WO2013158617A1 - Endostatin peptides and use thereof - Google Patents

Abstract

This invention relates to peptides that bind to and inhibit vascular endothelial growth factor receptor 3 (VEGFR-3). The invention also provides methods and compositions for inhibiting VEGF-C binding to VEGFR-3, and inhibiting VEGFR-3 -mediated cell growth and migration. The methods and compositions can be used for inhibiting lymphangiogenesis and treating lymphangiogenesis-related disease.

Description

ENDOSTATIN PEPTIDES AND USE THEREOF

[0001] This application claims the benefit of priority to U.S. Provisional Patent

Application Serial No. 61/624,851, filed April 16, 2012, the disclosure of which is

incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grant numbers

EY021886, EY10101 and EY01792 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] A number of vascular endothelial growth factors (VEGFs) have been identified that play a role in vasculogenesis and angiogenesis. Of these, VEGF-C has been reported to regulate both angiogenesis and lymphangio genesis. Lymphangiogenesis, the formation of lymphatic vessels, is important in cancer metastasis. See e.g., U.S. 7,611,711. Penetration of lymphatic vessels by tumor cells and migration through the lymphatic vessels to regional lymph nodes and ultimately to other parts of the body is a common step of lymphatic metastasis. One VEGF-C receptor, vascular endothelial growth factor receptor 3 (VEGFR-3), is expressed on all endothelial cells during early embryogenesis and restricted to lymphatic vessels later in life.

[0004] Endostatin is an angiogenesis inhibitor that inhibits VEGF- and beta fibroblast growth factor (bFGF)-induced vascular endothelial cell migration and proliferation in vitro. Endostatin also reduces VEGF- and bFGF-induced tumor progression in vivo. Endostatin is a 20-kDa proteolytic fragment of the C-terminal non-collagenous domain (NC-1) of Collagen XVIII. Endostatin is a tightly packed globular protein having two pairs of disulfide bonds (Becker et al., 2006, Fertil Steril. 85:71-7). The formation of the disulfide bonds is important in maintaining the native conformation, stability, and activity of endostatin.

[0005] In endothelial or tumor cell lines, endostatin inhibits angiogenesis by binding to specific cell surface receptors and interfering with growth factor signaling (Abdollahi et al., 2004, Mol. Cell 13:649-63). Putative endostatin receptors include VEGFR-2 and -3, integrins a5 and aV, and glypican-1 and -4. Activation of these receptors by endostatin induces endothelial cell apoptosis and inhibits endothelial cell proliferation and migration. [0006] Other collagen XVIII proteolytic fragments that comprise endostatin have also been shown to possess anti-angiogenic properties. For example, neostatin 7, which is cleaved from the C-terminus of collagen XVIII by the protease MMP-7 and is 60 amino acids longer (at the N-terminus) than endostatin, inhibits bFGF -induced corneal neovascularization

(Kojima et al, 2008, FEBB Lett., 582:2515-20). Nostatin 14, which is cleaved from the C- terminus of collagen XVIII by MMP-14 and is 14 amino acids longer at the N-terminus than endostatin, also has been shown to inhibit bFGF -induced angiogenesis (Chang et al, 2005, FEBS Lett., 579:3601-6).

[0007] The domain(s) in endostatin involved in receptor binding or important for the antitumor and antiangiogenesis effects are still not well characterized. It has been reported that the N-terminus 27-amino acid peptide of endostatin that contains a zinc-binding domain is responsible for its antitumor, antimigration and antipermeability activities (Tjin et al., 2005, Cancer Res. 65:3656-63). The disulfide bond formed between Cys 135 and Cys 165 near the C-terminus of endostatin may be important in maintaining the antitumor activity of endostatin. (Tijan et al, supra). Although endostatin has been considered as a promising cancer therapeutic, the results of clinical trials using endostatin as a cancer treatment were not conclusive. Thus, a more effective endostatin therapeutic is needed.

SUMMARY OF THE INVENTION

[0008] The invention provides reagents and methods for inhibiting endothelial cell growth and treating VEGFR-3 -mediated diseases, disorders and conditions. In one aspect, the invention provides isolated peptides wherein the peptide binds to vascular endothelial growth factor receptor 3 (VEGFR-3) and inhibits VEGFR-3 activity. In certain embodiments, the peptides are endostatin peptides. In certain particular embodiments, the peptide of the invention is no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27 or 26 amino acids long. In certain other particular embodiments, the peptide consists of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO: 22.

[0009] In another aspect, the invention provides isolated peptides of endostatin

comprising an amino acid sequence of the general formula

Xl-X2-X3-X4-Cys-X5-X6-X7-X8-X9-X10-Xl l-Cys-X12-X13-X14-Ser (SEQ ID NO: 12), wherein XI is Arg, Lys, Gin, Asn, or Ser; X2 is Ala, Val, Leu, Cys or He; X3 is Ala, Val, Leu, Gly or He; X4 is Ser, Thr, Ala, Cys, or Gly; X5 is His, Asn, Gin, Lys, Cys or Arg; X6 is His, Asn, or Gin; X7 is Ser, Ala, Glu, Thr, or Cys; X8 is Tyr, Glu, Trp, Phe, Thr or Ser; X9 is He, Gly, Ser, Leu, Val, Met, Ala, Phe or Norleucine; XI 0 is Val, Leu, He, Met, Phe, Ala, or

Norleucine; XI 1 is He, Leu, Gin, Val, Met, Ala, Phe or Norleucine; XI 2 is He, Met, Leu, Val, Ala, Phe or Norleucine; X13 is Glu, Asn, or Asp; and X14 is Asn, Thr or Gin. In each particular embodiment of these peptides, the peptide binds to vascular endothelial growth factor receptor 3 (VEGFR-3) and inhibits VEGFR-3 activity. In certain particular

embodiments, XI is Arg, Lys or Ser; X2 is Ala or Cys; X3 is Ala or Gly; X4 is Ser or Gly; X5 is His or Cys; X6 is Asn or His; X7 is Ser, Glu or Ala; X8 is Tyr or Glu; X9 is He, Gly or Ser; XI 0 is Val or Leu; XI 1 is Leu or Gin; XI 2 is He or Met; XI 3 is Glu or Asn; and XI 4 is Asn or Thr. In certain other particular embodiments, the peptides comprises the general formula Leu-Glu-Gln-Xl-X2-X3-X4-Cys-X5-X6-X7-X8-X9-X10-Xl l-Cys-X12-X13-X14-Ser-Phe- Met-Thr-Ser-Phe-Ser-Lys (SEQ ID NO: 13). In certain other particular embodiments, the peptides comprises the general formula Leu-Gly-Gln-Xl-X2-X3-X4-Cys-X5-X6-X7-X8-X9- X10-X11-Cys-X12-X13-X14-Ser-Phe-Met-Thr- Ala-Ser-Lys (SEQ ID NO: 14). In certain particular embodiments, the peptide of the invention is no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27 or 26 amino acids long. In certain other particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In certain other particular embodiments, the peptide consists of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0010] In further embodiments, the invention provides peptides that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identical or similar to the peptide having the amino acid sequence of SEQ ID NO:5. In further embodiments, the invention provides peptides that are at least 85%>, 86%>, 87%>, 88%>, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identical or similar to the peptide having the amino acid sequence of SEQ ID NO:6. In yet further embodiments, the invention provides peptides that are at least 85%>, 86%>, 87%>, 88%>, 89%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identical or similar to the peptide having the amino acid sequence of SEQ ID NO:22.

[0011] In certain particular embodiments, either or both Cys residues of any one of the general formula or either or both Cys residues of the sequence set forth in SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 22 are involved in disulfide bond formation. In certain embodiments, the aforementioned cysteine residues form an intra-molecular disulfide bond; while in other embodiments, either or both of the aforementioned cysteine residues form one or more inter-molecular disulfide bonds. Peptides linked by one or more intermolecular disulfide bonds can function as a dimer, trimer or multimer, all of which are contemplated to fall within the scope of the invention.

[0012] All the peptides of the invention can be further modified. In certain embodiments, the peptide is modified at the N-terminus, the C-terminus, any of the amino acid side chain or any combination thereof. In certain embodiments, the modification increases the stability and half-life of the peptide, and/or provides a way for the peptide to be conveniently and efficiently detected, or for any other advantageous purpose. In certain particular

embodiments, the peptide of the invention is modified at the N-terminus by an acetyl group and/or modified at the C-terminus by an amide group. In certain particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:5, wherein the N-terminus is modified by an acetyl group and the C-terminus is modified by an amide group (SEQ ID NO: 19). In certain other particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:6, wherein the N-terminus is modified by an acetyl group and the C- terminus is modified by an amide group (SEQ ID NO:20). In yet certain other particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:22, wherein the N-terminus is modified by an acetyl group and the C-terminus is modified by an amide group (SEQ ID NO:27).

[0013] In a further aspect, the invention provides methods of detecting VEGFR-3 in a cell comprising the steps of (a) contacting the cell with the isolated peptide of the invention, wherein the peptide is conjugated to a detectable label; and (b) detecting a signal from the label. Suitable detection methods are well known in the art including without limitation immunological assays, radiometric assays and enzymatic assays. In certain particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:5. In certain other particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:6. In yet certain other particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:22.

[0014] In a further aspect, the invention provides a pharmaceutical composition comprising the peptide of the invention and a pharmaceutical acceptable carrier, excipient or diluent. In certain optional embodiments, the pharmaceutical composition further comprises one or more anti-angiogenesis agents and/or cytotoxic agents, including without limitation angiostatin, prolactin, and IFN-a.

[0015] In yet another aspect, the invention provides methods of inhibiting proliferation of a cell that expresses VEGFR-3 comprising the step of contacting the cell with the isolated peptide of the invention or the pharmaceutical composition of the invention in an amount effective to inhibit proliferation of the VEGFR-3 -expressing cell. In a related aspect, the invention provides methods of inhibiting migration of a cell that expresses VEGFR-3 comprising the step of contacting the cell with the isolated peptide of the invention or the pharmaceutical composition of the invention in an amount effective to inhibit migration of the VEGFR-3 -expressing cell. In another related aspect, the invention provides methods of inhibiting VEGFR-3 -mediated proliferation and migration of a cell comprising the step of contacting the cell with the isolated peptide of the invention or the pharmaceutical composition of the invention in an amount effective to inhibit VEGFR-3 -mediated proliferation or migration of the cell. In yet another related aspect, the invention provides methods of inhibiting VEGF-C binding to VEGFR-3 in a cell comprising the step of contacting the cell with the isolated peptide of the invention or the pharmaceutical composition of the invention in an amount effective to inhibit binding of VEGF-C to

VEGFR-3. In certain particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:5. In certain other particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:6. In yet certain other particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:22.

[0016] In accordance with the above-referenced aspects, suitable target cells include without limitation, endothelial cells, lymphatic endothelial cells, vascular endothelial cells, endocardium cells, endothelial progenitor cells, or hematopoietic progenitor cells.

[0017] In an additional aspect, the invention provides methods of inhibiting angiogenesis or lymphangio genesis in a mammal in need thereof comprising the step of administering to the mammal a pharmaceutical composition of the invention in an amount effective to inhibit angiogenesis or lymphangiogenesis. In certain particular embodiments, the mammal is suffering from a disease or disorder, non-limiting examples thereof that include immune and non-immune inflammation, rheumatoid arthritis, Crohn's disease, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, loss of vision due to invasion of blood vessel, corneal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, solid tumors, tumor metastases, leukemias, angiofibromas, hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyogenic granulomas, Osier- Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, lymphedema, transplant rejection, hemophiliac joints, or wound granulation. Applicable tumor or tumor metastases includes without limitation colon tumors, liver tumors, spleen tumors, kidney tumors, lymph node tumors, gastrointestinal tract tumors, pancreatic tumors, stomach tumors, blood cell tumors, lymphatic vessel tumors, brain tumors, lung tumors, breast tumors, endometrium tumors, prostate tumors, skin tumors, bone marrow tumors, or head and neck tumors or metastases thereof. In certain particular embodiments, the mammal is a human.

[0018] In yet another aspect, the invention provides methods of treating an angiogenesis- related or lymphangiogenesis-related disease in a mammal in need thereof comprising the step of administering to the mammal the pharmaceutical composition of the invention in an amount effective to treat the angiogenesis-related or lymphangiogenesis-related disease. Applicable angiogenesis-related or lymphangiogenesis-related disease includes without limitation immune and non-immune inflammation, rheumatoid arthritis, Crohn's disease, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, loss of vision due to invasion of blood vessel, corneal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, solid tumors, tumor metastases, leukemias, angiofibromas, hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyogenic granulomas, Osier-Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, lymphedema, transplant rejection, hemophiliac joints, or wound granulation. In certain particular embodiments, the mammal is a human.

[0019] Specific embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows a photographic image of western blot analysis of a pull-down assay using an anti-VEGFR-3 antibody to pull down VEGFR-3 after the receptor had been incubated with recombinant VEGF-C at indicated concentrations in the presence of a GST- endostatin-containing fragment.

[0021] Figure 2A shows a comparison of the amino acid sequences from the C-terminus of endostatin and the cysteine loops of VEGF-C and -D. Figure 2B depicts a photograph of western blot analysis showing that both mEP (mammalian endostatin peptide, SEQ ID NO:5, lane 1) and mVEGF-C (mouse VEGF-C, lane 2) bound to recombinant VEGFR-3 in a pulldown assay.

[0022] Figure 3 shows results demonstrating that cysteine-to-alanine substitutions within the mEP cysteine loop abolished binding of the peptide to recombinant VEGFR-3. Figure 3 A shows the sequences of synthetic peptides containing Cys-to-Ala substitutions as indicated. Figure 3B shows the results of circular dichroism analysis of wild type and mutant endostatin peptides. Figure 3C depicts the results of MALDI-TOF of mEP showing a single peak with an m/z ratio of 3163.25. Figure 3D shows a photograph of western blot analysis representing the result of protein pull-down assays using an anti-VEGFR-3 antibody to pull down

VEGFR-3 after the receptor had been incubated with 50 μg mEP (lane 1), mEP-CA (lane 2), mEP-AC (lane 3), or mEP-AA (lane 4) and recombinant VEGFR-3.

[0023] Figure 4A depicts a representative sensorgram of fitted kinetic data showing the interaction of mEP with various concentrations of VEGFR-3 -Fc by surface plasmon resonance (SPR). Different concentrations of VEGFR-3 are indicated on the right. Figure 4B shows the fitted curve as the solid line super-imposed on the responses.

[0024] Figure 5 A shows a bar graph depicting inhibition of human lung lymphatic endothelial cells (hLECs) proliferation by mEP as determined by bromodeoxyribouridine (BrdU) incorporation assay. Figure 5B shows microphoto graphs depicting inhibition of VEGF-C-stimulated hLECs migration by mEP.

[0025] Figure 6 shows a photograph image of western blot analysis of a pull-down assay using an anti-VEGFR-3 antibody to pull down VEGFR-3 after the receptor had been incubated with an endostatin fragment.

[0026] Figure 7 shows a photograph image of western blot analysis of a pull-down assay using an anti-VEGFR-3 antibody to pull down VEGFR-3 after the receptor had been incubated with a mouse VEGF-A, VEGF-C, placental growth factor ( PLGF) mouse or an endostatin fragment. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0027] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0028] Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press) and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA).

[0029] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "an isolated peptide" means one or more isolated peptides.

[0030] All embodiments disclosed herein can be combined unless the context clearly dictates otherwise.

[0031] In one aspect, the invention provides isolated peptides comprising the amino acid sequence LEQKAASCHNSYIVLCIENSFMTSFSK (SEQ ID NO:5),

LGQSAASCHHAYIVLCIENSFMTASK (SEQ ID NO:6), or KAASCHNSYIVLCI (SEQ ID No. 22) wherein the peptide binds to vascular endothelial growth factor receptor 3 (VEGFR-3) and inhibits VEGFR-3 activity.

[0032] In another aspect, the invention provides isolated peptides of endostatin

comprising an amino acid sequence of the general formula Xl-X2-X3-X4-Cys-X5-X6-X7- X8-X9-X10-X11-Cys-X12-X13-X14-Ser (SEQ ID NO: 12), wherein XI is Arg, Lys, Gin, Asn, or Ser; X2 is Ala, Val, Leu, Cys or He; X3 is Ala, Val, Leu, Gly or He; X4 is Ser, Thr, Ala, Cys, or Gly; X5 is His, Asn, Gin, Lys, Cys or Arg; X6 is His, Asn, or Gin; X7 is Ser, Ala, Glu, Thr, or Cys; X8 is Tyr, Glu, Trp, Phe, Thr or Ser; X9 is He, Gly, Ser, Leu, Val, Met, Ala, Phe or Norleucine; XI 0 is Val, Leu, He, Met, Phe, Ala, or Norleucine; XI 1 is He, Leu, Gin, Val, Met, Ala, Phe or Norleucine; XI 2 is He, Met, Leu, Val, Ala, Phe or Norleucine; XI 3 is Glu, Asn, or Asp; and X14 is Asn, Thr or Gin. In each of these embodiments of this aspect of the invention, the peptide binds to vascular endothelial growth factor receptor 3 (VEGFR-3) and inhibits VEGFR-3 activity.

[0033] The binding of the peptides of the invention to VEGFR-3 can be detected and analyzed by any suitable method known in the art. In certain embodiments, binding can be analyzed using an antibody-based method such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence microscopy, immunohistochemistry or a pull-down assay. For example, in a binding reaction a peptide of the invention, optionally a detectably labeled embodiment thereof, is incubated with an antibody specific for a receptor and the presence of the receptor in the reaction detected by the antibody indicates binding of the peptide to the receptor. As another example, binding can also be analyzed by a pull-down assay, which is well-known in the art and further exemplified in the example section. In one variation of the assay, a receptor can be incubated with a peptide conjugated to a solid substrate such as an agarose bead. The receptor that binds to the peptide can be isolated from the rest of the reaction by separating (or pulled down) the solid substrate from the rest of the reaction. It is understood that variations of such immunology-based binding assay are well known in the art, and are all contemplated for use in detecting the binding of the peptides of the invention to the receptor. For example, an antibody specific for the peptide (or specific for an antigenic moiety conjugated to the peptide) can also be used for analyzing the binding of the peptide to the receptor. In certain embodiments, the peptide is conjugated with an epitope tag or a fusion moiety that is recognizable by an antibody. Such epitope tags and fusion moieties are well known in the art and include without limitation a (poly)His tag, a GST tag, an Fc fusion or a GFP fusion.

[0034] In certain particular embodiments, either the peptide or the receptor can be immobilized on a solid matrix such as a 96-well plate, an agarose bead or the like. In certain embodiments, binding of peptides to a receptor is analyzed by detecting one or more detectable labels conjugated to either the peptides or the receptor or both. Such labels include without limitation radiolabels, dyes, fluorescent labels, enzymes that generate colorimetric or fluorometric signals, chemiluminescent labels, enzymatic substrates, or members of a binding pair (for example, biotin and avidin) or the like. The use of such labels is well known in the art and is described for example in U.S. 3,850,752, U.S. 3,996,345, U.S.

3,939,350, and U.S. 4,277,437. Alternatively, binding can also be analyzed by surface plasmon resonance (SPR) or a pull down assay as exemplified below in the Examples section. The binding affinity of the peptides of the invention to VEGFR-3 can be determined by any suitable method known in the art, including without limitation SPR.

[0035] As described in more detail in the Examples section, the inventors of the instant application unexpectedly discovered that mutations/substitutions of either or both Cys residues in the peptide sequences of SEQ ID NO: 5 or SEQ ID NO: 6 or either or both Cys residues indicated in the general formula (SEQ ID NO: 12) abolished binding of the peptides to VEGFR-3. Without being bound by any specific mechanisms, in certain particular embodiments, the aforementioned cysteine residues stabilize the secondary structure of the endostatin peptide and are likely important for the activity of the endostatin peptide. In certain embodiments, the aforementioned cysteine residues form an intramolecular disulfide bond within the peptide of the invention. In certain other embodiments, either or both cysteine residues form one or more intermolecular disulfide linkages with another (identical or different) peptide of the invention. The stability of the peptides and secondary structure analysis of the peptides can be determined by any suitable methods known in the art. For example, secondary structure of a polypeptide can be determined spectroscopically, including without limitation circular dichroism analysis, infrared spectroscopy or nuclear magnetic resonance (NMR). It is understood that peptide variants with conservative amino acid substitutions will likely preserve the secondary structure and activity of the peptides.

[0036] The term "polypeptide" as used herein refers generally to a single linear chain of amino acids. The terms "peptide" and "fragment" refer to a linear chain of amino acids usually comprising no more than 100 amino acids, no more than 80 amino acids, no more than 70 amino acids, no more than 50 amino acids, no more than 27 amino acids or no more than 26 amino acids long. In addition, the term "fragment" as used herein also refers to peptide that is short of the full length protein. Thus, an endostatin peptide refers to a fragment of the full-length endostatin protein. In certain embodiments, the terms "peptide" and "fragment" specifically encompass an endostatin peptide or fragment that binds to VEGFR-3 and inhibits VEGFR-3 activity, including without limitation peptides having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO: 22. Thus, the term "peptides of the invention," "inhibitory peptides of the invention," or "endostatin peptides of the invention" refers to peptides described herein that bind to VEGFR-3 and inhibits

VEGFR-3 activity.

[0037] The term "protein" as used herein refers to one or more polypeptides generally comprising no more than 50 amino acids or no more than 100 amino acids long. In addition, a protein can comprise a single chain (e.g., a monomer), or more than one chain (i.e., a dimer, trimer or multimer of polypeptides that are linked by one or more covalent or non-covalent bonds). The terms "protein" and "polypeptide" are sometimes used interchangeably in this art and throughout this application. A polypeptide, peptide or protein may also comprise one or more chemical modifications known in the art and/or described herein.

[0038] A protein, polypeptide or peptide can be produced by naturally-occurring organisms and specifically non-recombinant cells, or by genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or sequences that have deletions, additions, and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass the endostatin protein, or species thereof that have deletions, additions, and/or substitutions of one or more amino acids of endostatin and that retain at least one functional property of the endostatin protein. In certain embodiments, the endostatin protein is a mammalian endostatin protein, and particularly, human or mouse endostatin protein.

[0039] The term "naturally-occurring" or "native" as used herein refers to an object that can be found in nature, for example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a source in nature and which has not been modified by man. The term "naturally occurring" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by man. Similarly, "recombinant," "non-naturally occurring" or "non-native" as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.

[0040] The term "identity," as known in the art, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as the case may be, as determined by comparing the sequences thereof. In the art, "identity" also means the degree of sequence relatedness between nucleic acid molecules or

polypeptides, as the case may be, as determined by the match between polymers comprising two or more nucleotide or two or more amino acid sequences. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms").

[0041] The term "similarity" is used in the art with regard to a concept relating to identity as defined herein, but in contrast to "identity," "similarity" refers to a measure of relatedness that includes both identical matches and conservative substitution matches. For example, if two polypeptide sequences have 10/20 identical amino acids, and the remainder are all non- conservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% (15/20).

Therefore, in cases where there are conservative substitutions, the percent similarity between two polypeptides will be higher than the percent identity between those two polypeptides.

[0042] Identity and similarity of related nucleic acids and polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk, A.M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, (Smith, D.W., ed.), 1993, Academic Press, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, PART 1, (Griffin, A.M., and Griffin, H.G., eds.), 1994, Humana Press, New Jersey; von Heinje, G., SEQUENCE

ANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.), 1991, M. Stockton Press, New York; Carillo et al, 1988, SIAMJ. Applied Math., 48: 1073; and Durbin et al, 1998, BIOLOGICAL SEQUENCE ANALYSIS, Cambridge University Press.

[0043] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al, 1984, Nucl. Acid. Res., 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al, 1990, J. Mol. Biol, 215:403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, MD 20894; Altschul et al, 1990, supra). The well-known Smith Waterman algorithm may also be used to determine identity.

[0044] In certain embodiments, the parameters for a polypeptide sequence comparison include the following: Algorithm: Needleman et al, 1970, J. Mol. Biol, 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al, 1992, supra; Gap Penalty: 12; Gap Length Penalty: 4; Threshold of Similarity: 0. The GAP program may be useful with the above parameters. For nucleotide sequences, parameters can include a gap penalty of 50 and a gap length penalty of 3, which is a penalty of 3 for each symbol in each gap. In certain embodiments, the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

[0045] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See IMMUNOLOGY-A SYNTHESIS, 2nd Edition, (E. S. Golub and D. R. Gren, Eds.), Sinauer Associates: Sunderland, MA, 1991, incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the invention. Examples of unconventional amino acids include: 4- hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ- N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[0046] Naturally occurring residues may be divided into classes based on common side chain properties:

1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, He;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe

[0047] Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

[0048] Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced, inter alia, into homologous regions of the molecule, or into the non-homologous regions of the molecule. [0049] In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);

asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0050] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et ah, 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included.

[0051] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0052] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as "epitopic core regions." [0053] A skilled artisan will be able to determine using well-known techniques suitable sequence variants of the polypeptides set forth herein. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In other embodiments, the skilled artisan can identify residues and portions of the molecules that are conserved among similar polypeptides. In further embodiments, even amino acid residues important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity thereof or without adversely affecting the polypeptide structure.

[0054] In certain embodiments, the inhibitory peptides of the invention comprise one or more modifications including without limitation phosphorylation, glycosylation,

hydroxylation, sulfonation, amidation, acetylation, carboxylation, palmitylation, pegylation, introduction of non-hydrolyzable bonds, and disulfide formation. The modification may improve the stability and/or activity of the peptides. In certain embodiments, the peptide comprises an intramolecular disulfide bond formed between two cysteine residues of the peptide. In certain other embodiments, the cysteine residues form one or more intermolecular disulfide bonds between two or more peptides to create dimers, timers or multimers.

[0055] In certain particular embodiments, the inhibitory peptides are stable and resistant to protease degradation. The inhibitory peptides can be made or modified to increase stability, shelf life, protease resistance and/or to reduce toxicity by one or more post translational modifications and/or other chemical modifications. In certain particular embodiments, the peptides of the invention have end modifications such as an amide at the C- terminus and/or an acetyl group at the N-terminus. The peptides can also be modified to form peptide derivatives by forming covalent or noncovalent linkage(s) to other chemical moieties. Covalent linkage can be made by linking the chemical moieties to functional groups on the side chains of the peptide or at the N- and/or C-terminus. In certain particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:5, wherein the N-terminus is modified by an acetyl group and the C-terminus is modified by an amide group (SEQ ID NO: 19). In certain other particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:6, wherein the N-terminus is modified by an acetyl group and the C-terminus is modified by an amide group (SEQ ID NO:20). In yet certain other particular embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:22, wherein the N-terminus is modified by an acetyl group and the C-terminus is modified by an amide group (SEQ ID NO:27).

[0056] The peptides of the invention can be synthesized recombinantly using

recombinant DNA techniques. Thus, in another aspect, the invention provides

polynucleotides that encode the inhibitory peptides of the invention. In a related aspect, the invention provides vectors, particularly expression vectors that comprise the polynucleotides encoding the peptides of the invention. In certain embodiments, the vector provides replication, transcription and/or translation regulatory sequences that facilitate recombinant synthesis of the desired peptides in a eukaryotic cell or prokaryotic cell. Accordingly, the invention also provides host cells for recombinant expression of the peptides and methods of harvesting and purifying the peptides produced by the host cells. Production and purification of recombinant polypeptide is routine practice to one of skilled in the art. The peptides can be purified by any suitable methods known in the art including without limitation gel filtration and affinity purification. When the peptides of the invention are produced in the form of a fusion protein, the fusion moiety (or the epitope tag) can optionally be cleaved off using a protease before further analysis.

[0057] Alternatively, the inhibitory peptides of the invention can be advantageously synthesized by any of the chemical synthesis techniques known in the art, particularly solid- phase synthesis techniques, for example, using commercially-available automated peptide synthesizers. See, for example, Stewart and Young, 1984, SOLID PHASE PEPTIDE SYNTHESIS, 2d. ed., Pierce Chemical Co.; Tarn et al, 1983, J. Am. Chem. Soc, 105:6442; Merrifield, 1986, Science, 232: 341-347; and Barany and Merrifield, THE PEPTIDES, Gross and

Meienhofer, eds, Academic Press, New York, 1-284; Barany et al, Int. J. Peptide Protein Res., 30, 705-739 (1987); and U.S. Pat. No. 5,424,398), each incorporated herein by reference.

[0058] The peptides of the invention bind to VEGFR-3 and can be used as a reagent for detecting VEGFR-3 in a sample. Thus, in another aspect, the invention provides methods of detecting VEGFR-3 in a sample or a cell comprising the step of contacting the sample or cell with the peptide of the inventions and detecting the binding of the peptide with VEGFR-3. In certain embodiments, the sample is a cell and the detection of VEGFR-3 in the cell indicates that the cell is likely a lymphatic or blood vessel endothelial cell. The detection of VEGFR-3 in a cell, especially in a cell surrounding a tumor, can also have diagnostic relevance in that the presence, quantity and distribution of VEGFR-3 in and around a tumor can be indicative of the progression and aggressiveness of the tumor. Such analysis can be extremely important and valuable in determining the most effective cancer treatment. Thus, in a related aspect, the invention provides methods of optimizing cancer treatment in a mammal in need thereof comprising the steps of obtaining a tumor biopsy sample from the mammal, detecting the presence or amount of VEGFR-3 in the tumor sample using the peptide of the invention, and increasing the dosage of cancer treatment when VEGFR-3 is detected in the tumor sample.

[0059] In a further aspect, the invention provides methods of inhibiting VEGFR-3 - mediated proliferation or migration of a cell, or methods of inhibiting VEGF-C binding to VEGFR-3 in a cell, comprising the step of contacting the cell with the isolated peptide of the invention or the pharmaceutical composition of the invention in an amount effective to inhibit VEGFR-3 -mediated proliferation or migration of the cell. Both in vitro and in vivo applications of the methods are contemplated.

[0060] In yet another aspect, the invention provides methods of treating an angiogenesis- related or lymphangiogenesis-related disease in a mammal in need thereof comprising the step of administering to the mammal the pharmaceutical composition of the invention in an amount effective to treat the angiogenesis-related or lymphangiogenesis-related disease. "Treating" a mammal having a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disease; (b) arresting the development of the disease or disorder; (c) inhibiting worsening of the disease or disorder; (d) limiting or preventing recurrence of the disease or disorder in patients that have previously had the disease or disorder; (e) causing regression of the disease or disorder; (f) improving or eliminating the symptoms of the disease or disorder; and (g) improving survival. In certain preferred embodiments, the mammal is a human and the disease or disorder is an

angiogenesis-related or lymphangiogenesis-related disease or disorder.

[0061] As used herein, the term "amount effective," "effective amount" or a

"therapeutically effective amount" refers to an amount of the peptide of the invention or a pharmaceutical composition comprising the inventive peptide sufficient to achieve the stated desired result, for example, treating or limiting development of angiogenesis-related or lymphangiogenesis-related disease. The amount of the peptide which constitutes an

"effective amount" or "therapeutically effective amount" may vary depending on the severity of the disease, the condition, weight, or age of the patient to be treated, the frequency of dosing, or the route of administration, but can be determined routinely by one of ordinary skill in the art. A clinician may titer the dosage or route of administration to obtain the optimal therapeutic effect. Typical dosages range from about 0.1 μg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In certain embodiments, the dosage may range from 0.1 μg/kg up to about 100 mg/kg, or 1 μg/kg up to about 100 mg/kg, or 5 μg/kg up to about 100 mg/kg.

[0062] The pharmaceutical compositions provided herein can be specially formulated for oral administration in solid or liquid form or for intravenous injection. Optimal

pharmaceutical compositions can be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18₄ Edition, (A. R. Gennaro, ed.), 1990, Mack Publishing Company.

[0063] The peptides of the invention can be incorporated in a conventional systemic dosage form, such as a tablet, capsule, soft gelatin capsule, elixir or injectable formulation. The dosage forms may also include the necessary physiologically acceptable carrier material, excipient, lubricant, buffer, surfactant, antibacterial, bulking agent (such as mannitol), antioxidants (ascorbic acid or sodium bisulfite) or the like.

[0064] Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as

ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine,

polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;

monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt- forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, Triton, trimethamine, lecithin, cholesterol, or tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, or sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, Id.

[0065] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Pharmaceutical compositions can comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.

Pharmaceutical compositions of the invention may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, Id.) in the form of a lyophilized cake or an aqueous solution. Further, the peptides of the invention may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0066] Administration routes for the pharmaceutical compositions of the invention include orally, through injection by intravenous, intraperitoneal, intracerebral (intra- parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. The

pharmaceutical compositions may be administered by bolus injection or continuously by infusion, or by implantation device. The pharmaceutical composition also can be

administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration. [0067] The pharmaceutical compositions of the invention can be delivered parenterally. When parenteral administration is contemplated, the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired compound identified in a screening method of the invention in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the compound identified in a screening method of the invention is formulated as a sterile, isotonic solution, appropriately preserved. Preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the product which may then be delivered via a depot injection. Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation. Implantable drug delivery devices may be used to introduce the desired molecule.

[0068] The compositions may also be formulated for inhalation. In these embodiments, the peptides of the invention is formulated as a dry powder for inhalation, or inhalation solutions may also be formulated with a propellant for aerosol delivery, such as by nebulization. Pulmonary administration is further described in the International Application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins and is incorporated herein by reference.

[0069] The pharmaceutical compositions of the invention can be delivered through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The peptides of the invention that are administered in this fashion may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. A capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the peptides of the invention disclosed herein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

[0070] These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0071] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, this may be accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration may be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0072] Once the pharmaceutical composition of the invention has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0073] The present invention also provides kits for producing a single-dose

administration unit. Kits according to the invention may each contain both a first container having a dried peptide of the invention and a second container having an aqueous formulation, including for example single and multi-chambered pre-filled syringes (e.g., liquid syringes, lyosyringes or needle-free syringes).

[0074] Any and every embodiment described above applies to any and every aspect of the invention, unless the context clearly indicates otherwise. All embodiments within and between different aspects can be combined unless the context clearly dictates otherwise

[0075] The Examples, which follow, are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention. EXAMPLES

Example 1

VEGF-C interferes with the binding of endostatin-containing fragments to VEGFR-3

[0076] VEGF-C competition assays for binding to VEGFR-3 were performed against endostatin as follows. Binding of endostatin-containing fragments to recombinant VEGFR- 3-Fc was analyzed in the presence of various concentrations of VEGF-C. GST-endostatin- containing fragments, VEGFR-3, and a range of concentrations of VEGF-C were incubated and receptor-ligand complexes analyzed using a pull-down assay. The results (set forth in Fig. 1) showed that VEGF-C competed with GST-endostatin-containing fragments for binding to recombinant VEGFR-3-Fc. A GST-endostatin containing fragment was able to bind VEGFR-3-Fc in the absence of VEGF-C and at low concentrations of VEGF-C (Fig. 1, lanes 1 and 2). However, this binding was diminished when VEGF-C was added to the assay mixture at higher concentrations (Fig. 1, lane 3). These results demonstrated that VEGF-C competed with endostatin in binding to VEGFR-3.

Example 2

A Conserved Cysteine-Rich Motif in Endostatin-Peptide and VEGF-C

[0077] To understand the basis of the competitive binding of endostatin and VEGF-C to VEGFR-3, the primary sequences of the 28-kDa endostatin-containing fragment of collagen XVIII, VEGF-C, and VEGF-D were compared using EMBOSS Needle-Pairwise Sequence Alignment (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). The alignment identified a conserved motif [(R/K)xxxCxNSxx(V/L)xCxxxS] (SEQ ID NO: 15) between residues 8-16 of the C-terminal end of endostatin and VEGF-C (Figure 2A). Western blot analysis confirmed that both synthesized mEP (mammalian endostatin peptide) and VEGF-C peptides bound recombinant VEGFR-3 (Figure 2B). Thus, the conserved motif present in the mEP peptide and VEGF-C suggests that this motif is important for competitive binding of the peptides to VEGFR-3. Example 3

Conserved Cysteine Residues of mEP Are Required for VEGFR-3 Binding

[0078] To address whether this conserved motif as disclosed in Example 2 above was required for endostatin binding to VEGFR-3, binding of wild-type peptide or peptides containing alanine substitutions for either one or both conserved cysteine residues, designated as mEP (SEQ ID NO:5), mEP-CA (SEQ ID NO:7), mEP-AC (SEQ ID NO:8), and mEP-AA (SEQ ID NO:9), was tested in a pull down assay. See Figure 3A. The sequence of the peptides are shown below:

mEP: LEQKAASCHNSYIVLCIENSFMTSFSK (SEQ ID NO:5);

mEP-CA: LEQKAASCHNSYIVLAIENSFMTSFSK (SEQ ID NO:7);

mEP-AC: LEQKAASAHNSYIVLCIENSFMTSFSK (SEQ ID NO: 8); and

mEP-AA: LEQKAASAHNSYIVLAIENSFMTSFSK (SEQ ID NO:9)

[0079] The peptides were synthesized by solid-phase synthesis on a Symphony Peptide Synthesizer (Protein Technologies, Tucson, AZ) using Fmoc-RinkAmide-MBHA resin (AnaSpec, San Jose, CA; Novabiochem, Gibbstown, NJ), and purified by reversed-phase CI 8 HPLC (McKern et al, 1993, Biomed Chromatogr. 7 : 15-9).

[0080] For these pull down assays, a total of 10 mg of each peptide was coupled to Sepharose CL4B beads in 10 ml of slurry resin. Recombinant mouse VEGFR-3-Fc proteins (R&D Systems, Minneapolis, MN) were pre-absorbed against Sepharose 4B in RIPA buffer for 1 h at 4°C. Unconjugated Sepharose beads bound to VEGFR-3-Fc was removed by centrifugation, and then 50 ng VEGFR-3-Fc in RIPA buffer (50 mM Tris-HCl, pH 7.4, and 150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxycholate, and 1% NP-40) was incubated with 50 μg mEP-conjugated Sepharose 4B beads for 2 h at 4°C. Pull-down assays were performed using 50 μg of Sepharose-conjugated peptide. Pull-down complexes were washed three times with PBS and analyzed by western blotting with rat anti -mouse VEGFR-3 antibody (1 : 1 ,000, eBiosciences, San Diego, CA) and horseradish peroxidase-conjugated anti-rat IgG antibody (1 :20,000, Cell Signaling Technology, Danvers, MA). The ECL detection system

(Amersham, Piscataway, NJ) detected bound VEGFR-3-Fc.

[0081] Analysis of these peptides using the receptor pull-down assay set forth above indicated that binding of the mEP to VEGFR-3 was abolished when either Cys was substituted with alanine (Figure 3D). The structures of the wild type and Cys-to-Ala- substituted peptides were analyzed using circular dichroism. In these assays, mEP, mEP-CA, -AC, and -AA peptides were dissolved separately in a 50:50 solution of acetonitrile and water to a final concentration of 200 μg/ml and scanned with a J-710 spectropolarimeter (Jasco, Easton, MD) at 25°C calibrated with dlO camphor sulfonic acid in 1-mm path-length fused-quartz cuvette. The resulting spectra were corrected with scans of the solvent mixture and smoothed. As shown in Figure 3B, ellipticity between 205 and 225 nm shown for the wild-type peptide was diminished by either or both of the Cys-to-Ala substitutions of the peptide. Specifically, the pronounced trough observed between -205 nm and -225 nm indicated secondary structure in the mEP peptide; and the substitution peptides mEP-CA, - AC and - AA lacked this feature. The results indicated that the cysteine residues are important for maintaining the secondary structure of the endostatin peptides.

[0082] For mass spectrometry (MS) analysis, peptides were spotted and dried onto a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) target for analysis in positive -ion reflector mode with delayed extraction over the m/z range 700-4000 using a Voyager DE-PRO Mass Spectrometer (Applied Biosystems, Foster City, CA) equipped with a nitrogen laser. MALDI-TOF analysis of mEP showed a single relative absorbance peak with an m/z of 3163.25 (Figure 3C).

Example 4

Binding Kinetics of mEP to VEGFR-3

[0083] The binding kinetics of mEP to VEGFR-3 was determined by surface plasmon resonance (SPR). The wild-type peptide mEP or a control peptide derived from an internal region of endostatin was injected and immobilized on a carboxymethylated dextran biosensor (CM5) chip. The CM5 chips were prepared by amine-coupling performed on a Biacore T100 instrument at 25°C at a flow rate of 10 IJmin in 10 mM HEPES, 150 mM NaCl, pH 7.4, and with the surface activated by injecting both l-ethyl-3-(3-dimethylaminopropyl) carbodimide and N-hydroxysuccinimide for 15 minutes. A total of 25 μg of mEP was dissolved in immobilization solution (10 mM sodium acetate, pH 5.0) and injected for 10 minutes over the CM5 chip. Ethanolamine was injected for 7 minutes to block residual activated groups. After immobilization, the instruments were first primed with analysis running buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl₂, 0.1% Tween 20, 0.1% Brij-35, 5% dimethyl sulfoxide, pH 8.0).

[0084] Binding affinities of the peptides were determined from experiments in which VEGFR-3-Fc at a range of concentrations (0, 0.01, 0.05, 0.25, 0.5, 1.0, 2.5, 5.0, and 10 μΜ) was passed over the immobilized peptides. The sensorgrams from the different

concentrations of VEGFR-3-Fc were simultaneously fitted and constrained the kinetic rate constants to a single value for each set of curves by the BIAevaluation 3.1 software. The results show that VEGFR-3-Fc bound mEP at all concentrations (Figure 4A). From this

7 -1 -1

analysis, the binding parameters were determined to be: Ka = 1.41 x 10' M^~V, Kd = 0.6718 s^"1, and ^ = 4.78 lO^~8 M (Figure 4B).

Example 5 mEP Inhibits Proliferation and Migration of Endothelial Cells

[0085] To test whether mEP binding to VEGFR-3 could inhibit or decrease VEGF- dependent lymphatic endothelial cell proliferation and migration, bromodeoxyuridine (BrdU) incorporation assays were conducted to assess the proliferation of human lung lymphatic endothelial cells (h-LECs) in the presence of VEGF-C with or without mEP.

[0086] h-LECs were seeded at 5 x 10⁴ cells/well in 96-well plates coated with collagen, serum starved overnight, and then incubated with 100 ng/mL VEGF-C and 0, 20, or 100 μg/mL mEP for 24 h at 37°C. A 25-amino acid peptide based on the internal endostatin sequence (FDGRDVLRHPAWPQKSVWHGSDPSG) (SEQ ID NO: 10) and a computer- generated, scrambled peptide (CVCYMEAEKHIFSNILTSSALKLFQNSS) (SEQ ID NO: l 1) were used as controls. Cells were then incubated in fresh media containing 10 μιηοΙ/L BrdU for 120 min and analyzed by ELISA, according to the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim, Germany). As shown in Figure 5A, compared to VEGF-C alone, VEGF-C-stimulated cell proliferation was reduced by one -third (i.e., more than 3-fold) in the presence of either 20 or 100 μg/mL mEP, respectively (Figure 5 A).

[0087] Cell migration was measured using a monolayer scratch wounding assay as follows. Confluent cell cultures in 96-well plates were cultured in serum-free medium for 24 h and then rinsed with Dulbecco's modified Eagle's medium. Each well was scratched with a sterile pipette tip, and cells were then washed twice with PBS, incubated in experimental medium for 24 h, rinsed twice with PBS, and fixed with 4% paraformaldehyde. The number of cells that migrated beyond the scratch was counted in at least three fields per well, and three wells were examined for each experiment and condition.

[0088] The scratch migration assay results show that mEP suppressed lymphatic cell migration. As shown in Figure 5B, in the absence of either ligand, the cells did not migrate into the denuded area (Figure 5B). VEGF-C stimulation led to approximately 50% repopulation of the scratch surface. In the presence of both mEP and VEGF-C, however, only a few cells migrated beyond the borders of the scratch. These results indicate that mEP can inhibit VEGF-C-induced proliferation and migration of endothelial cells.

Example 6

Endostatin short peptide binding to VEGFR3

[0089] To determine if short endostatin peptides were able to mimic the VEGF receptor ligand and act as an inhibitor, binding of various short peptides was tested in a pull down assay. See Figures 6 and 7. The sequence of the peptides are shown below:

Endol— KAASCHNSYIVLCIENSFM (SEQ ID No. 1)

Endo2— SCHNSYIVLCIENSFM (SEQ ID No. 3)

Endo3~ KAASCHNSYIVLCI (SEQ ID No. 21)

Endo4 - SCHNSYIVLCI (SEQ ID No. 22)

VEGFA mouse GCCNDEALECV (SEQ ID No. 23)

VEGFC mouse GCCNSEGLQCM (SEQ ID No. 24)

PLGF mouse GCCGDEGLHCV (SEQ ID No. 25)

Scramble peptide VSCYLIINCSH (SEQ ID No. 26)

[0090] The peptides were synthesized by solid-phase synthesis on a Symphony Peptide Synthesizer (Protein Technologies, Tucson, AZ) using Fmoc-RinkAmide-MBHA resin (AnaSpec, San Jose, CA; Novabiochem, Gibbstown, NJ), and purified by reversed-phase CI 8 HPLC (McKern et al, 1993, Biomed Chromatogr. 7 : 15-9).

[0091] For these pull down assays, a total of 10 mg of each peptide was coupled to Sepharose CL4B beads in 10 ml of slurry resin. Recombinant mouse VEGFR-3-Fc proteins (R&D Systems, Minneapolis, MN) were pre-absorbed against Sepharose 4B in RIPA buffer for 1 h at 4°C. Unconjugated Sepharose beads bound to VEGFR-3-Fc was removed by centrifugation, and then 100 ng VEGFR-3-Fc in RIPA buffer (50 mM Tris-HCl, pH 7.4, and 150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxycholate, and 1% NP-40) was incubated with 5 μg conjugated Sepharose 4B beads for 2 h at 4°C. Pull-down assays were performed using 5 μg of Sepharose-conjugated peptide. Pull-down complexes were washed three times with PBS and analyzed by western blotting with rat anti -mouse VEGFR-3 antibody (1 : 1 ,000, eBiosciences, San Diego, CA) and horseradish peroxidase-conjugated anti-rat IgG antibody (1 :20,000, Cell Signaling Technology, Danvers, MA). The ECL detection system

(Amersham, Piscataway, NJ) detected bound VEGFR-3-Fc.

[0092] Analysis of these peptides using the receptor pull-down assay set forth above indicated that the short endostatin peptides were able to bind to the VEGF receptor to VEGFR-3. Figures 6 and 7.

[0093] It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An isolated peptide comprising the amino acid sequence

LEQKAASCHNSYIVLCIENSFMTSFSK (SEQ ID NO:5)

LGQSAASCHHAYIVLCIENSFMTASK (SEQ ID NO:6).

2. The isolated peptide of claim 1, wherein the peptide is no more than 50 amino acids in length.

3. An isolated peptide of endostatin comprising an amino acid sequence of the general formula Xl-X2-X3-X4-Cys-X5-X6-X7-X8-X9-X10-Xl l-Cys-X12-X13-X14-Ser (SEQ ID NO: 12), wherein

XI is Arg, Lys, Gin, Asn, or Ser;

X2 is Ala, Val, Leu, Cys or He;

X3 is Ala, Val, Leu, Gly or He;

X4 is Ser, Thr, Ala, Cys, or Gly;

X5 is His, Asn, Gin, Lys, Cys or Arg;

X6 is His, Asn, or Gin;

X7 is Ser, Ala, Glu, Thr, or Cys;

X8 is Tyr, Glu, Trp, Phe, Thr or Ser;

X9 is He, Gly, Ser, Leu, Val, Met, Ala, Phe or Norleucine;

X10 is Val, Leu, He, Met, Phe, Ala, or Norleucine;

XI I is He, Leu, Gin, Val, Met, Ala, Phe or Norleucine;

XI 2 is He, Met, Leu, Val, Ala, Phe or Norleucine;

XI 3 is Glu, Asn, or Asp; and

X14 is Asn, Thr or Gin,

and wherein the peptide binds to vascular endothelial growth factor receptor 3 (VEGFR-3) and inhibits VEGFR-3 activity.

4. The isolated peptide of claim 3, wherein

XI is Arg, Lys or Ser;

X2 is Ala or Cys; X3 is Ala or Gly;

X4 is Ser or Gly;

X5 is His or Cys;

X6 is Asn or His;

X7 is Ser, Glu or Ala;

X8 is Tyr or Glu;

X9 is He, Gly or Ser;

XI 0 is Val or Leu;

XI 1 is Leu or Gin;

XI 2 is He or Met;

XI 3 is Glu or Asn; and

XI 4 is Asn or Thr.

5. The isolated peptide of claim 3 or 4, wherein the two Cys residues of the general formula form a disulfide bond.

6. The isolated peptide of any one of claims 3-5, wherein the peptide is at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 98% or at least 99% identical to the peptide having the amino acid sequence of SEQ ID NO:5.

7. The isolated peptide of any one of claims 3-5, wherein the peptide is at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 98% or at least 99% identical to the peptide having the amino acid sequence of SEQ ID NO:6.

8. The isolated peptide of any one of claims 3-6, wherein the peptide comprises the amino acid sequence of SEQ ID NO:5.

9. The isolated peptide of any one of claims 3-6 and 7, wherein the peptide comprises the amino acid sequence of SEQ ID NO:6.

10. The isolated peptide of any one of claims 3-9, wherein the peptide is no more than 50 amino acids in length.

11. An isolated peptide comprising the amino acid sequence KAASCHNSYIVLCI (SEQ ID No. 22).

12. The isolated peptide of claim 11, wherein the peptide is at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 98% or at least 99% identical to the peptide having the amino acid sequence of SEQ ID NO:22.

13. The isolated peptide of any one of claims 1-12, wherein the peptide is modified at the N- and/or C -terminus.

14. The isolated peptide of claim 13, wherein the peptide is modified at the N-terminus by an acetyl group and/or modified at the C-terminus by an amide group.

15. A pharmaceutical composition comprising the isolated peptide of any one of claims 1- 14 and a pharmaceutical acceptable carrier, excipient or diluent.

16. A method of inhibiting proliferation of a cell that expresses VEGFR-3 comprising the step of contacting the cell with the isolated peptide of any one of claims 1-14 or the pharmaceutical composition of claim 15 in an amount effective to inhibit proliferation of the VEGFR-3 -expressing cell.

17. A method of inhibiting migration of a cell that expresses VEGFR-3 comprising the step of contacting the cell with the isolated peptide of any one of claims 1-14 or the pharmaceutical composition of claim 15 in an amount effective to inhibit migration of the VEGFR-3 -expressing cell.

18. A method of inhibiting VEGFR-3 -mediated proliferation or migration of a cell

comprising the step of contacting the cell with the isolated peptide of any one of claims 1-14 or the pharmaceutical composition of claim 15 in an amount effective to inhibit VEGFR-3 -mediated proliferation or migration of the cell.

19. A method of inhibiting VEGF-C binding to VEGFR-3 in a cell comprising the step of contacting the cell with the isolated peptide of any one of claims 1-14 or the pharmaceutical composition of claim 15 in an amount effective to inhibit binding of VEGF-C to VEGFR-3.

20. The method of any one of claims 16-19, wherein the cell is an endothelial cell, a

lymphatic endothelial cell, a vascular endothelial cell, an endocardium cell, an endothelial progenitor cell, or a hematopoietic progenitor cell.

21. A method of inhibiting angiogenesis or lymphangiogenesis in a mammal in need

thereof comprising the step of administering to the mammal the pharmaceutical composition of claim 15 in an amount effective to inhibit angiogenesis or

lymphangiogenesis.

22. The method of claim 21, wherein the mammal is suffering from immune and nonimmune inflammation, rheumatoid arthritis, Crohn's disease, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, loss of vision due to invasion of blood vessel, corneal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, solid tumors, tumor metastases, leukemias, angiofibromas, hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyogenic granulomas, Osier- Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, lymphedema, transplant rejection, hemophiliac joints, or wound granulation.

23. The method of claim 22, wherein the tumor or tumor metastasis is a colon tumor, liver tumor, spleen tumor, kidney tumor, lymph node tumor, gastrointestinal tract tumor, pancreas tumor, stomach tumor, blood cell tumor, lymphatic vessel tumor, brain tumor, lung tumor, breast tumor, endometrium tumor, prostate tumor, skin tumor, bone marrow tumor, or head and neck tumor or metastasis thereof.

24. A method of treating an angiogenesis-related or lymphangiogenesis-related disease in a mammal in need thereof comprising the step of administering to the mammal the pharmaceutical composition of claim 15 in an amount effective to treat the angiogenesis-related or lymphangiogenesis-related disease.

25. The method of claim 24, wherein the angiogenesis-related or lymphangiogenesis- related disease is immune and non-immune inflammation, rheumatoid arthritis, Crohn's disease, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, loss of vision due to invasion of blood vessel, corneal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, solid tumors, tumor metastases, leukemias, angiofibromas, hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyogenic granulomas, Osier- Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, lymphedema, transplant rejection, hemophiliac joints, or wound granulation.

26. The method of any one of claims 21-25, wherein the mammal is a human.

27. A method of detecting VEGFR-3 in a cell comprising the steps of

(a) contacting the cell with the isolated peptide of any one of claims 1-15, wherein the peptide is conjugated to a detectable label; and

(b) detecting the signal of the label.

28. The method of claim 27, wherein the signal is detected by an immunological assay or an enzymatic assay.