MXPA99011041A - Angiostatin fragments and method of use - Google Patents

Abstract

Se proporcionan fragmentos de un inhibidor de proliferación celular endotelial y el método de uso de los mismos. El inhibidor de proliferación endotelial es una proteína derivada del plasminógeno o más específicamente es un fragmento de angiostatina. Los fragmentos de angiostatina corresponden, en general a estructuras kringle que se presentan dentro del inhibidor de proliferación celular endotelial. La actividad inhibidora de la célula endotelial de estos fragmentos proporciona un medio para inhibir la angiogénesis de tumores y para tratar enfermedades mediadas por angiogénesis.

Description

ANGIOSTATIN FRAGMENTS AND METHOD OF USING THESE Field of the Invention The present invention relates to endothelial inhibitors, called angiostatin, which reversibly inhibit the proliferation of endothelial cells. More particularly, the present invention relates to angiostatin proteins that can be isolated from body fluids, such as blood or urine, or can be synthesized by recombinant, enzymatic or chemical methods. Angiostatin is capable of inhibiting angiogenesis related to diseases and modulating angiogenic processes. In addition, the present invention relates to tests and diagnostic equipment for the measurement of angiostatin, with histochemical equipment for the localization of angiostatin, with the DNA sequences coding for angiostatin and molecular probes to verify the biosynthesis of angiostatin, with antibodies that are specific for angiostatin, with the development of agonists and protein antagonists for the angiostatin receptor, with agonists and antagonists specific to the angiostatin receptor, and with cytotoxic agents linked to angiostatin proteins.

Background of the Invention As used herein, the term "angiogenesis" means the generation of new blood vessels in a tissue or organ. Under normal physiological conditions, humans or animals experience angiogenesis only in restricted, very specific situations. For example, angiogenesis is observed normally in wound healing, in fetal and embryonic development and the formation of the corpus luteum, endometrium and placenta. The term "endothelium" means a thin layer of flat epithelial cells that lines the serous cavities, lymphatic vessels and blood vessels. It is thought that controlled and uncontrolled angiogenesis both proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which cover the lumen of the blood vessels, then project through the basement membrane. The angiogenic stimulants induce the entodetal cells to migrate through the eroded basal membrane. Igneous cells form a "bud" outside the blood vessel of origin, where the endothelial cells undergo mitosis and proliferate.

The endothelial buds fuse together to form capillary rings, creating the new blood vessel. Unregulated, persistent angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells and supports the pathological damage observed in those conditions. The various states of pathological diseases in which unregulated angiogenesis is present have been grouped as angiogenic dependent or angiogenic associated diseases. The hypothesis that the growth of a tumor depends on angiogenesis was first proposed in 1971. (Folkman J., Tumor Angiogenesis: Therapeutic Implications., N. Engl. Jour. Med. 285: 1182 1186, 1971). In its simplest terms it states that: "Once the tumor has" contracted ", each increase in the population of the tumor cells must be preceded by an increase in new capillaries that converge on the tumor." It is commonly understood that "contracting" a tumor indicates a prevascular phase of tumor growth, in which a population of tumor cells occupying a volume of a few cubic millimeters and not exceeding a few million cells, can survive or exist in guest microvessels. The expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels. For example, lung micrometastases in the initial prevascular phase in mice would be detectable except by high power microscopy over sections or histological sections. Examples of indirect evidence supporting this concept include: (1) The rate of growth of tumors implanted in transparent subcutaneous chambers in mice, it is slow and linear before neovascularization, and rapid and almost exponential after neovascularization. (Algire GH, et al.) Vascular reactions of normal and malignant tumors in vivo I. Vascular reactions of mice to wounds and to normal and neoplastic transplants, J. Na ti.Cancer Inst. 6: 73-85, 1945) (2) The growth of tumors in isolated perfused organs, where blood vessels do not proliferate, is limited to 1-2 mm3 but expands rapidly to >; 1000 times this volume when they are transplanted to mice and are neovascularized. (Folkman J, et al., Tumor behavior in isolated perfused organs: In vi tro growth and metastasis of biopsy material in canine rabbit and intestinal thyroid segments Annals of Surgery 164: 491-502, 1966) (3) The tumor growth in the avascular cornea proceeds slowly and at a linear speed, but changes to an exponential growth after neovascularization. (Gimbrone, MA, Jr. et al., Tumor neovascularization and growth: An experimental model using the rabbit cornea, J. Na ti, Cancer Institute 52: 41-427, 1974) (4) Tumors suspended in the fluid aqueous from the anterior chamber of the eye of the rabbit, remain viable, avascular and limited in size to < 1 mm3. Once they are implanted on the vascular bed of the iris, they are neovascularized and grow rapidly, reaching 16,000 times their original volume in 2 weeks. (Grimbrone MA Jr., et al., Latency of a tumor in vivo by preventing neovascularization J. Exp. Med. 136: 261-276) (5) When tumors are implanted on the chorioallantoic membrane of the embryo of chicken, they grow slowly during an avascular phase of > 72 hours, but do not exceed an average diameter of 0.93 • + 0.29 mm. The rapid expansion of the tumor occurs within 24 hours after the onset of neovascularization, and by day 7 these vascularized tumors reach an average diameter of 8.0 + 2.5 mm. (Knighton D., Avascular and Vascular Phases of Tumor Growth in the Chicken Embryo Bri Tish J. Cancer, 35: 347-356, 1977) (6) Vascular forms of metastases in rabbit liver reveal heterogeneity in size of metastases, but they show a relatively uniform cutoff of the size at which vascularization is present. The tumors are generally avascular up to 1 mm in diameter, but are neovascularized beyond that diameter. (Lien., Et al., Blood supply of experimental liver metastases, II, A microcirculatory study of normal and tumorous liver vessels with the use of perfused silicone rubber, Surgery 68: 334-340, 1970) (7 ) In transgenic mice, which develop carcinomas in the beta cells of the pancreatic islets, the pre-vascular hyperplastic islets are limited in size to < lmm At 6-7 weeks of age, 4-10% of the islets are neovascularized, and from these islets large vascularized tumors arise, more than 1000 times the size of the pre-vascular islets. (Folkman J, et al., Induction of angiogenesis during the transition from hyperplasia to neoplasia Na ure 339: 58-61, 1989) (8) A specific antibody against VEGF (vascular endothelial growth factor) reduces density of microvessels and produces a "significant or dramatic" inhibition of the growth of three human tumors, which depend on VEGF as their sole mediator of angiogenesis (in nude mice). The antibody does not inhibit the growth of tumor cells in vi tro. (Kim KJ, et al., Inhibition of angiogenesis induced by vascular endothelial growth factor suppresses tumor growth in vivo Na ture 362: 841-844, 1993) (9) Anti-bFGF monoclonal antibody causes 70 % inhibition of mouse tumor growth, which depends on the secretion of bFGF as the sole mediator of angiogenesis. The antibody does not inhibit the growth of tumor cells in vi tro. (Hori A, et al., Suppression of the growth of a solid tumor by immunoneutralizing monoclonal antibody against the growth factor of human basic fibroblasts Cancer Research, 51: 6180-6184, 1991) (10) Intraperitoneal injection of bFGF increases the growth of a primary tumor and its metastasis by stimulation of the growth of capillary endothelial cells in the tumor. The tumor cells themselves lack receptors for bFGF, and bFGF is not a mitogen for tumor cells in vi tro. (Gross JL, et al.) Modulation of the growth of a solid tumor in vivo by bFGF, Proc. Amer. Assoc. Canc. Res. 31:79, 1990) (11) A specific inhibitor of angiogenesis (AGM-1470) it inhibits tumor growth and metastasis in vivo, but is much less active in inhibiting proliferation in vi tro. It inhibits the proliferation of vascular cells up to half of 4 logs less than the concentration that inhibits the proliferation of tumor cells. (Ingber D, et al., Angioinhibinos: Synthetic analogs of fumagil which inhibits angiogenesis and suppresses tumor growth Na ure, 48: 555-557, 1990). There is indirect ccal evidence that tumor growth depends on angiogenesis. (12) Human retinoblastomas that are metastatic to vitreous develop into avascular spheroids, which are restricted to less than 1 mm3, despite the fact that they are viable and incorporate 3H-thymidine (when they are removed from the enucleated eye and analyzed in vi tro). (13) Carcinoma of ovarian metastasis to the peritoneal membrane as small white avascular seeds (1-3 mm3). These implants rarely grow more, until one or more of them become vascularized. (14) The intensity of neovascularization in breast cancer (Eidner N, et al., Tumor angiogenesis correlates with invasive breast metastasis N. Engl. J. Med. 324: 1-8, 1991, and Weidner N, et al., Tumor Angiogenesis: A New and Significant Independent Prognosis, Indicator of the Initial Stage of Breast Carcinoma, J Na ti, Cancer Inst. 84: 1875-1887, 1992) and in Prostate Cancer (Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis and invasive prostate carcinoma American Journal of Pa thology, 143 (2): 401-409, 1993) highly correlate with risk of future metastases. - (15) Metastasis of human cutaneous melanoma is rare before neovascularization. The appearance of neovascularization leads to an increase in the thickness of the lesion and an increase in the risk of metastasis. (Srivastava A, et al., The meaning of the prognosis of tumor vascularity of intermediate-thickness skin melanoma (thickness 0.76-4.0 mm) Amer. J. Pa thol 133: 419-423, 1988) (16) Cancer of the bladder region, the urinary level of the angiogenic protein, bFGF, is a more sensitive indicator of the state and extent of the disease than its cytology. (Nguyen M, et al., Elevated levels of an angiogenic protein, basic fibroblast growth factor, in urine of * patients with bladder cancer, J. Na ti, Cancer Inst. 85: 241-242, 1993) Thus, it is clear that angiogenesis plays a major role in the metastasis of a cancer. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In the disease state, the prevention of angiogenesis could avoid what is caused by the invasion of the new microvascular system. Therapies aimed at the control of angiogenic processes could lead to the abrogation or mitigation of these diseases. What is needed, therefore, is a composition and method that can inhibit the undesirable growth of blood vessels, especially in tumors. A method is also needed to detect, measure, and locate the composition. The composition should be capable of overcoming the activity of endogenous growth factors and pre-elastoic tumors and preventing the formation of capillaries in tumors, thereby inhibiting the growth of tumors. The composition, fragments of the composition, and antibodies specific for the composition, should also be capable of modulating the formation of capillaries in other angiogenic processes, such as wound hea and reproduction. The composition and method to inhibit angiogenesis should, preferably not be toxic and produce few side effects. A method is also needed to detect, measure, and locate the defined sites for the composition, as well as the biosynthesis sites of the composition. The composition and fragments of the composition should be capable of being conjugated with other molecules for radioactive and non-radioactive marking purposes.

Brief Description of the Invention In accordance with the present invention, there are provided compositions and methods that are effective to modulate angiogenesis, and inhibit undesirable angiogenesis, especially angiogenesis related to tumor growth. The present invention includes a protein, which has been named "angiostatin", defined by its ability to overcome the angiogenic activity of growth factors such as bFGF, in vi tro, and by its homologous amino acid sequence and structural similarity with a internal portion of the plasminogen starting approximately at a plasminogen amino acid 98. Angiostatin comprises a protein having a molecular weight between approximately 38 kilodaltons and 45 kilodaltons, as determined by reductive electrophoresis on polyacrylamide gel and having an amino acid sequence substantially similar to that of a murine plasminogen fragment starting with the amino acid of number 98 of an intact murine plasminogen molecule (SEQ ID NO: 2). The amino acid sequence of angiostatin varies slightly between species. For example, in human angiostatin, the amino acid sequence is substantially similar to the sequence of the plasminogen fragment described above, although an active human angiostatin sequence can start at amino acid number 97 or 99 of the amino acid sequence of intact human plasminogen . In addition, human plasminogen fragments have anti-angiogenic activity similar to that shown in a mouse tumor model. It should be understood that the number of amino acids in the active angiostatin molecule may vary, and that all amino acid sequences having endothelial inhibitory activity are contemplated to be included in the present invention. The present invention provides methods and compositions for treating diseases and processes mediated by unwanted and uncontrolled angiogenesis, by administration to a human or animal, of a composition comprising angiostatin or substantially pure angiostatin derivative in a dose sufficient to inhibit Angiogenesis The present invention is particularly useful for suppressing tumor growth. The administration of angiostatin to animals with prevascularized metastatic tumors will prevent the growth of these tumors. The present invention also encompasses DNA sequences encoding angiostatin, containing expression vectors in the DNA sequence with which they encode angiostatin, and cells containing one or more expression vectors of DNA sequences encoding angiostatin. . The present invention also encompasses methods of gene therapy, wherein the DNA sequences encoding angiostatin are introduced into a patient to modify angiostatin levels produced in vivo. The present invention also includes methods and diagnostic equipment for the detection and measurement of angiostatin in biological fluids and tissues, and for the localization of angiostatin in tissues and cells. The method and diagnostic equipment can be of any configuration known to those skilled in the art. The present invention also includes antibodies specific for the angiostatin molecule and portions thereof, and antibodies that inhibit the binding of antibodies specific for angiostatin. These antibodies can be polyclonal antibodies, monoclonal antibodies. The antibodies specific for angiostatin can be used in diagnostic equipment to detect the presence and quantity of angiostatin, which diagnoses or predicts the occurrence or recurrence of cancer and other diseases mediated by angiogenesis. Antibodies specific for angiostatin can also be administered to humans or animals to passively immunize the human or animal against angiostatin, thereby reducing angiogenic inhibition. The present invention also includes methods and diagnostic equipment for detecting the presence and amount of antibodies that bind to angiostatin in body fluids. The diagnostic method and equipment may be in any configuration well known to those skilled in the art. The present invention also includes specific angiostatin receptor-receptor antibodies that bind to the angiostatin receptor and transmit the appropriate signal to the cell and act as agonists or antagonists. The present invention also includes angiostatin protein fragments and analogs that can be labeled isotopically or with other molecules or proteins for use in the detection and visualization of angiostatin binding sites with techniques, including, but not limited to, tomography. of positron emission, autoradiography, flow cytometry, radioreceptor binding assays, and immunohistochemistry. These angiostatin proteins and analogues also act as agonists and antagonists at the angiostatin receptor, thereby increasing or blocking the biological activity of angiostatin. Such proteins are used in the isolation of the angiostatin receptor. The present invention also includes angiostatin, angiostatin fragments, angiostatin antiserum, or angiostatin receptor agonists and angiostatin receptor antagonists derived from cytotoxic agents for therapeutic and research applications. Moreover, angiostatin, angiostatin fragments, angiostatin antiserum, angiostatin receptor agonists, and angiostatin receptor antagonists are combined with pharmaceutically acceptable excipients, and optionally sustained release compounds or compositions, such as degradable polymers, to form compositions therapeutic The present invention includes molecular probes for ribonucleic acid and deoxyribonucleic acid involved in the transcription and translation of angiostatin. These molecular probes provide means to detect and measure the biosynthesis of angiostatin in tissues and cells. Accordingly, the object of the present invention is to provide a composition containing an angiostatin. Another object of the present invention is to provide a method for treating diseases and processes that are mediated by angiogenesis. Still another object of the present invention is to provide a diagnostic or prognostic method and equipment for detecting the presence and amount of angiostatin in a body fluid or tissue. Yet another object of the present invention is to provide a method and composition for treating diseases and processes that are mediated by angiogenesis, including, but not limited to, hemangioma, solid tumors, tumors formed by the blood, leukemia, metastasis, telangiectasia. , psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, platelet neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, neovascularization diabetic, macular degeneration, scarring of epidermis, peptic ulcer, Helicobacter-related diseases, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstruation, placentation, and cat scratch fever. Another object of the present invention is to provide a composition for treating or suppressing cancer growth. Another object of the present invention is to provide compounds that modulate or mimic the production or activity of enzymes that produce angiostatin in vivo or in vi tro. Still another object of the present invention is to provide angiostatin or angiostatin antibodies by direct injection of angiostatin DNA into a human or animal in need of such angiostatin or angiostatin antibodies. It is an object of the present invention to provide a method for detecting and quantifying the presence of an antibody specific for angiostatin in a body fluid. Still another object of the present invention is to provide a composition consisting of antibodies to angiostatin that are selective for the specific regions of the angiostatin molecule that do not recognize plasminogen. Another object of the present invention is to provide a method for the detection or prognosis of cancer. Another object of the present invention is to provide a composition for use to visualize and quantify angiostatin binding sites in vivo and in vi tro. Still another object of the present invention is to provide a composition for use in the detection and quantification of angiostatin biosynthesis. Still another object of the present invention is to provide a therapy for cancer that has minimal side effects. Still another object of the present invention is to provide a composition comprising angiostatin or an angiostatin protein linked to a cytotoxic agent., to treat or suppress the growth of a cancer. Another object of the present invention is to provide a method for targeted delivery of compositions related to angiostatin to specific sites.

Still another object of the present invention is to provide compositions and methods useful for gene therapy for the modulation of angiogenic processes. These and other objects, features and advantages of the present invention will become apparent upon review of the following detailed description of the described embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows SEQ ID NO: 1, the amino acid sequence of the whole murine plasminogen. Figure 2 shows the start of the murine angiostatin sequence (SEQ ID NO: 2) and compares the murine sequence with the human plasmogenic protein fragments (SEQ ID NO: 3), from Rhesus monkey (SEQ ID NO:) , porcine (SEQ ID NO: 5) and bovine (SEQ ID NO: 6) corresponding. The sequence of a mouse is listed, followed by human, rhesus, porcine and bovine. Figure 3 shows the BrdU labeling index of tumor cells in the lung in the presence or absence of a primary tumor. Figure 4 shows the Matrigel analysis of the influence of a primary Lewis lung tumor on bFGF-directed angiogenesis in vivo.

Figure 5 shows a dose response curve for serum derived from mice with lung carcinoma of Lewis (LLC-Low) versus the serum of normal mice. Bovine endothelial cells were tested in a 72-hour proliferation assay led by bFGF. Figure 6 shows that both tumors of high and low metastasis contain mitogenic endothelial activity in their ascites, but only the tumor line of low metastasis has endothelial inhibitory activity in the serum. Figure 7 shows a C4 Inverted Phase Cromatographic profile of partially purified serum or urine from animals with tumor. Figure 8 shows the superficial pulmonary metastasis after 13 days of treatment of mice with intact plasminogen molecule, an active fraction of a preparation of lysine I binding site of human plasminogen, concentrated urine of mice with tumor and concentrated urine of normal mice. Figure 9 shows lung weight after day 13 of treatment of mice with human plasminogen intact plasminogen molecule, lysine I binding sites active fraction, concentrated urine from mice with tumor and concentrated urine from normal mice.

Figure 10 is a schematic representation of the pTrcHis vector. Figure 11 describes an immunostain of human angiostatin expressed from E.coli from a 10L scaled fermentation, tested with a monoclonal antibody against the Kringle region of human plasminogen 1-3. The arrow shows recombinant human angiostatin. A) shows the recombinant angiostatin eluted with 0.2 M aminocaproic acid; B) shows the last wash with 1 X of PBS in the lysine column; and C) shows the cleared lysate of broken cells. Figure 12. It is a graph describing the percent inhibition of bovine endothelial cell growth as a function of the dilution of a standard; Al, A2, Bl, B2, and E are recombinant clones that express anti-angiogenesis activity of human angiostatin; the Cl, C2, Dl and D2 controls are clones of negative controls that contain a vector only without the human DNA sequence encoding angiostatin. Figure 13 shows an inhibitory effect on the proliferation of recombinant human angiostatin on bovine capillary endoletial cells in vi tro. Figure 14 shows the rate of growth proliferation and the apoptotic index after removal of the primary tumor and treatment with saline or a fumagillin analog with antiangiogenic activity. Figure 15 shows the inhibition and growth of a T241 primary tumor in mice, by treatment with human angiostatin in vivo with a single injection of 40 mg / kg / day. Figure 16 shows the inhibition and growth of a primary LLC-LM tumor in mice by treatment with human angiostatin in vivo at two doses of 10 mg / kg per dose (80 mg / kg / day). Figure 17 shows the effect of the removal of a primary tumor of Lewis lung carcinoma on the growth of pulmonary metastases. Figure 18 shows growth proliferation and apoptotic index after tumor dissection. Figure 19 shows the effect of administration of the angiostatin protein to mice implanted with T241 fibrosarcoma cells on the total volume of the tumor as a function of time. Figure 20 shows the effect of administration of the angiostatin protein to mice that have implanted lung carcinoma cells from Lewis (LM) on the total volume of the tumor as a function of time.

Figure 21 shows the effect of administering the angiostatin protein to mice having cellular reticulum sarcoma cells implanted on the total volume of the tumor as a function of time. Figure 22 shows the effect of administering the angiostatin protein to immunodeficient SCID mice having human PC-3 prostate carcinoma cells implanted on the total tumor volume, as a function of time over a period of 24 days. Figure 23 shows the effect of administration of the angiostatin protein to immunodeficient SCID mice having human MDA-MB brain carcinoma cells implanted on the total tumor volume as a function of time over a period of 24 days. Figure 24 is a schematic representation of the cloning of the mouse DNA sequence encoding the mouse angiostatin protein derived from the mouse plasminogen cDNA. Mouse angiostatin encompasses the kringle regions of mouse plasminogen 1-4. PCR stands for polymerase chain reaction; Pl is the oligonucleotide value of the 5 'end for PCR; P2 is the first value of the 3 'end oligonucleotide for PCR; SS designates the signal sequence; ATG is the codon of the start of the translation; TAA is the codon for the purpose of translation; HA represents the epitope mark of hemagglutinin (YPYDVPDYASL); Kl, K2, K3 and K4 represent the kringle regions of mouse plasminogen 1, 2, 3 and 4 respectively. CMV is the cytomegalovirus promoter; T7 is the bacterial phage promoter; PA represents the preactivation proteins; and SP6 is the Sp 6 promoter. Figure 25 describes the number of cells as a function of days for non-transfected cells (mock); cells transfected with the vector alone, without the DNA sequence coding for angiostatin (Vector 5), and two clones expressing angiostatin (AST 31 and AST 37). Panel (a) represents the results of the transfection of T241 cells. Panel (b) represents the results of LL2 cells. Figure 26 shows the results of a culture medium derived from E. coli cells containing angiostatin clones on the number of cells. The non-transfected cells (mock); cells transfected with the vector alone, without the DNA sequence coding for angiostatin (Vector 5), and three clones expressing angiostatin (AST 25, AST 31 and AST 37).

Panel (a) represents the results of the incubation of the culture medium of control clones (mock) and angiostatin (expression and non-expression) on the number of cells. Panel (b) represents the results of the incubation of the culture medium of control clones (mock), vector only (vector 6) and angiostatin clones expressing mouse angiostatin on the number of cells. Panel (c) represents the results of the incubation of the purified culture medium of the control (mock) clones and angiostatin expressing mouse angiostatin on the number of cells, where the culture medium was purified on a lysine column. sepharose to produce the components that bind to lysine. Figure 27 shows the effect on total tumor volume as a function of the time of T241 fibrosarcoma cell implantation in mice, where the fibrosarcoma cells have been transfected with a vector containing a DNA sequence encoding angiostatin protein , and where the vector is able to express the angiostatin protein. "Non-transfected" represents undisturbed T241 fibrosarcoma cells implanted in mice. "Vector 6" represents T241 fibrosarcoma cells transfected with the vector alone, which does not contain the DNA sequence encoding the angiostatin protein, implanted in mice. The "Clona 25, Clona 31 and Clone 37"represent three angiostatin-producing clones of T241 fibrosarcoma cells transfected with a vector containing the DNA sequence encoding the angiostatin protein implanted in mice, Figure 28 shows a schematic representation of the structure of human plasminogen and its kringle fragments Human plasminogen is a single chain protein containing 791 amino acids with a side of N-linked glycosylation at Asn289. The non-human plasminogen protease region consisting of 561 N-terminal amino acids exists in five separate domains, known as kringles as shown in the circles (Kl, K2, K3, K4 and K5), together with the proteins that separate those structures. Each kringle bound to triple disulfide contains 80 amino acids. Angiostatin covers the first 4 of these domains kringle (Kl-4), kringle 3 (Kl-3) and kringle 4 (K4), obtained by digestion of human plasminogen with elastase. The rest of the kringle fragments are recombinant proteins expressed in E. coli. SS = signal sequence. PA = preactivation protein. Figure 29 shows an SDS-PAGE analysis of recombinant and native kringle fragments purified from plasminogen, under reducing conditions. (A) Individual recombinant kringle fragments purified from bacterial lysates of E. coli were loaded onto gel with 15% SDS, followed by staining with Coomassie blue. Approximately 5 μg of each protein was loaded per lane, (lane 2 = ringle 1 (Kl); lane 3 = kringle 2 (K2); lane 4 = kringle 3 (K3); lane 5 = kringle 4 (K4); lane 1 = molecular weight markers). (B) The purified large kringle fragments were stained with Coomassie blue. Kringles 1-4 (lane 2) and kringles 1-3 (lane 3) were obtained by the digestion of human plasminogen with elastase and purified by lysine-Sepharose chromatography. The recombinant fragment of kringles 2-3 (lane 4) was expressed in E. coli and duplicated again in vi tro. Molecular weight markers are indicated on the left (lane 1). Figure 30 shows an inhibition of endothelial cell proliferation by individual recombinant kringle fragments of angiostatin. The kringle fragments were tested on bovine capillary endothelial cells in the presence of 1 ng / ml of bFGF for 72 hours. (A) Proliferative effects of two kringles antiendothelial cells that bind to lysine, rKl and rK4. The high-affinity lysine binding kringle, Kl (-o-), inhibited the proliferation of BCE cells in a dose-dependent manner. The intermediate affinity lysine binding kringle, K4 (- • -), showed very little inhibitory effect at high concentrations. (B) Inhibition of proliferation of BCE cells by K2 and K3 that do not bind to lysine. Both K2 (- • -) and K3 (-D-) inhibited the proliferation of BCE cells in a dose-dependent manner. The data represents the mean +/- SEM of triplicates. Figure 31 shows an anti-endothelial proliferation activity of large kringle fragments of angiostatin. The proteolytic fragments, Kl-4 (angiostatin) (-o-) and Kl-3 (- • -), inhibited the proliferation of BCE cells in a dose-dependent manner. The recombinant K2-3 (- • -) fragments exhibited a less potent inhibition than that of Kl-3 and Kl-4. The data represent the mean of three determinations (+/- SEM) as percentages of the inhibition. Figure 32 shows an additive inhibitory activity of recombinant kringle 2 and kringle 3. (A) The intact rK2-3 fragment (also see Figure 31) showed a weak inhibitory effect only at the concentration of 320 nM. At the same concentration, additive inhibition was observed when mutant cysteine fragments of rK2 were replaced by serine at position 169) and K3 (cysteine replaced by serine at position 297) were tested together on BCE cells. Each value represents the mean +/- SEM of triplicates. (B) Schematic structure of the amino acid sequence of K2 and K3. It was previously reported that an interchain kringle disulfide bond was present between the cysteine169 of K2 and the cysteine297 of K3 (Sdhndel, S., Hu, C.-K., Marti, D., Affolter, M., Schaller, J ., Llinas, M., and Rickli, EE (1996) Biochem. In print). Figure 33 shows an inhibition of endothelial proliferation by combined kringle fragments. The test was carried out with a concentration of 320 nM for each kringle fragment. The values represent the mean of three determinations (+/- SEM) as percentages of inhibition. (A) Inhibitory effects of - fragments of the combination of several individual kringles. (B) Combined inhibitory activity of combined kringle fragments. Figure 34 shows an inhibitory activity of angiostatin on endothelial cells after reduction and alkylation. (A) SDS-PAGE analysis of reduced forms (lane 2) and non-reduced forms (lane 1) of human angiostatin. Purified human angiostatin was reduced with DTT followed by alkylation of the protein with an excess amount of iodoacetamide. The treated samples were dialyzed and assayed on BCE cells. (B) Inhibition of proliferation of BCE cells by reduced and unreduced forms of angiostatin at a concentration of 320 nM. The data represent the average of the +/- SEM triplicate inhbition. Figure 35 shows the alignment of an amino acid sequence of putative kringle domains of human angiostatin. The sequences of four kringle domains were aligned according to their conserved cysteines. The identical and conserved amino acids are shaded. The amino acids in the kringle 4 in the boxes, show positive double charge cells adjacent to conserved cysteine residues of 22 and 80. Figur 36 shows the binding characteristics of lysine and the reactivity of angiostatin expressed. Figure 36A shows a Coomassie stained gel (40 μl load) Figure 36B shows an immunoblot (20 μl load) of similar gel The lane: 1 shows broth from stirred flasks of induced cultures showing angiostatin protein at about 50 kD and a few other proteins The broth from the induced cultures was diluted 1: 1 with buffer and loaded directly onto lysine-sepharose, Lane 2 shows the unbound fraction that passed through the lysine column. the angiostatin protein expressed by P. pastoris binds to the lysine column, lane: 3 shows the specific elution with 0.2 M aminocaproic acid showing that the angiostatin protein expressed by P. pastoris binds to lysine and can be purified in a single step until homogeneity on lysine-sepharose.Also, the angiostatin protein expressed by P. pastoris is recognized by a conformationally-dependent monoclonal antibody (VAP) ) directed against kringles 1 to 3. Figure 37 shows the angiostatin protein expressed by P. pastoris observed as a doublet migrating at 40 kD and 51.5 kD on Coomassie-stained gels of unreduced, denatured SDS-PAGE. The removal of the only complex N-linked chain of the angiostatin protein expressed with N-glycanase specific for higher mannose structures results in a single band of 49.5 kD. Panel A and panel B show a gel stained with Coomassie and a similar gel immunochamber respectively. Lane: 1 shows an angiostatin protein expressed by purified P. pastoris. Lane: 2 shows an angiostatin protein expressed by purified P. pastoris incubated under digestion conditions without N-glycanase. Lane 3 shows angiostatin protein expressed by purified P. pastoris digested with N-glycanase. Figure 38A shows 4 μg of angiostatin protein expressed by P. pastoris purified as a doublet on a Coomassie gel. Figure 38B shows that the purified recombinant inhibits BCE proliferation. The cell count of the BCE assay obtained after 72 hours is shown, in the presence (•) or absence (O) of bFGF, and the presence of bFGF with PBS as a control (?), And in the presence of bFGF with angiostatin protein expressed by P. pastoris (?). Figure 38C shows that the inhibition is not dose dependent. Figure 39 shows that purified angiostatin expressed by P. pastoris was given systemically (subcutaneously) to mice with primary tumors. Figures 39A and B show the number of metastases and lung weights respectively of mice treated daily with saline or angiostatin expressed by P. pastoris or angiostatin protein derived from plasminogen. In contrast to the lungs of mice treated with saline, the lungs of the mice treated with angiostatin protein expressed by P. pastoris or with angiostatin protein derived from plasminogen were not vascularized and the metastases were potentially suppressed. Figure 40 shows that the lungs of mice treated with angiostatin expressed by P. pastoris were pink with micrometastases, whereas the lungs of the control group with saline were completely covered with vascularized metastases.

Detailed Description The present invention includes compositions and methods for the detection and treatment of diseases and processes that are mediated by or associated with angiogenesis. The composition of angiostatin, which can be isolated from bodily fluids including, but not limited to, serum, urine and ascites, or synthesized by chemical or biological methods (eg cell culture, recombinant gene expression, protein synthesis, enzymatic catalysis in vi tro of plasminogen or plasmin to produce active angiostatin). Recombinant techniques include the genetic amplification of DNA sources using the polymerase chain reaction (PCR), and the genetic amplification of RNA sources using inverted transcriptase / PCR. Angiostatin inhibits the growth of blood vessels in tissues such as non-vascularized or vascularized tumors. The present invention also encompasses a composition comprising a vector containing a DNA sequence encoding angiostatin, wherein the vector is capable of expressing angiostatin when present in a cell, a composition comprising a cell containing a vector, wherein the vector contains a DNA sequence encoding angiostatin or fragments or analogs thereof, and wherein the vector is capable of expressing angiostatin when present in the cell, and a method comprising, implanting in a human or non-human animal a cell containing a vector, wherein the vector contains a DNA sequence encoding angiostatin, and wherein the vector is capable of expressing angiostatin when present in the cell.

Moreover, the present invention encompasses angiostatin, angiostatin fragments, angiostatin antiserum, angiostatin receptor agonists or angiostatin receptor antagonists that are combined with pharmaceutically acceptable excipients, and optionally sustained release compounds or compositions, such as biodegradable polymers, to form therapeutic compositions. In particular, the invention includes a composition comprising an antibody that specifically binds to angiostatin, where the antibody does not bind to plasminogen. More particularly, the present invention includes a protein designated angiostatin having a molecular weight of about 38 to 45 kilodaltons (kD) which is capable of overcoming the angiogenic activity of endogenous growth factors such as bFGF, in vitro. Angiostatin is a protein having a molecular weight of between about 38 kilodaltons and 45 kilodaltons, as determined by reductive electrophoresis on polyacrylamide gel and having an amino acid sequence substantially similar to that of a murine plasminogen fragment that begins at amino acid number 98 of an intact murine plasminogen molecule. The numbering of the amino acids here corresponds to the conventional numbering system starting with the methionine of the plasminogen molecule. The term "substantially similar", when used with reference to the amino acid sequences of angiostatin, means an amino acid sequence having anti-angiogenic activity and having a molecular weight of about 38 kD to 45 kD, which also has a high degree of of sequence homology with the mouse plasminogen protein fragment starting approximately at amino acid number 98 in mouse plasminogen and weighing 38 kD to 45 kD. A high degree of homology means at least one amino acid homology of 60%, desirably an amino acid homology of at least about 70%, and more desirably an amino acid homology of at least about 80%. The term "endothelial inhibitory activity" as used herein, means the ability of a molecule to inhibit angiogenesis in general and, for example,, to inhibit the growth of bovine capillary endothelial cells in culture in the presence of fibroblast growth factor. The amino acid sequence of the complete murine plasminogen molecule is shown in Figure 1 and SEQ ID N0: 1. The sequence for the angiostatin protein may start at approximately amino acid 98. Active human angiostatin, however, may also start in a variety of alternative positions. The examples demonstrate that the genetic constructs encoding the active angiostatin protein can start at amino acid 93 or 102, for example. The amino acid sequence of the first 339 amino acids of mouse angiostatin is shown in Figure 2 (SEQ ID NO: 2), and compared to the sequences of the corresponding plasminogen protein fragments for human plasminogen (SEQ ID NO. : 3), of Rhesus monkey (SEQ ID NO: 4), porcine (SEQ ID NO: 5) and bovine (SEQ ID NO: 6). Since those sequences are identical for well over 50% of the amino acids, it should be understood that the amino acid sequence of angiostatin is substantially similar between species. The total number of amino acids in angiostatin is not known precisely, but is defined by the molecular weight of the active molecule. The amino acid sequence of the angiostatin of the present invention may vary depending on the species from which the plasminogen molecule is derived. Thus, although the angiostatin of the present invention which is derived from human plasminogen has a slightly different sequence from that of angiostatin derived from the mouse, it has anti-angiogenic activity as shown in a mouse tumor model. Angiostatin has been shown to be capable of inhibiting the growth of endothelial cells in vi tro. Angiostatin does not inhibit the growth of cell lines derived from other cell types. Specifically, angiostatin has no effect on Lewis lung carcinoma cell lines, mink lung epithelium, 3T3 fibroblasts, bovine aortic smooth muscle cells, bovine retinal pigment epithelium, MDCk (canine renal epithelium) cells, W138 cells ( human fetal lung fibroblasts), EFN cells (murine fetal fibroblasts) and LM cells (murine connective tissue). Endogenous angiostatin in a mouse having a tumor is effective to inhibit metastasis at a systemic concentration of about 10 mg angiostatin / kg body weight. Angiostatin has a specific three-dimensional conformation that is defined by the kringle regions of the plasminogen molecule. (Robbins, K. C, "The plasmingen-enzyme plasmin system" Hemostasis and Thrombosis, Basic Principles and Practice, 2nd Edition, ed by Colman R. W. et al., J.B. Lippincott Company, pp. 340-357, 1987). There are five such kringle regions, which are conformationally related motifs and have substantial sequence homology, in the NH2 terminal portion of the plasminogen molecule. It is believed that the three-dimensional conformation of functional angiostatin encompasses the kringle 1 to 5 regions of plasminogen. Each kringle region of the plasminogen molecule contains approximately 80 amino acids and contains 3 disulfide bonds. It is known that the reason for cysteine exists in other biologically active proteins. These proteins include, but are not limited to, prothrombin, hepatocyte growth factor, diffusion factor, and macrophage-stimulating protein. (Yoshimura, T, et al., "The cloning, sequencing and expression of human macrophage stimulating protein" (MSP, MSTI) confirms MSP as a member of the kringle protein family and locates the MSP gene on the chromosome 3"J. Biol. Chem., Vol. 268, No. 21, pp. 15461-15468, 1993.) It was contemplated that any isolated protein or protein having a three-dimensional conformation similar to that of kringle or cysteine motif. that has anti-angiogenic activity in vivo, is part of the present invention.

The present invention also includes the detection of angiostatin in body fluids and tissues for the purpose of diagnosing and predicting diseases such as cancer. The present invention also includes the detection of angiostatin receptor and binding sites in cells and tissues. The present invention also includes methods for treating or preventing angiogenic diseases and processes including, but not limited to, arthritis and tumors by stimulating the production of angiostatin, and / or by administration of substantially purified angiostatin, or agonists or antagonists of angiostatin, and / or angiostatin antiserum or antiserum directed against angiostatin antiserum to a patient. Additional treatment methods include the administration of angiostatin, angiostatin fragments, angiostatin analogs, angiostatin antiserum or angiostatin receptor agonists and antagonists linked to cytotoxic agents. It should be understood that angiostatin may be of animal or human origin. Angiostatin can also be produced synthetically by chemical reaction or by recombinant techniques in conjunction with expression systems. Angiostatin can also be produced by the enzymatic cleavage of plasminogen or isolated plasmin to generate proteins that have anti-angiogenic activity. Angiostatin can also be produced by compounds that mimic the action of endogenous enzymes that cleave plasminogen to angiostatin. The production of angiostatin can also be modulated by compounds that affect the activity of enzymes that cleave plasminogen. Passive therapy using antibodies that specifically bind to angiostatin can be used to modulate angiogenic dependent processes such as reproduction, development, and wound healing and tissue repair. In addition, the antiserum directed to the Fab regions of angiostatin antibodies can be administered to block the ability of the endogenous angiostatin antiserum to bind to angiostatin. The present invention also encompasses gene therapy whereby the gene coding for angiostatin is regulated in a patient. Several methods for transferring or releasing DNA to cells for the expression of the protein product of the gene, in other circumstances known as gene therapy, are described in Genetic Transfer in Mammalian Somatic Cells in vivo, N. Yang, Crit. Rev. Biotechn. 12 (4): 335-356 (1992), which is incorporated herein by reference. Gene therapy encompasses DNA sequence incorporations in somatic cells or bacterial line cells for use in ex vivo or in vivo therapy. Genetic therapy is used to replace genes, increase normal or abnormal genetic function, to fight infectious diseases and other pathologies. Strategies to treat such medical problems with gene therapy include therapeutic strategies such as identifying the defective gene and then adding a functional gene to replace the function of the defective gene or increase a slightly functional gene; or prophylactic strategies, such as adding a gene for the protein product 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 angiostatin can be placed in a patient and thus prevent the occurrence of angiogenesis; or a gene that makes the tumor cells more susceptible to radiation could be inserted and then the tumor radiation would cause a greater mortality of the tumor cells. Many protocols for transferring angiostatin DNA or angiostatin regulatory sequences are contemplated in this invention. Transfection of promoter sequences, different from those normally found to be specifically associated with angiostatin, or other sequences that would increase the production of the angiostatin protein, were also considered as genetic therapy methods. An example of this technology is found in Transkaryotic Therapies, Inc., of Cambridge, Massachusetts, which uses homologous recombination to insert a "genetic switch" that activates an erythropoietin gene in cells. See Genetic Engineering News, April 15, 1994. Such "genetic switches" could be used to activate angiostatin (or the angiostatin receptor) in cells that do not normally express angiostatin (or the angiostatin receptor). Genetic transfer methods for gene therapy fall into three broad categories: physical (eg, electroporation, direct gene transfer and particle bombardment), chemistry (carriers of lipids or other non-viral vectors) and biological (vector derived from virus and receptor absorption). For example, non-viral vectors can be used, which include liposomes coated with DNA. Such liposome / DNA complexes can be injected directly intravenously into the patient. It is believed that the liposome / DNA complexes are concentrated in the liver, where they release DNA to macrophages and Kupffer cells. These cells have a long life and thus provide the long-term expression of the released DNA. Additionally, vectors or "naked" DNA of the gene can be injected directly into the desired organ, tissue or tumor to direct the delivery of the therapeutic DNA. Genetic therapy methodologies can also be described by the site of release. Fundamental ways to release genes include ex vivo gene transfer, in vivo gene transfer, and in vitro genetic transfer. In ex vivo gene transfer, the cells are taken from the patient and grown in a cell culture. The DNA is transfected into the cells, the transfected cells expand to a number and then reimplanted in the patient. In in vitro genetic transfer, transformed cells are cells that grow in culture, such as tissue culture cells, and are not particular cells of a particular patient. These "laboratory cells" are transfected, the transfected cells are selected and expanded for implantation in a patient or for other uses.

In vivo gene transfer involves introducing the DNA into the patient's cells when the cells are inside the patient. The methods include using virally mediated gene transfer using a non-infectious virus to release a gene in the patient or injecting naked DNA at a site in the patient and the DNA is absorbed by a percentage of cells in which the product protein is expressed. genetic. Additionally, the other methods described herein, such as the use of "genetic cannon", can be used for the insertion of angiostatin DNA in situ or angiostatin regulatory sequences. Chemical methods of gene therapy may involve a compound based on a lipid, not necessarily a liposome, to transport DNA through the cell membrane. Lipofectins or cytofectins, positive lipid-based ions that bind negatively charged DNA, form a complex that can cross the cell membrane and provide DNA inside the cell. Another chemical method uses the "" receptor-based endocytosis, which involves a specific ligand medium to a cell surface receptor and envelops and transports it through the cell membrane. The ligand binds to the DNA and the whole complex is transported to the cell. The genetic complex of the ligand is injected into the bloodstream and then the target cells it has in the receptor, will bind specifically to the ligand and transport the ligand-DNA complex to the cell. Many genetic 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 lymphocytes and tumor infiltrates, hepatocytes, epidermal cells, myocytes, or other somatic cells. Those altered cells are then introduced into the patient to provide the genetic product of 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, regulatory elements or cis-acting promoters known to be tissue-specific may be used. Alternatively, this can be achieved by using the release in si tu of the DNA or viral vectors to specific anatomical sites in vivo. For example, genetic transfer to blood vessels in vivo was achieved by implanting transduced endothelial cells in place at selected sites on arterial walls. The virus infected the surrounding cells, which also expressed the gene product. A viral vector can also be released directly to the site in vivo, by means of a catheter for example, thus allowing only certain areas to be infected by the virus, and providing site-specific, long-term genetic expression. In vivo gene transfer using retroviral vectors has also been demonstrated in mammalian tissues and liver tissue by injection of altered virus into the 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 polioviruses or Sindibis viruses, adenoviruses, adenovirus partners, herpes virus, SV 40, vaccinia and other DNA viruses. Murine retroviral vectors of defective reproduction are the most widely used vectors for genetic transfer. Retroviruses of murine leukemia are composed of a single strand of DNA complexed with a core nuclear protein and polymerase enzymes (pol), encapsulated by a protein core (gag) and surrounded by a glycoprotein (env) that determines the range of hosts. The genomic structure of the retroviruses includes the gag, pol and env genes enclosed by the 5 'and 3' terminal repeats (LTR). The retroviral vector systems exploit the fact that a minimum vector contains the LRT 5 'and 3' and the packaging signal are sufficient to ~ allow the packaging of the vector, infection and integration of target cells, with the proviso that the viral structural proteins are delivered in trans in the packaging cell line. The fundamental advantages of retroviral vectors for gene transfer include efficient gene expression in most cell types, precise integration of a single copy of the vector into the chromosomal DNA of the target cell and ease of manipulation of the retroviral genome. The adenovirus is composed of double-stranded DNA, linear, complexed with nuclear proteins and surrounded by capsular proteins. Advances in 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. The adenoviral base vectors will express proteins of the gene product at high levels. Adenoviral vectors have high infectivity efficiencies, even with low virus titers. Additionally, the virus is completely ineffective as a cell-free virion, so that the injection of producer cell lines is not necessary. Another potential advantage of adenoviral vectors is the ability to achieve long-term expression of heterologous genes in vivo. Mechanical methods of DNA release include fusogenic lipid vesicles such as liposomes or other vesicles for membrane fusion, lipid particles of DNA incorporating cationic lipids such as lipofectin, polylysine-mediated DNA transfer, direct DNA injection, such as microinjection of DNA in bacterial or somatic cells, particles coated with pneumatically released DNA, such as gold particles used in a "genetic cannon", and inorganic chemical methods such as transfection with calcium phosphate. Another method, ligand-mediated gene therapy, involves complexing the DNA with specific ligands to form ligand-DNA conjugates, to direct the DNA to a specific cell or tissue. It has been found that injecting plasmid DNA into muscle cells produces a high percentage of cells, which are transfected and have sustained expression of marker genes. The DNA of the plasmid may or may not be integrated into the genome of the cells. The non-integration of the transfected DNA would allow the transfection and expression of the proteins of the gene product in non-proliferative tissue, terminally differentiated over a prolonged period of time without danger of insertions, deletions or mutant alterations in the cellular or mitochondrial genome. The long-term, but not necessarily permanent, transfer of therapeutic genes into specific cells can provide treatments for genetic diseases or for prophylactic use. The DNA could be reinjected periodically to maintain the level of genetic product without mutations in the genomes of the recipient cells. The non-integration of hexogenic DNA can allow the presence of several different hexogen DNA constructs within a cell with all the constructs expressing several genetic products. Particle-mediated gene transfer methods were used for the first time to transform plant tissues. With a particle bombardment device, or a "genetic cannon", a driving force is generated to accelerate high density particles coated with DNA (such as gold or tungsten) at a high speed that allows the penetration of organs, tissues or target cells. Bombardment of particles can be used in the in vi tro system, or with ex vivo or in vivo techniques to introduce DNA into cells, tissues and organs. Electroporation for gene transfer uses an electric current to make the cells or tissues susceptible to gene transfer mediated by electroporation. A short electric impulse with a given field strength is used to increase the permeability of a membrane, in such a way that the DNA molecules can penetrate 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 and organs. Gene transfer mediated by the carrier in vivo can be used to transfect foreign DNA into cells. The DNA-carrier complex can be conveniently introduced into body fluids or blood flow and then specifically directed to the site toward the target organ or tissue in the body. Liposomes and polycations such as polylysine, lipofectins or cytofectins can be used. Liposomes can be developed, which are specific for a cell or specific for an organ and in this way the foreign DNA carried by the liposome will be absorbed by the target cells. The injection of immunoliposomes that are targeted to a specific receptor on certain cells can be used as a convenient method for inserting the DNA into cells that contain the receptor. Another carrier system that has been used is the conjugated system asialoglycoprotein / polylysine to transport DNA to hepatocytes for genetic transfer in vivo. The transfected DNA can also be complexed with other types of carriers, so that the DNA is transported to the recipient cell and then it resides in the cytoplasm or in the nucleoplasm. DNA can be coupled to nuclear carrier proteins in specifically designed vesicular complexes and carried directly to the nucleus. Genetic regulation of angiostatin can be achieved by administering compounds that bind to the angiostatin gene, or control regions associated with the angiostatin gene, or its corresponding RNA transcript to modify the rate of transcription or translation. Additionally, cells transfected with a DNA sequence encoding angiostatin can be administered to a patient to provide an in vivo source of angiostatin. For example, the cells can be transfected with a vector containing a nucleic acid sequence coding for angiostatin. The term "vector" as used herein means a carrier that may contain or be associated with specific nucleic acid sequences, which functions to transport the specific nucleic acid sequences to the cell. Examples of vectors include infectious plasmids and microorganisms such as viruses, or non-viral vectors such as ligand-DNA conjugates, liposomes, lipid-DNA complexes. It may be desirable for a recombinant DNA molecule comprising an angiostatin DNA sequence to be operably linked to an expression control sequence to form an expression vector capable of expressing angiostatin. The transfected cells may be cells derived from normal tissue of a patient, the diseased tissue of a patient, or may be cells belonging to the patient. For example, tumor cells removed from a patient can be transfected with a vector capable of expressing the angiostatin protein of the present invention, and reintroduced into the patient. The transfected tumor cells produce angiostatin levels in the patient that inhibit tumor growth. Patients can be humans or non-human animals. The cells can also be transfected by non-vector, or physical or chemical methods known in the art such as electroporation, ionoporation or via a "genetic cannon". Additionally, the angiostatin DNA can be injected directly, without the aid of a carrier, into a patient. In particular, the angiostatin DNA can be injected into the skin, muscle or blood. The genetic therapy protocol for transfecting angiostatin in a patient can be through the integration of angiostatin DNA into the genome of the cells, into the minichromosomes or as a DNA construct that reproduces or does not reproduce separately in the cytoplasm or nucleoplasm of the cell. The expression of angiostatin may continue for a prolonged period of time or may be periodically reinjected to maintain a desired level of the angiostatin protein in the cell, tissue or organ or a given blood level. Angiostatin can be isolated on a column of CLAR C4 (see Table 3). The angiostatin protein elutes from 30 to 35% in an acetonitrile gradient. On an electrophoresis on polyacrylamide gel with sodium dodecyl sulfate (PAGE) under reducing conditions, the protein band with activity elutes as a single peak at approximately 38 kilodaltons. The inventors have shown that a growing primary tumor is associated with the release into the bloodstream of inhibitors specific for the proliferation of endothelial cells, including angiostatin, which can suppress angiogenesis within a metastasis and therefore inhibit the growth of the metastasis itself. The source of angiostatin associated with the primary tumor is unknown. The compound can be produced by degradation of plasminogen by a specific protease, or angiostatin could be produced by the expression of a specific gene encoding angiostatin. The angiogenic phenotype of a primary tumor depends on the production of angiogenic proteins and excess endothelial cellulase inhibitors, which are elaborated by normal cells, but which are believed to be deregulated during the transformation towards neoplasia. Although the production of angiostatin can be deregulated in an individual tumor cell in relation to the production of its type of stem cell, the total amount of inhibitor made by the whole tumor may be sufficient to enter the circulation and suppress endothelial growth in sites remote from micrometastasis. The metastasis remains in the circulation for a significantly longer time than the angiogenic proteins released by a primary tumor. In this way, the angiogenic protein seems to act locally, whereas angiostatin acts globally and circulates in the blood with a relatively long half-life. The half-life of angiostatin is approximately 12 hours to 5 days. Although we did not expect to join the following hypothesis, it is believed that when the tumor becomes angiogenic it releases one or more angiogenic proteins (eg, AFGF, bFGF, VEGF, IL-8, GM-CSF, etc.), which act locally , by targeting the endothelium in the vicinity of a primary tumor from an extravascular direction, and without circulating (or circulating with a short half-life). These angiogenic proteins must be produced in an amount sufficient to overcome the action of the endothelial cell inhibitor (inhibitors of angiogenesis) so that a primary tumor continues to expand its population. Once such a primary tumor is growing well, it continues to release inhibitors of endothelial cells into the circulation. According to this hypothesis, these inhibitors act remotely at a distance from the primary tumor, the capillary endothelium targets a metastasis from an intravascular direction, and continue to the circulation. In this way, just at the moment when a remote metastasis can begin to initiate angiogenesis, the capillary endothelium in its vicinity could be inhibited by incoming angiostatin. Once a primary tumor has reached a sufficient size to cause the angiostatin to be continuously released into the circulation, it is difficult for a second tumor implant (or a micrometastasis) to begin or for an increase in its own angiogenesis. If a second tumor implant occurs (for example, in the subcutaneous space, or in the cornea, or intravenously to the lung) briefly after the primary tumor is implanted, the primary tumor will not be able to suppress the secondary tumor (because angiogenesis in the secondary tumor will already be in progress). If two tumors are implanted simultaneously (for example, in opposite flanks), the inhibitors can have an inhibitory effect equivalent to each other. The angiostatin of the present invention may be: (i) administered to humans or animals with tumors as antiangiogenic therapy; (ii) Verified in serum, urine, or tissues of humans or animals as prognostic markers; and (iii) Used as the basis for analyzing the serum and urine of cancer patients for similar angiostatic molecules. It was contemplated as part of the present invention that angiostatin can be isolated from a body fluid, such as blood or urine from patients or angiostatin can be produced by recombinant DNA methods or chemical protein synthesis methods that are well known for those skilled in the art. Protein purification methods are well known in the art and a specific example of a method for purifying angiostatin, and testing the activity of the inhibitor is provided in the examples below. The isolation of endogenous human angiostatin is achieved using similar techniques. An example of a method for producing angiostatin using recombinant angiostatin techniques involves the steps of (1) identifying and purifying angiostatin as discussed above, and as described more fully below, (2) determining the amino acid sequence N- terminal of the purified inhibitor (3) synthetically generate 5 'and 3' DNA oligonucleotide primers for the angiostatin sequence, (4) amplify the angiostatin gene sequence using polymerase, (5) insert the amplified sequence into an appropriate vector such as an expression vector, (6) inserting the vector containing the gene into a microorganism or other expression system capable of expressing the inhibitor gene, and (7) isolating the recombinantly produced inhibitor. Suitable vectors include viral, bacterial and eukaryotic expression vectors (such as yeast). The above techniques are described more fully in laboratory manuals such as "Molecular Cloning: A Laboratory Manual" Second Edition by Sambrook et al., Cold Spring Harbor Press, 1989. The DNA sequence of human plasminogen has been published (Browne, MJM et al., "Expression of plasminogen and recombinant human aglycosplasminogen in HeLa cells" Fibrinolysis Vol .5 (4), 257-260, 1991) and is incorporated herein by reference. The gene for angiostatin can also be isolated from cells or tissues (such as tumor cells) that express high levels of angiostatin (1) by isolating messenger RNA from tissue, (2) using inverted transcriptase to generate the corresponding DNA sequence and then (3) using the polymerase chain reaction (PCR) with the appropriate primers to amplify the DNA sequence encoding the amino acid sequence of the active angiostatin. Another method to produce angiostatin, or biologically active fragments thereof, is by protein synthesis. Once a biologically active fragment of an angiostatin is found using the assay system described more fully below, it can be sequenced, for example, by automated protein sequencing methods. Alternatively, once the gene or DNA sequence coding for angiostatin is isolated, for example by the methods described above, the DNA sequence can be determined using manual or automated sequencing methods well known in the art. The "nucleic acid sequence in turn provides information related to the amino acid sequence, in this way, if the biologically active fragment is generated by specific methods, such as tryptic digestions, or if the fragment is sequenced at the N-terminal, the remaining amino acid sequence can be determined from the corresponding DNA sequence.

Once the amino acid sequence of the protein is known, the fragment can be synthesized by techniques well known in the art, as exemplified by "Protein Synthesis in Solid Phase: A Practical Method" E. Atherton and R.C. Sheppard, IRL Press, Oxford, England. Similarly, multiple fragments can be synthesized, which are then ligated to form longer fragments. These synthetic protein fragments can also be made with amino acid substitutions at specific sites to test the agonist and antagonist activity in vi tro and in vivo. Protein fragments that possess high binding affinity for the tissues can be used to isolate the angiostatin receptor on affinity columns. The isolation and purification of the angiostatin receptor is a fundamental step towards the elucidation of the mechanism of action of angiostatin. The isolation of an angiostatin receptor and the identification of angiostatin agonists and antagonists will facilitate the development of drugs to modulate angiostatin receptor activity, the final pathway to biological activity. The isolation of the receptor allows the construction of nucleotide probes to verify the location and synthesis of the receptor, using the hybridization technology itself and the solution. In addition, the gene for the angiostatin receptor can be isolated, incorporated into an expression vector and transfected into cells, such as tumor cells of a patient to increase the ability of a cell, tissue or tumor type to bind to the angiostatin and inhibit local angiogenesis. Angiostatin is effective in treating diseases or processes that are mediated by, or imply, angiogenesis. The present invention includes the method for treating an angiogenesis-mediated disease with an effective amount of angiostatin, or a biologically active fragment thereof, or combinations of angiostatin fragments that collectively possess anti-angiogenic activity, or angiostatin agonists and antagonists. Diseases mediated by angiogenesis, include, but are not limited to, solid tumors; blood-borne tumors such as leukemias; tumor metastasis; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachoma and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, rematurity of prematurity, macular degeneration, rejection of corneal graft, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber syndrome; myocardial angiogenesis; plaque neovascularization; telangectasia; hemophiliac joints; angiofibroma; and granulation of wounds. Angiostatin is useful in the treatment of diseases of excessive or abnormal stimulation of endothelial cells. Those diseases include, but are not limited to, intestinal adhesion, Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars, ie, keloids. Angiostatin can be used as a birth control agent by preventing the vascularization required from the implantation of the embryo. Angiostatin is useful in the treatment of diseases that have angiogenesis as a pathological consequence, such as cat scratch disease (Jochele minalia quintosa) and ulcers (Helicobacter pylori). Angiostatin synthetic protein fragments have a variety of uses. The protein that binds to the angiostatin receptor with high specificity and avidity is radioactively labeled and used for the visualization and quantification of binding sites using autoradiographic and membrane binding techniques. This application provides a diagnosis and important research tools.

The knowledge of the binding properties of the angiostatin receptor facilitates the investigation of the transduction mechanism linked to the receptor. further, labeling angiostatin proteins with short-lived isotopes allows the visualization of receptor binding sites in vivo using positron emission tomography or other modern radiographic techniques to localize tumors with angiostatin binding sites. The systematic substitution of amino acids within those synthesized proteins produces high affinity protein agonists and antagonists for the angiostatin receptor that increase or decrease the binding of angiostatin to its receptor. Such agonists are used to suppress the growth of micrometastases, thus limiting the spread of cancer. Angiostatin antagonists are applied in situations of inadequate vascularization, to block the inhibitory effects of angiostatin and to promote angiogenesis. For example, this treatment may have therapeutic effects to promote wound healing in diabetics. The angiostatin proteins are used to develop affinity columns for the isolation of the angiostatin receptor from cultured tumor cells. The isolation and purification of the angiostatin receptor is followed by the sequencing of amino acids. Using this information, the gene or genes that code for the angiostatin receptor can be identified and isolated. Next, cloned nucleic acid sequences are developed to be inserted into vectors capable of expressing the receptor. These techniques are well known to those skilled in the art. The transfection of the nucleic acid sequences encoding the angiostatin receptor in tumor cells, and the expression of the receptor by the transfected tumor cells increases the response of those cells to endogenous or exogenous angiostatin and, therefore, decreases the speed of metastatic growth. Cytotoxic agents such as ricin, are linked to angiostatin, and protein fragments of high affinity angiostatin, so they provide a tool for the destruction of cells that bind to angiostatin. These cells can be found in many places, including, but not limited to, micrometastases and primary tumors. The proteins bound to the cytotoxic agents are infused in a manner designed to maximize release to the desired site. For example, high affinity angiostatin fragments linked to ricin are released through a cannula into the vessels supplying the target site or directly into the target. Such agents are also released in a controlled manner through osmotic pumps coupled to an infusion cannula. A combination of angiostatin antagonists with angiogenesis stimulators can be co-applied to increase tissue vascularization. This therapeutic regimen provides an effective means to destroy metastatic cancer. Angiostatin can be used in combination with other compositions and methods for the treatment of diseases. For example, a tumor can be treated conventionally with surgery, radiation or chemotherapy combined with angiostatin and then angiostatin can then be administered to the patient to extend the lethargy of the micrometastases and to stabilize and inhibit the growth of any residual primary tumor. Additionally, angiostatin, angiostatin fragments, angiostatin antiserum, angiostatin receptor agonists, angiostatin receptor antagonists, or combinations thereof, are combined with pharmaceutically acceptable excipients, and optionally a sustained release matrix, such as biodegradable polymers, to form therapeutic compositions. A sustained release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid / base hydrolysis or by dilution. Once inserted in the body, the matrix acts on enzymes and body fluids. The sustained release matrix is desirably chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (glycolic acid polymer), polylactide polyanhydrides co-glycolide (copolymers of lactic acid and glycolic acid), poly (ortho) ) esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of any one of the polylactide, polyglycolide or polyglycide co-glycolide (copolymers of lactic acid and glycolic acid). The therapeutic compositions that modulate the angiogenesis of the present invention may be a solid, liquid or aerosol and may be administered by any known route of administration. Examples of solid therapeutic compositions include pills, creams and implantable dosage units. The pills can be administered orally, the therapeutic creams can be administered topically. The implantable dosage units can be administered locally, for example at the site of a tumor, or they can be implanted for the systemic release of the therapeutic composition that modulates angiogenesis, for example subcutaneously. Examples of liquid composition include formulations adapted to be injected subcutaneously, intravenously, intraarterially, and formulations for topical and infraocular administration. Examples of aerosol formulations include the inhaler formulation for administration to the lungs. The angiostatin of the present invention can also be used to generate antibodies that are specific for the inhibitor and its receptor. The antibodies can be polyclonal antibodies or monoclonal antibodies. Those antibodies that bind specifically to angiostatin or angiostatin receptors can be used in methods and diagnostic kits that are well known to those skilled in the art to detect or quantify angiostatin or angiostatin receptors in a body fluid or tissue. . The results of these tests can be used to diagnose or predict the occurrence or recurrence of cancer and other diseases mediated in an angiogenic manner. Angiostatin can also be used in a diagnostic method and equipment to detect and quantify antibodies capable of binding to angiostatin. These devices would allow the detection of circulating angiostatin antibodies, which indicate the diffusion of micrometastases in the presence of angiostatin secreted by primary tumors in situ. Patients who have such antibodies to antiangiostatin are more likely to develop tumors and multiple cancers, and are more likely to have cancer recurrences after treatments or periods of remission. The Fab fragments of these antiangiostatin antibodies can be used as antigens to generate antisera from the Fab antiangiostatin fragment, which can be used to neutralize antiangiostatin antibodies. Such a method would reduce the removal of circulating angiostatin by antiangiostatin antibodies, thereby effectively elevating angiostatin levels in the circulation.

Another aspect of the present invention is a method for blogging the action of excess endogenous angiostatin. This can be done by passively immunizing a human or animal with antibodies specific for the undesirable angiostatin in the system. This treatment can be important in the treatment of ovulation, menstruation, placentation and abnormal vasculogenesis. This provides a useful tool to examine the effects of the removal of angiostatin on metastatic processes. The Fab fragment of angiostatin antibodies contains the binding site for angiostatin. This fragment is isolated from angiostatin antibodies using techniques known to those skilled in the art. The Fab fragments of the angiostatin antiserum are used as antigens to generate the production of anti-Fab fragment serum. Infusion of this antiserum against Fab fragments of angiostatin prevents angiostatin from binding to angiostatin antibodies. The therapeutic benefit is obtained by neutralizing endogenous antiangiostatin antibodies by blocking the binding of angiostatin to the Fab fragments of antiangiostatin. The net effect of this treatment is to facilitate the ability of endogenous circulating angiostatin to reach target cells, thus decreasing the spread of metastasis. It should be understood that the present invention contemplated including any angiostatin derivatives having endothelial inhibitory activity. The present invention includes all of the angiostatin protein, angiostatin protein derivatives and biologically active fragments of the angiostatin protein. These include proteins with angiostatin activity that have amino acid substitutions or have sugars or other molecules linked to the amino acid functional groups. The present invention also includes the genes encoding angiostatin and the angiostatin receptor, and the proteins that are expressed by those agents. Proteins and protein fragments with the angiostatin activity described above can be provided as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable formulations using the formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes. In general, the combinations can be administered by the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral route (for example intravenous, intraspinal, subcutaneous or intramuscular). In addition, angiostatin can be incorporated into biodegradable polymers that allow sustained release of the compound, with the polymers implanted in the vicinity of where release is desired, for example, at the site of a tumor or implanted so that angiostatin is released. systematically and slowly. Osmotic minipumps can also be used to provide controlled release of high concentrations of angiostatin through a cannula to the site of interest, such as directly in a metastatic growth or in the vascular supply to that tumor. Biodegradable polymers and their use are described, for example, in detail in Bren et al., < J. Neurosurg. 74: 441-446 (1991), which is incorporated herein by reference in its entirety. The dose of angiostatin of the present invention will depend on the condition or condition of disease being treated and other clinical factors such as the weight and condition of the human or animal and the route of administration of the compound. To treat humans or animals, between about 0.5 mg / kilogram to 500 mg / kilogram of angiostatin can be administered.

Depending on the half-life of angiostatin in the particular animal or human, angiostatin can be administered several times a day to once a week. It should be understood that the present invention has application for human and veterinary use. The methods of the present invention contemplate a single administration, as well as multiple administrations, given simultaneously or for an extended period of time. Formulations of angiostatin include those suitable for oral, rectal, ophthalmic (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intracranial administration) , intratracheal and epidural). The angiostatin formulations can be conveniently presented in unit dosage forms and can be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carriers or excipients. 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, forming the product. Formulations suitable for parenteral administration include sterile, aqueous injection solutions, which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the intended recipient's blood; and sterile aqueous and non-aqueous suspensions, which may include suspending agents and thickening agents. The formulations may be present in unit doses or containers with multiple doses, for example, sealed ampoules and flasks, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, eg, water for injection, immediately prior to use. . The extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the type described above. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, 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 invention may include other agents conventional in the art, having considered the type of formulation in question. Optionally, cytotoxic agents or otherwise be combined with angiostatin protein, or biologically functional protein fragments thereof, can be incorporated to provide a dual therapy to the patient. The proteins that inhibit angiogenesis of the present invention can be synthesized in a standard microchemical facility and its purity verified by HPLC and mass spectrometry. Methods of protein synthesis, purification by HPLC, and mass spectrometry are common to those skilled in the art. Angiostatin proteins and angiostatin receptor proteins are also produced in E. coli expression systems or recombinant yeasts, and purified by column chromatography. Different protein fragments of the intact angiostatin molecule can be synthesized for use in various applications, including, but not limited to the following; as antigens for the development of specific antiserum, as active agonists and antagonists at angiostatin binding sites, as proteins to be linked to, or used in combination with, cytotoxic agents for the targeted killing of cells that bind to angiostatin . The amino acid sequences comprising these proteins are selected on the basis of their position on the outer regions of the molecule and are accessible to bind antisera. The amino and carboxyl termini of angiostatin, as well as the middle region of the molecule are represented separately among the fragments to be synthesized. The protein sequences are compared to known sequences using protein sequence databases, such as GenBank, Brookhaven Protein, SWISS-PROT, and PIR to determine potential sequence homology. This information facilitates the elimination of sequences that exhibit a high degree of sequence homology with other molecules, thereby increasing the potential for high specificity in the development of angiostatin antisera, agonists and antagonists. Angiostatin and proteins derived from angiostatin can be coupled to other molecules using standard methods. The amino and carboxyl terminals of angiostatin contain both tyrosine and lysine residues and are labeled isotopically and non-isotopically with many techniques, for example radioactive labeling using conventional techniques (tyrosine-chloramine T residues, iodogen, lactoperoxidase, lysine residues- Bolton-Hunter reagent). These coupling techniques are well known to those skilled in the art. Alternatively, tyrosine or lysine is added to fragments that do not have those residues to facilitate labeling of reactive amino and hydroxyl groups on the protein. The coupling technique is chosen on the basis of the functional groups available on the amino acids including, but not limited to the amino, sulfhydral, carboxyl, amide, phenol and imidazole. Several reagents used to effect such couplings include, among others, glutaraldehyde, diazotized benzidine, carbodiimide and p-benzoquinone. The angiostatin proteins are chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, chemiluminescent compounds, bioluminescent compounds and others for a variety of applications. The efficiency of the coupling reaction is determined using different techniques appropriate for the specific reaction. For example, radiolabeling of an angiostatin protein with 125 I is achieved using chloramine T and Na125I of high specific activity. The reaction is terminated with sodium metadisulfite and the mixture is desalted on disposable columns. The labeled protein is eluted from the column and the fractions are collected. Aliquots of each fraction are removed and the radioactivity is measured in a gamma counter. In this way, unreacted Na125I is separated from the labeled angiostatin protein. The protein fractions with the highest specific radioactivity are stored for later use, such as in an analysis of the ability to bind angiostatin antiserum. Another application of protein conjugation is for the production of polyclonal antisera. For example, angiostatin proteins containing lysine residues are ligated to purified bovine serum albumin using glutaraldehyde. The efficiency of the reaction is determined by measuring the incorporation of the radiolabeled protein. The glutaraldehyde and the unreacted protein are separated by dialysis. The conjugate is stored for later use. Antiserum can be generated against angiostatin, angiostatin analogs, angiostatin protein fragments and the angiostatin receptor. After the synthesis and purification of the protein, monoclonal and polyclonal antisera are produced using established techniques known to those skilled in the art. For example, polyclonal antisera can be created in rabbits, sheep, goats or other animals. Angiostatin proteins conjugated to a carrier molecule such as bovine serum albumin, or angiostatin itself, are combined with an adjuvant mixture, emulsified and injected subcutaneously at multiple sites on the back, neck, flanks and sometimes on the plants from the feet. Booster injections at regular intervals, such as every 2 to 4 weeks. Blood samples are obtained by venipuncture, for example using the marginal veins of the ear after dilation, approximately 7 to 10 days after each injection. The blood samples are allowed to coagulate overnight at 4 ° C and are centrifuged at approximately 2400 X g at 4 ° C for approximately 30 minutes. The serum is removed, divided into aliquots, and stored at 4 ° C for immediate use or -20 to -90 ° C for subsequent analysis. All serum samples for the generation of polyclonal antiserum or samples of media for the production of monoclonal antisera are analyzed to determine the antibody titer. The title is established through various means, for example, using spot blots and density analysis, and also with precipitation of protein-antibody complexes radioactively labeled using protein A, secondary antisera, cold ethanol or carbon-dextran followed by the measurement of the activity with a gamma counter. Higher titre antisera are also purified by affinity columns, which are commercially available. The angiostatin proteins are coupled to the gel in the affinity column. The antiserum samples are passed through the column and the antiangiostatin antibodies remain attached to the column. These antibodies are subsequently eluted, collected and evaluated for the determination of titer and specificity. Angiostatin antiserum of higher titre is tested to establish the following; a) optimal dilution of the antiserum for the highest specific binding of the antibody and the least non-specific binding, b) the ability to bind larger amounts of angiostatin protein in a standard displacement curve, c) potential for cross-reactivity with related proteins and proteins , including plasminogen and also angiostatin of related species, d) ability to detect angiostatin proteins in extracts of plasma, urine, tissues and in cell culture media. The equipment for the measurement of angiostatin, and the angiostatin receptor are also used as part of the present invention. Antisera that possess the highest titer and specificity and that can detect angiostatin proteins in extracts of plasma, urine, tissues and cell culture media are further examined to establish easy-to-use equipment for rapid, reliable measurement and localization. sensitive and specific angiostatin. These test equipment include, but are not limited to the following techniques; competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, spot blots, enzyme-linked assays including ELISA, microtiter plates, submersible strips and rods coated with antibody for rapid verification of urine and blood, and immunocytochemistry. For each equipment, the interval, sensitivity, precision, reliability, specificity and reproducibility of the assay are established. Intraassay variation is established at points of 20%, 50% and 80% on the standard displacement or activity curves.

An example of a test kit commonly used in research and in the clinic is the radioimmunoassay (RIA) equipment. A RIA of angiostatin is illustrated below. After radioiodination and successful purification of angiostatin or an angiostatin protein, the antiserum having the highest titre is added to several dilutions to tubes containing a relatively constant amount of radioactivity, such as 10,000 cpm, in a buffer system. suitable. Other tubes contain buffer or preimmune serum to determine non-specific binding. After incubation at 4 ° C for 24 hours, protein A is added and the tubes vortexed, incubated at room temperature for 90 minutes, and centrifuged at approximately 2000-2500 X to 4 ° C to precipitate antibody complexes bound to the labeled antigen. The supernatant is removed by aspiration and the radioactivity in the granules measured in a gamma counter. The dilution of antiserum that binds approximately 10 to 40% of the labeled protein after subtraction of the non-specific binding is further characterized. Next, a dilution range (from about 0.1 pg to 10 ng) of the angiostatin protein used for the development of the antiserum is evaluated by adding known amounts of the protein to tubes containing radiolabelled protein and antiserum. After an additional incubation period, for example, 24 to 48 hours, protein A is added and the tubes are centrifuged, the supernatant is removed and the radioactivity in the pellet is stabilized. The displacement of the binding of the angiostatin protein radioactively labeled by the unlabeled angiostatin protein (standard) provides a standard curve. Different concentrations of other protein fragments of angiostatin, plasminogen, angiostatin from different species and homologous proteins are added to the test tubes to characterize the specificity of angiostatin antiserum. Extracts of various tissues, including but not limited to primary and secondary tumors, Lewis lung carcinoma, cultures of angiostatin-producing cells, placenta, uterus and other tissues such as the brain, liver and intestine, are prepared using extraction techniques that have been used successfully to extract angiostatin. After lyophilization or Speed Vac of the tissue extracts, test buffer is added and different aliquots are placed in the RIA tubes. Extracts from known angiostatin-producing cells produce displacement curves that are parallel to the standard curve, whereas tissue extracts that do not produce angiostatin do not displace the radioactively labeled angiostatin from the angiostatin antiserum. In addition, extracts of urine, plasma and cerebrospinal fluid from animals with Lewis lung carcinoma were added to the test tubes in increasing amounts. The parallel displacement of the curves indicates the utility of the angiostatin assay for measuring angiostatin in tissues and body fluids. The tissue extracts containing angiostatin are further characterized by subjecting aliquots to HPLC in the inverted phase. The eluted fractions are collected, dried in a Speed Vac, reconstituted in RIA buffer and analyzed in the angiostatin RIA. The maximum amount of immunoreactivity in angiostatin is located in the fractions corresponding to the angiostatin elution position. The assay kit provides the instructions, antiserum, angiostatin or angiostatin protein, and possibly angiostatin and / or radiolabeled reagent for the precipitation of angiostatin-angiostatin antibody bound complexes. The equipment is useful for the measurement of angiostatin in biological fluids and tissue extracts from animals and humans with and without tumors. Other equipment is used for the localization of angiostatin in tissues and cells. This team of inhistochemistry of angiostatin provides the instructions, angiostatin antiserum, and possibly serum blocker and secondary antiserum linked to a fluorescent molecule, such as fluorescein isothiocyanate, or some other reagent used to visualize the primary antiserum. Immunohistochemical techniques are well known to those skilled in the art. The immunohistochemical equipment of angiostatin allows the localization of angiostatin in sections of tissue and cultured cells using simple and electronic microscopy. This is used for research and clinical purposes. For example, biopsies are made or tumors and tissue sections are collected with a microtome to examine angiostatin production sites. Such information is useful for diagnosis and possibly for therapeutic purposes in the detection and treatment of cancer. Another method for visualizing angiostatin biosynthesis sites involves radiolabeling nucleic acids for use in the hybridization of a probe to the angiostatin messenger RNA. Similarly, the angiostatin receptor can be localized visualized and quantified with immunohistochemical techniques. The invention is further illustrated by the following examples, which should not be constituted in any way in limitations imposed on the scope thereof. On the contrary, it should be clearly understood that various other modalities, modifications and equivalents thereof may be resorted to, which, after reading the description herein, may in themselves be suggested to those skilled in the art without departing from the spirit of the invention. the present invention and / or the scope of the appended claims.

Example 1 Selection of an animal tumor system in which the growth of the metastasis is inhibited by the primary tumor and is accelerated after the removal of the primary tumor. Selecting a variety of murine tumors capable of inhibiting their own metastasis, a Lewis lung carcinoma was selected, in which the primary tumor more efficiently inhibited lung metastasis.

Male six-week-old C57B16 / J syngeneic mice were injected (subcutaneous dorsum) with 1 x 10 6 tumor cells. Visible tumors appeared for the first time after 3-4 days. When the tumors were approximately 1500 mm3 in size, the mice were randomly divided into two groups. The primary tumor was completely removed in the first group and left intact in the second group after a sham operation. Although tumors from 500 mm3 to 3000 mm3 inhibited the growth of metastasis, the 1500 mm3 tumor was the largest primary tumor that could be safely resected with high survival and without local recurrence. 21 days later, all the mice were sacrificed and subjected to autopsy. Mice with an intact primary tumor had four +2 visible metastases, compared to fifty +5 metastases in the mice in which the tumor had been removed (p <0.0001). These data were confirmed by the weight of the lung, which is closely related to the tumor burden, as previously demonstrated. There was an increase of 400% in the wet weight of the lung in the mice from which the tumors were removed compared to the mice in which the tumor remained intact (p <0.0001).

This experimental model has reproducible data and the described experiment is reproducible. This tumor was labeled as "Lewis lung carcinoma-low metastasis" (LLC-Low). The tumor also suppressed the metastasis in an almost identical pattern in SCID mice, which are deficient in B and T lymphocytes.

Example 2 Isolation of a variant of Lewis lung carcinoma tumor that is highly metastatic, whether or not the primary tumor is removed. A highly metastatic variant of Lewis lung carcinoma arose spontaneously from the LLC-Low cell line of Example 1 in a group of mice and has been isolated according to the methods described in Example 1 and repeatedly transplanted. This tumor (LLC-High) forms more than 30 visible lung metastases whether or not the primary tumor is present.

Example 3 Size of the metastases and sites of proliferation of tumor cells within them. Effect of the primary tumor that inhibits metastasis (CLL-Low). C57B16 / J mice were used in all experiments. Mice were inoculated subcutaneously with LLC-Low cells, and 14 days later the primary tumor was removed in half of the mice. At 5, 10 and 15 days after having removed a tumor, the mice were sacrificed. Histological sections of pulmonary metastases were obtained. Mice with an intact primary tumor had micrometastases in the lung, which were not neovascularized. These metastases were restricted to a diameter of 12-15 cell layers and did not show a significant increase in size, even 15 days after tumor removal. In contrast, the animals to which the primary tumor was removed revealed vascularized metastases as early as 5 days after the operation. These metastases experienced an additional 4-fold increase in volume on day 15 after the tumor was removed (as reflected by the weight and histology of the lung). Approximately 50% of the animals that had their primary tumor removed died of lung metastasis before the end of the experiment. All animals with an intact primary tumor survived at the end of the experiment. The reproduction rate of the tumor cells within the metastasis was determined by counting nuclei stained with -BrdU that had previously been injected into the mice. The high percentage of tumor cells that incorporate BrdU in avascular metastasis, small, of animals with an intact primary tumor was equivalent to the incorporation of BrdU of tumor cells in the large vascularized metastases of mice from which a primary tumor had been removed (Figure 3). These findings suggest that the presence of a primary tumor had no direct effect on the speed of reproduction of the tumor cells within the metastasis. In Figure 3, the panel on the left shows the BrdUd labeling index of the tumor cells in the lung in the presence or absence of a primary tumor. Before immunohistochemical staining, sections were permeabilized with 0.2 M HCl for 10 minutes and digested with 1 μg / ml proteinase K (Boehringer Mannheim GmbH, Mannheim, Germany) in 0.2 Tris-HCl, 2 mM CaCl2 at 37 ° C during 15 minutes. The marking index was established by counting the percentage of positive nuclei at a power of 250. The right panel of Figure 3 describes a total lung weight analysis of tumors with intact primary tumors removed 5, 10 and 15 days after the operation. . The animals were sacrificed 6 hours after the intraperitoneal injection of BrdU (0.75 mg / mouse).

Example 4 Inhibition of angiogenesis of lung metastases in the presence of an intact primary tumor. To measure the degree of vascularization in lung metastases, tissues were stained with antibodies against von Willebran factor (a specific endothelial marker, available from Dako Inc., Carpenteria, CA). Metastases from animals with intact tumors formed a thin fist (8-12 layers of tumor cells) around the existing pulmonary vessels. Except for the endothelial cells of the vessel lining, no or few cells were positive for von Willebrand factor. In contrast, lung metastases from animals 5 days after the removal of the primary tumor were not only larger but also infiltrated with capillary sites containing endothelial cells, which stained strongly for von Willebrand factor. In the immunohistochemical analysis of the presence of endothelial cells in the lung metastases, a pulmonary metastasis with the primary pulmonary tumor intact 19 days after the inoculation, had a fist of tumor cells around a pre-existing microvaso in the lung. The metastasis was limited to 8 to 12 cell layers. There was no evidence of 9a neovascularization around the microvaso, and did not contain any new microvaso. This was typical of the maximal size of an avascular preangiogenic metastasis. In an immunohistochemical analysis of the tissue collected 5 days after a primary tumor was resected (19 days after the inoculation of a primary tumor), the metastases surrounded a preexisting vessel in the lung. In contrast, in the sample where a primary tumor was resected, the tumor was neovascularized. Thus, an intact primary tumor inhibits the formation of new capillary blood vessels in metastases, but the proliferation of tumor cells within a metastasis is not affected by the primary tumor.

Example 5 A primary tumor inhibits the angiogenesis of a second tumor implanted in the mouse cornea. The growth of this second tumor is inhibited. 0.25 to 0.5 mm2 of Lewis lung tumor (LLC-Low) was implanted in the cornea of a mouse on day 0. (Muthukkaruppan, Angigenesis in the mouse cornea, Science 205: 1416-1418, 1979). A primary tumor was formed by inoculating IxlO6 LLC-Low cells subcutaneously on the dorsum, either 4 6 7 days before corneal implantation; or the day of the corneal implant; or 4 or 7 days after the corneal implant. The control mice received the corneal implant but not subcutaneously. Other control mice received the corneal implant and an inoculation of LLC-Alto tumor cells on the back 4 days before the cornea implant. The corneas were evaluated daily by stereomicroscopy by slotted lamp for the growth of the corneal tumor (measured by an ocular micrometer) and the growth of new capillaries from the edges of the corneal limbus. In control mice that do not have a primary subcutaneous tumor, most of the corneas (6/8) developed new neovascularization beginning on day 6 to day 7 after corneal implantation and continuing until day 10. On day 10, the tumors of vascularized cornea reached approximately a quarter of the volume of the entire eye. In the presence of the primary subcutaneous CLL-low tumor, the corneal implants were not vascularized if the primary tumor remained in place for at least 4 days or more before the corneal implant (Table 1). In the absence of neovascularization, the horny tumors grew slowly as white, thin, avascular discs within the cornea. However, if the primary tumor was not implanted until 4 days after the corneal implant, the corneas were vascularized and 3/3 corneal tumors grew at rates similar to the controls without tumor. In the presence of the primary subcutaneous LLC-High tumor, most of the corneas (2/3) developed neovascularization beginning on day 7 after cochrane implantation and continuing until day 10. On day 10, the vascularized corneal tumors again reached approximately fourth of the volume of the whole eye.

Table 1 Inhibition of tumor angiogenesis in the cornea by a primary subcutaneous tumor. [All primary tumors are LLC-Low except (*) which is LLC-High].

Day of the implant of the eye 0 0 0 0 0 0 0 Implantation day of -7 -4 -4 * or none +4 +7 primary tumor Number of mice with 2/10 0/9 2/3 2/3 6/8 3/3 2/3 new horny vessels in the day 10 It would be expected that 0/10 corneas would show neovascularization when the primary subcutaneous LLC-Low tumor was implanted 7 days prior to tumor implantation in the eye (ie -7). However, 2 of the tumors (2/10) became necrotic because they were too large (> 3 cm3).

EXAMPLE 6 The primary intact tumor inhibits angiogenesis induced by a secondary subcutaneous implantation of basic fibroblast growth factor (bFGF). Although the experiments described in the Examples V and VI show that a primary tumor inhibits angiogenesis in a secondary metastasis; these studies do not reveal whether the primary tumor: (i) inhibits endothelial proliferation (or angiogenesis) directly, or (ii) indirectly by deregulation of activity angiogenic of metastatic tumor cells. To distinguish between these two possibilities, a focus of subcutaneous angiogenesis was induced by means of a matrigel implant containing the growth factor of the basic fibroblasts (bFGF) (Passaniti A, et al., A simpler quantitative method, to evaluate angiogenesis and anti-angiogenic agents using reconstituted basement membrane, heparin and fibroblast growth factor, Lab. Invest. 67: 519, 1992). Matrigel (an extract of basal membrane proteins), containing 25 or 50 ng / ml of bFGF in the presence of heparin, was injected subcutaneously on the ventral surface of normal and tumor-bearing mice (LLC-Low). The mice were sacrificed 4 days later and the concentration of hemoglobin in the gel was measured to quantify the formation of blood vessels. Previously it had been shown that the number of new vessels entering the matrigel correlates with the concentration of hemoglobin. (Folkman J., Angiogenesis and its inhibitors in "Important Advances in Oncology 1985", VT DeVita, S. Hellman and S. Rosenberg, editors, J.B. Lippincott, Philadelphia 1985). Some gels were also prepared for the histological examination. In normal mice, matrigel granules containing 50 ng / ml bFGF were completely red. They were heavily invaded by new capillaries, and contained 2.4 g / dl of hemoglobin. The matrigel lacking the bFGF is transparent and gray and contained only 0.4 g / dl of hemoglobin (a difference of 6 times). In contrast, the matrigel of mice with a primary tumor contained only 0.5 g / dl (Figure 4). The almost complete inhibition of angiogenesis in this experiment suggests that the presence of a Lewis primary pulmonary tumor can inhibit the angiogenesis induced by bFGF directly.

Example 7 The transfer of serum from an animal with tumor to an animal from which the primary tumor has been removed suppresses the metastasis. Mice were implanted with Lewis lung carcinoma as described above. 15 days later, when the tumors were approximately 1500 mm3, the mice were randomly divided into 4 groups. Three groups underwent complete surgical resection of the primary tumor; in one group the tumors were left in place (after a sham surgical procedure). The mice in the three resection groups then received daily intraperitoneal injections of saline, serum from normal non-tumor mice, or serum from mice with Lewis lung carcinomas of 1500 mm3. The group of mice with intact tumors received intraperitoneal saline injections. All mice were treated for 21 days, after which the animals were euthanized and lung metastases counted (Table 2).

Table 2 Primarlo Tumor Removed Intact Primary Tumor Treatment Solution Serum Injection Serum (Saline injections mice mice of Solution Intraperitoneal) normal with Saline tumor Number of Metastases 55 ± 5 50 ± 4 7 ± 2 3 ± 1 pulmonary: Those results were confirmed by the weight of the lung. P = < 0.0001 for the difference between the two groups [(55 & 50) vs (7 & 3)]. Similar results were obtained using angiostatin from the urine of animals with tumor.

EXAMPLE 8 Bovine Capillar Endothelial Cell (BCE) Assay. BCE cells were used between 9 and 14 passes only. On day 0, BCE cells were cultured on gelatinised well plates (1.5% gelatin in PBS at 37 ° C, 10% C02 for 24 hours and then rinsed with 0.5 ml of PBS) at a concentration of 12,500 cells / water well. The cell counts were made using a hemocytometer. Cells were cultured in 500μl of DMEM with 10% inactivated bovine sheep serum (56 ° C for 20 minutes) and 1% glutamine-pen-strep (GPS). BCE cells are challenged as follows: The media was removed and replaced with 250μl of DMEM / 5% BCS / 1% GPS. The sample to be tested was then added to the wells. (The amount varies depending on the samples to be tested). The plates are placed at 37 ° C / 10% C02 for approximately 10 minutes. 250μl of DMEM / 5% BCS / 1% GPS was added with 2 ng / ml of bFGF to each well. The final media are 500μl DMEM / 5% BCS / 1% GPS / with lng / ml bFGF. The plate is returned to an incubator at 37 ° C / 10% C02 for 72 hours. At day 4, cells are counted by stirring the medium and then all wells are trypsinized (0.5 ml trypsin / EDTA) for 2 to 3 minutes. The suspended cells are then transferred to flask flasks with 9.5 ml. of Hemetal and are counted using a Coulter counter. One unit of activity is the amount of serum containing angiostatin which is capable of producing the maximum average inhibition of capillary endothelial proliferation when the endothelial cells are incubated in 1 ng / ml of bFGF for 72 hours.

Example 9 Serum from mice with a low metastatic Lewis lung tumor (CLL-Low) inhibits the proliferation of capillary endothelial cells in vi tro. Bovine capillary endothelial cells were stimulated with growth factor of the base fibroblasts (1 ng / ml of bFGF), in a proliferation test of 72 hours. Serum from mice with tumor added to those cultures inhibited endothelial cell proliferation in a dose-dependent and reversible manner. The normal serum was not inhibitory (Figure 5). Endothelial cell proliferation was similarly inhibited (relative to controls) by serum obtained from nu / nu mice and SCID mice with tumor. After the primary tumor was removed, the activity of angiostatin disappeared in the serum for 3-5 days. Serum with tumor also inhibited bovine aortic endothelial cells and endothelial cells derived from a spontaneous mouse hemangioendothelioma, (Obeso, et al., "Methods in Laboratory Research, A cell line derived from Hemangioendothelioma; its use as a model for the study of Endothelial Cell Biology ", Lab Invest., 63 (2), pgs 259-269, 1990) but did not inhibit Lewis lung tumor cells, 3T3 fibroblasts, aortal smooth muscle cells, pulmonary epithelium of mink, or fetal lung fibroblasts of human W138.

Example 10 The serum of mice carrying the Lewis lung tumor (LLC-High) that does not inhibit metastasis, does not inhibit the proliferation of endothelial cells in vitro. Serum from mice carrying the primary LLC-Alto tumor does not significantly inhibit the proliferation of bovine capillary endothelial cells stimulated with bFGF in relation to the controls. Also, when this serum was subjected to the first two steps of purification (chromatography with heparin-Sepharose and gel filtration), no angiostatin activity was found in any of the fractions.

Example 11 The ascites of Lewis lung carcinoma (low metastasis) also generate angiostatin serum. Mice received intraperitoneal injections of tumor cells (106) of LLC-Low or LLC-High, and a week later, 1-2 ml of blood ascites were obtained from each of 10-20 mice. A seeding of the mesenteric tumor was observed. The mice were then euthanized. The serum was obtained by cardiac puncture. Serum was also obtained from normal non-tumor-bearing mice as a control.

Serum and ascites were centrifuged to remove cells, and the supernatant was assayed on bovine capillary endothelial cells stimulated by bFGF (1 ng / ml) (see Example IX). The ascites originating from both types of tumors stimulated a significant proliferation of capillary endothelial cells (e.g., 100% proliferation) over the controls after 72 hours (Figure 6). In contrast, serum from poorly metastatic mice inhibited endothelial cell proliferation (inhibition of 79% of controls). The serum of the highly metastatic line was stimulator by 200%. These data show that ascites of the metastatic low line contain a predominance of an endothelial growth stimulator over angiostatin. This condition is analogous to a solid primary tumor. In addition, the activity of angiostatin appears in the serum, although it does not seem to oppose stimulatory activity. This pattern is similar to the solid primary tumor (CLL-Low). Ascitos of highly metastatic tumor (LLC-Alto) also seem to contain a predominant stimulator of endothelial cells, but angiostatin can not be identified in the serum.

Example 12 Fractionation of serum angiostatin by column chromatography and analysis of growth inhibitory fractions by SDS-PAGE. To purify angiostatins, sera from tumor-bearing mice were pooled. The inhibitory activity, tested according to the in vitro inhibitory activity assay described above, was subjected to sequential chromatography using heparin-Sepharose, Biogel AO .5 mm agarose, and several cycles of inverted phase high resolution liquid chromatography (HPLC). C4 The SDS-PAGE of the HPLC fraction contained endothelial inhibitory activity, revealed a discrete band of apparent reduced Mr of 38,000 Daltons, which was purified approximately 1 million times (see Table 3) at a specific activity of approximately 2xl07. At different stages of the purification, the pooled fractions were tested with specific antibodies to determine the presence of known endothelial inhibitors. Platelet factor 4, thrombospondin, or growth factor transforming beta, were not found in partially purified or purified fractions.

Table 3 Specific Activity (units * / mg) Purification base Serum 1.69 1 Hepapna Sepharose 14.92 8 .8 Bio-Gel A0. 5th 69.96 41.4 C AR / C4 M.07 1.2X106 * A unit of activities is that amount of serum containing angiostatin which is capable of producing a maximum average inhibition of capillary endothelial proliferation when the endothelial cells are incubated in 1 ng / ml of bFGF for 72 hours.

EXAMPLE 13 Fractionation of angiostatin from urine by column chromatography and analysis of growth inhibitory fractions by SDS-PAGE Purification of serum endothelial cell inhibitors is hampered by the small volume of serum obtainable from each mouse and for the large amount of protein in the serum. Urine from tumor-bearing mice was analyzed and found to contain an inhibitor of endothelial cell proliferation that is absent from the urine of non-tumor-bearing mice and mice with LLC-High tumors. The purification of the inhibitory activity of the cells Endothelial cells were carried out by the same strategy that was used for the purification of the serum (described above) (Figure 7). Figure 7 shows the C4 inverted phase chromatography of partially purified serum or urine from tumor bearing animals. All fractions were assayed on bovine capillary endothelial cells with bFGF in a 72 hour proliferation assay as described in Example IX. A discrete inhibition peak was observed in both cases eluting 30-35% of acetonitrile in fraction 23. Electrophoresis on polyacrylamide gel with SDS of the inhibitory fraction of the third cycle of C4 inverted phase chromatography of the serum of the carrier animals of tumor showed a single band at approximately 38, 000 Daltons.

Example 14 Characterization of Circulating Angiostatin Endothelial inhibition was tested according to the procedure described in Example 9. Angiostatin was isolated on a C4 column of HPLC Synchropak (Synchrom, Inc. Lafayette, IN). The inhibitor was eluted in an acetonitrile gradient of 30 to 35%.

On an electrophoresis gel on polyacrylamide gel with sodium dodecyl sulfate (PAGE) under reducing conditions (b-mercaptoethanol (5% volume / volume), the protein band with activity eluted at 38 kilodaltons. protein with activity eluted at 28 kilodaltons The activity was found at similar points, whether the initial mass was isolated from urine or serum, no activity was detected with any other band, activity associated with the bands was low when heated (100 ° C for 10 minutes) or treated with trypsin.When the band with activity was extracted with a water / chloroform mixture (1: 1), the activity was found in the aqueous phase only.

Example 15 Purification of human plasminogen inhibitor fragments: The lysine binding site of plasminogen I was obtained from Sigma Chemical Company. The preparation was purified from human plasminogen after digestion with elastase. The lysine I binding site obtained in this way is a population of proteins that contains, in aggregate form, at least the first three triple ring structures (numbers 1 to 3) in the A chain of plasmin (Kringle 1 + 2 + 3). (Sotrrup-Jensen, L., et al., In Progress in Chemical Fibrinolysis and Thrombolysis, Vol. 3, 191, Davidson, JF, et al., Raven Press, New York 1978 and William, B., et al., Biochemi ca et Biophysi ca Acta, 579, 142 (1979)). The plasminogen l lysine binding site (Sigma Chemical Company, St. Louis, MO) was resuspended in water and applied to a C4 inverted phase column that had been equilibrated with water grade CLAP / 0.1% TFA. The column was eluted with a water / 0.1% TFA to acetonitrile / 0.1% TFA gradient and the fractions were collected in polypropylene tubes. An aliquot of each was evaporated in a speed vac, suspended with water, and applied to BCE in a proliferation assay. This procedure was repeated twice for the inhibitory fractions using a similar gradient for elution. The inhibitory activity eluted at 30-35% acetonitrile in the final run of column C4. The SDS-PAGE of the inhibitory fraction revealed three discrete bands of apparent reduced molecular mass of 40, 42.5 and 45 kd. SDS-PAGE under non-reducing conditions revealed three bands of molecular mass of 30, 32.5 and 35 kd, respectively.

EXAMPLE 16 Extraction of SDS-PAGE inhibitory activity Purified purified human plasminogen inhibitory fractions were purified by SDS-PAGE under non-denaturing conditions. The areas of the gel corresponding to the bands observed in neighboring lanes loaded with the same samples were cut by silver staining of the gel and incubated in 1 ml of phosphate buffered saline at 4 ° C for 12 hours in polypropylene tubes. The supernatant was removed and dialysed twice against saline for 6 hours (MWCO = 6-8000) and twice against distilled water for 6 hours. The dialysate was evaporated by vacuum centrifugation. The product was suspended in saline and applied to bovine capillary endothelial cells stimulated with a 1 mg / ml growth factor of the basic fibroblasts in a 72-hour trial. The protein extracted from each of the three bands inhibited the capillary endothelial cells.

Example 17 Studies of Plasminogen Fragment Treatment Mice were implanted with Lewis lung carcinoma and resected when the tumors were 1500-2000 mm3. On the day of the operation, the mice were randomly divided into 6 groups of 6 mice each. Mice received daily intraperitoneal injections with the three purified inhibitory fragments of human plasminogen, complete human plasminogen, urine from tumor-bearing animals, urine from normal mice or saline. A group of tumor bearing animals that had only one simulated procedure was treated with saline injections. Immediately after the removal of the primary tumor, the mice received an intraperitoneal injection of 24 μg (1.2 mg / kg / day / mouse) of inhibitory plasminogen fragments as a loading dose. They then received daily intraperitoneal injections of 12 μg of the inhibitory fragment (0.6 mg / kg / day / mouse) for the duration of the experiment. Control mice received the same dose of complete plasminogen molecule after tumor removal. For urine treatments, the urine of normal and tumor-bearing mice was filtered, exhaustively dialysed, lyophilized and then resuspended in sterile water to obtain a concentration of 250 times. 0.8 ml of dialyzed urine concentrate was given to the mice, either from tumor carriers or normal mice, in two intraperitoneal injections on the day of primary tumor removal as a loading dose. They then received daily intraperitoneal injections of 0.4 ml of dialyzed and concentrated urine during the course of the experiment. The treatments continued for 13 days, at which point all mice were sacrificed and subjected to autopsy. The results of the experiment are shown in Figures 8 and 9. Figure 8 shows lung and superficial metastasis after 13 days of treatment. The superficial pulmonary metastases refer to the number of metastases observed in the lungs of the mice at autopsy. A stereomicroscope was used to count the metastases. Figure 8 shows the average number of superficial lung metastases that were counted and the standard error of the mean. As shown, the group of mice with the primary tumor present did not show metastasis. Mice in which the primary tumor was resected and were treated with saline showed extensive metastases. Mice treated with the human-derived plasminogen fragment showed no metastasis. Mice treated with complete plasminogen showed extensive metastases indicating that the whole plasminogen molecule has no endothelial inhibitory activity. Those mice treated with dialyzed and concentrated urine from tumor bearing mice showed no metastasis. Mice treated with concentrated urine from normal mice showed extensive metastases. When the lung weight was measured, similar results were obtained (Figure 9).

EXAMPLE 18 Amino acid sequence of murine and human angiostatin The amino acid sequence of angiostatin isolated from mouse urine and angiostatin isolated from the preparation of the human lysine binding site fragment I was determined in a protein sequencer of Applied Biosystem Model 477A. The amino acid fractions with phenylthioidantoin were identified with a CLI online ABI Model 120A. The amino acid sequence was determined from the N terminal sequence and the tryptic digests of murine and human angiostatin indicate that the sequence of angiostatin is similar to the sequence starting at amino acid number 98 of murine plasminogen. Thus, the amino acid sequence of angiostatin is a molecule comprising a protein having a molecular weight of between about 38 kilodaltons and 45 kilodaltons as determined by the electrophoresis on reducing polyacrylamide gel and having a sequence of amino acid substantially similar to that of a murine plasminogen fragment starting at amino acid number 98 of an intact murine plasminogen molecule. The start of the amino acid sequence of murine angiostatin (SEQ ID NO: 2) is shown in Figure 1. The length of the amino acid sequence may be slightly longer or shorter than that shown in Figure 1. The analysis of N-terminal amino acids and tryptic digestions of the active fraction of the human lysine binding site I (See Example 15) shows that the sequence of the fraction starts at approximately amino acid 97 or 99 of human plasminogen and human angiostatin is homologous to murine angiostatin. The start of the amino acid sequence of human angiostatin (starting at amino acid 98) is shown in Figure 2 (SEQ ID NO: 3). The amino acid sequence of human and murine angiostatin is compared in Figure 2 with the corresponding internal amino acid sequences of plasminogen from other species, including porcine, bovine and Rhesus monkey plasminogen, indicating the presence of angiostatin in these species.

Example 19 Expression of human angiostatin in E. coli The vector pTrcHisA (Invitrogen) was used (Figure 10) to obtain a high level, regulated transcription of the trc promoter to increase the efficiency of the translation of aucariotics genes in E. coli. The angiostatin was expressed fused to a polyhistidine tail that binds to the N-terminal nickel for one step purification using metal affinity resins. The site of recognition of the cleavage of enterokinase in the fusion protein allows the subsequent removal of the N-terminal histidine fusion protein from the purified recombinant protein. It was found that the recombinant human angiostatin protein binds to lysine; it cross-reacts with monoclonal antibodies specific for the kringle 1, 2 and 3 regions, and inhibits the proliferation of endothelial cells directed by bFGF in vi tro. To construct the insert, the gene fragment coding for human angiostatin was obtained from human liver mRNA, which was reverse transcribed and amplified using the polymerase chain reaction (PCR) and specific primers. The product of 1131 base pairs codes for amino acids 73 to 470 of human plasminogen. The amplified fragment was cloned and the Xhol / Kpnl site of pTrcHisA, and the resulting construct was transformed into E. coli XL-1B host cells (available from Stratagene). A clone control containing the plasmid vector pTrcHisA alone was transformed into E. coli XL-1B host cells as well. This clone is known as the vector control clone. Both clones were purified identically according to what is described below. The clones were selected with expression in the following manner. Collages detached from E. coli transformed with the gene coding for angiostatin were grown on nitrocellulose filters impregnated with IPTG and placed on an LB agar plate. After the induction of expression with IPTG, the colonies were used on nitrocellulose filters. Released nitrocellulose was blocked, rinsed and probed with separate monoclonal antibodies (Acm Dcd and Vap).; donated by S.G. McCance and F.J. Castellino, University of Notre Dame) which recognize the specific conformations of angiostatin. The colonies with expression strongly recognized by the mAbs were selected. To identify the optimal time for maximum expression, cells were collected at various times before and after induction by IPTG and exposed to repeated freeze-thaw cycles followed by SDS-PAGE analysis, immuining and probing with Dcd mAbs and Vap. Of those, the clone pTrcHisA / HAsAH4 was selected. Induction with IPTG was for 4 hours, after which the cell pellet was harvested and resuspended in 50 mM Tris at pH 8.0, 2 mM EDTA, 5% glycerol and 200 mg / ml lysozyme and stirred for 30 minutes at 4 ° C. The suspension was centrifuged at 14,000 rpm for 25 minutes and the pellet was resuspended in 50 mM Tris at pH 8.0, 2 mM EDTA, 5% glycerol and 0.1% DOC. This suspension was stirred for 1 hour at 4 ° C, and then centrifuged at 14,000 rpm for 25 minutes. The supernatant fraction in this step contains expressed angiostatin. It was found that human angiostatin expressed in E. coli possesses the physical properties of native angiostatin, ie the ability to bind to lysine. The angiostatin expressed in E. coli was thus purified on a lysine-sepharose column (Pharmacia or Sigma) in a single step. Elution of angiostatin from the column was with 0.2M epsilon-amino-n-caproic acid, pH7.5. After these experiments, the scaling was carried out on fermentation batches of 10 L of clones pTrcHisA / HAsH4. The cells obtained from this scaling induction were pelleted and resuspended in 50 mM Tris pH7.5, broke at 10,000 psi + three times cooling to 10 ° C between the passes. The lysate obtained was clarified by centrifugation at 10,000 rpm for 30 minutes at 4 ° C, and the expressed angiostatin was isolated on lysine-sepharose (Figure 11). Human angiostatin expressed in purified E. coli was exhaustively dialyzed with water and lyophilized. The expressed human angiostatin was resuspended in media (DMEM, 5% BCS, Gentamicin / penicillin / streptomycin) at an estimated concentration of 3 ug / ml, and the bovine capillary endothelial cell (BCE) assay was used in vi tro, in accordance to that described in EXAMPLE 8, page 39. Similarly, the control clone containing the vector was treated only in the same way as the clone pTrcHisA / HAsH. This was induced identically with IPTG, and the bacterial lysate used to bind lysine, eluted with 0.2 M aminocaproic acid, exhaustively dialyzed and lyophilized. This control preparation was resuspended in media also at an estimated concentration of 3 ug / ml. The samples of recombinant angiostatin, and controls obtained from different batches of induction and fermentation, as well as separate purification, were all encoded in EntreMed, Maryland. The ECB tests were performed with those samples coded blindly at the Children's Hospital of Boston. The results of the ECB assays of recombinant human angiostatin showed that human angiostatin expressed in E.coli inhibited the proliferation of BCE cells due to bFGF (used at 1 ng / ml) (Figure 12). The standard recombinant angiostatin in the media (at approximately 3 ug / ml) was used at a dilution of 1: 5, 1:10 and 1:20. The percent inhibition was calculated as follows: number of cells with angiostatin-number of cells per day 0 1- number of cells with bFGF only-number of cells per day 0 Percent inhibition of BCE cell proliferation was comparable to or greater than that of angiostatin derived from plasminogen at similar concentrations. The results of a repeated test of the ECB assay are described in Figure 13, where a 1: 5 dilution of the standard, recombinant angiostatin, gives percentage inhibitions similar to those obtained with plasminogen-derived angiostatin. Figure 13 shows the surprising result that the recombinant human angiostatin protein inhibits more than 60%, and up to 75% of BCE proliferation in culture.

Example 20 Angiostatin maintains the delay of micrometastases by increasing the rate of apoptosis. After subcutaneous inoculation of mice C57 BL6 / J in Lewis lung carcinoma cells (lOxlO6), primary tumors of approximately 1.5 cm3 were developed. The animals were subjected to surgical removal of the primary tumor or similar surgery. At 5, 10 and 15 days after surgery, the mice were sacrificed and their lungs prepared for histological examination. Animals with resected primary tumor showed massive proliferation of micrometastases compared to controls operated in a similar manner (Figure 14). These changes were accompanied by a significant increase in lung weight. Tumor cell proliferation analysis, measured by bromo-deoxyuridine (BrdU) uptake showed no difference between animals with intact primary tumors or tumors resected at 5, 9 and 13 days indicating that the increase in tumor mass could not explained by an increase in proliferation (Figure 15). Consequently, the cell death of these animals was examined. Apoptosis, a process of cell death is independent of changes in gene expression and contributes to the elimination of cells during development and in rapidly proliferating tissues, such as the small intestine, was examined with fragmented DNA immunohistochemically labeled with the technique of terminal deoxynucleotidyl transferase (TdT). The apoptotic index was determined at each time of slaughter. The removal of the primary tumors caused a statistically significant increase of (approximately 3 to 4 times) in the apoptotic index in all the times examined (Figure 15). Supportive evidence was obtained by treating the mice with primary tumors removed with an exogenous suppressor of angiogenesis. This substance, TNP-1470 (O-chloroacetylcarbamoyl fumagillol, formerly called AGM-1470), is an analogue of fumagillin with reported antiangiogenic activity. Subcutaneous injection of TNT-1470 (30 mg / kg twice daily) produced results that were strikingly similar to those described for animals that had intact primary tumors. These animals had a lower lung weight equivalent to a proliferative index and an apoptotic index compared to the controls injected with saline (Figure 16). These data indicate metastases that remain inactive when the proliferation of tumor cells is balanced by a rate equivalent to cell death. The removal of the primary tumor causes a rapid increase in the growth of the metastases, probably linked to the removal of the angiogenesis inhibitors (angiostatin) which control the etastatic growth by increasing apoptosis in tumor cells. These effects are similar to those observed after the removal of primary tumors and the administration of an exogenous inhibitor of angiogenesis. Taken together, these data suggest that a primary tumor releases angiostatin, which maintains the inactivity of micrometastases.

Example 21 Treatment of primary tumors with angiostatin in vivo. Angiostatin from human plasminogen was purified by limited digestion with elastase according to that described in Example 15 above. Angiostatin was resuspended in phosphate-buffered saline to be administered in six week old C57BI6 / J mice. Animals were implanted subcutaneously with lxlO6 tumor cells from either Lewis lung carcinoma or T241 fibrosarcoma. Treatment with angiostatin begins after four days when the tumors are 80-160 mm3__dLe_ size. Mice received injections of angiostatin in either a single injection of 40 mg / kg or two injections of 80 mg / kg via the intraperitoneal (ip) or subcutaneous (sc) routes. The animals were sacrificed at various times after a treatment that lasted up to 19 days. Angiostatin, administered at a daily dose of 40 mg / kg ip, produced a highly significant inhibition of T241 primary tumor growth (Figure 17). This inhibitory effect on growth was evidently visible within 2 days and a magnitude was increased throughout the course of the study time. On day 18, mice treated with angiostatin had tumors that were approximately 38% of the volume of controls injected with saline. This difference was statistically significant (p <; 0.001, Student's t test).

Treatment with angiostatin (total dose of 80 mg / kg / day, administered twice daily at 40 mg / kg ip or sc) also significantly reduced the growth rate of primary LLC-LM tumors (Figure 17). This inhibitory effect was evident at 4 days and one magnitude was increased at all the times subsequently examined. The last day of the experiment (day 19), the mice treated with angiostatin had a mean tumor volume that was only 20% that of the controls injected with saline, which was significantly different (p <0.001 Student's t test). In another series of experiments, angiostatin was administered (50 mg / kg ql2h) to mice implanted with T241 fibrosarcoma, Lewis lung carcinoma (LM) or reticulum cell sarcoma cells. For each type of tumor cell, the mice that received angiostatin had a substantially reduced tumor size. Figure 19 demonstrates that for T241 fibrosarcoma, mice treated with angiostatin had average tumor volumes that were only 15% in the mice treated on day 24. Figure 20 demonstrates that for Lewis lung carcinoma (LM), mice treated with angiostatin had mean tumor volumes that were only 13% of untreated mice at day 24. Figure 21 demonstrates that for reticulum sarcoma, mice treated with angiostatin had mean tumor volumes that were only 19% of the untreated mice at day 24. The data represent the average of 4 mice at each point in time. These results demonstrate that angiostatin is an extremely potent inhibitor of the growth of three different primary tumors in vivo.

Example 22 Treatment of primary tumors derived from human cells in mice with angiostatin in vivo. The effect of angiostatin on two human tumor cell lines, PC-3 human prostate carcinoma and MDA-MB human breast carcinoma was studied. Immunodeficient SCID mice were implanted with human tumor cells, and mice treated with 50 mg / kg angiostatin every 12 hours essentially as described in Example 21. The results demonstrate that the angiostatin protein of the present invention is a potent inhibitor of growth of human tumor cells. Figure 22 shows that for PC-3 human prostate carcinoma, mice treated with angiostatin had only 2% of the mean tumor volume, compared to untreated control mice at day 24. Figure 23 shows that for carcinoma of human breast MDA-MB, mice treated with angiostatin had only 8% of the mean tumor volume compared to control mice not treated at day 24.

Example 23 Genetic Therapy - Effect of Angiostatin Gene Transfection on Tumor Volume A 1380 base pair DNA sequence was generated for angiostatin derived from mouse plasminogen cDNA (obtained from the Americam Type Culture Collection (ATCC )), which codes for amino acids 1-460 of mouse plasminogen, using PCR and inserted into an expression vector. The expression vector was transfected into T241 fibrosarcoma cells and the transfected cells were implanted in mice. Control mice received untransfected T241 cells,. or T241 cells transfected with the vector alone (i.e. transfected cells that do not express angiostatin). Clones from the transfected cells expressing angiostatin were used in the experiment. The mean volume of the tumor was determined over time. The results show the surprising and dramatic reduction in the mean volume of the tumor in mice for the clones of cells expressing angiostatin compared to the nontransfected control cells and that do not express it. The mouse DNA sequence encoding the mouse angiostatin protein was derived from the mouse plasminogen cDNA. Mouse angiostatin encompasses the 1-4 kringle regions of mouse plasminogen. The scheme for the construction of this clone is shown in Figure 24. The mouse angiostatin protein clones were transfected into T241 fibrosarcoma cells using the LIPOFECTIN ™ transfection system (available from Life Technologies, Gaithesburg, MODERATION). The LIPOFECTINMR reagent is a 1: 1 (w / w) liposome formulation of N- [l- (2,3-dioleyloxy) propyl] -n, n, n-trimethylammonium chloride of cationic lipid (DOTMA), and diolecoyl phosphotidylethanolamine (DOPE) in water filtered through the membrane. The procedure for the transient transfection of the cells is as follows: 1. T241 cells are grown in 60 cm2 tissue culture vessels, seeded at "1-2 x 105 cells in 2 ml of appropriate growth medium supplemented with serum. 2. Incubate the cells at 37 ° C in an incubator with C02 until the cells are 40-70% confluent. This will usually take 18-24 h, but the time will vary between cell types. The confluence of T241 tumor cells was approximately 70%. 3. Prepare the following solutions in 12 x 75 ml sterile tubes: Solution A: For each transfection, dilute 5 μg of DNA in 100 μl of serum-free OPTI-MEM I Reduced Serum Medium (available from Life Technologies) (Deionized water grade tissue culture can also be used). Solution B: For each transfection, dilute μg of LIPOFECTIN in 100 μl of OPTI-MEM. 4. Combine the two solutions, mix gently, and incubate at room temperature for 10-15 minutes. 5. Wash the cells twice with serum-free medium. 6. For each transfection, add 0.8 ml of serum free medium to each tube containing LIPOFECTINMR-DNA reagent complexes. Mix gently and coat the complex on the cells. 7. Incubate the cells for approximately 12 h at 37 ° C in a C02 incubator. 8. Replace the DNA containing medium with 1 mg / ml selection medium containing serum and incubate the cells at 37 ° C in an incubation with C02 for a total of 48-72 h. 9. Test the cell extracts to determine the genetic activity at 48-72 h after transfection. The transfected cells can be assayed for the expression of the angiostatin protein using antibodies specific for angiostatin. Alternatively, after about 10-14 days, G418 resistant colonies appear on T241 cells transfected with CMV angiostatin. Also, a number of clones were observed in clones transfected with vector only but not in untransfected clones. The G418 resistant clones were selected for their angiostatin expression, using an immunofluorescence method. Interestingly, cell growth in T241 cells and Lewis lung cells transfected with angiostatin was not inhibited or otherwise adversely affected, as shown in Figures 25 and 26. Figure 27 describes the results of the transfection experiment. The three transfected T241 clones expressing angiostatin produced median tumor volumes in the mice that were substantially reduced relative to the tumor volume in the control mice. The mean volume of the tumor of the mice implanted with Clone 37 was only 13% of the control, while with Clone 31 and Clone 25 the tumor volumes were only 21% and 34% of the volumes of the control tumor, respectively. These results demonstrate that the DNA sequences encoding angiostatin can be transfected into cells, that the transfected DNA sequences are capable of expressing angiostatin protein by the implanted cells, and that the expressed angiostatin functions in vivo to reduce the growth of the tumor.

Example 24 Location of the in vivo site of angiostatin expression To localize the in vivo site of angiostatin protein expression, the total RNA of various cell types, Lewis lung carcinoma cells (mouse), T241 fibrosarcoma (mouse) and Burkitt's lymphoma (human), both fresh tumor and cell culture after several passes to determine the presence of angiostatin transcripts. Northern analysis of the samples showed absence of any hybridization signal with the sequence of all the samples except that of normal mouse liver RNA showing a single signal of approximately 2.4 kb corresponding to the mouse plasminogen. Northern analysis of human samples shows an absence of any hybridization signal with the sequence of human angiostatin for all samples except that of human liver RNA normal showing a single signal of approximately 2.4 kb corresponding to human plasminogen. Analysis of the polymerase chain reaction for reverse transcription (RT-PCR) showed an absence of any product from all samples probed with mouse angiostatin sequences except for the normal mouse liver. The RT-PCR analysis showed an absence of any product from all human samples probed with human angiostatin sequences except for the normal human liver (expected size of 1050 bp for the mouse and 1134 bp for the human). In this way, it seems that mouse angiostatin transcripts (assuming an identity with amino acids 97 to 450 of mouse plasminogen) are not produced by all previous mouse samples and human angiostatin transcripts (assuming an identity with amino acids 93 to 470 of human plasminogen) are not produced by previous human samples. The positive signals obtained in normal mouse / human liver are from plasminogen hybridization.

Example 25 Expression of Angiostatin in Yeast The fragment of the gene encoding amino acids 93 to 470 of human plasminogen was cloned into the Xhol / EcoRI site of pHIL-SI (Invitrogen), which allows secreted expression of protein using the signal of PHOl secretion in the yeast Pichia pastoris. Similarly, the fragment of the gene coding for amino acids 93 to 470 of human plasminogen was cloned into the SnaBI / EcoRI site of pPIC9 (Invitrogen) which allows secreted expression of proteins using the secretion signal of factor a in the yeast Pichia pastoris. The human angiostatin proteins expressed in these systems will have many advantages over those expressed in E. coli such as protein processing, protein folding and post-translational modification, including glycosylation. The expression of the gene in P. pastoris is described in (Sreekrishna, K. et al. (1988).) The high level of expression of heterologous proteins in methylotropic yeast Pichia pastoris J. Basic Microbiol. 29 (4): 265-278 , and Clare, JJ et al. (1991) Production of epidermal growth factor in yeast: High level of secretion using strains of Pichia pastoris that contains multiple copies of the gene, Gene 105: 205-212, both of which are incorporated here as reference.

EXAMPLE 26 Expression of angiostatin proteins in transgenic animals and plants Transgenic animals such as bovine or porcine family are created, which express the angiostatin gene transcript. The transgenic animal expresses angiostatin protein, for example in the milk of these animals. Additionally, edible transgenic plants were constructed, which express the transcript of the gene. angiostatin. The construction of transgenic animals expressing foreign DNA is described in Smith H. Transgenic phytochrome: functional, ecological and biotechnological applications, Semin. Cell. Biol. 1994 5 (5): 315-325, which is incorporated herein by reference.

Example 27 Characterization of Angiostatin Fragments That Inhibit the Proliferation of Endothelial Cells. The following example characterizes the activity of the individual and combined angiostatin fragments. The data suggest that there is a functional difference between the individual kringle structure, and the potent antiendothelial activity and consequently the antiangiogenic, which can be obtained from such fragments of angiostatin protein. As used herein, "angiostatin fragment" means a protein derived from angiostatin, or plasminogen, which has an inhibitory activity on the proliferation of endothelial cells. The fragments of angiostatin are useful for treating diseases or conditions mediated by angiogenesis. For example, fragments of angiostatin can be used to inhibit or suppress tumor growth. The amino acid sequence of such angiostatin fragment, for example, can be selected from a portion of murine plasminogen (SEQ ID NO: 1), murine angiostatin (SEQ ID NO: 2); human angiostatin (SEQ ID NO: 3), Rhesus angiostatin (SEQ ID NO: 4), porcine angiostatin (SEQ ID NO: 5), and bovine angiostatin (SEQ ID NO: 6), unless otherwise indicated by the context in which it is used. As used herein, "kringle 1" means a protein derived from a plasminogen having endothelial cell inhibitory activity or anti-angiogenic activity, and having an amino acid sequence comprising a homologue of the kringle sequence 1, exemplified by, but not limited to the murine of kringle 1 (SEQ ID NO: 7), human kringle 1 (SEQ ID NO: 8), kringle 1 Rhesus (SEQ ID NO: 9), kringle 1 porcine (SEQ ID NO: 10), and bovine kringle 1 (SEQ ID NO: 11), unless otherwise indicated by the context in which it is used. The murine kringle (SEQ ID NO: 7) corresponds to the positions of amino acids 103 to 181 (inclusive) of the murine plasminogen of SEQ ID NO: l, and corresponds to the amino acids of positions 6 to 84 (inclusive) of the murine angiostatin of SEQ ID NO: 2. The human kringle 1 (SEQ ID NO: 8), the rhesus kringle 1 (SEQ ID NO: 9) ), the porcine kringle (SEQ ID NO: 10), and the bovine kringle 1 (SEQ ID NO: 11) correspond to the positions of amino acids 6 to 84 (inclusive) of the angiostatin of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. As used herein, "kringle 2" means a plasminogen protein derivative having an endothelial cell inhibitory activity or anti-angiogenic activity, having an amino acid sequence comprising a homolog of the kringle sequence 2 (SEQ ID NO: 12), human kringle 2 (SEQ ID NO: 13), kringle 2 Rhesus (SEQ ID NO: 14), kringle 2 porcine (SEQ ID NO: 15), and kringle 2 bovine (SEQ ID NO: 16), at least that is indicated otherwise by the context in which it is used. The murine kringle 2 (SEQ ID NO: 12) corresponds to the positions of amino acids 185 to 262 (inclusive) the murine plasminogen of SEQ ID NO: 1, and corresponds to amino acid positions 88 to 165 (inclusive ) of murine angiostatin of SEQ ID NO: 2. Human kringle 2 (SEQ ID NO: 13), kringle 2 Rhesus (SEQ ID NO: 14), kringle 2 porcine (SEQ ID NO: 15), and kringle 2 bovine (SEQ ID NO: 16) corresponds to the amino acids at positions 88 to 165 (inclusive) of angiostatin of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. As used herein, "kringle 3" means a plasminogen protein derivative having an endothelial cell inhibitory activity or anti-angiogenic activity having an amino acid sequence comprising a kringle 3 homologue, exemplified for, but not limited to, murine kringle 3 (SEQ ID NO: 17), human kringle 3 (SEQ ID NO: 18), kringle 3 Rhesus (SEQ ID NO: 19), kringle 3 porcine (SEQ ID NO: 20), and kringle 3 bovine ( SEQ ID NO: 21). The murine kringle 3 (SEQ ID NO: 17) corresponds to the amino acids at positions 275 to 352 (inclusive) of the murine plasminogen of SEQ ID NO: 1, and corresponds to the amino acids at positions 178 to 255 (inclusive ) of murine angiostatin of SEQ ID NO: 2. Human kringle 3 (SEQ ID NO: 18), kringle 3 Rhesus (SEQ ID NO: 19), kringle 3 porcine (SEQ ID NO: 20), and kringle 3 bovine (SEQ ID NO: 21) correspond to the amino acids at positions 178 to 255 (inclusive) of the angiostatin of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. As used herein, "kringle 4" means a plasminogen protein derivative having an inhibitory activity or anti-angiogenic activity, having an amino acid sequence comprising a homologue of the kringle sequence 4, exemplified for, but not limited to, the kringle 4 of murine (SEQ ID NO: 22) and kringle 4 human (SEQ ID NO: 23), unless otherwise indicated by the context in which it is used. The murine kringle 4 (SEQ ID NO: 22) corresponds to the amino acids at positions 377 to 454 (inclusive) of the murine plasminogen of SEQ ID NO: 1.

As used herein, "kringle 2-3" means a plasminogen protein derivative having an endothelial cell inhibitory activity or anti-angiogenic activity, having an amino acid sequence comprising a sequence homologous to that of kringle 2-3, exemplified for, but not limited to - kringle 2-3 of murine (SEQ ID NO: 24), kringle 2-3 human (SEQ ID nO: 25), kringle 2-3 Rhesus (SEQ ID NO: 26), kringle 2-3 porcine (SEQ ID NO: 27), and kringle 2-3 bovine (SEQ ID NO: 28), unless that is indicated otherwise by the context in which it is used. The murine 2-3 kringle (SEQ ID NO: 24) corresponds to the amino acids of positions 185 to 352 (inclusive) of the murine plasminogen of SEQ ID NO: 1, and corresponds to the amino acids of positions 88 to 255 (inclusive) of murine angiostatin. of SEQ ID NO: 2. The human kringle 2-3 of the (SEQ ID NO: 25), kringle 2-3 Rhesus (SEQ ID NO: 26), kringle 2-3 porcine (SEQ ID NO: 27), and kringle 2-3 bovine (SEQ ID NIO: 28) corresponds to the amino acids at positions 88 to 255 (inclusive) of the angiostatin of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. As used herein "kringle 1-3" means a plasminogen protein derivative having an endothelial cell inhibitory activity or anti-angiogenic activity, and having an amino acid sequence comprising a sequence homologous to that of kringle 1-3, exemplified by, but not limited to that of murine kringle 1-3 (SEQ ID NO: 29), human kringle 1 (SEQ ID NO: 30), kringle 1-3 of Rhesus (SEQ ID NO: 31), kringle 1- 3 porcine (SEQ ID NO: 32), and kringle 1-3 of bovine (SEQ ID NO: 33), unless otherwise indicated by the context in which it is used. The murine kringle 1-3 (SEQ ID NO: 29) corresponds to the amino acids of positions 103 to 352 (inclusive) of the murine plasminogen of SEQ ID NO: 1, and corresponds to the amino acids of positions 6 to 255 (inclusive ) of murine angiostatin of SEQ ID NO: 2. The kringle 1-3 human (SEQ ID NO: 30), kringle 1-3 Rhesus (SEQ ID NO: 31), kringle 1-3 porcine (SEQ ID NO: 32) and kringle 1-3 bovine SEQ ID NO: 33) corresponds to the amino acids of positions 6 to 255 (inclusive) of angiostatin of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. As used herein "kringle 1-2" means a plasminogen protein derivative having an endothelial cell inhibitory activity or anti-angiogenic activity, and having an amino acid sequence comprising a sequence homologous to that of kringle 1-2, exemplified by, but not limited to that of murine kringle 1-2 (SEQ ID NO: 34), human kringle 1 (SEQ ID NO: 35), kringle 1-2 of Rhesus (SEQ ID NO: 36), kringle 1- 2 porcine (SEQ ID NO: 37), and kringle 1-2 bovine (SEQ ID NO: 38), unless otherwise indicated by the context in which it is used. The murine 1-2 kringle (SEQ ID NO: 34) corresponds to the amino acids of positions 103 to 262 (inclusive) of the murine plasminogen of SEQ ID NO: 1, and corresponds to the amino acids of positions 6 to 165 (inclusive ) of murine angiostatin of SEQ ID NO: 2. The kringle 1-2 human (SEQ ID NO: 35), kringle 1-2 of Rhesus (SEQ ID NO: 36), kringle 1-2 porcine (SEQ ID NO: 37) and kringle 1-2 bovine SEQ ID NO: 38) corresponds to the amino acids of positions 6 to 165 (inclusive) of the angiostatin of SEQ ID NO: 3, SEQ ID NO:, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. As used herein, "kringle 1-4" means a plasminogen protein derivative having an endothelial cell inhibitory activity or anti-angiogenic activity, and has an amino acid sequence comprising a homologue of the kringle sequence 1-4, exemplified by, but not limited to, that of murine kringle 1-4 (SEQ ID NO: 39), and human kringle 1-4 (SEQ ID NO: 40), unless otherwise indicated by the context in which is used. The murine 1-4 kringle (SEQ ID NO: 39) corresponds to the amino acids at positions 103 to 454 (inclusive) of the murine plasminogen of SEQ ID NO: 1. The amino acid sequences of the kringle, 1, kringle 2, kringle 3, kringle 4, kringle 2-3, kringle 1-3, kringle 1-2, and kringle 1-4 are homologous respectively of the specific kringle sequences identified above. Preferably, the amino acid sequences have a degree of homology to the described sequences of at least 60%, more preferably at least 70%, and most preferably at least 80%. It should be understood that a variety of substitutions, additions, deletions or other modifications of amino acids can be made to the fragments listed above to improve or modify the endothelial cell proliferation inhibitory activity or anti-angiogenic activity of angiostatin fragments. Such modifications do not intend to exceed the scope and spirit of the claims. For example, to avoid homodimerization by the formation of interkringle disulfide bonds, the C4 cysteine residues in the recombinant human kringle 2 (SEQ ID NO: 13) and C42 in the recombinant kringle 3 (SEQ ID NO: 18) were made to mutate to serinas. In addition, it should be understood that a variety of substitutions, additions, deletions or other modifications of amino acids can be made in the angiostatin fragments identified above, which do not significantly alter the inhibitory activity of endothelial cell proliferation of the fragments, and which, therefore, are not intended to exceed the scope of the claims. "Not significantly altering" means that the angiostatin fragment has at least 60%, more preferably at least 70%, and more preferably at least 80% of the inhibitory activity of endothelial cell proliferation compared to the closest homologous angiostatin fragment described herein.

Construction and Gene Expression A PCR-based method was used to generate cDNA fragments that encode kringle 1 (Kl), kringle 2 (K2), kringle 3 (K3), kringle 4 (K4) and kringle 2-3 (K2-3) of human plasminogen (HPg). The kringle 1 (rKl), kringle 2 (rK2), kringle 3 (rK3), kringle 4 (rK4) and kringle 2-3 (rK2-3) were expressed in E. coli as described above (Menhart, N. , Shel, LC, Kelly, RF, and Castellino, FJ (1991) Biochem, 30. 1948-1957; Marti, D., Schaller, J., Ochensberger, B., and Rickli, EE (1994) Eur. J. Biochem 219, 455-462; Shondel, S., Hu, C.-K,.

S., Affolter, M., Schaller, J., Linas, M., and Ricki, E.E. (1996) Biochem. . in print; Réjante, M.R., Byeon, I.-J.L., and Llinas, M. (1991) Biochem, 30, 11081-11092). To avoid homodimerization by interkringle disulfide bridge formation as shown in Figure 32B, the cysteine residues C169 and rK2 and C297 in rK3 were mutated to serines as seen in SEQ ID NOs: 13 and 18, in the positions 4 and 42, respectively (Sóhndel, S., Hu, C.-K, Marti, D., Affolter, M., Schaller, J., Llinas, M., and Ricki, E. E. (1996) Biochem. In print). RK3 and rK2-3 had a N-terminal hexahistidine label which was used for purification of the protein (not shown).

Proteolytic digestion The fragments of Kl-3, Kl-4 and K4 were prepared by digestion of Lys-HPg (Abbott Labs) with porcine elastase (Sigma) as described above.

(Powell, J.R., and Castellino, F.J. _ (1983) Biochem, 22, 923-927). Briefly, 1.5 mg of elastase was incubated at room temperature with 200 mg of human plasminogen in 50 mM Tris-HCl at pH 8.0 overnight with shaking. The reaction was terminated by the addition of diisopropyl fluorophosphate (DFP) (Sigma) to one with an end of lmM. The mixture was stirred for an additional 30 minutes at room temperature and dialyzed overnight against 50 mM Tris-HCl, pH 8.0.

Purification of Protein Recombinant Kl was expressed in E. coli DH5a bacterial cells using the plasmid vector pSTII. This protein was purified to homogeneity by chromatography using lysine-Sepharose 4B (Pharmacia) and Mono Q (BioRad) columns. Bacterial cells of E. coli (strain HB101) expressing rK2 and rK3 were grown to a DOeoo of about 0.8 to 3 ° C in YT 2 x medium containing 10 mg / ml ampicillin and 25 mg / ml kanamycin . IPTG (isopropyl-b-D-thiogalactopyranoside) was added to a final concentration of lmM and the cells were grown for an additional 4.5 hours at 37 ° C to induce the production of recombinant proteins. The cells were harvested by centrifugation and the pellets were stored at -80 ° C. The . Thawed cell lysates were resuspended in the extraction buffer (6 M guanidine hydrochloride in 0.1M sodium phosphate, pH 8.0). The suspension was centrifuged at 15,000 x g for 30 minutes and b-mercaptoefanol was added to the supernatant at a final concentration of 10 mM. The supernatant was then loaded onto an agarose column with Ni2 + -NTA (15 cm x 5 cm) pre-equilibrated with the extraction buffer. The column was washed successively with extraction buffer at pH 8.0 and pH 6.3, respectively. The recombinant K2 and K3 were eluted with extraction buffer at pH 5.0. The proteolytically cleaved fragments of Kl-3, Kl-4 and K4 were purified using a lysine-Sepharose 4B column (2.5 cm x 15 cm) equilibrated with 50 mM Tris-HCl, pH 8.0 until an absorbance of 0.005 was reached. 180 nm. The absorbed kringle fragments were eluted with Tris buffer with an e-aminocaproic acid content of 200 mM, pH 8.0. The eluted samples were then dialyzed overnight against 20 mM Tris-HCl, pH 5.0, and were applied to a Mono-S column of BioRad equilibrated with the same buffer. The fragments of K4, Kl-3 and K-4 were eluted with gradients gradients of 0-20%, 20-50% and 50-70% of 20 mM phosphate / 1 M KCl, pH 5.0. Most of the Kl-3 and Kl-4 fragments were eluted from the column with 0.5 M KCl as determined by SDS-PAGE. All fractions were dialysed overnight against 20 mM Tris-HCl, pH 8.0. After dialysis, the Kl-3 and Kl-4 fragments were further purified using a heparin-Sepharose column (5 cm and 10 cm) (Sigma) pre-equilibrated with 20 mM Tris-HCl buffer, pH 8.0. The Kl-3 fragment was eluted with 350 mM KCl and the Kl-4 was recovered from the fraction through the flow. The purified kringle fragments were analyzed on gels with SDS followed by silver staining, by Western immunoblot analysis with polyclonal antibodies antiK4 and human K-3, and by analysis of amino terminal sequencing.

In vitro refolding The refolding of rK2, rK3 and rK2-3 was carried out according to a standard protocol (Cleary, S., Mulkerrin, M.G., and Kelley, R.R. (1989) Biochem, 28 1884-1891). The purified proteins were adjusted to pH 8.0 and dithiothreitol (DTT) was added to a final concentration of 5 mM. After an overnight incubation, the solution was diluted with 4 volumes of 50 mM Tris-HCl, pH 8.0, with a reduced glutathione content of 1.25 mM. After 1 hour of incubation, oxidized glutathione was added to a final concentration of 1.25 mM and incubated for 6 hours at 4 ° C. The renaturized protein was initially dialyzed against H20 for 2 days and for an additional 2 days against saline buffered with 50 mM phosphate, pH 8.0. The solution was then loaded onto a lysine-Bio-Gel column (2 cm x 13 cm) equilibrated with the same phosphate-buffered saline solution. The column was washed with phosphate buffered saline and the protein was eluted with a phosphate buffer containing 50 mM 6-AHA (6-aminohexanoic acid). The reverse phase HPLC was carried out on a column of Butyral from Aquapore (2.1 x 100 mm, with a pore width of 30 nm, 7 mm, Applied Biosystems) and Hewlett Packard liquid chromatography with acetonitrile gradients was used.

Reduction and Alkylation Reduction and alkylation of the kringle fragments were carried out according to a standard protocol (CaO, Y., and Pattersson, R.F., (1990) Growth, Factors 3, 1013). Approximately 20-80 mg of purified proteins in 300-500 ml of DME medium in the absence of serum were incubated at room temperature with 15 ml of 0.5 M DTT for 15 minutes. After incubation, 30 ml of 0.5 M iodoacetamide was added to the reaction. The protein solution was dialyzed at 4 ° C overnight at initially against 20 volumes of DMEM. The solution was further dialyzed at 4 ° C for an additional 4 hours against 20 volumes of fresh DMEM. After dialysis, the samples were analyzed on SDS gel and assayed for their inhibitory activities on endothelial cell proliferation.

Endothelial Proliferation Assay Bovine capillary endothelial cells (BCE) were isolated as described previously (Folkman, J., Haudenschild, CC, and Zetter, BR (1979) Proc. Na ti. Acad. Sci USA 76, 5217-5121 ) and were maintained in DMEM supplemented with 10% heat inactivated bovine sheep serum (BCS), antibiotics, and 3 ng / ml recombinant human bFGF (Scios Nova, Mountainview, CA). The monolayers of BCE cells that grew in 6-well plates were dispersed in a 0.05% trypsin solution. The cells were suspended with DMEM containing 10% BCS. Approximately 12,500 cells in 0.5 ml were added to each well of 24-well gelatinized tissue culture plates and incubated at 37 ° C (in 10% ~ C02) for 24 hours. The medium was replaced with 500 ml of fresh DMEM containing 5% BCS and samples of individual kringle fragments combined in triplicate were added to each well. After 30 minutes of incubation, bFGF was added to a final concentration of 1 ng / ml. After 72 hours of incubation, the cells were trypsinized, resuspended in Hematall (Fisher Scientific, Pittsburg, PA) and counted with a Coulter counter.

Purification Characterization of the Human Plasminogen kringle Fragment The cDNA fragments encoding individual kringles (Kl, K2, K3, and K4) and the kringles 2-3 (K2-3) of human plasminogen were amplified by a method based on the PCR (Figure 28). The cDNA fragments amplified by PCR were cloned into a bacterial expression vector. The expressed recombinant proteins of Escherichia coli were refolded in vi tro and purified to a homogeneity > 98% using chromatography coupled to HPLC (Figure 29). Under reducing conditions, the recombinant K2, K3 and K4 migrated with molecular weights of 12-13 kDa (Figure 29A, lanes 2-4), which correspond to the estimated molecular weights of each kringle fragment. The recombinant Kl that migrates with a molecular weight greater than 17 kDa was identified by gel electrophoresis with SDS. The fragments of Kl-4 and Kl-3 were obtained by proteolytic digestion of human Lys-plasminogen (Lys-HPg) with elastase as described previously (Powell, JR, and Castellino, FJ (1983) Biochem, 22, 923- 927) Arch. Biochem. Biophys). Those two fragments (Figure 29B, lanes 1 and 2) with the estimated molecular weights of 43 kDa and 35 kDa, respectively, were also purified to homogeneity. Analysis of the N-terminal amino acid sequence in the purified fragments yielded an identical sequence, -YLSE-, followed by SEQ ID NO: 30 and SEQ ID NO: 40, for Kl-3 and Kl-4, respectively . The N-terminal sequence for K4 produced -VVQD- with approximately 20% of -VQD-, followed by SEQ ID NO: 23, each of which was estimated from the expected sequence beginning with the valine176 and the valina177 of human angiostatin (SEQ ID N0: 3).

Antiproliferative Activity of Cells Individual kringles endothelial fragments The individual recombinant kringle fragments of angiostatin were tested to determine the inhibitory activities on the growth of bovine capillary endothelial cells (BCE) stimulated by bFGF. As shown in Figure 30A, rK1 inhibited the proliferation of BCE cells in a dose-dependent manner. The concentration of rK1 required to reach 50% inhibition (ED50) was approximately 320 nM (Table 4). In contrast, rK4 exhibited little or no inhibitory effect on the proliferation of endothelial cells. Recombinant K2 and rK3, two fragments of kringle that do not bind to lysine, also produced a dose-dependent inhibition of endothelial cell proliferation (Figure 30B). However, the inhibitory potency of rK2 was substantially lower than that of rK1 and rK3 (ED50 = 460) (Figure 30 and Table 4). No cytotoxicity or a distinct morphology associated with apoptotic endothelial cells such as rounding, detachment and fragmentation of the cells could be detected, even after incubation with a high concentration of those kringle fragments. These data suggest that the endothelial anticlotting activity of angiostatin can be shared by the Kl, K2, and K3 fragments, and to a lesser degree by K4.

Table 4 Inhibitory activity on the proliferation of capillary endothelial cells. Fragments ED50 (nM) Kringle 1 320 Table 4 (continued) Inhibitory activity on the proliferation of capillary endothelial cells. Fragments ED50 (nM) Kringle 2 Kringle 3 460 Kringle 4 Kringle 2-3 Kringle 1-3 70 Kringle 1-4 (Angiostatin) 135 Antiproliferative activity of the Endothelial Cells of Fragments Kl-3 and Kl-4. To evaluate the antiproliferative effect of endothelial cells from combined kringle fragments, human Kl-4 and Kl-3 and rK2-3 fragments were tested on BCE cells. According to previous findings (O'Reilly, MS, Holmgren, L., Shing, Y., Chen, C, Rosenthal, RA, Moses, J., Lane, WS, Cao, Y., Sage, EH, and Folkman, J. (1994) Cell 79, 315-328), the proliferation of BCE cells, as shown in Figure 31, was significantly inhibited by the angiostatin-like fragment Kl-4 (ED5o = 135 nM) (Table 4). An increase in anti-endothelial growth activity was obtained with the Kl-3 fragment (ED50 = 70 nM) (Table 4). Inhibition of proliferation of endothelial cells occurred in a dependent manner. These results indicate that the removal of K4 from angiostatin enhances the endothelial anticlotting activity.

Additive Inhibition by rK2 and rK3 The fragments of rK2-3 showed only weak inhibitory activity, which was similar to that of rK2 (Figure 31). However, both rK2 and rK3 inhibited the proliferation of endothelial cells (Figure 30B). This finding suggests that the inhibitory effect of K3 was hindered in the structure of K2-3. Previous structural studies showed that an interkringle disulfide bond was present between K2 (cysteine169) and K3 (cysteine297) of human plasminogen, which corresponds to cysteine91 and cysteine219 of SEQ ID N0: 3 (Sóhndel, S., Hu, C.-K., Marti, D., Affolter, M., Schaller, J., Llinas, M., and Rickli, EE (1966) Biochem. In print) . See Figure 32B. The inhibitory effect of rK2 and rK3 in combination was tested. Interestingly, additive inhibition was observed when the individual rK2 and rK3 fragments were added to the BCE cells. See Figure 32A. These results imply that it is preferable to open the interdisulfide bridge between K2 and K3 to obtain the maximum inhibitory effect of K2-3.

The appropriate Plegami Kringle structures for the Anti-Endothelial Activity of Angiostatin It was required to study whether the folding of the kringle structures for endothelial antiproliferation activity, native angiostatin was reduced with DTT and tested on bovine capillary endothelial cells. After the reduction, the angiostatin was further alkylated with iodoacetamide and analyzed by SDS gel electrophoresis. As shown in Figure 34A, the DTT treated protein migrated to a higher position with a molecular weight of approximately 42 kDa (lane 2) compared to the native angiostatin with a molecular weight of 33 kDa (lane 1), suggesting that angiostatin was completely reduced. The antiproliferation activity of angiostatin was largely abolished after reduction (Figure 34B). From these results, we conclude that the correct folding of angiostatin through the intrakringle disulfide bonds is preferable to maintain its potent effect on the inhibition of endothelial cell proliferation. The alignment of the amino acid sequence of the kringle domains of human plasminogen shows that Kl, K2, K3 and K4 present an identical approximate architecture and a remarkable sequence homology (identification of 56-82%) as seen in the Figure 35. Among these structures, the kringle that binds to lysine with high K1 affinity is the most potent inhibitor segment of endothelial cell proliferation. Of interest, the fragment that binds to lysine with intermediate affinity, K4, lacks inhibitory activity. These data suggest that the lysine binding site of the kringle structures can not be directly involved in the inhibitory activity. The conservation of amino acids and the functional divergence of these kringle structures provides an ideal system for studying the role of orientation caused by the reproduction of DNA during evolution. Similar divergent activities were found related to the regulation of angiogenesis exhibited by a group of structurally related proteins in the families of the chemokine -XXXX- and the growth hormone prolactin (Maione, TE, Gray, GS, Petro, AJ, Hunt, AL, and Donner, SI (1990) Science 247, 77-79.; Koch, A.E., Polverini, P.J., Kunkel, S.L., Harlow, L.A., DiPietro, L.A., Einer, V.M., Einer, S.J., and Strieter, R.M. (1992) Science 258, 1798-1801; Cao, Y., Chen, C, Weatherbee, J.A., Tsang, M., and Folkman, J. (1995) J. Exp. Med. 182, 2069-2077; Strieter, R.M., Polverini, P.J., Arenberg, D.A., and Kunkel, S.L. (1995) Shock 4, 155-160; Jackson, D., Volpert, O.V., Bouck, N., and Linzer, D.I.H. (1994) Science 266, 1581-1584). Additional sequence analysis reveals that K4 contains two lysine residues positively charged adjacent to cysteines 22 and 78 (Figure 35). Nuclear magnetic resonance (NMR) 1H analysis shows that these 4 plants, together with lysine 57, form the nucleus of positively charged domain in K4 (Llinas M, unpublished data), while other kringle structures lack such a charged domain positively It is still not studied if this enriched domain of lysine contributes to the loss of the kringle 4 inhibitory activity of human plasminogen. Previously it was reported that K4 stimulates the proliferation of other cell types and increases the release of intracellular calcium (Donate, LE, Gherardi, E., Srinivasan, N., Sowdhamini, R., Aporicio, S., and Blundell, TL (1994) Prot. Sci. 3, 2378-2394). The fact that the removal of K4 from angiostatin enhances the inhibitory activity between endothelial cells suggests that this structure may prevent some of the inhibitory effects of Kl-3. The underlying mechanism of how angiostatin and its related kringle fragments specifically inhibit the growth of endothelial cells remains uncharacterized. It is not yet clear whether inhibition is mediated by a receptor that is specifically expressed in proliferating endothelial cells, or if angiostatin is internalized by endothelial cells and subsequently inhibits cell proliferation. Alternatively, angiostatin can interact with an endothelial cell adhesion receptor such as the integrin avb3, which blocks integrin-mediated angiogenesis (Brooks, PC, Montgomery, AM, Rosenfeld, M., Reisfeld, RA, Hu, T Klier, G., and Chereh, DA (1994) Cell 79, 1157-1164). Of interest, Friedlander et. to the. (Friedlander, M., Brooks, PC, Shaffer, RW, Kincaid, CM, Varner, JA, and Cheresh, DA (1995) 270, 1502) recently reported that in vivo angiogenesis in cornea or chorioallantoic membrane (induced by bFGF and tumor necrosis factor) was dependent on the integrin avb3. However, angiogenesis stimulated by VEGF, transforming growth factor a, or by phorbol esters was dependent on avb5. Antibodies to the individual integrins specifically blocked one of these pathways, and a cyclic protein antagonist of both integrins blocked the angiogenesis induced by each cytokine (Friedlander, M., Brooks, PC, Shaffer, RW, Kincaid, CM, Varner, JA , and Cheresh, DA (1995) 270, 1502). Because angiogenesis induced by bFGF and VEGF are inhibited by angiostatin, it can block a common pathway for these integrin-mediated angiogenesis. A growing number of endogenous inhibitors of angiogenesis have been identified in the last few decades (Folkman, J. (1995) N. Engl. J. Med. 333, 1757-1763). Of the nine characterized endothelial cell suppressors, several inhibitors are proteolytic fragments. For example, the N-terminal 16 kDa fragment of human prolactin inhibits cell proliferation and blocks angiogenesis in vivo (Clapp, C, Martial, J.A., Guz an, R.C., Rentierdelrue, F., and Weiner, R.I. (1993) Endocrinology 133, 1292-1299). In a recent article, D 'Angelo et. to the. reported that the anti-angiogenic 16 kDa N-terminal fragment inhibited the activation of mitogen-activated protein kinase (MAPK) by VEGF and bFGF in capillary endothelial cells (D'Angelo, G., Struman, I., Martial, J. , and Weiner, R. (1995) Proc. Na ti, Acad. Sci. 92, 6374-6378). Similar to angiostatin, the intact mother molecule of prolactin does not inhibit the proliferation of endothelial cells, nor is it an inhibitor of angiogenesis. Platelet factor 4 (PF-4) inhibits angiogenesis at high concentrations (Maione, TE, Gray, GS, Petro, AJ, Hunt, AL, and Donner, SI (1990) Science 247, 77-79; CaO, Y. , Chen, C, Weatherbee, JA, Tsang, M., and Folkman, J. (1995) J. Exp. Med. 182, 2069-2077). However, the truncated N-proteolytically cleaved PF-4 fragment exhibits a 30 to 50 fold increase in its antiproliferative activity on the intact PF-4 molecule (Gupta, SK, Hessel, T., and Singh, JP (1995) Proc. Na ti, Acad. Scí. 92, 7799-77803). Smaller protein fragments of fibronectin, murine epidermal growth factor, and thrombospondin have also been shown to specifically inhibit the growth of endothelial cells (Homandberg, GA, Williams, JE, Grant, D., Schumacher, B., and Eisenstein, R (1985) Am. J. Pa thol., 120, 327-332; Nelson, J., Alien; .E., Scott, WN, Bailie, JR, Walker, B., McFerran, NV and Wilson, DJ (1995) ) Cancer Res. 55, 3772-3776; Tolsma, SS, Volpert, OV, Good, DJ, Frazer, WA, Polverini, PJ, and Bouck, N. (1993) J. Cell. Biol. 122, 497-511) . The proteolytic processing of a large protein can change the conformational structure of the original molecule or expose new epitopes that are antiangiogenic. In this way, proteases can play a critical role in the regulation of angiogenesis. To date, little is known about the regulation of these protease activities in vivo. The data also show that the disulfide bond mediated by the folding of the kringle structures in angiostatin is preferable to retain its inhibitory activity on the growth of endothelial cells. Kringle structures analogous to those of plasminogen are also found in a variety of other proteins. For example, apolipoprotein (a) has up to 37 repeats of kringle 4 of plasminogen (McLean, JW, Tomlinson, JE, Kuang, W.-J., Eaton, DL, Chen, EY, Fless, GM, Scanu, AM, and Lawn, RM (1987) Na ture 330, 132-137). The amino terminal portion of prothrombin also contains two kringles that are homologous to those of plasminogen (Walz, D.A., Hewett-Emmett, D., and Seegers, W.H. (1977) Proc. Na ti. Acad. Sci. 74, 1969-1973). Urokinase has been shown to possess a kringle structure that shares extensive homology with plasminogen (Gunzler, WA, J., SG, Otting, F., Kim, S.-MA, Frankus, E., and Flohe, L. ( 1982) Hoppe-Seyler 's A. Physiol, Chem. 363, 1155-1165). In addition, the tensoactive protein B and hepatocyte growth factor (HGF) also contains kringle structures (Johansson, J., Curstedt, T., and Jórnvall, H. (1991) Biochem., 30, 6917-6921; Lukker, NA, Presta, LG, and Godowski, PJ (1994) Prot. Engin., 1, 895-903).

EXAMPLE 28 Suppression of Metastasis and Proliferation of Endothelial Cells by Angiostatin Fragments The following example characterizes the activity of additional angiostatin fragments. The data suggest that potent antiendothelial and tumor suppressor activity of such angiostatin protein fragments can be obtained. As used herein, "kringle 1-4BKLS" means a plasminogen protein derivative having an endothelial cell inhibitory activity, and having an amino acid sequence comprising a sequence homologous to that of kringle 1-4BKLS, exemplified by, but not limited to that of the murine 1-4BKLS kringle (SEQ ID NO: 41), and the human 1-4BKLS kringle (SEQ ID NO: 42), unless otherwise indicated by the context in the which one is used. The murine 1-4BKLS kringle (SEQ ID NO: 41) corresponds to the amino acids at positions 93 to 470 (inclusive) of the murine plasminogen of SEQ ID NO: 1. This example demonstrates that an "angiostatin fragment" can be a fragment of plasminogen and encompasses a sequence of amino acids greater than that of angiostatin presented in SEQ ID NO: 3, for example, and still has therapeutic activity inhibiting the proliferation of endothelial cells or anti-angiogenic activity. The amino acid sequence of kringle 1-4BLKS is homologous to the specific kringle sequences 1-4BLKS identified above. Preferably, the amino acid sequences have a degree of homology to the described sequences of at least 60%, more preferably at least 70%, and most preferably at least 80%. It should be understood that a variety of substitutions, deletions or other modifications of the amino acids can be made to the above-listed fragments to improve or modify the inhibitory activity of the endothelial cells of the fragments. Such modifications are not intended to exceed the scope and spirit of the claims. In addition, it should be understood that a variety of substitutions, additions or silent deletions of amino acids can be made in the kringle fragments identified above, which do not significantly alter the inhibitory activity of the endothelial cells of the fragments, and which, therefore, can therefore, they do not intend to exceed the scope of the claims.

Cloning of Angiostatin in Pichia pastoris The sequences coding for angiostatin were amplified by PCR using Vent polymerase (New England Biolabs) and primers # 154 (5 '-ATCGCTCGAGCGTTATTTGAAAAGAAAGTG-3') (SEQ ID N0: 43) and # 151 (5 '-ATCGGAATCAAGCAGGACAACAGGCGG- 3 ') (SEQ ID NO: 44) containing the XhoL and Eco Rl binders respectively and using the pTrcHis / HAs plasmid as standard. This plasmid contained the sequences coding for amino acids 93 to 470 of human plasminogen (SEQ ID NO: 42) for cloning in the Xho I / ECo Rl site of the expression vector pHIL-Sl using the native secretion signal PHO 1 of P. pastoris This same sequence was amplified in the same way using the primers # 156 (5'-ATCGTACGTATTATTTGAAAAGAAAGTG-3 ') (SEQ ID N0: 45) and # 151 containing the linkers Sna Bl and Eco Rl respectively, for on-site cloning Sna Bl / Eco Rl of the expression vector pPlC9 with the secretory signal of alpha factor. The products of the amplifications were purified in gel, the binders were deferred with the appropriate enzymes, and again purified using a gel cleaning (Bio 101). Those gene fragments were ligated to the appropriate vectors. The resulting clones were selected and the plasmid preparations of the clones were obtained and linearized to generate His + Muts and His + Mut + recombinant strains when transformed into the GS115 host strain of P. pastoris. The integration was conformed by PCR. Both His + and His + Mut + recombinants were induced with methanol and selected for their high angiostatin expression using SDS-PAGE stained with Coomassie and immunoblots using mouse monoclonal antibody against kringles 1 to 3 (Castellino, Enzyme Research Laboratories, Inc., South Bend, IN). Of these, the P. pastoris clone transformed with GS115 pHIL-Sl / HAsl8 was selected and characterized phenotypically as His + Muts.

Expression of PHIL-Sl / HAsl8 The angiostatin expression of pHIL-Sl / HAsl8 was typical for the clone His + Mut ?. In induction in baffled shake flasks, IO of DOeoo cells was cultured in 150 ml of buffered methanol complex medium containing 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34 % of nitrogen base of yeast with ammonium sulfate, 0.0004% of biotin and 0.5% of methanol, in a flask with deflectors of. The cells were constantly shaken at 30 ° C, 250 rpm. The methanol was fed in batches at 24 hour intervals by the addition of absolute methanol to a final concentration of 0.5%. After 120 hours the cells were centrifuged at 5000 rpm for 10 minutes, and the supernatants were stored at -70 ° C until used.

Purification of angiostatin by broth Fermentation of P. pastoris by Lysine-Sepharose Chromatography All procedures were carried out at 4 ° C. The crude fermentation broth, typically 200 ml, containing angiostatin was clarified by centrifugation at 14,000 x g and concentrated by means of a membrane with a 30 kDa molecular weight cut off Centriprep (amicon) to about a quarter of the original volume. A volume of 50 mM phosphate buffer, pH 7.5, was added to the concentrated sample, which was concentrated by Centriprep to a quarter of the volume of the original sample. The sample was again diluted: volume with 50 mM sodium phosphate buffer, pH 7.5. 60 g of lysine-sepharose 4B (Pharmacia) were resuspended in 500 ml of 50 mM ice-cooled phosphate buffer, pH 7.5, and used to pack a 48 x 100 mm column (~ 180 ml packed volume). The column was washed overnight with 7.5 column volumes (CV) of 50 mM sodium phosphate buffer, pH 7.5, at a flow rate of 1.5 ml / min. The mixture was pumped over the column at a flow rate of 1.5 ml / min and the column was washed with 1.5. 50 mM sodium phosphate CV, pH 7.5, at a flow rate of 1.5 ml / min. The column was then washed with 1.5 CV of buffered saline, phosphate, ph 7.4, at a flow rate of 3 ml / min. The angiostatin was then diluted with 0.2 M e-amino-n-caproic acid, pH 7.4 at a flow rate of 3 ml / min. Fractions containing significant absorbance were diluted and dialysed for 24-48 hours against deionized water and lyophilized. A typical recovery of a total protein load of 100 mg is 10 mg angiostatin. The columns were regenerated using 5 column volumes of 50 mM sodium phosphate / 1 M NaCl, pH 7.5.

Proliferation Assay of Bovine Capillar Endothelial Cells Bovine capillar endothelial cells were obtained as described above. Cells were maintained in DMEM containing 3 mg / ml of recombinant human bFGF (Scios Nova, Mountainview, Ca), supplemented with 10% heat-inactivated bovine sheep serum, 100 U / ml penicillin, 100 mg / ml streptomycin, and 0.25 mg / ml fungizone (Bio Whittaker) in 75 cm2 cell culture flasks. The test was carried out as described above.

Animal studies C57BI / 6J six-to-eight-week-old male mice (Jackson Laboratories) were inoculated subcutaneously with murine line of Lewis-Low metastatic lung carcinoma (CLL-LM) (1 x 106 cells / injection). Approximately 14 days after implantation, when the primary tumor reached 1.5 cm3, the animals were anesthetized with methoxyflurane and the primary tumors were surgically excised. The incision site was closed with simple interrupted sutures. Half of the animals in this group received a loading dose (3 mg / kg by the subcutaneous route) of the recombinant or plasminogen derived from angiostatin subcutaneously, immediately after surgery, followed by daily inoculations of 1.5 mg / kg for 14 days . A control group of mice received an equal volume of PBS daily for 14 days after surgery. All mice were sacrificed 14 days after removal of the primary tumor (28 days after tumor implantation), the lungs were removed and weighed, and superficial metastases were counted with stereomicroscopy.

Characteristics of the Fragments of Recombinant Human Angiostatin A gene fragment encoding human angiostatin includes kringles 1 to 4 of human plasminogen containing a total of 26 cysteines, was expressed in P. pastoris, the methyltropic yeast. Angiostatin expressed in P. pas tori s binds to lysine-sepharose and can be specifically eluted by e-aminocaproic acid. This demonstrates that kringles that bind to fully functional e-aminocaproic acid, which are physical properties of kringle 1 and 4 of plasminogen (Sottrup-Jensen, L. et al., Progress in Chemical Fibrinolysis and Thrombolysis, Vol. 3 ( 1978) Ravens Press, NY p.191), can be expressed and secreted by P. pa s tori s and purified by techniques that do not require refolding (Figure 36A and B). The expressed angiostatin of P. pas toris, as well as the angiostatin purified by the plasminogen elastase incision recognized by a conformationally dependent monoclonal antibody against kringle 1 to 3 (Castellino, Enzyme Research Laboratories, Inc., South Bend, IN) ( Figure 36B). This antibody does not recognize the reduced forms of plasminogen or angiostatin. The angiostatin expressed by P. pastoris is observed as a doublet that migrates at 49 kDa and 51.5 kDa on non-reduced and denatured Coomassie SDS-PAGE gels. The proteins expressed in P. pastor! they are translationally modified with the majority of the N-linked glycosylation of the superior mannose type and insignificant 0-linked glycosylation. To evaluate the possibility of glycosylation of angiostatin expressed by P. pas toris, we digested recombinant angiostatin with endoglycosidase H specific for upper mannose structures, causing the 51.5 kDa band to migrate identically with the band at 49 kDa (Figures 37A and B). Digestion of O-glycanase with treatment with anterior neuraminidase to remove sialic acid residues does not change the doublet migration pattern (data not shown). These results indicate that angiostatin was expressed in P. pastoris in two ways: (1) with an N-linked complex chain probably of the structure: (man) 2-150-Man Man-GlcNAc GlcNAc-Asn- (Man) 1 -2-Man and (2) without any glycosylation.

In Vitro Capillar Endothelial Cell Inhibition To determine whether recombinantly expressed angiostatin had the potential of anti-angiogenic activity, BCEs were cultured in the presence of bFGF to determine whether the addition of purified recombinant angiostatin would inhibit ECB proliferation. "Angiostatin expressed in purified P. pastoris inhibited bFGF-directed proliferation of bovine endothelial cells in vi tro (Figure 38B) in A dose-dependent form (Figure 38C) At 1 μg / ml of recombinant angiostatin, the inhibition was 80% 50% inhibition was equivalent to that obtained with angiostatin derived from the incision with plasminogen elastase human.

Suppression of In Vivo Metastasis The transplantable murine line LLC was used (LM) from which Angiostat was first identified. When implanted subcutaneously in syngeneic C57B1 / 6J mice, these tumors grew rapidly, producing tumors > 1.5 cm3 within 14 days. After resection of the primary tumor, the micrometastases in the lungs grew exponentially, until completely covering the surface of the lung. These metastases are highly vascularized at day 14 after the resection of the primary tumor. If the primary tumor is left, the micrometastases remain inactive and are not visible macroscopically. Recombinant angiostatin was administered systemically to mice after resection of the primary tumor to test growth suppression of metastases. Angiostatin expressed in P. pastoris administered systemically at 30 ug / mouse / day inhibited the growth of metastases according to what was quantified by the recording of superficial metastases (Figure 39A) and the total weight of the lung (Figure 39B). The weights of the lungs of the mice from which the primary tumors were resected and which received daily doses of recombinant angiostatin or angiostatin obtained from the incision with plasminogen elastase were comparable to those of the normal mice (190 to 200 mg). The lungs of the mice from which the primary tumors were resected and subsequently treated with daily doses of angiostatin, recombinant were roses with minimal numbers of nonvascularized mircrometastases (Figure 40). In contrast, mice treated with saline after resection of the primary tumor had lungs covered with vascularized metastases (Figure 41). Also of notable importance was the absence of systemic or local toxicity caused by angiostatin expressed in P. pastoris at the dose and regimen used in this study. There was no evidence of inflammation or hemorrhage in all treated mice. The angiostatin protein expressed by P. pastoris possesses two important physical characteristics of the natural protein: (1) it is recognized by a conformationally dependent monoclonal antibody directed against the kringle 1 to 3 of human plasminogen (Figure 36B) and (2) binds to lysine (Figure 36A and B). These properties indicate that the recombinant angiostatin protein was expressed with a conformation that mimics the native molecule. The angiostatin protein expressed by P. pastoris inhibits the proliferation of bovine capillary endothelial cells stimulated by bFGF in vi tro (Figure 38). When administered systemically, recombinant agiostatin maintained metastatic Lewis lung carcinoma in other lethal circumstances in a suppressed state (Figures 39A and B and Figure 40). Preliminary data show the absence of a detectable transcript for angiostatin in Lewis lung tumors freshly resected from tumors in LLC cells after 4 passages in culture in vi tro. The plasminogen produced by the liver is kept in circulation at a stable plasma concentration of 1.6 ± 0.2 μM. It is possible that LLC-LM tumors produce an enzyme that cleaves plasminogen, bound or in circulation, to produce angiostatin. Alternatively, inflammatory cells attracted to the tumor site could produce such an enzyme. It is intriguing that the P. pastoris plasminogen as well as the human native are produced in a glycosylated and non-glycosylated form. In the case of human plasminogen, a single transcript for a single gene can produce both forms. The molecular mechanism of the differential conformational modifications of human plasminogen, as well as those observed in TPA, is unknown. Angiostatin is highly expressed by P. pastoris. The supernatants contain 100 mg / ml of protein. Therefore, the amounts required for clinical trials should be the correct ones for production and purification using standard technology well known to those skilled in the art. The development of this expression system and the demonstration of the in vi tro and in vivo activity of purified recombinant angiostatin against metastasis, provided the rationale for assessing the ability of its fragments to inhibit tumor growth and prolong life in patients with cancer and others suffering from angiogenically mediated diseases.

EXAMPLE 29 Angiostatin Kringle Protein Fragment 1-5 The following example describes the method for the production of angiostatin kringle 1-5 protein fragment. Since agui is used, "kringle 1-5" means a plasminogen protein derivative having an endothelial cell inhibitory activity or anti-angiogenic activity, and having an amino acid sequence comprising a sequence homologous to that of kringle 1-5. , exemplified by, but not limited to that of murine kringle 1-5 which corresponds to the amino acids at positions 102 to 560 (inclusive) of the murine plasminogen of SEQ ID NO: l. The kringle 5 itself is represented in the murine plasminogen sequence of SEQ ID NO: l in the amino acids at positions 481-560 (inclusive). The corresponding amino acid and nucleotide sequences of plasminogen are provided in Forsgren et al. , "Cloning and molecular characterization of a full-length cDNA clone for human plasminogen", FEBS 213: 2, p. 254-260 (1987), which is incorporated herein by reference. The amino acid sequences of kringle 1-5 are respectively homologous to the specific kringle sequence 1-5 identified above. Preferably, the amino acid sequences have a degree of homology to the described sequences of at least 60%, more preferably at least 70%, and most preferably at least 80%. It should be understood that a variety of substitutions, additions, deletions or other amino acid modifications can be made to the fragments listed above to improve or modify the endothelial cell proliferation inhibitory activity or the anti-angiogenic activity of the angiostatin fragments. Such modifications are not intended to exceed the scope and spirit of the claims. For example, to avoid homodimerization by the formation of interkringle disulfide bridges, the cysteine residues can be made to mutate to serines. Furthermore, it should be understood that a variety of substitutions, additions, deletions or other modifications of amino acids can be made in the angiostatin fragments identified above, which do not significantly alter the inhibitory activity of endothelial cell proliferation of the fragments, and which, therefore, do not intend to exceed the scope of the claims. "Not significantly altered" means that the angiostatin fragment has at least 60%, more preferably at least 70%, and more preferably at least 80% of the inhibitory activity of endothelial cell proliferation, as compared to that of the closest homologous angiostatin fragment described here. The angiostatin kringle 1-5 protein fragment can be produced according to the following method: 1) Convert purified human plasminogen (Plg) to Lys Plg using the enzyme plasmin. 2) Digest Lys Plg with TPA or urokinase. This will result in heavy (A) and light (B) chains, but still linked by two disulfide bonds. 3) Those linkages can be specifically reduced by common reducing agents, such as beta mercaptoethanol to result in heavy chain A and light chain B separated. 4) Then block the cysteines so that they do not form bonds again, converting the A and B chains into S-carboxymethyl derivatives (Robbins, KC, Barnabe P, Arzadon L, Summaria LJ Biol. Chem. 247 (21): 6757 -6762 (1972). "The primary structure of human plasminogen I. The terminal NH2 sequence of human plasminogen and the heavy (A) and light (B) S-carboxymethyl chains derived from plasmin"). 5) Run the product from step 4 on a lysine-Sepharose column to purify the Kl-5 from the rest.

QED The angiostatin kringle 1-5 protein fragment can be used to inhibit the proliferation of endothelial cells and angiogenesis in vi tro and in vivo. In particular, the angiostatin kringle 1-5 protein fragment can be used to inhibit angiogenesis in a cancerous tumor. It should be understood that the foregoing relates only to the preferred embodiments of the present invention, and that numerous modifications or alterations may be made thereto without departing from the spirit and scope of the invention, as set forth in the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Folkman, M. Judah O'Reilly, Michael (ii) TITLE OF THE INVENTION: Angiostatin Fragments and Method of Use of These (iii) SEQUENCE NUMBER: 7 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Jones & Askew (B) STREET: 191 Peachtree Street, 37th Floor (C) CITY: Atlanta (D) STATE: Georgia (E) COUNTRY: E.U. (F) C.P .: 30303-1769 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) INFORMATION OF THE APPORTER / AGENT (A) NAME: Warren, William L. (B) REGISTRATION NUMBER: 36,714 (C) REFERENCE NUMBER / FILE: 05213-0126 (ix) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: 404-818-3700 (B) TELEFAX: 404-818-3799 (C) TELEX: 910 / 371-7168 (2) ~ INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 812 amino acids (B) TYPE: amino acid (C) HEBRA: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Murine (vii) IMMEDIATE SOURCE: (B) ) CLONA: Plasminogen (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: Met Asp His Lys Glu Val lie Leu Leu Phe Leu Leu Leu Leu Lys Pro 1 5 10 15 Gly Gln Gly Asp Ser Leu Asp Gly Tyr lie Be Thr Gln Gly Ala Be 25 30 Leu Phe Ser Leu Thr Lys Lys Gln Leu Wing Wing Gly Gly Val Ser Asp 40 45 Cys Leu Wing Lys Cys Glu Gly Glu Thr Asp Phe Val Cys Arg Ser Phe 50 55 60 Gln Tyr His Ser Lys Glu Gln Gln Cys Val lie Met Wing Glu Asn Ser 65 70 75 80 Lys Thr Ser Ser He He Arg Met Arg Asp Val He Leu Phe Glu Lys 85 90 95 Arg Val Tyr Leu Ser Glu Cys Lys Thr Gly He Gly Asn Gly Tyr Arg 100 '105 110 Gly Thr Met Ser Arg Thr Lys Ser Gly Val Wing Cys Gln Lys Trp Gly 115 120 125 Wing Thr Phe Pro His Val Pro Asn Tyr Ser Pro Ser Thr His Pro Asn 130 135 140 Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln 145 150 155 160 Gly Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys 165 170 175 Asn He Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys Ser Gly Glu Lys 180 185 190 Tyr Glu Gly Lys He Ser Lys Thr Met Ser Gly Leu Asp Cys Gln Ala 195 200 205 Trp Asp Ser Gln Ser Pro His Wing His Gly Tyr He Pro Wing Ala Lys Phe 210 215 220 Pro Ser Lys Asn Leu Lys Met Asn Tyr Cys His Asn Pro Asp Gly Glu 225 230 235 240 Pro Arg Pro Pro Cys Phe Thr Thr Asp Pro Thr Lys Arg Trp Glu Tyr 245 250 255 Cys Asp He Pro Arg Cys Thr Thr Pro Pro Pro Pro Pro Ser Thr 260 265 270 Tyr Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser 275 280 285 Val Thr Val Ser Gly Lys Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro 290 295 300 His Arg His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu 305 310 315 320 Glu Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Wing Pro Trp Cys Tyr 325 330 335 Thr Thr Asp Ser Gln Leu Arg Trp Glu Tyr Cys Glu He Pro Ser Cys 340 345 350 Glu Being Ser Wing Being Pro Asp Gln Being Asp Being Ser Val Pro Pro Glu 355 360 365 Glu Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser 370 375 380 Tyr Arg Gly Thr Be Ser Thr Thr He Thr Gly Ser Asp Gly Gln Ser 385 390 395 400 Trp Wing Wing Met Phe Pro His Arg His Ser Lys Thr Pro Glu Asn Phe 405 410 415 Pro Asp Wing Gly Leu Glu Net Asn Tyr Cys Arg Asn Pro Asp Gly Asp 420 425 430 Lys Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr 435 440 445 Cys Asn Leu Lys Arg Cys Ser Glu Thr Gly Gly Ser Val Val Glu Leu 450 455 460 Pro Thr Val Ser Gln Glu Pro Ser Gly Pro Ser Asp Ser Glu Thr Asp 465 470 475 480 Cys Met Tyr Gly Asn Gly Lys Asp Tyr Arg Gly Lys Thr Wing Val Thr 485 490 495 Wing Wing Gly Thr Pro Cys Gln Gly Trp Wing Wing Gln Glu Pro His Arg 500 505 510 His Ser He Phe Thr Pro Gln Thr Asn Pro Arg Wing Asp Leu Glu Lys 515 520 525 Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Asn Gly Pro Pro Cys Tyr 530 535 ~ 540 Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp He Pro Leu Cys 545 550 555 560 Wing Being Wing Being Ser Phe Glu Cys Gly Lys Pro Gln Val Glu Pro Lys 565 570 575 Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Wing Asn Pro His Ser 580 '585 590 Trp Pro Trp Gln He Ser Leu Arg Thr Arg Phe Thr Gly Gln His Phe 595 '600 605 Cys Gly Gly Thr Leu He Wing Pro Glu Trp Val Leu Thr Wing Ala His 610 615 620 Cys Leu Glu Lys Ser Ser Arg Pro Glu Phe Tyr Lys Val He Leu Gly 625 630 635 _ 640 Ala His Glu Glu Tyr He Arg Gly Leu Asp Val Gln Glu He Ser Val 645 650"655 Ala Lys Leu He Leu Glu Pro Asn Asn Arg Asp He Ala Leu Leu Lys 660 665 670 Leu Ser Arg Pro Wing Thr He Tyr Asp Lys Val He Pro Wing Cys Leu 675 680 685 Pro Ser Pro Asn Tyr Met Val Wing Asp Arg Thr He Cys Tyr He Thr 690 695 700 Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Wing Gly Arg Leu Lys Glu 705 710 715 720 Wing Gln Leu Pro Val He Glu Asn Lys Val Cys Asn Arg Val Glu Tyr 735 725 730 Leu Asn Asn Arg Val Lys Ser Thr Glu Leu Cys Wing Gly Gln Leu Wing 740 745 750 Gly Gly Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys 755 760 765 Phe Glu Lys Asp Lys Tyr He Leu Gln Gly Val Thr Ser Trp Gly Leu 770 775 780 Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg 785 790 795 800 Phe Val Asp Trp He Glu Arg Glu Met Arg Asn Asn 805 810 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 339 amino acids (B) TYPE: amino acid (C) HEBRA: (D) TOPOLOGY; linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Murine (vii) IMMEDIATE SOURCE: (B) ) CLONA: angiostatin fragment (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Val Tyr Leu Ser Glu Cys Lys Thr Gly He Gly Asn Gly Tyr Arg Gly 1 5 10 15 Thr Met Ser Arg Thr Lys Ser Gly Val Wing Cys Gln Lys Trp Gly Wing 20 25 30 Thr Phe Pro His Val Pro Asn Tyr Ser Pro Thr His Pro Asn Glu 35 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asn 65 70 75 80 He Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys Ser Gly Glu Lys Tyr 85 90 '95 Glu Gly Lys He Ser Lys Thr Met Ser Gly Leu Asp Cys Gln Wing Trp 100 105 110 -Asp Ser Gln Ser Pro His Wing His Gly Tyr He Pro Wing Lys Phe Pro 115 120 125 Ser Lys Asn Leu Lys Met Asn Tyr Cys His Asn Pro Asp Gly Glu Pro 130 135 140 Arg Pro Trp Cys Phe Thr Thr Asp Pro Thr Lys Arg Trp Glu Tyr Cys 145 150 155 160 Asp He Pro Arg Cys Thr Thr Pro Pro Pro Pro Pro Pro Thr Tyr 165 170 175 Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val 180 185 190 Thr Val Ser Gly Lys Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His 195 200 205 Arg His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Wing Pro Trp Cys Tyr Thr 225 230 235 240 Thr Asp Ser Gln Leu Arg Trp Glu Tyr Cys Glu He Pro Ser Cys Glu 245 250 255 Being Ser Wing Being Pro Asp Gln Being Asp Being Val Pro Pro Glu Glu 260 265 270 Tyr Gln Being Asp Gly Gln Being Tyr Gln Thr Pro Val Val Gln Glu Cys 275 280 285 Arg Gly Thr Ser Ser Thr Thr He Thr Gly Lys Lys Cys Gln Ser Trp 290 295 300 Wing Wing Met Phe Pro His Arg His Ser Lys Thr Pro Glu Asn Phe Pro 305 310 315"32Q Asp Wing Gly Leu Glu Met Asn Tyr Cys Arg Asn Pro Asp Gly Asp Lys 325 330 335 Gly Pro Trp (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 339 amino acids (B) TYPE: amino acid (C) HEBRA: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: ( B) CLONA: angiostatin fragment (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly 1 5 10 15 Thr Met Ser Lys Thr Lys Asn Gly He Thr Cys Gln Lys Trp Ser Ser 25 30 Thr Sr Pro His Arg Pro Arg Phe Ser Pro Wing Thr His Pro Ser Glu 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp 65 70 75 80 He Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr 85 90 - "95 Asp Gly Lys Be Ser Lys Thr Met Be Gly Leu Glu Cys Gln Ala Trp 100 105 110 Asp Ser Gln Ser Pro His Wing His Gly Tyr He Pro Ser Lys Phe Pro 115 120 125 Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu 130 135 140 Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys 145 150 '155 160 Asp He Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr 165 170 175 Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Wing Val 180 '' 185 190 Thr Val Ser Gly His Thr Cys Gln His Trp Ser Wing Gln Thr Pro His 195 200 205 Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys ^ His Thr 225 230 235 240 Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys He Pro Ser Cys Asp 245 250 255 Be Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu 260 265 270 Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr 275 280 285 Arg Gly Thr Be Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp 290 295 300 Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro 305 310 315 ~ 320 Asn Wing Gly Leu Thr Net Asn Tyr Cys Arg Asn Pro Asp Wing Asp Lys 325 330 335 Gly Pro Trp (2) INFORMATION FOR SEQ ID NO: 4: -. (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 339 amino acids (B) TYPE: amino acid (C) HEBRA: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Rhesus monkey (vii) IMMEDIATE SOURCE: (B) CLONA: angiostatin fragment (xi) SEQUENCE DESCRITION: SEQ ID NO: 4: Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly 1 5 10 15 Thr Met Ser Lys Thr Arg Thr Gly He Thr Cys Gln Lys Trp Ser Ser 20 25 30 Thr Ser Pro His Arg Pro Thr Phe Ser Pro Wing Thr His Pro Ser Glu 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Gly Gln Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Glu Glu Arg Phe Asp Tyr Cys Asp 65 70 75 80 He Pro Glu Cys Glu Asp Glu Cys Met His Cys Ser Gly Glu Asn Tyr 85 90 95 Asp Gly Lys He Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Wing Trp 100 105 110 Asp Ser Gln Ser Pro His Wing His Gly Tyr He Pro Ser Lys Phe Pro 115 120 125 Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro 130 135 140 Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys 145 150 155 160 Asp He Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr 165 _ 170"_ 175 Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asp Val Wing Val 180 185 190 Thr Val Ser Gly His Thr Cys His Gly Trp Ser Wing Gln Thr Pro His 195 200 205 Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Lys Wing Pro Trp Cys Tyr Thr 225 230 235 240 Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys He Pro Ser Cys Glu 245 250 255 Being Ser Pro Pro Being Thr Glu Pro Leu Asp Pro Thr Wing Pro Pro Glu 260 265 270 Leu Thr Pro Val Val Gln Glu Cys Tyr His Gly Asp Gly Gln Ser Tyr 275 280 285 Arg Gly Thr Be Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp 290 295 300 Ser Ser Met Thr Pro His Trp His Glu Lys Thr Pro Glu Asn Phe Pro 305 310 315 320 Asn Wing Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Wing Asp Lys 325 330 - 335 Gly Pro Trp (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 339 amino acids (B) TYPE: amino acid (C) HEBRA: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Porcine (vii) IMMEDIATE SOURCE: (B) CLONA: Fragment of angiostatin (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: He Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly 1 5 10 15 Thr Thr Ser Lys Thr Lys Ser Gly Val He Cys Gln Lys Trp Ser Val 25 30 Ser Pro Pro His Pro Lys Tyr Pro Pro Glu Lys Phe Pro Leu Ala 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Lys Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Glu Thr Arg Phe Asp Tyr Cys Asp 65 70 75 80 He Pro Glu Cys Glu Asp Glu Cys Met His Cys Ser Gly Glu His Tyr 85 90 ~ 95 Glu Gly Lys He Ser Lys Thr Met Ser Gly He Glu Cys Gln Ser Trp 100 105 110 Gly Ser Gln Ser Pro His Wing His Gly Tyr Leu Pro Ser Lys Phe Pro 115 120 125 Asn Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro 130 135 140 Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Phe Cys 145 150 155 160 Asp He Pro Arg Cys Thr Thr Pro Pro Pro Thr Ser Gly Pro Thr Tyr 165 170 175 Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val 180 185 190 Thr Wing Ser Gly His Thr Cys Gln Arg Trp Ser Wing Gln Ser Pro His 195 200 205 Lys His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Wing Pro Trp Cys Tyr Thr 225 230 235 240 Thr Asp Ser Glu Val Arg Trp Asp Tyr Cys Lys He Pro Ser Cys Gly 245 250 255 Being Ser Thr Thr Ser Thr Glu His Leu Asp Ala Pro Val Pro Pro Glu 260 265 270 Gln Thr Pro Val Wing Gln Asp Cys Tyr Arg Gly Asn Gly Glu Ser Tyr 275 280 285 Arg Gly Thr Be Ser Thr Thr He Thr Gly Arg Lys Cys Gln Ser Trp 290 295 300 Val Ser Met Thr Pro His Arg His Glu Lys Thr Pro Gly Asn Phe Pro 305 310 315 320 Asn Wing Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Wing Asp Lys 325 330 335 Ser Pro Trp (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 339 amino acids (B) TYPE: amino acid (C) HEBRA: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iü) HYPOTHETIC: "NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Bovine (vii) IMMEDIATE SOURCE: (B) CLONA: Fragment of angiostatin (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: He Tyr Leu Leu Glu Cys Lys Thr Gly Asn Gly Gln Thr Tyr Arg Gly 1 5 10 15 Thr Thr Wing Glu Thr Lys Ser Gly Val Thr Cys Gln Lys Trp Ser Wing 20 25 30 Thr Ser Pro His Val Pío Lys Phe Ser Pro Glu Lys Phe Pro Leu Ala 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asp 65 70 75 80 He Pro Glu Cys Glu Asp Lys Cys Met His Cys Ser Gly Glu Asn Tyr 85 90 95 Glu Gly Lys He Wing Lys Thr Met Ser Gly Arg Asp Cys Gln Wing Trp 100 '105 110 Asp Ser Gln Ser Pro His Wing His Gly Tyr He Pro Ser Lys Phe Pro 115 120 125 Asn Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro 130 135 140 Arg Pro Trp Cys Phe Thr Thr Asp Pro Gln Lys Arg Trp Glu Phe Cys 145 150 155 160_ Asp He Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Lys Tyr 165 170 175 Gln Cys Leu Lys Gly Thr Gly Lys Asn Tyr Gly Gly Thr Val Wing Val 180 185 190 Thr Glu Ser Gly His Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His 195 200 205 Lys His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Lys Wing Pro Trp Cys Tyr Thr 225 230 235 240 Thr Asn Ser Glu Val Arg Trp Glu Tyr Cys Thr He Pro Ser Cys Glu 245 250 255 Be Ser Pro Leu Be Thr Glu Arg Met Asp Val Pro Val Pro Pro Glu 260 265 270 Gln Thr Pro Val Pro Gln Asp Cys Tyr His Gly Asn Gly Gln Ser Tyr 275 280 285 Arg Gly Thr Be Ser Thr Thr He Thr Gly Arg Lys Cys Gln Ser Trp 290 295 300 Ser Ser Met Thr Pro His Arg His Leu Lys Thr Pro Glu Asn Tyr Pro 300 310 315 320 Asn Wing Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Wing Asp Lys 325 330 335 Ser Pro Trp It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the ent description of the invention.

Claims (15)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A therapeutic composition for inhibiting endothelial cell proliferation, characterized in that it comprises a pharmaceutically acceptable excipient and a fragment of plasminogen having a kringle region 1-5 of a plasminogen molecule.

2. The composition according to claim 2, characterized in that the plasminogen fragment is derived from murine plasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogen or bovine plasminogen.

3. The composition according to claim 2, characterized in that the plasminogen fragment corresponds to approximately 98 to 560 amino acids of a human plasminogen molecule.

4. A composition, characterized in that it comprises an isolated nucleotide sequence encoding a fragment of plasminogen having a kringle region 1-5 of a plasminogen molecule.

5 . The composition according to claim 4, characterized in that the plasminogen fragment is derived from murine plasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogen or bovine plasminogen.

6 The composition according to claim 4, characterized in that the plasminogen fragment corresponds to about 98 to 560 amino acids of a human plasminogen molecule.

A method for inhibiting endothelial cell proliferation, characterized in that it comprises administering to an endothelial cell an amount that inhibits the proliferation of a fragment of plasminogen having a kringle region 1-5 of a plasminogen molecule.

8. The method according to claim 7, characterized in that the plasminogen fragment is derived from murine plasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogen or bovine plasminogen.

9. The method according to claim 7, characterized the plasminogen fragment corresponds to approximately 98 to 560 amino acids of a human plasminogen molecule.

10. A method for treating a mammal with an angiogenically mediated disease, characterized in that it comprises administering to the mammal an effective amount for the treatment of a fragment of plasminogen having a kringle region 1-5 of a plasminogen molecule.

The method according to claim 10, characterized in that the plasminogen fragment is derived from murine plasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogen or bovine plasminogen.

The method according to claim 10, characterized the plasminogen fragment corresponds to approximately 98 to 560 amino acids of a human plasminogen molecule.

13. The method according to claim 10, characterized the angiogenically mediated disease is a cancer.

14. The composition according to claim 13, characterized in that it also comprises a cell that contains a vector.

15. A method for expressing a fragment of plasminogen having an endothelial cell proliferation inhibitory activity and having an amino acid sequence substantially similar to the kringle region 1-5 of a plasminogen molecule, characterized in that it comprises transfecting into a mammalian cell a vector, wherein the vector contains a DNA sequence encoding the plasminogen fragment, and wherein the vector is capable of expressing the angiostatin fragment when present in the cell.