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EP0672064A1 - Morphogenese induite par l'op-3 - Google Patents

Morphogenese induite par l'op-3

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Publication number
EP0672064A1
EP0672064A1 EP94901244A EP94901244A EP0672064A1 EP 0672064 A1 EP0672064 A1 EP 0672064A1 EP 94901244 A EP94901244 A EP 94901244A EP 94901244 A EP94901244 A EP 94901244A EP 0672064 A1 EP0672064 A1 EP 0672064A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
seq
species
tissue
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94901244A
Other languages
German (de)
English (en)
Inventor
Hermann Oppermann
Engin Ozkaynak
Thangavel Kuberasampath
David C. Rueger
Roy H. L. Pang
Charles M. Cohen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Stryker Corp
Original Assignee
Creative Biomolecules Inc
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Filing date
Publication date
Application filed by Creative Biomolecules Inc filed Critical Creative Biomolecules Inc
Publication of EP0672064A1 publication Critical patent/EP0672064A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K6/00—Preparations for dentistry
    • A61K6/50—Preparations specially adapted for dental root treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2310/00—Prostheses classified in A61F2/28 or A61F2/30 - A61F2/44 being constructed from or coated with a particular material
    • A61F2310/00005—The prosthesis being constructed from a particular material
    • A61F2310/00365—Proteins; Polypeptides; Degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates generally to the field of tissue morphogenesis and more particularly to a novel protein that induces tissue morphogenesis in mammals.
  • Cell differentiation is the central characteristic of morphogenesis which initiates in the embryo, and continues to various degrees throughout the life of an organism in adult tissue repair and regeneration mechanisms.
  • the degree of morphogenesis in adult tissue varies among different tissues and is related, among other things, to the degree of cell turnover in a given tissue.
  • tissues can be divided into three broad categories: (1) tissues with static cell populations such as nerve and skeletal muscle where there is no cell division and most of the cells formed during early development persist throughout adult life; (2) tissues containing conditionally renewing populations such as liver where there is generally little cell division but, in response to an appropriate stimulus, cells can divide to produce daughters of the same differentially defined type; and (3) tissues with permanently renewing populations including blood, testes and stratified squamous epithelia which are characterized by rapid and continuous cell turnover in the adult.
  • the terminally differentiated cells have a relatively short life span and are replaced through proliferation of a distinct subpopulation of cells, known as stem or progenitor cells.
  • TGF transforming growth factor
  • This "family" of proteins sharing substantial amino acid sequence homology within their morphogenically active C-terminal domains, including a conserved six or seven cysteine skeleton, are capable of inducing tissue-specific morphogenesis in a variety of organs and tissues, including bone, cartilage, liver, dentin, periodontal ligament, cementum, nerve tissue and the epithelial mucosa of the gastrointestinal tract.
  • the proteins apparently bind to surface receptors or otherwise contact and interact with progenitor cells, predisposing or stimulating the cells to proliferate and differentiate in a morphogenically permissive environment.
  • the morphogens are capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascularization, connective tissue formation, and nerve ennervation as required by the naturally occurring tissue.
  • proteins useful in tissue morphogenesis are proteins originally identified as bone inductive proteins, such as the OP-1, (also referred to in related applications as “OPl”), OP-2 (also referred to in related applications as “OP2”), and the CBMP2 proteins, as well as amino acid sequence-related proteins such as BMP5, BMP6 and its murine homolog, Vgr-1, DPP and 60A (from Drosophila), Vgl (from Xenopus), and GDF-1 (from mouse) see, for example, U.S. Patent No. 5,011,691 to Oppermann et al., Lee (1991) PNAS 8: 4250-4254, and harton et al. (1991) PNAS 8 ⁇ _: 9214-9218.
  • TGF- ⁇ superfamily members comprise a distinct subfamily of proteins different from other members of the TGF- ⁇ superfamily in that the family of morphogenic proteins are able to induce the full cascade of events that result in tissue morphogenesis, including stimulating cell proliferation and cell differentiation of progenitor cells, and supporting the growth and maintenance of differentiated cells.
  • the morphogenic proteins apparently can act as endocrine, paracrine or autocrine factors.
  • the endogenous morphogens may be synthesized by the cells on which they act, by neighboring cells, or by cells of a distant tissue, the secreted protein being transported to the cells to be acted on.
  • the family of morphogenic proteins induce true tissue morphogenesis, rather than inducing formation of fibrotic (scar) tissue as, for example, TGF- ⁇ does.
  • the morphogens are synthesized in the cell as a precursor molecule approximately three times larger than the mature protein that is processed to yield mature disulfide-linked dimers comprising the C-terminal domain of the precursor sequence.
  • the proteins are inactive when reduced e.g., in monomeric form, but are active as oxidized homodimeric species as well as when oxidized in combination with other morphogens under conditions to produce heterodimers.
  • the proteins useful in tissue morphogenesis typically require a suitable environment enabling cells to migrate, proliferate and differentiate in a tissue- specific manner into, e.g., cartilage-producing chondroblasts, bone-producing osteoblasts, hemopoietic cells, or liver cells, depending on the nature of the local environment.
  • the proliferation and differentiation of cells induced by the morphogenic proteins requires a suitable local environment, including a suitable substratum on which the cells can anchor.
  • the proliferating and differentiating cells also require the presence of appropriate signals to direct their tissue-specificity, such as cell surface markers.
  • the morphogenic properties of OP-3 include the ability to induce proliferation and differentiation of progenitor cells, and the ability to support and maintain the differentiated phenotype through the progression of events that results in the formation of adult tissue.
  • Another object is to provide methods for the expression and isolation of morphogenically active species of OP-3 using recombinant DNA techniques.
  • Yet another object is to provide generic sequences defining useful morphogens. Still another object is to provide tissue- specific acellular matrices that may be used in combination with OP-3, and methods for their preparation. Other objects include utilizing OP-3 in a variety of applications including methods for increasing a progenitor cell population in a mammal; methods for stimulating progenitor cells to differentiate _in vivo or _in vitro and to maintain their differentiated phenotype; methods for inducing tissue- specific growth ij vivo, and methods for the replacement of diseased or damaged tissue in vivo.
  • OP-3 novel substantially pure genetic sequence encoding a novel substantially pure protein referred to herein as "OP-3" now has been discovered.
  • This novel protein is a member of the morphogenic protein family previously described by Applicants (see, for example, US92/01968 ( 092/15323) , and US92/07432 ( O93/05751) .
  • the invention provides methods for utilizing OP-3 to induce the developmental cascade of tissue morphogenesis in a mammal. Specifically, methods are provided for utilizing OP-3 to induce the proliferation of uncommitted progenitor cells, to induce the differentiation of these stimulated progenitor cells in a tissue-specific manner under appropriate environmental conditions, and to support the growth and maintenance of these differentiated cells.
  • the protein also may be used to stimulate the "redifferentiation" of cells that have strayed from their differentiated phenotypes. Accordingly, OP-3 can be utilized to initiate and maintain the developmental cascade of tissue morphogenesis in an appropriate, morphogenically permissive environment.
  • useful OP-3 morphogens include proteins encoded by the DNA sequence provided in Seq. ID No. 1 ("mOP-3") and allelic and species variants thereof, as well as other naturally-occurring and biosynthetic amino acid sequence variants, including chimeric proteins, that are morphogenically active as defined herein.
  • “Morphogenically active fragment” is understood to include all proteins and protein fragments encoded by part or all of the sequence of Seq. ID No. 1 and which have morphogenic activity as defined herein.
  • a morphogen is a dimeric protein comprising a pair of polypeptide chains, wherein each polypeptide chain comprises at least the C-terminal six cysteine skeleton defined by residues 303 to 399 of Seq. ID No. 1 (or residues 335-431 of OPl, Seq. ID no.
  • the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells.
  • the morphogens of this invention comprise a morphogenically active dimeric species comprising a pair of polypeptide chains, wherein at least one of the polypeptide chains comprises the amino acid sequence defined by residues 303 to 399 of Seq. ID No. 1 including allelic, species and other amino acid sequence variants thereof.
  • at least one polypeptide chain comprises the sequence defined by residues 298-399, residues 261-399 or residues 264-399 of Seq. ID No. 1.
  • the amino acid sequence of both polypeptide chains may be defined by part or all of the amino acid sequence of Seq. ID No.
  • the other polypeptide chain preferably comprises at least the sequence defining the C-terminal six cysteine skeleton of any of the other known morphogen family members, including OP-1, OP-2, CBMP2A, CBMP2B, BMP3, BMP5, BMP6, Vgr-1, Vgl, 60A, DPP and GDF- 1, described, for example, in US92/07432 (WO93/05751) , including allelic, species and other amino acid sequence variants thereof, including chimeric variants.
  • Other useful sequences include biosynthetic constructs, such as are described in U.S. Pat. No. 5,011,691.
  • morphogens of this invention comprise morphogenically active proteins encoded by part or all of the genetic sequence listed in Seq. ID No. 1, including allelic, species and other amino acid sequence variants thereof.
  • the invention comprises morphogens encoded by nucleic acids that hybridize to part or all of the pro region of the OP-3 protein, bases 120 to 848 of Seq ID No. 1, under stringent hybridization conditions.
  • stringent hybridization conditions are defined as hybridization in 40% formamide, 5 x SSPE, 5 x Denhardt's Solution, and 0.1% SDS at 37°C overnight, and washing in 0.1 x SSPE, 0.1% SDS at 50°C.
  • morphogenically active fragments of OP-3 are useful in the replacement of diseased or damaged tissue in a mammal, including, but not limited to, damaged lung tissue resulting from emphysema; cirrhotic tissue, including cirrhotic kidney or liver tissue; damaged heart or blood vessel tissue, as may result from cardiomyopathies and/or atherothrombotic or cardioembolic strokes; damaged stomach and other mucosal tissues of the gastrointestinal tract resulting from ulceric perforations and/or their repair; damaged nerve tissue as may result from physical injury, degenerative diseases such as Alzheimer's disease, multiple sclerosis, or strokes; damaged cartilage and bone tissue as may result from metabolic bone diseases and other bone remodeling disorders; damaged dentin, periodontal and/or cementum tissue as may result from disease or mechanical injury; and in the replacement of damaged tissue as a result of inflammation and/or chronic inflammatory disease.
  • morphogenically active fragments of OP-3 are provided to a tissue-specific locus ii vivo, to induce the developmental cascade of tissue morphogenesis at that site.
  • Cells stimulated ex vivo by contact with OP-3 also may be provided to the tissue locus.
  • the existing tissue provides the necessary matrix requirements, providing a suitable substratum or scaffold for the proliferating and differentiating cells in a morphogenically permissive environment, as well as providing the necessary signals for directing the tissue-specificity of the developing tissue.
  • the proteins or stimulated cells also may be combined with a formulated matrix and implanted as a device at a locus jLn vivo.
  • the formulated matrix should be a biocompatible, preferably biodegradable acellular matrix having the characteristics described below. Where the necessary signals for directing the tissue-specificity of the developing tissue are not provided endogenously, the matrix preferably also is tissue-specific.
  • the members of the morphogen protein family also can control the body's cellular and humoral inflammatory response to a foreign object or an initial tissue injury.
  • the loss of tissue function results from the tissue destructive effects and the subsequent formation of scar tissue associated with the body's immune/inflammatory response to an initial or repeated injury to the tissue.
  • the degree of scar tissue formation generally depends on the regenerative properties of the injured tissue, and on the degree and type of tissue damage.
  • morphogenically active fragments of OP-3 may be used to prevent or to substantially inhibit the formation of scar tissue, including alleviating immune response-mediated tissue damage, by providing OP-3 or cells stimulated by exposure to OP-3 protein, to a newly injured tissue locus.
  • the OP-3 protein also may be provided as a prophylactic, provided to a site in anticipation of tissue injury, such as part of a surgical or other clinical procedure likely to produce tissue damage, and to induce an inflammatory/immune response.
  • OP-3 may be used as part of a transplant procedure, to enhance the tissue viability of the organ and/or tissue to be transplanted.
  • the morphogen may be provided to the organ and/or tissue to be transplanted prior to harvest, during its transport, and/or during transplantation into the recipient host as described below.
  • OP-3 also may be used to increase or regenerate a mesenchymal progenitor or stem cell population ij vitro or in a mammal.
  • progenitor cells may be isolated from an individual's bone marrow, stimulated ex vivo with morphogenic OP-3 for a time and at a concentration sufficient to induce the cells to proliferate, and returned to the bone marrow.
  • Other sources of progenitor cells that may be suitable include biocompatible cells obtained from a cultured cell line, stimulated in culture, and subsequently provided to the body.
  • OP-3 may be provided by systemic (e.g., oral or parenteral) administration, or it may be injected or otherwise provided to a progenitor cell population in an individual to induce its mitogenic activity in. vivo.
  • a morphogenically active fragment of OP-3 may be provided to the cells in vivo, e.g., by systemic injection, to induce mitogenic activity.
  • a particular population of hemopoietic stem cells may be increased by exposure to OP-3, for example by perfusing (plasmaphoresing) an individual's blood to extract the cells of interest, stimulating these cells ex vivo, and returning the stimulated cells to the blood.
  • the morphogens of this invention also can inhibit proliferation of epithelial cell populations.
  • the ability to inhibit epithelial cell proliferation may be exploited to reduce tissue damage associated with psoriasis and dermatitis, and other inflammatory skin diseases, as well as ulcerative diseases of the gastrointestinal tract, such as, for example, in the healing of ulcers, including gastric ulcers, and the ulcerations induced in oral mucocitis and inflammatory bowel disease.
  • Morphogens may be used to particular advantage as a cytoprotective agent in clinical therapies likely to effect proliferating epithelial populations, such as cancer radiotherapies and chemotherapies that typically induce oral mucositis, hair loss and/or skin disorders.
  • morphogenic OP-3 may be used to support the growth and maintenance of differentiated cells, inducing existing differentiated cells to continue expressing their phenotype. It is anticipated that this activity will be particularly useful in the treatment of tissue disorders where loss of function is caused by reduced or lost metabolic function in which cells become senescent or quiescent, such as may occur in aging cells and/or may be manifested in osteoporosis and a number of nerve degenerative diseases, including Alzheimer's disease.
  • Application of OP-3 directly to the cells to be treated, or providing it systemically, as by oral or parenteral administration, can stimulate these cells to continue expressing their phenotype, thereby significantly reversing the effects of the dysfunction.
  • a morphogenically active fragment of OP-3 also may be used in gene therapy protocols to stimulate the growth of quiescent cells, thereby potentially enhancing the ability of these cells to incorporate exogenous DNA.
  • a morphogenically active fragment of OP-3 also may be used to induce "redifferentiation" of cells that have strayed from their differentiation pathway, such as can occur during tumorgenesis. It is anticipated that this activity will be particularly useful in treatments to reduce or substantially inhibit the growth of neoplasms. The method also is anticipated to induce the de- and/or re-differentiation of these cells. As described supra, a morphogenically active OP-3 fragment may be provided to the cells directly or systemically, stimulating these cells to revert back to a morphology and phenotype characteristic of untransformed cells.
  • OP-3 may be used to stimulate cell adhesion molecule (CAM) expression levels in a cell.
  • CAMs are molecules defined as carrying out cell-cell interactions necessary for tissue formation. CAMs are believed to play a fundamental regulatory role in tissue development, including tissue boundary formation. embryonic induction and migration, and tissue stabilization and regeneration. Altered CAM levels have been implicated in a number of tissue disorders, including congenital defects, neoplasias, and degenerative diseases.
  • N-CAM expression is associated with normal neuronal cell development and differentiation, including retinal formation, synaptogenesis, and nerve- muscle tissue adhesion. Inhibition of one or more of the N-CAM isoforms is known to prevent proper tissue development. Altered N-CAM expression levels also are associated with neoplasias, including neuroblastomas (see infra), as well as with a number of neuropathies, including normal pressure hydrocephalous and type II schizophrenia.
  • Application of the morphogen directly to the cells to be treated, or providing the morphogen to the mammal systemically, for example, parenterally, or indirectly by oral administration, may be used to induce cellular expression of one or more CAMs, particularly N-CAMs and LI.
  • CAMs also have been postulated as part of a morphoregulatory pathway whose activity is induced by a to date unidentified molecule (See, for example,
  • the matrices utilized in the methods of the invention may be derived from organ-specific tissue, or they may be formulated synthetically.
  • OP-3 or a collection of progenitor cells stimulated by OP-3
  • the existing tissue at that locus, whether diseased or damaged has the capacity of acting as a suitable matrix or scaffold for the differentiation and proliferation of migrating progenitor cells.
  • a formulated matrix may be provided externally together with the stimulated progenitor cells or morphogenically active OP-3 fragment, as may be necessary when the extent of injury sustained by the damaged tissue is large.
  • the matrix should be a biocompatible, suitably modified acellular matrix having dimensions such that it allows the differentiation and proliferation of migratory progenitor cells, and is capable of providing a morphogenically permissive environment.
  • the matrix also preferably allows cellular attachment and is biodegradable. Where the necessary tissue-directing signals can not be provided endogenously, the matrix preferably also is tissue-specific.
  • Formulated matrices may be generated from dehydrated organ-specific tissue prepared, for example, by treating the tissue with solvents to substantially remove the intracelluiar, non-structural components from the tissue.
  • the matrix may be formulated synthetically using a biocompatible, preferably in vivo biodegradable, structural molecule, and may be formulated with suitable tissue-specific cell attachment factors.
  • the molecule may be a naturally occurring one such as collagen, laminin or hyaluronic acid, or a synthetic polymer comprising, for example, polylactic acid, polybutyric acid, polyglycolic acid, and copolymers thereof.
  • preferred structural polymers comprise tissue-specific collagens.
  • preferred cell attachment factors include glycosaminoglycans and proteoglycans.
  • the matrix further may be treated with an agent or agents to increase the number of pores and micropits on its surfaces, so as to enhance the influx, proliferation and differentiation of migratory progenitor cells from the body of the mammal.
  • the invention thus relates to compositions and methods for the use of morphogenically active fragments of OP-3, a novel species variant of the generic family of morphogens disclosed in USSN 667,274 and USSN 752,764, as a tissue morphogen.
  • Morphogenically active OP-3 and protein fragments can be isolated from naturally-occurring sources, or they may be constructed biosynthetically using conventional recombinant DNA technology.
  • Active OP-3 useful in the compositions and methods of this invention may include forms having varying glycosylation patterns, varying N-termini and active truncated forms, e.g., produced by recombinant DNA techniques.
  • Active OP-3 proteins also include chimeric constructs as described below, comprising both an OP-3 active domain and a non-OP-3 sequence as, for example, the pro domain and/or the N-terminal region of the mature protein.
  • OP-3 protein can be expressed from intact or truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions.
  • Useful host cells include procaryotes, including E_ ⁇ coli, and eucaryotic cells, including mammalian cells, such as CHO, COS, melanoma or BSC cells, or the insect/baculovirus system.
  • recombinant DNA techniques may be utilized to produce large quantities of OP-3 capable of inducing tissue-specific cell differentiation and tissue morphogenesis in a variety of mammals, including humans.
  • Figure 1 is a nucleotide sequence comparison of the mouse cDNA sequence of OP-2 and OP-3. Exon boundaries are indicated by bars beneath the sequence; diamonds indicate nucleotide differences within exons 2 and 3; and
  • Figure 2 is an immunoblot comparing mammalian cell expression of an OP1/OP3 chimeric protein construct (lanes 4-8) with that of authentic, recombinant OPl (lane 1) .
  • the invention provides a novel genetic sequence, mOP-3, encoding a novel protein, OP-3, having morphogenic properties.
  • the genetic sequence originally was identified in a mouse cDNA library, and the invention provides methods for identifying and isolating the gene from other species.
  • the methods described herein also may be used to isolate the OP-3 gene from other libraries, including genomic libraries.
  • the invention also provides means for producing the OP-3 genetic sequence and the encoded protein.
  • the invention further provides methods and compositions for inducing the developmental cascade of tissue morphogenesis in a mammal utilizing morphogenically active fragments of OP-3.
  • the methods and compositions provided herein may be utilized in a range of applications, including stimulating the proliferation and/or differentiation of progenitor cells and inducing the repair and regeneration of damaged tissue.
  • the morphogenic OP-3 species of the invention are novel members of the family of morphogens disclosed in US92/01968 (W092/15323) and US92/07432 (WO93/05751) .
  • OP-3 may be isolated from natural sources or constructed biosynthetically utilizing conventional recombinant DNA technology or constructed synthetically using standard chemical techniques.
  • Morphogenically active fragments of OP-3 are useful for initiating and maintaining the tissue-specific developmental cascade in a variety of tissues, including, but not limited to, bone, cartilage, dentin, neural tissue, liver, periodontal ligament, cementum, lung, heart, kidney and numerous tissues of the gastrointestinal tract.
  • OP-3 can induce the proliferation and differentiation of these progenitor cells.
  • tissue-specific signals to direct the differentiation of these cells, and a morphogenically permissive environment, OP-3 is capable of reproducing the cascade of cellular and molecular events that occur during embryonic development to yield functional tissue.
  • the protein can induce the de novo formation of cartilage and endochondral bone, including inducing the proliferation and differentiation of progenitor cells into chondrocytes and osteoblasts, inducing appropriate mineralization and bone remodeling, inducing formation of an appropriate bone tissue vascular supply and inducing formation of differentiated bone marrow (see Example 7 below.)
  • OP-3 proteins useful in the compositions and methods of this invention, including a description of how to make them, and methods and means for their therapeutic administration.
  • numerous, nonlimiting examples which (1) illustrate the suitability of these proteins as tissue morphogens and therapeutic agents, and (2) provide assays with which to test the morphogens encompassed by the invention in different tissues.
  • Example 9 Also provided in Example 9 is a method for screening compounds to identify morphogen stimulating agents capable of stimulating endogenous OP-3 expression and/or secretion.
  • OP-3 stimulating agents then may be used in any of the therapeutic applications described herein in place of, or in addition to, OP-3 protein administration.
  • a protein is morphogenic if it is capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new, organ-specific tissue and comprises at least the conserved C-terminal six cysteine skeleton or its functional equivalent (see supra).
  • the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells.
  • the morphogens may be purified from naturally-sourced material or recombinantly produced from procaryotic or eucaryotic host cells, preferably as described therein.
  • novel morphogenic sequences may be identified following the procedures disclosed therein.
  • Particularly useful morphogens identified to date include OP-1, OP-2, CBMP2A and CBMP2B (the morphogenically active domains of proteins referred to in the art as BMP2A and BMP2B, or BMP2 and BMP4, respectively), BMP3, BMP5, BMP6, Vgr-1, GDF-1, Vgl, DPP and 60A, including their allelic and species variants, as well as other amino acid sequence variants, including chimeric morphogens.
  • Morphogenically active biosynthetic constructs such as those disclosed in U.S. Pat. No. 5,011,691, (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16) also are envisioned to be useful.
  • the OP-3 proteins useful in the invention include any morphogenically active fragment of the OP-3 amino acid sequence present in Seq. ID No. 1, or allelic, species or other amino acid sequence variants thereof.
  • the morphogenically active fragment of OP-3 also may include any morphogenically active protein encoded by part or all of the nucleic acid sequence presented in Seq. ID No. 1.
  • the morphogenic protein also may comprise a protein encoded by part or all of a nucleic acid which hybridizes to at least part of the nucleic acid sequence encoding the "pro" region of the OP-3 protein, e.g., bases 120-848 of Seq. ID No. 1, under stringent conditions.
  • the mOP-3 gene encodes a protein ("mOP-3") first expressed as an immature translation product that is 399 amino acids in length.
  • This precursor form referred to herein as the "prepro” form, (Seq. ID. No. 1, amino acid residues 1-399) includes an
  • N-terminal signal peptide sequence typically less than about 20 residues, followed by a "pro" domain that is cleaved to yield the mature sequence.
  • the "pro" form of the protein includes the pro domain and the mature domain, and forms a soluble species that appears to be the primary form secreted from cultured mammalian cells.
  • the signal peptide anticipated to include residues 1-17 for mOP3, is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given sequence using the method of Von Heijne ((1986) Nucleic Acids Research 14:4683-4691).
  • the preferred form of morphogenically active OP-3 protein comprises a processed sequence, including fragments thereof, appropriately dimerized and disulfide bonded. Where a soluble form of the protein is desired, the protein preferably comprises both the mature domain, or an active portion thereof, and part or all of the pro domain.
  • the pro domain By amino acid sequence homology with other, known morphogens, the pro domain likely is cleaved at residues 257-260 of Seq. ID No. 1, which represent the canonical Arg-Xaa-Xaa-Arg cleavage site, to yield a mature sequence 139 amino acids in length (Seq. ID No. 1, residues 261-399). Alternatively, the pro domain may be cleaved at residues 260-263 to yield a shorter sequence 135 amino acids in length (Seq. ID No. 1, amino acid residues 264-399).
  • All morphogens including OP-1, OP-2 and the OP-3 proteins disclosed herein, comprise at least a conserved six cysteine skeleton in the amino acid sequence C-terminal domain and, preferably, a conserved seven cysteine skeleton (see, for example, US92/01968 (W092/15323) .
  • the conserved six cysteine skeleton in mOP-3 (Seq. ID No. 1) is defined by amino acid residues 303-399; the conserved seven cysteine skeleton is defined by amino acid residues 298-399.
  • the OP-3 proteins like the OP-2 proteins, also has one additional cysteine residue (residue 338 of Seq. ID No. 1) in the conserved C-terminal domain.
  • the mature sequence of OP-3 shares significant amino acid sequence homology with the morphogens identified to date. Specifically, the seven cysteine fragment shows greater than 79% amino acid identity with the corresponding mOP-2 and hOP-2 sequences, and greater than 66% identity with the corresponding OP-1 sequences. Like OP-2, OP-3 has an eighth cysteine within the seven cysteine domain (e.g., at position 338 of Seq. ID No. 1). In addition, OP-3 is unique among the morphogens identified to date in that the residue at position 9 in the conserved seven cysteine domain (e.g., residue 315 of Seq. ID No. 1) is a serine, whereas other morphogens typically have a tryptophan at this location (see Table I below, and Table II in PCT/US92/07358 (WO93/04692) , for example.)
  • amino acid sequence homology is understood to mean amino acid sequence similarity, and homologous sequences share identical or similar amino acids, where similar amino acids are conserved amino acids as defined by Dayoff et al.. Atlas of Protein Sequence and Structure; vol.5, Suppl.3, pp.345-362 (M.O. Dayoff, ed., Nat'l BioMed. Research Fdn., Washington D.C. 1978.)
  • a candidate sequence sharing 70% amino acid homology with a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acid in the reference sequence, or constitute a conserved amino acid change thereto.
  • amino acid sequence identity is understood to require identical amino acids between two aligned sequences.
  • a candidate sequence sharing 60% amino acid identity with a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 60% of the amino acids in the candidate sequence are identical to the corresponding amino acid in the reference sequence.
  • all homologies and identities calculated use OP-3 as the reference sequence.
  • sequences are aligned for homology and identity calculations using the method of Needleman et al. (1970) J.Mol. Biol. 48:443-453 and identities calculated by the Align program (DNAstar, Inc.) In all cases, internal gaps and amino acid insertions in the candidate sequence as aligned are ignored when making the homology/identity calculation.
  • useful OP-3 variants include, but are not limited to, amino acid sequences derived from Seq. ID No. 1 and wherein the cysteine at position 338 is replaced with another amino acid, preferably a tyrosine, histidine, isoleucine or serine and conservative substitutions thereof, e.g., such as defined by Dayoff et al., Atlas of Protein Sequence and Structure; vol. 5, Suppl. 3, pp.345-362 (M.O. Dayoff, ed., Nat'l BioMed. Research Fdn., Washington D.C. 1979.). Still other useful OP-3 variants include proteins wherein the serine at position 315 is replaced with another amino acid, preferably a tryptophan and conservative substitutions thereof.
  • Generic Sequence 7 (Seq. ID No. 12) and Generic Sequence 8 (Seq. ID No. 13) disclosed below, accommodate the homologies shared among perferred morphogen protein family members identified to date, including OP-1, OP-2, OP-3, CBMP2A, CBMP2B, BMP3, 60A, DPP, Vgl, BMP5, BMP6, Vrg-1, and GDF-1.
  • the amino acid sequences for these proteins are described herein (see Sequence Listing and Table I below) and/or in the art, as well as in PCT publication US 92/07358, filed August 28, 1992, for example.
  • the generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six and seven cysteine skeletons (Generic Sequences 7 and 8, respectively), as well as alternative residues for the variable positions within the sequence.
  • the generic sequences allow for an additional cysteine at position 41 (Generic Sequence 7) or position 46 (Generic
  • Sequence 8 providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and containing certain critical amino acids which influence the tertiary structure of the proteins.
  • Generic Sequence 8 (Seq. ID No. 13) includes all of Generic Sequence 7 and in addition includes the following sequence at its N-terminus:
  • each "Xaa” in Generic Seq. 8 is a specified amino acid defined as for Generic Seq. 7, with the distinction that each residue number described for Generic Sequence 7 is shifted by five in Generic Seq. 8.
  • Table I compares the C-terminal amino acid sequences defining the seven cysteine skeleton of human OP-1, mouse OP-1, human OP-2, mouse OP-2, and mouse OP-3 (mOP-3, Seq. ID No. 1).
  • the sequences are aligned essentially following the method of Needleman et al. (1970) J. Mol. Biol., 4_8:443-453, calculated using the Align Program (DNAstar, Inc.)
  • three dots indicates that the amino acid in that position is the same as the amino acid in hOP-1.
  • Three dashes indicate that no amino acid is present in that position, and are included for purposes of illustrating homologies.
  • significant amino acid sequence homology exists between mouse OP-3 and mouse and human OP-1 and OP-2.
  • the morphogens described herein may be provided to an individual by any suitable means, preferably directly or systemically, e.g., parenterally or orally.
  • the morphogen is to be provided directly (e.g., locally, as by injection, to a desired tissue site), or parenterally, such as by intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal or by aerosol administration
  • the morphogen preferably comprises part of an aqueous solution.
  • the solution is physiologically acceptable so that in addition to delivery of the desired morphogen to the patient, the solution does not otherwise adversely affect the patient's electrolyte and volume balance.
  • the aqueous medium for the morphogen thus may comprise normal physiologic saline (0.9% NaCl, 0.15M), pH 7-7.4.
  • the aqueous solution containing the morphogen can be made, for example, by dissolving the protein in 50% ethanol, or acetonitrile containing 0.1% trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent solvents.
  • One volume of the resultant solution then is added, for example, to ten volumes of phosphate buffered saline (PBS), which further may include 0.1-0.2% human serum albumin (HSA).
  • PBS phosphate buffered saline
  • HSA human serum albumin
  • the resultant solution preferably is vortexed extensively.
  • a given morphogen may be made more soluble by association with a suitable molecule.
  • association of the mature dimer with the pro domain of the morphogen increases solubility of the protein significantly.
  • the pro form of OP-3 comprises a species that is soluble in physiologically buffered solutions.
  • the endogenous protein is thought to be transported (e.g., secreted and circulated) to particular tissues in this form.
  • This soluble form of the protein may be obtained from the culture medium of morphogen-secreting mammalian cells.
  • a soluble species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain.
  • Another molecule capable of enhancing solubility and particularly useful for oral administrations, is casein. For example, addition of 0.2% casein increases solubility of the mature active form of OP-1 by 80%.
  • Other components found in milk and/or various serum proteins also may be useful.
  • Useful solutions for oral or parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences,
  • Formulations may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like.
  • Formulations for direct administration may include glycerol and other compositions of high viscosity.
  • Biocompatible, preferably bioresorbable polymers including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, polylactide, polyglycolide and lactide/glycolide copolymers, may be useful excipients to control the release of the morphogen ij vivo.
  • Formulations for inhalation administration may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • the morphogens described herein may be administered orally. Oral administration of proteins as therapeutics generally is not practiced as most proteins readily are degraded by digestive enzymes and acids in the mammalian digestive system before they can be absorbed into the bloodstream. However, the morphogens described herein typically are acid-stable and protease-resistant (see, for example, U.S. Pat. No. 4,968,590.)
  • at least one morphogen, OP-1 has been identified in bovine mammary gland extract, colostrum and milk, as well as saliva.
  • the OP-1 purified from mammary gland extract is morphogenically active.
  • this protein induces endochondral bone formation in mammals when implanted subcutaneously in association with a suitable matrix material, using a standard in vivo bone assay, such as is disclosed in U.S. Pat. No. 4,968,590.
  • endogenous morphogen also is detected in human serum.
  • the compounds provided herein also may be associated with molecules capable of targeting the morphogen to a desired tissue.
  • molecules capable of targeting the morphogen to a desired tissue For example, tetracycline and diphosphonates (bisphosphonates) are known to bind to bone mineral, particularly at zones of bone remodeling, when they are provided systemically in a mammal. Accordingly, these molecules may be included as useful agents for targeting OP-3 to bone tissue.
  • an antibody or other binding protein that interacts specifically with a surface molecule on the desired target tissue cells also may be used.
  • Such targeting molecules further may be covalently associated to the morphogen, e.g., by chemical crosslinking, or by using standard genetic engineering means to create, for example, an acid labile bond such as an Asp-Pro linkage.
  • Useful targeting molecules may be designed, for example, using the single chain binding site technology disclosed, for example, in U.S. Pat. No. 5,091,513.
  • the morphogen family members share significant sequence homology in the C-terminal active domains.
  • the sequences diverge significantly in the sequences which define the pro domain and the N-terminal 39 amino acids of the mature protein.
  • the pro domain and/or N-terminal sequence may be morphogen-specific.
  • the various morphogens identified to date are differentially expressed in the different tissues. Accordingly, without being limited to any given theory, it is likely that, under natural conditions in the body, selected morphogens typically act on a given tissue. Accordingly, part or all of morphogen-specific sequences may serve as targeting molecules for the morphogens described herein.
  • the pro domains may interact specifically with one or more molecules at the target tissue to direct the morphogen associated with the pro domain to that tissue.
  • another useful targeting molecule for targeting OP-3 to bone tissue may include part or all of a morphogen-specific sequence, such as part or all of a pro domain and/or the N-terminus of the mature protein.
  • Particularly useful are the morphogen-specific sequences of OP-1, BMP2 or BMP4, all of which proteins are found naturally associated with bone tissue (see, for example, US Pat. No. 5,011,691).
  • the morphogen-specific sequences of GDF-1 may be used to target morphogenic OP-3 to nerve tissue, particularly brain tissue where GDF-1 appears to be primarily expressed (see, for example, Lee, (1991) PNAS, 8 :4250-4254.
  • pro forms of the proteins may be obtained from the culture medium of morphogen-secreting mammalian cells.
  • a suitable species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain.
  • Chimeric OP-3 proteins comprising, for example, non-OP-3 pro domains and/or non-OP-3 N-termini, may be synthesized using standard recombinant DNA methodology and/or automated chemical nucleic acid synthesis methodology well described in the art and as disclosed below.
  • the OP-3 proteins provided herein may be administered alone or in combination with other molecules known to have a beneficial effect on tissue morphogenesis, including molecules capable of tissue repair and regeneration and/or inhibiting inflammation.
  • useful cofactois for stimulating bone tissue growth in osteoporotic individuals include but are not limited to, vitamin D ⁇ , calcitonin, prostaglandins, parathyroid hormone, dexamethasone, estrogen and IGF-I or IGF-II.
  • Useful cofactors for nerve tissue repair and regeneration may include nerve growth factors.
  • Other useful cofactors include symptom-alleviating cofactors, including antiseptics, antibiotics, antiviral and antifungal agents and analgesics and anesthetics.
  • compositions can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients and carriers.
  • compositions may be prepared for parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops or aerosols.
  • the composition may include the morphogen dispersed in a fibrinogen-thrombin composition or other bioadhesive such as is disclosed, for example in PCT US91/09275, (WO92/10567) .
  • the composition then may be painted, sprayed or otherwise applied to the desired tissue surface.
  • compositions can be formulated for parenteral or oral administration to humans or other mammals in therapeutically effective amounts, e.g., amounts which provide appropriate concentrations of OP-3 to target tissue for a time sufficient to induce morphogenesis, including particular steps thereof, as described above.
  • OP-3 is to be used as part of a transplant procedure
  • the morphogen may be provided to the living tissue or organ to be transplanted prior to removal of tissue or organ from the donor.
  • OP-3 may be provided to the donor host directly, as by injection of a formulation comprising OP-3 into the tissue, or indirectly, e.g., by oral or parenteral administration, using any of the means described above.
  • the organ or living tissue may be placed in a preservation solution containing OP-3.
  • the recipient also preferably is provided with the morphogen just prior to, or concommitant with, transplantation.
  • OP-3 may be administered directly to the tissue at risk, as by injection to the tissue, or it may be provided systemically, either by oral or parenteral administration, using any of the methods and formulations described herein and/or known in the art.
  • OP-3 comprises part of a tissue or organ preservation solution
  • any commercially available preservation solution may be used to advantage.
  • useful solutions known in the art include
  • an organ preservation solution usually possesses one or more of the following properties: (a) an osmotic pressure substantially equal to that of the inside of a mammalian cell, (solutions typically are hyperosmolar and have K+ and/or Mg++ ions present in an amount sufficient to produce an osmotic pressure slightly higher than the inside of a mammalian cell); (b) the solution typically is capable of maintaining substantially normal ATP levels in the cells; and (c) the solution usually allows optimum maintenance of glucose metabolism in the cells.
  • Organ preservation solutions also may contain anticoagulants, energy sources such as glucose, fructose and other sugars, metabolites, heavy metal chelators, glycerol and other materials of high viscosity to enhance survival at low temperatures, free oxygen radical inhibiting and/or scavenging agents and a pH indicator.
  • energy sources such as glucose, fructose and other sugars, metabolites, heavy metal chelators, glycerol and other materials of high viscosity to enhance survival at low temperatures, free oxygen radical inhibiting and/or scavenging agents and a pH indicator.
  • OP-3 is envisioned to be useful in enhancing viability of any organ or living tissue to be transplanted.
  • the morphogens may be used to particular advantage in lung, heart, liver, kidney or pancreas transplants, as well as in the transplantation and/or grafting of bone marrow, skin, gastrointestinal mucosa, and other living tissues.
  • the concentration of the compounds described in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g.. hydrophobicity) of the compounds employed, and the route of administration.
  • the preferred dosage of drug to be administered also is likely to depend on such variables as the type and extent of tissue loss or defect, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound, the presence and types of excipients in the formulation, and the route of administration.
  • the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 0.001 to 10% w/v compound for parenteral administration. Typical dose ranges are from about 10 ng/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.1 ⁇ g/kg to
  • morphogen-induced pathological lesions are induced when mature morphogen (e.g., OP-1, 20 ⁇ g ) is administered daily to normal growing rats for 21 consecutive days.
  • mature morphogen e.g., OP-1, 20 ⁇ g
  • 10 ⁇ q systemic injections of morphogen (e.g., OP-1) injected daily for 10 days into normal newborn mice does not produce any gross abnormalities.
  • a morphogenically active fragment of OP-3 may be implanted surgically, dispersed in a biocompatible, preferably in vivo biodegradable matrix appropriately modified to provide a structure or scaffold in which the OP-3 may be dispersed and which allows the differentiation and proliferation of migrating progenitor cells.
  • the matrix also may provide signals capable of directing the tissue specificity of the differentiating cells, as well as providing a morphogenically permissive environment, being essentially free of growth inhibiting signals.
  • the formulated matrix may be shaped as desired in anticipation of surgery or may be shaped by the physician or technician during surgery.
  • the material may be used in topical, subcutaneous, intraperitoneal, or intramuscular implants to repair tissue or to induce its growth de novo.
  • the matrix preferably is biodegradable _in vivo, being slowly absorbed by the body and replaced by new tissue growth, in the shape or very nearly in the shape of the implant.
  • the matrix also may be particulate in nature.
  • Suitable biocompatible, ij vivo biodegradable acellular matrices may be prepared from naturally- occurring tissue.
  • the tissue is treated with suitable agents to substantially extract the cellular, nonstructural components of the tissue.
  • the agents also should be capable of extracting any morphogenesis inhibiting components associated with the tissue.
  • the resulting material is a porous, acellular matrix, substantially depleted in nonstructurally-associated components.
  • the matrix also may be further treated with agents that modify the matrix, increasing the number of pores and micropits on its surfaces. Those skilled in the art will know how to determine which agents are best suited to the extraction of nonstructural components for different tissues.
  • soft tissues such as liver and lung may be thin-sectioned and exposed to a nonpolar solvent such as, for example, 100% ethanol, to destroy the cellular structure of the tissue and extract nonstructural components.
  • the material then may be dried and pulverized to yield nonadherent porous particles or it may be maintained as a gel-like solution.
  • Structural tissues such as cartilage and dentin where collagen is a primary proteinaceous component may be demineralized and extracted with guanidinium hydrochloride, essentially following the method of Sampath et al. (1983) PNAS j30_:6591-6595.
  • pulverized and demineralized dentin is extracted with five volumes of 4M guanidinium-HCl, 50mM Tris-HCl, pH 7.0 for 16 hours at 4°C.
  • the suspension then is filtered.
  • the insoluble material that remains is collected and used to fabricate the matrix.
  • the material is mostly collagenous in matter. It is devoid of morphogenic activity.
  • the matrix particles may further be treated with a collagen fibril-modifying agent that extracts potentially unwanted components from the matrix, and alters the surface structure of the matrix material.
  • Useful agents include acids, organic solvents or heated aqueous media. A detailed description of these matrix treatments are disclosed, for example, in U.S. Patent No.
  • the currently most preferred agent is a heated aqueous fibril-modifying medium such as water, to increase the matrix particle surface area and porosity.
  • the currently most preferred aqueous medium is an acidic aqueous medium having a pH of less than about 4.5, e.g., within the range of about pH 2 - pH 4 which may help to "swell" the collagen before heating. 0.1% acetic acid, which has a pH of about 3, currently is most preferred. 0.1 M acetic acid also may be used.
  • aqueous medium lg matrix/30ml aqueous medium
  • Preferred treatment times are about one hour, although exposure times of between about 0.5 to two hours appear acceptable.
  • the temperature employed is held constant at a temperature within the range of about 37°C to 65°C.
  • the currently preferred heat treatment temperature is within the range of about 45°C to 60°C.
  • the matrix is filtered, washed, lyophilized and used for implant.
  • the matrix also is preferably neutralized prior to washing and lyophilization.
  • a currently preferred neutralization buffer is a 200mM sodium phosphate buffer, pH 7.0.
  • the matrix preferably first is allowed to cool following thermal treatment, the acidic aqueous medium (e.g., 0.1% acetic acid) then is removed and replaced with the neutralization buffer and the matrix agitated for about 30 minutes. The neutralization buffer then may be removed and the matrix washed and lyophilized.
  • Other useful fibril-modifying treatments include acid treatments (e.g., trifluoroacetic acid and hydrogen fluoride) and solvent treatments such as dichloromethane, acetonitrile, isopropanol and chloroform, as well as particular acid/solvent combinations.
  • acid treatments e.g., trifluoroacetic acid and hydrogen fluoride
  • solvent treatments such as dichloromethane, acetonitrile, isopropanol and chloroform, as well as particular acid/solvent combinations.
  • the treated matrix may be washed to remove any extracted components, following a form of the procedure set forth below:
  • TBS Tris- buffered saline
  • UTBS Tris- buffered saline
  • RT room temperature
  • suitable matrix materials may be obtained commercially.
  • an extracellular matrix extract such as MatrigelTM, (Collaborative Research, Inc., Bedford) derived from mouse sarcoma cells, may be used to advantage.
  • tissue-specific matrices may be formulated synthetically.
  • These porous biocompatible, in vivo biodegradable synthetic matrices are disclosed in PCT publication US91/03603, published December 12, 1991 (W091/18558) .
  • the matrix comprises a porous crosslinked structural polymer of biocompatible, biodegradable collagen and appropriate, tissue-specific glycosammoglycans as tissue-specific cell attachment factors.
  • Collagen derived from a number of sources may be suitable for use in these synthetic matrices, including insoluble collagen, acid-soluble collagen, collagen soluble in neutral or basic aqueous solutions, as well as those collagens which are commercially available.
  • Glycosammoglycans or mucopolysaccharides are hexosamine-containing polysaccharides of animal origin that have a tissue specific distribution, and therefore may be used to help determine the tissue specificity of the morphogen-stimulated differentiating cells. Reaction with the GAGs also provides collagen with another valuable property, i.e., inability to provoke an immune reaction (foreign body reaction) from an animal host.
  • GAGs are made up of residues of hexosamines glycosidically bound and alternating in a more-or-less regular manner with either hexouronic acid or hexose moieties (see, e.g., Dodgson et al. in
  • GAGs include hyaluronic acid, heparin, heparin sulfate, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, and keratin sulfate.
  • Other GAGs are suitable for forming the matrix described herein, and those skilled in the art will either know or be able to ascertain other suitable GAGs using no more than routine experimentation. For a more detailed description of mucopolysaccharides, see Aspinall,
  • chondroitin-6-sulfate can be used where endochondral bone formation is desired.
  • Heparin sulfate may be used to formulate synthetic matrices for use in lung tissue repair.
  • Collagen can be reacted with a GAG in aqueous acidic solutions, preferably in diluted acetic acid solutions.
  • a GAG aqueous acidic solutions
  • coprecipitates of tangled collagen fibrils coated with GAG results.
  • This tangled mass of fibers then can be homogenized to form a homogeneous dispersion of fine fibers and then filtered and dried.
  • Insolubility of the collagen-GAG products can be raised to the desired degree by covalently cross- linking these materials, which also serves to raise the resistance to resorption of these materials.
  • any covalent cross-linking method suitable for cross-linking collagen also is suitable for cross- linking these composite materials, although crosslinking by a dehydrothermal process is preferred.
  • the crosslinked particles When dry, the crosslinked particles are essentially spherical, with diameters of about 500 ⁇ m. Scanning electron miscroscopy shows pores of about 20 ⁇ m on the surface and 40 ⁇ m on the interior.
  • the interior is made up of both fibrous and sheet-like structures, providing surfaces for cell attachment.
  • the voids interconnect, providing access to the cells throughout the interior of the particle.
  • the material appears to be roughly 99.5% void volume, making the material very efficient in terms of the potential cell mass that can be grown per gram of microcarrier.
  • Another useful synthetic matrix is one formulated from biocompatible, ij vivo biodegradable synthetic polymers, such as those composed of glycolic acid, lactic acid and/or butyric acid, including copolymers and derivatives thereof.
  • biocompatible, ij vivo biodegradable synthetic polymers such as those composed of glycolic acid, lactic acid and/or butyric acid, including copolymers and derivatives thereof.
  • polymers are well described in the art and are available commercially.
  • polymers composed of polyactic acid e.g., MW 100 kDa
  • 80% polylactide/20% glycoside or poly 3-hydroxybutyric acid e.g., MW 30 kDa
  • the polymer compositions generally are obtained in particulate form.
  • the steps preferably are performed under conditions which avoid hydrolysis of the polymers (e.g., non-aqueous conditions such as in an ethanol- trifluoro-acetic acid solution) .
  • OP-3 proteins described herein can be combined and dispersed in a suitable matrix using any of the methods described below:
  • Matrix is added to the morphogen dissolved in guanidine-HCl. Samples are vortexed and incubated at a low temperature. Samples are then further vortexed. Cold absolute ethanol is added to the mixture which is then stirred and incubated. After centrifugation (microfuge, high speed) the supernatant is discarded. The matrix is washed with cold concentrated ethanol in water and then lyophilized. 2. Acetonitrile Trifluoroacetic Acid Lyophilization
  • a preparation of a morphogenically active fragment of OP-3 in physiological saline also may be vortexed with the matrix and lyophilized to produce morphogenically active material.
  • Tissue morphogenesis requires a morphogenically permissive environment.
  • a control mechanism such as a feedback control mechanism, which regulates the control of cell growth and differentiation.
  • TGF- ⁇ , and MIS are capable of inhibiting cell growth when present at appropriate concentrations.
  • osteogenic devices comprising a bone-derived carrier (matrix) that has been demineralized and guanidine-extracted to substantially remove the noncollagenous proteins does allow endochondral bone formation when implanted in association with an osteoinductive morphogen. If, however, the bone-derived carrier is not demineralized but rather is washed only in low salt, for example, induction of endochondral bone formation is inhibited, suggesting the presence of one or more inhibiting factors within the carrier.
  • matrix bone-derived carrier
  • OP-3 proteins useful in the methods and compositions of this invention may be purified from natural sources or produced using standard recombinant methodology. General considerations for the recombinant production of OP3 morphogens are described below.
  • a genetic sequence encoding the morphogenic OP-3 protein was identified using a 0.3 kb EcoRI-BamHl OP-2 fragment from a mouse OP-2 cDNA as a hybridization probe, specific to the mid-pro region of OP-2
  • the 32 P-labeled probe was prepared using the random hexanucleotide priming method, and the hybridizations were performed using the following conditions: 40% formamide, 5 X SSPE, 5 X Denhardt's Solution, 0.1% SDS, at 37°C overnight, and washing in 0.1 X SSPE, 0.1% SDS at 50°C.
  • the isolated mOP-3 DNA sequence in accordance with other known morphogens, encodes a protein comprising a "pro" region (defined essentially by residues 20-260 or 20-263 of Seq. ID No. 1) and a mature region (defined essentially by residues 261-399 or 264-399 of Seq. ID No. 1), including a functional domain comprising the conserved cysteine skeleton.
  • OP-3 is marked by an eighth cysteine within the seven cysteine domain (e.g., at position 338 of Seq. ID No.l).
  • the extra cysteine likely helps stabilize the folded structure, possibly by providing inter-molecular disulfide bonding.
  • the extra cysteine also allows for heterodimer formation between OP-3 and another morphogen comprising the "eighth" cysteine, like OP-2 for example, or a modified OP-1, wherein an extra cysteine has been inserted at the appropriate location.
  • the extra cysteine also may allow tetramer formation.
  • the extra cysteine does not inhibit synthesis or reduce the stability of the translated sequence significantly as expressed proteins comprising the extra cysteine are readily detected by SDS gel electrophoresis.
  • a primary glycosylation site occurs just C terminal to the extra cysteine in both OP-2 and OP-3, which may provide a protective effect.
  • the cDNA sequences for both human and mouse OP-2 are provided in Seq. ID Nos. 7 and 9, and the genomic sequence for human OP-2 is provided in Seq. ID No. 11, wherein the exons defining the coding region of these proteins are indicated. The exon boundaries also are indicated in Fig. 1, described below.
  • the human OP-2 locus was isolated from a genomic library (Clontech, EMBL-3 #HL1067J) on three overlapping phage clones, using standard cloning procedures.
  • the OP-2 coding information was spread over 27 kb and, like OP-1, contains 7 exons. A comparison of exon-intron boundaries in the 7 cysteine domain showed matching locations with those of OP-1.
  • the first OP-2 exon contains 334 bp of coding sequence (111 amino acids), including the signal peptide, and is followed by the largest intron (14.6 kb).
  • the second exon (190 bp, 64 amino acids) is separated by a short intron (0.4 kb) from exon 3 (149 bp, 49 amino acids). It follows a large third intron of 9.5 kb.
  • the fourth exon (195 bp, 65 amino acids) encodes the maturation site ("OP-2-Ala”) and is followed by a 0.8 kb intron.
  • exon 5 (80 bp, 27 amino acids) encodes the first cysteine of mature OP-2 and is followed by intron 5 (0.5 kb in length), exon 6 (111 bp, 37 amino acids) is separated by a 2.5 kb intron from the seventh, last exon with 147 bp (49 amino acids) of coding sequence.
  • exon-intron boundaries are conserved between human OP-1 and OP-2, two different members of the morphogen family of proteins.
  • the exon- intron boundaries between human and mouse OP-2, two species variants of a morphogen are anticipated to be conserved as well.
  • Figure 1 shows the alignment of the murine OP-2 and murine OP-3 coding regions of the cDNA.
  • the exon boundaries are indicated by bars beneath the sequence. Both sequences have the same number of nucleotides.
  • the nucleotide sequence is about 80% conserved in the N-terminal and C-terminal regions. In the figure, nucleotide identity between the sequences is indicated by stippling. In addition, the central region of the sequence is highly conserved and this conserved region falls into the boundaries of exon 2 and 3. There are only three nucleotide differences in this region, indicated in the figure by diamonds. The high degree of conservation in the nucleotide sequences indicates that OP-2 and OP-3 likely share the nucleotide sequence of exon 2 and 3.
  • the different proteins may result from alternatively spliced transcripts, or they may arise from independent genes which share part of their coding sequence.
  • Intron 1 which lies upstream of exon 2 in OP-2 (see Seq. ID No.11) is large (14.6kb) and could include the start of the OP-3 gene and/or its first exon sequence.
  • one or more of the introns of these morphogens may include sequences having a transcription regulatory function.
  • OP-3 genetic sequences from other species and other libraries may be isolated.
  • a probe to the N-terminal region of the mature protein, or the 3' noncoding region flanking and immediately following the stop codon also may be used to screen for other OP-3 species variants. These sequences vary substantially among the morphogens and represent morphogen-specific sequences.
  • OP-3 Mammalian cell expression of OP-3 readily can be achieved using COS (simian kidney ATCC, CRL- 1650) or CHO (Chinese hamster ovary) cells (e.g., CHO- DXBII, from Lawrence Chasin, Columbia University, NY).
  • COS simian kidney ATCC, CRL- 1650
  • CHO Choinese hamster ovary
  • Other useful eukaryotic cell systems include the insect/baculovirus system or the mammalian complement system.
  • the OP-3 DNA is subcloned into an insertion site of a suitable, commercially available pUC-type vector (e.g., pUC-19, ATCC #37254, Rockville, MD), along with a suitable promoter/enhancer sequences and 3' termination sequences.
  • a suitable, commercially available pUC-type vector e.g., pUC-19, ATCC #37254, Rockville, MD
  • promoter/enhancer sequences are the CMV-MIE promoter (human cytomegalovirus major intermediate-early promoter, preferably the intron-free or "short" form of the promoter) and the mouse mammary tumor virus promoter (mMTV) boosted by the rous sarcoma virus LTR enhancer sequence (e.g., from Clontech, Inc., Palo Alto).
  • the plasmid also preferably contains a selectable marker, most preferably an amplifiable marker such as DHFR, e.g., under SV40 early promoter control (ATCC #37148).
  • a selectable marker most preferably an amplifiable marker such as DHFR, e.g., under SV40 early promoter control (ATCC #37148).
  • Transfection, cell culturing, gene amplification and protein expression conditions are standard conditions, well known in the art, such as are described, for example in Ausubel et al., ed.. Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989).
  • transfected cells are cultured in medium containing 0.1-0.5% dialyzed fetal calf serum (FCS), stably transfected high expression cell lines obtained by subcloning and evaluated by standard Northern blot.
  • Southern blots also are used to assess the state of integrated OP-3 sequences and the extent of their copy number amplification.
  • Chimeric OP-3 morphogens e.g., comprising an OP-3 active domain and, for example, part or all of a pro domain from another, different morphogen may be constructed using standard recombinant DNA technology and/or an automated DNA synthesizer to construct the desired sequence.
  • Useful chimeras include those wherein the non-OP-3 sequence is joined to the OP-3 sequence encoding the mature OP-3 protein, and the non- OP-3 sequence encodes part or all of the sequence between the signal peptide processing site and the
  • the non-OP-3 sequence may be joined to an OP-3 sequence encoding, for example, the 6 or 7 cysteine skeletons, wherein the non-OP-3 sequence includes the sequence encoding the N-terminus of the mature protein.
  • the non-OP-3 sequences may be composed of sequences from one or morphogens and/or may comprise novel biosynthetic sequences.
  • a biosynthetic gene construct encoding a chimeric OP1-OP3 polypeptide chain is demonstrated in the immunoblot presented in Fig. 2.
  • a vector carrying the construct under CMV promoter control was transfected into CHO cells (CHO-DXB11) using standard procedures and as described herein.
  • a chimeric gene was constructed by replacing the conserved seven cysteine domain of OP-1 with that of OP-3.
  • the resulting chimeric gene contains the entire pre-pro-domain of human OP-1 and the region of mature OP-1 between the maturation site and the first cysteine of the conserved C-terminal seven cysteine domain, fused to the conserved seven cysteine domain of mouse OP-3, but with two arginine residues in place of the native lysine residues found in OP-3 at the start of the seven cysteine domain.
  • the gene fusion was accomplished by splicing the Sad site of OP-3 (near the first cysteine of the seven cysteine domain) with a newly created Sad site in OP-1, created at the matching residues by silent mutagenesis.
  • the Sad site encodes the Glu-Leu dipeptide in the sequence Cys-Arg-Arg-His-Glu-Leu of OP-1 and Cys-Lys-Lys-His-Glu-Leu of OP-3, respectively.
  • the chimeric gene was placed downstream of the CMV (Cytomegalovirus) MIE "short" (intron-free) promoter and upstream of the SV40 transcriptional terminator in a pUC vector.
  • This plasmid was cotransfected with DNA encoding the DHFR marker and viral trans-activating elements (e.g., VA1, E1A) into a CHO dhfr(-) host and subjected to Methotrexate selection and one round of amplification at 1 mM Methotrexate including subcloning. 10 ⁇ l of "spent" culture supernatant (3 days old) was analysed by "Western blot" (immunoblot) , as follows.
  • the 10 ⁇ l harvested medium was briefly heated with concentrated SDS sample buffer, containing ⁇ -mercapto ethanol (5%) and directly analysed by electrophoresis on a 15% SDS- polyacrylamide gel (in the buffer system of Laemmli) along with a set of prestained molecular weight standards (Bio-rad, Richmond, CA) . Proteins were transferred from the gel to Immobilon membrane by the "Western blot" procedure.
  • the chimeric OP-l/OP-3 protein was detected by reaction with rabbit serum raised against a synthetic peptide representing the first 17 amino acids of mature OP-1, starting with serine-threonine-glycine-serine-. Authentic recombinant OP-1, expressed in CHO cells was included for comparison.
  • lanes were as follows: lane 1: OP-1; lanes 4, 5, 6, 7, and 8: chimeric OP-l/OP-3; lanes 9 and 10: prestained molecular weight standards.
  • the apparent mobility of the recombinant proteins, at approximately 20 kDa on this gel, is due to glycosylation of the OP-1 and OP-3 proteins which may also be the cause of the multiple species observed.
  • the expressed protein then can be purified as follows. For a typical 2L preparation of transfected mammalian cells conditioned in 0.5% FCS, for example, the total protein is typically about 700 mg.
  • the amount of OP-3 in the media estimated by Western blot, is between about 0.1-5.0 mg.
  • OP-3 media then is diluted in a low salt, physiologically buffered 6M urea solution, and loaded onto an S-Sepharose column, which acts as a strong cation exchanger. OP-3 binds to the column in low salt, and serum proteins are removed.
  • the column subsequently is developed with an NaCl gradient, e.g., O.lM NaCl-l.OM NaCl, in 6M urea, 20mM HEPES, pH 7.0. Most contaminants are removed at the start of the gradient, and OP-3 is eluted primarily at a higher salt concentration.
  • the sample then is loaded onto a phenyl-Sepharose column (hydrophobic interaction chromatography). OP-3 binds phenyl-Sepharose in the presence of high concentrations of a weak chaotropic salt (e.g., IM (NH 4 ) 2 S0 4 in a physiologically buffered 6M urea solution) .
  • a weak chaotropic salt e.g., IM (NH 4 ) 2 S0 4 in a physiologically buffered 6M urea solution
  • the column is developed with a decreasing ammonium sulfate gradient, e.g., 0.6M-0.0M (NH 4 ) 2 S0 4 gradient in a physiologically buffered, 6M urea solution. Again, most contaminants are removed at the start of the gradient, and OP-3 elutes primarily at low or no ammonium sulfate concentrations.
  • a decreasing ammonium sulfate gradient e.g., 0.6M-0.0M (NH 4 ) 2 S0 4 gradient in a physiologically buffered, 6M urea solution.
  • the 0P-3 eluted from the phenyl-Sepharose column then is dialyzed against water, and prepared for loading onto a reverse phase chromatography column (e.g., C-18 HPLC), for example, by dialyzing against 30% acetonitrile, 0.1% TFA.
  • a reverse phase chromatography column e.g., C-18 HPLC
  • An alternative chromatography protocol is to perform the S-Sepharose chromatography in the absence of 6 M urea.
  • the bound proteins then are eluted with salt step elutions (e.g., 0.1-0.6M NaCl).
  • Remaining OP-3 then can be eluted in the presence of 6M urea.
  • the 6M urea elution also may be used in place of the non-urea elution to achieve maximum recovery in one step.
  • OP-3 may be eluted from the phenyl- Sepharose column in 38% ethanol-0.01% TFA, thereby eliminating the need to dialyze the eluent before applying it to the C-18 column.
  • C-18 columns may be used (e.g., three), to further enhance purification and concentration of the protein.
  • OP-3 also will bind hydroxyapatite efficiently, typically in the absence of 6 M urea and at low phosphate concentrations (less than 5 mM phosphate). Bound OP-3 can be removed from the column with an elution gradient of about .001-0.5M step elution of phosphate in a physiologically buffered solution. Additionally, urea (6M) may be added during the elution step.
  • IMAC immobilized metal-ion affinity chromatography
  • a physiologically buffered phosphate solution may be used to advantage.
  • a currently preferred form of the OP-3 morphogen useful in therapeutic formulations, having improved solubility in aqueous solutions and consisting essentially of amino acids is a dimeric morphogenic protein comprising at least the 100 amino acid peptide sequence having the pattern of seven or more cysteine residues characteristic of the morphogen family complexed with a peptide comprising part or all of a pro region of a member of the morphogen family, or an allelic, species or other sequence variant thereof.
  • the dimeric morphogenic protein is complexed with two peptides.
  • the dimeric morphogenic protein preferably is noncovalently complexed with the pro region peptide or peptides.
  • the pro region peptides also preferably comprise at least the N-terminal eighteen amino acids that define the OP-3 morphogen pro region (e.g., residues 18-35 of Seq. ID No. 1). In a most preferred embodiment, peptides defining substantially the full length pro region are used.
  • soluble forms of morphogens include dimers of the uncleaved pro forms of these proteins, as well as "hemi-dimers" wherein one subunit of the dimer is an uncleaved pro form of the protein, and the other subunit comprises the mature form of the protein, including truncated forms thereof, preferably noncovalently associated with a cleaved pro domain peptide.
  • useful pro domains include the full length pro regions, as well as various truncated forms hereof, particularly truncated forms cleaved at proteolytic Arg-Xaa-Xaa-Arg cleavage sites.
  • possible pro sequences cleaved at Arg-Xaa-Xaa-Arg sites include sequences defined by residues 18-260 of Seq. ID No. 1 (anticipated full length form); or by residues 18-263.
  • currently preferred pro sequences are those encoding the full length form of the pro region for OP-3 or another, known morphogen.
  • Other pro sequences contemplated to have utility include biosynthetic pro sequences, particularly those that incorporate a sequence derived from the N-terminal portion of one or more morphogen pro sequences.
  • useful sequences encoding the pro region may be obtained from genetic sequences encoding known morphogens.
  • chimeric pro regions can be constructed from the sequences of one or more known morphogens.
  • Still another option is to create a synthetic sequence variant of one or more known pro region sequences.
  • useful pro region peptides include polypeptide chains comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a DNA or RNA sequence encoding at least the N-terminal eighteen amino acids of the pro region sequence for OP-3 e.g., nucleotides 120-173 of Seq. ID No. 1.
  • useful pro region peptides include polypeptide chains comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a DNA or RNA sequence encoding at least the N-terminal eighteen amino acids of the pro region sequence for OPl or OP2, e.g., nucleotides 136-192 and 152-211 of Seq. ID No. 3 and 7, respectively.
  • Morphogens are expressed from mammalian cells as soluble complexes. Typically, however the complex is disassociated during purification, generally by exposure to denaturants often added to the purification solutions, such as detergents, alcohols, organic solvents, chaotropic agents and compounds added to reduce the pH of the solution.
  • denaturants such as detergents, alcohols, organic solvents, chaotropic agents and compounds added to reduce the pH of the solution.
  • the method is rapid, reproducible and yields isolated soluble morphogen complexes in substantially pure form.
  • Soluble OP-3 morphogen complexes can be isolated from conditioned media using a simple, three step chromatographic protocol performed in the absence of denaturants.
  • the protocol involves running the media (or body fluid) over an affinity column, followed by ion exchange and gel filtration chromatographies.
  • the affinity column described below is a Zn-IMAC column.
  • the present protocol has general applicability to the purification of a variety of morphogens, all of which are anticipated to be isolatable using only minor modifications of the protocol described below.
  • An alternative protocol also envisioned to have utility an immunoaffinity column, created using standard procedures and, for example, using antibody specific for a the OP-3 pro domain (complexed, for example, to a protein A-conjugated Sepharose column. ) Protocols for developing immunoaffinity columns are well described in the art, (see, for example. Guide to Protein Purification, M. Deutscher, ed.. Academic Press, San Diego, 1990, particularly sections VII and XI.)
  • OP-1 was expressed in mammalian CHO (Chinese hamster ovary) cells as described in the art (see, for example, international application
  • US90/05903 (WO91/05802) . )
  • the CHO cell conditioned media containing 0.5% FBS was initially purified using Immobilized Metal-Ion Affinity Chromatography (IMAC).
  • IMAC Immobilized Metal-Ion Affinity Chromatography
  • the soluble OP-1 complex from conditioned media binds very selectively to the Zn-IMAC resin and a high concentration of imidazole (50 mM imidazole, pH 8.0) is required for the effective elution of the bound complex.
  • the Zn-IMAC step separates the soluble OP-1 from the bulk of the contaminating serum proteins that elute in the flow through and 35 mM imidazole wash fractions.
  • the Zn-IMAC purified soluble OP-1 is next applied to an S-Sepharose cation-exchange column equilibrated in 20 mM NaP0 4 (pH 7.0) with 50 mM NaCl.
  • This S-Sepharose step serves to further purify and concentrate the soluble OP-1 complex in preparation for the following gel filtration step.
  • the protein was applied to a Sephacryl S-200HR column equilibrated in TBS.
  • soluble morphogens also may be isolated from one or more body fluids, including serum, cerebro-spinal fluid or peritoneal fluid.
  • IMAC was performed using Chelating-Sepharose (Pharmacia) that had been charged with three column volumes of 0.2 M ZnS0 4 .
  • the conditioned media was titrated to pH 7.0 and applied directly to the ZN-IMAC resin equilibrated in 20 mM HEPES (pH 7.0) with 500 mM NaCl.
  • the Zn-IMAC resin was loaded with 80 mL of starting conditioned media per mL of resin. After loading, the column was washed with equilibration buffer and most of the contaminating proteins were eluted with 35 mM imidazole (pH 7.0) in equilibration buffer.
  • the soluble OP-1 complex then is eluted with 50 mM imidazole (pH 8.0) in 20 mM HEPES and 500 mM NaCl.
  • the 50 mM imidazole eluate containing the soluble OP-1 complex was diluted with nine volumes of 20 mM NaPO. (pH 7.0) and applied to an S-Sepharose (Pharmacia) column equilibrated in 20 mM NaP0 4 (pH 7.0) with 50 mM NaCl.
  • the S-Sepharose resin was loaded with an equivalent of 800 mL of starting conditioned media per mL of resin. After loading the S-Sepharose column was washed with equilibration buffer and eluted with 100 mM NaCl followed by 300 mM and 500 mM NaCl in 20 mM NaP0 4 (pH 7.0).
  • the 300 mM NaCl pool was further purified using gel filtration chromatography. Fifty mis of the 300 mm NaCl eluate was applied to a 5.0 X 90 cm Sephacryl S-200HR (Pharmacia) equilibrated in Tris buffered saline (TBS), 50 mM Tris, 150 mM NaCl (pH 7.4). The column was eluted at a flow rate of 5 mL/minute collecting 10 mL fractions.
  • the apparent molecular of the soluble OP-1 was determined by comparison to protein molecular weight standards (alcohol dehydrogenase (ADH, 150 kDa), bovine serum albumin (BSA, 68 kDa), carbonic anhydrase (CA, 30 kDa) and cytochrome C (cyt C, 12.5 kDa).
  • ADH alcohol dehydrogenase
  • BSA bovine serum albumin
  • CA carbonic anhydrase
  • cytochrome C cytochrome C
  • the identity of the mature OP-1 and the pro-domain was determined by N-terminal sequence analysis after separation of the mature OP-1 from the pro-domain using standard reverse phase C18 HPLC.
  • the soluble OP-1 complex elutes with an apparent molecular weight of 110 kDa. This agrees well with the predicted composition of the soluble OP-1 complex with one mature OP-1 dimer (35-36 kDa) associated with two pro-domains (39 kDa each). Purity of the final complex can be verified by running the appropriate fraction in a reduced 15% polyacrylamide gel.
  • the complex components can be verified by running the complex-containing fraction from the S-200 or S-
  • N-terminal sequencing of the isolated pro domain from mammalian cell produced OP-1 revealed 2 forms of the pro region, the intact form (beginning at residue 30 of Seq. ID No. 16) and a truncated form, (beginning at residue 48 of Seq. ID No. 16.)
  • N-terminal sequencing of the polypeptide subunit of the isolated mature species reveals a range of N-termini for the mature sequence, beginning at residues 293, 300, 313, 315, 316, and 318, of Seq. ID No. 16, all of which are active as demonstrated by the standard bone induction assay.
  • soluble complexes may be formulated from purified pro domains and mature dimeric species.
  • Successful complex formation apparently requires association of the components under denaturing conditions sufficient to relax the folded structure of these molecules, without affecting disulfide bonds.
  • the denaturing conditions mimic the environment of an intracelluiar vesicle sufficiently such that the cleaved pro domain has an opportunity to associate with the mature dimeric species under relaxed folding conditions.
  • the concentration of denaturant in the solution then is decreased in a controlled, preferably step-wise manner, so as to allow proper refolding of the dimer and pro regions while maintaining the association of the pro domain with the dimer.
  • Useful denaturants include 4-6M urea or guanidine hydrochloride (GuHCl), in buffered solutions of pH 4-10, preferably pH 6-8.
  • the soluble complex then is formed by controlled dialysis or dilution into a solution having a final denaturant concentration of less than 0.1-2M urea or GuHCl, preferably 1-2 M urea of GuHCl, which then preferably can be diluted into a physiological buffer.
  • Protein purification/renaturing procedures and considerations are well described in the art, and details for developing a suitable renaturing protocol readily can be determined by one having ordinary skill in the art.
  • One useful text one the subject is Guide to Protein Purification, M. Lieber, ed.. Academic Press, San Diego, 1990, particularly section V. Complex formation also may be aided by addition of one or more chaperone proteins.
  • the stability of the highly purified soluble morphogen complex in a physiological buffer can be enhanced by any of a number of means.
  • a physiological buffer e.g., tris-buffered saline (TBS) and phosphate-buffered saline (PBS)
  • TBS tris-buffered saline
  • PBS phosphate-buffered saline
  • a pro region that comprises at least the first 18 amino acids of the pro sequence (e.g., residues 18-35 of Seq. ID NO. 1 for OP-3), and preferably is the full length pro region.
  • Residues 18-35 show sequence homology to the N-terminal portion of other morphogens and are believed to have particular utility in enhancing complex stability for all morphogens.
  • additives include basic amino acids (e.g., L-arginine, lysine and betaine); nonionic detergents (e.g., Tween 80 or Nonidet P-120); and carrier proteins (e.g., serum albumin and casein).
  • useful concentrations of these additives include 1-100 mM, preferably 10-70 mM, including 50 mM, basic amino acid;, 0.01-1.0%, preferably 0.05-0.2%, including 0.1% (v/v) nonionic detergent;, and 0.01-1.0%, preferably 0.05-0.2%, including 0.1% (w/v) carrier protein.
  • rat osteoblast-enriched primary cultures preferably are used. Although these cultures are heterogeneous in that the individual cells are at different stages of differentiation, the culture is believed to more accurately reflect the metabolism and function of osteoblasts ij vivo than osteoblast cultures obtained from established cell lines.
  • all chemicals referenced are standard, commercially available reagents, readily available from a number of sources, including Sigma Chemical, Co., St. Louis; Calbiochem, Corp., San Diego and Aldrich Chemical Co., Milwaukee.
  • Rat osteoblast-enriched primary cultures are prepared by sequential collagenase digestion of newborn suture-free rat calvaria (e.g., from 1-2 day-old animals, Long-Evans strain, Charles River Laboratories, Wilmington, MA), following standard procedures, such as are described, for example, in Wong et al., (1975) PNAS 21:3167-3171.
  • Rat osteoblast single cell suspensions then are plated onto a multi-well plate (e.g., a 24 well plate) at a concentration of 50,000 osteoblasts per well in alpha MEM (modified Eagle's medium, Gibco, Inc., Long Island) containing 10% FBS (fetal bovine serum) , L-glutamine and penicillin/streptomycin. The cells are incubated for 24 hours at 37°C, at which time the growth medium is replaced with alpha MEM containing 1% FBS and the cells incubated for an additional 24 hours so that cells are in serum-deprived growth medium at the time of the experiment.
  • the cultured cells are divided into three groups: (1) wells which receive, for example, 0.1, 1.0, 10.0, 40 and 80.0 ng of OP-3; (2) wells which receive 0.1, 1.0, 10.0 and 40 ng of a local-acting growth factor (e.g., TGF- ⁇ ); and (3) the control group, which receive no growth factors.
  • the cells then are incubated for an additional 18 hours after which the wells are pulsed with 2 ⁇ Ci/well of 3 H-thymidine and incubated for six more hours.
  • the excess label then is washed off with a cold solution of 0.15 M NaCl and then 250 ⁇ l of 10% tricholoracetic acid is added to each well and the wells incubated at room temperature for 30 minutes.
  • the cells then are washed three times with cold distilled water, and lysed by the addition of 250 ⁇ l of 1% sodium dodecyl sulfate (SDS) for a period of 30 minutes at 37°C
  • SDS sodium dodecyl sulfate
  • the resulting cell lysates are harvested using standard means well known in the art, and the incorporation of 3 H-thymidine into cellular DNA determined by liquid scintillation as an indication of mitogenic activity of the cells.
  • OP-3 is anticipated to stimulate 3 H-thymidine incorporation into DNA, and thus promote osteoblast cell proliferation.
  • the effect of TGF- ⁇ is transient and biphasic. At high concentrations, TGF- ⁇ has no significant effect on osteoblast cell proliferation.
  • the in vitro effect of OP-3 on osteoblast proliferation also may be evaluated using human primary osteoblasts (obtained from bone tissue of a normal adult patient and prepared as described above) and on human osteosarcoma-derived cell lines. In all cases OP-3 is anticipated to induce cell proliferation in accordance with the morphogen's ability to induce endochondral bone formation (see Example 7, below).
  • Useful naive stem cells include pluripotential stem cells, which may be isolated from bone marrow or umbilical cord blood using conventional methodologies, (see, for example. Faradji et al.,
  • a cultured mesodermal cell line may be useful.
  • Another method for obtaining progenitor cells and for determining the ability of OP-3 fragments to stimulate cell proliferation is to capture progenitor cells from an in vivo source.
  • a biocompatible matrix material able to allow the influx of migratory progenitor cells may be implanted at an m. vivo site long enough to allow the influx of migratory progenitor cells.
  • a bone-derived, guanidine-extracted matrix formulated as disclosed for example in Sampath et al. ((1983) PNAS 80:6591-6595), or U.S. Patent No. 4,975,526, may be implanted into a rat at a subcutaneous site, essentially following the method of Sampath et al. After three days the implant is removed, and the progenitor cells associated with the matrix dispersed and cultured.
  • Progenitor cells are incubated in vitro with OP-3 under standard cell culture conditions well described in the art and described hereinabove. In the absence of external stimuli, the progenitor cells do not, or only minimally, proliferate on their own in culture. However, progenitor cells cultured in the presence of a morphogenically active fragment of OP-3 are anticipated to proliferate. Cell growth can be determined visually or spectrophotometrically using standard methods well known in the art.
  • Morphogenically active fragments of OP-3 can be utilized to induce cell differentiation.
  • the ability of OP-3 to induce cell differentiation can be demonstrated by culturing early mesenchymal cells in the presence of OP-3 and then studying the histology of the cultured cells by staining with toluidine blue using standard cell culturing and cell staining methodologies well described in the art.
  • rat mesenchymal cells destined to become mandibular bone when separated from the overlying epithelial cells at stage 11 and cultured in vitro under standard tissue culture conditions, e.g., in a chemically defined, serum-free medium, containing for example, 67% DMEM (Dulbecco's modified Eagle's medium), 22% F-12 medium, lOmM Hepes pH 7, 2mM glutamine, 50 ⁇ g/ml transferrin, 25 ⁇ g/ml insulin, trace elements, 2mg/ml bovine serum albumin coupled to oleic acid, with HAT (0.1 mM hypoxanthine, lO ⁇ M aminopterin, 12 ⁇ M thymidine, will not continue to differentiate.
  • DMEM Disulbecco's modified Eagle's medium
  • F-12 medium containing for example, 67% DMEM (Dulbecco's modified Eagle's medium), 22% F-12 medium, lOmM Hepes pH 7, 2
  • Stage 11 mesenchymal cells cultured jji vitro in the presence of OP-3, e.g., 10-100 ng/ml, are anticipated to continue to differentiate ij vitro to form chondrocytes just as they continue to differentiate j-n vitro if they are cultured with the cell products harvested from the overlying endodermal cells.
  • This experiment may be performed with different mesenchymal cells to demonstrate the cell differentiation capability of OP-3 in different tissues.
  • osteoblast differentiation As another example of morphogen-induced cell differentiation, the ability of OP-3 to induce osteoblast differentiation may be demonstrated in vitro using primary osteoblast cultures, or osteoblast-like cells lines, and assaying for a variety of bone cell markers that are specific markers for the differentiated osteoblast phenotype, e.g., alkaline phosphatase activity, parathyroid hormone-mediated cyclic AMP (cAMP) production, osteocalcin synthesis, and enhanced mineralization rates.
  • cAMP parathyroid hormone-mediated cyclic AMP
  • the cultured cells in serum-free medium are incubated with, a range of OP-3 concentrations, for example, 0.1, 1.0, 10.0, 40.0 or 80.0 ng OP-3/ml medium; or with a similar range of TGF- ⁇ concentrations.
  • 72 hours after the incubation period the cell layer is extracted with 0.5 ml of 1% Triton X-100.
  • the resultant cell extract then, is centrifuged, and 100 ⁇ l of the extract is added to 90 ⁇ l of paranitrosophenylphospate (PNPP)/glycerine mixture and incubated for 30 minutes in a 37°C water bath and the reaction stopped with 100 ⁇ l NaOH.
  • PNPP paranitrosophenylphospate
  • the samples then are run through a plate reader (e.g., Dynatech MR700 plate reader, and absorbance measured at 400 nm, using p-nitrophenol as a standard) to determine the presence and amount of alkaline phosphate activity.
  • Protein concentrations are determined by the Biorad method.
  • Rat osteoblasts are prepared and cultured in multi- well plates as described above.
  • six sets of 24 well plates are plated with 50,000 rat osteoblasts per well.
  • Each plate then is incubated for different lengths of time: 0 hours (control time), 24 hours, 48 hours, 96 hours, 120 hours and 144 hours. After each incubation period, the cell layer is extracted with 0.5 ml of 1% Triton X- 100.
  • the resultant cell extract is centrifuged, and alkaline phosphatase activity determined as for Example 3.1, using paranitroso-phenylphosphate (PNPP).
  • PNPP paranitroso-phenylphosphate
  • OP-3 stimulates the production of alkaline phosphatase in osteoblasts in dose-dependent manner so that increasing doses of OP-3 further increase the level of alkaline phosphatase production, and moreover, the OP-3-stimulated elevated levels of alkaline phosphatase in the treated osteoblasts is anticipated to last for an extended period of time.
  • PNPP paranitroso-phenylphosphate
  • Rat osteoblasts are prepared and cultured in a multiwell plate as described above.
  • the cultured cells then are divided into three groups: (1) wells which receive, for example, 1.0, 10.0 and 40.0 ng OP-3/ml medium); (2) wells which receive for example, TGF- ⁇ , at similar concentration ranges; and (3) a control group which receives no growth factors.
  • the plate is then incubated for another 72 hours. At the end of the 72 hours the cells are treated with medium containing 0.5% bovine serum albumin (BSA) and ImM 3-isobutyl-l- methylxanthine for 20 minutes followed by the addition into half of the wells of human recombinant parathyroid hormone (hPTH, Sigma, St.
  • BSA bovine serum albumin
  • hPTH human recombinant parathyroid hormone
  • Osteocalcin is a bone-specific protein synthesized by osteoblasts which plays an integral role in the rate of bone mineralization in vivo. Circulating levels of osteocalcin in serum are used as a marker for osteoblast activity and bone formation in vivo. Induction of osteocalcin synthesis in osteoblast- enriched cultures can be used to demonstrate OP-3 morphogenic efficacy in vitro.
  • Rat osteoblasts are prepared and cultured in a multi-well plate as above.
  • the medium is supplemented with 10%FBS, and on day 2, cells are fed with fresh medium supplemented with fresh 10 mM ⁇ -glycerophosphate (Sigma, Inc.). Beginning on day 5 and twice weekly thereafter, cells are fed with a complete mineralization medium containing all of the above components plus fresh L(+)-ascorbate, at a final concentration of 50 ⁇ g/ml medium.
  • OP-3 then is added to the wells directly, e.g., in 50% acetonitrile (or 50% ethanol) containing 0.1% trifluoroacetic acid (TFA), at no more than 5 ⁇ l morphogen/ml medium. Control wells receive solvent vehicle only.
  • the cells then are re-fed and the conditioned medium sample diluted 1:1 in standard radioimmunoassay buffer containing standard protease inhibitors and stored at -20° C until assayed for osteocalcin.
  • Osteocalcin synthesis is measured by standard radioimmunoassay using a commercially available osteocalcin-specific antibody.
  • Mineralization is determined on long term cultures (13 day) using a modified von Kossa staining technique on fixed cell layers: cells are fixed in fresh 4% paraformaldehyde at 23° C for 10 min, following rinsing cold 0.9% NaCl.
  • OP-3 stimulates osteocalcin synthesis in osteoblast cultures.
  • the increased osteocalcin synthesis in response to OP-3 is dose dependent and shows a significant increase over the basal level after 13 days of incubation.
  • the enhanced osteocalcin synthesis also can be confirmed by detecting the elevated osteocalcin mRNA message (20-fold increase) using a rat osteocalcin-specific probe.
  • the increase in osteoclacin synthesis correlates with increased mineralization in long term osteoblast cultures as determined by the appearance of mineral nodules.
  • OP-3 increases the initial mineralization rate significantly compared to untreated cultures. 3.5 Morphogen-Induced CAM Expression
  • CAM expression particularly N-CAM expression
  • CAMs are morphoregulatory molecules identified in all tissues as an essential step in tissue development.
  • N-CAMs which comprise at least 3 isoforms (N-CAM-180, N-CAM-140 and N-CAM-120, where "180", “140” and “120” indicate the apparent molecular weights of the isoforms as measured by SDS polyacrylamide gel electrophoresis) are expressed at least transiently in developing tissues, and permanently in nerve tissue. Both the N-CAM-180 and N- CAM-140 isoforms are expressed in both developing and adult tissue. The N-CAM-120 isoform is found only in adult tissue. Another neural CAM is LI.
  • NG108-15 cells are a transformed hybrid cell line (neuroblastoma x glioma, America Type Tissue Culture (ATCC), Rockville, MD), exhibiting a morphology characteristic of transformed embryonic neurons.
  • ATCC America Type Tissue Culture
  • NG108-15 cells exhibit a fibroblastic, or minimally differentiated, morphology and express only the 180 and 140 isoforms of N-CAM normally associated with a developing cell. Following morphogen treatment these cells exhibit a morphology characteristic of adult neurons and express enhanced levels of all three N-CAM isoforms.
  • NG108-15 cells are cultured for 4 days in the presence of increasing concentrations of OP-3 using standard culturing procedures, and standard Western blots performed on whole cell extracts.
  • N-CAM isoforms are detected with an antibody which crossreacts with all three isoforms, mAb H28.123, obtained from Sigma Chemical Co., St. Louis, the different isoforms being distinguishable by their different mobilities on an electrophoresis gel.
  • Control NG108-15 cells (untreated) express both the 140 kDa and the 180 kDa isoforms, but not the 120 kDa, as determined by Western blot analyses using up to 100 ⁇ g of protein.
  • OP-3 Treatment of NG108-15 cells with OP-3 results in a dose-dependent increase in the expression of the 180 kDa and 140 kDa isoforms, as well as the induction of the 120 kDa isoform induced.
  • OP-3-induced CAM expression correlates with cell aggregation, as determined by histology.
  • OP-3 morphogens described herein also can induce redifferentiation of transformed cells to a morphology characteristic of untransformed cells.
  • the examples provided below detail morphogen-induced redifferentiation of a transformed human cell line of neuronal origin (NG108-15); as well as mouse neuroblastoma cells (N1E-115), and human embryo carcinoma cells, to a morphology characteristic of untransformed cells.
  • NG108-15 is a transformed hybrid cell line produced by fusing neuroblastoma x glioma cells (obtained from ATTC, Rockville, MD), and exhibiting a morphology characteristic of transformed embryonic neurons, e.g., having a fibroblastic morphology.
  • the cells have polygonal cell bodies, short, spike-like processes and make few contacts with neighboring cells (see copending USSN 922,813).
  • Incubation of NG108-15 cells, cultured in a chemically defined, serum-free medium, with 0.1 to 300 ng/ml of morphogen (e.g; OP-3) for four hours is anticipated to induce an orderly, dose-dependent change in cell morphology.
  • morphogen e.g; OP-3
  • NG108-15 cells are subcultured on poly-L-lysine coated 6 well plates. Each well contains 40-50,000 cells in 2.5 ml of chemically defined medium. On the third day, 2.5 ⁇ l of morphogen (e.g., OP-3) in 60% ethanol containing 0.025% trifluoroacetic is added to each well. Morphogenic OP-3 of varying concentrations are tested (typically, concentration ranges of 0-300 ng/ml are tested). The media is changed daily with new aliquots of morphogen.
  • morphogen e.g., OP-3
  • OP-3 is anticipated to induce a dose-dependent redifferentiation of the transformed cells, including a rounding of the soma, an increase in phase brightness, extension of the short neurite processes, and other significant changes in the cellular ultrastructure.
  • treated cells should begin to form epithelioid sheets that then become highly packed, multi-layered aggregates, as determined visually by microscopic examination.
  • morphogen-induced redifferentiation occurs without any associated changes in DNA synthesis, cell division, or cell viability, making it unlikely that the morphologic changes are secondary to cell differentiation or a toxic effect of the morphogen.
  • OP-3 maintains cell stability and viability after inducing redifferentiation.
  • the OP-3 morphogens described herein accordingly provide useful therapeutic agents for the treatment of neoplasias and neoplastic lesions of the nervous system, particularly in the treatment of neuroblastomas, including retinoblastomas, and gliomas.
  • the ability of OP-3 to induce the "redifferentiation" of transformed human cells may be demonstrated using the following assay. Specifically, the effect of OP-3 on human EC cells (embryo carcinoma cells, e.g., NTERA-Z CL.Dl,
  • ATCC ATCC, Rockville, MD
  • these cells can be maintained as undifferentiated stem cells, and can be induced to grow in serum free media (SFM).
  • SFM serum free media
  • the cells proliferate rampantly and are anchorage-independent.
  • EC cells grow as flattened cells, becoming anchorage dependent and forming aggregates.
  • growth rate is reduced approximately 10 fold.
  • the cells are induced to differentiate.
  • concentrations of OP-3 e.g., 0-300 ng/ml
  • cultured cells e.g., 40-50,000 cells in 2.5 ml chemically defined medium
  • OP-3 is anticipated to stimulate redifferentiation of these cells to a morphology characteristic of untransformed embryo cells.
  • Morphogenically active fragments of OP-3 also may be used to maintain a cell's differentiated phenotype. This application is particularly useful for inducing the continued expression of phenotype in senescent or quiescent cells.
  • morphogens The phenotypic maintenance capability of morphogens is determined readily. A number of differentiated cells become senescent or quiescent after multiple passages in vitro under standard tissue culture conditions well described in the art (e.g.. Culture of Animal Cells: A Manual of Basic Techniques, C.R. Freshney, ed., Wiley, 1987). However, if these cells are cultivated in vitro in association with a morphogen such as OP-3, cells are stimulated to maintain expression of their phenotype through multiple passages. For example, the alkaline phosphatase activity of cultured osteoblasts, such as.cultured osteosarcoma cells and calvaria cells, is significantly reduced after multiple passages in vitro.
  • a morphogen such as OP-3
  • osteoblasts are cultured as described in Example 2. The cells are divided into groups, incubated with varying concentrations of OP-3 (e.g., 0-300 ng/ml) and passaged multiple times (e.g., 3-5 times) using standard methodology. Passaged cells then are tested for alkaline phosphatase activity, as described in Example 3 as an indication of differentiated cell metabolic function. Osteoblasts cultured in the absence of OP-3 should have reduced alkaline phosphatase activity, as compared to OP-3-treated cells.
  • concentrations of OP-3 e.g., 0-300 ng/ml
  • Passaged cells then are tested for alkaline phosphatase activity, as described in Example 3 as an indication of differentiated cell metabolic function. Osteoblasts cultured in the absence of OP-3 should have reduced alkaline phosphatase activity, as compared to OP-3-treated cells.
  • Phenotypic maintenance capability also may be demonstrated in vivo, using a rat model for osteoporosis, as disclosed in international application PCT/US92/07432 (WO93/05751) .
  • Long Evans female rats (Charles River Laboratories, Wilmington, MA) are Sham-operated (control animals) or ovariectomized using standard surgical techniques, to produce an osteoporotic condition resulting from decreased estrogen production.
  • rats are systemically provided with phosphate buffered saline (PBS) or morphogen, (e.g., OP-3, 1-100 ⁇ g) for 21 days (e.g., by daily tail vein injection.)
  • PBS phosphate buffered saline
  • morphogen e.g., OP-3, 1-100 ⁇ g
  • the rats then are sacrificed and serum alkaline phosphatase levels, serum calcium levels, and serum osteocalcin levels are determined, using standard methodologies as described therein and above. Elevated levels of osteocalcin and alkaline phosphatase should be observed in the rats treated with an effective amount of OP-3.
  • histomorphometric analysis on the tibial diasypheal bone is anticipated to show improved bone mass in OP-3-treated animals as compared with untreated, ovariectomized rats.
  • the bone mass of 0P-3- animals is anticipated to be comparable to (e.g., approaches) that of the sham-operated (e.g., nonovarectomized) rats.
  • Progenitor cells may be stimulated to proliferate in vivo or ex_ vivo.
  • the cells may be stimulated in vivo by injecting or otherwise providing a sterile preparation containing the morphogenically active fragment of OP-3 into the individual.
  • the hemopoietic pluripotential stem cell population of an individual may be stimulated to proliferate by injecting or otherwise providing an appropriate concentration of OP-3 to the individual's bone marrow.
  • Progenitor cells may be stimulated ex vivo by contacting progenitor cells of the population to be enhanced with a morphogenically active fragment of OP-3 under sterile conditions at a concentration and for a time sufficient to stimulate proliferation of the cells. Suitable concentrations and stimulation times may be determined empirically, essentially following the procedure described in Example 2, above. A morphogen concentration of between about 0.1-100 ng/ml and a stimulation period of from about 10 minutes to about 72 hours, or, more generally, about 24 hours, typically should be sufficient to stimulate a cell population of about 10 4 to 10 6 cells. The stimulated cells then are provided to the individual as, for example, by injecting the cells to an appropriate in vivo locus. Suitable biocompatible progenitor cells may be obtained by any of the methods known in the art or described hereinabove.
  • OP-3 may be used to repair diseased or damaged mammalian tissue.
  • the tissue to be repaired preferably is assessed first, and excess necrotic or interfering scar tissue removed as needed, e.g., by ablation or by surgical, chemical, or other methods known in the medical arts.
  • OP-3 then may be provided directly to the tissue locus as part of a sterile, biocompatible composition, either by surgical implantation or injection.
  • the morphogen also may be provided systemically, as by oral or parenteral administration.
  • a sterile, biocompatible composition containing progenitor cells stimulated by a morphogenically active fragment of OP-3 may be provided to the tissue locus.
  • the existing tissue at the locus provides the appropriate matrix to allow the proliferation and tissue-specific differentiation of progenitor cells.
  • a damaged or diseased tissue locus particularly one that has been further assaulted by surgical means, provides a morphogenically permissive environment.
  • Systemic provision of OP-3 should be sufficient for certain applications (e.g., in the treatment of osteoporosis and other disorders of the bone remodeling cycle, as an example).
  • the tissue may not be capable of providing a sufficient matrix for cell influx and proliferation.
  • it may be necessary to provide OP-3 or progenitor cells stimulated by OP-3 to the tissue locus in association with a suitable, biocompatible, formulated matrix, prepared by any of the means described below.
  • the matrix preferably is in vivo biodegradable.
  • the matrix also may be tissue-specific and/or may comprise porous particles having dimensions within the range of 70-850 ⁇ m, most preferably 150-420 ⁇ m.
  • OP-3 also may be used to prevent or substantially inhibit immune/inflammatory response-mediated tissue damage and scar tissue formation following an injury.
  • OP-3 is provided to a newly injured tissue locus, to induce tissue morphogenesis at the locus, preventing the aggregation of migrating fibroblasts into non- differentiated connective tissue.
  • OP-3 preferably is provided as a sterile pharmaceutical preparation injected into the tissue locus within five hours of the injury. Where an immune/inflammatory response is unavoidably or deliberately induced, as part of, for example, a surgical or other aggressive clinical therapy, OP-3 preferably is provided prophylactically to the patient, prior to, or concomitant with, the therapy.
  • a particularly useful mammalian tissue model system for demonstrating and evaluating the morphogenic activity of a protein is the endochondral bone tissue morphogenesis model known in the art and described, for example, in U.S. Pat. No. 4,968,590.
  • the ability to induce endochondral bone formation includes the ability to induce the proliferation of progenitor cells into chondroblasts and osteoblasts, the ability to induce cartilage matrix formation, cartilage calcification, and bone remodeling, and the ability to induce formation of an appropriate vascular supply and hematopoeitic bone marrow differentiation.
  • the local environment in which the morphogenic material is placed is important for tissue morphogenesis.
  • "local environment” is understood to include the tissue structural matrix and the environment surrounding the tissue.
  • the cells stimulated by morphogens need signals to direct the tissue-specificity of their differentiation. These signals vary for the different tissues and may include cell surface markers.
  • vascularization of new tissue requires a local environment which supports vascularization.
  • compositions may be injected or surgically implanted in a mammal, following any of a number of procedures well known in the art.
  • surgical implant bioassays may be performed essentially following the procedure of Sampath et al. (1983) PNAS 8 :6591-6595 and U.S. Pat No. 4,968,590. Histological sectioning and staining is preferred to determine the extent of morphogenesis i vivo, particularly in tissue repair procedures.
  • Excised implants are fixed in Bouins Solution, embedded in paraffin, and cut into 6-8 ⁇ m sections. Staining with toluidine blue or hemotoxylin/eosin demonstrates clearly the ultimate development of the new tissue. Twelve day implants are usually sufficient to determine whether the implants contain newly induced tissue.
  • the stages include: (1) leukocytes on day one; (2) mesenchymal cell migration and proliferation on days two and three;
  • cartilage matrix formation on day seven (4) cartilage matrix formation on day seven; (5) cartilage calcification on day eight; (6) vascular invasion, appearance of osteoblasts, and formation of new bone on days nine and ten; (7) appearance of osteoclastic cells, and the commencement of bone remodeling and dissolution of the implanted matrix on days twelve to eighteen; and (8) hematopoietic bone marrow differentiation in the resulting ossicles on day twenty-one.
  • tissue morphogenesis In addition to histological evaluation, biological markers may be used as markers for tissue morphogenesis.
  • Useful markers include tissue-specific enzymes whose activities may be assayed (e.g., spectrophotometrically) after homogenization of the implant. These assays may be useful for quantitation and for rapidly obtaining an estimate of tissue formation after the implants are removed from the animal. For example, alkaline phosphatase activity may be used as a marker for osteogenesis.
  • Incorporation of systemically provided OP-3 may be followed using tagged fragments (e.g., radioactively labelled) and determining their localization in the new tissue, and/or by monitoring their disappearance from the circulatory system using a standard labeling protocol and pulse-chase procedure.
  • OP-3 also may be provided with a tissue-specific molecular tag, whose uptake may be monitored and correlated with the concentration of OP-3 provided.
  • tissue-specific molecular tag whose uptake may be monitored and correlated with the concentration of OP-3 provided.
  • ovary removal in female rats results in reduced bone alkaline phosphatase activity, and renders the rats predisposed to osteoporosis (as described in Example 5). If the female rats now are provided with OP-3, a reduction in the systemic concentration of calcium should be seen, which correlates with the presence of the provided OP-3 and which is anticipated to correspond with increased alkaline phosphatase activity.
  • a method for inducing morphogenesis of substantially injured liver tissue following a partial hepatectomy utilizing OP-3 is presented. Variations on this general protocol may be used to test morphogen activity of OP-3 in other different tissues.
  • the general method involves excising an essentially nonregenerating portion of a tissue and providing OP-3, preferably as a soluble pharmaceutical preparation to the excised tissue locus, closing the wound, and examining the site at a future date.
  • OP-3 preferably as a soluble pharmaceutical preparation to the excised tissue locus
  • OP-3 e.g., 1 mg/ml
  • a biocompatible solution for example, (e.g., a purified recombinant mature form of OP-3, is solubilized in 50% ethanol, or compatible solvent, containing 0.1% trifluoroacetic acid, or compatible acid.
  • the mature protein may be solubilized by association with a pro domain.
  • the injectable OP-3 solution is prepared, e.g., by diluting one volume of OP-3 solvent-acid stock solution with 9 volumes of 0.2% rat serum albumin in sterile PBS (phosphate-buffered saline).
  • OP-3 phosphate buffered saline/rat serum albumin
  • Placebo samples are injection buffer only.
  • five rats in each group preferably are used. The wound is closed and the rats are allowed to eat normal food and drink tap water.
  • the rats are sacrificed and liver regeneration is observed visually, to evaluate the effects of the OP-3 on liver regeneration most effectively.
  • the OP-3 fragment-injected group is anticipated to show, e.g., complete liver tissue regeneration with no sign remaining of any cut in the liver.
  • the control group into which only PBS is injected show only minimal regeneration with the incision remaining in the sample.
  • Previous experiments with other morphogens show these morphogens alone induce liver tissue regeneration.
  • Pulp treatments used may include: a morphogenically active fragment of OP-3 dispersed in a carrier matrix; carrier matrix alone, and no treatment. Twelve teeth per animal (four for each treatment) are prepared, and two animals are used. At four weeks, teeth are extracted and processed histologically for analysis of dentin formation, and/or ground to analyze dentin mineralization.
  • demineralized teeth and OP-3 into surgically prepared canine tooth sockets is anticipated to stimulate new periodontal tissue formation, including new cementum and periodontal ligament, as well as new alveolar bone and dentin tissue, as described for OP-1 in international application PCT/US92/08742, filed 9/15/93.
  • untreated teeth or teeth treated with carrier vehicle alone do not induce periodontal tissue growth.
  • the induction of regenerative effects on central nervous system (CNS) repair, by a morphogenically active fragment of OP-3 may be demonstrated using a rat brain stab model.
  • CNS central nervous system
  • male Long Evans rats are anesthetized and the head area prepared for surgery.
  • the calvariae is exposed using standard surgical procedures and a hole drilled toward the center of each lobe using a 0.035K wire, just piercing the calvariae.
  • 25 ⁇ l solutions containing either morphogen (e.g., OP-3, 25 ⁇ g) or PBS then is provided to each of the holes by Hamilton syringe. Solutions are delivered to a depth approximately 3 mm below the surface, into the underlying cortex, corpus callosum and hippocampus. The skin then is sutured and the animal allowed to recover.
  • glial fibrillary acidic protein a marker protein for glial scarring, to qualitatively determine the degree of scar formation. Sections also are probed with OP-3-specific antibody to determine the presence of the protein. Reduced levels of glial fibrillary acidic protein are anticipated to be observed in the tissue sections of animals treated with OP-3, evidencing the ability of the morphogen to inhibit glial scar formation, thereby stimulating nerve regeneration.
  • OP-3 to stimulate peripheral nervous system axonal growth over extended distances may be demonstrated using the following model.
  • Neurons of the peripheral nervous system can sprout new processes on their own following injury, but without guidance these sproutings typically fail to connect appropriately and die. Where the break is extensive, e.g., greater than 5 or 10 mm, regeneration is poor or nonexistent.
  • Previous experiments with other morphogens, e.g., OP-1 show that morphogens stimulate peripheral nervous system axonal growth over extended distances, allowing repair and regeneration of damaged peripheral neural pathways.
  • OP-3 stimulation of nerve regeneration is demonstrated using the rat sciatic nerve model.
  • the rat sciatic nerve can regenerate spontaneously across a 5 mm gap, and occasionally across a 10 mm gap, provided that the severed ends are inserted in a saline-filled nerve guidance channel.
  • nerve regeneration across at least a 12mm gap is tested.
  • the central 12 mm of the tube consists of an OP-3 gel prepared by mixing 1 to 5 ⁇ g of substantially pure recombinantly produced OP-3 protein with approximately 100 ⁇ l of MATRIGELTM (from Collaborative Research, Inc., Bedford, MA), an extracellular matrix extract derived from mouse sarcoma tissue, and containing solubilized tissue basement membrane, including laminin, type IV collagen, heparin sulfate, proteoglycan and entactin, in phosphate- buffered saline.
  • the morphogen-filled tube then is implanted directly into the defect site, allowing 4 mm on each end to insert the nerve stumps. Each stump is abutted against the morphogen gel and is secured in the silicone tube by three stitches of commercially available surgical 10-0 nylon through the epineurium, the fascicle protective sheath.
  • Frozen sections then are cut throughout the graft site, and examined for axonal regeneration by immunofluorescent staining using anti-neurofilament antibodies labeled with flurocein (obtained, for example, from Sigma Chemical Co., St. Louis).
  • Determining the tissue distribution of morphogens may be used to identify different morphogens expressed in a given tissue, as well as to identify new, related morphogens. Tissue distribution also may be used to identify useful morphogen-producing tissue for use in screening and identifying candidate morphogen- stimulating agents.
  • the morphogens (or their mRNA transcripts) readily are identified in different tissues using standard methodologies and minor modifications thereof in tissues where expression may be low. For example, protein distribution may be determined using standard Western blot analysis or immunofluorescent techniques, and antibodies specific to the morphogen or morphogens of interest. Similarly, the distribution of morphogen transcripts may be determined using standard Northern hybridization protocols and transcript-specific probes.
  • any probe capable of hybridizing specifically to a transcript, and distinguishing the transcript of interest from other, related transcripts may be used. Because the morphogens described herein share such high sequence homology in their active, C-terminal domains, the tissue distribution of a specific morphogen transcript may best be determined using a probe specific for the pro region of the immature protein and/or the N-terminal region of the mature protein. Another useful sequence is the 3' non-coding region flanking and immediately following the stop codon. These portions of the sequence vary substantially among the morphogens of this invention, and accordingly, are specific for each protein.
  • OP-3-specific probe sequence is one derived from a portion of the 3' untranslated sequence, e.g., nucleotides 1310-1674 of Seq. ID No. 1, which shares little or no homology with other morphogen sequences, including OP-2.
  • the chosen fragment then is labelled using standard means well known and described in the art.
  • morphogen transcripts can be identified in mammalian tissue, using standard methodologies well known to those having ordinary skill in the art. A detailed description of a suitable hybridization protocol is described in Ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn. 179:116-123, and Ozkaynak, et al. (1992) J. Biol. Chemistry
  • RNA is prepared from various tissues (e.g., murine embryo and developing and adult liver, kidney, testis, heart, brain, thymus, stomach) by a standard methodology such as by the method of Chomczyaski et al. ((1987) Anal. Biochem 162:156-159) and described below.
  • Poly (A)+ RNA is prepared by using oligo (dT)-cellulose chromatography (e.g.. Type 7, from Pharmacia LKB Biotechnology, Inc.). Poly (A)+ RNA (generally 15 ⁇ g) from each tissue is fractionated on a 1% agarose/formaldehyde gel and transferred onto a Nytran membrane (Schleicher & Schuell).
  • the membrane is baked at 80°C and the RNA is cross-linked under UV light (generally 30 seconds at 1 mW/cm 2 ) .
  • the appropriate probe is denatured by heating.
  • the hybridization is carried out in a lucite cylinder rotating in a roller bottle apparatus at approximately 1 rev/min for approximately 15 hours at 37°C using a hybridization mix of 40% formamide, 5 x Denhardts, 5 x SSPE, and 0.1% SDS.
  • the non-specific counts are washed off the filters in 0.1 x SSPE, 0.1% SDS at 50°C
  • OP-3-specific 0.5 kb probe was made from a Stul- Bglll fragment of OP-3 cDNA.
  • the fragment contains the 3' untranslated sequence from nucleotides 1310-1674, plus an additional 140 bases.
  • the fragment was labelled using standard techniques and the hybridization performed as described.
  • OP-3 like OP-2, appears to be expressed primarily in early embryonic tissue. Specifically, Northern blots of murine embryos show abundant OP-3 expression in 8-day embryos, demonstrated by a strong band at 2.9 kb and a weaker band at 2.3 kb.
  • Candidate compound(s) which may be administered to affect the level of endogenous OP-3 morphogen may be found using the following screening assay, in which the level of OP-3 production by a cell type which produces measurable levels of the morphogen is determined with and without incubating the cell in culture with the compound, in order to assess the effects of the compound on the cell. This can be accomplished by detection of the morphogen either at the protein or RNA level. A detailed description also may be found in international application PCT/US92/07359, (WO93/05172) .
  • kidneys may be explanted from neonatal or new born or young or adult rodents (mouse or rat) and used in organ culture as whole or sliced (1-4 mm) tissues.
  • Primary tissue cultures and established cell lines, also derived from kidney, adrenals, urinary, bladder, brain, mammary, or other tissues may be established in multiwell plates (6 well or 24 well) according to conventional cell culture techniques, and are cultured in the absence or presence of serum for a period of time (1-7 days).
  • Cells may be cultured, for example, in Dulbecco's Modified Eagle medium (Gibco, Long Island, NY) containing serum (e.g., fetal calf serum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or in defined medium (e.g., containing insulin, transferrin, glucose, albumin, or other growth factors) .
  • serum e.g., fetal calf serum at 1%-10%, Gibco
  • serum-deprived medium e.g., fetal calf serum at 1%-10%, Gibco
  • defined medium e.g., containing insulin, transferrin, glucose, albumin, or other growth factors
  • Samples for testing the level of morphogen production includes culture supernatants or cell lysates, collected periodically and evaluated for morphogen production by immunoblot analysis (Sambrook et al., eds., 1989, Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, NY), or a portion of the cell culture itself, collected periodically and used to prepare polyA+ RNA for RNA analysis. To monitor de novo morphogen synthesis, some cultures are labeled according to conventional procedures with an
  • an immunoassay may be performed to detect the morphogen using a polyclonal or monoclonal antibody specific for that protein.
  • OP-3 may be detected using a polyclonal antibody specific for OP-3 in an ELISA, as follows.
  • a 100 ⁇ l aliquot of an appropriate dilution of each of the test samples of cell culture supernatant is added to each well in triplicate and incubated at 37°C for 30 min. After incubation, 100 ⁇ l biotinylated rabbit anti-OP-3 serum (stock solution is about 1 mg/ml and diluted 1:400 in BSB containing 1% BSA before use) is added to each well and incubated at 37°C for 30 min. The wells are then washed four times with BSB containing 0.1% Tween 20. 100 ⁇ l strepavidin-alkaline (Southern Biotechnology Associates, Inc.
  • Polyclonal antibody may be prepared as follows. Each rabbit is given a primary immunization of 100 ug/500 ⁇ l recombinantly-produced OP-3 protein or protein fragment in 0.1% SDS mixed with 500 ⁇ l Complete Freund's Adjuvant. The antigen is injected subcutaneously at multiple sites on the back and flanks of the animal. The rabbit is boosted after a month in the same manner using incomplete Freund's Adjuvant. Test bleeds are taken from the ear vein seven days later. Two additional boosts and test bleeds are performed at monthly intervals until antibody against OP-3 is detected in the serum using an ELISA assay. Then, the rabbit is boosted monthly with 100 ⁇ g of antigen and bled (15 ml per bleed) at days seven and ten after boosting.
  • Monoclonal antibody specific for a given morphogen may be prepared as follows. A mouse is given two injections of OP-3 protein or a protein fragment specific for OP-3. The protein preferably is recombinantly produced. The first injection contains lOO ⁇ g of OP-3 in complete Freund's adjuvant and is given subcutaneously. The second injection contains 50 ⁇ g of OP-3 in incomplete adjuvant and is given intraperitoneally. The mouse then receives a total of 230 ⁇ g of OP-3 in four intraperitoneal injections at various times over an eight month period.
  • the mouse is boosted intraperitoneally with OP-3 (e.g., 100 ⁇ g) and may be additionally boosted with an OP-3-specific peptide (e.g., corresponding to the N-terminus of the mature protein) conjugated to bovine serum albumin with a suitable crosslinking agent.
  • This boost can be repeated five days (IP) , four days (IP) , three days (IP) and one day (IV) prior to fusion.
  • the mouse spleen cells then are fused to commercially available myeloma cells at a ratio of 1:1 using PEG 1500 (Boeringer Mannheim, Germany), and the fused cells plated and screened for OP-3-specific antibodies using OP-3 as antigen.
  • the cell fusion and monoclonal screening steps readily are performed according to standard procedures well described in standard texts widely available in the art.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORIGINAL SOURCE
  • CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG TTT GAT CTT TCC AAG ATC 537 His Pro Arg Tyr His His His Arg Glu Phe Arg Phe Asp Leu Ser Lys He 150 155 160 CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TTC CGG ATC TAC AAG GAC 585 Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Asp 165 170 175
  • AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTCT 1593
  • CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTGCGC TGCTCGGACC 180 GCGGCCACAG CCGGACTGGC GGGTACGGCG GCGACAGAGG CATTGGCCGA GAGTCCCAGT 240
  • GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CCGACCAGCT 60
  • GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG ACG 641 Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr 170 175 180
  • CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA AAATTCTGGT 1439 CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA TCACCCCGCC CTCTCCATCC 1499
  • CTCTGCACCA TTCATTGTGG CAGTTGGGAC ATTTTTAGGT ATAACAGACA CATACACTTA 1739
  • MOLECULE TYPE DNA (genomic)
  • CAGGAGGTGG CACGGCAGGG CTGGAGGGCT CCCTATGAGT GGCGGAGACG GCCCAGGAGG 120
  • CGCCGCTCTT CATGCTGGAC CTGTACCACG CCATGGCCGG CGACGACGAC GAGGACGGCG 780 CGCCCGCGGA GCGGCGCCTG GGCCGCGCCG ACCTGGTCAT GAGCTTCGTT AACATGGGTG 840
  • CAGGAGCAGT CCAACAGGTG CCTTCCCCTT GGCCCGGGTG CCCACCTAAC CCCCCACCTC 1320
  • GACTTCAAGT CCCTGCCCTC CATCCTGCTG TGGGGACTTG GACATGGTCA CTGAGACTCA 1500
  • CAACGGGCCC CACGCTCCCA ACAGCCTTTC GTGGTCACTT TCTTCAGGGC CAGTCCGAGT 1980
  • CCCATCCGCA CCCCTCGGGC AGTGAGGCCA CTGAGGAGGA GGCAGCCGAA GAAAAGCAAC 2040
  • CAGGGGTCTC CAGAGAGGAG GAGGCACAGG ATGGCCGAGG GTCCTGCTGG GCTGTTTACT 2640 GGAGCATAAA GATGCTCATA GGCTGAAGGA CAGGGGAGGA CTGGGCACAG TGTCACTCTA 2700
  • GCTCCTGCCC AGCCTTTTCC GCCGGGGTTC CTGGGTGGAT TCAAGCCTCT TGGGGGAGAC 3420
  • CTCTGTGCTC TACTATGACA GCAGCAACAA
  • CGTCATCCTG CGCAAGCACC GCAACATGGT 6240
  • each Xaa is independently selected from a group of one or more specified amino acids as defined in the specification.

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Abstract

L'invention se rapporte à: (1) des séquences d'acide nucléique et d'acide aminé que renferme une nouvelle protéine morphogène; (2) des procédés produisant et exprimant la protéine sous une forme biologiquement active; et (3) des procédés d'utilisation de la protéine afin d'induire une morphogenèse tissulaire chez un mammifère, y compris des procédés augmentant une population de cellules géniteurs chez un mammifère, des procédés stimulant les cellules géniteurs pour différencier et maintenir leur phénotype différencié in vivo ou in vitro, des procédés induisant une croissance spécifique aux tissus in vivo et des procédés remplaçant les tissus malades ou endommagés in vivo.
EP94901244A 1992-11-03 1993-11-02 Morphogenese induite par l'op-3 Withdrawn EP0672064A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US40510 1987-04-20
US97109192A 1992-11-03 1992-11-03
US971091 1992-11-03
US2933593A 1993-03-04 1993-03-04
US29335 1993-03-04
US4051093A 1993-03-31 1993-03-31
PCT/US1993/010520 WO1994010203A2 (fr) 1992-11-03 1993-11-02 Morphogenese induite par l'op-3

Publications (1)

Publication Number Publication Date
EP0672064A1 true EP0672064A1 (fr) 1995-09-20

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EP94901244A Withdrawn EP0672064A1 (fr) 1992-11-03 1993-11-02 Morphogenese induite par l'op-3

Country Status (5)

Country Link
EP (1) EP0672064A1 (fr)
JP (1) JPH08503198A (fr)
AU (1) AU681362B2 (fr)
CA (1) CA2147598A1 (fr)
WO (1) WO1994010203A2 (fr)

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Also Published As

Publication number Publication date
JPH08503198A (ja) 1996-04-09
AU5590094A (en) 1994-05-24
WO1994010203A2 (fr) 1994-05-11
CA2147598A1 (fr) 1994-05-11
AU681362B2 (en) 1997-08-28
WO1994010203A3 (fr) 1994-08-18

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