EP1242451A2 - Compositions and methods for inhibiting endothelial cell proliferation - Google Patents

Abstract

Compositions and methods for regulating angiogenic activity wherein the compositions comprise proteins belonging to the family of kringle domain containing proteins and peptides and active fragments thereof are provided. More specifically, compositions and methods comprising kringle domain containing proteins and peptides such as hepatocyte growth factor (HGF) and/or macrophage stimulating protein (MSP), and biologically active fragments thereof are provided. HGF protein fragments of the present invention exhibit potent antiangiogenic activity on human and other animal cells, particularly endothelial cells. More particularly, compositions comprising HGF fragments, and/or HGF fragment homologs, may be combined with a pharmaceutically acceptable excipient or carrier and used to inhibit angiogenesis and angiogenesis-related diseases such as cancer, arthritis, macular degeneration, and diabetic retinopathy.

Description

COMPOSITIONS AND METHODS FOR INHIBITING ENDOTHELIAL CELL PROLIFERATION

TECHNICAL FIELD

This application relates to novel uses for knngle domam-containing proteins and peptides such as hepatocyte growth factor (HGF), also known as scatter factor, and macrophage stimulating protem (MSP), as regulators of angiogenesis useful for treating angiogenesis-related diseases including angiogenesis-dependent cancer. More specifically, the present invention relates to knngle domain containing proteins and peptides including active fragments of HGF and/or MSP, capable of regulating, and preferably inhibiting angiogenesis. The invention further relates to novel HGF and/or MSP fragment compositions and methods for curing angiogenesis- dependent disorders such as cancer, arthritis, blindness, diabetic retmopathy, macular degeneration, psoriasis and artherosclerosis. In addition, the present invention relates to knngle domain containing proteins and peptides antibodies that, for example, block the interaction of HGF (and fragments thereof) to the c-met receptor, the molecular probes for monitoring biosynthesis to antibodies that are specific for knngle domain containing protems and peptides, to the development of peptide agonists and antagonists to knngle domam containing protems and peptides, and to cytotoxic agents linked to receptors peptides.

BACKGROUND OF THE INVENTION

Knngle domam containing protems and peptides are unique in that they are characterized by triple disulfide loops structures. Examples of such proteins comprise hepatocyte growth factor, macrophage stimulating protem, tissue plasmmogen activator, apohpoprotem (a), prothrombin, urokinase, and ANGIOSTATIN protems.

Hepatocyte growth factor (HGF) is a mesenchyme derived glycoprotem and is named for its ability to induce kidney epithelial cells in a collagen matrix to form branching networks of tubules. (Grant et al PNAS 1993; 90: 1937- 1941) As characterized in the art, HGF is considered to be a potent angiogemc molecule that pπmanly acts on endothehal cells promoting cell motihty, proliferation, protease production, invasion and organization into capillary-like tubules. (Rosen et al. Adv. Cancer Res. 1995; 67:257-79)

HGF contains 29% identity with plasmmogen and withm the first four knngle domains HGF contains 44% similarity with plasmmogen. HGF contains approximately 44% identity with macrophage stimulating protem (MSP). In addition, all of the cystenes and most of the aromatic ammo acids such as trytophanes and prolmes are conserved between HGF and plasmmogen. It is produced as a preproprecursor of 728 ammo acids that is cleaved to a large heterodimeπc molecule made up of an α-cham (69kDa) and a β-cham (34kDa). (Lokker et al. Prot. Engin., 1994, 7.895-903) HGF is a basic hepann-bmdmg glycoprotem consisting of a heavy (58kDa) and a light (31kDa) subu t. (Grant et al.) The biological effects of HGF are triggered by the interaction of HGF with its high-affinity receptor c-Met The c- Met is a receptor-type tyrosme kinase contammg a 145kDa β-cham that transverses the membrane once and an extracellular 50kDa α-cham. HGF-Met signaling has been implicated in supporting significant roles in the pathogenesis and biology of human cancers. Specifically it is thought that by autocrme or paracπne mechanisms HGF-Met signaling promotes tumor cell growth, invasion and angiogenesis

As mentioned above, the structure of HGF is similar to that of plasmmogen The α-cham is distinguished by the presence of an N-termmal hairpm loop followed by four knngle domains, and the β-cham by a non-functional serine protease-like domain. Structural-functional studies have demonstrated that the hairpm loop and knngle domains are important in the binding of HGF to its receptors and proteoglycans. (To et al. Oncl Rep., 1998, 5: 1013-1024)

Macrophage Stimulating Protein

Macrophage stimulating protem (MSP) is a 78 KD plasma protem

(71 1aa) that is secreted by the liver into the circulation as smgle-cham, biologically inactive pro-MSP. After the proteolytic cleavage at a single site MSP becomes biologically active disulfide-hnked alpha beta-cham heterodimeπc molecule. MSP is a growth and motihty factor which interacts to its transmembrane tyrosme kmase called RON to induce activation of signal transduction pathway that mediates its biological effects

Both MSP and HGF are plasmmogen-related growth and motihty factors that interact with cell-surface protein tyrosme kinase receptors. Each one is a heterodimeπc protem comprising a disulfide-hnked alpha chain and a serine protease-hke beta chain. Despite structural similarities between MSP and HGF, the pnmary receptor binding site is located on the alpha chain of HGF but on the beta chain of MSP.

Evaluation studies indicated that HGF and HGF 1 /MSP evolved along with plasmmogen and other members of the krmgle-seπne protemase superfamily from an ancestral gene that contained a single copy of the knngle domain, a serine protease domam and an activation peptide connecting the two domains. So the knngle domains from plasmmogen, HGF and HGF1/MSP may still possess similar biological functions such as anti-angiogenesis

Angiogenesis and Cancer

The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971 by Judah Folkman (N. Engl Jour Med. 285" U 82 1 186, 1971) In its simplest terms the hypothesis proposes that expansion of tumor volume beyond a certain phase requires the induction of new capillary blood vessels. For example, pulmonary micrometastases m the early prevascular phase m mice would be undetectable except by high power microscopy on histological sections Further indirect evidence supporting the concept that tumor growth is angiogenesis dependent is found in U.S. Patent Nos. 5,639,725, 5,629,327, 5,792,845, 5,733,876, and 5,854,205, all of which are incorporated herein by reference. To stimulate angiogenesis, tumors upregulate their production of a variety of angiogemc factors, including the fibroblast growth factors (aFGF and bFGF) (Kandel et al., 1991 ) and vascular endothehal cell growth factor/vascular permeability factor (VEGF/NPF) and HGF. However, many malignant tumors also generate inhibitors of angiogenesis, including AΝGIOSTATIΝ protem and thrombospondm. (Chen et al., 1995; Good et al., 1990; O'Reilly et al., 1994) It is postulated that the angiogemc phenotype is the result of a net balance between these positive and negative regulators of neovasculanzation. (Good et al., 1990; O'Reilly et al., 1994; Parangi et al., 1996; Rastmejad et al., 1989). Several other endogenous inhibitors of angiogenesis have been identified, although not all are associated with the presence of a tumor. These include, platelet factor 4 (Gupta et al., 1995, Maione et al., 1990), mterferon-alpha, mterferon-mducible prote 10 (Angiohllo et al., 1995, Stπeter et al., 1995), which is induced by mterleukιn-12 and/or mterferon-gamma (Noest et al., 1995), gro-beta (Cao et al, 1995), and the 16 kDa Ν-termmal fragment of prolactm (Clapp et al , 1993) One example of an angiogenesis inhibitor that specifically inhibits endothehal cell proliferation is AΝGIOSTATIΝ protein (O'Reilly et al , 1994) ANGIOSTATIN protem is a approximately 38 kiloDalton (kDa) specific inhibitor of endothehal cell proliferation ANGIOSTATIN protem is an internal fragment of plasmmogen contaimng at least three of the five krmgles of plasmmogen ANGIOSTATIN protem has been shown to reduce tumor weight and to inhibit metastasis in certain tumor models (O'Reilly et al , 1994). Another angiogenesis inhibitor is ENDOSTATIN protem, which is a carboxy fragment of collagen or XVIII (O'Reilly et al., 1997).

What is needed is the discovery and development of additional anti- angiogemc agents that may be used alone or m combination with known angiogemc agents in order to treat cancer and hyperprohferative disorders

SUMMARY OF THE INVENTION

The present mvention comprises novel uses for knngle domain- contammg prote s and peptides such as hepatocyte growth factor (HGF), also known as scatter factor, and macrophage stimulating protem (MSP), as regulators of angiogenesis useful for treating angiogenesis-related diseases including angiogenesis- dependent cancer More specifically, the present invention relates to knngle domain containing prote s and peptides including active fragments of HGF and/or MSP, capable of regulating, and preferably inhibiting angiogenesis The invention further relates to novel HGF and/or MSP fragment compositions and methods for curing angiogenesis-dependent disorders such as cancer, arthritis, blindness, diabetic retmopathy, macular degeneration, psoriasis and artherosclerosis In addition, the present invention relates to knngle domain containing proteins and peptides antibodies that, for example, block the interaction of HGF (and fragments thereof) to the c-met receptor, the molecular probes for monitoring biosynthesis to antibodies that are specific for knngle domam containing protems and peptides, to the development of peptide agonists and antagonists to knngle domam containing proteins and peptides, and to cytotoxic agents linked to receptors peptides.The present invention generally relates to knngle domam containing protems and peptides and active fragments thereof, as angiogenesis inhibitors and methods of use thereof Examples of such protems include hepatocyte growth factor (HGF), macrophage stimulating protem (MSP), apohpoprotem, prothrombm urokmase and tissue plasmmogen activator. Whole HGF is a potent and specific regulator of endothehal cell function and angiogenesis, however, as demonstrated herein, HGF fragments comprising knngle domains serve the reverse effects of whole HGF Systemic therapy with active HGF fragments, causes suppression of tumor-mduced angiogenesis, and exhibits strong antitumor activity.

HGF, also known as scatter factor, has a molecular weight of approximately 87 kiloDaltons as determined by ammo acid composition As described herein, novel HGF fragments are capable of inhibiting endothehal cell function in cultured endothehal cells, tumor cells, smooth muscle cells, and other variety of cells.

The present invention provides methods and compositions for treating diseases and processes mediated by undesired and uncontrolled angiogenesis by administering to a human or animal with the undesired angiogenesis a composition comprising knngle domain containing proteins and peptides such as novel HGF and/or MSP active fragments of the present invention, or derivatives thereof, in a dosage sufficient to regulate, and preferably inhibit, angiogenesis More specifically, the present invention is directed knngles 1-3, and/or krmgles 2-3 of HGF and/or MSP The present invention is particularly useful for treating or for repressing the growth of tumors Administration of the presently identified novel HGF and/or MSP fragments to a human or animal with metastasized tumors prevents the growth or expansion of those tumors The invention further provides methods and compositions for regulating endothehal cell function in vivo as well as in vitro The present invention also includes knngle domain-containing protems and peptides such as HGF peptide fragments that can be labeled isotopically or with other molecules or protems for use the detection and visualization of HGF binding sites with state of the art techniques, including, but not limited to, positron emission tomography, autoradiography, flow cytometry, radioreceptor binding assays, and lmmunohistochemistry

The present invention also includes knngle domain containing protems and peptides , knngle domain containing protems and peptides fragments, or knngle domain containing protems and peptides receptor agonists and antagonists linked to cytotoxic agents for therapeutic and research applications The present invention also includes HGF, HGF fragments, or HGF receptor agonists and antagonists linked to cytotoxic agents for therapeutic and research applications

The present invention also includes MSP, MSP fragments, or MSP receptor agonists and antagonists linked to cytotoxic agents for therapeutic and research applications In addition, knngle domain containing protems and peptides may act as agonists and antagonists of knngle domam containing protem receptors, thereby enhancing or blocking the biological activity of such protems Such protems and peptides are used in the isolation of the receptors such as the HGF receptor A surpnsmg discovery is that various active fragments of HGF, can serve as sustained release anti-angiogenesis compounds when administered to a tumor-bearmg animal

The present invention also relates to methods of using knngle domam containing protems and peptides and fragments thereof, corresponding nucleic acid sequences, and antibodies that bind specifically to the inhibitor and its peptides, to diagnose endothehal cell-related diseases and disorders

The invention further encompasses a method for identifying receptors specific HGF fragments, and the receptor molecules identified and isolated thereby

An important medical method is a new form of birth control, wherem an effective amount of a knngle domain from a knngle domain containing protem such as HGF or MSP is administered to a female such that uterine endometπal vasculaπzation is inhibited and embryo implantation cannot occur or be sustained

A particularly important aspect of the present mvention is the discovery of a novel and effective method for treating angiogenesis-related diseases, particularly angiogenesis-dependent cancer, in patients, and for curing angiogenesis-dependent cancer in patients The method unexpectedly provides the medically important result of inhibition of tumor growth and reduction of tumor mass The method relates to the co-administration of an active HGF (or MSP) fragment of the present invention and another anti-angiogenesis compound, such as ANGIOSTATIN protem (EntreMed, Inc Rockville, MD) or ENDOSTATIN protem (EntreMed, Inc Rockville, MD) Accordingly, the present mvention also includes formulations contammg HGF knngle fragments, MSP knngle fragments, ANGIOSTATIN protem, and/or ENDOSTATIN protem, which are effective for treating or curing angiogenesis- dependent diseases Accordingly, it is an object of the present invention to provide compositions and methods compnsmg knngle domain contammg protems and peptides such as HGF, including active HGF fragments, useful for the treatment of angiogemc disorders

Another object of the present invention is to provide compositions and methods comprising knngle domam containing protems and peptides, or knngle domain containing protems and peptides fragments in combination with other antiangiogemc compounds such as ANGIOSTATIN protem or ENDOSTATIN™ protem, useful for the treatment of angiogemc disorders.

Another object of the present invention is to provide compositions and methods comprising HGF, or HGF knngle domain containing fragments in combination with other antiangiogemc compounds such as ANGIOSTATIN protein or ENDOSTATIN™ protem, useful for the treatment of angiogemc disorders.

Another object of the present mvention is to provide compositions and methods comprising MSP, or MSP knngle domain containing fragments, in combination with other antiangiogemc compounds such as ANGIOSTATIN protem or ENDOSTATIN protem, useful for the treatment of angiogemc disorders

It is another object of the present invention to provide compositions and methods of treating diseases and processes that are mediated by angiogenesis

It is yet another object of the present mvention to provide compositions and methods for treating diseases and processes that are mediated by angiogenesis including, but not limited to, hemangioma, solid tumors, leukemia, metastasis, telangiectasia psoriasis scleroderma, pyogenic granuloma, myocardial angiogenesis, plaque neovasculanzation, coronary collaterals, cerebral collaterals, artenovenous malformations, ischemic limb angiogenesis, cornea] diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovasculanzation, macular degeneration, wound healing, surgical adhesions, peptic ulcer, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstruation, and placentation

It is another object of the present invention to provide compositions and methods for treating or repressing the growth of a cancer Still another object of the present mvention is to provide compositions and methods comprising antibodies to knngle domam containing protems and peptides, or knngle domain contammg protems and peptides fragments, that are selective for specific regions of the knngle domain containing protems and peptides molecule Another object of the present mvention is to provide compositions and methods comprising antibodies to HGF, or HGF fragments, that are selective for specific regions of the HGF molecule

Still another object of the present invention is to provide compositions and methods comprising antibodies to MSP, or MSP fragments, that are selective for specific regions of the MSP molecule It is another object of the present invention to provide compositions and methods for the detection or prognosis of anti-angiogenesis activity.

It is yet another object of the present invention to provide a therapy for cancer that has minimal side effects. Still another object of the present invention is to provide compositions comprising kringle domain containing proteins and peptides such as HGF or HGF peptide fragments, linked to a cytotoxic agent for treating or repressing the growth of a cancer.

These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a structural comparison of HGF and plasminogen. Mature HGF is a heterodimeric molecule which is composed of an alpha-chain, containing the N-terminal hairpin domain and four kringle domains, and a beta-chain containing the serine protease-like domain. The alpha-chain has been identified as the receptor binding domain. HGF/NK1 , HGF NK2, HGF/NK3 and HGF/NK4 are defined as N-terminal hairpin domain plus the first kringle (NK1), or N-terminal haiφin domain plus the first 2 kringles (NK2) and so on HGF K2-3 comprises the second and the third kringles of HGF. Plasminogen is composed of kringle domains and serine protease domain. Angiostatin protein comprises the first 3 kringles of plasminogen (Kl-3).

Figure 2 shows protein sequence comparison between kringles 1-4 of HGF and plasminogen. Primary protein sequence comparison revealed that plasminogen kringle domain and HGF kringle domain has over 49% sequence similarity and all the cysteines are highly conserved.

Figure 3 provides the amino acid sequence for HGF (SEQ ID NO: l ). Figure 4 provides the amino acid sequence for HGF fragments (SEQ ID NOS:2 and 3).

Figure 5 provides the amino acid sequence for MSP (SEQ ID NO: 4). Figure 6 is a SDS-PAGE gel of recombinant HGF K2-3 expressed in Pichia pastori.

Figures 7(a) and 7(b) are graphs showing the HGF K2-3 inhibits VEGF as well as HGF stimulated HUVEC migration. Figure 8 is a graph showing HGF K2-3 inhibits HUVEC tube formation

Figure 9 is a graph showing that Angiostatin protem inhibits HGF stimulated HUVEC migration Figure 10 shows that Angiostatin protem does not inhibit HGF stimulated c-met phosphorylation

Figure 1 1 shows Western blots probed with anti-phosphotyrosme antibody (4G 10) or anti-c-met antibodies

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of specific embodiments included herein Although the present mvention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention The entire text of the references mentioned herein are hereby mcoφorated in their entireties by reference

Applicants have discovered a novel property for a class of protem molecules These protem molecules are characterized in that they typically include knngle domains, and/or protease domains, and have the suφπsing ability to regulate angiogemc function when added to proliferating endothehal cells "Hepatocyte Growth Factor" (HGF) and macrophage stimulating protein (MSP) along with tissue plasmmogen activator, apohpoprotem (a), prothrombm and urokmase, are examples of such proteins HGF and MSP belong to the plasmmogen-related knngle domain family, and as used herein, it is to be understood that the term HGF includes HGF analogs, homologs, HGF kringle domam fragments and active peptides thereof, and that the term MSP includes MSP analogs, homologs, and active peptides thereof

The term "Hepatocyte Growth Factor" (HGF) refers generally to a protein that is approximately 87 kiloDaltons in size as determined by ammo acid composition, more specifically to a protem that is approximately 43 kiloDaltons, and more preferably to a protein that is approximately 31 kiloDaltons HGF shares both structural (Figure 1) and sequence homology (Figure 2) with plasmmogen The amino acid sequence of a human HGF is provided in SEQ ID NO 1 as shown in Figure 3 The term HGF also includes precursor forms of the prepropeptide and propeptide as well as modified proteins and peptides that have a substantially similar ammo acid sequence, and which are capable of inhibiting proliferation of endothehal cells For example, silent substitutions of ammo acids, wherem the replacement of an ammo acid with a structurally or chemically similar ammo acid does not significantly alter the structure, conformation or activity of the protem, are well known m the art Such silent substitutions, additions and deletions, are intended to fall with the scope of the appended claims The term "Macrophage Stimulating Protem" (MSP) refers generally to a protein that is approximately 78 kilodaltons in size as determined by ammo acid composition, and shares both structural and sequence homology with plasminogen The ammo acid sequence of a human HGF is provided in SEQ ID NO 4 as shown in Figure 5 The term MSP also includes precursor forms of the prepropeptide and propeptide as well as modified protems and peptides that have a substantially similar ammo acid sequence, and which are capable of inhibiting proliferation of endothehal cells For example, silent substitutions of ammo acids, wherem the replacement of an ammo acid with a structurally or chemically similar ammo acid does not significantly alter the structure, conformation or activity of the protein, are well known in the art Such silent substitutions, additions and deletions, are intended to fall withm the scope of the appended claims

It will be appreciated that the terms "HGF" and "MSP" include shortened proteins or peptides wherein one or more ammo acid is removed from either or both ends of the protein, or from an internal region of the protein, yet the resulting molecule retains angiogemc regulating activity HGF and MSP also include lengthened protems or peptides wherein one or more ammo acid is added to either or both ends of the protem, or to an internal location in the protem, yet the resulting molecule retains angiogemc regulating activity Such molecules, for example with tyrosme added in the first position, are useful for labeling such as radioiodmation

125 with Iodine, for use in assays Labeling with other radioisotopes may be useful in providing a molecular tool for isolating and identifying the target cell containing

HGF of MSP receptors Other labeling, with molecules such as ricm, may provide a mechanism for destroying cells with HGF or MSP receptors The invention also contemplates that active peptides of HGF may be used alone or combined with other peptides and protems to form chimenc protems containing active HGF or MSP peptides Active HGF fragments of particular interest include krmgles 1 -3 or HGF as set forth in SEQ ID NO 2 (269 ammo acids, 31 kiloDaltons) and kringles 1 -4 of HGF as set forth m SEQID NO 3 (368 ammo acids, 43 kiloDaltons) Both SEQ ID NOS 2 and 3 were obtained from the Pichia production clone "Substantial sequence homology" means at least approximately 70% homology between ammo acid residue sequence in the knngle domain containing protem analog, homolog or derivative sequence and that of kringle domain containing protem, preferably at least approximately 80% homology, and more preferably at least approximately 90% homology.

Knngle domain containing protems and peptides such as HGF can be isolated from normal, hypeφlastic, primary and metatstatic tissue from a variety of species including humans. HGF and MSP can also be isolated from body fluids including, but not limited to, semen, serum, urine and ascites, or synthesized by chemical or biological methods (e g. peptide synthesis and in vitro enzymatic catalysis of precursor molecules to yield active HGF). Knngle domain containing proteins and peptides may be produced from recombinant sources, from genetically altered cells implanted into animals, from tumors, and from cell cultures as well as other sources. Recombinant techniques include gene amplification from DNA sources using the polymerase chain reaction (PCR), and gene amplification from RNA sources using reverse transc ptase/PCR. Though not wishing to be bound by the following theory, fragments of glycoprotems in the family of knngle domain contammg proteins and peptides such as HFG or MSP regulate angiogemc activity by specifically, and most likely reversibly, inhibiting endothehal cell proliferation. The inhibitor protem molecules of the present mvention are useful as birth control drugs, and for treating angiogenesis-related diseases, particularly angiogenesis-dependent cancers and tumors. The protem molecules are also useful for curing angiogenesis-dependent cancers and tumors. The unexpected and suφπsing ability of these novel compounds to treat and cure angiogenesis-dependent cancers and tumors answers a long-felt, unfulfilled need m the medical arts, and provides an important benefit to mankind Important terms that are used herein are defined as follows "Cancer" means angiogenesis-dependent cancers and tumors, i.e. tumors that require for their growth (expansion in volume and/or mass) an increase in the number and density of the blood vessels supplying them with blood. "Regression" refers to the reduction of tumor mass and size. As used herein, the term "angiogenesis" and related terms such as

"angiogemc" refer to activities associated with blood vessel growth and development, including, but not limited to, endothehal cell proliferation, endothehal cell migration and capillary tube formation.

As used herein, the term "antiangiogemc" refers to compositions and the like that are capable of inhibiting the formation of blood vessels, including but not limited to inhibiting endothehal cell proliferation, endothehal cell migration and capillary tube formation.

The process of angiogenesis is complex and involves a number of orchestrated steps that can be separately studied in vitro, such as FGF-2- and/or VEGF-stimulated endothehal cell proliferation and migration. For example, ANGIOSTATIN protem and ENDOSTATIN protem inhibit these processes (see U.S. Pat. No. 5,639,725 and U.S. Pat. No. 5,854,205). The inventors of the present invention have supnsmgly discovered antiangiogemc properties of protems belonging to the family of knngle domain containing proteins and peptides by demonstrating and systematically evaluating the effects of such proteins, for example HGF, on endothehal cell proliferation, migration, and invasion

Contrary to the teachings of the prior art wherem HGF is characterized as "potent angiogemc molecule, primarily act[ιng] on endothehal cells inducing cell motihty, proliferation, protease production, invasion, and organization into capillary- like tubes" (Rosen et al Adv. Cancer Res. 1995; 67:257-79), the inventors of the present invention demonstrate the opposite effect of novel HGF fragments as an anti- angiogenic molecule. Until the discovery of the novel HGF fragments as described herein, HGF was considered to be only related to the induction of angiogenesis- "HGF/SF has been shown to stimulate endothehal cell proliferation and migration, and induce angiogenesis in vivo HGF/SF may also potentiate new blood vessel formation by up-regulating the expression of vascular endothehal cell growth factor (VEGF) in vascular smooth muscle cells " (To et al. 1998) Accordingly the prior art m fact teaches away from the study of HGF related proteins or HGF fragments as exhibiting any angiogemc activity The effects of the novel HGF fragments of the present mvention on angiogemc activity are demonstrated in Human Umbilical Vein Endothehal Cells (HUVEC) Purified human HGF fragments demonstrate a potent and dose related inhibitory activity on FGF-2-shmulated proliferation of HUVEC cells. To determine if HGF fragments inhibit a variety of endothehal cells or simply display specificity for HUVECs, the ability of HGF to inhibit bovme adrenal cortex endothehal cell (BCE) and human microvascular dermal cell (HMVEC-d) proliferation is also demonstrated The effects of HGF on FGF-2-stιmulated endothehal cell proliferation are also conducted.

In order to demonstrate that HGF fragments exert antiangiogemc effects as opposed to general inhibition of cell proliferation, the inventors demonstrate experiments to show direct stimulatory or inhibitory effect on the proliferation of cancer cells.

The effects of HGF fragments on endothehal cell migration are demonstrated by the inventors to further confirm the antiangiogemc effects of such fragments. In order to evaluate the in vitro effects of HGF fragments on endothehal cell migration in response to FGF-2 or VEGF, confluent monolayers of HUVEC are scraped to remove a section of monolayer and cultured with FGF-2 or VEGF in the presence or absence of purified human HGF fragments.

The inventors further demonstrate antiangiogemc properties of HGF fragments by demonstrating effects on endothehal cell invasion. These experiments demonstrate that inhibition is dose dependent and not the result of toxicity as the endothehal cells appear viable and no junctions are made by the cells. These findings further support the inhibitory effects of HGF fragments on endothehal cell invasion and further confirm HGF fragment antiangiogemc activity. Further studies are conducted to determine whether HGF fragments of the present invention function similarly to other knngle domain containing protems such as ANGIOSTATIN protein. For examples the inventors are conducting experiments to determine whether ANGIOSTATIN protein binds the HGF receptor c-met and acts as an antagonist to compete with HGF binding, thus inhibiting tumorgenesis. The inventors are using a tumor cell line with expresses c-met receptor to determine whether ANGIOSTATIN protem blocks downstream effects (cell migration, proliferation, moφhology, cytokine production level, phosphorylations, etc.) of HGF.

Corresponding experiments described above for HGF fragments, are also conducted for MSP.

Protocols and methods for conducting the above-described experiments are well-known to those skilled in the art and are described in further detail in the Examples below, and in United States Patent Apphcation Serial No. 09/316,802. Though not wishing to be bound by the following theory, it is believed that the antiangiogemc properties of the present HGF fragments are related to the kringle activity of the protem. More specifically it is believed that the antiangiogemc activity is most likely located within krmgles 1-3 (SEQ ID NO:2) or withm krmgles 1 -4 (SEQ ID NO:3). It is also believed that the antiangiogemc properties of the MSP and fragments thereof are related to the knngle activity of the protein and that such antiangiogemc activity is located withm krmgles 1-3 or withm krmgles 1-4 of MSP. In conducting the above-described experiments, the inventors of the present invention supnsmgly demonstrate for the first time that certain novel HGF fragments are endothehal cell-specific inhibitors of angiogenesis that exhibit potent anti-prohferative and anti-migratory activity on a variety of cultured endothehal cells. Furthermore, these novel HGF fragments inhibit the endothehal-cell specific angiogenesis process of capillary tube formation m matπgel.

Based on the novel findings of the inventors, the present invention is directed to methods and compositions comprising the administration of protems belonging to the knngle domam containing protem family for the regulation of antiangiogemc processes. More particularly, the methods and compositions of the present mvention comprise the administration of novel HGF or MSP fragments for inhibiting angiogenesis and for reducing related cancer or tumor growth.

The novel antiangiogemc HGF or MSP fragments of the present invention can be made by automated protem synthesis methodologies well-known to one skilled m the art. Alternatively, HGF or MSP and peptide fragments thereof, may be isolated from larger known prepropeptides that share a common or similar amino acid sequence.

Protems and peptides derived from these and other sources, including manual or automated protem synthesis, may be quickly and easily tested for antiangiogemc activity using a biological activity assay such as the human umbilical vein endothehal cell proliferation assay (HUVEC) and the bovme capillary endothehal cell proliferation assay (BCE). Such assays are described in U.S Patent No 5,639,725 which is incoφorated herein by reference. Other bioassays for inhibiting activity include the chick CAM assay, the mouse corneal assay, and the effect of administering isolated or synthesized protems on implanted tumors The chick CAM assay is described by O'Reilly, et al. in "Angiogemc Regulation of Metastatic Growth" Cell, vol. 79 (2), October 21 , 1994, pp. 315-328, which is hereby incoφorated by reference m its entirety. Applicants' invention also encompasses nucleic acid sequences that correspond to, and code for antiangiogemc knngle domain containing protems and peptides, and to monoclonal and polyclonal antibodies that bind specifically to such protem molecules. The biologically active protein molecules, nucleic acid sequences corresponding to the proteins, and antibodies that bind specifically to the protems of the present invention are useful for modulating angiogemc processes in vivo, and for diagnosing and treating endothehal cell-related diseases, for example by gene therapy. Nucleic acid sequences that correspond to, and code for, knngle domain containing protems and peptides such as HGF fragments and HGF fragment analogs, can be prepared based upon the knowledge of the ammo acid sequence, and the art recognized correspondence between codons (sequences of three nucleic acid bases), and ammo acids Because of the degeneracy of the genetic code, wherein the third base in a codon may vary yet still code for the same ammo acid, many different possible coding nucleic acid sequences are derivable for any particular protem or peptide fragment

Nucleic acid sequences are synthesized using automated systems well known in the art Either the entire sequence may be synthesized or a series of smaller ohgonucleotides are made and subsequently hgated together to yield the full length sequence Alternatively, the nucleic acid sequence may be derived from a gene bank using ohgonucleotides probes designed based on the N-termmal amino acid sequence and well known techniques for cloning genetic material The present invention also encompasses gene therapy whereby genes encoding knngle domain containing protems and peptides such as HGF or MSP fragments, are regulated in a patient Various methods of transferring or delivering DNA to cells for expression of the gene product protein, otherwise referred to as gene therapy, are disclosed in Gene Transfer into Mammalian Somati. Cells in vivo, N Yang, Cπt Rev Biotechn 12(4) 335-356 (1992) Gene therapy encompasses mcoφoration of DNA sequences into somatic cells or germ line cells for use m either ex vivo or in vivo therapy Gene therapy functions to replace genes, augment normal or abnormal gene function, and to combat infectious diseases and other pathologies

Strategies for treating these medical problems with gene therapy include therapeutic strategies such as identifying the defective gene and then adding a functional gene to either replace the function of the defective gene or to augment a slightly functional gene, or prophylactic strategies, such as adding a gene for the product protem that will treat the condition or that will make the tissue or organ more susceptible to a treatment regimen As an example of a prophylactic strategy, a gene such as that for a desired HGF fragment may be placed in a patient and thus prevent occurrence of angiogenesis, or a gene that makes tumor cells more susceptible to radiation could be inserted and then radiation of the tumor would cause increased killing of the tumor cells

Many protocols for transfer of knngle domain containing protein DNA, or corresponding regulatory sequences are envisioned m this invention Transfection of promoter sequences, other than ones normally found specifically associated with such proteins or other sequences which would increase production of these protems are also envisioned as methods of gene therapy An example of this technology is found in Transkaryotic Therapies, Inc , of Cambridge, Massachusetts, using homologous recombination to insert a "genetic switch" that turns on an erythropoietm gene in cells See Genetic Engineering News, April 15, 1994 Such "genetic switches" could be used to activate HGF (or HGF receptors) in cells not normally expressing HGF (or the HGF receptor)

Gene transfer methods for gene therapy fall into three broad categories physical (e.g , electroporation, direct gene transfer and particle bombardment), chemical (hpid-based earners, or other non-viral vectors) and biological (virus-derived vector and receptor uptake) For example, non-viral vectors may be used which include hposomes coated with DNA Such hposome/DNA complexes may be directly injected intravenously into the patient It is believed that the hposome/DNA complexes are concentrated m the liver where they deliver the DNA to macrophages and Kupffer cells These cells are long lived and thus provide long term expression of the delivered DNA Additionally, vectors or the "naked" DNA of the gene may be directly injected into the desired organ, tissue or tu oi for targeted delivery of the therapeutic DNA

Gene therapy methodologies can also be described by delivery site Fundamental ways to deliver genes include ex vivo gene transfer, in vivo gene transfer, and in vitro gene transfer In ex vivo gene transfer, cells are taken from the patient and grown m cell culture The DNA is transfected into the cells, the transfected cells are expanded in number and then reimplanted in the patient In in vitro gene transfer, the transformed cells are cells growing in culture, such as tissue culture cells, and not particular cells from a particular patient These "laboratory cells" are transfected, the transfected cells are selected and expanded for either implantation into a patient or for other uses

In vivo gene transfer involves introducing the DNA into the cells of the patient when the cells are withm the patient Methods include using virally mediated gene transfer using a nonmfectious virus to deliver the gene in the patient or injecting naked DNA into a site in the patient and the DNA is taken up by a percentage of cells in which the gene product protem is expressed Additionally, the other methods descnbed herein, such as use of a "gene gun," may be used for in vitro insertion of DNA encoding HGF knngle domains or HGF regulatory sequences Chemical methods of gene therapy may involve a hpid based compound, not necessarily a hposome, to ferry the DNA across the cell membrane Lipofectms or cytofectms, hpid-based positive ions that bind to negatively charged DNA, make a complex that can cross the cell membrane and provide the DNA into the interior of the cell. Another chemical method uses receptor-based endocytosis, which involves binding a specific ligand to a cell surface receptor and enveloping and transporting it across the cell membrane. The ligand binds to the DNA and the whole complex is transported into the cell. The hgand gene complex is injected into the blood stream and then target cells that have the receptor will specifically bind the ligand and transport the hgand-DNA complex into the cell.

Many gene therapy methodologies employ viral vectors to insert genes into cells. For example, altered retrovirus vectors have been used in ex vivo methods to introduce genes into peripheral and tumor-infiltrating lymphocytes, hepatocytes, epidermal cells, myocytes, or other somatic cells These altered cells are then introduced into the patient to provide the gene product from the inserted DNA.

Viral vectors have also been used to insert genes into cells using in vivo protocols. To direct tissue-specific expression of foreign genes, cis-acting regulatory elements or promoters that are known to be tissue specific can be used. Alternatively, this can be achieved using in situ delivery of DNA or viral vectors to specific anatomical sites in vivo For example, gene transfer to blood vessels in vivo was achieved by implanting in vitro transduced endothehal cells in chosen sites on arterial walls The virus infected surrounding cells which also expressed the gene product. A viral vector can be delivered directly to the in vivo site, by a catheter for example, thus allowing only certain areas to be infected by the virus, and providing long-term, site specific gene expression. In vivo gene transfer using retrovirus vectors has also been demonstrated in mammary tissue and hepatic tissue by injection of the altered virus into blood vessels leading to the organs.

Viral vectors that have been used for gene therapy protocols include but are not limited to, retroviruses, other RNA viruses such as pohovirus or Sindbis virus, adenovirus, adeno-associated virus, heφes viruses, SV 40, vaccinia and other DNA viruses Replication-defective murme retroviral vectors are the most widely utilized gene transfer vectors. Murme leukemia retroviruses are composed of a single strand RNA complexed with a nuclear core protem and polymerase (pol) enzymes, encased by a protem core (gag) and surrounded by a glycoprotein envelope (env) that determines host range. The genomic structure of retroviruses include the gag, pol, and env genes enclosed at by the 5' and 3' long terminal repeats (LTR) Retroviral vector systems exploit the fact that a minimal vector containing the 5' and 3' LTRs and the packaging signal are sufficient to allow vector packaging, infection and integration into target cells providing that the viral structural protems are supplied in trans in the packaging cell line. Fundamental advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, precise single copy vector integration into target cell chromosomal DNA, and ease of manipulation of the retroviral genome

The adenovirus is composed of linear, double stranded DNA complexed with core protems and surrounded with capsid protems. Advances m molecular virology have led to the ability to exploit the biology of these organisms to create vectors capable of transducing novel genetic sequences into target cells in vivo Adenoviral-based vectors will express gene product peptides at high levels Adenoviral vectors have high efficiencies of mfectivity, even with low titers of virus. Additionally, the virus is fully infective as a cell free vinon so injection of producer cell lines are not necessary. Another potential advantage to adenoviral vectors is the ability to achieve long term expression of heterologous genes in vivo. Mechanical methods of DNA delivery include fusogemc hpid vesicles such as hposomes or other vesicles for membrane fusion, hpid particles of DNA mcoφoratmg cationic hpid such as hpofectm, polylysine-mediated transfer of DNA, direct injection of DNA, such as micromjection of DNA into germ or somatic cells, pneumatically delivered DNA-coated particles, such as the gold particles used in a "gene gun," and inorganic chemical approaches such as calcium phosphate transfection Another method, hgand-mediated gene therapy, involves complexmg the DNA with specific ligands to form hgand-DNA conjugates, to direct the DNA to a specific cell or tissue.

It has been found that injecting plasmid DNA into muscle cells yields high percentage of the cells which are transfected and have sustained expression of marker genes The DNA of the plasmid may or may not integrate into the genome of the cells Non-mtegration of the transfected DNA would allow the transfection and expression of gene product protems in terminally differentiated, non-prohferative tissues for a prolonged period of time without fear of mutational insertions, deletions, or alterations in the cellular or mitochondnal genome Long-term, but not necessarily permanent, transfer of therapeutic genes into specific cells may provide treatments for genetic diseases or for prophylactic use. The DNA could be reinjected periodically to maintain the gene product level without mutations occurring in the genomes of the recipient cells. Non-mtegration of exogenous DNAs may allow for the presence of several different exogenous DNA constructs withm one cell with all of the constructs expressing various gene products. Particle-mediated gene transfer methods were first used in transforming plant tissue. With a particle bombardment device, or "gene gun," a motive force is generated to accelerate DNA-coated high density particles (such as gold or tungsten) to a high velocity that allows penetration of the target organs, tissues or cells. Particle bombardment can be used in in vitro systems, or with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs.

Electroporation for gene transfer uses an electrical current to make cells or tissues susceptible to electroporation-mediated gene transfer. A brief electric impulse with a given field strength is used to increase the permeability of a membrane in such a way that DNA molecules can penetrate into the cells This technique can be used in in vitro systems, or with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs

Carrier mediated gene transfer in vivo can be used to transfect foreign DNA into cells The carπer-DNA complex can be conveniently introduced into body fluids or the bloodstream and then site specifically directed to the target organ or tissue m the body Both hposomes and polycations, such as polylysine, lipofectms or cytofectms, can be used. Liposomes can be developed which are cell specific or organ specific and thus the foreign DNA earned by the hposome will be taken up by target cells Injection of lmmunohposomes that are targeted to a specific receptor on certain cells can be used as a convenient method of inserting the DNA into the cells bearing the receptor. Another carrier system that has been used is the asialoglycoportem/polylysme conjugate system for carrying DNA to hepatocytes for in vivo gene transfer.

The transfected DNA may also be complexed with other kinds of earners so that the DNA is carried to the recipient cell and then resides in the cytoplasm or in the nucleoplasm. DNA can be coupled to carrier nuclear proteins in specifically engineered vesicle complexes and carried directly into the nucleus

Gene regulation of knngle domam containing protems and peptides such as HGF kringle fragments may be accomplished by administering compounds that for example bind to HGF genes, or control regions associated with the HGF genes, or corresponding RNA transcript to modify the rate of transcription or translation Additionally, cells transfected with a DNA sequence encoding HGF may be administered to a patient to provide an in vivo source of HGF fragments For example, cells may be transfected with a vector containing a nucleic acid sequence encoding HGF The term "vector" as used herein means a carrier that can contain or associate with specific nucleic acid sequences, which functions to transport the specific nucleic acid sequenc es into a cell. Examples of vectors include plasmids and infective microorganisms such as viruses, or non-viral vectors such as hgand-DNA conjugates, liposomes, hpid- DNA complexes. It may be desirable that a recombinant DNA molecule comprising HGF DNA sequence is operatively linked to an expression control sequence to form an expression vector capable of expressing HGF. The transfected cells may be cells derived from the patient's normal tissue, the patient's diseased tissue, or may be non-patient cells.

For example, tumor cells removed from a patient can be transfected with a vector capable of expressing HGF protem of the present invention, and re- introduced into the patient. The transfected tumor cells produce HGF levels in the patient that inhibit the growth of the tumor. Patients may be human or non-human animals. Cells may also be transfected by non-vector, or physical or chemical methods known in the art such as electroporation, lonoporation, or via a "gene gun." Additionally, HGF DNA may be directly injected, without the aid of a carrier, into a patient. In particular, HGF DNA may be injected into skin, muscle or blood.

The gene therapy protocol for transfectmg a knngle domain containing protein such as HGF into a patient may either be through integration of HGF DNA into the genome of the cells, into mimchromosomes or as a separate replicating or non-rephcating DNA construct in the cytoplasm or nucleoplasm of the cell. HGF expression may continue for a long-period of time or may be reinjected periodically to maintain a desired level of HGF protem m the cell, the tissue or organ or a determined blood level.

The present invention includes methods of treating or preventing angiogemc diseases and processes including, but not limited to, arthritis and tumors by stimulating the production of knngle domain containing proteins and peptides such as HGF fragments, and/or by administering substantially purified HGF fragments, or HGF fragment agonists or antagonists, and/or HGF fragment antisera to a patient. Additional treatment methods include administration of HGF fragments, HGF antisera, or HGF receptor agonists and antagonists linked to cytotoxic agents. It is to be understood that HGF can be animal or human in origin. HGF can also be produced synthetically by chemical reaction or by recombinant techniques in conjunction with expression systems. HGF, and fragments thereof, can also be produced by enzymatically cleaving different molecules, including HGF precursors, containing sequence homology or identity with segments of HGF to generate peptides having anti-angiogenesis activity. The present invention further includes methods of treating or preventing angiogemc diseases and processes including, but not limited to, arthritis and tumors by stimulating the production of kringle domain containing protems and peptides such as MSP fragments, and/or by administering substantially purified MSP fragments, or MSP fragment agonists or antagonists, and/or HGF fragment antisera to a patient Additional treatment methods include administration of HGF, MSP fragments, MSP antisera, or MSP receptor agonists and antagonists linked to cytotoxic agents. It is to be understood that MSP can be animal or human in ongm. MSP can also be produced synthetically by chemical reaction or by recombinant techniques in conjunction with expression systems. MSP, and fragments thereof, can also be produced by enzymatically cleaving different molecules, including MSP precursors, containing sequence homology or identity with segments of MSP to generate peptides having anti-angiogenesis activity.

Antibodies that specifically bind kringle domain containing proteins and peptides such as HGF fragments can be employed to modulate endothehal- dependent processes such as reproduction, development, and wound healing and tissue repair. In addition, antisera directed to the Fab regions of HGF antibodies for example can be administered to block the ability of endogenous HGF antisera to bind HGF fragments Antibodies specific kringle domain containing protems and peptides such as HGF fragments, and HGF fragment analogs, are made according to techniques and protocols well known m the art. The antibodies may be either polyclonal or monoclonal. The antibodies are utilized m well know lmmunoassay formats, such as competitive and non-competitive immunoassays, including ELISA, sandwich immunoassays and radioimmunoassays (RIAs), to determine the presence or absence of the endothehal proliferation inhibitors of the present invention in body fluids. Examples of body fluids include but are not limited to semen, blood, serum, peritoneal fluid, pleural fluid, cerebrospmal fluid, uterine fluid, saliva, and mucus

The protems, nucleic acid sequences and antibodies of the present mvention are useful for diagnosing and treating endothehal cell-related diseases and disorders A particularly important endothehal cell process is angiogenesis, the formation of blood vessels. Angiogenesis-related diseases may be diagnosed and treated using the endothehal cell proliferation inhibiting protems of the present invention Angiogenesis-related diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-Webber Syndrome; myocardial angiogenesis blindness; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation. The endothehal cell proliferation inhibiting proteins of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothehal cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. They are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers {Helicobacter pylorii).

The angiogenic regulating proteins of the present invention can be used as a birth control agent by reducing or preventing uterine vascularization required for embryo implantation. Thus, the present invention provides an effective birth control method when an amount of a kringle domain containing protein composition comprising for example inhibitory HGF fragments sufficient to prevent embryo implantation is administered to a female. In one aspect of the birth control method, an amount of the inhibiting protein sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a "morning after" method. While not wanting to be bound by this statement, it is believed that inhibition of vascularization of the uterine endometrium interferes with implantation of the blastocyst. Similar inhibition of vascularization of the mucosa of the uterine tube interferes with implantation of the blastocyst, preventing occurrence of a tubal pregnancy. Administration methods may include, but are not limited to, pills, injections (intravenous, subcutaneous, intramuscular), suppositories, vaginal sponges, vaginal tampons, and intrauterine devices. It is also believed that administration of the anti-angiogenic compositions of the present invention will interfere with normal enhanced vascularization of the placenta, and also with the development of vessels within a successfully implanted blastocyst and developing embryo and fetus.

Conversely, blockade of kringle domain containing protein receptors with corresponding analogs which act as receptor antagonists, may promote angiogenic activity such as endothelialization and vascularization. Such effects may be desirable in situations of inadequate vascularization of the uterine endometrium and associated infertility, wound repair, healing of cuts and incisions, treatment of vascular problems m diabetics, especially retinal and penpheral vessels, promotion of vascularization in transplanted tissue including muscle and skin, promotion of vascularization of cardiac muscle especially following transplantation of a heart or heart tissue and after bypass surgery, promotion of vascularization of solid and relatively avascular tumors for enhanced cytotoxm delivery, and enhancement of blood flow to the nervous system, including but not limited to the cerebral cortex and spmal cord

The present invention also relates to methods of using angiogemc peptide fragments of kringle domain containing protems and peptides such as HGF, nucleic acid sequences corresponding to HGF fragments, and antibodies that bind specifically to HGF fragments and related peptides, to diagnose endothehal cell- related diseases and disorders

The invention further encompasses a method for identifying kringle domam containing protem-specific receptors, and the receptor molecules identified and isolated thereby The present invention also provides a method for quantitation of such receptors

A particularly important aspect of the present invention is administration of HGF fragments either alone or in combination w ith one or more anti-angiogenic agents, such as ENDOSTATIN protein, ANGIOSTATIN protem, or METASTATIN™ protem (Enfremed, Inc , Rockville, MD), in an amount sufficient to inhibit tumor growth and cause sustainable regression of tumor mass to microscopic size Accordingly, the present invention also includes formulations effective for treating or curing angiogenesis-dependent cancers and tumors

More particularly, recombinant HGF fragments, from insect cells or E coli, for example, can potently inhibit angiogenesis and the growth of metastases It is contemplated as part of the present invention that HGF fragments can be isolated from a body fluid such as semen, blood or urine of patients, or that HGF fragments can be produced by recombinant DNA methods or synthetic peptide chemical methods that are well known to those of ordinary skill m the art Protem purification methods are well known in the art

One example of a method of producing a desired knngle domain containing protem such as HGF fragments using recombinant DNA techniques entails the steps of (1) identifying an HGF fragment as discussed above, and as more fully described below, (2) synthetically generating a DNA ohgonucleotide probe that corresponds to the protem sequence, (3) conducting PCR from human liver cDNA (4) inserting the gene into an appropriate vector such as an expression vector, (5) inserting the gene-contammg vector into a microorganism or other expression system capable of expressing the inhibitor gene, and (6) isolating the recombmantly produced inhibitor The above techniques are more fully described in laboratory manuals such as "Molecular Cloning: A Laboratory Manual" Latest Edition by Sambrook et al., Cold Spring Harbor Press, 1989.

Yet another method of producing desired protems of the present invention such as HGF fragments is by peptide synthesis. For example, once a biologically active fragment of HGF is found, it can be sequenced, for example by automated peptide sequencing methods. Alternatively, once the gene or DNA sequence which codes for HGF fragment is isolated, for example by the methods described above, the DNA sequence can be determined, which in turn provides information regarding the amino acid sequence Thus, if the biologically active fragment is generated by specific methods, such as tryptic digests, or if the fragment is N-terminal sequenced, the remaining ammo acid sequence can be determined from the corresponding DNA sequence.

Once the ammo acid sequence of the peptide is known, for example the N-termmal 20 amino acids, the fragment can be synthesized by techniques well known in the art, as exemplified by "Solid Phase Peptide Synthesis: A Practical Approach" E. Atherton and R.C. Sheppard, IRL Press, Oxford England. Similarly, multiple fragments can be synthesized which are subsequently linked together to form larger fragments. These synthetic peptide fragments can also be made with ammo acid substitutions at specific locations in order to test for agonistic and antagonistic activity in vitro and in vivo.

The synthetic peptide fragments of kringle domain containing protems and peptides such as HGF have a variety of uses. The peptide that binds to the HGF receptor with high specificity and avidity is radiolabeled and employed for visualization and quantitation of binding sites using autoradiographic and membrane binding techniques. Knowledge of the binding properties of the HGF receptor facilitates investigation of the transduction mechanisms linked to the receptor. Different peptide fragments of the mtact HGF molecule can be synthesized for use m several applications including, but not limited to the following; as antigens for the development of specific antisera, as agonists and antagonists active at HGF binding sites, as peptides to be linked to cytotoxic agents for targeted killing of cells that bind HGF. The ammo acid sequences that comprise these peptides are selected on the basis of their position on the exterior regions of the molecule and are accessible for binding to antisera. Peptides can be synthesized in a standard microchemical facility and purity checked with HPLC and mass spectrophotometry. Methods of peptide synthesis, HPLC purification and mass spectrophotometry are commonly known to those skilled in these arts.

HGF kringle containing fragments or peptides can also be produced in recombinant E. coli, or in insect or yeast expression systems, mammalian cell expression systems and transgenic expression systems and purified with column chromatography.

HGF peptides can be chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules and other compounds for a variety of applications. The efficiency of the coupling reaction is determined using different techniques appropriate for the specific reaction.

Systematic substitution of amino acids within the synthesized peptides yields high affinity peptide agonists and antagonists to HGF receptors that enhance or diminish HGF binding to its receptor. Such agonists are used to suppress the growth of primary and metastatic tumors, thereby limiting the spread of cancer. Antagonists to HGF are applied in situations of inadequate vascularization, to block the inhibitory effects of HGF and possibly promote angiogenesis. This treatment may have therapeutic effects to promote wound healing in diabetics.

HGF peptides are employed to develop affinity columns for isolation of the HGF receptor from cultured cells. Isolation and purification of the HGF receptor is followed by amino acid sequencing. Next, nucleotide probes are developed for insertion into vectors for expression of the receptor. These techniques are well known to those skilled in the art. These techniques can be helpful in defining minimal structures of HGF for receptor engagement. Cytotoxic agents, such as ricin, are linked to the kringle domain containing proteins and peptides of the present invention such as HGF kringle fragments and high affinity HGF peptide fragments, thereby providing a tool for destruction of cells that bind HGF. These cells may be found in many locations, including but not limited to, metastases and primary tumors. Peptides linked to cytotoxic agents are infused in a manner designed to maximize delivery to the desired location. For example, ricin-linked high affinity HGF fragments are delivered through a cannula into vessels supplying the target site or directly into the target. Such agents are also delivered in a controlled manner through osmotic pumps coupled to infusion cannulae. A combination of HGF antagonists may be co-applied with stimulators of angiogenesis to increase vascularization of tissue. Antiserum against knngle domam containing protems and peptides such as HGF fragments can be generated. After peptide synthesis and purification, both monoclonal and polyclonal antisera are raised using established techniques known to those skilled in the art. For example, polyclonal antisera may be raised in rabbits, sheep, goats or other animals. HGF peptides conjugated to a carrier molecule such as bovme serum albumin, are combined with an adjuvant mixture, emulsified and injected subcutaneously at multiple sites on the back, neck, flanks, and sometimes in the footpads. Booster injections are made at regular intervals, such as every 2 to 4 weeks. Blood samples are obtained by vempuncture, for example using the marginal ear veins after dilation, approximately 7 to 10 days after each injection The blood samples are allowed to clot overnight at 4°C and are centrifuged at approximately 2400 X g at 4°C for about 30 minutes.

All serum samples from generation of polyclonal antisera or media samples from production of monoclonal antisera are analyzed for determination of titer Titer is established through several means, for example, using dot blots and density analysis, and also with precipitation of radiolabeled peptide-antibody complexes using protein A, secondary antisera, cold ethanol or charcoal -dextran followed by activity measurement with a gamma counter. The highest titer antisera are also purified on affinity columns which are commercially available HGF peptides are coupled to the gel in the affinity column. Antiserum samples are passed through the column and anti-HGF fragment antibodies remain bound to the column These antibodies are subsequently eluted, collected and evaluated for determination of titer and specificity

The highest titer HGF fragment antisera is tested to establish the following; a) optimal antiserum dilution for highest specific binding of the antigen and lowest non-specific binding, b) the ability to bind increasing amounts of HGF peptide in a standard displacement curve, c) potential cross-reactivity with related peptides and protems, including HGF related species, d) ability to detect HGF peptides in extracts of, semen, plasma, urme, tissues, and in cell culture media. According to the present invention, knngle domain containing protem compositions such as HGF fragment compositions, may be used m combination with other compositions and procedures for the treatment of diseases For example, a tumor may be treated conventionally with surgery, radiation or chemotherapy combined with or without HGF fragment compositions and then such compositions may be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize any residual primary tumor It is to be understood that the present mvention is contemplated to include any derivatives of kringle domain contammg protems and peptides that have angiogemc activity. The present invention includes the entire HGF protein including the knngle domains, derivatives of the HGF protem and biologically-active fragments of the HGF protem. These include protems with HGF activity that have ammo acid substitutions or have sugars or other molecules attached to ammo acid functional groups. The present invention also includes genes that code for HGF and HGF receptors, HGF kringle domains, and to protems that are expressed by those genes. The present invention includes the entire MSP protem, derivatives of the MSP protem, MSP knngle fragments, and biologically-active fragments of the MSP protein These include protems with MSP activity that have amino acid substitutions or have sugars or other molecules attached to amino acid functional groups. The present invention also includes genes that code for MSP and MSP receptors as well as MSP kringle domam fragments, and to protems that are expressed by those genes.

The kringle domain containing protein having antiangiogemc activity described above can be provided as isolated and substantially purified protems and protem fragments m pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill m the art These formulations can be administered by standard routes In general, the combinations may be administered by the topical, transdermal, lntrapeπtoneal, mtracramal, mtracerebroventricular, mtracerebral, mtravagmal, mtrauterme, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) route In addition, the protems may be incoφorated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the HGF is slowly released systemically. Osmotic mmipumps may also be used to provide controlled delivery of high concentrations of HGF knngle fragments through cannulae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor. The biodegradable polymers and their use are described, for example, in detail m Brem et al., J Neurosurg 74 441-446 (1991)

The formulations of the present mvention include those suitable for oral, rectal, ophthalmic (including intravitreal or mtracameral), nasal, topical (including buccal and sublmgual), mtrauterme, vaginal or parenteral (including subcutaneous, mtrapentoneal, intramuscular, intravenous, mtradermal, mtracramal, mtratracheal, and epidural) administration. The formulations may conveniently be presented in unit dosage fonn and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carπer(s) or excιpιent(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored m a freeze-dned (lyophihzed) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

The dosage of the compositions of the present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. For example, for treating humans or animals, between approximately 0.5 to 500 mg/kilogram is typical broad range for administering a HGF protein, or a composition comprising kringle domain fragments of HGF. Depending upon the half-life of the protein in the particular animal or human, the protein can be administered between several times per day to once a week It is to be understood that the present invention has application for both human and veterinary use The methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present mvention may include other agents conventional in the art having regard to the type of formulation in question. This mvention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled m the art without departing from the spirit of the present invention and/or the scope of the appended claims.

EXAMPLE 1 Purified human recombinant HGF K2-3 expressed in Pichia pastori (Figure 6)

The HGF K2-3 gene was cloned into pPICZaA and expressed m X33 P pastoris strain. The fermentation supernatant was purified through ion exchange and hydrophobic interaction chromatography. 1 1 μg of purified HGF K2-3 was analyzed on the denaturing and non-reducing SDS-PAGE gel stained with Coomassie blue

EXAMPLE 2 HGF K2-3 inhibits VEGF as well as HGF stimulated HUVEC migration (Figures 7a and 7b)

Micro Chemotaxis Assay Apparatus: Chamber. Neuro Probe Standard 48 Well Chemotaxis Chamber Cat #

AP48 (301-229-8598)

Filter Membrane: Poretics Membrane Polycarbonate PVP Free 8 micron 25 X 80 mm (Osmo cs Inc.800-444-8212 Cat #10474)

Forceps: VWR Scientific 800- 234-9300 Cat# 25681 -269. For ease of handling membranes use these forceps

Preparation of Filter Membrane:

Coat membranes with Rat tail Collagen Type 1 (BD Collaborative Res Cat # 40236) at 100 mg/ml in 29 ml of .2N Acetic Acid. For .2N Acetic Acid add 345 ml of Glacial Acetic Acid (99%) in 29.66 sterile H20 (BRL).

1. Dilute collagen (100 mg/ml) in .2N Acetic Acid in 50 ml conical tube. Mix well.

2. Place collagen solution in either a petn dish or small plastic staining box Submerge membranes individually (10 membranes). 3. Agitate slowly on rocker at room temperature for 48 hours 4. Air dry filters in laminar flow hood by laying out filters on open petπ dishes.

5. Store dry filters in covered container at room temperature (2 weeks).

Assay:

Assay Media: Medium -200 supplemented with 1% L-glutamine (BioWhittaker) and 0.1 % BSA (Sigma Cat# A8412)

1. Harvest HUVEC p2-p7 from flasks by trypsinization. Neutralize trypsm with growth medium Remove trypsm/versene/media by centrifugation at 1000 φm for 5 minutes Resuspend in 10 ml of assay media. Do not over trypsmize cells or over centrifuge cells.

2. Count cells by dilution with Trypan Blue solution. Determine viability. Resuspend cells at 2 X 105/ml m assay media.

3. Before adding cells to chambers, premcubate cells with test proteins in 17X100 mm (14ml) polypropylene round bottom tube (Falcon 35-2059) for 30 minutes at 37°C with 5% CO2. Do not snap top of tube. Incubate control cells with assay media.

4. Approximately 5 minutes before addition of cells prepare chamber. To the first three columns of the bottom chamber add approximately 28 ml of assay media alone. This volume varies with individual chambers from 25-30 ml. Adjust volume so that a slight positive meniscus is formed over well. Fill rest of wells with chemoattractant (e.g., VEGF165 R&D Cat #293-VE) 2-10 ng/ml. Reconstitute VEGF in assay media Optimum concentration depends on cells. The last three columns are stimulated controls. 5. All manipulations with membranes are done with forceps. Cut off small corner of membrane and orient cut off corner with NP trademark Place membrane on top of lower chamber shiny side up. Do not adjust membrane as this will contaminate wells.

6. Gently place sihcone gasket on top of membrane. Then place the top portion of the chamber oriented with the trademark. With firm even pressure screw on the thumb nuts tightly. Note: Cells will leak if this is not done correctly.

7. Add 50 ml of either control cells or treated cells to upper chambers. To avoid bubbles lift pipette tip while adding cells. Be careful not to puncture membrane. 8. Place chamber in 150mm petn dish with moist paper towel. Incubate chamber or 6 h at 37°C with 5% CO2. 9. After 6 hours, gently remove thumb nuts and upper chamber. Peel off membrane which is stuck to the gasket with forceps.

10. Fix and stain filter membrane with Diff-Quik (Dade Int. Cat# 84132-10) Fix for 2 minutes; Solution I for 2 minutes; Solution II for 3 minutes. 11. Rmse membrane 2X in distilled H2O Place membrane on top of 3

X 2 glass microscope slide (VWR Cat # 28351-100). With a wet Kim- Wipe remove non-migratmg cells while holding onto membrane to prevent movement After removal of cells allow membrane to dry Place 4 small drops of super-glue (use pipette tip on each corner of the slide. Place additional slide on top. View under microscope to determine total number of migrated cells

The migration of HUVECs was evaluated with the micro chemotaxis chamber (modified Boyden chamber, Neuro Probe, Inc , Gaithersburg, MD) described above which allows for the measurement of cell movement and directionality HUVECs pretreated with HGF K2-3 or basal media (M-200 containing 0.1% BSA) for 30' at 37°C were placed into the upper chamber The lower chamber contained basal media with (a) VEGF (5 ng/ml) or (b) HGF (50 ng/mL) or without. Cells were allowed to migrate through an 8 mm polycarbonate PVP free filter coated with 100 μg/ml of rat tail collagen type 1 (Collaborative Biomedical Products, Bedford, MA) The chamber was then incubated as above for 6 h The non-migrated cells were removed and the filter was fixed and stained with Diff-Quik (Dade Diagnostics, Aquado, Puerto Rico) The numbers of migrated cells were determined using the Image-Pro Plus analysis system (Media Cybernetics, Silver Spring, MD). BSA at 100 μg/ml showed no inhibition effect on the growth factor stimulated HUVEC migration (data not shown).

EXAMPLE 3 HGF K2-3 inhibits HUVEC tube formation. (Figure 8)

HUVECs were trypsimzed and resuspend at 1X10 5 cells/ml in the assay media [medium -200 supplemented with LSGS and 5% heat inactivated FBS (Hyclone)]. 100 ml of HGFK2-3 dilution in assay and 100 ul of HUVECs were plated with onto a Matngel substratum(Collaboratιve Biomedical Products, Bedford, MA) for 16 h at 37°C. After incubation, endothehal cells were examined microscopically and evaluated for tube formation by counting the number of junctions. BSA at 200 μg/ml showed no inhibition effect (data not shown) EXAMPLE 4 Angiostatin protein inhibits HGF stimulated HUVEC migration. (Figure 9)

The migration of HUVECs was evaluated with a micro chemotaxis chamber (modified Boyden chamber, Neuro Probe, Inc., Gaithersburg, MD). HUVECs prefreated with Angiostatin protem or basal media (M-200 containing 0.1 %

BSA) for 30' at 37°C were placed into the upper chamber. The lower chamber contained basal media with HGF (50 ng/ml) or without. Cells were allowed to migrate through an 8 mm polycarbonate PVP free filter coated with 100 mg/ml of rat tail collagen type 1 (Collaborative Biomedical Products, Bedford, MA). The chamber was then incubated as above for 6 h. The non-migrated cells were removed and the filter was fixed and stained with Diff-Quik (Dade Diagnostics, Aquado, Puerto Rico). The numbers of migrated cells were determined using the Image-Pro Plus analysis system

(Media Cybernetics, Silver Spring, MD). BSA at 100 ug/ml showed no inhibition effect on the growth factor stimulated HUVEC migration (data not shown).

EXAMPLE 5 Angiostatin protein does not induce c-met phosphorylatwn (Figu e 1 )

A549 cells were starved in serum free media plus 0.1 % BSA for 6 hours and then treated with Angiostatin protem or HGF for 10 mm. at 37 o C. The cells were lysed and the supernatant was then immunoprecipated with 15 ul of rabbit anti-c-met antibodies and analyzed with Western blots probed with anti-phosphotyrosine antibody (4G10) or anti-c-met antibodies.

EXAMPLE 6 Angiostatin protein does not inhibit HGF stimulated c-met phosphorylation (Figure

H) The HUVECs were starved in serum free media plus 0.1% BSA for 6 hours and then treated with Angiostatin protem at 4° C for 30 mm. The cold media was replaced with fresh media (37° C) with the same concentration of Angiotstatm and then induced with 20 ng/ml HGF for 10 mm. at 37° C. The cells were lysed and the supernatant was then immunoprecipated with 15 ul of rabbit anti-c-met anti-bodies and analyzed with Western blots probed with anti-phosphotyros e antibody (4G10) or anti-c-met antibodies

Claims

We Claim

1 A method of inhibiting angiogenesis in an animal comprising administering to the animal an angiogenesis inhibiting amount of a composition compnsmg knngle domain-containing protems and peptides

2 The method of Claim 1, where kringle domain containing protems and peptides comprise hepatocyte growth factor, macrophage stimulating protem, and biologically active fragments thereof

3 The method of Claim 2, wherem the hepatocyte growth factor has the amino acid sequence as set forth in SEQ ID NO 1 , or an antiangiogemc fragment thereof

4 The method of Claim 1 , further comprising a pharmaceutically acceptable excipient

5 The method of Claim 1 , wherem the composition further comprises the peptide having the ammo acid sequence set forth m SEQ ID NO 2

6 The method of Claim 1 , wherem the composition further comprises the peptide having the ammo acid sequence set forth in SEQ ID NO 3

7 The method of Claim 1 , wherem the animal has an angiogenesis-mediated disease selected from the group consisting of angiogenesis- dependent cancers, benign tumors, rheumatoid arthritis, psoriasis, ocular angiogenesis diseases, Osier- Webber Syndrome, myocardial angiogenesis, plaque neovasculanzation, telangiectasia, hemophiliac joints, angiofibroma, wound granulation, intestinal adhesions, atherosclerosis, scleroderma, hypertrophic scars, cat scratch disease and Hehcobacter pylori ulcers

8 A method of inhibiting cell proliferation compnsmg, administering to a cell undergoing proliferation a proliferation inhibiting amount of a composition comprising biologically active fragments of HGF to inhibit cell proliferation

9. The method of Claim 8, wherem the cell proliferation comprises endothehal cell proliferation and smooth muscle cell proliferation.

10. The method of Claim 8, wherem the kringle domain containing protems and peptides comprise hepatocyte growth factor, macrophage stimulating protem, and biologically active fragments thereof.

1 1 The method of Claim 8, wherem the hepatocyte growth factor has the ammo acid sequence set forth in SEQ ID NO: 1, or an antiproliferative fragment thereof.

12. The method of Claim 1 1 , wherem the composition further comprises the peptide having the ammo acid sequence set forth in SEQ ID NO: 2

13 The method of Claim 1 1 , wherem the composition further comprises the peptide having the amino acid sequence set forth m SEQ ID NO: 3

14 The method of Claim 1 1 , wherein the cell proliferation is related to an angiogenesis-mediated disease.

15 The method of Claim 14, wherem the angiogenesis-mediated disease is selected from the group consisting of angiogenesis-dependent cancers, benign tumors, rheumatoid arthritis, psoriasis, ocular angiogenesis diseases, Osler- Webber Syndrome, myocardial angiogenesis, plaque neovasculanzation, telangiectasia, hemophiliac joints, angiofibroma, wound granulation, intestinal adhesions, atherosclerosis, scleroderma, hypertrophic scars, cat scratch disease and Helicobacter pylori ulcers.

16 A method of diagnosing a disease or determining the prognosis of a disease mediated by angiogenesis comprising obtaining a biological sample and determining the levels of HGF in the sample.

17 The method of Claim 16, wherem the knngle domain containing protems and peptides comprise hepatocyte growth factor, macrophage stimulating protem and biologically active fragments thereof.

18. The method of Claim 17 wherein the hepatocyte growth factor comprises the amino acid sequence as set forth in SEQ ID NO: 1, or an anti- angiogenic fragment thereof.

19. The method of Claim 17 wherein the macrophage stimulating protein comprises the amino acid sequence as set forth in SEQ ID NO: 4, or an anti- angiogenic fragment thereof.

20. The method of Claim 16, wherein the angiogenesis-mediated disease is selected from the group consisting of angiogenesis-dependent cancers, benign tumors, rheumatoid arthritis, psoriasis, ocular angiogenesis diseases, Osier- Webber Syndrome, myocardial angiogenesis, plaque neovasculanzation, telangiectasia, hemophiliac joints, angiofibroma, wound granulation, intestinal adhesions, atherosclerosis, scleroderma, hypertrophic scars, cat scratch disease and Helirobacter pylori ulcers.