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WO1999016465A1 - Methode d'inhibition de l'angiogenese tumorale chez un sujet vivant - Google Patents

Methode d'inhibition de l'angiogenese tumorale chez un sujet vivant Download PDF

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Publication number
WO1999016465A1
WO1999016465A1 PCT/US1997/017485 US9717485W WO9916465A1 WO 1999016465 A1 WO1999016465 A1 WO 1999016465A1 US 9717485 W US9717485 W US 9717485W WO 9916465 A1 WO9916465 A1 WO 9916465A1
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Prior art keywords
integrin
vegf
tumor
vivo
tumor angiogenesis
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PCT/US1997/017485
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English (en)
Inventor
Donald R. Senger
Michael Detmar
Kevin P. Claffey
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Beth Israel Deaconess Medical Center
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Priority to AU46569/97A priority Critical patent/AU4656997A/en
Priority to PCT/US1997/017485 priority patent/WO1999016465A1/fr
Publication of WO1999016465A1 publication Critical patent/WO1999016465A1/fr
Priority to US09/532,310 priority patent/US6596276B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is concerned with angiogenesis broadly and with tumor angiogenesis directly; and is focused on means and methods for inhibiting tumor angiogenesis involving vascular endothelial growth factor ("VEGF”) and integrin heterodimer surface receptors found in the vasculature of a living subject.
  • VEGF vascular endothelial growth factor
  • integrin heterodimer surface receptors found in the vasculature of a living subject.
  • Angiogenesis the formation of new capillaries and blood vessels, is a complex process first recognized in studies of wound healing and then with investigations of experimental tumors.
  • Angiogenesis involves extracellular matrix remodeling, endothelial cell migration and proliferation, and functional maturation of endothelial cells into mature blood vessels [Brier, G. and K. Alitalo, Trends Cell Biol. 6: 454-456 (1996)].
  • the process generally has been studied for more than 50 years, the existence and in-vivo effects of several discrete angiogenic factors have been identified just over a decade ago [Folkman, J. and M. Klagsbum, Science 235: 444-447 (1985)].
  • the process of angiogenesis is a normal host response to injury; and as such is an integral part of the host body's homeostatic mechanisms.
  • tumor angiogenesis is the specific development in-vivo of an adequate blood supply for a solid tumor mass; and the growth of a tumor in-vivo beyond the size of a few millimeters in diameter is believed to be dependent upon the existence, maintenance, and continued development of sufficient and functional blood vasculature in-situ.
  • tumor survival and growth has been linked with new capillary and new blood vessel formation. Histological examination of such neoplasms has revealed that tumor cells typically surround blood capillaries in a cylindrical configuration with a radius not exceeding about 200 micrometers - the critical travel distance for diffusion of molecular oxygen [Folkman, J., Cancer Res. 46: 467-473 (1986)].
  • tumor angiogenesis originates at least in part from the sprouting of new capillaries and blood vessels directly from the pre-existing and functional normal vasculature; and possibly also from stem cells existing in the blood. Tumor angiogenesis thus involves endothelial cell penetration of the vascular basement membrane in a pre-existing blood vessel; followed by endothelial cell proliferation; and then by an invasion of the extracellular matrix surrounding the blood vessel to form a newly created vascular spout [Vernon, R. and E.H. Sage, Am. J. Pathol. 147: 873-883 (1995); Auspunk, D.H. and J. Folkman, Microvasc. Res. 14'. 53-65 (1977)].
  • a number of different biologically active and physiologically functional molecular entities appear to be individual factors of angiogenesis.
  • biologically active classes of substances known as vascular endothelial growth factor and the integrin protein family of cell surface receptors.
  • vascular endothelial growth factor and the integrin protein family of cell surface receptors.
  • integrin protein family of cell surface receptors Each of these two classes will be summarily reviewed as to their conventionally known properties and functions.
  • Vascular endothelial growth factor also known as vascular permeability factor, is a 34-45 kilodalton dimeric glycoprotein; is a cytokine; and is a potent inducer of microvascular hyperpermeability.
  • VEGF is believed to be responsible for the vascular hyperpermeability and consequent plasma protein-rich fluid accumulation that occurs in-vivo with solid tumors and ascites tumors [Senger et al.. Science 219: 983-985 (1983); Dvorak et al.. J. Immunol. 122: 166 (1979); Nagy et al., Biochem. Biophvs. Acta.
  • VEGF increases microvascular permeability with a potency which is typically 50,000 times that of histamine [Senger et al., Cancer Res. 50: 1774-1778 (1990].
  • VEGF vascular endothelial growth factor
  • EC vascular endothelial cells
  • VEGF exerts a number of other effects on endothelial cells in-vitro. These include: an increase in intracellular calcium; a stimulation of inositol triphosphate formation; a provocation of von Willebrand factor release; and a stimulation of tissue factor expression [Brock et al., Am. J. Pathol. 138: 213 (1991 ); Clauss et al., J. Ex£. Med. 172: 1535 (1990)].
  • Vascular endothelial growth factor elicits potent angiogenic effects by stimulating endothelial cells through two receptor tyrosine kinases, Flt-1 and KDR/Flk-1 [Dvorak et al.. Am. J. Pathol. 146: 1029-1039 (1995); Mustonen, T. and K. Alitalo, J. Cell. Biol. 129: 895-898 (1996)].
  • Flt-1 Flt-1 and KDR/Flk-1
  • VEGF angiogenic activity has been demonstrated in several experimental models including the chick chorioallantoic membrane [Whiting et al.. Anat. Embrvol. 186: 251-257 (1992)]; rabbit ischemic hind limb [Takeshita et al., J. Clin. Invest. 93: 662-670 (1994)]; tumor xenografts in mice [Potgens et al.. Biol. Chem. Hoppe. Seyler 376: 57-70 (1995); Claffey et a , Cancer Res. 56: 172-181 (1996)]; and a primate model of iris neovascularization [Tolentino et al.. Arch. Qphthalmol.
  • VEGF appears not only to promote angiogenesis in a variety of experimental systems, but also appears to be overexpressed in a diversity of settings in which neovascularization is prominent.
  • VEGF is typically synthesized and secreted in-vivo by a variety of cultured tumor cells, transplantable animal tumors, and many different primary and metastatic human tumors [Dvorak et al., J. Exp. Med. 174: 1275- 1278 (1991 ); Senger et al., Cancer Res. 46: 5629-532 (1986); Plate et al.. Nature 359: 845-848 (1992); Brown et al.. Am. J. Pathol. 143: 1255-1262 (1993)]. Solid tumors, however, must generate a vascular stroma in order to grow beyond a minimal size [Folkman, J. and Y. Shing, J. Biol. Chem. 267: 10931-10934 (1992)].
  • VEGF today is believed able to be a central mediator of angiogenesis generally as well as of tumor angiogenesis in particular.
  • Monoclonal antibody directed against VEGF has been shown to suppress growth and decrease the density of blood vessels in experimental tumors [Kim et al., Nature 362: 841-844 (1993)].
  • Integrins are a specific family of cell surface receptors which function in-vivo as adhesive molecules for a large variety of different compounds and ligands.
  • each integrin entity chemically is a heterodimeric glycoprotein; and is structurally composed of two different non-covalently linked protein subunits, each of the individual subunit moieties being chosen from among the alternative members forming a discrete 130-210 kilodalton "alpha" ( ⁇ ) subunit group and the individual members forming another distinct 95-130 kilodalton "beta” ( ⁇ ) subunit group.
  • the overall structure of an integrin receptor molecule generally is illustrated by Fig.
  • the alpha and beta subunits are joined in a non- covalent linkage to form a unitary whole - e , the heterodimer.
  • Each subunit has a transmembrane segment (shown in Fig. A as a dark area); a small C- terminal cytoplasmic domain (shown in Fig. A as a stippled area); and a large N-terminal extracellular domain.
  • the beta ( ⁇ ) subunits as a group typically contain sequences of extensive intrachain disulphide bonding, including four repeated regions of a forty amino acid cysteine-rich segment (shown in Fig. A as a crosshatched area).
  • alpha ( ⁇ ) subunit members of the group are cleaved posttranslationally to provide a heavy chain and a light chain linked by internal disulphide bonding to form the complete subunit entity.
  • integrin molecular structure see Hynes, R.O., Cell 48: 549-554 (1987) and the references cited therein; Hynes, R.O., Cell 69: 11-25 (1992); Ruoslahti et al.. Kidney Intematl. 45: S17-S22 (1994); and INTEGRINS: Molecular and Biological Responses to the Extracellular Matrix, (Cheresh & Mecham, editors), Academic Press, 1994.
  • each alpha subunit group and each beta subunit group has its own distinctive members, each of which can become non-covalently linked to more than one member of the corresponding subunit type.
  • the alpha subunit group comprises not less than fourteen (14) different entities; while the beta subunit group comprises not less than eight (8) different members.
  • Fig. B A representative listing and correlation of the presently recognized possible combinations and permutations of individual ⁇ and ⁇ subunits is shown by Fig. B. [reproduced in part from INTEGRINS: Molecular And Biological Responses to the Extracellular Matrix, (Cheresh & Mecham, editors), Academic Press, 1994, (preface page xii)].
  • the recognized biological role and in-vivo function of the integrin protein family are as cell surface receptors for cell-to-cell or cell-to-matrix interactions.
  • Many of the individual integrin heterodimers comprising the family as a whole were first identified by their ability to bind with one specific ligand or matrix glycoprotein extracellularly. In this manner, the individual integrin heterodimers (each comprised of different ⁇ and ⁇ subunits) have demonstrated a variety of unique and alternative specific binding affinities and capacities for a diverse range of singular extracellular ligands in-vivo.
  • the conventionally known range of such extracellular ligands presently includes: laminin, collagen, fibronectin, vitronectin, epiligin, entactin, merosin, kalinin, invasin, tenascin, osteopontin, thrombospondin, adenovirus penton base, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1 ), and von Willebrand factor.
  • IAM-1 intercellular adhesion molecule-1
  • VCAM-1 vascular cell adhesion molecule-1
  • von Willebrand factor A representative listing of the individual ⁇ and ⁇ subunits composing the integrin unit with the corresponding specific bind affinity ligand is presented by Fig. C [also reproduced in part from INTEGRINS: Molecular and Biological Responses to the Extracellular Matrix, (Cheresh and Mecham, editors), Academic Press, 1994, (preface page xii)].
  • integrin unit in 1987 was also alternatively titled "platelet membrane glycoprotein la-lla complex” or GPIa/lla; and as “human very late activation protein 2 complex” or VLA-2; and also as “fibroblast extracellular matrix receptor II", a misnomer of its true binding affinity (as shown by Fig. C).
  • T lymphocyte help lymphocyte cytotoxicity
  • ligands are- FN. fibronectin. LM, laminin, VN, vitronectin; FB. fibnnogen; VWF. von Willebrand factor; TSP. thrombospondin, C3b ⁇ , inactivated form of C3b component of complement
  • LM laminin, VN, vitronectin
  • FB fibnnogen
  • VWF von Willebrand factor
  • TSP thrombospondin
  • C3b ⁇ inactivated form of C3b component of complement
  • the integrin protein family as a whole being cell surface receptors for specific extracellular matrix ligands, has been implicated in the processes of extracellular matrix remodeling, in endothelial cell migration, and in the function maturation of new endothelial cells into mature blood vessels - the complex process of angiogenesis generally. See for example, Hynes, R.O., Cell 69: 11-25 (1992); Ruoslahti et al., Kidney Intematl. 45: S17S22 (1994); and Schwartz et al.. Ann. Rev. Cell Dev. Biol. H: 549599 (1995).
  • a first aspect of the invention provides a method for inhibiting tumor angiogenesis mediated by vascular endothelial growth factor (VEGF) and integrin cell surface receptors expressed in the vasculature of a living subject, said method comprising the steps of: allowing mobile VEGF secreted by a tumor mass present within the body of a living subject to become bound in-vivo to the surface of endothelial cells in a tumor-associated blood vessel; permitting said bound VEGF to induce the expression of specified integrin heterodimers on the endothelial cell surface of the tumor-associated blood vessel in-vivo, said induced and expressed integrin heterodimers being selected from the group consisting of integrins composed of a- and O _ integrin subunits; and then administering at least one antagonistic antibody preparation effective against said induced and expressed specified integrin heterodimers on the endothelial cell surface to the living subject such that tumor angiogenesis is inhibited in-vivo, said antagonistic preparation comprising at least
  • a second aspect of the invention provides an alternative method for inhibiting tumor angiogenesis mediated by vascular endothelial growth factor (VEGF) and integrin cell surface receptors expressed in the vasculature of a living subject, said alternative method comprising the steps of: allowing mobile VEGF secreted by a tumor mass present within the body of a living subject to become bound in-vivo to the surface of endothelial cells in a tumor-included blood vessel; permitting said bound VEGF to induce the expression of specified integrin heterodimers on the endothelial cell surface of the tumor-included blood vessel in-vivo, said induced and expressed integrin heterodimers being selected from the group consisting of integrins composed of ⁇ i, and ⁇ 2 integrin subunits; and then administering at least one antagonistic antibody preparation effective against said induced and expressed specified integrin heterodimers on the endothelial cell surface to the living subject such that tumor angiogenesis is inhibited in-vivo, said antagonist
  • Fig. A is an illustration of the general structure of an integrin heterodimer functional as a cell surface receptor in-vivo
  • Fig. B is an illustrative correlation showing the presently known possible combinations and permutations between alpha and beta integrin subunits
  • Fig. C is an illustrative correlation of the different extracellular matrix ligands able to be bound in-vivo by different combinations of alpha and beta integrin subunits;
  • Fig. 1 is a statement of the amino acid sequence for the four major variant forms of VEGF
  • Fig. 2 is a statement of the amino acid sequence for the ⁇ -i integrin subunit
  • Fig. 3 is a statement of the amino acid sequences for the ⁇ 2 integrin subunit
  • Figs. 4A and 4B illustrate the qualitative results and densitometric qualities of northern analyses of integrin subunit mRNAs in human dermal endothelial cells stimulated with VEGF for up to 24 hours;
  • Fig. 5 illustrates integrin expression at the surface of dermal microvascular endothelial cells following stimulation with VEGF for 72 and 96 hours;
  • Figs. 6A-6E illustrate the results of ligand-cell cell attachment assays performed with different ligands, dermal microvascular endothelial cells, and specified integrin-blocking monoclonal antibodies;
  • Figs. 7A-7D illustrate the spreading of dermal microvascular endothelial cells on type I collagen gels
  • Figs. 8A-8D illustrate the inhibition of VEGF-driven angiogenesis in- vivo by a combination of monoclonal antibodies specific for ⁇ i and 0.2 integrin subunits.
  • VEGF vascular endothelial growth factor
  • the present invention is a method for inhibiting tumor angiogenesis mediated by vascular endothelial growth factor (VEGF) and specified integrin cell surface receptors induced and expressed in the vasculature of a living subject.
  • VEGF vascular endothelial growth factor
  • this unique inhibition methodology provides both the means and the manipulations for inhibiting new capillary and blood vessel formation effectively and reliably; and also provides multiple advantages and unforeseen benefits both to the physician/clinician as well as for the patient afflicted with a solid tumor mass in-vivo.
  • Some of the uncontemplated advantages and unforeseen benefits include the following:
  • the methodology recognizes for the first time that tumor angiogenesis, mediated in-vivo by vascular endothelial growth factor (VEGF), induces the expression of specified integrin heterodimers, namely ⁇ -
  • VEGF vascular endothelial growth factor
  • the method for inhibiting tumor angiogenesis in-vivo is thus based on this unforeseen recognition of this specific inter-relationship and on a dependence upon VEGF previously secreted by the tumor mass and its in-vivo effect as an inducing agent in order to induce the expression of these specific integrin heterodimers as a requisite forerunner of tumor angiogenesis.
  • the present methodology relies upon and utilizes the novel inducement and expression of specific integrin heterodimers comprised of either the ⁇ -j or ⁇ 2 subunits as the basis and the mechanism of action for inhibiting tumor angiogenesis.
  • the present invention is thus unique in its focus and in its dependence upon the new expression of integrin cell surface receptors constituted of either ⁇ -
  • the present methodology is able to inhibit new capillary and new blood vessel formation both within the tumor mass itself as well as in the immediately adjacent blood vasculature surrounding the perimeter of the tumor mass itself.
  • the method for inhibiting new blood vessel formation is effective for tumor-included blood vessels - those blood vessels and capillaries found within the perimeter edge and substance of the solid tumor mass; and also for tumor-associated blood vessels - those blood vessels lying outside the tumor but within about 0.5 millimeters distance of the tumor mass itself.
  • the present methodology is thus effective and functional in inhibiting both tumor-included and tumor-associated angiogenesis.
  • the present invention has been demonstrated to be effective in inhibiting tumor angiogenesis under in-vivo test conditions. As the experiments and empirical data presented hereinafter clearly evidence and show, the present methodology is effective in inhibiting new blood vessel formation in and around the tumor mass in a reproducible, reliable, and clinically verifiable manner. The present invention is thus deemed to be suitable as a therapeutic technique and clinical tool by which to treat human and animal subjects afflicted with a solid tumor mass in their bodies.
  • tumor angiogenesis is the specific development in-vivo of an adequate blood supply for a solid tumor mass. Since the growth of a solid tumor mass in-vivo is believed to be dependent upon the existence, maintenance, and continuing development of a sufficient and functional blood supply and vasculature in-situ, the present invention's goal and objective is to inhibit and prevent the development of the blood supply required by a pre-existing tumor to survive and continue growth. Accordingly, it is the purpose of the present inhibitory methodology to prevent tumor angiogenesis.
  • any solid tumor mass lying in any part of the body and in any particular tissue or cell type is suitable as the intended target for inhibition of angiogenesis.
  • a tumor is a neoplasm - an abnormal mass of cells typically exhibiting uncontrolled and progressive growth.
  • Neoplasms are broadly classified into two categories: (1 ) according to the cell type from which they originate; and (2) according to their biologic behavior - whether they are benign or malignant.
  • neoplasm is a solid mass of abnormal cells in which there is a distinct or discrete tumor matrix, stroma, and included and/or associated blood vasculature, that neoplasm is a proper and suitable target for inhibition of tumor angiogenesis using the present methodology.
  • the tumor may be a "benign" neoplasm - that is, mild, favorable, or kindly (the opposite of malignant).
  • Benign neoplasms are usually well circumscribed and are often encapsulated; and, by definition, do not invade locally and do not metastasize.
  • a "malignant" tumor is a neoplasm having the tendency to become clinically progressively worse and to result in the death of the subject.
  • malignant denotes the properties of tumor invasiveness and metastasis.
  • the term "metastasis” is defined as the process by which malignant cells are disseminated from the tumor of origin (the primary tumor) to form a new growth (the secondary tumor) at a distant site; it is the discontinuous extension of a malignant neoplasm.
  • the primary tumor the tumor of origin
  • the secondary tumor the new growth
  • it is a primary purpose and goal of the present invention to inhibit tumor angiogenesis both in benign and in malignant tumors generally wherever they may be found as a discrete tumor mass.
  • the present inhibitory methodology is directed to solid tumors found clinically within the living patient in-situ; and the entire broad class of human and animal solid mass tumors is deemed suitable for such therapeutic treatment wherever the tumor may be found within the body. Equally important, and especially for purposes of malignant tumors and neoplasms, the present inhibitory methodology is suitable for use with the tumor regardless of what kind, type, grade, age, size, stage, or cell origin may apply to the tumor in question. Thus, all types of primary and metastatic solid tumors can be treated in-vivo.
  • Representative examples are breast cancer, endometrial cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, glioblastoma of the brain, malignant melanoma, Kaposi's sarcoma, and squamous cell carcinoma of the skin.
  • the present method for inhibiting tumor angiogenesis is deemed to be a broadly applicable and clinically valuable therapeutic treatment.
  • VEGF vascular endothelial growth factor
  • the newly induced and expressed integrin proteins now serving as cell surface receptors on the endothelial cells of tumor-included and tumor-associated blood vessels are unexpectedly integrin heterodimers comprising the ⁇ -
  • and the ⁇ *2 subunits in this context has never before been appreciated or utilized for the purpose of inhibiting tumor angiogenesis.
  • the present invention not only identifies these events as working principles by which tumor angiogenesis proceeds in-situ; but also utilizes these singular findings as the basis of manipulations by which to control and inhibit tumor angiogenesis.
  • the present invention thus recognizes and utilizes in a unique way the fact that VEGF and induced expression of ⁇ -
  • the invention recognizes also for the first time that both VEGF and integrin heterodimers comprising the ⁇ -
  • the present invention employs the fact that the newly induced and expressed ⁇ -j ⁇ and ⁇ 2 ⁇ integrin heterodimers present at the surface of endothelial cells of tumor- included and tumor-associated blood vessels can be antagonized using particular antagonistic agents in order to neutralize, block, and deny the functional value of these newly expressed integrin heterodimers as collagen and laminin-1 receptors specifically.
  • the Manipulative Steps Comprising The Present Methodology is a methodology which comprises three manipulative steps. Each of the steps comprising the inhibitory treatment reflects and recognizes the underlying principles by which tumor angiogenesis is now understood to proceed; and utilizes these principles; and controls as well as manipulates the progression of events in order to achieve an inhibition of tumor angiogenesis in an effective and reliable manner. Each of the essential manipulative steps will be described individually hereinafter.
  • Step 1 Allowing Endogenous Mobile VEGF to Become Bound In-Vivo.
  • the endogenous VEGF which becomes bound in- vivo to the surface receptors of endothelial cells of tumor-included and tumor- associated blood vessels is that tumor-secreted and initially mobile VEGF which subsequently concentrates and binds selectively to the endothelium of tumor-included and/or tumor-associated blood vessels in a far greater degree than is found in normal blood vasculature and normal organs and tissues.
  • tumor-included blood vasculature are those blood vessels lying within the tumor stroma and are included within the matrix substance of the solid tumor mass.
  • tumor-associated blood vessels are those blood vessels lying immediately adjacent to and within about 0.5 millimeters from the solid tumor mass and its microvasculature. Tumor-associated blood vessels include both pre-existing and those newly induced by angiogenesis. Both types provide endothelial cells ("EC") which bear surface receptors for VEGF such as Flt-1 and KDR as well as heparin-containing proteoglycans on the cell surface.
  • EC endothelial cells
  • VEGF is predominantly synthesized by tumor cells and, generally to a lesser degree, by tumor-associated stromal cells.
  • the VEGF bound in- vivo on the endothelium cell surface is primarily the result and consequence of previously mobile VEGF that had been synthesized and secreted by the nearby tumor cells.
  • VEGF vascular endothelial growth factor
  • the entirety of the VEGF which is the inducing agent of the present methodology is and must be solely that VEGF which becomes bound in-vivo to the endothelial cells of at least one tumor-included or tumor-associated blood vessel.
  • VEGF vascular endothelial growth factor
  • circulating and unbound VEGF is uninvolved and is unrelated to the means of action, the utility, and the purposes of the present invention. It is, therefore, an essential requirement of the present invention that the VEGF in question become bound in-vivo in each and every instance to the surface of the endothelium in a blood vessel lying either within or immediately adjacent to the solid tumor mass itself.
  • mobile VEGF in fact can be prevented from becoming bound to the surface of endothelial cells in tumor- included and tumor-associated blood vessels in-vivo; and bound VEGF can also be prevented from serving as an inducing agent.
  • the prevention and neutralization of effects in-vivo for mobile VEGF are described by U.S. Patent Nos. 4,456,550 and 5,036,003.
  • the in-vivo targeting of bound VEGF function is described by Strawn et al.. Cancer Res. 56: 3540-3545 (1996).
  • the present invention does not interrupt and does not prevent the singular cellular consequences stemming from VEGF becoming bound to the surface of endothelial cells in-vivo.
  • the endogenous VEGF be allowed to bind to the endothelial cells of tumor-included and/or tumor-associated blood vessels; and that such bound VEGF be allowed to act in-situ as an inducing agent in order that new integrin heterodimers be synthesized and expressed at the cell surface of the endothelial cells.
  • This requirement satisfies and is in accordance with the first and second underlying principles as described previously herein.
  • endogenous VEGF is a dimeric protein which is produced in-vivo in at least four major variant forms as a result of alternative splicing of mRNA [Houck et al., Mol. Endocrinol. 5: 1806-1814 (1991 ); Keck et al., Science 246: 1309-1312 (1989); Leung et al., Science 246: 1306-1309 (1989); Tischer et al., Biochem. Biophvs. Res. Commun. 165: 1198-1206 (1989)].
  • the variants of human VEGF include monomer, single strands of VEGF which are respectively 121 , 165, 189, and 206 amino acid residues in length.
  • Fig. 1 The precise amino acid sequencing in the primary structure for the four molecular species of VEGF is shown by Fig. 1 (reproduced from Ferrara et al., Endocrine Reviews 13: 18 (1992)] wherein the identity of each individual amino acid residue in sequence is given by the single-letter code system, as conventionally known and employed routinely in this field.
  • the different amino acid segments include omissions in some instances, particularly in the center area of the molecular structure, thereby causing the shorter length strands.
  • the secreted and released variants of VEGF are generally two of the four: the 121 length variant is secreted and soluble; the 165 length variant is soluble and is the prevalent form which is released.
  • the 189 length variant and the 206 length variant are forms also synthesized and secreted by the tumor cell but are mostly retained by the extracellular matrix of the cell.
  • Step 2 Allowing The Expression Of Integrin Heterodimers Comprising ⁇ -j and 012 Subunits It is a requisite of the present methodology that the bound VEGF be permitted to induce the expression of specified integrin heterodimers on the endothelial cell surface of the tumor-included or tumor-associated blood vessel in-vivo. It is also required that the newly induced and expressed integrin heterodimers serving as cell surface receptors be comprised of ⁇ -j and/or 0:2 subunits.
  • this step incorporates the underlying second and third principles as described previously herein; and also specifies that the newly induced and expressed integrin heterodimers be composed of either ⁇ - j and/or 0:2 subunits as a requisite result and consequence. It will be appreciated also that the existence of inducable ⁇
  • Fig. 2 The composition and amino acid sequence of the human ⁇
  • Fig. 3 the specific amino acid composition and sequence of the human o_2 integrin subunit is shown by Fig. 3 herein, which has been reproduced in part from Takada, Y. and M.E. Hemler, J. Cell Biol. 109: 397- 407 (1989).
  • Fig. 3 the specific amino acid composition and sequence of the human o_2 integrin subunit is shown by Fig. 3 herein, which has been reproduced in part from Takada, Y. and M.E. Hemler, J. Cell Biol. 109: 397- 407 (1989).
  • the reader is directed to both of these scientific publications, each of which is expressly incorporated by reference herein.
  • the typical integrin heterodimer induced by VEGF at the endothelial cell surface is the ⁇ - ⁇ ⁇ -
  • both of these expressed integrin heterodimers are specific receptors for collagens or laminin-1 alone.
  • These ⁇ i ⁇ i and ⁇ 2 ⁇ integrin heterodimers are markedly different in their ligand binding specificities and affinities from all other ⁇ subunit and ⁇ subunit combinations.
  • the ⁇ -] and 0.2 integrin subunits do not have a recognition capability for peptides or other kinds of substances carrying the RGD recognition sequence - a trait which is typical of other integrin proteins.
  • subunit and the o_2 subunit are unique and unusual even among the alpha subunit family.
  • Step 3 Administering At Least One Antagonistic Antibody Preparation against The Induced And Expressed ⁇ i And 012 Integrin Subunits
  • the third and final manipulation is the administration to the subject of at least one antagonistic preparation effective against the newly induced and expressed specified integrin heterodimers on the endothelial cell surface such that tumor angiogenesis is inhibited in-vivo.
  • the preferred agent is a function-blocking antibody preparation comprised of monoclonal and/or polyclonal antibodies which are specific for epitopes on either or both of the ⁇ and ⁇ 2 integrin subunits.
  • the function-blocking antibody antagonist is a function-blocking antibody preparation comprised of monoclonal and/or polyclonal antibodies which are specific for epitopes on either or both of the ⁇ and ⁇ 2 integrin subunits.
  • the preferred function-blocking antibody antagonist will demonstrate two characteristics: It will have the capability of binding specifically to one or more epitopes present within a spatially exposed region of the ⁇
  • the other essential characteristic of the specific function-blocking antibody is - that upon binding to the particular alpha integrin subunit, ( ⁇ -
  • the antigenic determinants recognized by the function-blocking antibodies are provided by the amino acid residues comprising the ⁇ i or o_2 integrin subunits as shown by Figs. 2 and 3 respectively herein.
  • this specific binding capability can be demonstrated not only by a whole intact antibody, but also by F(ab') 2 fragments as well as by Fab fragments derived from the whole antibody structure.
  • the F(ab') 2 fragment represents a divalent binding fragment of the whole antibody; while the Fab binding portion is a univalent binding unit having a minimum of antibody structure.
  • the user has the option to chose whether the function-blocking antibody antagonist(s) is obtained from monoclonal, or polyclonal or broad antisera sources. Equally important, the user will decide whether the antibody or antibody fragments should be isolated and purified prior to use; whether they should be altered into humanized antibody form; or whether the antibody antagonist can be employed as a heterogeneous mixture of different entities and varying binding affinities, only some of which will have the requisite affinity and specific binding capability for an exposed epitope on the ⁇ -] or 0.2 integrin subunit expressed in-situ.
  • and 012 integrins is left to the discretion and needs of the user.
  • and/or 012 integrin subunits or different fragments thereof theoretically can serve as immunogens insofar as antibodies obtained with such immunogens will be evaluated and selected for their specific binding and function-blocking properties. It will be noted and appreciated also that the range and variety of the intended sites for epitope binding within the induced and expressed ⁇ -
  • peptide lengths of at least 10-20 residues are generally preferred.
  • or ( 2 integrin structure (shown by Figs. 2 and 3) available for use as a source of antigenic determinants each provide far longer amino acid residue segments for this purpose.
  • an extended segment length of amino acid residues were purposely employed as the immunogen, a larger number of different antigenic determinants becomes available, given the range of residue choices. Accordingly, the number of potential epitopes becomes enormous; yet each of these epitopes is a potential specific binding site for the antibody antagonist(s).
  • peptide immunogens it is intended and envisioned that at least one peptide segment of suitable length (preferably at least 10-20 residues) be chosen as the immunogen in order to provide the antigenic determinants and the production of specific antibodies using a living host animal.
  • the chosen antigenic or haptene segment must be prepared.
  • the desired amino acid segment can be synthetically prepared using conventionally known solid phase peptide synthesis methods [such as Merrifield, RB, J. Am- Chem. Soc. 85: 2149 (1963)].
  • the chosen segment be purified (such as by gel filtration) and desirably analyzed for content and purity (such as by sequence analysis and/or mass spectroscopy).
  • the chosen peptide segment is typically coupled to a protein carrier to form the immunogen.
  • a protein carrier Conventionally suitable protein carriers available for this purpose are available in great variety from many diverse sources. The only requirements regarding the characteristics and properties of the carrier are: first, that the protein carrier be in fact antigenic alone or in combination with the synthesized chosen amino acid residue sequence; and second, that the carrier protein be able to present the antigenic determinants of the residue sequence such that antibodies specific against the amino acid residues are produced in a living host animal.
  • the preferred choice of protein carrier for immunization purposes include keyhold limpet hemocyanin (KLH), coupled by glutaraldehyde (GLDH), sulfo-m-maleimidobenzo
  • MBS M-hydroxysuccinimide ester
  • BDB bisdiazobenzidine
  • any other carrier protein compatible with the host to be immunized is also suitable for use.
  • carrier proteins include bovine serum albumin, thyroglobulin, and the like.
  • the polyclonal antisera and/or monoclonal antibodies and/or genetically engineered antibodies should be evaluated and verified for their ability to bind specifically with an epitope existing within a spatially exposed region the on, or ⁇ .2 integrin subunits and for the capability to functionally block the abilities of the ⁇ -
  • cleavage with papain will produce two Fab fragments plus the Fc fragment
  • cleavage of the antibodies with pepsin produces the divalent F(ab') 2 fragment and the Fc' fragment - all as conventionally known It will be expressly understood, however, that regardless of whether the antibody binding portion represents polyclonal antisera, monoclonal antibodies, the F(ab') 2 fragment, Fab fragments, humanized antibodies, or other antibody species - all of these are suitable and intended for use so long as the specific function blocking capability is demonstrated after binding to at least one epitop
  • the Ha31/8 antibody reacts with the 180-kDa integrin - ⁇ chain (CD49a), which is a transmembrane glycoprotein that non-covalently associates with the integrin ⁇ 1 (CD49a/CD29 or VLa-1 ) complex VLA-1 is expressed on activated T cells, smooth muscle cells, and endothelial cells, and it is a receptor for collagen and laminin 1
  • CD49a 180-kDa integrin - ⁇ chain
  • VLa-1 transmembrane glycoprotein that non-covalently associates with the integrin ⁇ 1
  • VLA-1 is expressed on activated T cells, smooth muscle cells, and endothelial cells, and it is a receptor for collagen and laminin 1
  • the immunogen for the Ha31/8 clone was emulsified rat glomeruli, and the monoclonal antibody is specific for both rat and mouse CD49a 2,3 it has been reported that Ha31/8 antibody can block VLA-1 -mediated binding to collagen 3
  • the HMo_2 antibody recognizes integrin 0.2 chain (CD49b), the 150-kDa transmembrane glycoprotein that non-covalently associates with the integrin ⁇ -l subunit (Cd29) to form the integrin ⁇ 2 ⁇ complex known as VLA-2, which is a receptor for collagen and laminin 1 VLA-2 is expressed on some splenic CD4+T lymphocytes, ' '2 on intestinal intraepithelial and lamina prop ⁇ a lymphocytes 3 NK cells, 2 and platelets, 2 but it is not on thymocytes 1 nor Peyers patch, peripheral lymph nodes and mesentenc lymph nodes lymphocytes 3
  • the expression of VLA-2 is upregulated on lymphocytes in response to motigens 1
  • the HMc? ? antibody has been reported to partially block the interaction of T-cell blasts with collagen 1 >4
  • Antibody Class lgG 2a - purified by protein A affinity chromatography.
  • Immunogen Purified human Integrin ⁇ 1.
  • Source From hybridoma produced by fusing SP2/0 mouse myeloma cells with immunized Balb/c splenocytes, and propagated as mouse ascites.
  • Formulation Frozen liquid.
  • Quantity 200 ⁇ g/vial in 116 ⁇ l O.IM Tris-glycine, pH 7.4, containing 0.05% sodium azide.
  • compositions embodying the specifically binding and functionally- blocking antagonistic antibody for the present invention can be administered in any manner which preserves the function of the antibody and delivers it to the tumor site - such as intravenous, subcutaneous or other parenteral administration
  • the prepared antagonistic antibody can be introduced by any means or routing that inhibits tumor angiogenesis as described
  • the dosage to be administered to any patient will vary and be dependent upon the age, overall health, and weight of the human or animal recipient, the kind of concurrent treatment, if any, the frequency of concurrent treatment, and the physician's prognosis for the patient Generally, a range doses of antagonistic antibody from 0 1 milligrams to about 10 0 milligrams per kilogram of body weight, in twice weekly or three times weekly administrations is expected to be effective to yield the desired therapeutic result
  • the duration of antagonistic antibody dose administration is expected to be continued so long as a favorable clinical result is obtained It is believed that this treatment regimen will inhibit tumor angiogenesis in-vivo, and, in this manner, act to retard or halt the growth of the solid tumor in-situ However, it is as yet unclear whether or not this inhibitory treatment method will provide for complete regression of tumor For this reason especially, the treatment duration and dosage should be monitored accordingly
  • the antagonistic antibody preparation is typically to be given intravenously, subcutaneously, or other parenteral applications, the appropriate quantity of antibody will be prepared in sterile form exist in single or multiple dose formats, and typically be dispersed in a fluid carrier such as sterile physiological saline or 5% dextrose solutions commonly used with injectabies
  • EC Human dermal microvascular endothelial cells
  • RNA from EC was isolated, subjected to elect, ophoresis, and transferred to nylon membranes as previously described [Senger et al , Am J Path 149 293-305 (1996)1 32 P-labeled cDNA probes were prepared as described therein using purified cDNA inserts isolated from the following human c_2 integrin plasmid (clone 2 72F) and human c.3 integrin plasmid (clone 3 10) from the American Type Culture Collection (Rockville, MD), human ⁇ 1 integrin plasmid (clone 3RA), generously provided by Dr Eugene Marcantonio (Columbia U , New York, NY), and a plasmid containing a 2 5 kb human ⁇ -
  • Immunoprecipitates were subjected to electrophoresis; transferred to PVDF membrane; visualized with chemiluminescence; and protein bands were quantitated as described above.
  • Biotinylated protein standards purchased from Bio-Rad included myosin (Mr 200,000), ⁇ -galactosidase (Mr 116,000) and phosphorylase B (Mr 97,400).
  • 96 well plates (Corning Costar Corp., Cambridge, MA) were coated with matrix proteins at a concentration of 10 ⁇ g/ml for 1 hr followed by a coating of 100 mg/ml bovine serum albumin (Cat. #A9306, Sigma Chemical Co., St. Louis, MO) for 2 h to block the remaining protein binding sites.
  • the coating of matrix proteins included human placental collagen I and mouse EHS laminin-1 (Life Technologies, Grand Island, NY) and human placenta! collagens IV and V (Collaborative Biomedical, Bedford, MA). Cultured cells were prelabeled with fluorescent Cell Tracker Dye (Molecular Probes, Eugene, OR) at a concentration of 3 ⁇ M for 30 min.
  • mice IgG and mouse monoclonal blocking antibody specific for the human ⁇ integrin subunit were purified from control serum and P4C10 ascites (Life Technologies), respectively, using the MAPS II antibody purification kit (Bio-Rad).
  • Purified mouse monoclonal blocking antibodies specific for the human ⁇ -j integrin subunit (clone 5E8D9) and specific for the or ? integrin subunit (clone A2-IIE10) were purchased from Upstate Biotechnology (Lake Placid, NY)
  • Vitrogen bovine dermal collagen I, Collagen Corp , Palo Alto, CA
  • diluted 500 ⁇ g/ml with serum-free medium
  • serum-free medium 500 ⁇ g/ml
  • 1 2 x 10 ⁇ cells were added to each well containing antibodies (see above)
  • the assay employed was essentially as described previously by Passaniti et al [Lab Invest 67 519-528 (1992)] with the following modifications
  • Athymic NCr nude mice (7-8 weeks old, females) were injected subcutaneously midway on the right and left back sides with 0 25 ml Mat ⁇ gel (Collaborative Biomedical, Bedford, MA) at a final concentration of 10 mg/ml together with 2 5 x 106 VEGF-transfected SK-MEL-2 cells [Claffey et al , Cancer Res 56 172-181 (1996)] Soon after injection, the Matngel implant solidified and persisted without apparent deterioration throughout the six day assay interval
  • the animals were individually treated with one of the following purified, low endotoxin ( ⁇ 0 01 ng/ ⁇ g protein), hamster monoclonal antibodies ("MAbs", Pharmmgen, San Diego, CA) ⁇ i -blocking MAb (clone Ha31/8), ⁇ 2-block ⁇ ng MAb
  • Fig 4A shows the results of Northern analysis of integrin subunit mRNAs in human dermal microvascular EC stimulated with VEGF (20 ng/ml) for up to 24 h Ten micrograms of local cellular RNA was loaded in each well
  • Fig 4B shows the densitomet ⁇ c quantitation of the Northern analyses The signal associated with each integrin mRNA was normalized to the internal ⁇ -actm mRNA standard to adjust for minor differences in RNA loading
  • VEGF stimulation resulted in a > 6-fold induction of ⁇
  • VEGF-stimulated cells showed no induction of c.3 mRNA or ⁇ «
  • 0.5 mRNA was not induced by VEGF stimulation (data not shown)
  • Fig 5 shows integrin expression at the surface of dermal microvascular EC following stimulation with VEGF (20 ng/ml) for 72 h and 96 h Lysates from biotinylated cells were subjected to immunoprecipitation, and the immunoprecipitates were then subjected to electrophoresis in 7 5% polyacrylamide gels under
  • Figs 6A-6E show the results of ligand to cell attachment assays performed with dermal microvascular EC and integnn-blocking MAbs
  • Cultured cells were stimulated with VEGF (20 ng/ml, 72 h) prior to assay for maximal induction and expression of ⁇ -
  • Substrata were coated with matrix proteins, followed by a coating of BSA to block the remaining protein binding sites
  • Cells were allowed to attach for 45 minutes time in serum-free medium, control IgG and specific MAbs were employed at a concentration of 10 ⁇ g/ml
  • VEGF prestimulation promoted EC spreading on polymeric collagen as compared to unstimulated EC Similar results were obtained with EC embedded in type I collagen (data not shown)
  • ⁇ - j -blocking MAb in combination with 0.2-block ⁇ ng MAb completely inhibited spreading of the VEGF-stimulated cells Individually, the ⁇ -
  • mice angiogenesis model was employed together with specific hamster monoclonal MAbs which specifically block only the murine ⁇ -
  • the mouse angiogenesis model which is a modified version of one described previously in the scientific literature [Passaniti et al , Lab Invest 67 519-528 (1992)] involves subcutaneous injection of athymic nude mice with Mat ⁇ gel containing human SK-MEL-2 tumor cells stably transfected for expression of murine VEGF164 Untransfected SK-MEL-2 tumor cells are known to not provoke an angiogenic response, and therefore the angiogenic stimulus provided by the VEGF transfectants is entirely or predominantly attributable to VEGF Furthermore, the hamster monoclonal MAbs specific for murine ⁇ -
  • each animal received implants by subcutaneous injection, midway on the right and left back sides on day zero Isotype-matched control Ab (300 ⁇ g) or a combination of ⁇ -j MAb and 0.2 MAb (150 ⁇ g each) were administered to the individual mouse by mtrapentoneal injection on days 1 , 3, and 5, and 5 animals were employed in each group under test On day 6, all animals were sacrificed and dissected, the excised implants were photographed, and the excised tissues were fixed for histoiogical analyses which included immu ⁇ ostaining for the EC marker CD31 (PECAM-1 ) Thus, a total of 20 implants were analyzed, 10 implants were derived from animals treated with control MAb and 10 implants were derived from animals treated with ⁇ -
  • Fig 8 illustrates the inhibition of VEGF-dnven angiogenesis in vivo by a combination of ⁇ -
  • Figs 8A and 8B show the Mat ⁇ gel implants (M) together with overlying skin Note the reduced density of small blood vessels associated with implant from animal treated with ⁇ -
  • the larger pre-existing blood vessels appear unaffected
  • Figs 8C and 8D show the immunohistochemical staining for CD31 (blue color) which reveals that new blood vessels at the interface between the Mat ⁇ gel implant (M) and host dermis (D), and in association with large nerves (N), were markedly reduced in cross-sectional area at sizes > 90% in the - ⁇ MAb + c_2 MAb treated animals, in comparison with controls
  • the evidence of Figs 8A-8D also clearly demonstrate that the overlying skin adjacent to the implants showed substantially reduced numbers of small

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Abstract

La présente invention concerne une méthode d'inhibition de l'angiogénèse tumorale chez un sujet vivant. La méthode se base sur l'angiogénèse tumorale induite par le facteur de croissance endothéliale vasculaire et des récepteurs intégrines de surface cellulaire induits spécifiés et exprimés sur les cellules endothéliales de vaisseaux sanguins inclus dans les tumeurs et associés aux tumeurs. La méthodologie consiste également à administrer au moins une préparation antagoniste efficace contre les hétérodimères de l'intégrine spécifiés induits et exprimés sur la surface de cellules endothéliales des sujets vivants, dont la conséquence a pour résultat une inhibition efficace de l'angiogénèse tumorale in vivo.
PCT/US1997/017485 1997-09-30 1997-09-30 Methode d'inhibition de l'angiogenese tumorale chez un sujet vivant WO1999016465A1 (fr)

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US09/532,310 US6596276B1 (en) 1997-09-30 2000-03-22 Method for inhibiting tumor angiogenesis in a living subject

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6759047B1 (en) 1998-06-17 2004-07-06 Beth Israel Deaconess Hospital Corp. Anti-angiogenic proteins and methods of use thereof
US6962974B1 (en) 1998-06-17 2005-11-08 Beth Israel Deaconess Medical Center Anti-angiogenic proteins and fragments and methods of use thereof
WO2005118639A1 (fr) * 2004-06-02 2005-12-15 Valtion Teknillinen Tutkimuskeskus Procédé d'activation de la tyrosine phosphatase d'une protéine de lymphocyte t et procédés thapeutiques basés sur celui-ci
WO2007024921A3 (fr) * 2005-08-24 2007-06-14 Cell Matrix Therapies combinees visant a inhiber les interactions integrine-matrice extracellulaire
US7387779B2 (en) 1998-06-17 2008-06-17 Beth Israel Deaconess Medical Center Anti-angiogenic proteins and fragments and methods of use thereof
WO2008142198A3 (fr) * 2007-05-23 2009-07-02 Valtion Teknillinen Procédé pour inhiber ou stimuler l'angiogenèse dans un sujet
US7585865B2 (en) 2006-07-21 2009-09-08 The Penn State Research Foundation Protein kinase C zeta inhibition to treat vascular permeability
US7740841B1 (en) 2000-01-28 2010-06-22 Sunnybrook Health Science Center Therapeutic method for reducing angiogenesis
US8093229B2 (en) 2005-03-30 2012-01-10 Conforma Therapeutics Corporation Alkynyl pyrrolo[2,3-d]pyrimidines and related analogs as HSP90-inhibitors
US12037361B2 (en) 2021-08-31 2024-07-16 The Hong Kong Polytechnic University Methods for treating multidrug resistant breast cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BASSON et al., "Human Enterocyte Migration is Modulated In Vitro by Extracellular Matrix Composition and Epidermal Growth Factor", J. CLIN. INVEST., July 1992, Vol. 90, No. 1, pages 15-23. *
VINK et al., "Role of beta-1 Integrin in Organ Specific Adhesion of Melanoma Cells In Vitro", LAB. INVES., February 1993, Vol. 68, No. 2, pages 192-203. *
WARREN et al., "Regulation by Vascular Endothelial Growth Factor of Human Colon Cancer Tumorigenesis in a Mouse Model of Experimental Liver Metastasis", JOURNAL OF CLIN. INVEST., Volume 95, April 1995. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6962974B1 (en) 1998-06-17 2005-11-08 Beth Israel Deaconess Medical Center Anti-angiogenic proteins and fragments and methods of use thereof
US6759047B1 (en) 1998-06-17 2004-07-06 Beth Israel Deaconess Hospital Corp. Anti-angiogenic proteins and methods of use thereof
US7387779B2 (en) 1998-06-17 2008-06-17 Beth Israel Deaconess Medical Center Anti-angiogenic proteins and fragments and methods of use thereof
US7740841B1 (en) 2000-01-28 2010-06-22 Sunnybrook Health Science Center Therapeutic method for reducing angiogenesis
EP2301579A1 (fr) 2000-01-28 2011-03-30 Sunnybrook Health Science Centre Procédé thérapeutique pour la réduction de l'angiogenèse
WO2005118639A1 (fr) * 2004-06-02 2005-12-15 Valtion Teknillinen Tutkimuskeskus Procédé d'activation de la tyrosine phosphatase d'une protéine de lymphocyte t et procédés thapeutiques basés sur celui-ci
US7687464B2 (en) 2004-06-02 2010-03-30 Valtion Teknillinen Tutkimuskeskus Method for activating T cell protein tyrosine phosphatase for therapeutic applications
US8093229B2 (en) 2005-03-30 2012-01-10 Conforma Therapeutics Corporation Alkynyl pyrrolo[2,3-d]pyrimidines and related analogs as HSP90-inhibitors
WO2007024921A3 (fr) * 2005-08-24 2007-06-14 Cell Matrix Therapies combinees visant a inhiber les interactions integrine-matrice extracellulaire
US7585865B2 (en) 2006-07-21 2009-09-08 The Penn State Research Foundation Protein kinase C zeta inhibition to treat vascular permeability
US8211893B2 (en) 2006-07-21 2012-07-03 The Penn State Research Foundation Protein kinase C zeta inhibition to treat diabetic retinopathy
WO2008142198A3 (fr) * 2007-05-23 2009-07-02 Valtion Teknillinen Procédé pour inhiber ou stimuler l'angiogenèse dans un sujet
US12037361B2 (en) 2021-08-31 2024-07-16 The Hong Kong Polytechnic University Methods for treating multidrug resistant breast cancer

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