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Definitions

• the present invention relates to a method of using antagonists of EMMPRIN (Extracellular Matrix Metalloproteinase Inducer) to treat pathological processes associated with proliferative diseases, such as cancer, by specifically preventing or inhibiting the ability of proliferating tissue to develop a blood supply.
• EMMPRIN Extracellular Matrix Metalloproteinase Inducer
• the invention more specifically relates to methods of treating such diseases by the use of EMMPRIN antagonists such as antibodies directed toward EMMPRIN, including specified portions or variants, specific for at least one protein or fragment thereof, in an amount effective to inhibit angiogenesis.
• EMMPRIN Angiogenesis is the process of new vessel formation. In adults, angiogenesis occurs only locally and transiently under physiological conditions such as wound healing, menstruation and pregnancy.
• angiogenesis In contrast, excessive angiogenesis occurs in more than 70 disease conditions such as cancer, atherosclerosis, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, and psoriasis.
• insufficient angiogenesis underlies diseases such as coronary artery disease, stroke, and delayed wound healing.
• Matrix metalloproteinases (MMPs) a family of more than twenty endopeptidases that are capable of cleaving all of the extracellular matrix components, play critical roles in angiogenesis [Klagsbrun and Moses 1999].
• Angiogenesis initiates as the breakdown of blood vessel basement membrane by capillary endothelial cells activated by angiogenic stimulators derived from tumors, inflammation sites, or tissues undergo other pathological conditions.
• the activated endothelial cells express increased MMPs, which in turn, enable disseminated endothelial cells to migrate away from their parental vessels. Only after the cells escape, do they respond to various growth factors to proliferate, and eventually go through a complex differentiation process to form new vessels. Depletion of MMPs, such as MMP-2 or MMP-9, results in a significant inhibition of tumor angiogenesis, supporting the critical role of MMPs in this process. [Bergers et al. 2000; Fang et al. 2000]. Extracellular matrix metalloproteinase inducer (EMMPRIN) (also known as CD 147) is a
• TCSF tumor collagenase stimulating factor
• MMPs are expressed by peritumoral stromal cells.
• EMMPRIN The role of EMMPRIN in tumor growth and metastasis was directly illustrated using EMMPRIN-overexpressing human breast cancer cells.
• MDA MB 436 cells are normally slow growing cells when they are implanted into nude mice. However, when these cells were transfected with EMMPRIN, they adopted a more aggressive growth pattern, with both accelerated growth rate and metastatic phenotypes [Zucker et al. 2001].
• Angiogenesis is the process of generating new capillary blood vessels, and it results from activated proliferation of endothelial cells.
• Neovascularization is tightly regulated, and occurs only during embryonic development, tissue remodeling, wound healing and periodic cycle of corpus luteum development (Folkman and Cotran, Relation of vascular proliferation to tumor growth, Int. Rev. Exp. Pathol.'16, 207-248(1976)).
• Endothelial cells normally proliferate much more slowly than other types of cells in the body. However, if the proliferation rate of these cells becomes unregulated, pathological angiogenesis can result.
• Pathological angiogenesis is involved in many diseases.
• cardiovascular diseases such as angioma, angiofibroma, vascular deformity, atherosclerosis, synechia and edemic sclerosis
• opthalmological diseases such as neovascularization after cornea implantation, neovascular glaucoma, diabetic retinopathy, angiogenic corneal disease, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, and granular conjunctivitis are related to angiogenesis.
• Chronic inflammatory diseases such as arthritis; dermatological diseases such as psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis, venous ulcers, acne, rosacea (acne rosacea or erythematosa), warts (verrucas), eczema, hemangiomas, lymphangiogenesis are also angiogenesis-dependent. Vision can be impaired or lost because of various ocular diseases in which the vitreous humor is infiltrated by capillary blood. Diabetic retinopathy can take one of two forms, non-proliferative or proliferative.
• Proliferative retinopathy is characterized by abnormal new vessel formation (neovascularization), which grows on the vitreous surface or extends into the vitreous cavity. In advanced disease, neovascular membranes can occur, resulting in a traction retinal detachment. Vitreous hemorrhages may result from neovascularization. Visual symptoms vary. A sudden severe loss of vision can occur when there is intravitreal hemorrhage. Visual prognosis with proliferative retinopathy is more guarded if associated with severe retinal ischemia, extensive neovascularization, or extensive fibrous tissue formation. Macular degeneration, likewise takes two forms, dry and wet.
• exudative macular degeneration (wet form), which is much less common, there is formation of a subretinal network of choroidal neovascularization often associated with intraretinal hemorrhage, subretinal fluid, pigment epithelial detachment, and hyperpigmentation. Eventually, this complex contracts and leaves a distinct elevated scar at the posterior pole. Both forms of age-related macular degeneration are often bilateral and are preceded by drusen in the macular region. Another cause of loss of vision related to angiogenic etiologies are damage to the iris.
• vascular endothelial cells in the synovial cavity is activated by the inflammatory cytokines, and results in cartilage destruction and replacement with pannus in the articulation (Koch AK, Polverini PJ and Leibovich SJ, Arth; 15 Rhenium, 29, 471-479(1986); Stupack DG, Storgard CM and Cheresh DA,
• Psoriasis is caused by uncontrolled proliferation of skin cells. Fast growing cell requires sufficient blood supply, and abnormal angiogenesis is induced in psoriasis (Folkman J., J. Invest. Derrnatol., 59, 40- 48(1972)). There is now considerable evidence that tumor growth and cancer progression requires angiogenesis, the formation of new blood vessels in order to provide tumor tissue with nutrients and oxygen, to carry away waste products and to act as conduits for the metastasis of tumor cells to distant sites (Folkman, et al. N Engl J Med 285: 1181-1186, 1971 and Folkman, et al. N Engl J Med 333: 1757-
• vascular endothelial growth factor VEGF
• TNFalpha TNFalpha
• bFGF vascular endothelial growth factor
• cytokines including IL-6 and IL-12.
• integrins cell adhesion molecules
• VEGF vascular endothelial growth factor
• TNFalpha TNFalpha
• bFGF basic fibroblast growth factor
• cytokines including IL-6 and IL-12.
• IL-6 is elevated in tissues undergoing angiogenesis and can induce VEGF in A431 cells, a human epidermoid carcinoma cell line (Cohen, et al. J. Biol. Chem. 271 : 736-741 , 1996).
• angiogenesis is known to be a contributing factor in a number of pathological conditions including the ability of tumors to grow and metastasize, disorders of the eye including retinopathies, and disorders of the skin including Kaposi's Sarcoma.
• EMMPRIN directly stimulates VEGF production, stimulates endothelial cells, in addition to local fibroblast cells, to express MMPs and therefore facilitate tumor angiogenesis, growth, invasion and metastasis.
• the present invention relates to a method of using antagonists of EMMPRIN, including antibodies directed toward EMMPRIN, and specified portions or variants thereof specific for at least one EMMPRIN protein or fragment thereof, to inhibit angiogenesis in disease conditions associated with abnormal angiogenesis.
• Such EMMPRIN antagonists such as antibodies can act through their ability to prevent the ability of EMMPRIN from stimulating MMP expression by microvascular endothelial cells, the cells involved in angiogenesis, in a dose-dependent fashion. Secondly, such antagonists or antibodies can act by limiting EMMPRIN induction of VEGF in the local environment thereby reducing the angiongenic potential of the tissue. By interfering with angiogenesis, such antagonists can prevent events associated with the initiation or progression of cancer tissue including events involved with angiogenesis and the metastatic spread of cancer. Based on the aforementioned action of the EMMPRIN antagonists of the invention, these antagonists can be best described as anti-angiogenic EMMPRIN antagonists.
• EMMPRIN can directly stimulate MMP-1 expression by microvascular endothelial cells, the cells involved in angiogenesis, in a dose-dependent fashion. This stimulation is specifically inhibited by function- blocking anti-EMMPRIN monoclonal antibodies. Since MMPs are essential for angiogenesis, such EMMPRIN antagonists can be useful as therapeutics for such diseases as cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, and psoriasis. In one embodiment, the EMMPRIN antagonist is an capable of preventing the production of EMMPRIN by cells, such as an siRNA or a shRNA molecule.
• the EMMPRIN antagonist is an antibody that specifically binds EMMPRIN.
• a particular advantage of such antibodies is that they are capable of binding EMMPRIN in a manner that prevents its action systemically.
• the method of the present invention thus employs antibodies having the desirable neutralizing property which makes them ideally suited for therapeutic and preventative treatment of metastatic disease states associated with various forms of cancer in human or nonhuman patients. Accordingly, the present invention is directed to a method of treating a disease or condition which is dependent on angiogenesis in a patient in need of such treatment which comprises administering to the patient an amount of a neutralizing EMMPRIN antibody to inhibit angiogenesis.
• the antibody is known as CNT0146 and is a murine anti-human EMMPRIN of the IgGl k class which has distinguished capability of inhibiting EMMPRIN-induced MMP production, including inhibiting
• MMP-1 production in fibroblast stimulated with recombinant EMMPRIN as well as inhibiting MMP production in the co-culture of tumor cells and fibroblast cells.
• FIG. 1 Schematic illustration of the central role of EMMPRIN in diseases involving abnormal angiogenesis.
• Fig. 2. Recombinant EMMPRIN dose-dependently stimulated MMP-1 production by HMVEC-L cells.
• Fig. 3. Inhibition of EMMPRIN-induced MMP-1 production in HMVEC-L cells by a neutralizing anti-EMMPRIN monoclonal antibody.
• Fig. 4. is a set of bar graphs showing (A) the relative endothelial cell migration induced by
• Fig. 5 is a bar graph showing the average final tumor weights of tumors produced by MDA MB231 human breast tumor cells manipulated to express greater or lesser amounts of EMMPRIN than normal (WT) and (B) is a bar graph showing the relative reduction in the migration of endothelial cells induced by WT cells in the presence of increasing concentrations of anti-VEGF antibody.
• Fig. 5A is a bar graph showing the average final tumor weights of tumors produced by
• 5B is a micrograph showing the difference in angiogenic structures between tumors produced by implantation of mice with WT versus S1- 3 cells.
• 5C is a set of bar graphs shoinw the amount of human VEGF (left panel) and mouse VEGF (right panel) in tumors produced by MDA MB231 human breast tumor cell types.
• Fig. 6A is a bar graph showing the amount of human EMMMPRIN in tissue extracts from xenograft tumors derived from WT, Vector control, S1-3, or AS EMMPRIN engineered human tumor cells.
• B. is a photo of a zymography gel showing MMP expression profile in tissue extracts from the same tumors containing where 10 ⁇ g of total protein was added to each lane.
• C. is a bar graph showin the quantitative determination of human and mouse MMP-2 levels in xenograft tumors.
• D. is a pair of bar graphs showing quantitative determination of human (left panel) and mouse (right panel) MMP-9 levels in xenograft tumors.
• Fig. 7. Photographs showing increased angiogenesis evidenced by numerous new capillary blood vessels in tumors derived from sense cells expressing EMMPRIN, but not in tumors derived from WT OR AS cells.
• FIG. 8 shows photographs of tumors after immunohistochemical analysis of MMP, VEGF, EMMPRIN: A. H&E staining of MDA-MB-231 xenograft tumors; B. Mouse MMP-9 staining; C. Mouse EMMPRIN staining; D. Blood vessel staining with anti-CD31 antibodies. Left panels - Vector control tumors; right panels - S1-3 tumors. DETAILED DESCRIPTION OF THE INVENTION
• EMMPRIN expressed by cells in diseased tissues directly stimulates neighboring endothelial cells, which results in an increase in MMP expression, i.e., MMP-1.
• MMP-1 MMP-1
• FIG. 1 MMP-1
• These MMPs mediate the breakdown of basement membrane of existing blood vessels; promote endothelial cells to migrate away from parental vessels; stimulate the expression and release of angiogenic growth factors; enable endothelial cells to respond to angiogenesis stimulatory factors leading to cell proliferation; and facilitate the remodeling of extracellular matrix for endothelial cell differentiation and assembly of new vessels. All these changes lead to an increase in angiogenesis and further contribute to the overall disease progression.
• the anti-angiogenic EMMPRIN antagonists of the invention are useful in inhibiting and preventing angiogenesis in so far as they block the stimulatory effects of EMMPRIN on endothelial cells, reduce VEGF production by endothelial cell, reduce endothelial cell division, decrease endothelial cell migration, and impair the activity of the proteolytic enzymes secreted by the endothelium.
• a number of pathologies including various forms of solid primary tumors and the metastases, lesions of the eye and disorders of the skin are improved by treatment with EMMPRIN antagonists in the method of the present invention.
• Both benign and malignant tumors including various cancers such as, cervical, anal and oral cancers, stomach, colon, bladder, rectal, liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis, renal, brain/ens (e.g., gliomas), head and neck, eye or ocular, throat, skin melanoma, acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's Sarcoma, Kaposi's Sarcoma, basal cell carinoma and squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, Wilms Tumor, neuroblastoma, mouth/pharynx, esophageal, larynx, kidney and lymphoma, among others may be treated using anti- EMMPRIN antibodies of the present invention.
• a secondary tumor is a tumor which originated in a primary site elsewhere in the body, but has now spread to a distant organ.
• the common routes for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems, and tracking along tissue planes and body cavaties with, for example, peritoneal fluid or cerebrospinal fluid.
• Secondary hepatic tumors are one of the most common causes of death in cancer patients and are by far and away the most common form of liver tumor.
• tumors which are most likely to spread to the liver include: cancer of the stomach, colon, and pancreas; melanoma; tumors of the lung, oropharynx, and bladder; Hodgkin's and non- Hodgkin's lymphoma; tumors of the breast, ovary, and prostate.
• Secondary lung, brain, and bone tumors are common to advanced stage breast, prostate and lung cancers. Any cancer may metastasize to bone, but metastases from carcinomas are the most common, particularly those arising in the breast, lung, prostate, kidney, and thyroid.
• Carcinoma of the lung is very commonly accompanied by hematogenous metastatic spread to the liver, brain, adrenals, and bone and may occur early, resulting in symptoms at those sites before obvious pulmonary symptom. Metastases to the lungs are common from primary cancers of the breast, colon, prostate, kidney, thyroid, stomach, cervix, rectum, testis, and bone and from melanoma. Each one of the above-named secondary tumors may be treated by the antibodies of the present invention. In addition to tumors, numerous other non-tumorigenic angiogeneis-dependent diseases which are characterized by the abnormal growth of blood vessels may also be treated with the anti- angiogenic EMMPRIN antagonists of the present invention.
• non-tumorigenic angiogenesis-dependent diseases include corneal neovascularization, hypertrophic scars and keloids, proliferative diabetic retinopathy, rheumatoid arthritis, arteriovenous malformations (discussed above), atherosclerotic plaques, delayed wound healing, hemophilic joints, nonunion fractures, Osier-Weber syndrome, psoriasis, pyogenic granuloma, scleroderma, tracoma, menorrhagia (discussed above) and vascular adhesions.
• the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions, however, capillaries may enter the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. Blood vessels can enter the cornea in a variety of patterns and depths, depending upon the process which incites the neovascularization.
• pannus trachomatosus pannus leprosus
• pannus phylctenulosus pannus degenerativus
• glucomatous pannus The corneal stroma may also be invaded by branches of the anterior ciliary artery (called interstitial vascularization) which causes several distinct clinical lesions: terminal loops, a "brush-like" pattern, an umbel form, a lattice form, interstitial arcades (from episcleral vessels), and aberrant irregular vessels. Corneal neovascularization can result from corneal ulcers.
• corneal ulcers may produce corneal ulcers including for example corneal infections (trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (graft rejection and Stevens-Johnson's syndrome), alkali bums, trauma, inflammation (of any cause), toxic and Vitamin A or protein deficiency states, and as a complication of wearing contact lenses. While the cause of corneal neovascularization may vary, the response of the cornea to the insult and the subsequent vascular ingrowth is similar regardless of the cause. Several angiogenic factors are likely involved in this process, many of which are products of the inflammatory response.
• Topical therapy with EMMPRIN antibodies may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. Use in combination with a steroid is also contemplated.
• Neovascular glaucoma is a pathological condition wherein new capillaries develop in the iris of the eye.
• the angiogenesis usually originates from vessels located at the pupillary margin, and progresses across the root of the iris and into the trabecular meshwork.
• Fibroblasts and other connective tissue elements associate with the capillary growth and a fibrovascular membrane develops which spreads across the anterior surface of the iris eventually forming a scar.
• the scar formation prevents adequate drainage of aqueous humor resulting in an increase in intraocular pressure that may result in blindness.
• Neovascular glaucoma generally occurs as a complication of diseases in which retinal ischemia is predominant. In particular, about one third of the patients with this disorder have diabetic retinopathy.
• Other causes include chronic retinal detachment, end-stage glaucoma, carotid artery obstructive disease, retrolental fibroplasia, sickle-cell anemia, intraocular tumors, and carotid cavernous fistulas.
• Angiogenic Conditions of the Skin comprising the step of administering one of the above-described anti- angiogenic compositions to a hypertrophic scar or keloid.
• healing of wounds and scar formation occurs in three phases: inflammation, proliferation, and maturation.
• the first phase inflammation, occurs in response to an injury which is severe enough to cause tissue damage and vascular leaking.
• This phase which lasts 3 to 4 days, blood and tissue fluid form an adhesive coagulum and fibrinous network which serves to bind the wound surfaces together.
• a proliferative phase in which there is ingrowth of capillaries and connective tissue from the wound edges, and closure of the skin defect.
• the maturation process begins wherein the scar contracts and becomes less cellular, less vascular, and appears flat and white. This final phase may take between 6 and 12 months.
• Overproduction of connective tissue at the wound site causes a persistently cellular and possible red and raised scar to be formed. If the scar remains within the boundaries of the original wound it is referred to as a hypertrophic scar, but if it extends beyond the original scar and into the surrounding tissue, the lesion is referred to as a keloid. Hypertrophic scars and keloids are produced during the second and third phases of scar formation.
• Several wounds are particularly prone to excessive endothelial and fibroblastic proliferation, including burns, open wounds, and infected wounds.
• an anti-EMMPRIN antibody in the method of the present invention to inhibit angiogenesis in such conditions can thus inhibit the formulation of such keloid scars.
• Angiogenesis is characterized by the invasion, migration and proliferation of smooth muscle and endothelial cells.
• the ⁇ v ⁇ 3 integrin also known as the vitronectin receptor
• the ⁇ v ⁇ 3 integrin is known to play a role in various conditions or disease states including tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget's disease, humoral hypercalcemia of malignancy, angiogenesis, including tumor angiogenesis, retinopathy, including macular degeneration, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis and smooth muscle cell migration (e.g. restenosis).
• the adhesion receptor integrin ⁇ v ⁇ 3 binds vitronectin, fibrinogen, von Willebrand Factor, laminin, thrombospondin, and other like ligands. It was identified as a marker of angiogenic blood vessels in chick and man and plays a critical role in angiogenesis or neovascularization. Antagonists of ⁇ v ⁇ 3 inhibit this process by selectively promoting apoptosis of cells in neovasculature. Therefore, ⁇ v ⁇ 3 antagonists would be useful therapeutic targets for treating such conditions associated with neovascularization (Brooks et al., Science, Vol. 264, (1994), 569-571).
• tumor cell invasion occurs by a three step process: 1) tumor cell attachment to extracellular matrix; 2) proteolytic dissolution of the matrix; and 3) movement of the cells through the dissolved barrier. This process can occur repeatedly and can result in metastases at sites distant from the original tumor.
• the ⁇ v ⁇ 3 integrin has been shown to play a role in tumor cell invasion as well as angiogenesis.
• the antagonists of ⁇ v ⁇ 3 and neutralizing anti-EMMPRIN antibodies both target neovasculature but act through different mechanisms, the combination of anti-integrin antibodies with anti-EMMPRIN antibodies should result in a particularly potent and effective combination therapy with little normal tissue toxicity.
• a method of treating a disease or condition associated with angiogenesis which comprises administering a combination of an integrin antagonist and an anti-EMMPRIN antibody to inhibit angiogenesis in a patient in need of such treatment.
• Other antibodies which selectively bind integrins or integrin subunits, especially those that bind the alphaV subunit, are disclosed in U.S. Patents 5,985,278 and 6,160,099.
• Mabs that inhibit binding of alphaVbeta3 to its natural ligands containing the t peptide argininyl-glycyl- aspartate (RGD) are disclosed in US 5,766,591 and WO0078815.
• a preferred combination of antibodies is the anti-alphaVbeta3 and anti-alphaVbeta5 Mab described in applicant's co-pending application U.S. serial no. 09/092,026 and an anti-EMMPRIN antibody, as disclosed herein. Both of the foregoing applications are incorporated by reference into the present application and form part of the disclosure hereof.
• other known anti-angiogenesis agents such as thalidomide may also be employed in combination with an anti- EMMPRIN antibody.
• CAM chick chorio-allantoic membrane assay
• corneal micropocket assay of neovascularization.
• CAM chick chorio-allantoic membrane assay
• India ink can be injected into the heart of some embryos just before formalin fixation so that vessels are visible near the edge of the avascular zone in histological sections. Histologic cross-sections of the chorioallantoic are examined to determine whether the test substance prevents normal development of the capillaries. This method, described in U.S. Pat. No. 5,001 ,116 which is also specifically incorporated herein by reference, showed the test useful in the selection of anti-angiogenic compounds or combinations of compounds.
• the corneal micropocket assay of neovascularization may be practiced using rat or rabbit corneas.
• This in vivo model is widely accepted as being generally predictive of clinical effect, as described in many review articles and papers such as O'Reilly et. al. Cell 79: 315-328. Briefly, a plug or pellet containing the recombinant bFGF (Takeda Pharmaceuticals- Japan) is implanted into corneal micropockets of each eye of an anesthetized female New Zealand white rabbit, 2 mm from the limbus followed by topical application of erythromycin ointment onto the surface of the cornea. The animals are dosed with the test compounds and examined with a slit lamp every other day by a corneal specialist.
• bFGF Takeda Pharmaceuticals- Japan
• the corneal micropocket assay may be used to demonstrate the anti-angiogenesis effect of anti-EMMPRIN antibodies. This is evidenced by a significant reduction in angiogenesis, as represented by a consistently observed and preferably marked reduction in the number of blood vessels within the cornea.
• Tumor vessels are generally primitive, that is, contain only endothelial cells.
• Other cell types found in more mature vessels include: smooth muscle cells, retinal pigment epithelial cells, fibroblasts, and epithelial cells, as well as tumor cells such as hemangioendothelioma cells or carcinoma cells.
• angiogenesis inhibitor that specifically inhibits endothelial cell proliferation is ANGIOSTATIN® protein. (O'Reilly et al., 1994 supra).
• Various representative cell lines are available for testing.
• SMC bovine aortic smooth muscle
• RPE retinal pigment epithelial
• MLE mink lung epithelial
• LLC Lewis lung carcinoma
• EOMA hemangioendothelioma cells and 3T3 fibroblasts.
• cells are washed with PBS and dispersed in a 0.05% solution of trypsin.
• Optimal conditions for the cell proliferation assays are established for each different cell type. Generally, cells are trypsinized and re- seeded in growth medium in the presence and absence of EMMPRIN and anti-EMMPRIN neutralizing Mab. After approximately 72 hours, the change in cell number is assessed by using a vital stain such as a tetrazolium dye
• EMMPRIN antagonists refers to a substance which inhibits or neutralizes the angiogenic activity of EMMPRIN. Such antagonists accomplish this effect in a variety of ways.
• One class of EMMPRIN antagonists will bind to EMMPRIN protein with sufficient affinity and specificity to neutralize the angiogenic effect of EMMPRIN. Included in this class of molecules are antibodies and antibody fragments (such as for example, F(ab) or F(ab') 2 molecules).
• Another class of EMMPRIN antagonists are fragments of EMMPRIN protein, muteins or small organic molecules i.e.
• EMMPRIN antagonists include EMMPRIN antibody, EMMPRIN receptor antibody, modified EMMPRIN, antisense EMMPRIN and partial peptides of EMMPRIN or EMMPRINR.
• Anti-EMMPRIN Antibodies Neutralizing antibodies to soluble factors that mediate inflammation and tumor proliferation, such as TNFalpha, have proved to highly effective therapeutics.
• REMICADE infliximab
• PA an anti-TNFalpha MAb is prescribed for RA and Crohn's Disease and RITUXAN (rituximab) an anti-CD20 Mab sold by Genentech, San Bruno, CA is used to treat B-cell lymphoma.
• "Neutralizing" Mabs not only bind their target but also inhibit its biological activity, usually by preventing its interaction with its cognate cell surface receptor. In certain cases, the target protein will comprise more than one active domain and exhibit multiple actions due to binding to more than one ligand or receptor.
• EMMPRIN is such a molecule and exhibits two immunoglobulin-like domains in the extracellular portion of the molecule, the Ig-like C2-type domain from aa 22-103 of basigin isoform 2 (NCBI accession # NP_940991) domain and the Ig-like V-type domain at 105-199 of the same isoform (Biswas, Zhang, DeCastro, Guo, Nakamura, Kataoka and Nabeshima, (1995), Cancer Res 55: 434-9).
• Monoclonal antibodies raised to EMMPRIN from cancer cells are capable of inhibiting EMMPRIN-induced MMP production in fibroblast cells, indicating neutralizing activity (Ellis, Nabeshima and Biswas, (1989), Cancer Res 49: 3385-91). These antibodies were subsequent shown to bind to EMMPRIN in the region 34-99 which lies within the C2-type domain.
• CBL1 a murine IgM, anti-human lymphoblastoid monoclonal antibody that was raised in Balb/c mice immunized with the T cell acute lymphoblastic leukemia cell line (T-ALL) CEM.
• T-ALL T cell acute lymphoblastic leukemia cell line
• WO9945031 teaches that antibodies with activities similar to CBL1 share a consensus binding sequence located in a region more C-terminal than the V-type domain, that is RVSR (residues 201-204 of NP_940991) and of a panel of MAbs made to the extracellular domain of EMMPRIN only one, designated M-6/6, is capable of inhibiting OKT3-induced T- cell activation and binds to a region in the C2-type domain Koch, C. et al. (1999) Internat. Immunol. 11 : 777-786; Staffler, G. et al. (2003) J. Immunol. 171 : 1707-1714).
• any of the anti-EMMPRIN antibodies known in the art which are anti-angiongenic EMMPRIN antagonists may be employed in the method of the present invention.
• Murine monocolonal antibodies to EMMPRIN are known as in, for example, in Ellis et al, 1989 supra and Koch, et al. 1999 Internat. Immunol. 11 (5): 777-786.
• an "EMMPRIN antibody”, “anti-EMMPRIN antibody,” “anti- EMMPRIN antibody portion,” or “anti-EMMPRIN antibody fragment” and/or “anti-EMMPRIN antibody variant” and the like include any protein or polypeptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, or at least one portion of an EMMPRIN binding protein derived from a EMMPRIN protein or peptide, which can be incorporated into an antibody for use in the present invention.
• CDR complementarity determining region
• Such antibody optionally further affects a specific ligand, such as but not limited to where such antibody modulates, decreases, increases, antagonizes, agonizes, mitigates, alleviates, blocks, inhibits, abrogates and/or interferes with EMMPRIN angiogenic activity, in vitro, in situ and/or in vivo.
• a suitable anti-EMMPRIN antibody, specified portion or variant of the present invention can bind at least one EMMPRIN protein or peptide, or specified portions, variants or domains thereof.
• a suitable anti- EMMPRIN antibody, specified portion, or variant affects EMMPRIN angiogenic function in a variety of ways, such as but not limited to, RNA, DNA or protein synthesis, EMMPRIN release, EMMPRIN receptor signaling, EMMPRIN receptor binding, EMMPRIN production and/or synthesis.
• the term "antibody" is further intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antitbody or specified fragment or portion thereof, including single chain antibodies and fragments thereof.
• Functional fragments include antigen-binding fragments that bind to a mammalian EMMPRIN.
• antibody fragments capable of binding to EMMPRIN or portions thereof including, but not limited to Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the invention (see, e.g., Colligan, Immunology, supra).
• Fab e.g., by papain digestion
• Fab' e.g., by pepsin digestion and partial reduction
• F(ab')2 e.g., by pepsin digestion
• facb e.g., by plasmin digestion
• Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein.
• Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
• a combination gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and/or hinge region of the heavy chain.
• the various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
• the anti-EMMPRIN antibody may be a primate, rodent, or human antibody or a chimeric or humanized antibody.
• human antibody refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, CL, CH domains (e.g., CH1 , CH2, CH3), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
• antibodies designated primate monkey, baboon, chimpanzee, etc.
• rodent mouse, rat, rabbit, guinea pig, hamster, and the like
• other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies.
• chimeric antibodies of the invention can include any combination of the above.
• a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linker peptide that is not found in native human antibodies.
• a Fv can comprise a linker peptide, such as 2 to about 8 glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
• linker peptides are considered to be of human origin.
• Bispecific, heterospecific, heteroconjugate or similar antibodies can also be used that are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for at least one EMMPRIN protein, the other one is for any other antigen. Methods for making bispecific antibodies are known in the art.
• bispecific antibodies are based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low.
• Anti-EMMPRIN antibodies useful in the methods and compositions of the present invention can optionally be characterized by high affinity binding to EMMPRIN and optionally and preferably having low toxicity.
• an antibody, specified fragment or variant of the invention, where the individual components, such as the variable region, constant region and framework, individually and/or collectively, optionally and preferably possess low immunogenicity is useful in the present invention.
• the antibodies that can be used in the invention are optionally characterized by their ability to treat patients for extended periods with measurable alleviation of symptoms and low and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties, can contribute to the therapeutic results achieved.
• Low immunogenicity is defined herein as raising significant HAHA, HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low titres in the patient treated (less than about 300, preferably less than about 100 measured with a double antigen enzyme immunoassay) (Elliott et al., Lancet 344:1125-11 7 (1994), entirely incorporated herein by reference).
• Suitable antibodies include those that compete for binding to human EMMPRIN with the commercially available monoclonal antibody CD147-RDI/clone UM-8D6 (Research Diagnostics, Inc., Flanders, NJ).
• the neutralizing anti-EMMPRIN monoclonal antibodies described herein can be used to inhibit angiogenesis and thus prevent or impair tumor growth and prevent or inhibit metastases. Additionally, such monoclonal antibodies can be used to inhibit angiogenic inflammatory diseases amenable to such treatment, which may include but are not limited to rheumatoid arthritis, diabetic retinopathy, psoriasis, and macular degeneration.
• the individual to be treated may be any mammal and is preferably a primate, a companion animal which is a mammal and most preferably a human patient.
• the amount of monoclonal antibody administered will vary according to the purpose it is being used for and the method of administration.
• the anti-angiogenic anti-EMMPRIN antibodies may be administered by any number of methods that result in an effect in tissue in which angiogenesis is desired to be prevented or halted. Further, the anti-antiangiongenic anti-EMMPRIN antibodies need not be present locally to impart an anti- angiogenic effect, therefore, they may be administered wherever access to body compartments or fluids containing EMMPRIN is achieved. In the case of inflamed, malignant, or otherwise compromised tissues, these methods may include direct application of a formulation containing the antibodies. Such methods include intravenous administration of a liquid composition, transdermal administration of a liquid or solid formulation, oral, topical administration, or interstitial or inter-operative administration.
• Adminstration may be affect by the implantation of a device whose primary function may not be as a drug delivery vehicle as, for example, a vascular stent.
• methods are provided for treating corneal neovascularization (including corneal graft neovascularization), comprising the step of administering a therapeutically effective amount of an anti-angiogenic EMMPRIN antibody of the invention directly to the cornea or systemically to the patient, such that the formation of blood vessels is inhibited.
• methods for treating neovascular glaucoma comprising the step of administering a therapeutically effective amount of an anti- angiogenic neutralizing anti-EMMPRIN antibodies directly to the eye or systemically to the patient, such that the formation of blood vessels is inhibited.
• an anti-angiogenic EMMPRIN antibody of the invention alone, or in combination with another anti-angiogenic agent are directly injected into a hypertrophic scar or keloid in order to prevent the progression of these lesions.
• This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids such as burns.
• Treatment may be effective when begun after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development.
• Administration may also be oral or by local injection into a tumor or tissue but generally, the monoclonal antibody is administered intravenously.
• the dosage range is from about 0.05 mg/kg to about 12.0 mg/kg. This may be as a bolus or as a slow or continuous infusion which may be controlled by a microprocessor controlled and programmable pump device.
• DNA encoding preferably a fragment of said monoclonal antibody may be isolated from hybridoma cells and administered to a mammal.
• the DNA may be administered in naked form or inserted into a recombinant vector, e.g., vaccinia virus in a manner which results in expression of the DNA in the cells of the patient and delivery of the antibody.
• a recombinant vector e.g., vaccinia virus
• the monoclonal antibody used in the method of the present invention may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in
• the monoclonal antibody will typically be combined with a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier include water, physiological saline, or oils.
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Except insofar as any conventional medium is incompatible with the active ingredient and its intended use, its use in any compositions is contemplated.
• the formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
• sterile liquid carrier for example, water for injections
• Abs antibodies polyclonal or monoclonal aV integrin subunit alpha V b3 integrin subunit beta 3 bFGF basic fibroblast growth factor IFN interferon lg immunoglobulin IgG immunoglobulin G IL interleukin MMP-1 EMMPRIN extracellular matric metalloproteinase inducer EMMPRINR receptor sEMMPRINR soluble EMMPRIN receptor Mab monoclonal antibody VEGF vascular endothelial growth factor MMP matrix metallopoteinase
• EXAMPLE 1 Recombinant EMMPRIN stimulates MMP-1 production by human microvascular endothelial cells from the lung (HMVEC-L)
• HMVEC-L cells were obtained from Clonetics, Walkersville, Maryland (Cat# CC-2527, Lot# 8F1528). HMVEC-L cells were cultured under conditions recommended by the supplier.
• EMM-2 MV Endothelium Cell Growth Medium MV
• hEGF human epithelial growth factor
• hFGF-B human basic fibroblast growth factor
• VEGF vascular endothelial growth factor
• hlGF-1 human insulin-like growth factor-1
• ascorbic acid gentamicin
• DM - Fibroblast Basic Medium + 2% FBS Dilution Medium
• MMP-1 activity in the conditioned medium was carried out using Human MMP-1 Activity Kit (R&D Systems, Minneapolis, Minnesota) (Cat#F1 M00). Briefly, MMP-1 in 150 ⁇ l of standard or sample was captured by anti-MMP-1 antibodies immobilized at the bottom of each well. Captured MMP-1 was subsequently activated by 4-aminophenylmercuric acetate (APMA). MMP substrate added into each well was cleaved by active MMP-1 and the resulting fluorescence was determined using SpectraFluor Plus Plate Reader (TECAN, Zurich, Switzerland) (Cat* F129005, Ser# 94747) with the following parameters: excitation wavelength at 320 nm and emission wavelength at 405nm.
• APMA 4-aminophenylmercuric acetate
• HMVEC-L cells were challenged with different concentrations of recombinant EMMPRIN to stimulate MMP-1 production.
• EMMPRIN dose-dependently stimulated MMP-1 production in endothelial cells.
• HMVEC-L cells produced approximately 40 ng/ml MMP-1 when treated with 20 ⁇ g/ml EMMPRIN.
• This response of HMVEC-L to EMMPRIN stimulation was even stronger than that by NHLF cells, which produced only half of that amount of MMP-1 in response to the same treatment.
• the stimulation of MMP-1 production was first observed after one-day challenge and sustained for at least three days. Results shown in Fig. 2, for the first time, demonstrated that EMMPRIN can directly stimulate MMP-1 expression by microvascular endothelial cells, the cells directly involved in angiogenesis, in a dose-dependent fashion.
• EXAMPLE 2 Inhibition of EMMPRIN-induced MMP-1 production by an anti-EMMPRIN mAb in HMVEC-L cells to further confirm the specificity of EMMPRIN-induced MMP-1 production, monoclonal antibodies against human EMMPRIN were included in the assay 15 minutes after cells were stimulated with EMMPRIN. At 10 ⁇ g/ml, the CD147-RDI/clone UM-8D6 (Research Diagnostics, Inc., Flanders, NJ) significantly inhibited MMP-1 production by fibroblast cells induced by EMMPRIN (5 ⁇ g/ml) (Fig. 3).
• EMMPRIN Effects of EMMPRIN on Human Endothelial Cell Migration
• Human endothelial cells derived from primary tissue (umbilical cord) HUVEC cells were used in an in vitro system wherein endothelial cells are seeded in the top wells of the transwell system, in cell medium containing 1% FBS.
• FBS fetal bovine serum
• culturing medium with 10% FBS will serve as a chemotactic source to induce cell migration or invasion.
• the top and bottom wells are separated by a membrane with pores of 8 ⁇ m in diameter.
• the membrane is either uncoated or coated with various extracellular matrix proteins, i.e., collagen, fibronectin, vitronectin, or Matrigel, for determining cell migration or invasion, respectively.
• MDA-MB-231 human breast cancer cells were purchased from ATCC (Manassas, VA). Methods for transfection and establishment of MDA-MB-231 cells stably expressing different levels of EMMPRIN have been described previously (Tang, Y. et al. (2004) Mol. Cancer Res. 2:73-80). The cells were transfected with the cDNA corresponding to human EMMPRIN open reading frame sense (MDA MB231 S1-3) or an antisense strand of the same ORF (MDA MB231 AS1-5 and MDA MB231 AS2- 5) in pcDNA3.1 TOPO vector (Invitrogen, Carlsbad, CA).
• HUVEC Endothelial cell migration assay kit
• MDA MB231 cells Serum-free media conditioned by MDA MB231 cells: WT, Vector, S1-3, AS1 -5, or AS2-5 was used as the chemoattactant source in the bottom compartment of chamber.
• anti-VEGF mAb R&D Systems, Minneapolis, MN was added into the bottom compartment at various concentrations to neutralize VEGF biological activity.
• Fig. 4A shows the relative level of HUVEC cell migration induced by conditioned medium derived from the various MDA-MB-231 cell constructs. WT cell-induced migration was assigned 100%. Error bars represent standard deviations of triplicate data points. Significant differences by Students T- test ( * ) was at the p ⁇ 0.01 value compared to endothelial cell migration induced by WT cells. Fig.
• EMMPRIN Effects of EMMPRIN on HMVEC-L cell tube formation
• the role of EMMPRIN in angiogenesis can be shown using in vitro tube formation assays.
• HMVEC cells When seeded on Matrigel, HMVEC cells initiate a spontaneous differentiation process to form capillary-like tube structure. This in vitro differentiation mimics in vivo angiogenesis process and is often employed in angiogenesis studies.
• EMMPRIN will change the properties of endothelial cells by stimulating MMP expression, and therefore to stimulate cell migration and invasion.
• An enhanced tube formation will occur when these cells are stimulated with EMMPRIN.
• the specificity of EMMPRIN in tube formation will be investigated using monoclonal antibodies against human EMMPRIN.
• EXAMPLE 5 Effects of EMMPRIN on angiogenesis in vivo - Matrigel plug assay The role of EMMPRIN in angiogenesis will be directly investigated in vivo using Matrigel plug assays.
• Matrigel is a solubilized basement membrane preparation extracted from the Engel-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins. The major component is laminin, but Matrigel also contains trace amounts of fibroblast growth factor, TGF-beta, tissue plasminogen activator, and other growth factors that occur naturally in the EHS tumor.
• Matrigel is the basis for several types of tumor cell invasion assays and provides the necessary substrate for the study of angiogenesis.
• Matrigel forms a soft gel plug when injected subcutaneously into mice or rats and supports an intense vascular response when supplemented with angiogenic factors.
• Matrigel plugs containing suboptimal doses of angiogenic growth factors such as basic fibroblast growth factor (FGF), or vascular endothelial cell growth factor (VEGF) can be implanted into mice to induce angiogenesis in vivo.
• FGF basic fibroblast growth factor
• VEGF vascular endothelial cell growth factor
• EMMPRIN effects of EMMPRIN as tested in the Matrigel plug angiogenesis assay can be used to demonstrate the activity of EMMPRIN antagonists such as siRNA or anti-EMMPRIN antibodies in preventing angiongenesis.
• EXAMPLE 6 Effects of EMMPRIN on angiogenesis in vivo - corneal pocket assay Similarly, the role of EMMPRIN in angiogenesis will be directly investigated in vivo using corneal pocket assays.
• Polymer discs containing angiogenic growth factors, such as basic fibroblast growth factor (FGF), or vascular endothelial cell growth factor (VEGF) will be implanted into a corneal pocket in order to evoke vascular outgrowth from the peripherally located limbal vasculature.
• FGF basic fibroblast growth factor
• VEGF vascular endothelial cell growth factor
• EMMPRIN angiogenic growth factors supplemented with various doses of recombinant EMMPRIN. Since EMMPRIN will induce MMP production by endothelial cells, we expect to observe an increase in angiogenesis due to enhanced endothelial cell migration and invasion. The specificity of EMMPRIN in corneal pocket angiogenesis assay will be investigated using EMMPRIN antagonists such as siRNA or anti-EMMPRIN antibodies.
• EMMPRIN Effects of EMMPRIN on angiogenesis -stimulation of VEGF production and release mediated by MMP
• EMMPRIN also stimulates the expression of membrane-type matrix metalloproteinase 1 (MT1 -MMP) [Sameshima et al. 2000b].
• MT1-MMP membrane-type matrix metalloproteinase 1
• VEGF membrane-type matrix metalloproteinase 1
• EMMPRIN the link between EMMPRIN and VEGF, in both in vitro and in vivo settings can be demonstrated.
• VEGF vascular endothelial growth factor
• MDA-MB-231 human breast cancer cells were purchased from ATCC (Manassas, VA). Methods for transfection and establishment of MDA-MB-231 cells stably expressing different levels of EMMPRIN have been described previously (Tang, Y. et al. (2004) Mol. Cancer Res. 2:73-80). The cells were transfected with the cDNA corresponding to human EMMPRIN open reading frame sense (MDA MB231 S1-3) or an antisense strand of the same ORF (MDA MB231 AS1-5 and MDA MB231 AS2- 5) in pcDNA3.1 TOPO vector (Invitrogen, Carlsbad, CA).
• NHLF or NHDF normal human lung or dermal fibroblast cells
• HMVEC-L human microvascular endothelial cells from the lung
• HMVEC-L human umbilical vein endothelial cells
• ECM-2 Endothelial Growth Medium-2
• 100,000 cancer cells MDI MB231 WT, S1-3, AS1-5, or AS 2-5) were cultured together with 200,000 NHDF cells in a six-well culture plate in complete DMEM. After 24 h, the medium was replaced with serum-free DMEM and the cultures continued for 2 days.
• the medium was replaced with fresh serum free DMEM and the cultures maintained for an additional 3 days at which time the medium was collected and analyzed.
• the cells were lysed with Tris-buffered saline plus 1% NP40 to determine cell-associated EMMPRIN.
• the relative amount of EMMPRIN expressed in 10 ug of total cell protein was determined by Western blot analysis using scanning densitometry, by quantitative ELISA using anti-EMMPRIN antibody (RDI-147, Research diagnostics) as described (Tang et al. 2004) and on the cell surface by fluorescence activated cell analysis (FAC analysis).
• the FAC analysis confirmed that cell surface EMMPRIN was absent on cells transfected with antisense constructs (data not shown).
• MMP-2 and MMP-9 in serum-free medium or tumor extracts was determined by substrate SDS-PAGE zymography using 10 ug of total protein. Proteolytic activities on the gel were detected as clear bands on a blue background of undigested and stained gelatin.
• ELISA measurements of human or mouse MMP-2, MMP-9 and VEGF concentrations were performed using Quantikine ELISA kits from R&D Systems, according to the manufacturer's instructions. Each sample was analyzed in triplicates.
• MMP-2, MMP-9 or VEGF contained in 100 ⁇ l of standard or samples (equivalent of 50 ⁇ g of total protein) were captured by anti-MMP-2-, anti-MMP-9-, or anti-VEGF-antibodies immobilized on the bottom of assay wells. After washing, the MMP or VEGF specific antibody was used to quantitate the amount present. Results The ransfected cells had altered levels of total EMMPRIN when grown in cell culture conditions (Table 1 ). S1-3 cells had approximately twice the level of WT cells and 4-fold that of the AS cells. TABLE 1.
• Sense EMMPRIN cells were created that represent a cell population derived from a single cell clone that was stably transfected with a mammalian expression vector encoding the full-length human EMMPRIN.
• Antisense cells were generated by transfecting MDA MB 231 cells with a mammalian expression vector encoding the full-length human EMMPRIN in the antisense orientation.
• Sense cells constitutively express increased levels of EMMPRIN, and antisense cells express decreased levels of EMMPRIN due to inhibition of protein translation by the antisense RNA (See Example 7). These cells, together with wild- type cells, were implanted subcutaneously into nude mice. Tumor angiogenesis were assessed in tumors derived from these cells.
• Fig. 5B increased angiogenesis was evidenced by numerous new capillary blood vessels in tumors derived from sense cells, but not in tumors derived from wild-type and antisense cells.
• Human VEGF level was 2.6-fold higher in xenografts tissue produced by S1-3 cells
• EXAMPLE 9 Effect of tumor EMMPRIN on tumor tissue environment with respect to MMPs and VEGF Human breast tumor cells described in Example 3, were used to assess the effect of increased of decreased EMMPRIN on tumor tissue and tumor stroma (fibroblasts, endothelial cells, and other ancillary cells) in vivo.
• tumor tissue and tumor stroma fibroblasts, endothelial cells, and other ancillary cells
• mice On day 0, at approximately 6 weeks of age, mice were assigned to each of 5 groups consisting of 8 mice per group. Animals were inoculated with 10 7 cells in 0.1 mL of cell suspension subcutaneously in the right flank region. Tumor growth was monitored weekly by caliper measurement and tumor volume (mm 3 ) were calculated based on the formula [length x width x width]/2.
• EMMPRIN sense tumors, and lowered levels when EMMPRN expression was suppressed (Figure 6B).
• Visualization of tumor EMMPRIN-MMP systems in vivo Tumor The difference in angiogenic activity is between tumors produced by the cells overexpressing EMMPRIN and the WT or under expressing cells, AS, were clearly visible (Fig. 7).
• the effect of tumor EMMPRIN expression on host EMMPRIN-MMP system was further studied in immunohistochemical analysis of the xenograft tumors.
• MMP-9 and EMMPRIN Co-localization of MMP-9 and EMMPRIN around angiogenic blood vessels was further supported by overlapping distribution of mouse MMP-9, EMMPRIN and that of CD31 , a blood vessel marker (Fig. 8).
• MMP-9 MMP-9 was mainly detected in macrophage-like cells
• EMMPRIN was detected at very low levels in some fibroblast cells (Fig. 8).
• Anti-angiogenic anti-EMMPRIN antibodies can be prepared using standard procedures and screened using the properties described herein for anti-angiogenic anti-EMMPRIN antagonists.
• mice Three 12-14 week old Balb/c mice were obtained from Charles River Laboratories. Two mice each received combination intradermal and intraperitoneal injections of 25 ⁇ g rHuEMMPRIN (R&D Systems) (12.5 ⁇ g/site) in 75 ⁇ L PBS emulsified in an equal amount of Freund's complete adjuvant on day 0, and 25 ⁇ g rHuEMMPRIN in 75 ⁇ L PBS emulsified in an equal amount of Freund's incomplete adjuvant on days 14, 28 and 51.
• 25 ⁇ g rHuEMMPRIN R&D Systems
• the third mouse received an initial injection of 25 ⁇ g of rHuEMMPRIN + 0.33 x 10 5 U murine IFN ⁇ + 0.33 x 10 5 U murine IFN ⁇ (Biosource) in 100 ⁇ l PBS administered S.Q. at the base of the tail.
• the mouse received additional injections of 0.33 x 10 5 U IFN ⁇ + 0.33 x 10 5 U IFN ⁇ in 100 ⁇ L PBS administered S.Q. at the base of the tail.
• the mouse was boosted with 25 ⁇ g EMMPRIN + 100 ⁇ g anti-murine CD40 agonist Mab (R&D Systems) administered S.Q. at the base of the tail.
• mice were bled at various time-points throughout the immunization schedule. Blood collections were performed by retro-orbital puncture and serum was collected for titer determination by solid phase EIA. Once titer plateau was obtained, the mice received their final booster of 25 ⁇ g of EMMPRIN in PBS given intraveneously (IV). Three days later the mice were euthanized by C0 2 asphyxiation, and the spleens were aseptically removed and immersed in 10 mL cold PBS containing 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, and 0.25 ⁇ g/mL amphotericin B (PBS/PSA).
• PBS/PSA amphotericin B
• Lymphocytes were harvested by sterilely passing cells though a wire mesh screen immersed in cold PBS/PSA. The cells were washed once in cold PSA/PBS, counted using Trypan blue dye exclusion and resuspended in 10 mL PBS.
• EIAs Enzyme immunoassays
• EMMPIRN used as antigen protein was assayed by its ability to stimulate production of MMP-1 from EMMPRIN stimulated in fibroblast cells was performed as described (Guo, Zucker, Gordon, Toole and Biswas, (1997), J Biol Chem 272: 24-7)(24)), modified by using highly homogenous primary human fibroblast cells of less than three passages and modified stimulation conditions. Only highly pure fibroblast cells that were confirmed being negative for cytokeratin 18, cytokeratin 19, factor Vll-related antigen, and alpha actin were used in the assay.
• EMMPRIN stimulation was dependent on the passage of fibroblast cells.
• Cells of earlier passages responded more potently in producing increased amounts of MMP, compared to cells that have been cultured for more than three passages.
• a new cell challenge method was used. Instead of adding recombinant EMMPRIN to adherent cells, we preloaded recombinant EMMPRIN in testing wells. Suspended cells were then added into these wells and were directly exposed to recombinant EMMPRIN. This new challenge procedure ensures the maximal exposure of cell surface receptors, which are likely expressed on the basolateral surfaces and could be out of access in adherent cells, to EMMPRIN for optimal assay sensitivity.
• Recombinant EMMPRIN corresponding to the extracellular domain of human EMMPRIN protein was produced in NSO cells (R&D Systems, Minneapolis, MN). MMP-1 activity in serum-free medium conditioned by fibroblast cells treated with different amounts of recombinant EMMPRIN protein was quantitatively determined using an MMP-1 Activity Assay Kit according to product manual (R&D Systems, Minneapolis, MN). Briefly, MMP-1 contained in 150 ⁇ l of standards or samples was captured by anti-MMP-1 antibodies immobilized on the bottom of assay wells. Captured MMP-1 was subsequently activated by 4-aminophenylmercuric acetate (APMA).
• APMA 4-aminophenylmercuric acetate
• MMP substrate added into each well was cleaved by activated MMP-1 and the resulting fluorescence was determined using SpectraFluor Plus Plate Reader (TECAN, Research Triangle Park, NC) with the following parameters: excitation wavelength at 320 nm and emission wavelength at 405 nm.
• SpectraFluor Plus Plate Reader TECAN, Research Triangle Park, NC
• the antibody designated CNTO 146 met the initial selection criteria for an anti- angiongenic anti-EMMPRIN Mab. Inhibition of MMP-2 production in co-culture of tumor cells and fibroblast cells
• the co-culture assay was performed as previously described above using normal human dermal fibroblasts and human melanoma tumor cells (G361) were used and either the commercial antibody RDI CD147 or CNTO 146 were added to the cultures. Three days after the last change of serum free medium, the amount of MMP-2 was quantitated. The data showed that CNTO 146 was capable of inhibiting MMP-2 production in these co-cultures as did the commercial antibody.

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