psoriasis.

"In one preferred embodiment, the pharmaceutical composition will further comprise a carrier.

"The invention also provides a method of reducing FGF signaling activity in a mammal, the method comprising dosing the mammal with an effective amount of the pharmaceutical composition. In one preferred aspect of the invention, the pharmaceutical composition is administered to the mammal orally. In another preferred embodiment, the mammal is a human.

"In a different preferred aspect of the invention, the pharmaceutical composition is administered to the mammal topically. In a still different preferred aspect of the invention, the pharmaceutical composition is administered to the mammal intravenously.

"The method preferably involves a mammal that has, or is at risk for developing, a vascularized solid tumor or metastases from a primary tumor.

"In a further preferred embodiment, the method comprises the step of administering to the mammal another compound that inhibits tumor angiogenesis. In a preferred such aspect of the invention, the additional compound is chosen from a group comprising CV 3988, WEB 2086, INF-2.alpha., TNP-470, endostatin, SU 5416, SU 6668, batimistat, angiostatin, and celecoxib."

For additional information on this patent, see: Takada, Yoshikazu; Mori, Seiji. Compositions and Methods Related to anti-FGF Agents. U.S. Patent Number 8796209, filed March 30, 2012, and published online on August 5, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8796209.PN.&OS=PN/8796209RS=PN/8796209

Keywords for this news article include: Pharmaceuticals, Drugs, Heparin, Therapy, Genetics, Oncology, Chemicals, Glutamine, Integrins, Fibrinogen, Proteomics, Mutagenesis, Angiogenesis, Biochemistry, Inflammation, Cancer Vaccines, Tyrosine Kinase, Basic Amino Acids, Endothelial Cells, Membrane Proteins, Peptide Receptors, Cell Proliferation, Protein Precursors.

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