WO2005009457A1 - Rdp58 compositions and methods for inhibiting vascularization of cell populations - Google Patents

Abstract

The invention discloses compositions and methods for modulating angiogenesis, VEGF activity, MMP production, tumor growth, tumor survival, tumor invasiveness, and tumor metastasis. The invention further discloses compositions useful for the modulation of signal transduction pathways in mammalian cells and the modulation of physiological processes effected thereby.

Description

RDP58 COMPOSITIONS AND METHODS FOR INHIBITING VASCULARIZATION OF CELL POPULATIONS

STATEMENT OF RELATEDNESS

[001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/470,837, filed 15 May 2003, and U.S. Provisional Patent Application Serial No. 60/470,839, filed 15 May 2003, both of which are expressly incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

[002] The invention relates to angiogenesis, and concerns the mechanisms of tumor angiogenesis, tumor invasiveness, and tumor metastasis. The invention is directed to the modulation of angiogenesis, VEGF activity, MMP production, tumor growth, tumor survival, tumor invasiveness, and tumor metastasis, as well as to the treatment of diseases other than cancer involving angiogenesis.. The invention is further directed to the modulation of signal transduction pathways in mammalian cells and the modulation of physiological processes effected thereby.

BACKGROUND

[003] Ligands, cell surface receptors, and intracellular signal-transducing molecules form signal transduction pathways that relay extracellular signals to the interior of cells, where they may effect changes in gene expression and cell activity.

[004] The ability to modulate signal transduction, and alter cell activity thereby, is desirable for the treatment of developmental defects and pathological conditions. Many current therapies, for example cancer therapies, involve the use of therapeutics that target components of signal transduction pathways.

[005] One particular approach that holds promise for the treatment of a variety of diseases, including cancer, is the targeting of signal transduction pathways involved in angiogenesis.

[006] The mammalian vasculature begins as an embryonic network of vessels formed from endothelial precursor cells during vasculogenesis. This network expands during tissue growth by at least two distinct mechanisms of angiogenesis, particularly sprouting and non-sprouting angiogenesis. Sprouting angiogenesis involves the proteolytic degradation of extracellular matrix, migration and proliferation of endothelial cells, formation of a lumen, and functional maturation of the extended tubules. Non-sprouting angiogenesis, or "intussusception", involves the growth of interstitial columns of tissue in the lumen of pre-existing vessels, and the consequent division of these vessels. Blood vessels formed in the adult, including those that supply tumors, arise from pre-existing vessels by sprouting and non-sprouting angiogenesis in response to cues from tissue (see Risau, Nature, 386:671-674, 1997; Carmeliet et al., Nature, 407:249-257, 2000.) [007] Vascular endothelial growth factor (VEGF) and its receptors, VEGF-Rs, are essential for normal vasculogenesis and angiogenesis. VEGF is also an important mediator of angiogenesis attendant a variety of human diseases, including cancer. Two structurally related tyrosine kinases that bind to VEGF with high affinity have been identified: the flt-1 receptor (Shibuya et al., (1990) Oncogene 5:519-524; De Vries et al., (1992) Science 255:989-991) and the KDR/FLK-1 receptor. VEGF binding to VEGF receptors promotes endothelial cell migration and proliferation, two key requirements for angiogenesis. VEGF also increases vascular permeability and endothelial cell survival through its activation of VEGF-Rs.

[008] Both tumor cells and stromal cells are sources of VEGF. VEGF expression is upregulated in many human tumors, and anti-VEGF antibody has been shown to have a potent inhibitory effect on tumor growth in mice. Growth inhibitory effects on tumors have also been reported for a variety of inhibitors of VEGF-R expression and activity. ^'

[009] Several studies have shown that anti-VEGF treatment can be used in combination with chemotherapy or radiation therapy to improve cancer treatment. Further, clinical trials with several anti-VEGF agents are ongoing in cancer patients, and Avastin™ , a recombinant humanized antibody to VEGF, has been approved for use in the treatment of metastatic cancer of the colon or rectum.

[0010] VEGF is also highly expressed in a variety of hematological cancers, including multiple myeloma, T cell lymphoma, acute lymphoblastic leukemia, and Burkit lymphoma. Further, VEGF-R expression has been detected in numerous leukemia cell lines, and VEGF-R inhibitors have demonstrated inhibitory effects on leukemia cell lines.

[0011] In addition to cancer, VEGF inhibitors may be useful for the treatment of diseases involving angiogenesis. For example, studies have established the involvement of VEGF in intraocular neovascular syndromes, such as age-related macular degeneration (AMD) and the intraocular neovascularization attendant diabetes mellitus, and data support the potential clinical usefulness of VEGF inhibitors in these conditions. Moreover, clinical trials with VEGF inhibitors in AMD patients are ongoing. VEGF has also been implicated in the development of brain edema and in the angiogenesis attendant polycystic ovary syndrome, and data suggest that VEGF inhibition may be beneficial in the treatment of ischemia.

[0012] Angiogenesis has also been suggested to contribute to the accumulation of body fat in obese individuals. In support, adipose tissue has been shown to be highly angiogenic, preadipocytes reportedly migrate to sites of neovascularization, and adipose cells express the angiogenic factors VEGF and bFGF. These data suggest a strategy of VEGF antagonism to treat obesity.

[0013] VEGF-R signal transduction involves many intracellular protein kinases, including the extracellular signal regulated kinases (ERKs), and the stress activated protein kinases (SAPK)/Jun N- terminal kinases (JNKs) (see Davis, R.J., Cell 103: 239-252 (2000)).

[0014] VEGF activates JNK, and JNK antagonists reportedly inhibit VEGF activity. Among the genes regulated by the JNK pathway are MMPs and VEGF. The JNK signal transduction pathway is also activated in response to environmental stress and by the activation of several classes of cell surface receptors, including cytokine receptors, serpentine receptors, and receptor tyrosine kinases. JNK appears to mediate many of its effects by phosphorylating a number of transcription factors, such as c-Jun, CREB, Elk-1 , and ATF2.

[0015] The ERK kinases are rapidly activated in response to VEGF-R activation, and antagonists of ERK activity have been shown to inhibit VEGF-induced vessel formation. ERKs are also rapidly activated in response to ligand binding by growth factor receptors that are tyrosine kinases, such as the epidermal growth factor receptor, and receptors that are coupled to heterotrimeric guanine nucleotide binding proteins (G proteins), such as the thrombin receptor. The ERKs appear to integrate multiple intracellular signals transmitted by various second messengers. ERKs, like JNKs, phosphorylate and regulate the activity of enzymes and transcription factors. The particular substrates of ERKs include the EGF receptor, Rsk 90, phospholipase A, c-Myc, c-Jun and Elk-1/TCF.

[0016] Other second messenger systems in addition to the MAP kinase pathways are known to be induced by VEGF-R activation. For example, phosphatidylinositol 3'-kinase (PI3K) (Kazlauskas and Cooper (1989) Cell 58: 1121 ; Coughlin et al. (1989) Science 243, 1191 ) is an enzyme that phosphorylates the inositol ring of phosphatidyl inositol (PI) at the D-3 position (Whitman et al (1988) Nature 332, 644). PI3K activity is associated with a variety of activated tyrosine kinases and correlates with the presence of a tyrosine phosphorylated 85-kilodalton (kD) protein (p85) (Kaplan et al. (1987) Cell 50: 1021 ; Fukui and Hanafusa (1989) Mol. Cell. Biol. 9, 1651). Purified PI3K is a heterodimeric complex that contains p85 and a 110-Kd protein (p110) (Carpenter et al. (1990) J. Biol. Chem. 265, 19704). Phosphatidylinositol-kinases belong, together with specific phospholipases, to an enzyme group which catalyses the formation of intracellular second messenger substances from the membrane lipid phosphatidyl inositol (PI).

[0017] Among the enzymes activated by VEGF and lying downstream of PI3K is Akt (anti-apoptotic kinase; protein kinase B), itself a serine/threonine protein kinase. Akt has been implicated in VEGF- promoted endothelial cell survival. VEGF and βlll integrin also activate focal adhesion kinase (FAK), which contributes to endothelial cell survival.

[0018] With respect to additional VEGF activities, ERK, JNK, protein kinase C, c-src, and phospholipase C-gamma have all been implicated as mediators of VEGF-induced endothelial cell proliferation. FAK, c-src, Rho and Rac have been implicated in mediating VEGF's chemotactic signaling and endothelial cell migration.

[0019] The extracellular matrix (ECM) plays a critical role in cell growth, differentiation, survival and motility. For a tumor cell to metastasize from a primary tumor to a secondary location, it must degrade ECM components that physically impede cell migration. Matrix metalloproteinases (MMPs) are the key enzymes responsible for ECM breakdown. In addition, MMPs are known to cleave non- matrix molecules such as growth factors, cytokines, chemokines, and receptors. Many MMP genes have been identified in humans, and many have been implicated in cancer. MMP expression enhances tumor cell mobility and tumor invasiveness, and also contributes to the formation of a microenvironment that promotes tumor growth and leads to cancer progression. MMP production can affect the proliferation, survival, and migration of tumor cells and stromal cells.

[0020] Among the MMPs implicated in cancer are MMP2 and MMP9.

[0021] For example, MMP9 is a prognostic factor in patients with non-Hodgkin lymphoma, and patients expressing higher levels of MMP9 have a more aggressive form of the disease and lower survival rate than those expressing lower levels of MMP9 (Sakata K., Cancer. 2004 Jan 15;100(2):356-65). In addition, MMP9 is induced in premetastatic lung endothelial cells by VEGF, and the induction of MMP9 significantly promotes lung metastasis. Hiratsuka S Cancer Cell. 2002 Oct;2(4):289-300. MMP9 also appears to promote angiogenesis during tumor growth by cleaving non-matrix molecules (see for example Mira E., J Cell Sci. 2004 Apr 1 ;117(Pt 9):1847-57).

[0022] Additionally, the expression of MMP2 and MMP9 strongly correlates with glioma progression. Further, local invasion of tumour cells is characteristic of brain tumour progression and is associated with increased motility and a potential to hydrolyze ECM components.

[0023] In accordance with the reported involvement of VEGF signaling, angiogenesis, and MMP production in cancer, a number of MMP inhibitors and VEGF inhibitors are currently in clinical trials for the treatment of cancer.

SUMMARY

[0024] The present invention provides compositions and methods for modulating signal transduction in cells, as well as for modulating the processes controlled thereby. The "RDP58 compositions" provided herein are capable of modulating a variety of signals transduced by a variety of cell surface receptors in a variety of mammalian cell types.

[0025] In addition to other activities, the "RDP58 compositions" disclosed herein have potent anti- angiogenesis activity, and are capable of decreasing the expression of matrix metalloproteinases, particularly MMP2 and MMP9. These RDP58 compositions are useful for modulating angiogenesis, tumor vascularization, tumor growth, tumor survival, tumor invasiveness, and tumor metastasis. These RDP58 compositions are also useful for treating a variety of disorders other than cancer that involve angiogenesis.

[0026] In one aspect, the invention provides compositions and methods for decreasing angiogenesis. In another aspect, the invention provides compositions and methods for decreasing matrix metalloproteinase (MMP) production. In a further aspect, the invention provides compositions and methods for coordinately decreasing angiogenesis and decreasing MMP production.

[0027] In a preferred embodiment, the invention provides compositions and methods for decreasing the vascularization of a cell population in vivo. The methods comprise providing RDP58 to the vicinity of a cell population in vivo, wherein blood vessels are present in the vicinity of the cell population. The RDP58 composition provided decreases angiogenesis in the vicinity of the cell population, thereby decreasing vascularization of the population. [0028] In one embodiment, one or more cells of the cell population expresses an MMP, and the method further comprises decreasing MMP production in the cell population by contacting the cell population with the RDP58 composition. In a preferred embodiment, the MMP expressed by one or more cells of the population is MMP2 or MMP9.

[0029] In a preferred embodiment, the in vivo cell population is present in a tumor. The cell population may comprise tumor cells and/or cells of the tumor stroma. In one embodiment, one or more cells of the population expresses an MMP, and the method further comprises decreasing MMP production in the cell population by contacting the cell population with the RDP58 composition. In a preferred embodiment, the MMP expressed by one or more cells of the population is MMP2 or MMP9.

[0030] In one embodiment, the tumor is a metastatic tumor.

[0031] In one embodiment, the tumor is an invasive tumor.

[0032] In a preferred embodiment, the cell population comprises tumor cells.

[0033] In an alternative embodiment, discussed further below, the cell population comprises adipocytes.

[0034] In one aspect, the invention provides compositions and methods for decreasing the survival of a tumor. In one aspect, the invention provides compositions and methods for inhibiting the growth of a tumor.

[0035] In one embodiment, the methods comprise providing an RDP58 composition to the vicinity of a tumor, wherein blood vessels are present in the vicinity of the tumor, and wherein the RDP58 composition decreases angiogenesis in the vicinity of the tumor.

[0036] In one embodiment, the tumor comprises a cell expressing an MMP, and the methods additionally comprise contacting the tumor cell with the RDP58 composition, wherein the RDP58 composition decreases production of the MMP in the tumor.

[0037] In one aspect, the invention provides methods for decreasing VEGF-R activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The VEGF-R activity is preferably kinase activity as directed at a VEGF-R substrate, or binding activity as directed at a VEGF-R binding partner. The methods may be used to decrease endothelial cell proliferation, endothelial cell migration, endothelial cell survival, and angiogenesis.

[0038] In one aspect, the invention provides compositions and methods for decreasing Akt activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The Akt activity is preferably kinase activity as directed at an Akt substrate, or binding activity as directed at an Akt binding partner. The methods may be used to decrease endothelial cell survival, nitric oxide synthesis, and angiogenesis.

[0039] In one aspect, the invention provides compositions and methods for decreasing ERK activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The ERK activity is preferably kinase activity as directed at an ERK substrate, or binding activity as directed at an ERK binding partner. The methods may be used to decrease endothelial cell proliferation and angiogenesis.

[0040] In one aspect, the invention provides compositions and methods for decreasing JNK activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The JNK activity is preferably kinase activity as directed at a JNK substrate, or binding activity as directed at a JNK binding partner. The methods may be used to decrease endothelial cell proliferation and angiogenesis.

[0041] In one aspect, the invention provides compositions and methods for decreasing FAK activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The FAK activity is preferably kinase activity as directed at a FAK substrate, or binding activity as directed at a FAK binding partner. The methods may be used to decrease endothelial cell survival, endothelial cell migration, and angiogenesis.

[0042] In one aspect, the invention provides compositions and methods for decreasing PI3K activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The PI3K activity is preferably kinase activity as directed at a substrate of PI3K, or binding activity as directed at a PI3K binding partner. The methods may be used to decrease endothelial cell survival, nitric oxide synthesis, and angiogenesis.

[0043] In one aspect, the invention provides compositions and methods for decreasing PLCγ activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The PLCγ activity is preferably phospholipase activity as directed at a substrate of PLCγ, or binding activity as directed at a PLCγ binding partner. The methods may be used to decrease endothelial cell proliferation and angiogenesis.

[0044] In one aspect, the invention provides compositions and methods for decreasing Rac activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The Rac activity is preferably GTPase activity or binding activity as directed at a Rac binding partner. The methods may be used to decrease endothelial cell migration and angiogenesis.

[0045] In one aspect, the invention provides compositions and methods for decreasing Rho activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The Rho activity is preferably GTPase activity or binding activity as directed at a Rho binding partner. The methods may be used to decrease endothelial cell migration and angiogenesis.

[0046] In one aspect, the invention provides compositions and methods for decreasing src activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The src activity is preferably kinase activity as directed at a substrate of src, or binding activity as directed at a src binding partner. The methods may be used to decrease endothelial cell migration, endothelial cell proliferation, and angiogenesis.

[0047] In one aspect, the invention provides compositions and methods for decreasing angiogenesis. The methods involve contacting endothelial cells with an RDP58 composition, wherein the RDP58 composition decreases nitric oxide synthesis, endothelial cell proliferation, endothelial cell migration, or endothelial cell survival.

[0048] In one aspect, the invention provides methods for treating cancer. The methods involve providing an RDP58 composition to the vicinity of a tumor where blood vessels are present. In a preferred embodiment, the methods further involve contacting tumor cells and/or stromal cells that express MMP with the RDP58 composition. The RDP58 composition decreases angiogenesis in the vicinity of the tumor. In a preferred embodiment, the RDP58 composition decreases MMP production in the tumor cells or stromal cells.

[0049] In one aspect, the invention provides methods for decreasing tumor invasiveness and tumor metastasis. The methods involve contacting tumor cells that express MMP with an RDP58 composition. Contact with the RDP58 composition decreases MMP production in the tumor cells. In a preferred embodiment, the MMP is MMP9 or MMP2.

[0050] In one aspect, the invention provides compositions and methods for treating a variety of disorders other than cancer that involve angiogenesis.

[0051] In one aspect, the invention provides compositions and methods for treating obesity. In one embodiment, methods for decreasing the vascularization of an adipose cell population in vivo are provided. The methods comprise providing an RDP58 composition to the vicinity of an adipose cell population in vivo, wherein blood vessels are present in the vicinity of the adipose cell population. The RDP58 composition provided decreases angiogenesis in the vicinity of the adipose cell population, thereby decreasing vascularization of the adipose cell population.

[0052] In one aspect, the invention provides compositions and methods for treating intraocular neovascularizati on disorders, including adult macular degeneration and the intraocular neovascularizati on attendant diabetes mellitus. In a preferred embodiment, the invention provides methods for inh biting intraocular neovascularization, which may be used to treat intraocular neovascular disorders. The methods comprise providing an RDP58 composition intraocularly to the vicinity of blood vessels. The RDP58 composition provided decreases intraocular angiogenesis.

[0053] In one aspect, the invention provides compositions and methods for decreasing brain edema. In a preferred embodiment, the methods comprise providing an RDP58 composition to the vicinity of blood vessels of the brain. The RDP58 composition decreases blood vessel permeability and decreases angiogenesis in the vicinity of the blood vessels. In one embodiment, the methods are used to treat brain ischemia.

[0054] In one aspect, the invention provides composition and methods for decreasing angiogenesis attendant polycystic ovary syndrome. The methods comprise providing an RDP58 composition to the vicinity of an ovary, wherein blood vessels are present in the vicinity of the ovary. The RDP58 composition decreases angiogenesis in the vicinity of the ovary. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] The "RDP58 compositions" provided herein are capable of modulating the activity of a variety of signal transducing molecules, including MyD88, IRAK, TRAF, MEK, MEKK, Ras, Rac, CDC42, Rho, c-src, Akt, JNK, ERK, PI3K, p38MAPK, NF-/.B, AP-1 , paxillin, FAK, Fyn, Pyk2, PLCK, and p53, in mammalian cells.

[0056] The RDP58 compositions provided herein may be used to decrease the phosphorylation and activity of TRAF, IRAK, JNK, p38MAPK, ERK, Akt, MEK, MEKK, Ras, Rac, CDC42, Rho, c-src, PI3K, paxillin, FAK, Fyn, Pyk2, and PLCJ Further, RDP58 compositions may be used to decrease the DNA binding activity of NF-/ B and AP-1. Further, RDP58 compositions may be used to decrease the transcription promoting activity of NF-/eB and AP-1 , and modulate the transcription promoting activity of p53. RDP58 compositions may also be used to alter the activity of various transcription regulating factors that are modulated by signal transduction pathways impacted by RDP58 compositions, including but not limited to c-Jun, c-Fos, AP1, SP1 , C/EBP, c-myc, TCF, ATF, c-rel, CREB, Elk, MEF- 2, CHOP, p53, NF-z B, and the like.

[0057] The signal transduction pathways impacted by RDP58 compositions play a variety of roles in a variety of cell types in a variety of tissues. RDP58 compositions may be used to modulate a wide variety of cellular and physiological activities involving, for example, the immune system, the pulmonary system, the cardiovascular system, adipose tissue, the kidney, the nervous system including the central and peripheral and enteric nervous systems, the liver, bone and cartilage, various epithelia, the circulatory system, and the gastrointestinal system.

[0058] In addition to other activities, the RDP58 compositions disclosed herein have potent anti- angiogenesis activity and are capable of inhibiting the production of matrix metalloproteinases, particularly MMP2 and MMP9. These RDP58 compositions are useful for modulating angiogenesis, tumor vascularization, tumor growth, tumor survival, tumor invasiveness, and tumor metastasis, and for treating a variety of disorders other than cancer that involve angiogenesis.

[0059] In one aspect, the invention provides compositions and methods for decreasing angiogenesis. In another aspect, the invention provides compositions and methods for decreasing MMP production. In a further aspect, the invention provides compositions and methods for coordinately decreasing angiogenesis and decreasing MMP production.

[0060] In a preferred embodiment, the invention provides compositions and methods for decreasing the vascularization of a cell population in vivo. The methods comprise providing an RDP58 composition to the vicinity of a cell population in vivo, wherein blood vessels are present in the vicinity of the cell population. The RDP58 composition provided decreases angiogenesis in the vicinity of the cell population, thereby decreasing vascularization of the population.

[0061] "Providing an RDP58 composition to the vicinity of means providing an RDP58 composition within an effective distance of the reference site. "Effective distance" means a distance within which the RDP58 composition can exert a bioactivity, particularly the reduction of angiogenesis; the reduction of VEGF-R signaling; the reduction of Akt phosphorylation or Akt activity; the reduction of endothelial cell proliferation; the reduction of endothelial cell migration; the reduction of endothelial cell survival; the reduction of nitric oxide synthesis by endothelial cells; or the inhibition of MMP expression, at the reference site. Providing can be done, for example, by local delivery, oral delivery, systemic delivery, etc. The RDP58 composition need not be directly delivered within the effective distance of the reference site to be "provided to the vicinity" of the reference site. The effective distance will vary with the nature of the RDP58 composition, the amount and formulation of the RDP58 composition used, and the nature of the tissue, but will be readily determined with standardizing experiments.

[0062] By "decreasing angiogenesis" is meant decreasing partially or completely the growth of new blood vessels. "Angiogenesis" as used herein includes both sprouting and non-sprouting angiogenesis. Decreasing angiogenesis may be achieved by a number of cellular mechanisms, including decreasing endothelial cell proliferation, decreasing endothelial cell migration, and decreasing endothelial cell survival.

[0063] In one embodiment, one or more cells of the cell population expresses an MMP, and the method further comprises decreasing MMP production in the cell population by contacting the cell population with the RDP58 composition. In a preferred embodiment, the MMP expressed by one or more cells of the population is MMP2 or MMP9.

[0064] By "contacting with an RDP58 composition" is meant providing the RDP58 composition to the cell population in such a manner and in such an amount as to effect physical contact between the RDP58 composition and cells of the cell population.

[0065] By "decreasing MMP expression in the cell population" or "decreasing MMP production" is meant decreasing partially or completely the production of MMP protein by the cell population. This may be done, for example, by reducing the production of an MMP mRNA by reducing the transcription of an MMP gene.

[0066] It will be appreciated that MMPs such as MMP9 have been implicated in the production of pro-angiogenic signals and the promotion of angiogenesis. Without being bound by theory, RDP58 compositions may be used according to the present methods to decrease angiogenesis by both directly impairing the function of endothelial cells and by inhibiting the MMP-mediated production of pro-angiogenic signals in the microenvironment of blood vessels.

[0067] In a preferred embodiment, the in vivo cell population is present in a tumor. The cell population may comprise tumor cells and/or cells of the tumor stroma. In one embodiment, one or more cells of the population expresses an MMP, and the method further comprises decreasing MMP production in the cell population by contacting the cell population with the RDP58 composition. In a preferred embodiment, the MMP expressed by one or more cells of the population is MMP2 or MMP9.

[0068] By "present in a tumor" is meant that the cell population is physically localized in a tumor mass. The cell population may comprise tumor cells and/or non-tumor stromal cells. In a preferred embodiment, the cell population comprises tumor cells. In a preferred embodiment, the tumor cells express an MMP, preferably MMP9 or MMP2. [0069] MMP production enhances tumor cell mobility and tumor invasiveness, and also contributes to the formation of a microenvironment that promotes tumor growth and leads to cancer progression. Without being bound by theory, RDP58 compositions may be used according to the present methods to decrease vascularization of a tumor population, reduce the metastatic and invasive potential of the tumor, and decrease the ability of tumor cells, stromal cells, and cells in the vicinity of the tumor to establish and maintain a tumor-growth promoting microenvironment.

[0070] In one embodiment, the tumor is a metastatic tumor. "Metastatic tumor" includes a tumor that has metastasized. In this embodiment, the cell population may be in the primary tumor location, or a secondary tumor location. "Metastatic tumor" also includes tumors that have not yet metastasized but have been determined to have metastatic potential.

[0071] In one embodiment, the tumor is an invasive tumor. "Invasive tumor" includes a tumor that has dissolved or begun to dissolve a basement membrane supporting the tumor. Tumors that have dissolved a basement membrane and migrated or grown into adjacent tissue are also included.

[0072] In one aspect, the invention provides compositions and methods for decreasing the survival of a tumor. In one aspect, the invention provides compositions and methods for inhibiting the growth of a tumor.

[0073] In one embodiment, the methods comprise providing an RDP58 composition to the vicinity of a tumor, wherein blood vessels are present in the vicinity of the tumor, and wherein the RDP58 composition decreases angiogenesis in the vicinity of the tumor. By decreasing the vascularization of the tumor, the provision of an RDP58 composition to the vicinity of the tumor effectively keeps the tumor from growing and compromises the survival of tumor cells, which may die by necrosis or apoptosis.

[0074] In one embodiment, the tumor expresses an MMP, and the methods additionally comprise contacting the tumor with the RDP58 composition, wherein the RDP58 composition decreases expression of the MMP in the tumor.

[0075] As discussed above, MMP production enhances tumor cell mobility and tumor invasiveness, and also contributes to the formation of a microenvironment that is pro-angiogenic and promotes angiogensis and tumor growth. The present invention provides methods for the coordinated reduction of MMP production and reduction of angiogenesis using RDP58 compositions.

[0076] By "coordinated" is meant that the two effects are achieved together with the provision of an RDP58 composition to the vicinity of the reference cell population in vivo. Coordination does not depend on the ability of MMP to generate a pro-angiogenic signal because the present RDP58 compositions exert direct anti-angiogenic effects on endothelial cells.

[0077] In addition, the present disclosure establishes the ability of RDP58 compositions to inhibit VEGF signaling, CD40 signaling, IL-18 signaling, JNK activity, p38MAPK activity, AP1 activity, Akt activity, TRAF activity, NF- B activity, and PI3K activity. In one aspect, the invention provides methods for using RDP58 compositions to inhibit one or more of these signaling molecules in tumor cells to inhibit tumor growth and/or survival. The methods involve contacting tumor cells with an RDP58 composition, wherein contact with the RDP58 composition decreases the activity of one or more of these signaling molecules in the tumor cells.

[0078] The inhibition of JNK activity is desirable for the treatment of a variety of cancers, including but not limited to solid tumors such as breast, prostate, and lung cancers. JNK is implicated in the regulation of cell proliferation and migration, and the well known oncogene ras can transduce signals through JNK. JNK is also implicated in the regulation of protease expression in cancer cells, which provides for metastasis, and has additionally been implicated in metastasis. p38MAPK and AP-1 have also been implicated in the regulation of protease expression in cancer cells.

[0079] Additionally, Akt has been implicated in the survival of cancer cells.

[0080] Additionally, CD40 activation and TRAF overexpression have been linked to cancer (for example, Munzert et al., Blood 2002 Nov 15;100(10):3749-56; Izumi et al., Proc Natl Acad Sci, 1997 Feb 18;94(4): 1447-52; Izban et al., Mod Pathol 2000 Dec;13(12):1324-31; Durkop et al., Blood 93:617-623, 1999).

[0081] Additionally, PI3K signal transduction and VEGF-induced signals have been implicated in angiogenic processes that support the growth and expansion of cancer tissue.

[0082] PI3K activity has also been implicated in fibrosarcoma.

[0083] IL-18 signaling has been implicated in melanoma carcinoma.

[0084] The modulation of JNK and NF-/ B activities is also desirable for the sensitization of cancer cells to radiation and chemotherapy.

[0085] In one embodiment, the invention provides methods for decreasing VEGF signaling, CD40 signaling, IL-18 signaling, JNK activity, p38MAPK activity, AP1 activity, Akt activity, TRAF activity, NF- KB activity, or PI3K in tumor cells. The methods comprise contacting tumor cells with an RDP58 composition, wherein contact with the RDP58 composition decreases the activity of one or more of these signaling molecules or pathways in the tumor cells. The methods may be used to decrease tumor survival, inhibit tumor growth, inhibit tumor proliferation, and to increase tumor cell differentiation, and to treat cancer.

[0086] In one aspect, the invention provides methods for decreasing VEGF-R activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The VEGF-R activity is preferably kinase activity as directed at a VEGF-R substrate, or binding activity as directed at a VEGF-R binding partner. The methods may be used to decrease endothelial cell proliferation, endothelial cell migration, endothelial cell survival, and angiogenesis.

[0087] In one aspect, the invention provides compositions and methods for decreasing Akt activity in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The Akt activity is preferably kinase activity as directed at an Akt substrate, or binding activity as directed at an Akt binding partner. The methods may be used to decrease endothelial cell survival, nitric oxide synthesis, and angiogenesis. [0088] Without being bound by theory, it appears that RDP58 compositions impact VEGF activity in at least two ways. First, RDP58 compositions can modulate the expression of VEGF receptor and an endothelial cell's ability to respond to VEGF. Second, RDP58 compositions can inhibit PI3K activity and Akt activity, which activities lie downstream of VEGF receptor, and thereby inhibit the action of VEGF.

[0089] In other aspects, the invention provides methods for decreasing the activity of PI3K, FAK, PLCγ, Rac, Rho, ERK, and JNK in endothelial cells. The methods involve contacting endothelial cells with an RDP58 composition. The methods may be used to decrease endothelial cell survival, endothelial cell proliferation, endothelial cell migration, and angiogensis.

[0090] In one aspect, the invention provides compositions and methods for treating a variety of disorders other than cancer that involve angiogenesis.

[0091] In one aspect, the invention provides compositions and methods for treating obesity. In one embodiment, methods for decreasing the vascularization of an adipose cell population in vivo are provided. The methods comprise providing an RDP58 composition to the vicinity of an adipose cell population in vivo, wherein blood vessels are present in the vicinity of the adipose cell population. The RDP58 composition provided decreases angiogenesis in the vicinity of the adipose cell population, thereby decreasing vascularization of the adipose cell population.

[0092] In one aspect, the invention provides compositions and methods for treating intraocular neovascularization disorders, including adult macular degeneration and the intraocular neovascularization attendant diabetes mellitus. In a preferred embodiment, the invention provides methods for inhibiting intraocular neovascularization, which may be used to treat intraocular neovascular disorders. The methods comprise providing an RDP58 composition intraocularly to the vicinity of blood vessels. The RDP58 composition provided decreases intraocular angiogenesis.

[0093] In one aspect, the invention provides compositions and methods for decreasing brain edema. In a preferred embodiment, the methods comprise providing an RDP58 composition to the vicinity of blood vessels of the brain. The RDP58 composition decreases blood vessel permeability and decreases angiogenesis in the vicinity of the blood vessels. In one embodiment, the methods are used to treat brain ischemia.

[0094] In one aspect, the invention provides composition and methods for decreasing angiogenesis attendant polycystic ovary syndrome. The methods comprise providing an RDP58 composition to the vicinity of an ovary, wherein blood vessels are present in the vicinity of the ovary. The RDP58 composition decreases angiogenesis in the vicinity of the ovary.

[0095] As disclosed herein, RDP58 compositions are capable of modulating a variety of signal transduction pathways in a variety of cell types, making them useful for the treatment of diseases including but not limited to scleroderma, chronic thyroiditis, Grave's disease, ischemic stroke, myocardial infarction, glomerulonephritis, cancer (e.g., involving dysregulation of tumor suppressor genes, metastatic myeloma, Karposi's sarcoma, myeloma, etc.), periprostetic osteolysis, osteoporosis, peridontitis, cardiac hypertrophy, restenosis after angioplasty, hypertension, lung fibrosis, epilepsy, idiopathic aplastic anemia, asthma, intestinal bowel disorders, inflammatory disorders; autoimmune diseases, and the like. These conditions include degenerative disorders, including without limitation, Alzheimer's disease, Parkinson's disease, myasthenia gravis, and the like.

Treatment of Pulmonary Disease

[0096] The p38MAPK, JNK, and PI3K signal transduction pathways have been implicated in diseases of the pulmonary system, including but not limited to pulmonary fibrosis, allergic asthma and allergen-induced eosinophil and lymphocyte increase, airway inflammation in response to virus, bronchial asthma, pulmonary hypertension and chronic obstructive pulmonary disease (COPD), including emphysema. The inhibition of these signal transduction pathways is desirable for the treatment and prevention of these pulmonary diseases.

[0097] In particular, the inhibition of the p38MAPK pathway is desirable for the treatment of pulmonary fibrosis, while inhibition of NF- B and the JNK, p38MAPK, and PI3K pathways is desirable for the treatment of COPD and the smooth muscle proliferation thought to contribute thereto. The present invention provides methods for the treatment and prevention of pulmonary conditions, particularly those that are steroid refractory or resistant.

[0098] In one aspect, the invention provides methods for decreasing the activity of the p38MAPK, JNK, and PI3K signal transduction pathways to treat these diseases of the pulmonary system. The methods involve providing an RDP58 composition to affected regions of the pulmonary system and contacting cells therein that contribute to the etiology or progression of the pulmonary disease. Contact with the RDP58 composition decreases the activity of the p38MAPK, JNK, or PI3K signal transduction pathway in cells that contribute to the etiology or progression of the pulmonary disease.

Treatment of Cardiovascular Disease

[0099] The p38MAPK and JNK pathways, as well as NF-κB, have been implicated in diseases and processes of the cardiovascular system, including but not limited to hypertension, vascular smooth muscle cell proliferation and collagen synthesis, ischaemic heart disease, and vascular and cardiac hypertrophy. In addition, the Akt pathway has been implicated in cardiomyopathy in type II diabetes mellitus. The inhibition of these signal transduction pathways is desirable for the treatment and prevention of these diseases.

[00100] Additionally, the inhibition of p38MAPK is desirable for the inhibition of restenosis and neointimal hyperplasia after angioplasty; the inhibition of ischaemic cell death and the cell death accompanying reperfusion following heart attack; and the inhibition of cardiac hypertrophy, hypertension and stroke.

[00101] In one aspect, the invention provides methods for decreasing the activity of the p38MAPK, JNK, and Akt signal transduction pathways, as well as the activity of NF- B, to treat these cardiovascular diseases and conditions. The methods involve providing an RDP58 composition to affected regions of the cardiovascular system and contacting cells therein that contribute to the etiology or progression of the cardiovascular disease or condition. Contact with the RDP58 composition decreases the activity of the p38MAPK, JNK, or Akt signal transduction pathway, or the activity of NF-κB, in cells that contribute to the etiology or progression of the cardiovascular disease or condition.

Treatment of Kidney Disease

[00102] The inhibition of p38MAPK is desirable for the treatment of glomerulonephritis and the inhibition of glomerulosclerosis and interstitial fibrosis.

[00103] In one aspect, the invention provides methods for decreasing the activity of the p38MAPK signal transduction pathway to treat glomerulonephritis and inhibit glomerulosclerosis and interstitial fibrosis. The methods involve providing an RDP58 composition to affected regions of the kidney and contacting cells therein that contribute to the etiology or progression of glomerulonephritis, as well as those cells that contribute to glomerulosclerosis and interstitial fibrosis. Contact with the RDP58 composition decreases the activity of the p38MAPK signal transduction pathway in these cells.

Inhibition of Hepatic Cell Death

[00104] The inhibition of JNK, p38MAPK, and NF-/cB is desirable for the inhibition of hepatic cell death, such as the cell death observed in ischaemia reperfusion injury following stroke, heart attack, shock, and organ transplantation.

[00105] In one aspect, the invention provides methods for decreasing the activity of the JNK and p38MAPK signal transduct :iion pathways, and the activity of NF-/ 3, to inhibit hepatic cell death attendant a variety of cond litions, such as ischaemia reperfusion injury following stroke, heart attack, shock, and organ transplantation. The methods involve contacting hepatic cells with an RDP58 composition. Contact with the RDP58 composition decreases the activity of the JNK or p38MAPK signal transduction pathway, or decreases NF- d3 activity, in the hepatic cells.

Treatment of Diseases of the Gastrointestinal System

[00106] The inhibition of JNK and p38MAPK is desirable for the treatment of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis.

[00107] In one aspect, the invention provides methods for decreasing the activity of the JNK and p38MAPK signal transduction pathways to treat inflammatory bowel disease. The methods involve contacting cells of the gastrointestinal system that contribute to the etiology or progression of the inflammatory bowel disease with an RDP58 composition. Contact with the RDP58 composition decreases the activity of the JNK and p38MAPK signal transduction pathways in these cells.

Modulation of Tachykinin-lnduced Cellular Activities

[00108] The PI3K, p38MAPK, ERK, and JNK pathways play important roles in mediating and modulating signal transduction in response to tachykinins such as substance P, neurokinin A, neurokinin B, neuropeptide K, and neuropeptide gamma. For example, PI3K, p38MAPK, and JNK pathways mediate the induction of TNFσ and the exocytosis of histamine observed in mast cells in response to substance P. Further, at least some tachykinin receptors are also regulated by these signaling pathways. For example, ERK activation upregulates the expression of substance P receptor expression in nociceptve spinal neurons and contributes to pain hypersensitivity. The receptors for tachykinins are expressed in a variety of tissues and cell types (e.g., nervous system; immune system; endothelial cells; gingival tissue), and substance P is purported to play a role in the modulation of a broad spectrum of conditions, including pain (including migraine and fibromyalgia), asthma, interstitial cystitis (IC), inflammatory bowel disease, emesis, psoriasis, and central nervous system disorders. With respect to the nervous system, studies in mice have demonstrated that loss of substance P receptor (NK1 R) function results in marked reduction of anxiety-like behavior and associated physiological changes, and substance P receptor antagonists find use in the treatment of affective disorders in humans.

[00109] In one embodiment, the present invention provides methods for modulating tachykinin- induced cellular activities using RDP58 compositions. In a preferred embodiment, methods for reducing substance P-induced cellular activities are provided. Such methods find use in the treatment of pain and affective disorders in humans. The methods involve contacting the cells concerned with an RDP58 composition, wherein contact with the RDP58 composition decreases the activity of one or more of these signaling molecules or pathways in these cells.

Inhibition of CD40-, CD30,-, CD27-, and 4-1 BB-lnduced Cellular Activities

[00110JCD40, CD30, CD27, and 4-1 BB signaling involves TRAF activation that may be reduced by RDP58 compositions. Activities regulated by these factors include immunoglobulin class switching, affinity maturation in B cells, dendritic cell activation, B cell transformation (Brown et al., J. Exp. Med., 193:943-954, 2001), T-cell coactivation and B-cell Ig synthesis (Akiba et al., J Biol. Chem., 273:13353-13358, 1998). In addition, the inhibition of CD30 activation may inhibit HIV replication (Tsitsikov et al., Proc Natl. Acad. Sci, 94:1390-1395, 1997). Accordingly, in one embodiment, the invention provides methods using RDP58 compositions to inhibit CD40-, CD30,-, CD27-, and/or 4- 1 BB-induced cellular activities. Such methods find use in the treatment of autoimmune disorders, B- cell transformation, lymphoma, leukemia, and dendritic cell activation, among other conditions. The methods involve contacting the cells concerned with an RDP58 composition, wherein contact with the RDP58 composition decreases TRAF activity in these cells.

Inhibition of Leptin-lnduced Cellular Activities

[00111] Leptin is a ligand produced by adipocytes that regulates body fat content. Leptin also modulates estrogen levels, possibly through the regulation of aromatase, and may be involved in certain cancers, such as breast cancer. Leptin activates MAP kinases and the AP-1 transcriptional complex, thereby effecting changes in gene expression. In one embodiment, the present invention provides methods using RDP58 compositions to inhibit leptin-induced cellular activities, such as the induction of aromatase expression. Such methods find use in the treatment of cancer. The methods comprise contacting the cells concerned with an RDP58 composition, wherein contact with the RDP58 composition decreases the activity of the MAPK pathway, or decreases AP1 activity, in these cells.

Inhibition of LMP-1 Induced Cellular Activities [00112] The Epstein Barr virus (EBV) protein latent membrane protein 1 (LMP1) binds to and activates TRAF, and this association is required for the B-cell transforming activity of LMP1. In one embodiment, the present invention provides methods using RDP58 compositions to inhibit LMP1- induced cellular activities, such as the transformation of B cells. Such methods find use in the treatment of lymphomas, leukemias, and other cancers characterized by signaling activity like that of LMP1 expressing B cells. For example, Kaposi's sarcoma (see for example Glenn et al., J Virol 1999 Aug;73(8):6953-63). The methods comprise contacting the cells concerned with an RDP58 composition, wherein contact with the RDP58 composition decreases TRAF activity in these cells.

Inhibition of LPS-lnduced Cellular Activities

[00113] Cellular responses to bacterial lipopolysaccharide (LPS) are transduced by TLR, Myd88, IRAK, TRAF, and MAP kinase. In a preferred embodiment, the present invention provides methods using RDP58 compositions to inhibit LPS-induced cellular activities, such as the production of inflammatory cytokines, angiogenesis, and osteoclast differentiation. The methods comprise contacting the cells concerned with an RDP58 composition, wherein contact with the RDP58 composition decreases the activity of one or more of these signaling molecules or pathways in the cells.

Inhibition of BLP-lnduced Cellular Activities

[00114] Cellular responses to bacterial lipoproteins (BLP) are transduced by TLR, Myd88, IRAK, TRAF, and MAP kinase. In a preferred embodiment, the present invention provides methods using RDP58 compositions to inhibit BLP-induced cellular activities, such as the production of inflammatory cytokines. The methods comprise contacting the cells concerned with an RDP58 composition, wherein contact with the RDP58 composition decreases the activity of one or more of these signaling molecules or pathways in the cells.

Inhibition of TNFR-lnduced Cellular Activities

[00115] Members of the TNFR family are known to mediate diverse functions, including but not limited to apoptosis, osteoclastogenesis, and immune system cell regulation. In a preferred embodiment, the present invention provides methods using RDP58 compositions to inhibit TNFR-induced cellular activities, such as apoptosis, osteoclastogenesis, and immune system cell regulation. Such methods find use in the treatment of autoimmune and inflammatory disorders, developmental morphogenic disorders, cancer, and disorders involving an undesired imbalance between bone deposition and resorption. The methods comprise contacting the cells concerned with an RDP58 composition, wherein contact with the RDP58 composition decreases the activity of the TNFR pathway in the cells.

Inhibition of CpG-DNA-lnduced Cellular Activities

[00116] Cellular responses to bacterial DNA (CpG-DNA) are transduced by TLR, Myd88, IRAK, TRAF, and MAP kinase. In a preferred embodiment, the present invention provides methods using RDP58 compositions to inhibit CpG-DNA-induced cellular activities, such as the production of inflammatory cytokines. The methods comprise contacting the cells concerned with an RDP58 composition, wherein contact with the RDP58 composition decreases the activity of one or more of these signaling molecules or pathways in the cells.

Inhibition of TLR3-lnduced Cellular Activities

[00117] The TLR3 protein (toll-like receptor 3) is activated in response to. doubles stranded RNA (dsRNA), and RDP58 compositions are capable of inhibiting such signal transduction. In one embodiment, the invention provides methods using RDP58 compositions to inhibit TLR3-induced cellular activities, including responses to dsRNA. The methods comprise contacting the cells concerned with an RDP58 composition, wherein contact with the RDP58 composition decreases the activity of one or more of these signaling molecules or pathways in the cells.

Inhibition of Cellular Activities Induced by Lipid Raft Domain Complexes

[00118] The activation of particular signal transduction pathways involves the assembly of signaling complexes in lipid raft microdomains . Lipid rafts recruit and exclude particular components to form a microdomain organized for signal transduction. Many signal transduction proteins and receptors localize, either constitutively or transiently, to lipid raft microdomains. For example, the non-receptor tyrosine kinase c-src is found in microdomains, as is PI3K. Lipid raft-based signaling plays a role in a diverse array of cellular activities. For example, RANK-induced differentiation of osteoclasts involves lipid rafts; RANK activation leads to TRAF6 translocation to the raft microdomain and the subsequent activation of Akt.

[00119] Interestingly, RDP58 localizes to lipid raft microdomains. Further, RDP58 is observed in the Golgi complex, as are lipid raft markers that are translocated thereto from the cell surface. Moreover, drugs that perturb cholesterol availability, which is normally present in lipid rafts, also disrupt lipid raft microdomains and can modulate signal transduction, for example, signal transduction in lymphocytes that is mediated by GPI-anchored proteins.

[00120] In a preferred embodiment, the present invention provides methods using RDP58 compositions to inhibit the assembly of signaling complexes in lipid raft microdomains. In another preferred embodiment, methods of using RDP58 compositions to perturb the translocation of signal transduction molecules and/or receptors to lipid raft microdomains from the Golgi complex, and/or from lipid raft microdomains to the Golgi complex, are provided. In an especially preferred embodiment, the present invention provides methods using RDP58 compositions to inhibit signal transduction by TRAF proteins in lipid raft microdomains. In another preferred embodiment, the present invention provides methods using RDP58 compositions to inhibit PI3K activation in lipid raft microdomains. In another preferred embodiment, the present invention provides methods using RDP58 compositions to inhibit c-src activation in lipid raft microdomains. In an especially preferred embodiment, the present invention provides methods using RDP58 compositions to disrupt a TLR4- MyD88-IRAK-TRAF complex, such as that formed in response to LPS. The methods comprise contacting the cells concerned with an RDP58 composition. RDP58 Compositions

[00121] RDP58 compositions suitable for use in the methods disclosed herein will generally comprise at least one peptide, polypeptide or oligopeptide capable of decreasing angiogenesis, nitric oxide synthesis by endothelial cells, endothelial cell proliferation, endothelial cell survival, endothelial cell migration, MMP2 and/or MMP9 expression, or VEGFR signaling (including Akt phosphorylation).

[00122] In one embodiment, peptides are selected from the family of RDP58 peptides described in PCT Publication WO 98/46633, which are characterized therein as being capable of inhibiting the cytotoxic activity of lymphocytic cells, inhibiting the production of inflammatory cytokines and inflammatory responses associated with those cytokines, inhibiting the activity of heme-containing enzymes and delaying the onset of autoimmune disease in a mammal at risk of developing such a disease. As disclosed herein, it has now been found that such peptides also have the ability to modulate a variety of biochemical pathways and decrease angiogenesis.

[00123] Suitable peptides for use in the compositions and methods provided herein have a variety of characteristics, and may be identified in a number of ways.

[00124] Peptides may be identified by their ability to inhibit angiogenesis, nitric oxide synthesis by endothelial cells, endothelial cell proliferation, endothelial cell survival, endothelial cell migration, MMP2 and/or MMP9 expression, or VEGFR signaling (including Akt phosphorylation). For example, matrigel assays described in the present examples may be used to identify a peptide that inhibits angiogenesis and is suitable for use in an RDP58 composition.

[00125] In the preferred embodiment, the subject RDP58 peptides comprise one or more of the cytomodulating peptides disclosed in co-pending U.S. Patent Applications U.S.S.N 09/028,083 & U.S.S.N. 08/838,916 as well as corresponding International application WO 98/46633, the disclosures of which are expressly incorporated herein by reference. In a preferred embodiment, the RDP58 peptide comprises the core sequence Arg-nL-nL-nL-Arg-nL-nL-nL-Gly-Tyr, where nL is norieucine and all amino acids other than glycine are the D-stereoisomers. In another preferred embodiment, both Arg residues, the Gly residue, and the nL residue at position 7 (position 7 of 10, reading N-terminus to C-terminus) are L-stereoisomers.

[00126] In the preferred embodiment, the core sequence of the RDP58 peptide desirably comprises two basic amino acids separated by from three to four hydrophobic amino acids, particularly three hydrophobic amino acids, and particularly where the N-terminus is a basic amino acid. More desirably, the C-terminal amino acid is an aromatic amino acid, particularly tyrosine. Of particular interest is where at least one of the oligopeptide core terminal amino acids is an oligopeptide terminal amino acid, which may be in the monomeric or oligomeric form of the compound.

[00127] More particularly, the preferred RDP58 peptides for use in the compositions and methods of the present invention comprise oligopeptides having the core sequence B-X-X-X-B-X-X-X-J-Tyr, where B is a basic amino acid, preferably Lys or Arg, particularly Arg on at least one position, preferably at both positions; J is Gly, B or an aliphatic hydrophobic amino acid of from 5 to 6 carbon atoms, particularly Gly or B; and X is an aliphatic or aromatic amino acid. In one embodiment, at least three X amino acid residues are the same non-polar aliphatic amino acid, preferably at least four are the same non-polar aliphatic amino acid, more preferably at least five are the same non-polar aliphatic amino acid, and most preferably, all are the same non-polar aliphatic amino acid. In a preferred embodiment, the non-polar aliphatic amino acids are of from 5 to 6 carbon atoms, particularly 6 carbon atoms, particularly the non-polar aliphatic amino acids Val, lie, Leu, and nL. Thus, in some embodiments, X is any amino acid other than a charged aliphatic amino acid, and preferably any amino acid other than a polar aliphatic amino acid.

[00128] Of the six amino acids indicated by X in the B-X-X-X-B-X-X-X-J-Tyr peptide sequence, preferably at least 3 are aliphatic amino acids of from 5 to 6 carbon atoms, more preferably at least 4 are aliphatic amino acids of from 5 to 6 carbon atoms, most preferably at least 5 are aliphatic amino acids of 5-6 carbon atoms, more particularly 6 carbon atoms. In a preferred embodiment, the aliphatic amino acids are non-polar aliphatic amino acids of from 5 to 6 carbon atoms, particularly Val, lie, Leu, and nL. The other amino acids may be other uncharged aliphatic amino acids, particularly non-polar aliphatic amino acids or aromatic amino acids.

[00129] Compositions of particular interest will include an RDP58 peptide having the core sequence:

[00130] Arg-U-X-X-Arg-X-X-X-J-Tyr

[00131] wherein all of the symbols have been defined previously except U, which comprises an uncharged aliphatic amino acid or aromatic amino acid, particularly a non-polar aliphatic amino acid or aromatic amino acid.

[00132] The amino acids may be naturally occurring amino acids or D- isomers thereof. The peptides may have one or more D-stereoisomer amino acids, up to all of the amino acids. Additionally, the peptides may comprise oligomers of the subject peptides, particularly dimers thereof, or comprise a cyclic peptide, that is a ring structure, as further described below.

[00133] For the purposes of this invention, the amino acids (for the most part natural amino acids or their D-stereoisomers) will be broken down into the following categories:

[00134] 1. Aliphatic

[00135] (a) non- polar aliphatic:

[00136] Gly, Ala, Val, nL, lie, Leu

[00137] (b) polar aliphatic:

[00138] (1) uncharged:

[00139] Cys, Met, Ser, Thr, Asn, Gin

[00140] (2) charged:

[00141] Asp, Glu, Lys, Arg

[00142] 2. Aromatic:

[00143] Phe, His, Trp, Tyr [00144] wherein Pro may be included in the non-polar aliphatic amino acids, but will normally not be included. "nL" represents norieucine, where the non-polar aliphatic amino acids may be substituted with other isomers.

[00145] Exemplary RDP58 peptides include the following:

nL = norieucine

[00146] Other exemplary RDP58 peptides are disclosed in PCT application serial number PCT/US98/07231 , filed 10 April 1998, US Patent Application Serial No. 08/838,916, filed 11 April 1997, and US Patent Application Serial No. 09/028,083 filed 23 February 1998, each being expressly incorporated herein in its entirety by reference. Generally, the term "RD-58 peptide" as used herein is meant to encompass all of the foregoing peptide compounds.

[00147] In further embodiments, other known peptides such as HLA peptides and TCR peptides may be alternatively or additionally used in the subject invention as components of the subject RDP58 compositions. These include HLA-B a\ -domain, particularly the amino acids from 75 to 84 and variations of this sequence where not more than 2 amino acids are replaced (see, e.g., WO 95/13288 and Buelow et al., expressly incorporated herein by reference). Also included are sequences based on the human TCR-σ transmembrane region consisting of that sequence and sequences having not more than 2 mutations from that sequence (see Australian Application Nos. PN 0589 and PN 0590, filed January 16, 1995, expressly incorporated herein by reference). These sequences include 2 basic amino acids, where the 2 basic amino acids are separated by 4 aliphatic hydrophobic amino acids, although the application indicates that from 3 to 5 hydrophobic amino acids may be present. By mutation is intended each substitution of one amino acid for another or an insertion or deletion, each being counted as one mutation. Generally, the term "peptide" as used herein is meant to encompass all of the foregoing peptide compounds, as well as analogs, derivatives, fusion proteins and the like.

[00148] The subject peptides may be modified in a variety of conventional ways well known to the skilled artisan. For example, one or both, usually one terminus of the peptide, may be substituted with a lipophilic group, usually aliphatic or aralkyl, of from 8 to 36, usually 8 to 24 carbon atoms and fewer than two heteroatoms in the aliphatic chain, the heteroatoms usually being oxygen, nitrogen and sulfur. As further described below, the chain may be saturated or unsaturated, desirably having not more than 3 sites, usually not more than 2 sites of aliphatic unsaturation. Conveniently, commercially available aliphatic fatty acids, alcohols and amines may be used, such as caprylic acid, capric acid, lauric acid, myristic acid and myristyl alcohol, palmitic acid, palmitoleic acid, stearic acid and stearyl amine, oleic acid, linoleic acid, docosahexaenoic acid, etc. (see U.S. Patent No. 6,225,444, hereby incorporated by reference). Preferred are unbranched, naturally occurring fatty acids between 14-22 carbon atoms in length. Other lipohiiic molecules include glyceryl lipids and sterols, such as cholesterol. The lipophilic groups may be reacted with the appropriate functional group on the oligopeptide in accordance with conventional methods, frequently during the synthesis on a support, depending on the site of attachment of the oligopeptide to the support. Lipid attachment is useful where oligopeptides may be introduced into the lumen of the liposome, along with other therapeutic agents for administering the peptides and agents into a host. Increasing lipophilicity is also known to increase transport of compounds across endothelial cells and therefore useful in promoting uptake of such compounds from the intestine or blood stream into surrounding tissues.

[00149] The terminal amino group or carboxyl group of the peptide may be modified by alkylation, amidation, or acylation to provide esters, amides or substituted amino groups, where the alkyl or acyl group may be of from about 1 to 30, usually 1 to 24, preferably either 1 to 3 or 8 to 24, particularly 12 to 18 carbon atoms. This is done using conventional chemical synthetic methods. The peptide or derivatives thereof may also be modified by acetylation or methylation to alter the chemical properties, for example lipophilicity. Methods for acylating, and specifically for acetylating the free amino group at the N-terminus are well known in the art. For the C-terminus, the carboxyl group may be modified by esterification with alcohols or amidated to form -CONH2, CONHR, or CONR, wherein each R is a hybroxycarbyl (1-6 carbons). Methods of esterification and amidation are done using well known techniques. Other modifications include deamination of glutamyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of praline and lysine; phosphorylation of hydroxyl groups of serine or threonine; and methylation of amino groups of lysine, arginine, and histidine side chains (see T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co. San Francisco, CA, 1983). [00150] In additional embodiments, either or both the N- and C-terminus of the peptide may be extended by not more than a total of about 100, usually not more than a total of about 30, more usually not more than about 20 amino acids, often not more than about 9 amino acids, where the amino acids will have fewer than 25%, more usually fewer than 20% polar amino acids, more particularly, fewer than 20% which are charged amino acids. Thus, extensions of the above core sequences in either direction are mainly done with lipophilic, uncharged amino acids, particularly non- polar aliphatic amino acids and aromatic amino acids. The peptides may comprise L-amino acids, D- amino acids, or mixtures of D and L amino acids. Exceptions to the number of amino acid extensions are contemplated when the oligopeptides are expressed as fusion or chimeric proteins, as described below.

[00151] The peptides may also be in the form of oligomers, particularly dimers of the peptides, which may be head to head, tail to tail, or head to tail, there being not more than about 6 repeats of the peptide. The oligomer may contain one or more D-stereoisomer amino acids, up to all of the amino acids. The oligomers may or may not include linker sequences between the peptides. When linker sequences are used, suitable linkers include those comprising uncharged amino acids and (Gly)n, where n is 1-7, Gly-Ser (e.g., (GS)n, (GSGGS)n and (GGGS)n, where n is at least 1 ), Gly-Ala, Ala- Ser, or other flexible linkers, as known in the art. Linkers of Gly or Gly-Ser may be used since these amino acids are relatively unstructured, which allows interaction of individual peptides with cellular target molecules and limits structural perturbations between peptides of the oligomer.

[00152] Peptides may also be in a structurally constrained form such as cyclic peptides of from about 9-50, usually 12 to 36 amino acids, where amino acids other than the specified amino acids may be present as a bridge. Thus, for example, addition of terminal cysteines allows formation of disulfide bridges to form a ring peptide. In some instances, one may use other than amino acids to cyclize the peptide. Bifunctional crosslinking agents are useful in linking two or more amino acids of the peptide. Other methods for ring formation are described in Chen et al., Proc. Natl. Acad. Sci. USA 89:5872- 5876 (1992); Wu et al., Protein Engineering 6:471-478 (1993); Anwer, et al., Int. J. Pep. Protein Res. 36:392-399 (1990); and Rivera-Baeza, et al. Neuropeptides 30: 327-333 (1996); all references incorporated by reference. Alternatively, structurally constrained peptides are made by addition of dimerization sequences to the N- and C- terminal ends of the peptide, where interaction between dimerization sequences lead to formation of a cyclic type structure (see WO/0166565, incorporated by reference). In other instances, the subject peptides are expressed as fusions to other proteins, which provide a scaffold for constrained display on a surface exposed structure, such as a loop of a coiled- coil orβ-turn structure.

[00153] Depending upon their intended use, particularly for administration to mammalian hosts, the subject peptides may also be modified by attachment to other compounds for the purposes of incorporation into carrier molecules, changing peptide bioavailability, extend or shorten half-life, control distribution to various tissues or the blood stream, diminish or enhance binding to blood components, and the like. The subject peptides may be bound to these other components by linkers which are cleavable or non-cleavable in the physiological environment such as blood, cerebrospinal fluid, digestive fluids, etc. The peptides may be joined at any point of the peptide where a functional group is present, such as hydroxyl, thiol, carboxyl, amino, or the like. Desirably, modification will be at either the N-terminus or the C-terminus. For these purposes, the subject peptides may be modified by covalently attaching polymers, such as polyethylene glycol, polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidine, polyproline, poly(divinyI-ether-co-maleic anhydride), po!y(styrene-c- maleic anhydride), etc. Water soluble polymers, such a polyethylene glycol and polyvinylpyrrolidine are known to decrease clearance of attached compounds from the blood stream as compared to unmodified compounds. The modifications can also increase solubility in aqueous media and reduce aggregation of the peptides.

Peptide Conjugates and Fusion Proteins

[00154] In another aspect, the RDP58 peptide or other useful peptide is preferably conjugated to one or more small molecules for detection and isolation of the peptide, and to target or transport the peptide into specific cells, tissues, and organs. Small molecule conjugates include haptens, which are substances that do not initiate an immune response when introduced by themselves into an animal. Generally, haptens are small molecules of molecular weight less than about 2 kD, and more preferably less that about 1 kD. Haptens include small organic molecules (e.g., p-nitrophenol, digoxin, heroin, cocaine, morphine, mescaline, lysergic acid, tetrahydrocannabinol, cannabinol, steroids, pentamidine, biotin, etc.). Binding to the hapten, for example for purposes of detection or purification, are done with hapten specific antibodies or specific binding partners, such as avidin which binds biotin.

[00155] Small molecules that target the conjugate to specific cells or tissues may also be used. It is known that presence of a biotin-avidin complex increases uptake of such modified peptides across endothelial cells. Linkage of peptides to carbohydrate moieties, for example to a ?-glycoside through a serine residue on the peptide to form a β-0 linked glycoside, enhances transport of the glycoside derivative via glucose transporters (Polt, R. et al. Proc. Natl. Acad. Sci. USA 91 : 7144-7118 (1994); Oh et al. Drug Transport and targeting, In Membrane Transporters as Drug Targets, Amidon, G.L. and Sadee, W. eds., pg 59-88, Plenum Press, New York, 1999). Both of these types of modifications are encompassed within the scope of the present invention.

[00156] The peptides may have attached various label moieties such as radioactive labels and fluorescent labels for detection and tracing. Fluorescent labels include, but are not limited to, fluorescein, eosin, Alexa Fluor, Oregon Green, rhodamine Green, tetramethylrhodamine, rhodamine Red, Texas Red, coumarin and NBD fluorophores, the QSY 7, dabcyl and dabsyl chromophores, BIODIPY, Cy5, etc.

[00157] In one aspect, the peptides are joined to a wide variety of other peptides or proteins for a variety of purposes. The peptides may be linked to other peptides or proteins to provide convenient functionalities for bonding, such as amino groups for amide or substituted amine formation, e.g., reductive amination; thiol groups for thioether or disulfide formation; carboxyl groups for amide formation; and the like. Of particular interest are peptides of at least 2, more usually 3, and not more than ahnut 60 Ivsine αrouDs. Darticularlv oolvlvsines of from about 4 to 20, usually 6 to 18 lysine units, referred to as multiple antigenic peptide system (MAPS), where the subject peptides are bonded to the lysine amino groups, generally at least about 20%, more usually at least about 50%, of available amino groups, to provide a multipeptide product (Butz, S. et al. Pept. Res. 7: 20-23 (1994)). In this way, molecules having a plurality of the subject peptides are obtained where the orientation of the subject peptides is in the same direction; in effect one has a linking group to provide for tail to tail di- or oligomerization.

[00158] In another aspect, other naturally occurring or synthetic peptides and proteins may be used to provide a carrier immunogen for generating antibodies to the subject peptides, where the antibodies serve as reagents for detecting the peptides or for identifying other peptides having a comparable conformation. Suitable carriers for generating antibodies include, among others, hemocyanins (e.g., Keyhole Limpet hemocyanin - KLH); albumins (e.g., bovine serum albumin, ovalbumin, human serum albumin, etc.); immunoglobulins; thyroglobulins (e.g., bovine thyroglobulin); toxins (e.g., diptheria toxoid, tetanus toxoid); and polypeptides such as polylysine, as described above, or polyalanine- lysine. Although proteins are preferred carriers, other carriers, preferably high molecular weight compounds, may be used, including carbohydrates, polysaccharides, lipopolysaccharides, nucleic acids, and the like of sufficient size and immunogenicity. In addition, the resulting antibodies may be used to prepare anti-idiotypic antibodies which may compete with the subject peptides for binding to a target site. These anti-idiotypic antibodies are useful for identifying proteins to which the subject peptides bind.

[00159] In another aspect, the peptides are conjugated to other peptides or proteins for targeting the peptide to cells and tissues, or adding additional functionalities to the peptides. For targeting, the protein or peptide used for conjugation will be selected based on the cell or tissue being targeted for therapy (Lee, R. et al. Arthritis. Rheum. 46: 2109-2120 (2002); Pasqualini, R. Q. J. Nucl. Med. 43: 159-62 (1999); Pasgualini, R. Nature 380: 364-366 (1996); hereby incorporated by reference). For targeting to the central nervous system, suitable carrier proteins include, among others, antibodies against the transferrin receptor (see U.S. Patent No. 5,527,527, hereby incorporated by reference); cationized albumin; met-enkephalin (see U.S. Patent No. 5,442,043, 4902,505, and 4,801 ,575; incorporated by reference); and antibodies to human insulin receptor (see Pardridge, W.M. et al. Pharm. Res. 12: 807-816 (1995 ); incorporated by reference). The proteins may also compromise poly-amino acids including, but not limited to, polyarginine; and polylysine, polyaspartic acid, etc. , which may be incorporated into other polymers, such as polyethylene glycol, for preparation of vesicles or particles containing the conjugated peptides.

[00160] Targeting to the central nervous system is also done by coupling the peptides to conjugates of proteins and small molecules that are readily transported across the blood brain barrier. For instance, anti-transferrin receptor monoclonal antibody 0X26 coupled to streptavidin is selectively transported across the blood brain barrier. Consequently, conjugating the subject peptides to this antibody-streptavidin complex allows delivery of the attached peptide into the brain (Boado, et al. J. Pharma. Sci. 87: 1308-1315 (1998)). [00161] Targeting to tumors may be done using techniques well known in the art. For example, Antibodies with some selectivity for tumor cells, relative to normal cells, are known and may be used by coupling to the subject peptides (see Kyriakos et al., Cancer Res., 52: 835 (1992)). Cell lines for testing the conjugates and monoclonal antibodies (mAbs) useful for making conjugates according to the invention are readily available (see, for example, Kyriakos et al., above; Mattes et al., Cancer (Suppl.) 73: 787 (1994); Ong et al., Molec. Immunol. 30: 1455 (1993); Demignot et al, Cancer Immunol. Immunotherap. 33: 359 (1991 ); AN et al., Cancer Res. 50: 783S (1990); Halpern et al., Cancer Res. 43: 5347 (1983); Anderson-Berg et al., Cancer Res. 47: 1905 (1987), all of which are incorporated herein by reference). mAbs used in the experiments can be mouse IgG antibodies, but humanized and human antibodies fall within the scope of this invention.

[00162] In another aspect, the subject peptides may be expressed in conjunction with other peptides or proteins, so as to be a portion of the polypeptide chain, either internal, or at the N- or C- terminus to form chimeric proteins or fusion proteins. By "fusion polypeptide" or "fusion protein" or "chimeric protein" herein is meant a protein composed of a plurality of protein components that, while typically joined in the native state, are joined by the respective amino and carboxy termini through a peptide linkage to form a continuous polypeptide. Plurality in this context means at least two, and preferred embodiments generally three to twelve components, although more may be used. It will be appreciated that the protein components can be joined directly or joined through a peptide linker/spacer as outlined below.

[00163] Fusion polypeptides may be made to a variety of other peptides or proteins to display the subject peptides in a conformationally restricted form, for targeting to cells and tissues, for targeting to intracellular compartments, tracking the fusion protein in a cell or an organism, and screening for other molecules that bind the peptides. Proteins useful for generating fusion proteins include various reporter proteins, structural proteins, cell surface receptors, receptor ligands, toxins, and enzymes. Exemplary proteins include fluorescent proteins (e.g., Aequoria victoria GFP, Renilla reniformis GFP, Renilla muelleri GFP, luciferases, etc., and variants thereof); -galactosidase; alkaline phosphatase; E. coli. maltose binding protein; coat proteins of filamentous bacteriophage (e.g., minor coat protein, pill, or the major coat protein, pVIII, for purposes of phage display).

[00164] Fusion proteins also encompass fusions with fragments of proteins or other peptides, either alone or as part of a larger protein sequence. Thus, the fusion polypeptides may comprise fusion partners. By "fusion partners" herein is meant a sequence that is associated with the peptide that confers all members of the proteins in that class a common function or ability. Fusion partners can be heterologous (i.e., not native to the host cell) or synthetic (i.e., not native to any cell). The fusion partners include, but are not limited to, a) presentation structures, which provide the subject peptides in a conformationally restricted or stable form; b) targeting sequences, which allow localization of the peptide to a subcellular or extracellular compartment; c) stability sequences, which affects stability or protection from degradation to the peptide or the nucleic acid encoding it; d) linker sequences, which conformationally decouples the oligopeptide from the fusion partner; and e) any combination of the above. [00165] In one aspect, the fusion partner is a presentation structure. By "presentation structure" as used herein is meant a sequence that when fused to the subject peptides presents the peptides in a conformationally restricted form. Preferred presentation structures enhance binding interactions with other binding partners by presenting a peptide on a solvent exposed exterior surface, such as a loop. Generally, such presentation structures comprise a first portion joined to the N-terminus of the peptide and a second portion joined to the C-terminal end of the subject peptide. That is, the peptide of the present invention is inserted into the presentation structures. Preferably, the presentation structures are selected or designed to have minimal biological activity when expressed in the target cells.

[00166] Preferably, the presentation structures maximize accessibility to the peptides by displaying or presenting the peptide on an exterior loop. Suitable presentation structures include, but are not limited to, coiled coil stem structures, minibody structures, loops on ?-turns, dimerization sequences, cysteine linked structures, transglutaminase linked structures, cyclic peptides, helical barrels, leucine zipper motifs, etc.

[00167] In one embodiment, the presentation structure is a coiled-coil structure, which allows presentation of the subject peptide on an exterior loop (see Myszka et al. Biochemistry 33: 2362-2373 (1994)), such as a coiled-coil leucine zipper domain (see Martin et al. EMBO J. 13: 5303-5309 (1994)). The presentation structure may also comprise minibody structures, which is essentially comprised of a minimal antibody complementarity region. The minibody structure generally provides two peptide regions that are presented along a single face of the tertiary structure in the folded protein (see Bianchi et al. J. Mol. Biol. 236: 649-659 (1994); Tramontano et al. J. Mol. Recognit. 7: 9-24 (1994)).

[00168] In another aspect, the presentation structure comprises two dimerization sequences. The dimerization sequences, which can be same or different, associate non-covalently with sufficient affinity under physiological conditions to structurally constrain the displayed peptide; that is, if a dimerization sequence is used at each terminus of the subject oligopeptide, the resulting structure can display the subject peptide in a structurally limited form. A variety of sequences are suitable as dimerization sequences (see for example, WO 99/51625; incorporated by reference). Any number of protein-protein interaction sequences known in the art are useful.

[00169] In a further aspect, the presentation sequence confers the ability to bind metal ions to generate a conformationally restricted secondary structure. Thus, for example, C2H2 zinc finger sequences are used. C2H2 sequences have two cysteines and two histidines placed such that a zinc ion is chelated. Zinc finger domains are known to occur independently in multiple zinc-finger peptides to form structurally independent, flexibly linked domains (see Nakaseko, Y. et al. J. Mol. Biol. 228: 619-636 (1992)). A general consensus sequence is (5 amino acids)-C-(2 to 3 amino acids)-C-(4 to 12 amino acids)-H-(3 amino acids)-H-(5 amino acids). A preferred example would be -FQCEEC-random peptide of 3 to 20 amino acids-HIRSHTG. Similarly, CCHC boxes having a consensus sequence -C- (2 amino acids)-C-(4 to 20 random peptide)-H-(4 amino acids)-C- can be used, (see Bavoso, A. et al. Biochem. Biophys. Res. Commun. 242: 385-389 (1998)). Other examples include (1 ) -VKCFNC-4 to 20 random amino acids-HTARNCR-, based on the nucleocapsid protein P2; (2) a sequence modified from that of the naturally occurring zinc-binding peptide of the Lasp-1 LIM domain (Hammarstrom, A. et al. Biochemistry 35: 12723-32 (1996)); and (3) -MNPNCARCG-4 to 20 random amino acids- HKACF-, based on the NMR structural ensemble 1ZFP (Hammarstrom et al., supra).

[00170] In yet another aspect, the presentation structure is a sequence that comprises two or more cysteine residues, such that a disulfide bond may be formed, resulting in a conformationally constrained structure. That is, use of cysteine containing peptide sequences at each terminus of the subject peptides results in cyclic peptide structures, as described above. A cyclic structure reduces susceptibility of the presented peptide to proteolysis and increases accessibility to its target molecules. As will be appreciated by those skilled in the art, this particular embodiment is particulariy suited when secretory targeting sequences are used to direct the peptide to the extracellular space.

[00171] In another embodiment, the fusion partner is a targeting sequence. Targeting sequences comprise binding sequences capable of causing binding of the expressed product to a predeterimed molecule or class of molecules while retaining bioactivity of the expression product; sequences signaling selective degradation of the fusion protein or binding partners; and sequences capable of constitutively localizing peptides to a predetermined cellular locale. Typical cellular locations include subcellular locations (e.g, Golgi, endoplasmic recticulum, nucleus, nucleoli, nuclear membrane, mitochondria, secretory vesicles, lysoso es) and extracellular locations by use of secretory signals.

[00172] Various targeting sequences are known in the art. Targeting to nucleus is achieved by use of nuclear localization signals (NLS). NLSs are generally short, positively charged domains that direct the proteins in which the NLS is present to the cell's nucleus. Typical NLS sequences include the single basic NLS of SV40 large T antigen (Kalderon et al. Cell 39: 499-509 (1984)); human retinoic acid receptor-^ nuclear localization signal (NF-kB p50 and p65 (Ghosh et al. Cell 62: 1019-1029 (1990)); Nolan et al. Cell 64: 961-999 (1991 )); and the double basic NLS exemplified by nucleoplasmin (Dingwall et al. J. Cell Biol. 107: 641-649 (1988)).

[00173] In another aspect the targeting sequences are membrane anchoring sequences. Peptides are directed to the membrane via signal sequences and stably incorporated in the membrane through a hydrophobic transmembrane domain (designated as TM). The TM segment is positioned appropriately on the expressed fusion protein to display the subject peptide either intracellularly or extracellularly, as is known in the art. Membrane anchoring sequences and signal sequences include, but are not limited to, those derived from (a) class I integral membrane proteins such as IL-2 receptor /i-chain; Hatekeyama et al. Science 244: 551-556 (1989)) and inuslin receptors-chain (Hetekayama et al, supra); (b) class II integral membrane proteins such as neutral endopeptidase (Malfroy et al Biochem. Biophys. Res. Commun. 144: 59-66 (1987)); and (c) type III proteins such as human cytochrome P450 NF25 (Hetekayama et al, supra); and those from CD8, ICAM-2, IL-8R, and LFA-1.

[00174] Membrane anchoring sequences also include the GPI anchor, which results in covalent bond formation between the GPI anchor sequence and the lipid bilayer via a glycosyl-phosphatidylinositol. GPI anchor sequences are found in various proteins, including Thy-1 and DAF (see Homans et al. Nature 333: 269-272 (1988)). Similarly, acylation sequences allow for attachment of lipid moieties, e.q., isoprenylation (i.e., farnesyl and qeranvl-qeranvl; see Farnsworth et al. Proc. Natl. Acad. Sci. USA 91 : 11963-11967 (1994) and Aronheim et al. Cell 78: 949-61 (1994)), myristoylation (Stickney, J.T. Methods Enzymol. 332: 64-77 (2001)), or palmitoylation. In one aspect, the subject peptide will be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as that of a liposome.

[00175] Other intracellular targeting sequences are lysozomal targeting sequences (e.g., sequences in LAMP-1 and LAMP-2; Uthayakumar et al. Cell Mol. Biol. Res. 41 : 405-420 (1995) and Konecki et al. Biochem. Biophys. Res. Comm. 205: 1-5 (1994)); mitochondrial localization sequences (e.g., mitochondrial matrix sequences, mitochondrial inner membrane sequences, mitochondrial intermembrance sequences, or mitochondrial outer membrane sequences; see Shatz, G. Eur. J. Biochem. 165: 1-6 (1987)); endoplasmic recticulum localization sequences (e.g., calreticulin, Pelham, H. R. Royal Soc. London Transactions B: 1-10 (1992); adenovirus E3/19K protein, Jackson et al. EMBO J. 9: 3153-3162 (1990)); and peroxisome localization sequences (e.g., luciferase peroxisome matrix sequence, Keller et al. Proc. Natl. Acad. Sci. USA 4: 3264-3268 (1987)).

[00176] In another aspect, the targeting sequence is a secretory signal sequence which effects secretion of the peptide. A large number of secretory sequences are known to direct secretion of a peptide into the extracellular space when placed at the amino end relative to the peptide of interest, particularly for secretion of a peptide by cells, including transplanted cells. Suitable secretory signals included those found in IL-2 (Villinger et al. J. Immuno. 155: 3946-3954 (1995)), growth hormone (Roskam et al. Nucleic Acids Res. 7: 305-320 (1979)), preproinsulin, and influenza HA protein.

[00177] The fusion partner may further comprise a stability sequence, which confers stability to the fusion protein or the nucleic acid encoding it. Thus, for example, incorporation of glycines after the initiating methionine (e.g., MG or MGG) can stabilize or protect the fused peptide from degradation via ubiquitination as per the N-End rule of Varshavsky, thus conferring increased half-life in a cell.

[00178] Additional amino acids may be added for tagging the peptide for purposes of detection or purification. These sequences may comprise epitopes recognized by antibodies (e.g., flag tags) or sequences that bind ligands, such a metals ions. Various tag sequences and ligand binding sequences are well known in the art. These include, but are not limited to, poly-histidine (e.g., 6xHis tags, which are recognized by antibodies but also bind divalent metal ions); poly-histidine-glycine (poly-his-gly) tags; flu HA tag polypeptide; c-myc tag; Flag peptide (Hopp et al. BioTechnology 6: 1204-1210 (1988)); KT3 epitope peptide; tubulin epitope peptide (Skinner et al. J. Biol. Chem. 266: 15163-12166 (1991 )); and T7 gene 10 protein peptide tag (Lutz-Freyermuth et al. Proc. Natl. Acad. Sci. USA 87: 6363-6397 (1990)).

[00179] Fusion partners include linker or tethering sequences for linking the peptides and for presenting the peptides in an unhindered structure. As discussed above, useful linkers include glycine polymers (G)n where n is 1 to about 7, glycine-serine polymers (e.g., (GS)n, (GSGGS)n and (GGGS)n, where n is at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Preferably, the linkers are glycine or glycine-serine polymers since these amino acids are relatively unstructured, hydrophilic, and are effective for joining segments of proteins and nfintiries. [00180] If desired, various groups are introduced into the peptide during synthesis or during expression, which allows for linking to other molecules or to a surface. Thus, cysteines can be used to make thioethers or cyclic peptides, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like. When cysteine residues are introduced for cyclizing the peptide, formation of disulfide bonds are conducted in the presence of mild oxidizing agents. Chemical oxidants may be used, or the cysteine bearing peptides are exposed to oxygen to form the linkages, typically in a suitable solution such as a aqueous buffer containing DMSO. As described above, lipids may be attached either chemically or by use of appropriate lipidation sequences in the expressed peptide.

[00181] For conjugating various molecules to the peptides of the present invention, functional groups on the peptides and the other molecule are reacted in the presence of an appropriate conjugating (e.g., crosslinking) agent. The type of conjugating or crosslinking agent used will depend on the functional groups, such as primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids being used. Agents may be fixatives and crosslinking agents, which may be homobifunctional, heterobifunctional, or trifunctional crosslinking agents (Pierce Endogen, Chicago, IL). Commonly used fixatives and crosslinking agents include formaldehyde, glutaraldehyde, 1 ,1-bis(diazoacetyl)-2- phenylethane, N-hydroxysuccinimide esters, dissuccimidyl esters, maleimides (e.g., bis-N-maleimido- 1-8-octane), and carbodiimides (e.g., N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide; dicyclohexylcarbodiimide. Spacer molecules comprising alkyl or substituted alkyl chains with lengths of 2 - 20 carbons may be used to separate conjugates. Preferably, reactive functional groups on the peptide not selected for modification are protected prior to coupling of the peptide to other reactive molecules to limit undesired side reactions . By "protecting group" as used herein is a molecule bound to a specific functional group which is selectively removable to reexpose the functional group (see Greene, T.W. and Wuts, P.G.M. Protective Groups in Organic Synthesis (3rd ed.), John Wiley & Sons, Inc., New York, 1999). The peptides may be synthesized with protected amino acid precursors or reacted with protecting groups following synthesis but before reacting with crosslinking agent. Conjugations may also be indirect, for example by attaching a biotin moiety, which can be contacted with a compound or molecule which is coupled to streptavidin or avidin.

[00182] For peptides that have reduced activity in the conjugated form, the linkage between the peptides and the conjugated compound is chosen to be sufficiently labile to result in cleavage under desired conditions, for example after transport to desired cells or tissues. Biologically labile covalent bonds, e.g., imimo bonds and esters, are well known in the art (see U.S. Patent No. 5,108,921, hereby incorporated by reference). These modifications permit administration of the peptides in potentially a less active form, which is then activated by cleavage of the labile bond.

[00183] In the present invention, combinations of fusion partners may be used. Any number of combinations of presentation structures, targeting sequences, rescue sequences, tag sequences and stability sequences may be used with or without linker sequences.

Peptide Preparation and Salts [00184] The RDP58, TCR, or HLA peptides of the present invention may be prepared in a number of ways. Chemical synthesis of peptides are well known in the art. Solid phase synthesis is commonly used and various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc., Foster City, CA; Beckman; etc. Solution phase synthetic methods may also be used, although it is less convenient. By using these standard techniques, naturally occurring amino acids may be substituted with unnatural amino acids, particularly D- stereoisomers, and also with amino acids with side chains having different lengths or functionalities. Functional groups for conjugating to small molecules, label moieties, peptides, or proteins, or for purposes of forming cyclized peptides may be introduced into the molecule during chemical synthesis. In addition, small molecules and label moieties may be attached during the synthetic process. Preferably, introduction of the functional groups and conjugation to other molecules minimally affects the structure and function of the subject peptide.

[00185] The peptides of the present invention may be present in the form of a salt, generally in a salt form which is pharmaceutically acceptable. These include inorganic salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and the like. Various organic salts of the peptide may also be made with, including, but not limited to, acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid, benozic acid, cinnamic acid, salicylic acid, etc.

[00186] Synthesis of the peptides and derivatives thereof may also be carried out by using recombinant techniques. For recombinant production, one may prepare a nucleic acid sequence which encodes a single oligopeptide or preferably a plurality of the subject peptides in tandem with an intervening amino acid or sequence, which allows for cleavage to the single peptide or head to tail dimers. Where methionine or tryptophane is absent, an intervening methionine or tryptophane may be incorporated, which allows for single amino acid cleavage using CNBr or BNPS-Skatole (2-(2- nitrophenylsulfenyl)-3-methyl-3-bromoindolenine), respectively. Alternatively, cleavage is accomplished by use of sequences that are recognized by particular proteases for enzymatic cleavage or sequences that act as self-cleaving sites (e.g., 2A sequences of apthoviruses and cardioviruses; Donnelly, M.L. J. Gen. Virol. 78: 13-21 .(1997); Donnelly, M.L. J. Gen. Virol. 82: 1027- 41 (2001 ), hereby incorporated by reference). The subject peptide may also be made as part of a larger peptide, which can be isolated and the oligopeptide obtained by proteolytic cleavage or chemical cleavage. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. To prepare these compositions, a gene encoding a particular peptide, protein, or fusion protein is joined to a DNA sequence encoding the peptides of the present invention to form a fusion nucleic acid, which is introduced into an expression vector. Expression of the fusion nucleic acid is under the control of a suitable promoter and other control sequences, as defined below, for expression in a particular host cell or organism (see, Sambrook et al., Molecular Biology: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 2001 ; Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1988, updates up to 2002; incorporated by reference). Nucleic Acids, Expression Vectors, and Methods of Introduction

[00187] When the synthesis or delivery of the peptides is via nucleic acids encoding the subject peptides, the nucleic acids are cloned into expression vectors and introduced into cells or a host. The expression vectors are either self-replicating extrachromosomal vectors or vectors that integrate into the host chromosome, for example vectors based on retroviruses, vectors with site specific recombination sequences, or by homologous recombination. Generally, these vectors include control sequences operably linked to the nucleic acids encoding the peptides. By "control sequences" is meant nucleic acid sequences necessary for expression of the subject peptides in a particular host organism. Thus, control sequences include sequences required for transcription and translation of the nucleic acids, including, but not limited to, promoter sequences, enhancer or transcriptional activator sequences, ribosomal binding sites, transcriptional start and stop sequences; polyadenylation signals; etc.

[00188] A variety of promoters are useful in expressing the peptides of the present invention. The promoters may be constitutive, inducible, and/or cell specific and may comprise natural promoters, synthetic promoters (e.g. tTA tetracycline inducible promoters), or hybrids of various promoters. Promoters are chosen based on, among others, the cell or organism in which the proteins are to be expressed, the level of desired expression, and regulation of expression. Suitable promoters are bacterial promoters (e.g., pL I phage promoter, tac promoter, lac lac promoter, etc.); yeast based promoters (e.g., GAL4 promoter, alcohol dehydrogenase promoter, tryptophane synthase promoter, copper inducible CUPI promoter, etc.), plant promoters (e.g., CaMV S35, nopoline synthase promoter, tobacco mosaic virus promoter, etc), insect promoters (e.g., Autographa nuclear polyhedrosis virus, Aedes DNV viral p& and p61, hsp70, etc.), and promoters for expression mammalian cells (e.g., ubiquitin gene promoter, ribosomal gene promoter, ?-globin promoter, thymidine kinase promoter, heat shock protein promoters, and ribosomal gene promoters, etc.), and particularly viral promoters, such as cytomegalovirus (CMV) promoter, simian virus (SV40) promoter, and retroviral promoters.

[00189] By "operably linked" herein is meant that a nucleic acid is placed into a functional relationship with another nucleic acid. In the present context, operably linked means that the control sequences are positioned relative to the nucleic acid sequence encoding the subject peptides in such a manner that expression of the encoded peptide occurs. The vectors may comprise plasmids or comprise viral vectors, for example retroviral vectors, which are useful delivery systems if the cells are dividing cells, or lentiviral and adenoviral vectors if the cells are non-dividing cells. Particularly preferred are self- inactivating retroviral vectors (SIN vectors), which have inactivated viral promoters at the 3'-LTR, thereby permiting control of expression of heterologous genes by use of non-viral promoters inserted into the viral vector (see for example, Hoffman et al. Proc. Natl. Acad. Sci. USA 93: 5185 (1996). As will be appreciated by those in the art, modifications of the system by pseudotyping allows use of retroviral vectors for all eukaryotic cells, particulariy for higher eukaryotes (Morgan, R.A. et al. J. Virol. 67: 4712-21 (1993); Yang, Y. et al. Hum. Gene Ther. 6: 1203-13 (1995)).

[00190] In addition, the expression vectors also contain a selectable marker gene to allow selection of transformed host cells. Generally, the selection will confer a detectable phenotype that enriches for cells containing the expression vector and further permits differentiation between cells that express and do not express the selection gene. Selection genes are well known in the art and will vary with the host cell used. Suitable selection genes included genes that render the cell resistant to a drug, genes that permit growth in nutritionally deficient media, and reporter genes (e.g. ?-galactosidase, fluorescent proteins, glucouronidase, etc.), all of which are well known in the art and available to the skilled artisan.

[00191] There are a variety of techniques available for introducing nucleic acids into viable cells. By "introduced" into herein is meant that the nucleic acid enters the cells in a manner suitable for subsequent expression of the nucleic acid. Techniques for introducing the nucleic acids will vary depending on whether the nucleic acid is transferred in vitro into cultured cells or in vivo into the cells of the intended host organism and the type of host organism. Exemplary techniques for introducing the nucleic acids in vitro include the use of liposomes, Lipofectin®, electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, and biolistic particle bombardment. Techniques for transfer in vivo include direct introduction of the nucleic acid, use of viral vectors, typically retroviral vectors, and liposome mediated transfection, such as viral coated liposome mediated transfection. The nucleic acids expressing the peptides of the present invention may exist transiently or stably in the cytoplasm or stably integrate into the chromosome of the host (i.e., through use of standard regulatory sequences, selection markers, etc.). Suitable selection genes and marker genes are used in the expression vectors of the present invention.

[00192] In some situations, it is desirable to include an agent that targets the target cells or tissues, such as an antibody specific for a cell surface protein or the target cell, a ligand for a receptor on the target cell, a lipid component on the cell membrane, or a carbohydrate on the cell surface. If liposomes are employed, proteins that bind a cell surface protein which is endocytosed may be used for targeting and/or facilitating uptake. These include as non-limiting examples, capsid proteins or fragments thereof tropic for a particular cell types, antibodies for proteins which undergo internalization (see Wu et al. J. Biol. Chem. 262: 4429-4432 (1987); Wagner et al. Proc. Natl. Acad. Sci. USA 87: 3410-3414 (1990)), and proteins that direct localization (e.g., antibody to transferrin receptor for targeting to brain) or enhance in vivo half-life.

[00193] Expression is done in a wide range of host cells that span prokaryotes and eukaryotes, including bacteria, yeast, plants, insects, and animals. The peptides of the present invention may be expressed in, among others, E. coli., Saccharomyces cerevisiae, Saccharomyces pombe, Tobacco or Arabidopsis plants, insect Schneider cells, and mammalian cells, such as COS, CHO, HeLa, and the like, either intracellularly or in a secreted form by fusing the peptides to an appropriate signal peptide. Secretion from the host cell may be done by fusing the DNA encoding the peptide and a DNA encoding a signal peptide. Secretory signals are well known in the art for bacteria, yeast, insects, plants, and mammalian systems. Nucleic acids expressing the peptides may be inserted into cells, for example stem cells for tissue expression or bacteria for gut expression, and the cells transplanted into the host to provide an in vivo source of the peptides.

Purified Peptides [00194] In a preferred embodiment, the RDP58, TCR and HLA peptides of the present invention may be purified or isolated after synthesis or expression. By "purified" or "isolated" is meant free from the environment in which the peptide is synthesized or expressed and in a form where it can be practically used. Thus purified or isolated is meant that the peptide or its derivative is substantially pure, i.e., more than 90% pure, preferably more than 95% pure, and preferably more than 99% pure. The peptides and derivatives thereof may be purified and isolated by way known to those skilled in the art, depending on other components present in the sample. Standard purification methods include electrophoretic, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, size exclusion, reverse phase HPLC, and chromatofocusing. The proteins may also be purified by selective solubility, for instance in the presence of salts or organic solvents. The degree of purification necessary will vary depending on use of the subject peptides. Thus, in some instances no purification will be necessary.

[00195] For the most part, the compositions used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and usually at least about 99.5% by weight, relative to contaminants related to the method of product preparation, the purification procedure, and its intended use, for example with a pharmaceutical carrier for the purposes of therapeutic treatment. Usually, the percentages will be based upon total protein.

Pharmaceutical Formulations, Dosage Forms, Dosages, and Methods of Administration

[00196] The subject compositions, either alone or in combination, may be used in vitro, ex vivo, and in vivo depending on the particular application. In accordance, the present invention provides for administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmacologically effective amount of one or more of the subject peptides, or suitable salts thereof. The pharmaceutical composition may be formulated as powders, granules, solutions, suspensions, aerosols, solids, pills, tablets, capsules, gels, topical cremes, suppositories, transdermal patches, etc.

[00197] As indicated above, pharmaceutically acceptable salts of the peptides is intended to include any art recognized pharmaceutically acceptable salts including organic and inorganic acids and/or bases. Examples of salts include sodium, potassium, lithium, ammonium, calcium, as well as primary, secondary, and tertiary amines, esters of lower hydrocarbons, such as methyl, ethyl, and propyl. Other salts include organic acids, such as acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, salicylic acid, etc.

[00198] As used herein, "pharmaceutically acceptable carrier" comprises any of standard pharmaceutically accepted carriers known to those of ordinary skill in the art in formulating pharmaceutical compositions. Thus, the subject peptides, by themselves, such as being present as pharmaceutically acceptable salts, or as conjugates, or nucleic acid vehicles encoding such peptides, may be prepared as formulations in pharmaceutically acceptable diluents; for example, saline, phosphate buffer saline (PBS), aqueous ethanol, or solutions of glucose, mannitol, dextran, propylene glycol, oils (e.g., vegetable oils, animal oils, synthetic oils, etc.), microcrystalline cellulose, carboxymethyl cellulose, hydroxylpropyl methyl cellulose, magnesium stearate, calcium phosphate, gelatin, polysorbate 80 or the like, or as solid formulations in appropriate excipients. Additionally, the formulations may include bactericidal agents, stabilizers, buffers, emulsifiers, preservatives, sweetening agents, lubricants, or the like. If administration is by oral route, the oligopeptides may be protected from degradation by using a suitable enteric coating, or by other suitable protective means, for example internment in a polymer matrix such as microparticles or pH sensitive hydrogels.

[00199] Suitable formulations may be found in, among others, Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Philadelphia, PA, 1985 and Handbook of Pharmceutical Excipients, 3rd Ed, Kibbe, A.H. ed., Washington DC, American Pharmaceutical Association, 2000; hereby incorporated by reference in their entirety. The pharmaceutical compositions described herein can be made in a manner well known to those skilled in the art (e.g., by means conventional in the art, including mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes).

[00200] Additionally, the peptides may also be introduced or encapsulated into the lumen of liposomes for delivery and for extending life time of the peptide formulations ex vivo or in vivo. As known in the art, liposomes can be categorized into various types: multilamellar (MLV), stable plurilamellar (SPLV), small unilamellar (SUV) or large unilamellar (LUV) vesicles. Liposomes can be prepared from various lipid compounds, which may be synthetic or naturally occurring, including phosphatidyl ethers and esters, such as phosphotidylserine, phosphotidylcholine, phosphatidyl ethanolamine, phosphatidylinositol, dimyristoylphosphatidylcholine; steroids such as cholesterol; cerebrosides; sphingomyelin; glycerolipids; and other lipids (see for example, U.S. Patent No. 5,833,948).

[00201] Cationic lipids are also suitable for forming liposomes. Generally, the cationic lipids have a net positive charge and have a lipophilic portion, such as a sterol or an acyl or diacyl side chain. Preferably, the head group is positively charged. Typical cationic lipids include 1 ,2-dioleyloxy-3- (trimethylamino)propane; N-[1-(2,3,-ditetradecycloxy)propyl]-N,N-dimethyl-N-N- hydroxyethylammonium bromide; N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide; N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride; 3-[N- (N',N'-dimethylaminoethane) carbamoyl] cholesterol; and dimethyldioctadecylammonium.

[00202] Of particular interest are fusogenic liposomes, which are characterized by their ability to fuse with a cell membrane upon appropriate change in physiological condition or by presence of fusogenic component, particularly a fusogenic peptide or protein. In one aspect, the fusogenic liposomes are pH and temperature sensitive in that fusion with a cell membrane is affected by change in temperature and/or pH (see for example, U.S. Patent No. 4,789,633 and 4,873,089). Generally, pH sensitive liposomes are acid sensitive. Thus, fusion is enhanced in physiological environments where the pH is mildly acidic, for example the environment of a lysosome, endosome and inflammatory tissues. This property allows direct release of the liposome contents into the intracellular environment following endocytosis of liposomes (see Mizoue, T. Int. J. Pharm. 237: 129-137 (2002)).

[00203] Another form of fusogenic liposomes comprise liposomes that contain a fusion enhancing aαent. That is, when incorporated into the lioosome or attached to the lioids, the aαents enhance fusion of the liposome with other cellular membranes, thus resulting in delivery of the liposome contents into the cell. The agents may be fusion enhancing peptides or proteins, including hemaggulutinin HA2 of influenza virus (Schoen, P. Gene Ther. 6: 823-832 (1999)); Sendai virus envelope glycoproteins (Mizuguchi, H. Biochem. Biophys. Res. Commun. 218: 402-407 (1996)); vesicular stomatitis virus envelope glycoproteins (VSV-G) glycoprotein (Abe, A. et al. J Virol 72: 6159- 63 (1998)); peptide segments or mimics of fusion enhancing proteins; and synthetic fusion enhancing peptides (Kono, K. et al. Biochim. Biophys. Acta. 1164: 81-90 (1993); Pecheur, E.I. Biochemistry 37: 2361-71 (1998); U.S. Patent No. 6,372,720).

[00204] Liposomes also include vesicles derivatized with a hydrophilic polymer, as provided in U.S. Patent No. 5,013,556 and 5,395,619, hereby incorporated by reference, (see also, Kono, K. et al. J. Controlled Release 68: 225-35 (2000); Zalipsky, S. et al. Bioconjug. Chem. 6: 705-708 (1995)) to extend the circulation lifetime in vivo. Hydrophilic polymers for coating or derivation of the liposomes include polyethylene glycol, polyvinylpyrrolidone, polyvinylmethyl ether, polyaspartamide, hydroxymethyl cellulose, hydroxyethyl cellulose, and the like. In addition, as described above, attaching proteins that bind a cell surface protein which is endocytosed, e.g., capsid proteins or fragments thereof tropic for a particular cell types and antibodies for cell surface proteins which undergo internalization (see Wu et al, supra; Wagner et al., supra), may be used for targeting and/or facilitating uptake of the liposomes to specific cells or tissues.

[00205] Liposomes are prepared by ways well known in the art (see for example, Szoka, F. et al. Ann. Rev. Biophys. Bioeng. 9: 467-508 (1980)). One typical method is the lipid film hydration technique in which lipid components are mixed in an organic solvent followed by evaporation of the solvent to generate a lipid film. Hydration of the film in aqueous buffer solution, preferably containing the subject peptide or nucleic acid, results in an emulsion, which is sonicated or extruded to reduce the size and polydispersity. Other methods include reverse-phase evaporation (see Pidgeon, C. et al. Biochemistry 26: 17-29 (1987); Duzgunes, N. et al. Biochim. Biophys. Acta. 732: 289-99 (1983)), freezing and thawing of phospholipid mixtures, and ether infusion.

[00206] In another preferred embodiment, the carriers are in the form of microparticles, microcapsules, micropheres and nanoparticles, which may be biodegradable or non-biodegradable (see for example, Microencapsulates: Methods and Industrial Applications, Drugs and Phamaceutical Sciences, Vol 73, Benita, S. ed, Marcel Dekker Inc., New York, 1996; incorporated by reference). As used herein, microparticles, microspheres, microcapsules and nanoparticles mean a particle, which is typically a solid, containing the substance to be delivered. The substance is within the core of the particle or attached to the particle's polymer network. Generally, the difference between microparticles (or microcapsules or microspheres) and nanoparticles is one of size. As used herein, microparticles have a particle size range of about 1 to about >1000 microns. Nanoparticles have a particle size range of about 10 to about 1000 nm.

[00207] A variety of materials are useful for making microparticles. Non-biodegradable microcapsules and microparticles include, but not limited to, those made of polysulfones, poly(acrylonitrile-co-vinyl chloride), ethylene-vinyl acetate, hydroxyethylmethacrylate-methyl-methacrylate copolymers. These are useful for implantation purposes where the encapsulated peptide diffuses out from the capsules. In another aspect, the microcapsules and microparticles are based on biodegradable polymers, preferably those that display low toxicity and are well tolerated by the immune system. These include protein based microcapsulates and microparticles made from fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginate or poly-amino acids such as poly-lysine. Biodegradable synthetic polymers for encapsulating may comprise polymers such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly(caprolactone), polydioxanone trimethylene carbonate, polyhybroxyalkonates (e.g., poly(β-hydroxybutyrate)), poly(#-ethyl glutamate), poly(DTH iminocarbony (bisphenol A iminocarbonate), poly (ortho ester), and polycyanoacrylate. Various methods for making microparticles containing the subject compositions are well known in the art, including solvent removal process (see for example, U.S. Patent No. 4,389,330); emulsification and evaporation (Maysinger, D. et al. Exp. Neuro. 141 : 47-56 (1996); Jeffrey, H. et al. Pharm. Res. 10: 362-68 (1993)), spray drying, and extrusion methods.

[00208] Another type of carrier is nanoparticles, which are generally suitable for intravenous administrations. Submicron and nanoparticles are generally made from amphiphilic diblock, triblock, or multiblock copolymers as is known in the art. Polymers useful in forming nanoparticles include, but are limited to, poly(lactic acid) (PLA; see Zambaux et al., J. Control Release 60: 179-188 (1999)), poly(lactide-co-glycolide), blends of poly(lactide-co-glycolide) and polycarprolactone, diblock polymer poly(l-leucine-block-l-glutamate), diblock and triblock poly(lactic acid) (PLA) and poly(ethylene oxide) (PEO) (see De Jaeghere, F. et al., Pharm. Dev. Technol. ;5: 473-83 (2000)), acrylates, arylamides, polystyrene, and the like. As described for microparticles, nanoparticles may be non-biodegradable or biodegradeable. Nanoparticles may be also be made from poly(alkylcyanoacrylate), for example poly(butylcyanoacrylate), in which the peptide is absorbed onto the nanoparticles and coated with surfactants (e.g., polysorbate 80). Methods for making nanoparticles are similar to those for making microparticles and include, among others, emulsion polymerization in continuous aqueous phase, emulsification-evaporation, solvent displacement, and emulsification-diffusion techniques (see Kreuter, J. Nano-particle Preparation and Applications, In Microcapsules and nanoparticles in medicine and pharmacy," (M. Donbrow, ed.), pg. 125-148, CRC Press, Boca Rotan, FL, 1991 ; incorporated by reference).

[00209] Hydrogels are also useful in delivering the subject agents into a host. Generally, hydrogels are crosslinked, hydrophilic polymer networks permeable to a wide variety of drug compounds, including peptides. Hydrogels have the advantage of selective trigger of polymer swelling, which results in controlled release of the entrapped drug compound. Depending on the composition of the polymer network, swelling and subsequent release may be triggered by a variety of stimuli, including pH, ionic strength, thermal, electrical, ultrasound, and enzyme activities. Non-limiting examples of polymers useful in hydrogel compositions include, among others, those formed from polymers of poly(lactide- co-glycolide), poly(N-isopropylacrylamide); poly(methacrylic acid-g-polyethylene glycol); polyacrylic acid and poly(oxypropylene-co-oxyethylene) glycol; and natural compounds such as chrondroitan sulfate, chitosan, gelatin, or mixtures of synthetic and natural polymers, for example chitosan-poly(ethylene oxide). The polymers are crosslinked reversibly or irreversiblv to form αels embedded with the oligopeptides of the present invention (see for example, U.S. Patent No. 6,451 ,346; 6,410,645; 6,432,440; 6,395,299; 6,361 ,797; 6,333,194; 6,297,337 Johnson, O. et al., Nature Med. 2: 795 (1996); incorporated by reference in their entirety).

[00210] In one preferred embodiment, the gel polymers are acrylic acid polymers, preferably carbomers (e.g., carboxypolymethylene), such as Carbopol (e.g., Carbopol 420-430, 475, 488, 493, 910, 934P, 974P, and the like; Brock et al., Pharmacotherapy 14: 430-437 (1994)), which are nonlinear polymers of acrylic acid crosslinked with polyalkenyl polyether. Others types of carbomers include acrylic acids crosslinked with polyfunctional compounds, such as polyallysucrose. In addition to the advantage of hydrating and swelling to a gel, which entraps the subject compounds and limits their release, carbomer gels are mucoadhesive.

[00211] The concentrations of the peptides or nucleic acid encoding therefore will be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering the peptides ex vivo or in vivo for therapeutic purposes, the subject formulations are given at a pharmacologically effective dose. By "pharmacologically effective amount" or "pharmacologically effective dose" is an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease condition, including reducing or eliminating one or more symptoms of the disorder or disease.

[00212] The amount administered to the host will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the host, the manner of administration, the number of administrations, interval between administrations, and the like. These can be determined empirically by those skilled in the art and may be adjusted for the extent of the therapeutic response. Factors to consider in determining an appropriate dose include, but are not limited to, size and weight of the subject, the age and sex of the subject, the severity of the symptom, the stage of the disease, method of delivery of the agent, half-life of the agents, and efficacy of the agents. Stage of the disease to consider include whether the disease is acute or chronic, relapsing or remitting phase, and the progressiveness of the disease. Determining the dosages and times of administration for a therapeutically effective amount are well within the skill of the ordinary person in the art.

[00213] The toxicity and therapeutic efficacy are generally determined by cell culture assays and/or experimental animals, typically by determining a LD50 (lethal dose to 50% of the test population) and ED50 (therapeutically effectiveness in 50% of the test population). The dose ratio of toxicity and therapeutic effectiveness is the therapeutic index. Preferred are compositions, individually or in combination, exhibiting high therapeutic indices. Determination of the effective amount is well within the skill of those in the art, particularly given the detailed disclosure provided herein.

[00214] Generally, in the case where formulations are administered directly to a host, the present invention provides for a bolus or infusion of the subject composition that will be administered in the range of about 0.1-50, more usually from about 1-25 mg/kg body weight of host. The amount will generally be adjusted depending upon the half-life of the peptide where the half life will generally be at if-a-:t ΠΠR minute, more usuallv at least about 10 min, desirably in the range of about 10 min to 12 h. Short half-lives are acceptable, so long as efficacy can be achieved with individual dosages, continuous infusion, or repetitive dosages. Formulations for administration may be presented in unit a dosage form, e.g., in ampules, capsules, pills, or in multidose containers or injectables.

[00215] Dosages in the lower portion of the range and even lower dosages may be employed, where the peptide has an enhanced half-life or is provided as a depot, such as a slow release composition comprising particles, a polymer matrix which maintains the peptide over an extended period of time (e.g., a collagen matrix, carbomer, etc.), use of a pump which continuously infuses the peptide over an extended period of time with a substantially continuous rate, or the like. The host or subject may be any mammal including domestic animals, pets, laboratory animals, primates, particularly humans subjects.

[00216] In addition to administering the subject peptide compositions directly to a cell culture in vitro, to particular cells ex vivo, or to a mammalian host in vivo, nucleic acid molecules (DNA or RNA) encoding the subject peptides may also be administered thereto, thereby providing an effective source of the subject peptides for the application desired. As described above, nucleic acid molecules encoding the subject peptides may be cloned into any of a number of well known expression plasmids (see Sambrook et al., supra) and/or viral vectors, preferably adenoviral or retroviral vectors (see for example, Jacobs et al., J. Virol. 66:2086-2095 (1992), Lowenstein, Bio/Technology 12:1075-1079 (1994) and Berkner, Biotechniques 6:616-624 (1988)), under the transcriptional regulation of control sequences which function to promote expression of the nucleic acid in the appropriate environment. Such nucleic acid-based vehicles may be administered directly to the cells or tissues ex vivo (e.g., ex vivo viral infection of cells for transplant of peptide producing cells) or to a desired site in vivo, e.g. by injection, catheter, orally (e.g., hybrogels), and the like, or, in the case of viral-based vectors, by systemic administration. Tissue specific promoters may optionally be employed, assuring that the peptide of interest is expressed only in a particular tissue or cell type of choice. Methods for recombinantly preparing such nucleic acid-based vehicles are well known in the art, as are techniques for administering nucleic acid-based vehicles for peptide production.

[00217] For the purposes of this invention, the methods of administration are chosen depending on the condition being treated, the form of the subject compositions, and the pharmaceutical composition. Administration of the oligopeptides can be done in a variety of ways, including, but not limited to, cutaneously, subcutaneously, intravenously, orally, topically, transdermally, intraperitoneally, intramuscularly, nasally, and rectally (e.g., colonic administration). For example, microparticle, microsphere, and microencapsulate formulations are useful for oral, intramuscular, or subcutaneous administrations. Liposomes and nanoparticles are additionally suitable for intravenous administrations. Administration of the pharmaceutical compositions may be through a single route or concurrently by several routes. For instance, oral administration can be accompanied by rectal or topical administration to the affected area. Alternatively, oral administration is used in conjunction with intravenous or parenteral injections.

[00218] Delivery of the peptides to the CNS may also rely on disruptions to the blood brain barrier, such as intracranial infusion with hypertonic mannitol solutions. Alternatively, it may be preferable to administer the peptide in combination with agents that increase transport across the blood brain barrier. These compounds have the effect of increasing permeability across the blood brain barrier and may or may not be conjugated to the subject peptides. See U.S. Patent Nos. 5,112,596; 5,268,164 and 5,506,206, incorporated by reference). Administration of a pharmaceutically effective amount to the brain may also be achieved through the olfactory neural pathway, as provided in U.S. Patent No. 6,342,478, hereby incorporated by reference.

[00219] The delivery systems also include sustained release or long term delivery methods, which are well known to those skilled in the art. By "sustained release or" "long term release" as used herein is meant that the delivery system administers a pharmaceutically therapeutic amount of subject compounds for more than a day, preferably more than a week, and most preferable at least about 30 days to 60 days, or longer. Long term release systems may comprise implantable solids or gels containing the subject peptide, such as biodegradable polymers described above; pumps, including peristaltic pumps and fluorocarbon propellant pumps; osmotic and mini-osmotic pumps; and the like. Peristaltic pumps deliver a set amount of drug with each activation of the pump, and the reservoir can be refilled, preferably percutaneously through a port. A controller sets the dosage and can also provides a readout on dosage delivered, dosage remaining, and frequency of delivery. Fluorocarbon propellant pumps utilize a fluorocarbon liquid to operate the pump. The fluorocarbon liquid exerts a vapor pressure above atmospheric pressure and compresses a chamber containing the drug to release the drug. Osmotic pumps (and mini-osmotic pumps) utilize osmotic pressure to release the drug at a constant rate. The drug is contained in an impermeable diaphragm, which is surrounded by the osmotic agent. A semipermeable membrane contains the osmotic agent, and the entire pump is housed in a casing. Diffusion of water through the semipermeable membrane squeezes the diaphragm holding the drug, forcing the drug into bloodstream, organ, or tissue. These and other such implants are particularly useful in treating a disease condition, especially those manifesting recurring episodes or which are progressive in nature, by delivering the oligopeptides of the invention via systemic (e.g., intravenous or subcutaneous) or localized doses (e.g., intracerebroventricular) in a sustained, long term manner.

[00220] In one preferred embodiment, the method of administration is by oral delivery, in the form of a powder, tablet, pill, or capsule. Pharmaceutical formulations for oral administration may be made by combining one or more peptide with suitable excipients, such as sugars (e.g., lactose, sucrose, mannitol, or sorbitol), cellulose (e.g., starch, methyl cellulose, hydroxylmethyl cellulose, carbonxymethyl cellulose, etc.), gelatin, glycine, saccharin, magnesium carbonate, calcium carbonate, polymers such as polyethylene glycol or polyvinylpyrrolidone, and the like. The pills, tablets, or capsules may have an enteric coating, which remains intact in the stomach but dissolves in the intestine. Various enteric coating are known in the art, a number of which are commercially available, including, but not limited to, methacrylic acid-methacrylic acid ester copolymers, polymer cellulose ether, cellulose acetate phathalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, and the like. Alternatively, oral formulations of the peptides are in prepared in a suitable diluent. Suitable diluents include various liquid form (e.g., syrups, slurries, suspensions, etc.) in aqueous diluents such as water, saline, phosphate buffered saline, aαueous ethanol. solutions of sugars (e.g. sucrose, mannitol, or sorbitol), glycerol, aqueous suspensions of gelatin, methyl cellulose, hydroxylmethyl cellulose, cyclodextrins, and the like. As used herein, diluent or aqueous solutions also include infant formula. In some embodiments, lipohilic solvents are used, including oils, for instance vegetable oils, peanut oil, sesame oil, olive oil, corn oil, safflower oil, soybean oil, etc.); fatty acid esters, such as oleates, triglycerides, etc.; cholesterol derivatives, including cholesterol oleate, cholesterol linoleate, cholesterol myristilate, etc.; liposomes; and the like.

[00221] In one embodiment, administration is done rectally. This may use formulations suitable for topical application in the form of salves, tinctures, cremes, or for application into the lumen of the intestine by use of compositions in the form of suppositories, enemas, foams, etc. Suppositories may contain conventional suppository bases such as cocoa butter, carbowaxes, polyethylene glycols, or glycerides, which are solid or semi-solid at room temperature but liquid at body temperature.

[00222] In yet another preferred embodiment, the administration is carried out cutaneously, subcutaneously, intraperitonealy, intramuscularly or intravenously. As discussed above, these are in the form of peptides dissolved or suspended in suitable aqueous medium, as discussed above. Additionally, the pharmaceutical compositions for injection may be prepared in lipophilic solvents, which include, but is not limited to, oils, such as vegetable oils, olive oil, peanut oil, palm oil soybean oil, safflower oil, etc; synthetic fatty acid esters, such as ethyl oleate or triglycerides; cholesterol derivatives, including cholesterol oleate, cholesterol linoleate, cholesterol myristilate, etc.; or liposomes, as described above. The compositions may be prepared directly in the lipophilic solvent or preferably, as oil/water emulsions, (see for example, Liu, F. et al. Pharm. Res. 12: 1060-1064 (1995); Prankerd, R.J. J. Parent. Sci. Tech. 44: 139-49 (1990); U.S. Patent No. 5,651 ,991 ).

[00223] The delivery systems also include sustained release or long term delivery methods, which are well known to those skilled in the art. By "sustained release or" "long term release" as used herein is meant that the delivery system administers a pharmaceutically therapeutic amount of subject compounds for more than a day, preferably more than a week, and most preferable at least about 30 days to 60 days, or longer. Long term release systems may comprise implantable solids or gels containing the subject peptide, such as biodegradable polymers described above; pumps, including peristaltic pumps and fluorocarbon propellant pumps; osmotic and mini-osmotic pumps; and the like. Peristaltic pumps deliver a set amount of drug with each activation of the pump, and the reservoir can be refilled, preferably percutaneously through a port. A controller sets the dosage and can also provides a readout on dosage delivered, dosage remaining, and frequency of delivery. Fluorocarbon propellant pumps utilize a fluorocarbon liquid to operate the pump. The fluorocarbon liquid exerts a vapor pressure above atmospheric pressure and compresses a chamber containing the drug to release the drug. Osmotic pumps (and mini-osmotic pumps) utilize osmotic pressure to release the drug at a constant rate. The drug is contained in an impermeable diaphragm, which is surrounded by the osmotic agent. A semipermeable membrane contains the osmotic agent, and the entire pump is housed in a casing. Diffusion of water through the semipermeable membrane squeezes the diaphragm holding the drug, forcing the drug into bloodstream, organ, or tissue. These and other such implants are particularly useful in treating an inflammatory disease condition, especially those manifesting recurring episodes or which are progressive in nature, by delivering the oligopeptides of the invention via systemic (e.g., intravenous or subcutaneous) or localized doses in a sustained, long term manner.

[00224] The present invention also encompasses the therapeutic combinations disclosed herein in the form of a kit or packaged formulation. A kit or packaged formulation as used herein includes one or more dosages of a subject peptide, and salts thereof, in a container holding the dosages together with instructions for simultaneous or sequential administration to a patient. For example, the package may contain the peptides along with a pharmaceutical carrier combined in the form of a powder for mixing in an aqueous solution, which can be ingested by the afflicted subject. Another example of packaged drug is a preloaded pressure syringe, so that the compositions may be delivered colonically. The package or kit includes appropriate instructions, which encompasses diagrams, recordings (e.g., audio, video, compact disc), and computer programs providing directions for use of the combination therapy. The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.

[00225] U.S. provisional patent application Serial No. 60/431 ,420 is expressly incorporated herein in its entirety by reference. PCT application serial number PCT/US98/07231 , filed 10 April 1998, is expressly incorporated herein in its entirety by reference. US patent application serial no. 08/838,916, filed 11 April 1997 is expressly incorporated herein in its entirety by reference. US patent application serial no. 09/028,083 filed 23 February 1998 is expressly incorporated herein in its entirety by reference. All other publications and patent applications cited in this specification are herein incorporated in their entirety by reference.

EXPERIMENTAL

Angiogensis Assays and VEGFR Expression Assays

[00226] Angiogenesis assay was performed using a human vascular endothelial cells (HUVEC) co- culture assay kit (TCS cell works Ltd. Buckingham, UK) according to manufacturer's protocols. Briefly, cells were stimulated with VEGF-σ (5ng/ml) in the presence or absence of RDP58 (50 mM). Media was changed on day 4, 7 and 9. At day 11 cells were fixed and immunostained with anti-von Willebrand's factor antibodies (TCS cell works Ltd. Buckingham, UK).

[00227] RDP58 inhibited the ability of human vascular endothelial cells (HUVECs) to form a capillarylike network of tubules in vitro. HUVECs were co-cultured and treated with VEGF-c and/or RDP58. At day 11 , cells were fixed and immunostained with antibodies to a tubule-specific marker, von Willebrand's factor. Control human vascular endothelial cells formed capillary-like network of tubules. VEGF-σ caused a more pronounced formation of an anastomosing network of tubules. Suramine, a known inhibitor of angiogenesis, as well as RDP58, significantly inhibited both VEGF-σ-stimulated and unstimulated formation of capillary-like structures. [00228] RDP58 decreased LPS-induced expression of Flt-1 mRNA. Stimulation of RAW264J cells with LPS for 4 hours caused an 18-fold increase in the steady state mRNA levels of Flt-1. This increase was more pronounced (150-fold) in the cells treated for 24 hours. RDP58 abrogated this LPS-mediated induction of Flt-1 mRNA by 80% and 98% at 4 and 24 hours, respectively.

Matrigel Angiogensis Assays

[00229] Matrigel (Becton Dickson, Bedford, MA) was thawed on ice overnight and spread evenly over each well (750 ml) of a 6-well plate. The plates were incubated for 30 min at 37°C to allow Matrigel to solidify. HUVECS (1X10⁵) were suspended in 750 ml of EGM with VEGF (10ng/ml) and spread on Matrigel plates and then incubated at 37°C for 6 hours. Images were captured using a Zeiss Axiocam (Carl Zeiss Inc, Thomwood, NY). To determine antiangiogenic effects of RDP58, we tested the ability of RDP58 to inhibit HUVEC attachment, migration or differentiation into tubules in Matrigel assay. In this assay, untreated control HUVECs differentiated into tubule like structures. Both RDP58 as well as RDP2044 significantly inhibited tubule formation at 25μm and 50μM levels in vitro. In contrast, RDP2121 which is a derivative of RDP58 did not show any effect on HUVEC differentiation in vitro. RDP58 used has amino acid sequence r-nle-nle-nle-r-nle-nle-G-y wherein nle is norieucine, and all amino acids are D-stereoisomers except for glycine. RDP2044 is an RDP58 peptide having the sequence R-nle-nle-nle-R-nle-Nle-nle-G-y wherein nle is norieucine and all amino acids identified by lower case are D-isomers, and those identified by upper case (R, Nle, G) are L-stereoisomers. RDP2121 is an RDP58 peptide having the sequence nle-nle-nle-nle-r-r-nle-nle-r-G-r wherein nle is norieucine, and all amino acids are D-isomers except for G.

In Vivo Angiogensis Assay

[00230] C57BL/6 mice received 109 pfu i.v. of antiangiogenic adenoviruses 2 days before assay. Mice were anesthetized with avertin i.p. and the eye was treated with topical proparacaine-HCI (Allergan, Irvine, CA). Hydron/sucralfate pellets containing VEGF-A165 (R & D Systems) and RDP58, RDP2044, and RDP2121 were implanted into a corneal micropockel at 1 mm from the limbus of both eyes under an operating microscope (Zeiss) followed by intrastomal linear keratotomy by using a microknife (Medtroni Xomed, Jacksonville, FL). A corneal micropocket was dissected toward the limbus with a von Graefe knife #3 (2 X 30 mm), followed by pellet implantation and application of topical erythromycin. After 5 days, neovascularization was quantitated by using a slit lamp biomicroscope and the formula 2 p X (vessel length/10) X (clock hours). The results were as follows:

[00231] Results shown are the average of vascularized area in 8 corneas. RDP2044 inhibit VEGF induced angiogenesis by 30% and 50% respectively. In contrast, RDP58 and RDP2121 do not have a significant effect on angiogenesis in this assay (in this assay <35% is not significant). RDP58 used has amino acid sequence r-nle-nle-nle-r-nle-nle-G-y wherein nle is norieucine, and all amino acids are D-stereoisomers except for glycine. RDP2044 is an RDP58 peptide having the sequence R-nle- nle-nle-R-nle-Nle-nle-G-y wherein nle is norieucine and all amino acids identified by lower case are D- isomers, and those identified by upper case (R, Nle, G) are L-stereoisomers. RDP2121 is an RDP58 peptide having the sequence nle-nle-nle-r-r-nle-nle-r-G-r wherein nle is norieucine, and all amino acids are D-isomers except for G.

RDP58 Effects on VEGF-induced Phosphorylation of Akt

[00232] HUVEC cells in EGM were plated on type I collagen or fibronectin coated 96 well plates, after 24 hrs media was exchanged with EBM. After overnight culture, fresh EBM containing VEGF and/or RDP58 peptides added and cells were harvested at 15 and 30 min. Cells were lysed in buffer (50mM HEPES pH 7.5, 150mM NaCI, 5mM EDTA, 1 % NP-40) containing protease inhibitor (Roche, Mannheim, Germany) and phospatase inhibitor cocktails I and II (Sigma St. Louis, MO) for 20 min on ice. The cell lysates were centrifuged at 10,000g for 10 min at 4°C and proteins were quantitated using the BCA assay kit (Pierce, Rockford, CA). Equal amounts of total protein was run on 12% Novex Tris-Glycine gels (Invitrogen, Carlsbad, CA), transferred onto PVDF membranes (Sigma St. Louis, MO) and western blotting was done according to manufacturer suggested protocols using rabbit anti-phospho PKB/Akt1 antibodies (Biosource, Camarillo, CA) specific for threonine 308 and serine 473. The blots were incubated with anti-rabbit-HRP (Amersham Biosciences, Piscataway, NJ) secondary antibodies and developed using ECL plus reagent (Amersham Biosciences, Piscataway, NJ). To normalize for loading differences, blots were stripped and probed with rabbit b-actin (Sigma St. Louis, MO). The blots were scanned by Epson Expression 1680 scanner using the Epson TWAIN Pro software. VEGF induced the phosphorylation of Akt1 on serine 473 but not on threonine 308 residue. Both RDP58 and RDP2044 reduced the VEGF-mediated phosphorylation at serine 473. In contrast, RDP2121 had little or no effect on the level of Akt1 phosphorylation at either residue. RDP58 used has amino acid sequence r-nle-nle-nle-r-nle-nle-G-y wherein nle is norieucine, and all amino acids are D-isomers except for glycine. RDP2044 is an RDP58 peptide having the sequence R-nle-nle-nle-R-nle-Nle-nle-G-y wherein nle is norieucine and all amino acids identified with lower case letters are D-isomers, and those identified with capital letters (R, Nle, G) are L-isomers. RDP2121 is an RDP58 peptide having the sequence nle-nle-nle-nle-r-r-nle-nle-r-G-r wherein nle is norieucine, and all amino acids are D-isomers except for G.

RDP58 Effects on MMP Expression

[00233] Application of an LPS-soaked ligature around second left maxillary molars of adult rats in an in vivo model of periodontitis increased MMP9 expression in the rat peridontium. Local administration of RDP58 decreased the LPS-induced expression of MMP9 to nearly undetectable, control levels.

[00234] In cultured cell lines, RDP58 inhibits induction of MMP9 and MMP2. Tumor cell lines are cultured in MMP9 and/or MMP2 inducing media in the presence or absence of RDP58. Protein samples are prepared from the cultures and run for western blot analysis with anti-MMP9 and anti- MMP2 antibodies. Results show that RDP58 inhibits MMP9 and MMP2 induction in tumor cells.

Mechanism of RDP58 Action [00235] RAW cells (mouse macrophage cell line) were stimulated with LPS (1ug/ml), TNFσ (100 ng/ml), RANKL (250 ng/ml), IL-1 ? (100 ng/ml) or IL-18 (100 ng/ml) in the presence or absence of RDP58 (50 uM) for 30 min, after which total protein was isolated, run on protein gels, transferred to PVDF membranes and the membranes were probed with anti-phospho p38MAPK or anti-phospho- JNK1 ,2 antibodies (Sigma). Subsequently the membranes were stripped and reprobed with anti-total p38MAPK or anti-total JNK1.2 antibodies (Sigma) to normalize for loading differences. All stimuli increased the phosphorylation of p38MAPK and JNK1 ,2. RDP58 reduced the phosphorylation of p38MAPK and JNK1 ,2 caused by all stimuli used.

[00236] RAW cells were transiently transfected with NF-κB- and AP1-luciferase reporter constructs to assay NF-κB and AP1 activity, respectively. The transfected cells were stimulated with LPS (1ug/ml), TNFer (100 ng/ml), RANKL (250 ng/ml), IL-1jff (100 ng/ml) or IL-18 (100 ng/ml) in the presence or absence of RDP58 (50 uM) for 48 hrs, after which cells were lysed and luciferase activity was measured. All conditions stimulated NF-kB and AP1 activity. RDP58 reduced the induction of NF-kB and AP1 activity in all conditions.

[00237] THP1 cells (human monocyte cell line) and Jurkat E6 cells (human T-cell line) were stimulated with LPS (1 ug/ml) or PMA (1 OOpg/ml) + PHA (5ug/ml) respectively for 30 min, in the presence or absence of RDP58 (50 uM) after which total protein was isolated, run on protein gels, transferred to PVDF membranes and the membranes were probed with anti-phospho antibodies for the specified proteins. Subsequently the membranes were stripped and reprobed with anti-total p38MAPK, anti-total JNK1 ,2 or anti-actin antibodies to normalize for loading differences. All stimuli increased the phosphorylation of p38MAPK and JNK1.2. RDP58 reduced the phosphorylation of P38MAPK and JNK1 ,2 caused by all stimuli.

[00238] RAW cells and Jurkat E6 cells were transiently transfected with NF-κB- and AP1 -luciferase reporter constructs to assay NF-κB and AP1 activity, respectively. The transfected cells were stimulated with LPS (1 ug/ml) in the presence or absence of RDP58 (50 uM) for 48 hrs, after which cells were lysed and luciferase activity was measured. LPS stimulated NF-kB and AP1 activity. RDP58 reduced the LPS-induced activation of NF-kB and AP1.

[00239] CaCo2 cells (intestinal epithelial cell line) was matured for 72 hrs with IFN after which the cells were stimulated with LPS (1 ug/ml) in the presence or absence of RDP58 (50 uM) for 30 min, after which total protein was isolated, run on protein gels, transferred to PVDF membranes and the membranes were probed with anti-phospho p38MAPK or anti-phospho-JNK1 ,2 antibodies (Sigma). Subsequently the membranes were stripped and reprobed with anti-total p38MAPK or anti-total JNK1.2 antibodies (Sigma) to normalize for loading differences. LPS increased the phosphorylation of p38MAPK and JNK1 ,2. RDP58 reduced the LPS-induced phosphorylation of p38MAPK and JNK1.2.

[00240] CaCo2 cells were matured for 72 hrs with IFN after which the cells were stimulated with LPS (1 ug/ml) in the presence or absence of RDP58 (50 uM) for 24 hrs, after which the TNFσ released by these cells in the supernatant was measured by ELISA. LPS induced the production of TNFσ. RDP58 reduced the LPS-induced production of TNFσ.

[00241] BEAS-2b cells (human bronchial epithelium cell line) were stimulated with PMA+Histamine, TNF_7+IL4+IFN , or by neutrophil elastase in the presence or absence of RDP58 (50 uM) after which total protein was isolated, run on protein gels, transferred to PVDF membranes and the membranes were probed with anti-phospho ERK1.2, anti-phospho p38MAPK or anti-phospho-JNK1,2 antibodies (Sigma). Subsequently the membranes were stripped and reprobed with anti-total ERK1 ,2 anti-total p38MAPK or anti-total JNK1.2 antibodies (Sigma) to normalize for loading differences. All stimuli increased the phosphorylation of ERK1 ,2, p38MAPK and JNK1 ,2. RDP58 reduced the phosphorylation of ERK1.2, p38MAPK and JNK1.2 caused by all stimuli.

[00242] BEAS-2b cells were transiently transfected with NF-κ:B- and AP1 -luciferase reporter constructs to assay NF-κB and AP1 activity, respectively. The transfected cells were stimulated with PMA+Histamine, TNFσ+IL4+IFN or by neutrophil elastase in the presence or absence of RDP58 (50 uM) for 48 hrs, after which cells were lysed and luciferase activity was measured. All stimuli induced NF-kB activity, while AP1 activity was only induced by PMA+Histamine. RDP58 reduced NF-kB activation by all stimuli and reduced AP1 activation by PMA+Histamine.

[00243] THP1 cells were stimulated with LPS (1 ug/ml) in the presence or absence of RDP58 (50 uM) for 10 min, after which total protein was isolated and protein complexes were pulled down either with anti-TLR4, anti-MyD88, anti-IRAK or anti-TRAF6 antibodies. Subsequently the protein complexes were run on gels, transferred to PVDF membranes and the membranes were probed with antibodies against IRAK, TRAF6 and MyD88 to determine their presence in the complex. All stimuli increased the presence of IRAK, TRAF6 and MyD88 in complexes with each other and with TLR4. RDP58 reduced complex formation.

[00244] THP1 cells were stimulated with LPS (1 ug/ml) in the presence or absence of RDP58 (50 uM) for 10 min, after which total protein was isolated and TRAF6 was immuno-precipitated with anti- TRAF6 antibodies. Subsequently the immuno-precipitated protein was run on gels, transferred to PVDF membranes and the membranes were probed with anti-phosphoserine or anti- phosphothreonine antibodies to determine the phosphorylation status of the IP protein. The membranes were stripped and reprobed with anti-TRAF6 antibody to normalize for IP differences. LPS increased the Ser-Thr phosphorylation of TRAF6, while RDP58 inhibited the TRAF6 phosphorylation stimulated by LPS.

[00245] THP1 cells were stimulated with LPS (1 ug/ml) in the presence or absence of RDP58 (50 uM) for 10 min or 30 min, after which total protein was isolated and protein complexes were pulled down either with anti-IRAK or anti-TRAF6 antibodies. Subsequently the protein complexes were used in kinase reactions with myelin basic protein (a general ser-thr kinase substrate) as a substrate to determine the ser-thr kinase activity in the complexes. At both time points LPS increased the ser-thr kinase activity of both TRAF6 and IRAK complexes, while RDP58 completely inhibited the kinase activity in these complexes stimulated by LPS. Effects of RDP58 on Protein Kinase Phosphorylation and Activation

[00246] Jurkat E6 cells were stimulated for 5 min, 15 min, 30 min or 1 hr with PMA (100ng/ml) and PHA (5mg/ml). THP-1 cells were matured with 100ng/ml PMA for 16 hrs, after which they were stimulated with LPS (1 mg/ml). Total cell protein was isolated, quantitated and assayed by Western blot analysis with phospho-specific antibodies. Increased phosphorylation of PLCγ, Pyk, FAK, c-src, paxillin, Akt, JNK1 ,2, ERK1 ,2, and p38MAPK was observed at different time-points after stimuli. In these cell models, RDP58 was able to inhibit the induced phosphorylation and activation of a number of intracellular signal mediators, including PLCγ, Pyk, FAK, c-src, paxillin, Fyn, Akt and the stress- activated protein kinases p38 and JNK, but not of ERK1.2. Note that RDP58 did inhibit ERK1.2, activation in other cell models.

[00247] Immunohistochemistry with aπti-TLR antibodies and antibodies recognizing labeled RDP58 revealed that RDP58 colocalizes with TLR2 and TLR4.

[00248] Time course immunohistochemical analysis (5 min, 15 min, 30 min, 1 hour) using antibodies recognizing labeled RDP58 revealed that RDP58 is internalized in CaC02 cells and THP-1 cells.

[00249] Transcription factor activity regulation by RDP58. Expression assays in THP1 cells demonstrated that RDP58 inhibits p65, p50, c-rel, c-fos, and c-jun induction by combination of PMA and LPS. Expression assays in Jurkat E6 cells demonstrated that RDP58 inhibits p65, p50, c-rel, c- fos, and c-jun induction by combination of PMA and LPS. Luciferase reporter assays in RAW cells demonstrated that RDP58 inhibits NF-κB and AP1 activity induced by LPS. Luciferase reporter assays in Jurkat E6 cells demonstrated that RDP58 inhibts NF- B and AP1 activity induced by PMA and PHA.

[00250] Effects of RDP58 on constitutively active signal transduction mediators in macrophages. Using Elk-luciferase, Jun-luciferase, and CHOP-luciferase reporter constructs in RAW cells and Jurkat E6 cells, RDP58 was not able to decrease the transcriptional activity induced by constitutively active MEK1 , MEK3, MEKK, src, Rho, Ras, cdc42, or Rac in these cells.

[00251] Effects of RDP58 on constitutively active signal transduction mediators in T cells. Using Jun- luciferase and CHOP-luciferase reporter constructs in Jurkat E6 cells, RDP58 was not able to decrease the transcriptional activity induced by constitutively active MEK1 , MEK3, MEKK, src, Rho, Ras, cdc42, or Rac in these cells.

[00252] CaCo2 cells and THP1 cells were stimulated with LPS (1 ug/ml) in the presence or absence of RDP58 (50 uM) for 10 min, after which total protein was isolated and protein complexes were pulled down either with anti-TLR4, anti-MyD88, anti-IRAK or anti-TRAF6 antibodies. Subsequently the protein complexes were run on gels, transferred to PVDF membranes and the membranes were probed with anti-TRAF6. LPS induced TRAF6 association with TLR4, MyD88, and IRAK. The LPS- induced associations with TRAF6 were reduced by RDP58.

Claims

We claim:

1. A method for decreasing the vascularization of a cell population in vivo, comprising providing an RDP58 composition to the vicinity of said cell population in vivo, wherein vessels are present in said vicinity of said cell population in vivo, and wherein said RDP58 composition decreases angiogenesis in said vicinity of said cell population.

2. The method of claim 1 , wherein said cell population expresses an MMP, and further comprising decreasing expression of said MMP in said cell population by contacting said cell population with said RDP58 composition.

3. The method of claim 2, wherein said MMP is MMP9 or MMP2.

4. The method of claim 1, wherein said cell population is present in a tumor.

5. The method of claim 4, wherein said tumor expresses an MMP, and further comprising decreasing expression of said MMP in said tumor.

6. The method of claim 5, wherein said MMP is MMP9 or MMP2.

7. The method of claim 4, wherein said tumor is a metastatic tumor.

8. The method of claim 1 , wherein said RDP58 composition comprises a peptide having the sequence r-nle-nle-nle-r-nle-nle-nle-G-y.

9. The method of claim 8, wherein all amino acids of said peptide are D-stereoisomers with the exception of G, which is an L-stereoisomer.

10. The method of claim 8, wherein said peptide has the sequence R-nle-nle-nle-R-nle-Nle-nle-G-y, wherein residues identified by upper case are L-stereoisomers, and residues identified by lower case are D-stereoisomers.

11. A method for inhibiting the growth of a tumor, comprising: (i) providing an RDP58 composition to the vicinity of said tumor, wherein blood vessels are present in said vicinity of said tumor, and wherein said RDP58 composition decreases angiogenesis in said vicinity of said tumor.

12. The method of claim 11 , wherein said tumor expresses an MMP, and further comprising decreasing expression of said MMP in said tumor by contacting said tumor with said RDP58 composition.

13. The method of claim 11 , wherein said MMP is MMP2 or MMP9.