WO1995024474A1 - Proteine 10 osseuse morphogenique - Google Patents

Proteine 10 osseuse morphogenique Download PDF

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Publication number
WO1995024474A1
WO1995024474A1 PCT/US1994/005292 US9405292W WO9524474A1 WO 1995024474 A1 WO1995024474 A1 WO 1995024474A1 US 9405292 W US9405292 W US 9405292W WO 9524474 A1 WO9524474 A1 WO 9524474A1
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Prior art keywords
polypeptide
bmp
polynucleotide
dna
εaid
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PCT/US1994/005292
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English (en)
Inventor
Wei Wu He
Timothy A. Coleman
Gregg A. Hastings
Craig A. Rosen
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Human Genome Sciences, Inc.
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Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to AU67875/94A priority Critical patent/AU6787594A/en
Publication of WO1995024474A1 publication Critical patent/WO1995024474A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptide ⁇ encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is Bone Morphogenic Protein-10 (BMP-10). The invention also relates to inhibiting the action of such polypeptide ⁇ .
  • BMP-10 Bone Morphogenic Protein-10
  • BMP may be used to induce bone and/or cartilage formation and is therefore useful in wound healing and tissue repair.
  • BMP may be used for treating a number of bone defects and periodontal disease and various types of wounds.
  • a 32-36 kDa o ⁇ teogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor-beta super family, the bovine equivalent of human osteogenic protein-1 and bone morphogenic protein-2a. It is reported that recombinant human osteogenic protein-1 (HOP-1) induces new bone formation in vivo with a specific activity compatible with natural bovine osteogenic protein and stimulate ⁇ osteoblast proliferation and differentiation in vitro (Sampath, T.K., et al., J. Biol. Chem., 267:20352-62 (1992)).
  • HOP-1 human o ⁇ teogenic protein-1
  • DVR-4 (Bone Morphogenetic Protein-4) induces amphibian embryos to develop with an overall posterior and/or ventral character, and that DVR-4 induces ventral types of mesoderm in animal explant ⁇ .
  • DVR-4 i ⁇ therefore the first molecule reported both to induce po ⁇ teroventral mesoderm and to counteract dorsalizing ⁇ ignal ⁇ ⁇ uch as activin, (Jones, CM. et al, Development, 115:639-47 (1992)).
  • polypeptide of the present invention is of human origin.
  • polynucleotides (DNA or RNA) which encode such polypeptides.
  • a proce ⁇ for utilizing ⁇ uch polypeptide ⁇ , or polynucleotides encoding such polypeptide ⁇ for therapeutic purpo ⁇ e ⁇ , for example, for the promotion of de novo bone formation during surgical insertion of prothese ⁇ , for the treatment of non-union bone fractures, and for treatment of osteoporosis and periodontal disease.
  • an antibody again ⁇ t ⁇ uch polypeptide ⁇ may be u ⁇ ed diagnostically in the detection of lung di ⁇ order ⁇ by mea ⁇ uring the serum level of BMP-10 in patient ⁇ .
  • FIG. la depict ⁇ the cDNA ⁇ equence and corre ⁇ ponding deduced a ino acid ⁇ equence of the mature BMP-10 polypeptide.
  • the amino acid sequence i ⁇ represented by the ⁇ tandard three letter code for amino acid ⁇ .
  • FIG. lb shows the homology between the amino acid ⁇ equence of the active domain of BMP-10 (upper line) with that of BMP-3a (lower line).
  • FIG. 2 depict ⁇ the re ⁇ ult ⁇ of a Northern Blot Analy ⁇ i ⁇ which indicate ⁇ the expre ⁇ sion levels of the mRNA tran ⁇ cript for BMP-10 in human ti ⁇ ue ⁇ .
  • nucleic acid polynucleotide which encodes for the mature polypeptide having the deduced amino acid sequence of Figure la or for the mature polypeptide encoded by the cDNA of the clone deposited a ⁇ ATCC Deposit No. 75672 on February 9, 1994.
  • a polynucleotide encoding a polypeptide of the present invention was di ⁇ covered in a fetal lung cDNA library. It contains an open reading frame encoding a mature polypeptide of 119 amino acids and shows 80 % sequence identity to the BMP-3a gene product.
  • the polypeptide is a member of the bone morphogenic protein family which i ⁇ a ⁇ ubfamily of the tran ⁇ forming growth factor Beta (TGF-jS) ⁇ uperfa ily.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA include ⁇ cDNA, geno ic DNA, and synthetic DNA.
  • the DNA may be double- ⁇ tranded or ⁇ ingle-stranded, and if ⁇ ingle ⁇ tranded may be the coding ⁇ trand or non-coding (anti- ⁇ en ⁇ e) strand.
  • the coding sequence which encode ⁇ the mature polypeptide may be identical to the coding ⁇ equence ⁇ hown in Figure la or that of the depo ⁇ ited clone or may be a different coding ⁇ equence which coding ⁇ equence, a ⁇ a re ⁇ ult of the redundancy or degeneracy of the genetic code, encode ⁇ the ⁇ ame, mature polypeptide a ⁇ the DNA of Figure la or the depo ⁇ ited cDNA.
  • the polynucleotide which encode ⁇ for the mature polypeptide of Figure la or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such a ⁇ a leader or secretory sequence or a proprotein ⁇ equence; the coding sequence for the mature polypeptide (and optionally additional coding ⁇ equence) and non-coding ⁇ equence, ⁇ uch a ⁇ intron ⁇ or non-coding ⁇ equence 5' and/or 3' of the coding ⁇ equence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompa ⁇ ses a polynucleotide which includes only coding ⁇ equence for the polypeptide a ⁇ well as a polynucleotide which include ⁇ additional coding and/or non-coding ⁇ equence.
  • the pre ⁇ ent invention further relate ⁇ to variants of the hereinabove described polynucleotides which encode for fragments, analog ⁇ and derivative ⁇ of the polypeptide having the deduced amino acid ⁇ equence of Figure la or the polypeptide encoded by the cDNA of the depo ⁇ ited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the ⁇ ame mature polypeptide a ⁇ ⁇ hown in Figure la or the ⁇ ame mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variant ⁇ encode for a fragment, derivative or analog of the polypeptide of Figure la or the polypeptide encoded by the cDNA of the depo ⁇ ited clone.
  • nucleotide variant ⁇ include deletion variant ⁇ , ⁇ ub ⁇ titution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding ⁇ equence ⁇ hown in Figure la or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which doe ⁇ not substantially alter the function of the encoded polypeptide.
  • the present invention also includes polynucleotide ⁇ , wherein the coding ⁇ equence for the mature polypeptide may be fu ⁇ ed in the ⁇ ame reading frame to a polynucleotide ⁇ equence which aid ⁇ in expre ⁇ ion and ⁇ ecretion of a polypeptide from a ho ⁇ t cell, for example, a leader ⁇ equence which function ⁇ as a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotide ⁇ may also encode for a proprotein which i ⁇ the mature protein plus additional 5' amino acid re ⁇ idues.
  • a mature protein having a prosequence i ⁇ a proprotein and i ⁇ .an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
  • the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a pro ⁇ equence and a pre ⁇ equence (leader sequence).
  • the polynucleotide ⁇ of the present invention may also have the coding ⁇ equence fu ⁇ ed in frame to a marker ⁇ equence which allows for purification of the polypeptide of the pre ⁇ ent invention.
  • the marker ⁇ equence may be a hexa- hi ⁇ tidine tag ⁇ upplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag correspond ⁇ to an epitope derived from the influenza hemagglutinin protein ( il ⁇ on, I., et al.. Cell, 37:767 (1984)).
  • the pre ⁇ ent invention further relate ⁇ to polynucleotide ⁇ which hybridize to the hereinabove-de ⁇ cribed sequences if there i ⁇ at least 50% and preferably 70% identity between the sequence ⁇ .
  • the present invention particularly relates to polynucleotide ⁇ which hybridize under ⁇ tringent conditions to the hereinabove-de ⁇ cribed polynucleotide ⁇ .
  • ⁇ tringent conditions mean ⁇ hybridization will occur only if there i ⁇ at lea ⁇ t 95% and preferably at least 97% identity between the ⁇ equence ⁇ .
  • polypeptides which retain substantially the ⁇ ame biological function or activity a ⁇ the mature polypeptide encoded by the cDNA of Figure la or the depo ⁇ ited cDNA.
  • the depo ⁇ it( ⁇ ) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organism ⁇ for purpo ⁇ e ⁇ of Patent Procedure.
  • the ⁇ e depo ⁇ it ⁇ are provided merely a ⁇ convenience to those of ⁇ kill in the art and are not an admission that a deposit i ⁇ required under 35 U.S.C. ⁇ 112.
  • the sequence of the polynucleotides contained in the deposited materials, a ⁇ well a ⁇ the amino acid ⁇ equence of the polypeptide ⁇ encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any de ⁇ cription of sequences herein.
  • a license may be required to make, u ⁇ e or ⁇ ell the depo ⁇ ited material ⁇ , and no such license is hereby granted.
  • the pre ⁇ ent invention further relate ⁇ to a BMP-10 polypeptide which ha ⁇ the deduced amino acid ⁇ equence of Figure la or which ha ⁇ the amino acid sequence encoded by the deposited cDNA, a ⁇ well a ⁇ fragments, analog ⁇ and derivative ⁇ of such polypeptide.
  • fragment when referring to the polypeptide of Figure la or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity a ⁇ such polypeptide. Thu ⁇ , an analog include ⁇ a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure la or that encoded by the depo ⁇ ited cDNA may be (i) one in which one or more of the amino acid re ⁇ idue ⁇ are substituted with a conserved or non-conserved amino acid residue (preferably a con ⁇ erved amino acid re ⁇ idue) and such ⁇ ub ⁇ tituted amino acid re ⁇ idue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid re ⁇ idue ⁇ include ⁇ a ⁇ ub ⁇ tituent group, or (iii) one in which the mature polypeptide.
  • SUBSUME SHEET (RULE 26) derivative ⁇ and analog ⁇ are deemed to be within the ⁇ cope of tho ⁇ e ⁇ killed in the art from the teachings herein.
  • polypeptides and polynucleotide ⁇ of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • i ⁇ olated mean ⁇ that the material i ⁇ removed from it ⁇ original environment (e.g., the natural environment if it i ⁇ naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide pre ⁇ ent in a living animal i ⁇ not i ⁇ olated, but the ⁇ ame polynucleotide or polypeptide, ⁇ eparated from ⁇ ome or all of the coexi ⁇ ting material ⁇ in the natural ⁇ y ⁇ tem, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptide ⁇ could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the pre ⁇ ent invention al ⁇ o relate ⁇ to vector ⁇ which include polynucleotide ⁇ of the present invention, ho ⁇ t cell ⁇ which are genetically engineered with vector ⁇ of the invention and the production of polypeptide ⁇ of the invention by recombinant techniques.
  • Ho ⁇ t cells are genetically engineered (transduced or transformed or tran ⁇ fected) with the vector ⁇ of thi ⁇ invention which may be, for example, a cloning vector or an expre ⁇ ion vector.
  • the vector may be, for example, in the form of a pla ⁇ mid, a viral particle, a phage, etc.
  • the engineered ho ⁇ t cells can be cultured in conventional nutrient media modified a ⁇ appropriate for activating promoter ⁇ , ⁇ electing tran ⁇ formant ⁇ or amplifying the BMP-10 gene ⁇ .
  • the culture conditions ⁇ uch a ⁇ temperature, pH and the like, are tho ⁇ e previou ⁇ ly u ⁇ ed with the ho ⁇ t cell selected for expre ⁇ ion, and will be apparent to the ordinarily ⁇ killed arti ⁇ an.
  • polynucleotides of the present invention may be employed for producing polypeptides by recombinant
  • the polynucleotide may be included in any one of a variety of expre ⁇ ion vector ⁇ for expre ⁇ ing a polypeptide.
  • vector ⁇ include chromo ⁇ omal, nonchromo ⁇ omal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculoviru ⁇ ; yea ⁇ t pla ⁇ mid ⁇ ; vector ⁇ derived from combinations of plasmids and phage DNA, viral DNA such a ⁇ vaccinia, adenoviru ⁇ , fowl pox viru ⁇ , and pseudorabies.
  • any other vector may be u ⁇ ed as long as it i ⁇ replicable and viable in the ho ⁇ t.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedure ⁇ and others are deemed to be within the ⁇ cope of those skilled in the art.
  • the DNA sequence in the expres ⁇ ion vector i ⁇ operatively linked to an appropriate expression control sequence( ⁇ ) (promoter) to direct mRNA ⁇ ynthesi ⁇ .
  • promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P L promoter and other promoters known to control expres ⁇ ion of genes in prokaryotic or eukaryotic cell ⁇ or their viru ⁇ e ⁇ .
  • the expre ⁇ ion vector al ⁇ o contain ⁇ a ribo ⁇ ome binding ⁇ ite for tran ⁇ lation initiation and a tran ⁇ cription terminator.
  • the vector may al ⁇ o include appropriate ⁇ equence ⁇ for amplifying expre ⁇ ion.
  • the expre ⁇ ion vector ⁇ preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cell ⁇ such a ⁇ dihydrofolate reducta ⁇ e or neomycin re ⁇ i ⁇ tance for eukaryotic cell culture, or ⁇ uch as tetracycline or ampicillin resistance in E. coli.
  • -9- control sequence may be employed to transform an appropriate ho ⁇ t to permit the ho ⁇ t to expre ⁇ the protein.
  • bacterial cells such as E. coli. Streptomyces. Salmonella typhimurium; fungal cell ⁇ , ⁇ uch a ⁇ yea ⁇ t; in ⁇ ect cell ⁇ such as Dro ⁇ ophila and Sf9; animal cells such a ⁇ CHO, COS or Bowes melanoma; plant cells, etc.
  • bacterial cells such as E. coli. Streptomyces. Salmonella typhimurium
  • fungal cell ⁇ ⁇ uch a ⁇ yea ⁇ t
  • in ⁇ ect cell ⁇ such as Dro ⁇ ophila and Sf9
  • animal cells such a ⁇ CHO, COS or Bowes melanoma
  • plant cells etc.
  • the selection of an appropriate host is deemed to be within the scope of those ⁇ killed in the art from the teaching ⁇ herein.
  • the pre ⁇ ent invention also includes recombinant constructs comprising one or more of the sequence ⁇ a ⁇ broadly described above.
  • the construct ⁇ compri ⁇ e a vector, such as a plasmid or viral vector, into which a ⁇ equence of the invention ha ⁇ been in ⁇ erted, in a forward or reverse orientation.
  • the con ⁇ truct further compri ⁇ es regulatory ⁇ equence ⁇ , including, for example, a promoter, operably linked to the sequence.
  • regulatory ⁇ equence ⁇ including, for example, a promoter, operably linked to the sequence.
  • Large numbers of ⁇ uitable vector ⁇ and promoter ⁇ are known to tho ⁇ e of ⁇ kill in the art, and are commercially available.
  • the following vector ⁇ are provided by way of example.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pb ⁇ , pDIO, phagescript, psiX174, pblue ⁇ cript SK, pb ⁇ k ⁇ , pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLNEO, PSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • any other pla ⁇ mid or vector may be u ⁇ ed a ⁇ long a ⁇ they are replicable and viable in the ho ⁇ t.
  • Promoter region ⁇ can be ⁇ elected from any de ⁇ ired gene u ⁇ ing CAT (chloramphenicol tran ⁇ fera ⁇ e) vector ⁇ or other vector ⁇ with selectable markers.
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoter ⁇ include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoter ⁇ include CMV immediate early, HSV thymidine kina ⁇ e, early and late SV40, LTRs from retrovirus. and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to ho ⁇ t cells containing the above-described constructs.
  • the ho ⁇ t cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the con ⁇ truct into the ho ⁇ t cell can be effected by calcium pho ⁇ phate tran ⁇ fection, DEAE- Dextran mediated tran ⁇ fection, or electroporation. (Davi ⁇ , L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)).
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthe ⁇ izer ⁇ .
  • Mature protein ⁇ can be expressed in mammalian cell ⁇ , yea ⁇ t, bacteria, or other cell ⁇ under the control of appropriate promoter ⁇ .
  • Cell-free tran ⁇ lation ⁇ y ⁇ tem ⁇ can al ⁇ o be employed to produce ⁇ uch protein ⁇ u ⁇ ing RNA ⁇ derived from the DNA con ⁇ truct ⁇ of the pre ⁇ ent invention.
  • Appropriate cloning and expression vector ⁇ for u ⁇ e with prokaryotic and eukaryotic ho ⁇ t ⁇ are de ⁇ cribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancer ⁇ are ci ⁇ -acting element ⁇ of DNA, u ⁇ ually about from 10 to 300 bp that act on a promoter to increase its tran ⁇ cription. Examples including the SV40 enhancer on the late ⁇ ide of the replication origin bp 100 to 270, a cytomegaloviru ⁇ early promoter enhancer, the polyoma enhancer on the late ⁇ ide of the replication origin, and adenoviru ⁇ enhancers.
  • recombinant expres ⁇ ion vector ⁇ will include origin ⁇ of replication and ⁇ electable markers permitting tran ⁇ formation of the ho ⁇ t cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expres ⁇ ed gene to direct tran ⁇ cription of a down ⁇ tream ⁇ tructural ⁇ equence.
  • promoter ⁇ can be derived from operon ⁇ encoding glycolytic enzymes ⁇ uch a ⁇ 3-pho ⁇ phoglycerate kina ⁇ e (PGK), ⁇ -factor, acid pho ⁇ phata ⁇ e, or heat ⁇ hock proteins, among other ⁇ .
  • the heterologou ⁇ ⁇ tructural sequence is a ⁇ embled in appropriate phase with tran ⁇ lation initiation and termination sequences, and preferably, a leader ⁇ equence capable of directing ⁇ ecretion of tran ⁇ lated protein into the peripla ⁇ mic ⁇ pace or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteri ⁇ tic ⁇ , e.g., ⁇ tabilization or ⁇ implified purification of expressed recombinant product.
  • Useful expres ⁇ ion vector ⁇ for bacterial u ⁇ e are con ⁇ tructed by in ⁇ erting a ⁇ tructural DNA ⁇ equence encoding a desired protein together with suitable tran ⁇ lation initiation and termination ⁇ ignal ⁇ in operable reading pha ⁇ e with a functional promoter.
  • the vector will compri ⁇ e one or more phenotypic ⁇ electable marker ⁇ and an origin of replication to en ⁇ ure maintenance of the vector and to, if de ⁇ irable, provide amplification within the ho ⁇ t.
  • Suitable prokaryotic ho ⁇ t ⁇ for tran ⁇ formation include E. coli, Bacillu ⁇ ⁇ ubtili ⁇ . Salmonella typhimuriu and variou ⁇ ⁇ pecie ⁇ within the genera P ⁇ eudomona ⁇ , Streptomyce ⁇ , and Staphylococcu ⁇ , although others may al ⁇ o be employed a ⁇ a matter of choice.
  • u ⁇ eful expre ⁇ ion vector ⁇ for bacterial u ⁇ e can compri ⁇ e a selectable marker and bacterial origin of replication derived from commercially available pla ⁇ mid ⁇ compri ⁇ ing genetic element ⁇ of the well known cloning vector pBR322 (ATCC 37017).
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA).
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cell ⁇ are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical di ⁇ ruption, or u ⁇ e of cell lysing agents, such methods are well know to those skilled in the art.
  • mammalian cell culture ⁇ y ⁇ tem ⁇ can al ⁇ o be employed to expre ⁇ recombinant protein.
  • mammalian expres ⁇ ion ⁇ y ⁇ tem ⁇ include the COS-7 line ⁇ of monkey kidney fibrobla ⁇ t ⁇ , de ⁇ cribed by Gluzman, Cell, 23:175 (1981), and other cell line ⁇ capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell line ⁇ .
  • Mammalian expres ⁇ ion vector ⁇ will compri ⁇ e an origin of replication, a ⁇ uitable promoter and enhancer, and al ⁇ o any nece ⁇ ary ribosome binding ⁇ ite ⁇ , polyadenylation ⁇ ite, ⁇ plice donor and acceptor ⁇ ite ⁇ , tran ⁇ criptional termination ⁇ equence ⁇ , and 5' flanking nontran ⁇ cribed sequence ⁇ .
  • DNA ⁇ equence ⁇ derived from the SV40 ⁇ plice, and polyadenylation ⁇ ite ⁇ may be u ⁇ ed to provide the required nontran ⁇ cribed genetic element ⁇ .
  • the polypeptide ⁇ can be recovered and purified from recombinant cell culture ⁇ by method ⁇ including ammonium ⁇ ulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, pho ⁇ phocellulo ⁇ e chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It i ⁇ preferred to have low concentration ⁇ (approximately 0.15-5 mM) of calcium ion pre ⁇ ent during purification. (Price et al., J. Biol. Chem. , 244:917 (1969)). Protein refolding ⁇ tep ⁇ can be u ⁇ ed, a ⁇ nece ⁇ ary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification ⁇ tep ⁇ .
  • HPLC high performance liquid chromatography
  • the polypeptide ⁇ of the pre ⁇ ent invention may be a naturally purified product, or a product of chemical ⁇ ynthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic ho ⁇ t (for example, by bacterial, yea ⁇ t, higher plant, in ⁇ ect and mammalian cell ⁇ in culture).
  • a prokaryotic or eukaryotic ho ⁇ t for example, by bacterial, yea ⁇ t, higher plant, in ⁇ ect and mammalian cell ⁇ in culture.
  • the polypeptide ⁇ of the pre ⁇ ent invention may be glyco ⁇ ylated or may be non-glyco ⁇ ylated.
  • Polypeptide ⁇ of the invention may also include an initial methionine amino acid residue.
  • the BMP-10 may be employed to promote de novo bone formation which may be u ⁇ ed in the treatment of periodontal disease and other bone defects of the oral cavity.
  • BMP-10 may al ⁇ o be u ⁇ ed during ⁇ urgical in ⁇ ertion of prothe ⁇ e ⁇ .
  • prothe ⁇ i ⁇ i ⁇ held in place by ⁇ urgical cement.
  • the cement eventually loo ⁇ en ⁇ , however, making it nece ⁇ ary to perform another ⁇ urgery.
  • Thi ⁇ second surgery is a much more difficult procedure and is respon ⁇ ible for ⁇ urgeon ⁇ reluctantance to in ⁇ ert prothe ⁇ e ⁇ in young people.
  • BMP-10 may, therefore, be u ⁇ ed to coat the prothe ⁇ i ⁇ before in ⁇ ertion which re ⁇ ult ⁇ in bone formation around the prothe ⁇ is, making a ⁇ tronger union and allowing for the u ⁇ e of le ⁇ cement.
  • BMP-10 may also be employed in the treatment of o ⁇ teoporo ⁇ is, which i ⁇ characterized by exce ⁇ ive bone re ⁇ orption re ⁇ ulting in thin and brittle bone ⁇ . BMP-10 would ⁇ timulate bone formation to help alleviate this condition.
  • polypeptide ⁇ may al ⁇ o be employed in accordance with the pre ⁇ ent invention by expre ⁇ ion of ⁇ uch polypeptide ⁇ in vivo , which i ⁇ often referred to a ⁇ "gene therapy.”
  • cell ⁇ from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo , with the engineered cell ⁇ then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cell ⁇ may be engineered by procedure ⁇ known in the art by u ⁇ e of a retroviral particle containing RNA encoding a polypeptide of the pre ⁇ ent invention.
  • cell ⁇ may be engineered in vivo for expre ⁇ ion of a polypeptide in vivo by, for example, procedure ⁇ known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the pre ⁇ ent invention may be administered to a patient for engineering cells in vivo and expres ⁇ ion of the polypeptide in vivo .
  • the expres ⁇ ion vehicle for engineering cell ⁇ may be other than a retrovirus, for example, an adenovirus which may be u ⁇ ed to engineer cells in vivo after combination with a ⁇ uitable delivery vehicle.
  • the polypeptide ⁇ of the present invention may be employed in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier include ⁇ but i ⁇ not limited to ⁇ aline, buffered ⁇ aline, dextro ⁇ e, water, glycerol, ethanol, and combination ⁇ thereof.
  • a carrier include ⁇ but i ⁇ not limited to ⁇ aline, buffered ⁇ aline, dextro ⁇ e, water, glycerol, ethanol, and combination ⁇ thereof.
  • the formulation ⁇ hould suit the mode of admini ⁇ tration.
  • the invention al ⁇ o provide ⁇ a pharmaceutical pack or kit compri ⁇ ing one or more container ⁇ filled with one or more of the ingredient ⁇ of the pharmaceutical compo ⁇ ition ⁇ of the invention.
  • a ⁇ ociated with ⁇ uch container( ⁇ ) can be a notice in the form pre ⁇ cribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical ⁇ or biological products, which notice reflect ⁇ approval by the agency of manufacture, u ⁇ e or ⁇ ale for human administration.
  • the polypeptide ⁇ of the pre ⁇ ent invention may be employed in conjunction with other therapeutic compound ⁇ .
  • BMP-10 i ⁇ preferably u ⁇ ed topically, however, when it i ⁇ used sy ⁇ temically, the pharmaceutical compo ⁇ itions may be administered in a convenient manner such as by the oral, intravenous, intraperitoneal, intramuscular, subcutaneou ⁇ , intrana ⁇ al, or intradermal routes.
  • the amounts and dosage regimen ⁇ of pharmaceutical compo ⁇ ition ⁇ of BMP-10 administered to a subject will depend on a number of factors such as the mode of administration, the nature of the condition being treated, the body weight of the subject being treated and the judgment of the prescribing physician.
  • pharmaceutical compo ⁇ ition ⁇ of BMP-10 are given, for example, in appropriate do ⁇ e ⁇ of at lea ⁇ t about 10 ⁇ g/kg body weight and in mo ⁇ t ca ⁇ e ⁇ will not be admini ⁇ tered in an amount in excess of about 8 mg/kg body weight, and preferably i ⁇ given in do ⁇ e ⁇ of about 10 g/kg body weight to about 1 mg/kg daily, taking into account the route ⁇ of admini ⁇ tration, ⁇ ymptom ⁇ , etc.
  • BMP-10 may be prepared a ⁇ a gel matrix formulation and administered, for example, ectopically, which i ⁇ preferably admini ⁇ tered at the site of bone fracture at a dosage of 50 mg, by applying the matrix directly to the ⁇ ite of the fracture.
  • Thi ⁇ matrix may al ⁇ o be applied to protheses before in ⁇ ertion.
  • the ⁇ equence ⁇ of the present invention are also valuable for chromosome identification.
  • the ⁇ equence i ⁇ ⁇ pecifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagent ⁇ based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromo ⁇ ome ⁇ according to the present invention i ⁇ an important fir ⁇ t ⁇ tep in correlating tho ⁇ e ⁇ equence ⁇ with gene ⁇ a ⁇ ociated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
  • the ⁇ e primers are then used for PCR screening of somatic cell hybrid ⁇ containing individual human chromo ⁇ ome ⁇ . Only tho ⁇ e hybrid ⁇ containing the human gene corre ⁇ ponding to the primer will yield an amplified fragment.
  • mapping of ⁇ omatic cell hybrid ⁇ i ⁇ a rapid procedure for a ⁇ signing a particular DNA to a particular chromo ⁇ ome.
  • ⁇ ublocalization can be achieved with panel ⁇ of fragment ⁇ from specific chromo ⁇ ome ⁇ or pool ⁇ of large genomic clone ⁇ in an analogou ⁇ manner.
  • Other mapping ⁇ trategie ⁇ that can ⁇ imilarly be used to map to it ⁇ chromo ⁇ ome include in situ hybridization, pre ⁇ creening with labeled flow- ⁇ orted chromosomes and preselection by hybridization to con ⁇ truct chromosome ⁇ pecific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone ⁇ to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one ⁇ tep.
  • This technique can be u ⁇ ed with cDNA a ⁇ short a ⁇ 500 or 600 ba ⁇ e ⁇ ; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromo ⁇ omal location with ⁇ ufficient ⁇ ignal inten ⁇ ity for ⁇ imple detection.
  • FISH require ⁇ u ⁇ e of the clone ⁇ from which the EST wa ⁇ derived, and the longer the better.
  • a cDNA precisely localized to a chromosomal region as ⁇ ociated with the disea ⁇ e could be one of between 50 and 500 potential cau ⁇ ative gene ⁇ . (Thi ⁇ assumes 1 megaba ⁇ e mapping re ⁇ olution and one gene per 20 kb) .
  • Compari ⁇ on of affected and unaffected individual ⁇ generally involve ⁇ fir ⁇ t looking for ⁇ tructural alteration ⁇ in the chromo ⁇ ome ⁇ , ⁇ uch a ⁇ deletion ⁇ or tran ⁇ location ⁇ that are vi ⁇ ible from chromo ⁇ ome ⁇ pread ⁇ or detectable u ⁇ ing PCR ba ⁇ ed on that cDNA ⁇ equence.
  • complete sequencing of genes from ⁇ everal individual ⁇ i ⁇ required to confirm the pre ⁇ ence of a mutation and to di ⁇ tingui ⁇ h mutation ⁇ from polymorphisms.
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cell ⁇ expre ⁇ ing them can be u ⁇ ed a ⁇ an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodie ⁇ .
  • the present invention also includes chimeric, single chain, and humanized antibodie ⁇ , a ⁇ well a ⁇ Fab fragment ⁇ , or the product of an Fab expre ⁇ ion library. Variou ⁇ procedure ⁇ known in the art may be used for the production of such antibodie ⁇ and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptide ⁇ into an animal or by admini ⁇ tering the polypeptide ⁇ to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide ⁇ it ⁇ elf. In thi ⁇ manner, even a ⁇ equence encoding only a fragment of the polypeptide ⁇ can be used to generate antibodies binding the whole native polypeptide ⁇ . Such antibodie ⁇ can then be u ⁇ ed to i ⁇ olate the polypeptide from ti ⁇ ue expre ⁇ ing that polypeptide.
  • Example ⁇ include the hybridoma technique (Kohler and Mil ⁇ tein, 1975, Nature, 256:495-497), the trioma technique, the human B-ce.ll hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodie ⁇ (Cole, et al., 1985, in Monoclonal Antibodie ⁇ and Cancer Therapy, Alan R. Li ⁇ , Inc., pp. 77-96).
  • Antibodies specific to the polypeptide of the pre ⁇ ent invention may further be u ⁇ ed a potential marker for lung cancer.
  • the procedure would con ⁇ ist of drawing blood from a patient, adding the above- mentioned antibodie ⁇ to the blood ⁇ erum, then performing a ⁇ tandard immunoassay such as ELISA or a radioimmunoassay to detect the amount of antibody bound to BMP-10 which i ⁇ directly related to the amount of BMP-10 produced in the lung.
  • a ⁇ tandard immunoassay such as ELISA or a radioimmunoassay to detect the amount of antibody bound to BMP-10 which i ⁇ directly related to the amount of BMP-10 produced in the lung.
  • Tumor cell ⁇ are more active than cell ⁇ with a normal phy ⁇ iology and therefore, produce more BMP-10 which would be indicative of lung cancer.
  • pre ⁇ ent invention will be further de ⁇ cribed with reference to the following example ⁇ ; however, it i ⁇ to be understood that the pre ⁇ ent invention i ⁇ not limited to such example ⁇ . All part ⁇ or amounts, unle ⁇ otherwi ⁇ e ⁇ pecified, are by weight.
  • pla ⁇ mids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting pla ⁇ mid ⁇ herein are either commercially available, publicly available on an unre ⁇ tricted ba ⁇ i ⁇ , or can be con ⁇ tructed from available pla ⁇ mid ⁇ in accord with published procedure ⁇ .
  • equivalent pla ⁇ mid ⁇ to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzyme ⁇ used herein are commercially available and their reaction conditions, cofactors and other requirements were used a ⁇ would be known to the ordinarily ⁇ killed arti ⁇ an.
  • For analytical purpose ⁇ typically 1 ⁇ g of pla ⁇ mid or DNA fragment i ⁇ u ⁇ ed with about 2 unit ⁇ of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragments for pla ⁇ mid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and sub ⁇ trate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to i ⁇ olate the de ⁇ ired fragment
  • Oligonucleotides refer ⁇ to either a ⁇ ingle ⁇ tranded polydeoxynucleotide or two complementary polydeoxynucleotide ⁇ trand ⁇ which may be chemically ⁇ ynthe ⁇ ized. Such ⁇ ynthetic oligonucleotide ⁇ have no 5' pho ⁇ phate and thu ⁇ will not ligate to another oligonucleotide without adding a pho ⁇ phate with an ATP in the presence of a kina ⁇ e. A ⁇ ynthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase DNA ligase
  • the 5' oligonucleotide primer 5'- GATCGGATCCAAAGCCCGGAGGAAGCAG-3' contains a Bam HI re ⁇ triction enzyme ⁇ ite followed by 18 nucleotides of BMP-10 coding sequence starting from amino acid 4 (Lys); the 3' ⁇ equence 5'-GTACTCTAGATCACCGGCAGGCACAGGTG-3' contain ⁇ complementary ⁇ equence ⁇ to an Xba I ⁇ ite, a tran ⁇ lation ⁇ top codon and the la ⁇ t 16 nucleotide ⁇ of BMP-10 coding ⁇ equence.
  • the re ⁇ triction enzyme ⁇ ite ⁇ correspond to the re ⁇ triction enzyme ⁇ ite ⁇ on the bacterial expre ⁇ ion vector pQE-9 (Qiagen, Inc., 9259 Eton Ave., Chatsworth, CA 91311, Catalog No. 33093).
  • the plasmid vector encodes antibiotic resistance (Amp r ), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/0), a ribo ⁇ ome binding ⁇ ite (RBS), a 6- hi ⁇ tidine tag (6-Hi ⁇ ) and restriction enzyme cloning sites.
  • the pQE-9 vector was digested with Bam HI and Xba I and the insertion fragments were then ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS.
  • the ligation mixture was then used to transform the E. coli strain M15/rep4 available from Qiagen under the trademark ml5/rep4.
  • M15/rep4 contain ⁇ multiple copie ⁇ of the pla ⁇ mid pREP4, which expre ⁇ e ⁇ the lad repre ⁇ or and al ⁇ o confer ⁇ kanamycin re ⁇ istance (Kan r ) .
  • Transformant ⁇ are identified by their ability to grow on LB plate ⁇ containing both Amp and Kan.
  • Northern blot analy ⁇ i ⁇ wa ⁇ carried out to examine the level ⁇ of expression of BMP-10 in human tis ⁇ ue ⁇ .
  • Total cellular RNA ⁇ ample ⁇ were i ⁇ olated with RNAzolTM B system (Biotecx Laboratories, Inc., 6023 South Loop Ea ⁇ t, Houston, TX 77033).
  • About 10 ug of total RNA isolated from each human tis ⁇ ue ⁇ pecified was separated on 1% agarose gel and blotted onto a nylon filter (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Pre ⁇ (1989)).
  • the labeling reaction wa ⁇ done according to the Stratagene Prime- It kit with 50 ng DNA fragment.
  • the labeled DNA wa ⁇ purified with a Select-G-50 column from 5' Prime — 3 Prime, Inc., 5603 Arapahoe Road, Boulder, CO 80303.
  • the filter wa ⁇ then hybridized with radioactive labeled full length BMP-10 gene at 1,000,000 cp /ml in 0.5 M NaPo 4 and 7% SDS overnight at 65"C.
  • the filter ⁇ were wa ⁇ hed twice at room temperature and twice at 60 * C with 0.5 x SSC, 0.1% SDS, the filter ⁇ were then exposed at -70 * C overnight with intensifying screen.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • MOLECULE TYPE PROTEIN
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:2:
  • Lys Thr lie Gin Ly ⁇ Ala Arg Arg Ly ⁇ Gin Trp A ⁇ p Glu Pro Arg
  • Trp Asn Glu Trp lie lie Ser Pro Ly ⁇ Ser Phe A ⁇ p Ala Tyr Tyr

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Abstract

L'invention concerne un polypeptide de la protéine 10 osseuse morphogénique (BMP-10) humaine et l'ADN (ARN) codant ce polypeptide. Ledit polypeptide de la protéine 10 osseuse morphogénique humaine qui peut être produit par des techniques de recombinaison de l'ADN est utile pour induire la formation osseuse de novo. L'invention porte également sur un anticorps dirigé contre ledit polypeptide et pouvant être utilisé pour diagnostiquer le cancer des poumons.
PCT/US1994/005292 1994-03-10 1994-05-12 Proteine 10 osseuse morphogenique WO1995024474A1 (fr)

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AU67875/94A AU6787594A (en) 1994-03-10 1994-05-12 Bone morphogenic protein-10

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US20921494A 1994-03-10 1994-03-10
US08/209,214 1994-03-10

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Cited By (8)

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US5656593A (en) * 1991-03-11 1997-08-12 Creative Biomolecules, Inc. Morphogen induced periodontal tissue regeneration
EP1027430A1 (fr) * 1997-07-16 2000-08-16 Human Genome Sciences Serie de 64 proteines humaines secretees
EP1095159A1 (fr) * 1998-07-15 2001-05-02 Human Genome Sciences, Inc. Proteine morphogenetique osseuse
US6407060B1 (en) 1996-03-22 2002-06-18 Curis, Inc. Method for enhancing functional recovery following central nervous system ischemia or trauma
WO2005113590A2 (fr) * 2004-05-12 2005-12-01 Acceleron Pharma Inc. Propeptides de bmp10 et procédés correspondants
US7465706B2 (en) 2004-06-24 2008-12-16 Acceleron Pharma Inc. GDF3 propeptides and related methods
US7625867B2 (en) 2004-03-26 2009-12-01 Acceleron Pharma Inc. BMP-3 propeptides and related methods
US10336800B2 (en) 2014-07-10 2019-07-02 Cambridge Enterprise Limited Therapeutic use of bone morphogenetic proteins

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US5116738A (en) * 1986-07-01 1992-05-26 Genetics Institute, Inc. DNA sequences encoding
US5168050A (en) * 1990-05-24 1992-12-01 Genentech, Inc. Mammalian expression of the bone morphogenetic protein-2b using bmp2a/bmp2b fusion
US5284756A (en) * 1988-10-11 1994-02-08 Lynn Grinna Heterodimeric osteogenic factor

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Publication number Priority date Publication date Assignee Title
US5116738A (en) * 1986-07-01 1992-05-26 Genetics Institute, Inc. DNA sequences encoding
US5284756A (en) * 1988-10-11 1994-02-08 Lynn Grinna Heterodimeric osteogenic factor
US5168050A (en) * 1990-05-24 1992-12-01 Genentech, Inc. Mammalian expression of the bone morphogenetic protein-2b using bmp2a/bmp2b fusion

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MOLECULLAR REPRODUCTION AND DEVELOPMENT, Volume 32, issued 1992, WOZNEY et al., "The Bone Morphogenetic Protein Family and Osteogenesis", pages 160-167. *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5656593A (en) * 1991-03-11 1997-08-12 Creative Biomolecules, Inc. Morphogen induced periodontal tissue regeneration
US5733878A (en) * 1991-03-11 1998-03-31 Creative Biomolecules, Inc. Morphogen-induced periodontal tissue regeneration
US6407060B1 (en) 1996-03-22 2002-06-18 Curis, Inc. Method for enhancing functional recovery following central nervous system ischemia or trauma
EP1027430A1 (fr) * 1997-07-16 2000-08-16 Human Genome Sciences Serie de 64 proteines humaines secretees
EP1027430A4 (fr) * 1997-07-16 2001-09-19 Human Genome Sciences Serie de 64 proteines humaines secretees
EP1095159A1 (fr) * 1998-07-15 2001-05-02 Human Genome Sciences, Inc. Proteine morphogenetique osseuse
EP1095159A4 (fr) * 1998-07-15 2003-01-02 Human Genome Sciences Inc Proteine morphogenetique osseuse
US7625867B2 (en) 2004-03-26 2009-12-01 Acceleron Pharma Inc. BMP-3 propeptides and related methods
WO2005113590A3 (fr) * 2004-05-12 2006-03-02 Acceleron Pharma Inc Propeptides de bmp10 et procédés correspondants
WO2005113590A2 (fr) * 2004-05-12 2005-12-01 Acceleron Pharma Inc. Propeptides de bmp10 et procédés correspondants
US7741284B2 (en) 2004-05-12 2010-06-22 Acceleron Pharma Inc. BMP10 propeptides and related methods
US8287868B2 (en) 2004-05-12 2012-10-16 Acceleron Pharma Inc. BMP10 antibodies and related methods
US8772234B2 (en) 2004-05-12 2014-07-08 Acceleron Pharma, Inc. BMP10 propeptides and related methods
US7465706B2 (en) 2004-06-24 2008-12-16 Acceleron Pharma Inc. GDF3 propeptides and related methods
US8293238B2 (en) 2004-06-24 2012-10-23 Acceleron Pharma Inc. GDF3 antibodies and related methods
US8765670B2 (en) 2004-06-24 2014-07-01 Acceleron Pharma Inc. GDF3 propeptides and related methods
US10336800B2 (en) 2014-07-10 2019-07-02 Cambridge Enterprise Limited Therapeutic use of bone morphogenetic proteins
EP3669886A1 (fr) 2014-07-10 2020-06-24 Cambridge Enterprise, Ltd. Protéines morphogénétiques osseuses
EP3906936A1 (fr) 2014-07-10 2021-11-10 Cambridge Enterprise, Ltd. Protéines morphogénétiques osseuses
US11572396B2 (en) 2014-07-10 2023-02-07 Cambridge Enterprise Limited Therapeutic use of bone morphogenetic proteins

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