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WO1998037195A9 - Proteines morphogeniques - Google Patents

Proteines morphogeniques

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Publication number
WO1998037195A9
WO1998037195A9 PCT/US1998/003283 US9803283W WO9837195A9 WO 1998037195 A9 WO1998037195 A9 WO 1998037195A9 US 9803283 W US9803283 W US 9803283W WO 9837195 A9 WO9837195 A9 WO 9837195A9
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WO
WIPO (PCT)
Prior art keywords
human
bmp
protein
dan
binding
Prior art date
Application number
PCT/US1998/003283
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English (en)
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WO1998037195A1 (fr
Filing date
Publication date
Application filed filed Critical
Priority to CA002282302A priority Critical patent/CA2282302A1/fr
Priority to AU61779/98A priority patent/AU736328B2/en
Priority to IL13142098A priority patent/IL131420A0/xx
Priority to EP98906592A priority patent/EP0968285A1/fr
Priority to JP53688498A priority patent/JP4357003B2/ja
Publication of WO1998037195A1 publication Critical patent/WO1998037195A1/fr
Publication of WO1998037195A9 publication Critical patent/WO1998037195A9/fr

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Definitions

  • the field of this invention is proteins which regulate cell function, and in particular, antagonize bone morphogenic proteins
  • va ⁇ etv ot such regulators have been shown to have protound effects on basic cellular differentiation and developmental pathwa ⁇ s
  • the recently cloned cerberus protein induces the formation ot head structures in anterior endoderm ot vertebrate embryos Similarh , the noggin protein induces head structures in vertebrate embryos, and can redirect mesodermal tates from ventral fates, such as blood and me, to dorsal fates such as muscle and notochord and can redirect epidermal fates to anterior neural fates
  • the actiwties ot chordin are similar to those ot noggin reflecting a common mechanism or action - nameh antagonizing bone morphogenic proteins (BMP) and thereby preventing their function BMPs have diverse biological activities in different biological contexts, including the induction ot cartilage, bone and connective tissue, and roles
  • TGF ⁇ induces neural crest to torm smooth muscle
  • BMP2 induces the same cells to become neurons
  • dissociated animal cap cells proteosectoderm
  • a striking example results from implantation of a matrix impregnated with a BMP into muscle; when the effects are monitored histologically, BMP2, BMP4 and BMP7 induce endochondral bone formation, whereas a related molecule BMP12/GDF7 induces connective tissue similar to tendon. Similarly, BMP4 can induce cell death in the hindbrain neural crest, while the related protein dorsalin 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-screenmg-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-spec ⁇ f ⁇ c 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 h ⁇ b ⁇ dization probes and primers capable of specifically hybridizing with the disclosed genes specific binding agents such as specific antibodies, and methods ot making and using the subject compositions in diagnosis (e g , genetic hybridization screens tor 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
  • Preferred applications of the subject DAN and b57 proteins include modifying the physiology ot 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 spe ⁇ hcalh interacts with a component of the medium and /or the extracellular surface to eftect a change in the physiology of the cell.
  • Also preferred are methods tor screening tor 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 BRIEF DESCRIPTION OF THE FIGURES
  • 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 premcubated with a Gremlin COS supernatant at final concentration of 83nM or 21 nM Gremlin, mock-transtected 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 recombmant proteins comprising a DAN or b57 ammo acid sequence, or a functional DAN or b57 protein domain thereof having an assav-discernable DAN or b57-spec ⁇ f ⁇ c activity Accordingly, the proteins mav 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-spec ⁇ t ⁇ c activity or function and are functionally distinct from each other and from cerberus and noggm 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 b
  • DAN is an intracellular zinc finger protein
  • DAN or b57-specific 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, m animals (e.g., immune response, gene therapy, transgenics, 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, preterably a bone morphogenic protein such as BMP2 or BMP4, chaperone, or other regulator that directly modulates DAN or b57 activity or its localization: or non-natural binding target such as a specific immune protein such as an antibody, or a DAN or b57 specific agent such as those identified in assays described below.
  • a natural binding target such as a TGF ⁇ protein, a morphogenic protein, preterably a bone morphogenic protein such as BMP2 or BMP4, chaperone, or other regulator that directly modulates DAN or b57 activity or its localization
  • non-natural binding target such as a specific immune protein such as an antibody, or a DAN or b57 specific agent such as those identified in assays described below.
  • binding specificity is assayed by bioassay (e.g., the ability to induce neuronal tissue from injected embryonic ectoderm), TGF ⁇ protein binding equilibrium constants (usually at least about 10 7 M" 1 , preferably at least about 10 8 M" 1 , more preferably at least about 10 y M "1 ), by the ability of the subject protein to function as negative mutants in DAN or b57-express ⁇ ng cells, to elicit DAN or b57 specific antibody in a heterologous host (e.g , a rodent or rabbit), etc
  • 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 preferably at least about 5% by weight of the total protein in a given sample; a "pure" protein constitutes at least about 90%, and preterably 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 recombmant technology, or purified from cells.
  • the subject proteins find a wide variety of uses including use as immunogens, targets in screening assays, bioactive reagents tor 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 in 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; and 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.g., ectopic) bone formation, inhibit the growth of cells that require a morphogenic protein (e.g., BMP-dependent neuroblastomas and gliomas), alter morphogen-dependent cell fate /differentiation in culture, such as with cells for transplantation or infusion, etc.
  • a morphogenic protein e.g., BMP-dependent neuroblastomas and gliomas
  • alter morphogen-dependent cell fate /differentiation in culture such as with cells for transplantation or infusion, etc.
  • the proteins may be may be introduced, expressed, or repressed in specific populations of cells by any convenient way such as microinjection, promoter-specific expression of recombinant enzyme, targeted delivery of lipid vesicles, etc.
  • the invention provides natural and non-natural DAN and b57-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development.
  • DAN or b57-specific binding agents include b57-specific ligands such as BMPs, and receptors, such as somatically recombined protein receptors like specific antibodies or T-cell antigen receptors (See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and also includes other natural binding agents identified with assays such as one-, two- and three-hybrid screens, and non-natural binding agents identified in screens of chemical libraries such as described below. Agents of particular interest modulate DAN or b57 function.
  • 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 l.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 Weintraub, H. (1994) Genes Dev 8, 1434-47; Baker, J. C, and Harland, R. M. (1996) Genes & Development 10).
  • b57-specific function of the human gene product was determined by injection of purified, synthetic transcripts of cDNA clones into Xenopus embryos. This assay provides a bioassay for antagonists of BMP activity, as exemplified by the induction of ectopic body axes or enlarged heads.
  • Embryos injected with transcript from the human b57 clone had enlarged heads and partial ectopic body axes similar to the Xenopus b57 injected embryos, indicating a b57 specific biological function for 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 ot 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 ot total nucleic acid present m a given fraction, and usually recombmant, meaning thev 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 j oined on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preterably fewer than 2 kb, which is immediately flanked by a sequence other than that to which it is joined on a natural
  • the amino acid sequences of the disclosed DAN and b57 proteins are used to back translate DAN or b57 protein-encod g 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 vectors and may be incorporated into recombmant host cells, e g , for expression and screening, for transgenic animals, for functional studies such as the efficacy 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 NaCl, 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).
  • 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 Lised to generate allele-specific ongonucleotide (ASO) probes tor high-throughput clinical diagnoses.
  • DAN and b57 nucleic acids are also used to modulate cellular expression or mtracellular concentration or availability ot 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 may be constitutive or inducible and the vector may provide for stable extrachromosomal maintenance or integration.
  • single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding a given DAN or b57 protein may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the targeted protein.
  • An enhancement in DAN or b57 expression is effected by introducing into the targeted cell type DAN or b57 nucleic acids which increase the functional expression of the corresponding gene products.
  • nucleic acids may be DAN or b57 expression vectors, vectors which upregulate the functional expression of an endogenous allele, or replacement vectors for targeted correction of mutant alleles.
  • Techniques for introducing the nucleic acids into viable cells are known in the art and include retroviral-based transfection, viral coat protein-liposome mediated transfection, etc.
  • 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.
  • these screening methods involve assaying for compounds which modulate DAN or b57 interaction with a natural DAN or b57 binding target.
  • assays for binding agents are provided including protein-protein 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 may 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 in the assay.
  • 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 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 ot 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 ot the DAN or b57protem 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 tor 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 ot 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 ention is an isolated b57 protein comprising the ammo acid sequence as set forth in SEQ NO 2 or a fragment thereof having b57- specific activity
  • Another embodiment of the invention is a recombmant nucleic acid encoding b57 protein comprising the ammo 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 m the presence ot 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 lvsolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • Freund ' s complete and incomplete
  • mineral gels such as aluminum hydroxide
  • surface active substances such as lvsolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol
  • BCG Bacille Calmette-Guerin
  • 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 idiotype 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 papain 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.
  • 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 in 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 in 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,
  • Fibrodysplasia Ossificans Progressiva FOP. Since b 7 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 mav be used to block BMP activity in mammals
  • the present invention also provides tor 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 in vivo by any appropriate route including, but not limited to injection (e g , intravenous, mtrape ⁇ toneal, intramuscular, subcutaneous, endoneural, permeural, mtraspmal, mtravent ⁇ cular, intravitreal, mtrathecal etc ), bv 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 concentration of the e ingredient used in the formulation ⁇ , ill depend upon the effective dose required and the mode ot administration used The dose used should be sufficient to ach ⁇ e ⁇ e circulating plasma concentrations of active ingredient that are efficacious Effective doses mav be extrapolated from dose-response cur ⁇ es de ⁇ ed from in vitro or animal model test systems
  • the invention further provides for the use ot von Willebrand factor to regulate or modulate the activity of a BMP
  • ot 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 ot nine separate cysteine residues en this striking homology, it is expected that yon 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.
  • 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 Biosvstems, 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.
  • SEQ. NO. 1 - N ucleotide Sequence encoding human b57
  • 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.
  • 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 dorsalins, 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.
  • GDFs growth differentiation factors
  • TGF ⁇ s transforming growth factor beta and its homologue
  • noggin Smith and Harland, 1992; Zimmerman et al., 1996)
  • chordin Pieris et al., 1996)
  • follistatin Hemmati-Brivanlou et al., 1994.
  • noggin and chordin have been shown to bind to BMP2 and BMP4 and inhibit their biological actions by blocking the interaction with the BMP receptors.
  • hb57 has also been expressed in E. coli and refolded, and rabbit anti- b57 polyclonal antisera have been raised against it (Antisera # Q-1523-1 and Q- 1523-2 prepared under contract by Quality Controlled Biochemicals, Inc., Hopkinton, Massachusetts, 01748 USA).
  • hb57 Using recombinant hb57 proteins, we have tested hb57 for binding to hBMP2 and hBMP4, and the ability of noggin to antagonize this interaction We ha ⁇ e also tested the ability ot hb57 to block the biological activity of hBMP2 in a cell-based assay
  • 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. coh strain RFJ143 containing pRG622 was grown in LB medium and expression of hb57 was induced bv the addition ot 1 mM IPTG Induced cells were collected by cent ⁇ fugation resuspended in 10 volumes of 100 mM T ⁇ s- HC1, pH 8 5, 20 mM EDTA, and lvsed bv 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 Sephacrvl S-300 column equilibrated in 8 M urea, 20 mM MES, pH 6 0, 200 mM NaCl, 1 mM
  • hb57myc3 (1 ml of COS7-denved serum-free conditioned media) was co-mcubated with hBMP2 (1 ⁇ g/ml) or hBMP4 (1 ⁇ g/ml) in the absence or in the presence of human nogg 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 T ⁇ s pH 7.6, 150 mM NaCl, 0 1" ⁇ .
  • Binding was allowed to proceed for 1 hour, at 25°C, m a reaction volume of 1 1 ml, with continuous mixing to keep the Protein G-Sepharose in suspension, after which point the beads were spun down, washed once with TBST, moved to new eppendorf tubes, and washed 3 more times with TBST. Proteins bound to the beads were solubihzed by addition of 25 ⁇ l of Laemh SDS-PAGE sample buffer and loaded onto 4 to 12% NuPAGE/MES gradient gels (Novex), which were run under reducing conditions The proteins were subsequently transferred on Immobilon P and western blotted for the presence of BMP2 or BMP4 using polvclonal antisera raised against the respective proteins.
  • hb57myc3 binds to both hBMP2 (Fig. 1A, lane 1) and hBMP4 (figure IB, lane 1)
  • This interaction appears to be stable in 1 M NaCl (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 by binding to hBMP2 or hBMP4 and blocking their ability to bind to hb57myc3.
  • hBMP2 and hBMP4 were also tested for their ability to bind to hNG ⁇ B2Fc (an Fc-tagged form of the hNG mutein hNG ⁇ B2). Both hBMP2 and hBMP4 bound to hNG ⁇ B2Fc (Fig. 1A, lane 4; and Fig. IB, lane 4), in agreement with results obtained previously (Zimmerman et al., 1996), and the interaction was blocked by the addition of untagged hNG (Fig. 1A, lane 5; and Fig. IB, lane 5).
  • hb57 that had expressed in E. coh and refolded, was tested for its ability to block the biological activity of hBMP2 in the C2C12 mouse pluripotent mesenchymal precursor cell line.
  • the C2C12 cells have been shown to respond to BMP2 and BMP4 (Katagin et l., 1994)
  • BMP2 and BMP4 Katagin et l., 1994
  • One ot the hallmarks of the response is upregulation of expression of Alkaline Phoshatase, the activity of which can easily be measured in cells or cell lysates using a colorimet ⁇ c 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 of -70 ng/ml.
  • the ability ot hb57 to block this response was tested by co-incubatmg different amounts of hb57 (3 ⁇ g/ml, or 1 ⁇ g/ml, or 0.3 ⁇ g/ml) while performing a dose response with hBMP2 starting at 1 ⁇ g/ml.
  • hb57 As can be seen in figure 2, inclusion of 3 ⁇ g/ml hb57 leads to complete blocking of the hBMr2 response when used at 0.5 ⁇ g/ml. With hb57 at 1 ⁇ g/ml, there is -50% inhibition of the hBMP2 response when hBMP2 is at - 0.3 ⁇ g/ml and complete inhibition when hBMP2 is at -0.12 ⁇ g/ml.
  • an identically-treated plate was subjected to an MTT assay (Mosmann, 1983) which measures the proliferation of cells.
  • hb57 is a potent antagonist of BMP2 activity, and it appears to mediate this effect by directly binding to BMP2 and blocking its biological actions. Taken together with the binding results, it is postulated that hb57 should also block the activity of hBMP4.
  • 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 in 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.
  • At approx. 21nM Gremlin 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 b
  • Bindings were carried out in 20 mM T ⁇ s pH 7.6, 150 mM NaCl, 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.
  • C2C12 bioassay protocol (adapted from (Katagi ⁇ et al, 1994)).
  • Important Cells must be monodispersed during trypsinization and prior to plating. Clumps ot cells will give erroneous results and variability in the response.
  • the following dav add BMPs and other factors to each well. Important points: 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.
  • Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer Nature 3S2, 595-601
  • BMPs Bone morphogenetic proteins
  • Follistatin an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell 77, 283-95.
  • Bone morphogenetic protein-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 127, 1755-66.

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.
PCT/US1998/003283 1997-02-19 1998-02-19 Proteines morphogeniques WO1998037195A1 (fr)

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