EP0968285A1 - Proteines morphogeniques - Google Patents

Proteines morphogeniques

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Publication number
EP0968285A1
EP0968285A1 EP98906592A EP98906592A EP0968285A1 EP 0968285 A1 EP0968285 A1 EP 0968285A1 EP 98906592 A EP98906592 A EP 98906592A EP 98906592 A EP98906592 A EP 98906592A EP 0968285 A1 EP0968285 A1 EP 0968285A1
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EP
European Patent Office
Prior art keywords
human
bmp
protein
dan
binding
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Withdrawn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
EP98906592A
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German (de)
English (en)
Inventor
David M. Valenzuela
Aris Economides
Richard M. Harland
David Hsu
Neil Stahl
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.)
University of California
Regeneron Pharmaceuticals Inc
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University of California
Regeneron Pharmaceuticals Inc
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Application filed by University of California, Regeneron Pharmaceuticals Inc filed Critical University of California
Publication of EP0968285A1 publication Critical patent/EP0968285A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the field of this invention is proteins which regulate cell function, and in particular, antagonize bone morphogenic proteins.
  • Natural regulators of cellular growth, differentiation and function have provided important pharmaceuticals, clinical and laboratory tools, and targets for therapeutic intervention.
  • a variety of such regulators have been shown to have profound effects on basic cellular differentiation and developmental pathways.
  • the recently cloned cerberus protein induces the formation of head structures in anterior endoderm of vertebrate embryos.
  • the noggin protein induces head structures in vertebrate embryos, and can redirect mesodermal fates from ventral fates, such as blood and mesenchyme, to dorsal fates such as muscle and notochord and can redirect epidermal fates to anterior neural fates.
  • chordin The activities of chordin are similar to those of noggin, reflecting a common mechanism of action - namely antagonizing bone morphogenic proteins (BMP) and thereby preventing their function.
  • BMPs have diverse biological activities in different biological contexts, including the induction of cartilage, bone and connective tissue, and roles in kidney, tooth, gut, skin and hair development.
  • TGF ⁇ induces neural crest to form smooth muscle
  • BMP2 induces the same cells to become neurons.
  • dissociated animal cap cells proteosectoderm
  • BMP2 BMP4 and BMP7 induce endochondral bone formation
  • BMP12/GDF7 a related molecule BMP12/GDF7 induces connective tissue similar to tendon
  • BMP4 can induce cell death in the hindbram neural crest, while the related protein dorsahn does not
  • BMP antagonists that have specificity in blocking subsets of BMPs could change the balance of BMPs that are presented to a cell, thus altering cell fate
  • regulators of cellular function and BMP function in particular such as noggin and cerberus, provide valuable reagents with a host of clinical and biotechnological applications
  • the present invention relates to a new family of regulators of cellular function
  • the invention provides methods and compositions relating to DAN (Differential-screening-selected gene Aberrative in Neuroblastoma) and b57 proteins and related nucleic acids. Included are natural DAN and b57 homologs from different species, as well as proteins comprising a DAN or b57 domain and having DAN or b57-specific activity, particularly the ability to antagonize a bone morphogenic protein such as BMP2 or BMP4.
  • the proteins may be produced recombinantly from transformed host cells with the subject nucleic acids.
  • the invention provides isolated hybridization probes and primers capable of specifically hybridizing with the disclosed genes, specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g., genetic hybridization screens for b57 transcripts), therapy (e.g., gene therapy to modulate b57 gene expression) and in the biopharmaceutical industry (e.g., reagents for screening chemical libraries for lead pharmacological agents).
  • diagnosis e.g., genetic hybridization screens for b57 transcripts
  • therapy e.g., gene therapy to modulate b57 gene expression
  • biopharmaceutical industry e.g., reagents for screening chemical libraries for lead pharmacological agents.
  • Preferred applications of the subject DAN and b57 proteins include modifying the physiology of a cell comprising an extracellular surface by contacting the cell or medium surrounding the cell with an exogenous DAN or b57 protein under conditions whereby the added protein specifically interacts with a component of the medium and /or the extracellular surface to effect a change in the physiology of the cell.
  • Also preferred are methods for screening for biologically active agents which methods involve incubating a DAN or b57 protein in the presence of an extracellular DAN or b57 protein-specific binding target and a candidate agent, under conditions whereby, but for the presence of the agent, the protein specifically binds the binding target at a reference affinity; detecting the binding affinity of the protein to the binding target to determine an agent- biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that the agent modulates the binding of the protein to the binding target.
  • FIGURES 1A - IB - Demonstration that human b57 binds to BMP2 and BMP4.
  • FIGURE 2 Demonstration that human b57 blocks BMP2 biological activity.
  • FIGURE 3 - Xenopus b57 (also referred to as Gremlin) blocks the activity of BMP2.
  • BMP2 at 78pM, 156pM, 313 pM, 625 pM, 1.25 nM, 2.5 nM or 5 nM was preincubated with a Gremlin COS supernatant at final concentration of 83nM or 21 nM Gremlin, mock-transfected media, or fresh DMEM prior to addition to cells. Alkaline phosphatase activity was assayed 24 hours later. Approx. 83nM Gremlin completely blocks BMP2 activity. Approx. 21nM Gremlin partially blocks BMP2 doses tested.
  • the invention provides DAN and b57 proteins which include natural DAN and b57 proteins and recombinant proteins comprising a DAN or b57 amino acid sequence, or a functional DAN or b57 protein domain thereof having an assav-discernable DAN or b57-specific activity. Accordingly, the proteins may be deletion mutants of the disclosed natural DAN and b57 proteins and may be provided as fusion products, e.g., with non-b57 polypeptides.
  • the subject DAN and b57 protein domains have DAN or b57-specific activity or function and are functionally distinct from each other and from cerberus and noggin homologs.
  • Such domains include at least 6 and preferably at least 8 consecutive residues of a natural DAN or b57 protein (See DAN sequence reported by Enomoto, et al. (1994) Oncogene 9: 2785-2791 and human b57 sequence disclosed herein).
  • Preferred b57 proteins comprise a b57 sequence conserved across species.
  • DAN is an intracellular zinc finger protein
  • the natural DAN protein is structurally and functionally related to b57 and that both it and DAN proteins as described herein are extracellularly active as antagonists of certain morphogenic proteins such as BMPs DAN or b57-spec ⁇ f ⁇ c activity or function may be determined by convenient in vitro, cell-based, or in vivo assays - e g , in vitro binding assays, cell culture assays, in animals (e g , immune response, gene therapy, transgemcs, etc ), etc Binding assays encompass any assay where the specific molecular interaction of a DAN or b57 protein with a binding target is evaluated
  • the binding target may be a natural binding target such as a TGF ⁇ protein, a morphogenic protein, preferably a bone morphogenic protein such as BMP2 or BMP4, chaperone, or other regulator that directly modulates DAN or b57 activity or its localization, or
  • the claimed proteins may be isolated or pure - an isolated protein is one that is no longer accompanied by some of the material with which it is associated in its natural state, and that preferably constitutes at least about 0 5%, and more preferablv at least about 5% by weight of the total protein m a given sample, a "pure" protein constitutes at least about 90%, and preferably at least about 99% by weight of the total protein in a given sample
  • the subject proteins and protein domains may be synthesized, produced by recombinant technology, or purified from cells.
  • a wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e g , Molecular Cloning, A Laboratory Manual (Sambrook, et al , Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds Ausubel, et al , Greene Publ Assoc , Wiley-lnterscience, NY)
  • the subject proteins find a wide variety of uses including use as immunogens, targets in screening assays, bioactive reagents for modulating cell growth, differentiation and/or function, etc
  • the invention provides methods for modifying the physiology of a cell comprising an extracellular surface by contacting the cell or medium surrounding the cell with an exogenous DAN or b57 protein under conditions whereby the added protein specifically interacts with a component of the medium and /or the extracellular surface to effect a change m the physiology of the cell
  • the extracellular surface includes plasma membrane-associated receptors
  • the exogenous DAN or b57 refers to a protein not made by the cell or, if so, expressed at non-natural levels, times or physiologic locales
  • suitable media include in vitro culture media and physiological fluids such as blood, synovial fluid, etc
  • Effective administrations of subject proteins can be used to reduce undesirable (e , ectopic) bone formation, inhibit the growth of cells that require a morphogenic protein (e g , BMP
  • the invention provides b57 and DAN nucleic acids, which find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc , as well as use in detecting the presence of DAN and b57 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional DAN and b57 homologs and structural analogs
  • Xenopus and chick b57 sequence data was used to search the EST database of the I M A G E consortium and a human cDNA clone 272074 was discerned to contain homologous sequence homologous by DNA sequencing
  • the insert was cloned into CS105, a suitable vector for synthesis of synthetic mRNA (Turner, D L , and Wemtraub, H (1994) Genes Dev 8, 1434-47, Baker, J C , and Harland, R M (1996) Genes & Development 10) b57-spec ⁇ f ⁇ c function of the human gene product
  • Xenopus cerberus sequence data was used to search the EST database of the I M A G E consortium and human cDNA clone 272074 was discerned to contain homologous sequence
  • This clone was obtained from Genome Systems, Inc (St Louis, MO) and sequenced using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc , Foster City, CA)
  • the nucleotide sequence encoding human b57 is set forth herein as SEQ NO 1 and the deduced ammo acid sequence of human b57 is set forth herein as SEQ NO 2
  • the subject nucleic acids are of synthetic /non-natural sequences and/or are isolated, I e , no longer accompanied by some of the material with which it is associated in its natural state, preferably constituting at least about 0 5%, more preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleot ⁇ de(s) other than that which it is joined to on a natural chromosome
  • Nucleic acids comprising the nucleotide sequence of SEQ NO 1 or fragments thereof contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that to which it is joined on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is immediately flanked by a sequence other than that to which it is joined on a natural chromosome While the nucleic acids are usually
  • the amino acid sequences of the disclosed DAN and b57 proteins are used to back translate DAN or b57 protem-encoding nucleic acids optimized for selected expression systems (Holler, et al (1993) Gene 136 323-328, Martin, et al (1995) Gene 154 150-166) or used to generate degenerate ohgonucleotide primers and probes for use in the isolation of natural b57 encoding nucleic acid sequences ("GCG software, Genetics Computer Group, Inc , Madison, WI) DAN and b57 encoding nucleic acids may be part of expression ⁇ ectors and mav be incorporated into recombinant host cells, e g , tor expression and screening, for transgenic animals for functional studies such as the efficacv of candidate drugs for disease associated with b57 mediated signal transduction, etc Expression systems are selected and /or tailored to effect b57 protein structural and functional variants through alternative post-translational processing
  • the invention also provides for nucleic acid hybridization probes and replication/amplification primers having a DAN or b57 cDNA specific sequence and sufficient to effect specific hybridization with SEQ NO 1 Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5 x SSPE (0 18 M NaCI, 0 01 M NaP0 4 , pH7 7, 0 001 M EDTA) buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0 2 x SSPE, preferably hybridizing in a buffer comprising 50% formamide in 5 x SSPE buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0 2x SSPE buffer at 42°C DAN and b57 cDNA homologs can also be distinguished from other protein using alignment algorithms, such as BLASTX (Altschul, et al (1990) Basic Local Alignment Search Tool, J Mol Biol 215 403- 410)
  • DAN and b57 hybridization probes find use in identifying wild-type and mutant alleles in clinical and laboratory samples Mutant alleles are used to generate allele-specific ohgonucleotide (ASO) probes for high-throughput clinical diagnoses
  • DAN and b57 nucleic acids are also used to modulate cellular expression or intracellular concentration or availability of active DAN or b57 DAN and b57 inhibitory nucleic acids are typically antisense - single stranded sequences comprising complements of the disclosed natural b57 coding sequences
  • Antisense modulation of the expression of a given DAN or b57 protein may employ antisense nucleic acids operably linked to gene regulatory sequences
  • Cells are transfected with a vector comprising a DAN or b57 sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous DAN or b57 encoding mRNA Transcription of the antisense nucleic acid
  • the invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of DAN or b57 modulatable cellular function Generally, these screening methods involve assaying for compounds which modulate DAN or b57 interaction with a natural DAN or b57 binding target A wide variety of assays for binding agents are provided including protein-protem binding assays, immunoassays, cell based assays, etc Preferred methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds
  • In vitro binding assays employ a mixture of components including a DAN or b57 protein, which mav be part of a fusion product with another peptide or polypeptide, e g , a tag for detection or anchoring, etc
  • the assay mixtures comprise a natural DAN or b57 binding target, e g , a TGF ⁇ protein such as a BMP While native binding targets may be used, it is frequently preferred to use portions thereof as long as the portion provides binding affinity and avidity to the subject DAN or b57 conveniently measurable m the assav
  • the assay mixture also comprises a candidate pharmacological agent.
  • Candidate agents encompass numerous chemical classes, though typically they are organic compounds, preferably small organic compounds, and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents such as salts, buffers, neutral proteins, e.g., albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may also be included.
  • the mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding.
  • the mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the DAN or b57 specifically binds the cellular binding target, portion or analog with a reference binding affinity. Incubation periods are chosen for optimal binding but are also minimized to facilitate rapid, high throughput screening.
  • the agent-biased binding between the DAN or b57 and one or more binding targets is detected by any convenient way.
  • a separation step is often used to separate bound from unbound components. Separation may be effected by precipitation, immobilization, etc., followed by washing by, e.g., membrane filtration or gel chromatography.
  • one of the components usually comprises or is coupled to a label.
  • the label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc., or indirect detection such as an epitope tag, an enzyme, etc.
  • a variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g., through optical or electron density, radiative emissions, nonradiative energy transfers, or indirectly detected with antibody conjugates, etc.
  • a difference in the binding affinity of the DAN or b57 protein to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the DAN or b57protein to the corresponding binding target.
  • a difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.
  • the invention provides for a method for modifying the physiology of a cell comprising an extracellular surface in contact with a medium, said method comprising the step of contacting said medium with an exogenous DAN or b57 protein under conditions whereby said protein specifically interacts with at least one of a component of said medium and said extracellular surface to effect a change in the physiology of said cell.
  • the invention further provides for a method for screening for biologically active agents, said method comprising the steps of a) incubating a DAN or b57 protein in the presence of an extracellular DAN or b57 protein specific binding target and a candidate agent, under conditions whereby, but for the presence of said agent, said protein specifically binds said binding target at a reference affinity; b) detecting the binding affinity of said protein to said binding target to determine an agent-biased affinity, wherein a difference between the agent- biased affinity and the reference affinity indicates that said agent modulates the binding of said protein to said binding target.
  • One embodiment of the invention is an isolated b57 protein comprising the amino acid sequence as set forth in SEQ NO. 2 or a fragment thereof having b57- specific activity.
  • Another embodiment of the invention is a recombinant nucleic acid encoding b57 protein comprising the amino acid sequence as set forth in SEQ NO. 2 or a fragment thereof having b57- specific activity.
  • Still another embodiment is an isolated nucleic acid comprising a nucleotide sequence as set forth in SEQ NO. 1 or a fragment thereof having at least 18 consecutive bases of SEQ NO. 1 and sufficient to specifically hybridize with a nucleic acid having the sequence of SEQ NO. 1 in the presence of natural DAN and cerberus cDNA.
  • the present invention also provides for antibodies to the b57 protein described herein which are useful for detection of the protein in, for example, diagnostic applications. For preparation of monoclonal antibodies directed toward this b57 protein, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the monoclonal antibodies for diagnostic or therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad.
  • Chimeric antibody molecules may be prepared containing a mouse antigen-binding domain with human constant regions (Morrison et al, 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al.,
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dimtrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Gue ⁇ n) and Corynebactenum parvum
  • a molecular clone of an antibody to a selected b57 protein epitope can be prepared by known techniques Recombinant DNA methodology (see e g , Maniatis et al , 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof
  • Antibody fragments which contain the ldiotype of the molecule can be generated by known techniques
  • fragments include but are not limited to the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule, the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papam and a reducing agent
  • Antibody molecules may be purified by known techniques, e g , immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof
  • the invention further provides for a method of using a DAN or b57 protein or fragment thereof as an antagonist of the activity of a bone morphogenic protein (BMP)
  • BMP bone morphogenic protein
  • the invention provides for a method of antagonizing the function of a Bone Morphogenic Protein (BMP) which comprises contacting said BMP with b57
  • BMP Bone Morphogenic Protein
  • the method of the invention is carried out under conditions whereby the b57 binds to the BMP
  • the b57 is mammalian b57, and more preferably, human b57, or a fragment thereof capable of binding to the BMP
  • the human b57 is used to antagonize the function of BMP2 or BMP4
  • Antagonists to BMP's may be useful for preventing and treating BMP-related disorders of animals, especially of humans It was, therefore, an object of this invention to identify substances which effectively antagonize the function of BMP's in disease states in animals, preferably mammals, especially m humans It was another object of this invention to prepare novel compounds which inhibit BMP It was still another object of this invention to develop a method of antagonizing the functions of BMP's in disease states in mammals It was also an object of this invention to develop a method of preventing or treating disorders relating to the function of BMP's
  • BMPs In addition to their roles in normal bone formation, the BMPs appear to be involved in diseases in which they promote abnormal bone growth For example, BMPs have been reported to play a causative role m the disease known as Fibrodysplasia Ossificans Progressiva (FOP), in which patients grow an abnormal "second skeleton" that prevents any movement
  • FOP Fibrodysplasia Ossificans Progressiva
  • an object of the present invention is to provide a novel molecule for the treatment of diseases or disorders including, but not limited to,
  • b57 Fibrodysplasia Ossificans Progressiva (FOP) Since b57 is a blocker of BMP's, it offers hope as a therapeutic agent for this disease Additionally, abnormal bone growth can occur after hip replacement surgery and thus ruin the surgical outcome This is a more common example of pathological bone growth and a situation in which blockers of BMP's such as b57 may be therapeutically useful b57 may be useful as well as for treating other forms of abnormal bone growth, such as the pathological growth of bone following trauma, burns or spinal cord injury In addition, b57 may be useful for treating or preventing the undesirable actions of BMP's associated with the abnormal bone growth seen in connection with metastatic prostate cancer or osteosarcoma
  • the b57 nucleic acids, proteins, and peptides of the invention may be used to block BMP activity in mammals
  • compositions comprising a b57 molecule, as described herein and a suitable carrier
  • the active ingredient which may comprise the b57, should be formulated in a suitable carrier for systemic or local administration m vivo by any appropriate route including, but not limited to injection (e g , intravenous, mtrape ⁇ toneal, intramuscular, subcutaneous, endoneural, pe ⁇ neural, mtraspinal, mtraventncular, intravitreal, intrathecal etc ), by absorption through epithelial or mucocutaneous linings (e g , oral mucosa, rectal and intestinal mucosa, etc ), or by a sustained release implant, including a cellular or tissue implant
  • the active ingredient may be formulated in a liquid carrier such as saline, incorporated into hposomes, microcapsules, polymer or wax-based and controlled release preparations, or formulated into tablet, pill or capsule forms
  • the invention further provides for the use of von Willebrand factor to regulate or modulate the activity of a BMP
  • von Willebrand factor By aligning the carboxy- terminal domains of chicken (chB57), human (hB57) and Xenopus b57 (xB57), human and mouse DAN, mouse and Xenopus cerberus and human von Willebrand factor (VWF), (see Table 1) applicants have discovered a striking homology among these various proteins, including the conservation of nine separate cysteme residues Given this striking homology, it is expected that von Willebrand factor may also be useful for regulating or modulating the activity of a BMP
  • the following protocol may be used for a high throughput human b57 - BMP binding assay:
  • Blocking buffer 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.
  • BMP lO- 7 - 10- 4 M biotinylated BMP in PBS.
  • Xenopus cerberus sequence data was used as a probe to search the EST database of the I.M.A.G.E. consortium and human cDNA clone 272074 was discerned to contain homologous sequence.
  • This clone was obtained from Genome Systems, Inc. (St. Louis, MO) and sequenced using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA).
  • the nucleotide sequence encoding human b57 is set forth as SEQ. NO. 1 and the deduced amino acid sequence of human b57 is set forth as SEQ. NO. 2.
  • CAGACCATCC ACGAGGAAGG CTGCAACAGT CGCACCATCA TCAACCGCTT CTGTTACGGC
  • Human b57 belongs to a family of proteins that includes cerberus (Bouwmeester et al , 1996) and DAN (Enomoto et al., 1994) - see Table 1 These b57 relatives have been postulated to function as antagonists for different members of the bone morphogenetic protein (BMP) family
  • BMP bone morphogenetic protein
  • the BMP family has many different members displaying varying degrees of homology to each other and it includes not only the BMPs, but also the growth differentiation factors (GDFs), transforming growth factor beta and its homologues (TGF ⁇ s), the activins, the inhibins, the dorsahns, as well as nodal, vegetal, vegetal-related, and several new members (Furuta et al., 1997) BMPs have been shown to play important role in many different biological processes and thus the existence of naturally occurring antagonists of their activity is of great interest and pharmacological potential
  • BMP antagonists include noggm (Smith and Harland, 1992, Zimmerman et al , 1996), chordin (Piccolo et al , 1996), and folhstatin (Hemmati-B ⁇ vanlou et al., 1994) These do not belong to the cerberus/b57/DAN family, and they also do not share any homology with each other Nonetheless, noggm and chordin have been shown to bind to BMP2 and BMP4 and inhibit their biological actions by blocking the interaction with the BMP receptors
  • a DNA fragment encoding the gene for human b57 (hb57) was PCR amplified from an EST clone using the primers
  • Nl-hb57 (5'-GAGAGTCATGAAAAAGAAAGGGTCCCAAGGTGC-3') and Cl-hb57 (5'-GAGAGGCGGCCGCTCATTAATCCAAATCGATGGATATGCAAC-
  • E coli strain RFJ143 containing pRG622 was grown in LB medium and expression of hb57 was induced by the addition of 1 mM IPTG Induced cells were collected by centrifugation, resuspended in 10 volumes of 100 mM T ⁇ s- HC1, pH 8 5, 20 mM EDTA, and lysed by passage through a Niro-Soave Panda cell disrupter The cell lysate was centnfuged and the pellet was resuspended in 10 volumes of 9 M urea, 50 mM Tns-HCl, pH 8 5, 1 mM EDTA, 100 mM Na2S03, 10 mM Na2S406 and stirred for 16 hr at room temperature The solubihzed inclusion bodies were fractionated on a Sephacryl S-300 column equilibrated m 8 M urea, 20 mM MES, pH 6 0, 200 mM NaCI, 1 mM EDTA Fractions containing hb
  • hb57myc3 (1 ml of COS7-de ⁇ ved serum-free conditioned media) was co- cubated with hBMP2 (1 ⁇ g/ml) or hBMP4 (1 ⁇ g/ml) in the absence or in the presence of human noggm protein (hNG ⁇ B2, 10 ⁇ g/ml)
  • the formation of a stable complex between hb57 and the BMPs was determined by lmmunoprecipitating hb57 and associated proteins using an anti-myc monoclonal antibody (9E10, 1 ⁇ g/ml) bound to Protein G-Sepharose beads (Pharmacia)
  • the binding reaction was carried out in the serum-free conditioned media after it was made 20 mM Tns pH 7 6, 150 mM NaCI, 0 1% Tween 20 (TBST), 1 mg/ml bovine serum albumin (BSA), by addition of a lOx concentrate of these reagents Binding was allowed to proceed for 1 hour
  • hb57myc3 binds to both hBMP2 (Fig 1A, lane 1) and hBMP4 (figure IB, lane 1)
  • This interaction appears to be stable in 1 M NaCI (Fig 1A, lane 2, and Fig IB, lane 2), although some reduction of binding is seen under those conditions
  • Addition of 10 ⁇ g hNG completely blocks this interaction (Fig 1A, lane 3, and Fig IB, lane 3), presumably bv binding to hBMP2 or hBMP4 and blocking their ability to bind to hb57mvc3
  • hb57myc3 was omitted trom the reaction (Fig 1A, lane 6, and Fig IB, lane 6), indicating that there is no non-specific binding of hBMP2 or hBMP4 to the beads and that the observed binding is hb57-dependent
  • hb57 that had expressed in E coli and refolded was tested for its ability to block the biological activity of hBMP2 in the C2C12 mouse plunpotent mesenchymal precursor cell line
  • the C2C12 cells have been shown to respond to BMP2 and BMP4 (Katagi ⁇ et al , 1994)
  • One ot the hallmarks of the response is upregulation of expression of Alkaline Phoshatase, the activity of which can easily be measured m cells or cell lysates using a colo ⁇ metric substrate
  • C2C12 cells respond to hBMP2 with a maximal response obtained at -200 ng/ml hBMP2, and a minimal response obtained with -10 ng/ml hBMP2, with an apparent EC50 ot -70 ng/ml
  • the ability of hb57 to block this response was tested by co-mcubatmg different amounts of hb57 (3 ⁇ g/ml, or 1 ⁇ g/
  • Xenopus b57 was also examined for its ability to antagonize the activity of purified BMP-2 in a cytokine assay
  • the murine bone marrow stromal cell line W-20-17 provides a direct, quantitative bioassay for BMP activity by induction of alkaline phosphatase m response to BMP treatment (Thies, et al Endocrinology 130 1318-24 (1992))
  • Preincubation of purified BMP- 2 with a Gremlin COS supernatant at a final concentration of approx 83nM Gremlin completely blocked BMP-2 activity at doses from 78pM to 5nM
  • BMP-2 activity was reduced, but not eliminated (see Figure 3) Mock-transfected COS supernatant had no effect Similar results were obtained with BMP-4
  • Tissue Relative Level of Expression heart very low brain medium placenta undetectable lung undetectable liver low skeletal muscle low kidney low pancreas low spleen undetectable thymus undetectable prostate low testis very low ovary very low small intestine high colon (mucosa lining) high peripheral blood leukocytes undetectable stomach high thyroid very low spinal chord medium lymph node high trachea low adrenal gland low bone marrow very low 7 Materials and Methods For Examples 2 through 6
  • Bindings were carried out in 20 mM Tns pH 7 6, 150 mM NaCI, 0 1% Tween 20 (TBST), 1 mg/ml bovine serum albumin (BSA), at 25°C, with continuous mixing Protein G-Sepharose (G-Se) was used to capture either the anti-myc 9E10 monoclonal antibody or the Fc-tagged hNG ⁇ B2Fc Non-specifically bound proteins were removed from the beads by washing once with TBST, then moving the G-Se beads to new tubes and washing three more times with TBST Bound hBMP2 and hBMP4 were visualized by western blotting with anti- hBMP2 or ant ⁇ -hBMP4 polyclonal antisera
  • the antibodies were derived from rabbits immunized with recombinant hBMP2 or recombinant hBMP4
  • the antibody preparation was total serum from the bled rabbits
  • a C2C12 will respond to BMP2 and to BMP4 If the cells are incubated with these factors for three days a maximal response can be obtained at 1 to 2 ⁇ g/ml A response above background is seen at 10 ng/ml If the cells are incubated with the BMPs for 4 days, a response to as little as 1 ng/ml can be seen, and a maximal response is obtained at 300 ng/ml No change of medium is required during the 4 day incubation period b Incubation to both BMP2 and BMP4 together does not lead to an additive response above that expected for the equivalent amount of each factor alone c Dilution of BMP2 or BMP4 must be made either directly into the assay media
  • the response to BMP2 and BMP4 can be blocked by addition of an equimolar amount of human noggm 3 Incubate for 3 to 4 days, depending on levels of BMPs used
  • Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature 382, 595-601.
  • BMPs Bone morphogenetic proteins
  • Bone morphogenetic protem-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J Cell Biol 1995 Feb,128(4) following 713] Journal of Cell Biology 227, 1755-66

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Abstract

L'invention concerne des protéines b57 et des acides nucléiques apparentés. Elle concerne également des DAN naturels et des homologues de b57 provenant de plusieurs espèces, ainsi que des protéines comprenant un domaine de DAN ou de b57 et exerçant une activité spécifique, en particulier, d'antagonisme avec une protéine morphogénique osseuse. On peut préparer ces protéines par recombinaison à partir de cellules hôtes transformées avec les acides nucléiques en question. Elle concerne encore des sondes et des amorces d'hybridation isolées capables d'hybridation spécifique avec les gènes objets de l'invention, des agents de fixation spécifique et des procédés de préparation et d'utilisation des compositions décrites.
EP98906592A 1997-02-19 1998-02-19 Proteines morphogeniques Withdrawn EP0968285A1 (fr)

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AU3366099A (en) * 1998-03-26 1999-10-18 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The Drm, a secreted protein with cell growth inhibiting activity, and related methods and compositions
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