WO1998052038A1 - Utilisation d'un complexe recepteur de proteine morphogenetique osseuse (bmp) a des fins de selection - Google Patents

Utilisation d'un complexe recepteur de proteine morphogenetique osseuse (bmp) a des fins de selection Download PDF

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WO1998052038A1
WO1998052038A1 PCT/US1998/009519 US9809519W WO9852038A1 WO 1998052038 A1 WO1998052038 A1 WO 1998052038A1 US 9809519 W US9809519 W US 9809519W WO 9852038 A1 WO9852038 A1 WO 9852038A1
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Jan Susan Rosenbaum
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The Procter & Gamble Company
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Priority to EP98922179A priority Critical patent/EP0981745A1/fr
Priority to IL13292298A priority patent/IL132922A0/xx
Priority to CA002290755A priority patent/CA2290755A1/fr
Priority to JP54935398A priority patent/JP2002510198A/ja
Priority to AU74782/98A priority patent/AU7478298A/en
Publication of WO1998052038A1 publication Critical patent/WO1998052038A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to the field of bone formation and development and cellular differentiation Specifically, the present invention relates to the use of a type II receptor that is shared between activins and bone morphogenetic proteins, together with a bone morphogenetic protein type I receptor, for screening cellular differentiation actives.
  • the invention further relates to cells co-transfected with DNA coding for this receptor and DNA coding for a type I bone morphogenetic protein receptor.
  • Bone-related disorders range from bone fractures, to debilitating diseases such as osteoporosis. While in healthy individuals bone growth generally proceeds normally and fractures heal without the need for pharmacological intervention, in certain instances bones may become weakened or may fail to heal properly. For example, healing may proceed slowly in the elderly and in patients undergoing treatment with corticosteroids (e.g., transplant patients). Osteoporosis is a condition in which bone hard tissue is lost disproportionately to the development of new hard tissue.
  • Osteoporosis can generally be defined as the reduction in the quantity of bone, or the atrophy of skeletal tissue; marrow and bone spaces become larger, fibrous binding decreases, and compact bone becomes fragile.
  • Another bone related disorder is osteoarthritis, which is a disorder of the movable joints characterized by deterioration and abrasion of articular cartilage, as well as by formation of new bone at the joint surface.
  • Bone morphogenetic proteins have been demonstrated to play a role in bone formation and development (J. M. Wozney, Molec. Reproduct. and Develop., 32: 160-167 (1992); B.L.M Hogan, Genes & Dev. 10: 1580-1594 (1996)).
  • BMPs may not be limited to their role in bone.
  • the finding that the BMPs are found at significant concentrations in other tissues such as brain, kidney, stratified squamous epithelia, and hair follicle (N.A. Wall, M. Blessing, C.V.E. Wright, and B.L.M. Hogan, J. Cell Biol., 120: 493-502 (1993); E. Ozkaynak, P.N.J. Schnegelsberg, D.F. Jin, G.M. Clifford, F.D. Warren, E.A. Drier, and H. Oppermann, J. Biol. Chem., 267: 25220-25227 (1992); K.M. Lyons, CM.
  • BMPs have recently been found to promote nerve cell differentiation, to affect hair follicle formation, and have been implicated in cardiac and kidney development as well as the development of a variety of other organs (K. Basler, T. Edlund, T.M. Jessell, and T. Yamada, Cell, 73: 687-702 (1993); V.M. Paralkar, B.S.
  • a BMP initiates its biological effect on cells by binding to a specific BMP receptor expressed on the plasma membrane of a BMP-responsive cell.
  • a receptor is a protein, usually spanning the cell membrane, which binds to a ligand from outside the cell, and as a result of that binding sends a signal to the inside ofthe cell which alters cellular function.
  • the ligand is the protein BMP, and the signal induces the cellular differentiation.
  • purified BMP receptor compositions are useful in diagnostic assays for BMPs, as well as in raising antibodies to the BMP receptor for use in diagnosis and therapy.
  • purified soluble BMP receptor compositions may be used directly in therapy to bind or scavenge BMPs, thereby providing a means for regulating the activities of BMPs in bone and other tissues.
  • purified compositions of BMP receptor are needed.
  • compositions are obtainable in practical yields only by cloning and expressing genes encoding the receptors using recombinant DNA technology.
  • Efforts to purify BMP receptors for use in biochemical analysis or to clone and express mammalian genes encoding BMP receptors have been impeded by lack of a suitable source of receptor protein or mRNA.
  • Prior to the present invention few cell lines were known to express high levels of high affinity BMP receptors consisting of the receptor subunits described herein, which precluded purification of the receptor for protein sequencing or construction of genetic libraries for direct expression cloning.
  • Availability of the BMP receptor sequence will make it possible to generate cell lines with high levels of recombinant BMP receptor for biochemical analysis and use in screening experiments.
  • the BMPs are members of the TGF- ⁇ superfamily.
  • Other members of the TGF- ⁇ superfamily include TGF- ⁇ , activins, inhibins, M ⁇ llerian Inhibiting Substance, and the Growth and Differentiation Factors (GDFs) (B.L.M Hogan, Genes & Dev. 10: 1580-1594 (1996)).
  • GDFs Growth and Differentiation Factors
  • the receptors for various members of the TGF- ⁇ superfamily share similar structural features.
  • Receptors of the TGF- ⁇ ligand superfamily are typically classified into one of two sub-groups, designated as type I and type II. The type I and type II receptors are classified as such based on amino acid sequence characteristics.
  • Both the type I and type II receptors possess a relatively small extracellular ligand binding domain, a transmembrane region, and an intracellular protein kinase domain that is predicted to have serine/threonine kinase activity (Lin and Moustakas, Cellular and Molecular Biology, 40: 337-349 (1994); L.S. Mathews, Endocrine Reviews, 15: 310-325 (1994); L. Attisano, J.L. Wrana, F. L ⁇ pez-Casillas, and J. Massague, Biochimica et Biophysica Acta, 1222: 71-80 (1994); P. Ten Dijke, K. Miyazono, and C.-H.
  • the type I receptors cloned to date belong to a distinct family whose kinase domains are highly related and share > 85% sequence similarity (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)).
  • the intracellular juxtamembrane region of the type I receptors is characterized by an SGSGSG motif 35-40 amino acids from the transmembrane region, and the carboxy terminus of these receptors is extremely short (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); L. Attisano, J.L. Wrana, F. L ⁇ pez-Casillas, and J.
  • the extracellular domain of the type I receptors contains a characteristic cluster of cysteine residues, termed the “cysteine box”, located within 25-30 amino acids of the transmembrane region, and another cluster of cysteine residues, termed the "upstream cysteine box", located after the putative signal sequence (B. B. Koenig, et al., Molecular and Cellular Biology, 14; 5961-5974 (1994); L. Attisano, et al., Biochimica et Biophysica Acta, 1222: 71-80 (1994); J. Massague, F. Weis-Garcia, Cancer Surveys 27: 41-64 (1996)).
  • cysteine box located within 25-30 amino acids of the transmembrane region
  • upstream cysteine box located after the putative signal sequence
  • BRK-1 Bone Morphogenetic Protein Receptor Kinase- 1
  • ALK-2 ALK-6
  • BRK-1 is the mouse homologue of human ALK-3, and is also known as BMPR-IA or TFR-11 (see P. Ten Dijke, K. Miyazono, and C.-H.
  • BRK-1 has been shown to bind both BMP-2 and BMP-4 more efficiently than it binds BMP-7, as measured by affinity labeling (J.M. Graff, R.S. Thies, J.J. Song, A.J. Celeste, and D.A. Melton, Cell 79:169-179 (1994); B.B.
  • ALK-6 also known as BMPR-IB (see P. Ten Dijke, K. Miyazono, and C.-H. Heldin, Current Opinion in Cell Biology 8:139-145 (1996)) are similar to that of BRK-1, except that ALK-6 is also capable of binding GDF-5 (P.
  • ALK-6 is the mouse homologue of the chicken receptor Bone Morphogenetic Protein Receptor Kinase-2 (herein referred to as "BRK-2") (also referred to as RPK-1) (S. Sumitomo, T. Saito, and T. Nohno, DNA Sequence, 3: 297-302 (1993)).
  • BRK-2 Bone Morphogenetic Protein Receptor Kinase-2
  • RPK-1 RPK-1
  • ALK-2 also known as ActRI, Tsk7L, or SKR1 (see P. Ten Dijke, K. Miyazono, and C.-H. Heldin, Current Opinion in Cell Biology 8:139-145 (1996)); K. Matsuzaki, J. Xu, F. Wang, W.L. McKeehan, L. Krummen, and M. Kan, J.
  • the kinase domains ofthe type II receptors are only distantly related to one another.
  • the SGSGSG motif found in type I receptors is not found in type II receptors.
  • the "upstream cysteine box" of type I receptors is not present in type II receptors.
  • all of the activin type II receptors contain a proline-rich sequence motif in the intracellular juxtamembrane region, there is no characteristic sequence motif that is common to all type II receptors (L.S. Mathews, Endocrine Reviews, 15: 310-325 (1994)).
  • the length ofthe carboxy terminus of the type II receptors is considerably variable, with the longest known carboxy terminus being found in the nematode BMP type II receptor DAF-4 (M. Estevez, L. Attisano, J.L. Wrana, P.S. Albert, J. Massague, and D.L. Riddle, Nature, 365: 644-49 (1993)) that was cloned from C. elegans, and the mammalian BMP-specific type II receptor BRK-3 described in U. S. Patent application serial number 08/334,179 by Rosenbaum and Nohno, incorporated herein by reference, also known as BMP-RII (B.L. Rosenzweig, T. Imamura, T.
  • the extracellular domain of the type II receptors contains a single cysteine box located near the transmembrane region. Aside from the presence ofthe cysteine box, there is little sequence similarity amongst the extracellular domains of the type II receptors for TGF- ⁇ , activin, and BMPs.
  • BMPs bind to a heteromeric receptor complex consisting of a type I (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); P. ten Dijke, H.
  • crosslinking of BMP-2 or BMP-4 to the mammalian BMP type II receptor BRK-3 is not detectable in the absence of the type I receptor, and only a very low level of binding is detectable at the whole cell level in cells transfected with the type II receptor alone (T. Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S. Noji, D.H. Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995)).
  • the mammalian activin type II receptor has been reported to bind BMP-7 with high affinity and to signal in concert with the ALK-2 or BRK-2 type I receptors, but a signal was not produced when BRK-1 was the type I receptor, suggesting that mammalian ActRII and BRK-1 do not form a signaling receptor complex (H. Yamashita, P. ten Dijke, D. Huylebroeck, T.K. Sampath, M. Andries, J.C. Smith, C.-H. Heldin, and K. Miyazono, J. Cell Biology 130:217-226 (1995)).
  • the ActRIIB receptor exists in four distinct splice variants, described as ActRIIB b ActRIIB 2 , ActRIIB 3 , and ActRIIB 4 each of which is capable of binding activin ligand (L. Attisano, J.L. Wrana, S. Cheifetz, and J. Massague, Cell 68: 97- 108 (1992)).
  • the BRK-1 + ActRIIB 2 complex binds BMP-4 ligand with higher affinity than does the BRK-1 type I receptor alone, indicating that the BRK-1 + ActRIIB 2 complex represents a high affinity complex for BMP-4, analagous to what is observed with the BRK-2 + BRK-3 BMP receptor complex (T. Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S. Noji, D.H. Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522- 22526 (1995)), and that the BRK-1 + ActRIIB 2 complex therefore represents a distinct high affinity BMP receptor complex.
  • the present invention relates to a method for determining whether a compound is capable of binding to a BMP receptor kinase protein complex, the method comprising introducing a sample comprising the compound to the BMP receptor kinase protein complex and allowing the compound to bind to the BMP receptor kinase protein complex, wherein the BMP receptor kinase protein complex is comprised of a BMP type I receptor kinase protein and the BMP/Activin type II receptor kinase protein, comprising the eight amino acid juxtamembrane region, which is characteristic of ActRIIB] or ActRIIB 2 .
  • the invention further relates to a method for determining the concentration of a BMP receptor ligand in a clinical sample, the method comprising introducing the sample comprising the ligand to a BMP receptor kinase protein complex and allowing the ligand to bind to the BMP receptor kinase protein complex, wherein the BMP receptor kinase protein complex is comprised of a BMP type I receptor kinase protein and BMP/ Activin type II receptor kinase protein, ActRIIB] or ActRIIB 2 .
  • the invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for the BMP/Activin type II receptor kinase protein ActRIIB 2 and an expression vector comprising a DNA sequence that codes for a BMP type I receptor kinase protein.
  • the invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for a soluble BMP type I receptor kinase protein and a soluble BMP/Activin type II receptor kinase protein ActRIIB 2 .
  • the invention further relates to a method for determining a test compound produces a signal upon binding to a BMP receptor protein complex, the method comprising: (a) transfecting BMP receptor protein complex expressing cells with a 3TP-Lux luciferase reporter gene (J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang, and J.
  • a 3TP-Lux luciferase reporter gene J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang, and J.
  • the present invention answers the need for a method for determining whether a compound has BMP receptor affinity.
  • the method comprises introducing a sample comprising a test compound to a BMP receptor kinase protein complex and allowing the compound to bind to the BMP receptor kinase protein complex, wherein the receptor complex comprises a BMP type I receptor kinase protein and the BMP/Activin type II receptor kinase protein, generally refered to as ActRIIB, with anspecific eight amino acid juxtamembrane region, characteristic of a protein designated herein as "ActRIIB 2 ".
  • the invention also answers the need for a host cell that is co-transfected with an expression vector comprising a DNA sequence that codes for BMP/Activin type II receptor kinase protein ActRIIB 2 and an expression vector comprising a DNA sequence that codes for a BMP type I receptor kinase protein. Also provided is a method for determining the concentration of a BMP receptor ligand in a clinical sample, the method comprising introducing the sample comprising the ligand to a BMP receptor kinase protein complex and allowing the ligand to bind to the receptor complex, wherein the receptor complex is comprised of a BMP type I receptor kinase protein and BMP receptor kinase protein ActRIIB 2 .
  • the invention also answers the need for a host cell that is co- transfected with an expression vector comprising a DNA sequence that codes for a soluble BMP/Activin type II receptor kinase protein ActRIIB 2 and an expression vector comprising a DNA sequence that codes for a soluble BMP type I receptor kinase protein.
  • mouse ActRIIB means a protein having the amino acid sequence SEQ ID NO:4, as well as proteins having amino acid sequences substantially similar to SEQ ID NO:4, and which are biologically active in that they are capable of binding a BMP molecule (including, but not limited to BMP-2, BMP- 4, and/or BMP-7), or transducing a biological signal initiated by a BMP molecule binding to a cell, or crossreacting with antibodies raised against ActRIIB 2 protein, or peptides derived from the protein sequence of ActRIIB 2 , or forming a complex with a BMP type I receptor, or co-immunoprecipitating with a BMP type I receptor when antibodies specific for either ActRIIB 2 or a BMP type I receptor are used.
  • BMP molecule including, but not limited to BMP-2, BMP- 4, and/or BMP-7
  • mae BMP receptor kinase protein or “m-BRK-3” means a protein having amino acid sequence SEQ ID NO: 12 or a sequence substantially similar to that sequence. Also included in this definition are proteins of this ilk which are biologically active in that they are capable of binding a BMP molecule (including, but not limited to BMP-2, BMP-4, and/or BMP-7), or transducing a biological signal initiated by a BMP molecule binding to a cell, or crossreacting with antibodies raised against this protein, or peptides derived from the protein sequence of this protein, or forming a complex with a BMP type I receptor, or co- immunoprecipitating with a BMP type I receptor when antibodies specific for either this protein or a BMP type I receptor are used.
  • BMP molecule including, but not limited to BMP-2, BMP-4, and/or BMP-7
  • BMP receptor kinase protein BRK-3 or “BRK-3” refers individually and collectively to the receptor proteins h-BRK-3 (SEQ ID NO: 10), and m-BRK-3 (and soluble and incomplete fragments of any of these).
  • proteins of this ilk which are biologically active in that they are capable of binding a BMP molecule (including, but not limited to BMP-2, BMP -4, and/or BMP-7), or transducing a biological signal initiated by a BMP molecule binding to a cell, or crossreacting with antibodies raised against this protein, or peptides derived from the protein sequence of this protein, or forming a complex with a BMP type I receptor, or co-immunoprecipitating with a BMP type I receptor when antibodies specific for either this protein or a BMP type I receptor are used.
  • BMP molecule including, but not limited to BMP-2, BMP -4, and/or BMP-7
  • transducing a biological signal initiated by a BMP molecule binding to a cell or crossreacting with antibodies raised against this protein, or peptides derived from the protein sequence of this protein, or forming a complex with a BMP type I receptor, or co-immunoprecipitating with a B
  • BMP receptor kinase proteins substantially similar to h- BRK-3 and m-BRK-3 (and soluble and incomplete fragments as above).
  • Such receptor proteins, DNA sequences coding for the proteins, and recombinant expression vectors comprising said DNA are described and claimed in U. S. Patent application serial number 08/462,467 by Rosenbaum, incorporated herein by reference.
  • a "BMP Type I Receptor Kinase” is a protein capable of binding BMP-2, BMP -4 and/or other known BMPs, and bears sequence characteristics of a type I receptor including, but not limited to, an extracellular ligand binding domain containing a cysteine box and an upstream cysteine box, an SGSGSG motif, designated the GS domain, in the intracellular juxtamembrane region, an intracellular kinase domain that is greater than about 85% similar to other type I receptors for other ligands in the TGF- ⁇ superfamily, and/or a relatively short carboxy terminus.
  • BMP Type I Receptor Kinase also includes receptor proteins having the characteristics of a BMP type I receptor as described in the literature, such as in: B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); L. Attisano, et al., Biochimica et Biophysica Acta, 1222: 71-80 (1994); J. Massague, L. Attisano, and J. L. Wrana, Trends in Cell Biology, 4: 172- 178 (1994); and ten Dijke, et al., J. Biological Chemistry, 269: 16985-16988 (1994).
  • BMP type I receptors include, but are not limited to: BRK-1 (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994), the rat homologue of which is BMPR-Ia (K. Takeda, S. Oida, H. Ichijo, T. Iimura, Y. Maruoka, T. Amagasa, and S. Sasaki, Biochem. Biophys. Res. Communica., 204: 203-209 (1994)); BRK-2, also referred to as RPK-1 (S. Sumitomo, T. Saito, and T.
  • ALK-6 which has been shown to be a receptor for BMP-7 (ten Dijke et al, J. Biological Chemistry, 269: 16985-16988 (1994)); Uie Xenopus BMP type I receptor that binds BMP-2 and BMP-4 and which is involved in mesoderm induction (J.M. Graff, R.S.
  • Drosophila receptors from Drosophila that bind the decapentaplegic peptide, which is the Drosophila homologue of BMP-2 and BMP-4.
  • Drosophila receptors are designated 25D1, 25D2, and 43E (T. Xie, A.L. Finelli, and R.W. Padgett, Science, 263: 1756-1759 (1994); A. Penton, Y. Chen, K. Staehling-Hampton, j. L. Wrana, L. Attisano, J. Szidonya, J. A. Cassill, J.
  • Preferred BMP type I receptors useful in the present invention include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NOJ4 (BRK-1), SEQ ID NOJ6 (BRK-2).
  • soluble fragment refers to an amino acid sequence corresponding to the extracellular region of BRK-1 , BRK-2, or the ActRIIB, preferably Act RUB] or ActRIIB2, which is capable of binding BMPs. Soluble fragments include the complete extracellular domain of the receptor protein, prior to the start ofthe predicted transmembrane region.
  • Examples of such soluble fragments for ActRIIB2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO:4, wherein amino acids 1-134 are present.
  • Examples of such soluble fragments for ActRIIB2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO:4, wherein amino acids 1-134 are present. It is understood from these examples that by homology one can determine the essential and non-essential region, given these examples.
  • soluble fragments for BRK-1 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to amino acid residues 1-153 in SEQ ID NOJ4.
  • soluble fragments for BRK-2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to amino acid residues 1-126 in SEQ ID NOJ6.
  • incomplete receptor kinase fragment refers to an amino acid sequence corresponding to the extracellular, transmembrane, and intracellular juxtamembrane region of BRK-1, BRK-2, or the ActRIIB, preferably ActRIIB j or ActRIIB2, which is capable of binding BMPs in a manner similar to the full-length receptor, but which is incapable of signaling due to deletion of the intracellular kinase domain (otherwise known as a dominant negative receptor construct).
  • Examples of such incomplete receptor fragments for ActRIIB 2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO:4; wherein amino acids 1-161 are present and amino acids 162-191 are optionally present, for ActRIIB b with amino acid sequences substantially similar to SEQ ID NO:2, amino acids 1-161 are present and amino acids 162-215 are optionally present.
  • incomplete receptor fragments for BRK-1 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO: 14; wherein amino acids 1-177 are present and amino acids 178-229 are optionally present.
  • incomplete receptor fragments for BRK-2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NOJ6; wherein amino acids 1-149 are present and amino acids 150-199 are optionally present
  • a "BMP receptor kinase protein complex” is the combination of a BMP type I receptor and BMP receptor kinase protein ActRIIB 2 .
  • the combination of the type I and ActRIIB 2 receptors includes, but is not limited to, a combination of the type I and ActRIIB 2 receptors in solution (e.g., as soluble fragments); a combination of the receptors (e.g., as soluble fragments) attached to a solid support; or a combination of the receptors (e.g., as full-length or incomplete fragments) within a cell membrane of transfected cells.
  • nucleic acid sequences and analogs are “substantially similar” to the specific DNA sequence disclosed herein if the DNA sequences, as a result of degeneracy in the genetic code, encode an amino acid sequence substantially similar to the reference amino acid sequence.
  • substantially similar means a receptor protein that will react with antibodies generated against the ActRIIB 2 protein or peptides derived from the protein sequence of ActRIIB 2 .
  • Homologues are proteins that maintain the similar function and have substantially the same amino acid chain as those proteins listed in the sequence listings, however there may be innocuous substitutions in the chain that would not alter structure or function, for example, one hydrophobic amino acid for another, e.g., leucine for isoleucine; or an acidic amino acid for another, such as glutamic acid for aspartic acid, and the like.
  • these proteins include whole proteins with at least 90% homology as understood by the art, with deletions and/or insertions or fragments thereof.
  • a rat protein which is 95% homologous to that of a human based on the peptide sequence derived from the DNA or cDNA sequence, and a similarly derived bovine protein with the same function and similar homology, are both considered homologues.
  • homologous cDNAs cloned from other organisms give rise to homologous proteins.
  • proteins may be considered homologues based on the amino acid sequence alone. Practical limitations of amino acid sequencing would allow one to determine that a protein is homologous to another using for example comparison of the first 50 amino acids of the protein. Hence 90% homology in would allow for 5 differing amino acids in the chain of the first 50 amino acids of the homologous protein.
  • biologically active means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of binding detectable quantities of BMP-2 or BMP-4, or transmitting a BMP-2 or BMP-4 stimulus to a cell, for example, as a component of a hybrid receptor construct.
  • a biologically active BMP type I receptor: ActRIIB 2 receptor complex within the scope of the present invention means the receptor protein kinase complex is capable of binding [ ⁇ 5j]_ BMP-4 with nanomolar or subnanomolar affinity (Kj approximately equal to 10" ⁇ M).
  • the affinity is from about lxl0" 10 M to lxlO ⁇ M, with a proportion of binding sites exhibiting a K ⁇ less than 10' ⁇ M.
  • signal refers to a biological response caused by some external stimulus, preferably related to binding of a molecule, including a small molecule, peptide or the like, that may be detected if instrumentation is sensitive enough and the correct parameter is measured.
  • signaling include, the agonism or antagonism of an enzyme, the triggering or inhibition of a biochemical cascade, or the like.
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of influencing the activity of other portions of the same linear DNA sequence.
  • DNA for a signal peptide secretory leader
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
  • operably linked means contiguous and, in the case of secretory leaders, contiguous in reading frame.
  • bone morphogenetic protein 2 or "BMP-2” means a peptide encoded by a DNA sequence contained in ATCC No. 40345 (see ATCC/NIH REPOSITORY CATALOGUE OF HUMAN AND MOUSE DNA PROBES AND LIBRARIES, sixth Edition, 1992, p. 57, hereinafter "ATCC/NIH REPOSITORY CATALOGUE”). Isolation of BMP-2 is disclosed in U.S. Patent No. 5,013,649, Wang, Wozney and Rosen, issued May 7, 1991 ; U.S. Patent No. 5,166,058, Wang, Wozney and Rosen, issued November 24, 1992; and U.S. Patent No. 5,168,050, Hammonds and Mason, issued December 1 , 1992; each of which is incorporated herein by reference.
  • bone morphogenetic protein 4" or "BMP-4" means a peptide encoded by a DNA sequence contained in ATCC No. 40342 (see ATCC/NIH REPOSITORY CATALOGUE). Isolation of BMP-4 is disclosed in U.S. Patent No. 5,013,649, Wang, Wozney and Rosen, issued May 7, 1991, incorporated herein by reference.
  • bone morphogenetic protein 7 or "BMP-7” means a peptide encoded by a DNA sequence contained in ATCC No. 68020 and ATT 68182 (see ATCC/NIH Repository Catalogue), where the cDNA in ATCC 68182 is claimed to contain all of the nucleotide sequences necessary to encode BMP-7 proteins. Isolation of BMP-7 is disclosed in U S. Patent 5,141,905, issued August 25, 1992, to Rosen, et al., which is incorporated herein by reference.
  • DNA sequence refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector.
  • sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences (introns) which are typically present in eukaryotic genes. Genomic DNA containing the relevant sequences could also be used.
  • Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.
  • DNA sequences encoding the proteins provided by this invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.
  • recombinant means that a protein is derived from a DNA sequence which has been manipulated in vitro and introduced into a host organism. Such an organism may produce the protein naturally, or may be devoid of any mechanism for making the protein initially: preferably the host organism does not produce the protein in its normal state and hence is a "heterologous host.”
  • Recombinant proteins can be made using bacterial, fungal (e.g., yeast), or insect expression systems.
  • recombinant expression vector refers to a DNA construct used to express DNA which encodes a desired protein (for example, ActRIIB 2 ) and which includes a transcriptional subunit comprising an assembly of 1) genetic elements having a regulatory role in gene expression, for example, promoters and enhancers, 2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and 3) appropriate transcription and translation initiation and termination sequences.
  • a desired protein for example, ActRIIB 2
  • a transcriptional subunit comprising an assembly of 1) genetic elements having a regulatory role in gene expression, for example, promoters and enhancers, 2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and 3) appropriate transcription and translation initiation and termination sequences.
  • Possible vectors for use in the present invention include, but are not limited to: for mammalian cells, pJT4 or pJT6 (discussed further below), pcDNA-1 (Invitrogen, San Diego, Ca) and pSV-SPORT 1 (Gibco-BRL, Gaithersburg, MD); for insect cells, pBlueBac III or pBlueBacHis baculovirus vectors (Invitrogen, San Diego, CA); and for bacterial cells, pET-3 (Novagen, Madison, WI).
  • the DNA sequence coding for a ActRIIB 2 protein receptor kinase ofthe present invention can be present in the vector operably linked to regulatory elements.
  • the present invention relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for BMP receptor kinase protein ActRIIB 2 and an expression vector comprising a DNA sequence that codes for a BMP type I receptor kinase protein.
  • the expression vector for the mouse ActRIIB protein comprises a DNA sequence coding for the mouse ActRIIB 2 receptor protein, or a soluble or incomplete fragment thereof.
  • the DNA can be genomic or cDNA.
  • the mouse ActRIIB 2 protein is coded for by the nucleic acid sequence SEQ ID NO:3.
  • the host cells of the present invention are co-transfected with the plasmid construct pJT6-mActRIIB 2 and the plasmid construct pJT4-J159F (for BRK-1) or plasmid construct pJT3- BRK-2 (for BRK-2), thereby resulting in co-expression of mActRIIB 2 and BRK-1, or mActRIIB 2 and BRK-2, respectively.
  • Transfection with the recombinant molecules can be effected using methods well known in the art.
  • host cell means a cell comprising a recombinant expression vector described herein. Host cells may be stably transfected or transiently transfected within a recombinant expression plasmid or infected by a recombinant virus vector.
  • the host cells include prokaryotic cells, such as Escherichia coli, fungal systems such as Saccharomyces cerevisiae, permanent cell lines derived from insects such as Sf-9 and Sf-21 , and permanent mammalian cell lines such as Chinese hamster ovary (CHO) and SV40-transformed African green monkey kidney cells (COS).
  • the present invention relates to a method that is useful for identifying compounds capable of binding to a BMP receptor kinase protein.
  • the invention relates to a method that is useful for determining the concentration of a BMP receptor ligand (e.g., BMP-2, BMP-4, or BMP-7, or another as-yet identified BMP receptor ligand) in a clinical sample.
  • a sample comprising a putative ligand or a known ligand is introduced to a BMP receptor kinase protein complex, wherein the receptor complex is comprised of a BMP type I receptor kinase protein and BMP receptor kinase protein ActRIIB 2 .
  • the ActRIIB receptor kinase protein is m-ActRIIB 2 , having an amino acid sequence SEQ ID NO:4, or the soluble fragment thereof or the incomplete fragment thereof.
  • BMP concentration in a sample can be determined by radioreceptor assay, in which unlabeled BMP in the sample competes with labeled tracer BMP for binding to the ActRIIB 2 + BMP type I receptor complex. As the amount of BMP in the sample increases, it reduces the amount of labeled BMP which is able to bind to the receptor protein complex comprising ActRIIB 2 and the type I receptor. Comparison with a standard curve prepared with known concentrations of unlabeled BMP allows accurate quantitation of BMP concentration in the sample. Labeling of tracer BMP is preferably done by iodination with [125i]N a j
  • ActRJIB 2 can be expressed in the outer membrane of a stable cell line which also expresses the BMP type I receptor kinase, or supplied as a soluble fragment in solution with a soluble type I receptor fragment, or as a soluble fragment covalently attached to a solid support in conjunction with a type I receptor covalently attached to a solid support.
  • unlabeled BMP from the sample and labeled tracer BMP compete for binding to the receptor until equilibrium is reached.
  • the receptor-BMP complex is then isolated from free ligand, for example by washing (in the case of an adherent cell line), rapid filtration or centrifugation (in the case of a nonadherent cell line or receptor bound to a solid support), or precipitation ofthe receptor-ligand complex with antibodies, polyethylene glycol, or other precipitating agent followed by filtration or centrifugation (in the case of a soluble receptor).
  • the amount of labeled BMP in the complex is then quantitated, typically by gamma counting, and compared to known standards.
  • the methods of the present invention is also useful in high-throughput screens to identify compounds capable of binding to ActRIIB 2 , or a homologous receptor protein, that is complexed to a BMP type I receptor kinase protein.
  • the higher the affinity of the compound for the ActRIIB 2 /type I complex the more efficiently it will compete with the tracer for binding to the complex, and the lower the counts in the receptor-ligand complex.
  • This invention is useful for determining whether a ligand, such as a known or putative drug, is capable of binding to and/or activating the receptors encoded by the DNA molecules of the present invention.
  • Transfection of said DNA sequence into the cell systems described herein provides an assay system for the ability of ligands to bind to and/or activate the receptor complex encoded by the isolated DNA molecules.
  • Recombinant cell lines, such as those described herein are useful as living cell cultures for competitive binding assays between known or candidate drugs and ligands which bind to the receptor and which are labeled by radioactive, spectroscopic or other reagents.
  • Membrane preparations containing the receptor isolated from transfected cells are also useful for competitive binding assays.
  • Soluble receptors derived from the ligand binding domain of the receptor can also be employed in high-throughput screening of drug candidates. Functional assays of intracellular signaling can act as assays for binding affinity and efficacy in the activation of receptor function.
  • the recombinant cell lines may be modified to include a reporter gene operably linked to a response element such that a signal sent by the receptor turns on the reporter gene. Such a system is especially useful in high throughput screens directed at identification of receptor agonists.
  • These recombinant cell lines constitute "drug discovery systems", useful for the identification of natural or synthetic compounds with potential for drug development. Such identified compounds could be further modified or used directly as therapeutic compounds to activate or inhibit the natural functions of the receptor encoded by the isolated DNA molecule.
  • the soluble receptor protein complex of the present invention can be administered in a clinical setting using methods such as by intraperitoneal, intramuscular, intravenous, or subcutaneous injection, implant or transdermal modes of administration, and the like. Such administration can be expected to provide therapeutic alteration ofthe activity ofthe BMPs.
  • SEQ ID NO: 3 and SEQ ID NO: 7 represent the DNA sequences coding for m-ActRJIB 2 and mActRIIB 4 receptor proteins, respectively, that were isolated from mouse hair/skin samples. These sequences could be readily used to obtain the cDNA for ActRIIB 2 or ActRIIB 4 from other species, including, but not limited to, human, rat, rabbit, Drosophila, and Xenopus.
  • the present invention further relates to a method for determining whether a test compound produces a signal upon binding to a BMP receptor protein complex.
  • a method for determining whether a test compound produces a signal upon binding to a BMP receptor protein complex comprises employing the BMP receptor protein complex in a transcriptional reporter assay.
  • the method for determining whether a test compound produces a signal upon binding to the BMP receptor protein complex comprises (a) transfecting BMP receptor protein complex expressing cells with a 3TP-Lux luciferase reporter gene (J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang, and J.
  • a method for determining whether a test compound produces a signal upon binding to the BMP receptor portion complex comprises (a) labeling BMP receptor protein complex expressing cells with 3-P, wherein the cells have been transfected with a DNA sequence coding for BMP receptor kinase protein ActRIIB) or ActRIIB 2 and a DNA sequence coding for a BMP type I receptor kinase protein; (b) culturing (i) a first set of the cells in the presence of the test compound, and (ii) a second set of the cells in the absence of the test compound; (c) quantitating via autoradiography any phosphorylated proteins produced from step (b); and (d) comparing the amount of phosphorylated proteins quantitated in step (c) from the first set of cells to the amount of phosphorylated proteins quantitated in step (c) for the second set of cells.
  • a PCR probe is generated from degenerate primers JT65 (SEQ ID NO: 17) and JT69 (SEQ ID NO: 18) from conserved protein sequences in kinase domain II (JT65) and in kinase domain NIII (JT69) of the known receptor serine/threonine kinases.
  • This probe was subcloned into pBLUESCRIPT vector (Stratagene, La Jolla, CA) to give pBS2-54 and used to screen against a ⁇ IH3T3 library, which has been previously described in U. S. Patent application serial number 08/462,467 by Rosenbaum, incorporated herein by reference.
  • a partial ActRIIB clone (38-4-2) which contains approximately 2.3 kb of the ActRIIB sequence is isolated.
  • a cDNA library is constructed from mouse hair/skin tissues (E82 strain, 2 days after hair shaving).
  • Total RNA (1.8 mg) is isolated from the cells using a Total RNA Separator Kit (Clontech, Palo Alto, CA).
  • Messenger RNA (6.3 ⁇ g) is isolated from this total RNA (1 mg) using the mRNA Separator Kit (Clontech, Palo Alto, CA).
  • An aliquot of the mRNA (2 ⁇ g) is used to make cDNA library using the SUPER SCRIPT Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions.
  • the resulting library contains approximately 500,000 primary colonies, and is divided into 72 pools, each containing 7,000 colonies.
  • the initial screen of the library is accomplished by PCR. Plasmids used as templete are purified from each of the 72 pools, using QIAGEN columns (Qiagen, Chatsworth, CA). Positive pools are identified by PCR with primers designed to amplify all ActRIIB isoforms as described previously (Tsung-Chieh, J.Wu, M.H. Jih, L. Wang, and Y.-J. Y. Wan, Molecular Reproduction and Development 38: 9-15 (1994)); these primers are provided herein and referenced as ActRIIBTF (SEQ ID NO: 19) and ActRIIBTR (SEQ ID NO:20).
  • the PCR reaction was performed using the GENE-AMP PCR Kit with AMPLITAQ DNA Polymerase (Perkin Elmer, Applied Biosystems, Foster City, CA). An initial melting period at 94°C for 5 min was followed by 30 cycles of the following program: melting at 94°C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min.
  • AMPLITAQ DNA Polymerase Perkin Elmer, Applied Biosystems, Foster City, CA.
  • An initial melting period at 94°C for 5 min was followed by 30 cycles of the following program: melting at 94°C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min.
  • plates are streaked with the E. coli stocks from three positive pools (2,000 colonies/plate). A HYBOND nylon membrane is placed on top of the plate so that the bacterial colonies are transferred to the filter. The colonies are then allowed to recover at 37 °C for 2-3 hr.
  • the filter is soaked in 10% SDS for 3 min, then transferred to 1.5 M NaCl, 0.5 M NaOH for 5 min, neutralized in 1.5 M NaCl, 0.5 M Tris, pH 7.5 for 5 min, and washed in 3X SSC. To remove proteins, the blots are then shaken with 50 ⁇ g/ml of proteinase K (Boehringer Mannheim, Indianapolis, IN) in 0.1 M Tris, pH 7.6, 10 mM EDTA, 0J5 M NaCl, 0.02% SDS at 55 °C for 1 hr.
  • the mouse partial ActRIIB cDNA isolated from NIH3T3 library (clone 38-4-2) is cut with Mlu I and to give a 2.3 kb fragment.
  • the fragment is randomly labeled with ⁇ -[ 32 P]-dCTP having a specific activity of 3000 Ci/mmol (NEN Research Products, Boston, MA), using a PRIME-IT II Random Primer Labeling Kit (Stratagene, La Jolla, CA; a kit for random primer labeling of DNA, including Klenow DNA polymerase, primers, and buffers).
  • the labeled probe is allowed to hybridize to the filters for 18 hr at 42°C in hybridization buffer (Sigma, St.
  • Colonies which corresponded to labeled spots on the autoradiograph are streaked on plates for tertiary screening, which is performed exactly as described above for secondary screening. Three positive clones are isolated.
  • the inserts from the 3 positive clones are sequenced using the TAQ DYE DEOXY Terminator Cycle Sequencing Kit and an Applied Biosystems Model 373A Automated DNA Sequencer. Comparison of the sequences shows that clone A46- 3/pSPORT contains the complete coding of the ActRIIB 4 variant whereas clone A49- 8/pSPORT aligns with the ActRIIB 2 variant approximately 50 base pairs from the beginning of the coding region (see Example 2 below).
  • ActRIIB 2 For ActRIIB 2 , we have data confirming the sequence at ATG (base 44) through 900 bases, as well as the 3' end sequence (1460-1708). The sequencing data verifies this is the ActRIIB 2 variant as it contains the first insert (bp 413-436).
  • the ActRIIB 4 variant does not contain either of the inserts, as indicated by the DNA sequence of the A46-3/pSPORT clone shown in SEQID NO: 7 and the protein sequence shown in SEQ ID NO: 8.
  • the clone isolated was full length and in addition contained — 300 bases of unknown sequence 5' to the ATG at bp 44.
  • the pJT4 expression vector has been previously described in U. S. Patent application serial number 08/462,467 by Rosenbaum, incorporated herein by reference.
  • This expression vector has been optimized for transient expression in COS cells, and includes the cytomegalovirus early promoter and enhancer, which gives very efficient transcription of message; an "R" element from the long terminal repeat of the human T-cell leukemia virus-1, which has been shown to increase expression levels further; an intron splice site from SV40, which is believed to enhance message stability; a multiple cloning site; a polyadenylation signal derived from SV40, which directs the addition of a poly A tail to the message, as is required for most eukaryotic mRNA; and the SV40 origin of replication, which permits the replication of the plasmid to extremely high copy number in cells which contain the SV40 large T antigen, such as COS cells.
  • the vector contains an E. coli origin of replication and an amp
  • m-ActRIIB 4 is subcloned into the mammalian expression vector pJT6.
  • This vector is a derivative of pJT3, described in in U. S. Patent application serial number 08/462,467 by Rosenbaum, incorporated herein by reference, (see Example 4) in which the Not I site at the 5' end of the multiple cloning site has been deleted, and a spacer inserted between the Pst I and BamHI restriction sites in the multiple cloning site.
  • m-ActRIIB 4 cDNA is excised from A46-3/pSPORT using Not I and Sal I, then subcloned into pJT6 at the Not I and Sal I sites to generate pJT6-A46L.
  • a Sal I site is first placed at the 5' end of clone A46-3/pSPORT as follows.
  • a primer is synthesized which contains a Sal I site followed by nucleotides 1- 15 of the coding sequence of A46-3/pSPORT; the sequence of the primer is 5' ATC GTC GAC CAT GAC GGC GCC CTG G 3' (SEQ ID NO:21).
  • the fragment amplified from A46-3/pSPORT is inserted into pJT6 vector as follows.
  • the amplified fragment from A46-3/pSPORT is digested with Sal I and Apa I.
  • the insert from A49/pSPORT is digested with Apa I and Not I.
  • the vector pJT6 is digested with Sal I and Not I.
  • the three fragments are combined in a three-way ligation using T4 DNA ligase (3 hr, 25 °C) and used to transform electrocompetent E. coli, strain DH5-a, using a BIO-RAD Gene PULS ⁇ R (BIO-RAD, Hercules, CA) according to the manufacturer's instructions.
  • a positive colony is selected and is designated pJT6-A49. Sequencing of the 5' portion of the insert that was amplified by PCR shows a sequence identical to that of clone A46-3/pSPORT, indicating that no mutations are introduced during the amplification.
  • Example 4 Mammalian expression of mActRHB,, mActRIIB,. m-BRK-3. BRK-1. and BRK-2 Transient expression of the receptors in mammalian cells using the expression plasmids described above is carried out in COS-1 cells (ATCC CRL 1650) or RIB/L-17 cells (J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang, and J. Massague, Cell 71: 1003-1014 (1992)) for the binding and immunoprecipitation studies (Examples 6-8 below) or RIB/L-17 cells (J.L.
  • the cells are grown to approximately 70% -90% confluencey in DME high glucose media (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah), nonessential amino acids, and glutamine in T-175 flasks (Corning, San Diego, CA).
  • DME high glucose media Life Technologies, Gaithersburg, MD
  • fetal bovine serum HyClone, Logan, Utah
  • nonessential amino acids amino acids
  • glutamine glutamine
  • T-175 flasks Ses, San Diego, CA
  • the cells are washed twice with 37 C serum-free DME media, after which 10 ml of DNA mixture is added to each T-175 flask.
  • the DNA mixture contains DME, 10% Nu-Serum (Collaborative Biomedical Products.
  • the empty expression vector pJT6 or pJT4 (described in detail in U.S. Patent Application Serial Number 08/462,467 by Rosenbaum, incorporated herein by reference) is substituted for the corresponding receptor cDNA.
  • the cells are then incubated at 37 °C with the DNA mixture for 3 hr.
  • the solution is aspirated and the cells are incubated with 10 ml of a solution containing 10% dimethylsulfoxide (DMSO) in Dulbecco's phosphate buffered saline without calcium or magnesium (PBS; Life Technologies, Gaithersburg, MD).
  • DMSO dimethylsulfoxide
  • the DMSO solution is aspirated, the cells are washed with the growth media described above, and fresh media is returned to the plates.
  • the transfected cells are split into 12 well plates 24 hr post transfection for whole cell binding (Example 6) or 100 mm plates for affinity labeling and immunoprecipitation (Example 7). 36 to 72 hours after transfection the cells are suitable for binding analysis.
  • the conditions are identical to that described above for the COS-1 cells except that MEM media (Life Technologies, Gaithersburg, MD) is used in place of DME-high glucose media, and R1B/L-17 cells were transfected 24 hours after seeding in a T-175 flask (Corning, San Diego, CA) at 5-10 x 10 6 cells per flask.
  • MEM media Life Technologies, Gaithersburg, MD
  • R1B/L-17 cells were transfected 24 hours after seeding in a T-175 flask (Corning, San Diego, CA) at 5-10 x 10 6 cells per flask.
  • the conditions are identical to that described immediately above, except that 30 ⁇ g of the 3TP-Lux reporter plasmid (J.L. Wrana, L.
  • Recombinant human BMP-2 and BMP-4 dimers are produced and purified from CHO cells as previously described (B.B. Koenig et al. , Molecular and Cellular Biology, 14: 5961-5974 (1994)).
  • [ 125 I]-BMP-4 is prepared using IODOBEADS (Pierce, Rockford, IL; immobilized chloramine-T on nonporous polystyrene beads). Lyophilized BMP-4 (2 ⁇ g) is taken up in 50 ⁇ l of 10 mM acetic acid and added to 450 ⁇ l of phosphate-buffered saline (PBS) (Sigma, St. Louis, MO) on ice.
  • PBS phosphate-buffered saline
  • BMP-4 is labeled with 125j by the chloramine-T method (CA. Frolik, L.M. Wakefield, D.M. Smith, and M.B. Sporn, J. Biol. Chem., 259: 10995- 11000 (1984)).
  • BMP-4 (2 ⁇ g) is taken up in 5 ⁇ l of 30% acetonitrile, 0.1 % trifluoracetic acid (TFA) plus an additional 5 ⁇ l of 1.5 M sodium phosphate, pH 7.4.
  • TFA trifluoracetic acid
  • Example 6 Characterization of BMP-4 Binding Affinity to mActRIIB 2 in the presence of a BMP type I receptor Binding of BMP-4 to mActRIIB 2 in the presence of the BMP type I receptors can be demonstrated by whole cell binding of radiolabeled BMP-4, and by covalent crosslinking (affinity labeling) and immunoprecipitation of radiolabeled BMP-4 to the receptor. These two methods are described in detail in this Example and in Example 7 below.
  • COS-1 cells are transfected with pJT6-mActRIIB 2 for mActRIIB 2 expression, or pJT6-mActRIIB 4 for mActRIIB 4 expression; in the presence of pJT4-J159F for BRK-1 expression, or pJT3-BRK2 for BRK-2 expression, as described in Example 4. After transfection, cells are seeded into 12 well plates and the binding experiments are carried out at 24 to 36 hr. after plating.
  • binding buffer 50 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KC1, 5 mM MgSO4, 1.2 mM CaCl2, 2 mg/ml BSA
  • the buffer is then aspirated, and to each well is added 500 ⁇ l of binding buffer (4° C), containing [ 12 5i]-BMP-4 tracer (100 - 400 pM), as well as varying concentrations of unlabeled BMP-2, BMP-4, or other unlabeled ligand, depending on the assay.
  • BMP-4 is added to the binding buffer at a final concentration of 10 to 50 nM.
  • a protease inhibitor cocktail is also added, to give a final concentration of 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml antipain, 50 ⁇ g/ml aprotinin, 100 ⁇ g/ml benzamidine, 100 ⁇ g/ml soybean trypsin inhibitor, 10 ⁇ g/ml bestatin, 10 ⁇ g/ml pepstatin, and 300 ⁇ M phenylmethylsulfonyl fluoride (PMSF).
  • the cells are incubated for 4 hr at 4°C with gentle shaking.
  • the buffer is aspirated, and the cells are rinsed 4 times with 1 ml washing buffer (50 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KC1, 5 mM MgSO4, 1.2 mM CaCl2, 0.5 mg/ml BSA).
  • 1 ml washing buffer 50 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KC1, 5 mM MgSO4, 1.2 mM CaCl2, 0.5 mg/ml BSA.
  • 200 ⁇ l of RIPS buffer (20 mM Tris Base, lOOmM NaCl, 1 mM EDTA, 0.5 % NP-40, 0.5% Deoxycholic Acid, 0.1 % SDS, 10 mM Nal, 1 % BSA, pH 8.0) is added to each well and incubated at room temperature for 15 - 30 min.
  • the solubilized cells are then transferred to fresh tubes and counted in a Packard
  • ActRIIB 2 type II receptor represents a type II receptor that is capable of forming a high affinity binding complex for BMP-4 in the presence of the BRK-1 receptor; and as such BRK-1 + ActRIIB 2 represents a novel high affinity BMP receptor complex.
  • Receptors of the TGF- ⁇ receptor family have been shown to form complexes involving a type I and a type II receptor (L. Attisano, J.L. Wrana, F. Lopez- Casillas, and J. Massague, J. Biochim Biophys. Acta, 1222: 71-80 (1994)).
  • ActRIIB 2 In order to demonstrate that the eight amino acids in the extracellular juxtamembrane region of ActRIIB 2 but not in ActRIIB 4 (L. Attisano, J.L. Wrana, S. Cheifetz, and J.
  • the receptors are crosslinked to [ 125 I]-BMP-4, then subjected to immunoprecipitation with antibodies specific for the type I receptors BRK-1 and BRK-2 described below using previously described methods (T. Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S. Noji, D.H. Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995); B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)).
  • the BRK-1 rabbit polyclonal antibody #1353 is raised against the E. coli produced extracellular domain and produced as described previously (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)).
  • the BRK-2 rabbit polyclonal antibody JM#2 was raised against the intracellular juxtamembrane peptide A R P R Y S I G L ⁇ Q D ⁇ T Y I P P C (AA: 155-172) conjugated by standard methods to keyhole limpet, and used to immunize three New Zealand White rabbits (Berkeley Antibody Company, Richmond, CA 94806-1965).
  • antisera are evaluated for their ability to recognize the original peptide coated on plastic, using an antibody capture ⁇ LISA via the COOH cysteine.
  • antisera was IgG purified over a Pierce Immunopure Plus Immobilized Protein A column (Pierce Chemical Company, Rockford, IL) using the Immunopure (A) IgG Purification Kit (product # 44667) as described by the manufacturer.
  • RIB/L-17 cells which do not express receptors to the levels observed in the COS-1 cells (D. Vivien, L. Attisano, J.L. Wrana, and J. Massague, /. Biological Chemistry, 270:7134- 7141(1995)).
  • RIB/L-17 cells complex formation is observed between either the BRK-1 or the BRK-2 type I receptor and the BRK-3 type II receptor, as previously described in COS-1 cells (T. Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S. Noji, D.H. Wolsing, and J.
  • m-ActRHB + BRK-1 or m-ActRIIB 2 + BRK-2 in a ligand binding assay for the identification of BMP receptor agonists and antagonists
  • Identification of ligands that interact with mActRIIB 2 complexed to a type I BMP receptor can be achieved through the use of assays that are designed to measure the interaction of the ligands with this BMP receptor complex.
  • a receptor binding assay that uses the m-ActRIIB 2 + BRK-1 or mActRIIB 2 + BRK-2 complex and is adapted to handle large numbers of samples is carried out as follows.
  • COS-1 cells are transfected with the cDNAs for m-ActRIIB 2 , using the construct pJT6-mActRIIB 2 , and either BRK-1, using the construct pJT4-J159F for BRK-1 expression, or BRK-2, using the construct pJT3-BRK-2 for BRK-2 expression, as described in Example 4 above, except that the cells are grown in a 12 well culture dish or a 96-well microtitre plate.
  • the DNA mixture used to transfect the cells contains the receptors in the concentrations described above in Example 4.
  • the cells are washed once with binding buffer (50 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KCL, 5 mM MgSO4, 1.2 mM CaCl2, 2 mg/ml BSA), then equilibrated in the same buffer at 4°C for 60 min with gentle shaking. After equilibration, the buffer is aspirated, and to each well is added 4°C binding buffer containing [125i]BMP-4 tracer (100-400 pM) in the presence or absence of varying concentrations of test compounds (i.e. , putative ligands), for a period of 4 hours at 4°C with gentle shaking.
  • binding buffer 50 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KCL, 5 mM MgSO4, 1.2 mM CaCl2, 2 mg/ml BSA
  • BMP-2 is added at a final concentration of 10 nM.
  • a protease inhibitor cocktail is also added, to give a final concentration of 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml antipain, 50 ⁇ g/ml aprotinin, 100 ⁇ g/ml benzamidine, 100 ⁇ g/ml soybean trypsin inhibitor, 10 ⁇ g/ml bestatin, 10 ⁇ g/ml pepstatin, and 300 ⁇ M phenylmethylsulfonyl fluoride (PMSF).
  • PMSF phenylmethylsulfonyl fluoride
  • the buffer is aspirated, and the cells are rinsed 4 times with washing buffer (50 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KC1, 5 mM MgSO4, 1.2 mM CaCl2, 0.5 mg/ml BSA).
  • washing buffer 50 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KC1, 5 mM MgSO4, 1.2 mM CaCl2, 0.5 mg/ml BSA.
  • RIPS buffer (20 mM Tris Base, lOOmM NaCl, 1 mM EDTA, 0.5 % NP-40, 0.5 % Deoxycholic Acid, 0.1 % SDS, 10 mM Nal, 1 % BSA, pH 8.0.
  • the solubilized cells are then transferred to fresh mbes and counted in a Packard Model 5005 COBRA Gamma Counter (Packard Instruments, Meriden, CT).
  • Test compounds which interact with the mActRIIB 2 + BRK-1 or mActRIIB 2 + BRK-2 receptor complex are observed to compete for binding to the receptor complex with the [125i]BMP-4 tracer, such that less [125i]BMP-4 tracer is bound in the presence of the test compound in comparison to the binding observed when the tracer is incubated in the absence of the novel compound.
  • a decrease in binding of the [125i]BMP-4 tracer by >_ 30% at the highest concentration of the test compound that is studied demonstrates that the test compound binds to the mActRIIB 2 + BRK-1 or mActRIIB 2 + BRK-2 receptor complex.
  • Example 9 Demonstration of BMP-mediated signaling through mActRIIB 2 but not mActRIIB d in a Complex With Type I BMP Receptors Since several laboratories have previously demonstrated use of the R1B/L- 17 mink lung epithelial cells (L. Attisano, J. Carcamo, F. Ventura, F.M.B. Weis, J. Massague, and J.L. Wrana, Cell 75:671-680 (1993); J. Carcamo, F.M.B. Weis, F. Ventura, R. Wieser, J.L. Wrana, L. Attisano, and J.
  • R-1B/L17 cells are transfected with various BMP receptor pairs, the 3TP- Lux reporter plasmid (J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang, and J. Massague, Cell 71:1003-1014 (1992)), and a ⁇ -galactosidase reporter construct driven by the CMV promoter (Clontech, Palo Alto, CA) as described in Example 4, and, after 24 hours, are plated at 2x10 cells per well in six well standard tissue culture plates (Corning, San Diego, CA).
  • the 3TP- Lux reporter plasmid J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang, and J. Massague, Cell 71:1003-1014 (1992)
  • the growth media is replaced with 0.1 %FBS-MEM for 2 to 4 hours.
  • BMPs are then applied in increasing concentrations in 0.1 %FBS-MEM for 18 hours prior to cell harvesting.
  • Cells are harvested and assayed for luciferase activity using the Dual Light System (Tropix, Bedford, MA) as described by the manufacturer.
  • reported luciferase activity values are normalized to the ⁇ -galactosidase values reported for the same aliquot (determined using the Dual Light System (Tropix, Bedford, MA) as described by the manufacturer), and all data are expressed as arbitrary units.
  • Test compounds which are agonists of the BRK-1 +ActRIIB 2 receptor complex will cause an increase in reporter activity in RIB cells co-expressing the BRK-1 and ActRIIB2 receptors in combination with the 3TP-Lux and ⁇ - galactosidase reporter genes, quantited via the arbitrary light units the level of luciferase activity produced by activation of the luciferase enzyme normalized to the arbitrary light units produced by activation of the ⁇ -galactosidase enzyme as described for BMP-2 or BMP-4 in Example 9 above.
  • RIB/L-17 cells expressing co-expressing the BRK-1 and ActRIIB 2 receptors in combination with the 3TP-Lux and ⁇ -galactosidase reporter genes are produced as described in Examples 4 and 9 above are exposed to various concentrations of unknown agents, and the cells are evaluated for their response, quantited via the arbitrary light units the level of luciferase activity produced by activation of the luciferase enzyme normalized to the arbitrary light units produced by activation of the ⁇ - galactosidase enzyme as described for BMP-2 or BMP-4 in Example 9 above.
  • Those compounds which produce an activity in RIB/L-17 cells expressing BRK-1 + ActRIIB 2 receptors in combination with the 3TP-Lux and ⁇ -galactosidase genes, but not in RIB/L-17 cells which express only the 3TP-Lux and ⁇ -galactosidase genes are said to act as agonists of the BRK-1 + ActRIIB 2 receptor complex.
  • test compounds are added to RIB/L- 17 cells expressing BRK-1 + ActRIIB 2 receptors in combination with the 3TP- Lux and ⁇ -galactosidase genes in the presence of a fixed concentration of BMP-4 or another BMP receptor agonist.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • GGC CTG GCT GTT CGG TTT GAG CCA GGG AAG CCT CCT GGG GAT ACC CAT 1111 Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His 345 350 355
  • MOLECULE TYPE DNA (genomic)
  • GGC CTG GCT GTT CGG TTT GAG CCA GGG AAG CCT CCT GGG GAT ACC CAT 1159 Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His 360 365 370
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE
  • GGC CTT TGG GAG AAA TCA AAA GGG GAC ATA AAT CTT GTA AAA CAA GGA 657 Gly Leu Trp Glu Lys Ser Lys Gly Asp He Asn Leu Val Lys Gin Gly 70 75 80
  • GCT GTA AAA GTG TTT TCC TTT GCA AAC CGT CAG AAT TTT ATC AAC GAA 1137 Ala Val Lys Val Phe Ser Phe Ala Asn Arg Gin Asn Phe He Asn Glu 230 235 240

Abstract

L'invention porte sur l'utilisation d'un récepteur de type II partagé entre des activines et des protéines morphogénétiques osseuses, conjugué à un récepteur de protéine morphogénétique osseuse de type I, pour sélectionner des agents de différenciation cellulaire. L'invention concerne en outre des cellules contenant un ADN codant pour ce récepteur, et cet ADN. Un procédé pour déterminer la capacité d'un composé de se lier à un nouveau complexe BMP et la capacité d'un composé d'essai à produire un signal après sa liaison avec ce complexe protéine et récepteur de BMP, et un procédé pour déterminer la concentration d'un ligand de récepteur de BMP dans un échantillon clinique utilisant un nouveau complexe BMP sont également décrits, ainsi qu'une cellule hôte co-transfectée avec un vecteur d'expression contenant une séquence d'ADN qui code pour les composants de ce complexe, ou des fragments solubles de ce dernier.
PCT/US1998/009519 1997-05-16 1998-05-13 Utilisation d'un complexe recepteur de proteine morphogenetique osseuse (bmp) a des fins de selection WO1998052038A1 (fr)

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EP98922179A EP0981745A1 (fr) 1997-05-16 1998-05-13 Utilisation d'un complexe recepteur de proteine morphogenetique osseuse (bmp) a des fins de selection
IL13292298A IL132922A0 (en) 1997-05-16 1998-05-13 The use of a bone morphogenetic protein (bmp) receptor complex for screening
CA002290755A CA2290755A1 (fr) 1997-05-16 1998-05-13 Utilisation d'un complexe recepteur de proteine morphogenetique osseuse (bmp) a des fins de selection
JP54935398A JP2002510198A (ja) 1997-05-16 1998-05-13 スクリーニングのための骨形態形成タンパク質（ｂｍｐ）受容体複合体の使用
AU74782/98A AU7478298A (en) 1997-05-16 1998-05-13 The use of a bone morphogenetic protein (bmp) receptor complex for screening

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US4676897P 1997-05-16 1997-05-16
US60/046,768 1997-05-16

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WO2009114180A1 (fr) * 2008-03-13 2009-09-17 The General Hospital Corporation Inhibiteurs de la voie de signalisation bmp
JP2009261402A (ja) * 2001-04-24 2009-11-12 Johns Hopkins Univ 筋重量を増加させるためのフォリスタチンの使用方法
AU2015200950B2 (en) * 2004-07-23 2017-04-27 Acceleron Pharma Inc. ActRII receptor polypeptides, methods and compositions
US9682983B2 (en) 2013-03-14 2017-06-20 The Brigham And Women's Hospital, Inc. BMP inhibitors and methods of use thereof
US10513521B2 (en) 2014-07-15 2019-12-24 The Brigham And Women's Hospital, Inc. Compositions and methods for inhibiting BMP
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US8822411B2 (en) 1997-08-01 2014-09-02 The Johns Hopkins University School Of Medicine Truncated activin type II receptor and methods of use
JP2004504832A (ja) * 2000-07-27 2004-02-19 ザ　ジョーンズ　ホプキンス　ユニバーシティー　スクール　オブ　メディシン 増殖分化因子受容体、そのアゴニスト、およびアンタゴニスト、ならびにそれらの使用方法
JP2012235781A (ja) * 2000-07-27 2012-12-06 Johns Hopkins Univ School Of Medicine 増殖分化因子受容体、そのアゴニスト、およびアンタゴニスト、ならびにそれらの使用方法
JP2009261402A (ja) * 2001-04-24 2009-11-12 Johns Hopkins Univ 筋重量を増加させるためのフォリスタチンの使用方法
AU2015200950B2 (en) * 2004-07-23 2017-04-27 Acceleron Pharma Inc. ActRII receptor polypeptides, methods and compositions
WO2009114180A1 (fr) * 2008-03-13 2009-09-17 The General Hospital Corporation Inhibiteurs de la voie de signalisation bmp
US8507501B2 (en) 2008-03-13 2013-08-13 The Brigham And Women's Hospital, Inc. Inhibitors of the BMP signaling pathway
US9045484B2 (en) 2008-03-13 2015-06-02 The Brigham And Women's Hospital, Inc. Inhibitors of the BMP signaling pathway
US9682983B2 (en) 2013-03-14 2017-06-20 The Brigham And Women's Hospital, Inc. BMP inhibitors and methods of use thereof
US10017516B2 (en) 2013-03-14 2018-07-10 The Brigham And Women's Hospital, Inc. BMP inhibitors and methods of use thereof
US10513521B2 (en) 2014-07-15 2019-12-24 The Brigham And Women's Hospital, Inc. Compositions and methods for inhibiting BMP
US11440949B2 (en) 2016-10-05 2022-09-13 Acceleron Pharma Inc. TGF-beta superfamily type I and type II receptor heteromultimers and uses thereof

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AU7478298A (en) 1998-12-08
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CA2290755A1 (fr) 1998-11-19
IL132922A0 (en) 2001-03-19

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