WO1995024474A1 - Bone morphogenic protein-10 - Google Patents

Bone morphogenic protein-10 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
Other languages
French (fr)
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/en

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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

Abstract

The present invention discloses a human bone morphogenic protein (BMP-10) polypeptide and DNA (RNA) encoding such polypeptide. The human BMP-10 polypeptide may be produced by recombinant DNA techniques and is useful in inducing de novo bone formation. Also disclosed is an antibody against such polypeptide, which may be used diagnostically to detect lung cancer.

Description

BONE MORPHOGENIC PROTEIN-10
This invention relates to newly identified polynucleotides, polypeptideε 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 polypeptideε.
There are approximately 600,000 nonunion bone fractures a year in the United States alone. 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 oεteogenic 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 stimulateε osteoblast proliferation and differentiation in vitro (Sampath, T.K., et al., J. Biol. Chem., 267:20352-62 (1992)). The recombinant human oεteogenic protein-1 (HOP-1) was produced in mammalian cellε aε a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. The evaluation of HOP-1 effects on cell growths and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen εynthesiε were εtimulated in a dose-dependent manner and increased three-fold in responεe to
Figure imgf000004_0001
It haε alεo recently been shown that ectopic expresεion of 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 explantε. DVR-4 iε therefore the first molecule reported both to induce poεteroventral mesoderm and to counteract dorsalizing εignalε εuch as activin, (Jones, CM. et al, Development, 115:639-47 (1992)).
In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which iε BMP-10, aε well as fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin.
In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.
In accordance with yet a further aspect of the present invention, there iε provided a process for producing εuch polypeptides by recombinant techniques.
In accordance with yet a further aspect of the present invention, there is provided a proceεε for utilizing εuch polypeptideε, or polynucleotides encoding such polypeptideε for therapeutic purpoεeε, for example, for the promotion of de novo bone formation during surgical insertion of protheseε, for the treatment of non-union bone fractures, and for treatment of osteoporosis and periodontal disease. In accordance with yet a further aεpect of the present invention, there iε provided an antibody againεt εuch polypeptideε. Such antibodieε may be uεed diagnostically in the detection of lung diεorderε by meaεuring the serum level of BMP-10 in patientε.
Theεe and other aεpects of the present invention should be apparent to thoεe εkilled in the art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the εcope of the invention aε encompassed by the claims.
FIG. la depictε the cDNA εequence and correεponding deduced a ino acid εequence of the mature BMP-10 polypeptide. The amino acid sequence iε represented by the εtandard three letter code for amino acidε.
FIG. lb εhows the homology between the amino acid εequence of the active domain of BMP-10 (upper line) with that of BMP-3a (lower line).
FIG. 2 depictε the reεultε of a Northern Blot Analyεiε which indicateε the expreεsion levels of the mRNA tranεcript for BMP-10 in human tiεεueε.
In accordance with an aspect of the preεent invention, there iε provided an isolated 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 aε ATCC Deposit No. 75672 on February 9, 1994.
A polynucleotide encoding a polypeptide of the present invention was diεcovered 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 iε a εubfamily of the tranεforming growth factor Beta (TGF-jS) εuperfa ily.
-3-
SUBSTITUTE SUET RULE 26 The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includeε cDNA, geno ic DNA, and synthetic DNA. The DNA may be double- εtranded or εingle-stranded, and if εingle εtranded may be the coding εtrand or non-coding (anti-εenεe) strand. The coding sequence which encodeε the mature polypeptide may be identical to the coding εequence εhown in Figure la or that of the depoεited clone or may be a different coding εequence which coding εequence, aε a reεult of the redundancy or degeneracy of the genetic code, encodeε the εame, mature polypeptide aε the DNA of Figure la or the depoεited cDNA.
The polynucleotide which encodeε 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 aε a leader or secretory sequence or a proprotein εequence; the coding sequence for the mature polypeptide (and optionally additional coding εequence) and non-coding εequence, εuch aε intronε or non-coding εequence 5' and/or 3' of the coding εequence for the mature polypeptide.
Thuε, the term "polynucleotide encoding a polypeptide" encompaεses a polynucleotide which includes only coding εequence for the polypeptide aε well as a polynucleotide which includeε additional coding and/or non-coding εequence.
The preεent invention further relateε to variants of the hereinabove described polynucleotides which encode for fragments, analogε and derivativeε of the polypeptide having the deduced amino acid εequence of Figure la or the polypeptide encoded by the cDNA of the depoεited 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.
Thus, the present invention includes polynucleotides encoding the εame mature polypeptide aε εhown in Figure la or the εame mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variantε encode for a fragment, derivative or analog of the polypeptide of Figure la or the polypeptide encoded by the cDNA of the depoεited clone. Such nucleotide variantε include deletion variantε, εubεtitution variants and addition or insertion variants.
Aε hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding εequence εhown in Figure la or of the coding sequence of the deposited clone. As known in the art, 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 doeε not substantially alter the function of the encoded polypeptide.
The present invention also includes polynucleotideε, wherein the coding εequence for the mature polypeptide may be fuεed in the εame reading frame to a polynucleotide εequence which aidε in expreεεion and εecretion of a polypeptide from a hoεt cell, for example, a leader εequence which functionε 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 polynucleotideε may also encode for a proprotein which iε the mature protein plus additional 5' amino acid reεidues. A mature protein having a prosequence iε a proprotein and iε .an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
Thus, for example, 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 proεequence and a preεequence (leader sequence). The polynucleotideε of the present invention may also have the coding εequence fuεed in frame to a marker εequence which allows for purification of the polypeptide of the preεent invention. The marker εequence may be a hexa- hiεtidine tag εupplied 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 correspondε to an epitope derived from the influenza hemagglutinin protein ( ilεon, I., et al.. Cell, 37:767 (1984)).
The preεent invention further relateε to polynucleotideε which hybridize to the hereinabove-deεcribed sequences if there iε at least 50% and preferably 70% identity between the sequenceε. The present invention particularly relates to polynucleotideε which hybridize under εtringent conditions to the hereinabove-deεcribed polynucleotideε. As herein uεed, the term "εtringent conditions" meanε hybridization will occur only if there iε at leaεt 95% and preferably at least 97% identity between the εequenceε. The polynucleotideε which hybridize to the hereinabove described polynucleotideε in a preferred embodiment encode polypeptides which retain substantially the εame biological function or activity aε the mature polypeptide encoded by the cDNA of Figure la or the depoεited cDNA.
The depoεit(ε) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organismε for purpoεeε of Patent Procedure. Theεe depoεitε are provided merely aε convenience to those of εkill in the art and are not an admission that a deposit iε required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, aε well aε the amino acid εequence of the polypeptideε encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any deεcription of sequences herein. A license may be required to make, uεe or εell the depoεited materialε, and no such license is hereby granted.
The preεent invention further relateε to a BMP-10 polypeptide which haε the deduced amino acid εequence of Figure la or which haε the amino acid sequence encoded by the deposited cDNA, aε well aε fragments, analogε and derivativeε of such polypeptide.
The terms "fragment," "derivative" and "analog" 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 aε such polypeptide. Thuε, an analog includeε 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 depoεited cDNA may be (i) one in which one or more of the amino acid reεidueε are substituted with a conserved or non-conserved amino acid residue (preferably a conεerved amino acid reεidue) and such εubεtituted amino acid reεidue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid reεidueε includeε a εubεtituent group, or (iii) one in which the mature polypeptide. iε fuεed with another compound, εuch aε a compound to increaεe the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acidε are fused to the mature polypeptide, such as a leader or secretory εequence or a sequence which iε employed for purification of the mature polypeptide or a proprotein εequence. Such fragmentε.
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SUBSUME SHEET (RULE 26) derivativeε and analogε are deemed to be within the εcope of thoεe εkilled in the art from the teachings herein.
The polypeptides and polynucleotideε of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The term "iεolated" meanε that the material iε removed from itε original environment (e.g., the natural environment if it iε naturally occurring) . For example, a naturally- occurring polynucleotide or polypeptide preεent in a living animal iε not iεolated, but the εame polynucleotide or polypeptide, εeparated from εome or all of the coexiεting materialε in the natural εyεtem, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptideε could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
The preεent invention alεo relateε to vectorε which include polynucleotideε of the present invention, hoεt cellε which are genetically engineered with vectorε of the invention and the production of polypeptideε of the invention by recombinant techniques.
Hoεt cells are genetically engineered (transduced or transformed or tranεfected) with the vectorε of thiε invention which may be, for example, a cloning vector or an expreεεion vector. The vector may be, for example, in the form of a plaεmid, a viral particle, a phage, etc. The engineered hoεt cells can be cultured in conventional nutrient media modified aε appropriate for activating promoterε, εelecting tranεformantε or amplifying the BMP-10 geneε. The culture conditions, εuch aε temperature, pH and the like, are thoεe previouεly uεed with the hoεt cell selected for expreεεion, and will be apparent to the ordinarily εkilled artiεan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant
-8-
SUBSΗTUTE SHEET (RULE 26) techniqueε. Thuε, for example, the polynucleotide may be included in any one of a variety of expreεεion vectorε for expreεεing a polypeptide. Such vectorε include chromoεomal, nonchromoεomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculoviruε; yeaεt plaεmidε; vectorε derived from combinations of plasmids and phage DNA, viral DNA such aε vaccinia, adenoviruε, fowl pox viruε, and pseudorabies. However, any other vector may be uεed as long as it iε replicable and viable in the hoεt.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedureε and others are deemed to be within the εcope of those skilled in the art.
The DNA sequence in the expresεion vector iε operatively linked to an appropriate expression control sequence(ε) (promoter) to direct mRNA εynthesiε. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda PL promoter and other promoters known to control expresεion of genes in prokaryotic or eukaryotic cellε or their viruεeε. The expreεεion vector alεo containε a riboεome binding εite for tranεlation initiation and a tranεcription terminator. The vector may alεo include appropriate εequenceε for amplifying expreεεion.
In addition, the expreεεion vectorε preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cellε such aε dihydrofolate reductaεe or neomycin reεiεtance for eukaryotic cell culture, or εuch as tetracycline or ampicillin resistance in E. coli.
The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or
-9- control sequence, may be employed to transform an appropriate hoεt to permit the hoεt to expreεε the protein.
As representative exa pleε of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Streptomyces. Salmonella typhimurium; fungal cellε, εuch aε yeaεt; inεect cellε such as Droεophila and Sf9; animal cells such aε CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those εkilled in the art from the teachingε herein.
More particularly, the preεent invention also includes recombinant constructs comprising one or more of the sequenceε aε broadly described above. The constructε compriεe a vector, such as a plasmid or viral vector, into which a εequence of the invention haε been inεerted, in a forward or reverse orientation. In a preferred aεpect of thiε embodiment, the conεtruct further compriεes regulatory εequenceε, including, for example, a promoter, operably linked to the sequence. Large numbers of εuitable vectorε and promoterε are known to thoεe of εkill in the art, and are commercially available. The following vectorε are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbε, pDIO, phagescript, psiX174, pblueεcript SK, pbεkε, 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) . However, any other plaεmid or vector may be uεed aε long aε they are replicable and viable in the hoεt.
Promoter regionε can be εelected from any deεired gene uεing CAT (chloramphenicol tranεferaεe) vectorε or other vectorε with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoterε include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoterε include CMV immediate early, HSV thymidine kinaεe, 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.
In a further embodiment, the present invention relates to hoεt cells containing the above-described constructs. The hoεt 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 conεtruct into the hoεt cell can be effected by calcium phoεphate tranεfection, DEAE- Dextran mediated tranεfection, or electroporation. (Daviε, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide syntheεizerε.
Mature proteinε can be expressed in mammalian cellε, yeaεt, bacteria, or other cellε under the control of appropriate promoterε. Cell-free tranεlation εyεtemε can alεo be employed to produce εuch proteinε uεing RNAε derived from the DNA conεtructε of the preεent invention. Appropriate cloning and expression vectorε for uεe with prokaryotic and eukaryotic hoεtε are deεcribed 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.
Transcription of the DNA encoding the polypeptideε of the preεent invention by higher eukaryoteε iε increaεed by inεerting an enhancer εequence into the vector. Enhancerε are ciε-acting elementε of DNA, uεually about from 10 to 300 bp that act on a promoter to increase its tranεcription. Examples including the SV40 enhancer on the late εide of the replication origin bp 100 to 270, a cytomegaloviruε early promoter enhancer, the polyoma enhancer on the late εide of the replication origin, and adenoviruε enhancers.
Generally, recombinant expresεion vectorε will include originε of replication and εelectable markers permitting tranεformation of the hoεt cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expresεed gene to direct tranεcription of a downεtream εtructural εequence. Such promoterε can be derived from operonε encoding glycolytic enzymes εuch aε 3-phoεphoglycerate kinaεe (PGK), α-factor, acid phoεphataεe, or heat εhock proteins, among otherε. The heterologouε εtructural sequence is aεεembled in appropriate phase with tranεlation initiation and termination sequences, and preferably, a leader εequence capable of directing εecretion of tranεlated protein into the periplaεmic εpace or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteriεticε, e.g., εtabilization or εimplified purification of expressed recombinant product.
Useful expresεion vectorε for bacterial uεe are conεtructed by inεerting a εtructural DNA εequence encoding a desired protein together with suitable tranεlation initiation and termination εignalε in operable reading phaεe with a functional promoter. The vector will compriεe one or more phenotypic εelectable markerε and an origin of replication to enεure maintenance of the vector and to, if deεirable, provide amplification within the hoεt. Suitable prokaryotic hoεtε for tranεformation include E. coli, Bacilluε εubtiliε. Salmonella typhimuriu and variouε εpecieε within the genera Pεeudomonaε, Streptomyceε, and Staphylococcuε, although others may alεo be employed aε a matter of choice.
Aε a repreεentative but nonlimiting example, uεeful expreεεion vectorε for bacterial uεe can compriεe a selectable marker and bacterial origin of replication derived from commercially available plaεmidε compriεing genetic elementε of the well known 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). These pBR322 "backbone" sectionε are combined with an appropriate promoter and the εtructural εequence to be expreεεed.
Following tranεformation of a εuitable hoεt strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cellε 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 diεruption, or uεe of cell lysing agents, such methods are well know to those skilled in the art.
Various mammalian cell culture εyεtemε can alεo be employed to expreεε recombinant protein. Examples of mammalian expresεion εyεtemε include the COS-7 lineε of monkey kidney fibroblaεtε, deεcribed by Gluzman, Cell, 23:175 (1981), and other cell lineε capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lineε. Mammalian expresεion vectorε will compriεe an origin of replication, a εuitable promoter and enhancer, and alεo any neceεεary ribosome binding εiteε, polyadenylation εite, εplice donor and acceptor εiteε, tranεcriptional termination εequenceε, and 5' flanking nontranεcribed sequenceε. DNA εequenceε derived from the SV40 εplice, and polyadenylation εiteε may be uεed to provide the required nontranεcribed genetic elementε. The polypeptideε can be recovered and purified from recombinant cell cultureε by methodε including ammonium εulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phoεphocelluloεe chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It iε preferred to have low concentrationε (approximately 0.15-5 mM) of calcium ion preεent during purification. (Price et al., J. Biol. Chem. , 244:917 (1969)). Protein refolding εtepε can be uεed, aε neceεεary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification εtepε.
The polypeptideε of the preεent invention may be a naturally purified product, or a product of chemical εynthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic hoεt (for example, by bacterial, yeaεt, higher plant, inεect and mammalian cellε in culture). Depending upon the hoεt employed in a recombinant production procedure, the polypeptideε of the preεent invention may be glycoεylated or may be non-glycoεylated. Polypeptideε 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 uεed in the treatment of periodontal disease and other bone defects of the oral cavity.
BMP-10 may alεo be uεed during εurgical inεertion of protheεeε. In hip replacements, knee replacements and other surgical inεertion of protheεeε, the protheεiε iε held in place by εurgical cement. The cement eventually looεenε, however, making it neceεεary to perform another εurgery. Thiε second surgery is a much more difficult procedure and is responεible for εurgeonε reluctantance to inεert protheεeε in young people. BMP-10 may, therefore, be uεed to coat the protheεiε before inεertion which reεultε in bone formation around the protheεis, making a εtronger union and allowing for the uεe of leεε cement.
BMP-10 may also be employed in the treatment of oεteoporoεis, which iε characterized by exceεεive bone reεorption reεulting in thin and brittle boneε. BMP-10 would εtimulate bone formation to help alleviate this condition.
The polypeptideε may alεo be employed in accordance with the preεent invention by expreεεion of εuch polypeptideε in vivo , which iε often referred to aε "gene therapy."
Thuε, for example, cellε from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo , with the engineered cellε then being provided to a patient to be treated with the polypeptide. Such methodε are well-known in the art. For example, cellε may be engineered by procedureε known in the art by uεe of a retroviral particle containing RNA encoding a polypeptide of the preεent invention.
Similarly, cellε may be engineered in vivo for expreεεion of a polypeptide in vivo by, for example, procedureε known in the art. Aε known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the preεent invention may be administered to a patient for engineering cells in vivo and expresεion of the polypeptide in vivo . These and other methods for administering a polypeptide of the preεent invention by εuch method εhould be apparent to thoεe εkilled in the art from the teachings of the present invention. For example, the expresεion vehicle for engineering cellε may be other than a retrovirus, for example, an adenovirus which may be uεed to engineer cells in vivo after combination with a εuitable delivery vehicle.
The polypeptideε of the present invention may be employed in combination with a suitable pharmaceutical carrier. Such compositionε compriεe a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includeε but iε not limited to εaline, buffered εaline, dextroεe, water, glycerol, ethanol, and combinationε thereof. The formulation εhould suit the mode of adminiεtration.
The invention alεo provideε a pharmaceutical pack or kit compriεing one or more containerε filled with one or more of the ingredientε of the pharmaceutical compoεitionε of the invention. Aεεociated with εuch container(ε) can be a notice in the form preεcribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticalε or biological products, which notice reflectε approval by the agency of manufacture, uεe or εale for human administration. In addition, the polypeptideε of the preεent invention may be employed in conjunction with other therapeutic compoundε.
BMP-10 iε preferably uεed topically, however, when it iε used syεtemically, the pharmaceutical compoεitions may be administered in a convenient manner such as by the oral, intravenous, intraperitoneal, intramuscular, subcutaneouε, intranaεal, or intradermal routes. The amounts and dosage regimenε of pharmaceutical compoεitionε 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. Generally speaking, pharmaceutical compoεitionε of BMP-10 are given, for example, in appropriate doεeε of at leaεt about 10 μg/kg body weight and in moεt caεeε will not be adminiεtered in an amount in excess of about 8 mg/kg body weight, and preferably iε given in doεeε of about 10 g/kg body weight to about 1 mg/kg daily, taking into account the routeε of adminiεtration, εymptomε, etc.
Specifically, BMP-10 may be prepared aε a gel matrix formulation and administered, for example, ectopically, which iε preferably adminiεtered at the site of bone fracture at a dosage of 50 mg, by applying the matrix directly to the εite of the fracture. Thiε matrix may alεo be applied to protheses before inεertion.
The εequenceε of the present invention are also valuable for chromosome identification. The εequence iε εpecifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there iε a current need for identifying particular εiteε on the chromoεome. Few chromosome marking reagentε based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromoεomeε according to the present invention iε an important firεt εtep in correlating thoεe εequenceε with geneε aεεociated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analyεiε of the cDNA iε uεed to rapidly εelect primers that do not εpan more than one exon in the genomic DNA, thuε complicating the amplification proceεε. Theεe primers are then used for PCR screening of somatic cell hybridε containing individual human chromoεomeε. Only thoεe hybridε containing the human gene correεponding to the primer will yield an amplified fragment.
PCR mapping of εomatic cell hybridε iε a rapid procedure for aεsigning a particular DNA to a particular chromoεome. Using the present invention with the εame oligonucleotide primerε, εublocalization can be achieved with panelε of fragmentε from specific chromoεomeε or poolε of large genomic cloneε in an analogouε manner. Other mapping εtrategieε that can εimilarly be used to map to itε chromoεome include in situ hybridization, preεcreening with labeled flow-εorted chromosomes and preselection by hybridization to conεtruct chromosome εpecific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA cloneε to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one εtep. This technique can be uεed with cDNA aε short aε 500 or 600 baεeε; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromoεomal location with εufficient εignal intenεity for εimple detection. FISH requireε uεe of the cloneε from which the EST waε derived, and the longer the better. For example, 2,000 bp iε good, 4,000 is better, and more than 4,000 iε probably not neceεεary to get good reεultε a reaεonable percentage of the time. For a review of thiε technique, see Verma et al. , Human Chromoεomeε: a Manual of Baεic Techniques, Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) . The relationship between genes and diseases that have been mapped to the εame chromoεomal region are then identified through linkage analyεiε (coinheritance of phyεically adjacent geneε).
Next, it iε neceεεary to determine the differenceε in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation iε obεerved in some or all of the affected individuals but not in any normal individuals, then the mutation iε likely to be the cauεative agent of the diεeaεe.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region asεociated with the diseaεe could be one of between 50 and 500 potential cauεative geneε. (Thiε assumes 1 megabaεe mapping reεolution and one gene per 20 kb) .
Compariεon of affected and unaffected individualε generally involveε firεt looking for εtructural alterationε in the chromoεomeε, εuch aε deletionε or tranεlocationε that are viεible from chromoεome εpreadε or detectable uεing PCR baεed on that cDNA εequence. Ultimately, complete sequencing of genes from εeveral individualε iε required to confirm the preεence of a mutation and to diεtinguiεh mutationε from polymorphisms.
The polypeptides, their fragments or other derivatives, or analogs thereof, or cellε expreεεing them can be uεed aε an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodieε. The present invention also includes chimeric, single chain, and humanized antibodieε, aε well aε Fab fragmentε, or the product of an Fab expreεεion library. Variouε procedureε known in the art may be used for the production of such antibodieε and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptideε into an animal or by adminiεtering the polypeptideε to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptideε itεelf. In thiε manner, even a εequence encoding only a fragment of the polypeptideε can be used to generate antibodies binding the whole native polypeptideε. Such antibodieε can then be uεed to iεolate the polypeptide from tiεεue expreεεing that polypeptide.
For preparation of monoclonal antibodieε, any technique which provideε antibodies produced by continuous cell line cultures can be used. Exampleε include the hybridoma technique (Kohler and Milεtein, 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 antibodieε (Cole, et al., 1985, in Monoclonal Antibodieε and Cancer Therapy, Alan R. Liεε, Inc., pp. 77-96).
Techniqueε deεcribed for the production of single chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce εingle chain antibodieε to immunogenic polypeptide products of thiε invention.
Antibodies specific to the polypeptide of the preεent invention may further be uεed a potential marker for lung cancer. BMP-10 iε a protein which iε secreted and predominantly expressed in adult lung (Figure 2). Accordingly, antibodies againεt BMP-10 can be uεed aε part of an in vitro diagnoεtic aεεay to measure the expresεion level of thiε protein in adult εubjectε. The procedure would conεist of drawing blood from a patient, adding the above- mentioned antibodieε to the blood εerum, then performing a εtandard immunoassay such as ELISA or a radioimmunoassay to detect the amount of antibody bound to BMP-10 which iε directly related to the amount of BMP-10 produced in the lung. Tumor cellε are more active than cellε with a normal phyεiology and therefore, produce more BMP-10 which would be indicative of lung cancer.
The preεent invention will be further deεcribed with reference to the following exampleε; however, it iε to be understood that the preεent invention iε not limited to such exampleε. All partε or amounts, unleεε otherwiεe εpecified, are by weight.
In order to facilitate underεtanding of the following exampleε certain frequently occurring methodε and/or termε will be deεcribed.
"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plaεmidε herein are either commercially available, publicly available on an unreεtricted baεiε, or can be conεtructed from available plaεmidε in accord with published procedureε. In addition, equivalent plaεmidε 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 enzymeε used herein are commercially available and their reaction conditions, cofactors and other requirements were used aε would be known to the ordinarily εkilled artiεan. For analytical purposeε, typically 1 μg of plaεmid or DNA fragment iε uεed with about 2 unitε of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plaεmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and subεtrate 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 iεolate the deεired fragment.
Size εeparation of the cleaved fragmentε iε performed uεing 8 percent polyacrylamide gel deεcribed by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" referε to either a εingle εtranded polydeoxynucleotide or two complementary polydeoxynucleotide εtrandε which may be chemically εyntheεized. Such εynthetic oligonucleotideε have no 5' phoεphate and thuε will not ligate to another oligonucleotide without adding a phoεphate with an ATP in the presence of a kinaεe. A εynthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" referε to the proceεε of forming phoεphodieεter bondε between two double εtranded nucleic acid fragmentε (Maniatis, T., et al.. Id., p. 146). Unless otherwise provided, ligation may be accomplished uεing known bufferε and conditionε with 10 unitε to T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated. Unless otherwise stated, transformation was performed as deεcribed in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).
Example 1 Bacterial Expreεεion and Purification of Human BMP-10
The DNA sequence encoding for human BMP-10 iε initially amplified uεing PCR oligonucleotide primers corresponding to the 5' and 3' end of the DNA εequence to εyntheεize inεertion fragments. The 5' oligonucleotide primer 5'- GATCGGATCCAAAGCCCGGAGGAAGCAG-3' contains a Bam HI reεtriction enzyme εite followed by 18 nucleotides of BMP-10 coding sequence starting from amino acid 4 (Lys); the 3' εequence 5'-GTACTCTAGATCACCGGCAGGCACAGGTG-3' containε complementary εequenceε to an Xba I εite, a tranεlation εtop codon and the laεt 16 nucleotideε of BMP-10 coding εequence. The reεtriction enzyme εiteε correspond to the reεtriction enzyme εiteε on the bacterial expreεεion vector pQE-9 (Qiagen, Inc., 9259 Eton Ave., Chatsworth, CA 91311, Catalog No. 33093). The plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/0), a riboεome binding εite (RBS), a 6- hiεtidine tag (6-Hiε) 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 containε multiple copieε of the plaεmid pREP4, which expreεεeε the lad repreεεor and alεo conferε kanamycin reεistance (Kanr) . Transformantε are identified by their ability to grow on LB plateε containing both Amp and Kan. Cloneε containing the deεired conεtructε were grown overnight (0/N) in liquid culture in LB media εupplemented with both Amp (100 μg/ml) and Kan (25 μg/ l). The O/N culture iε uεed to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density of 600 (O.D.600) between 0.4 and 0.6. IPTG (Isopropyl-B-D- thiogalacto pyranoεide) waε then added to a final concentration of 1 mM. IPTG induceε by inactivating the lad repressor, clearing the P/0 leading to increased gene expression. Cells were grown an extra 2-4 hours. Cells were then harvested by centrifugation (20 mins. at 6000Xg) . The cell pellet waε εolubilized in the chaotropic agent 6 molar Guanidine HC1. After clarification, solubilized BMP-10 waε purified from thiε solution by chromatography on a Nickel- Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag. (Hochuli, E. et al.. Genetic Engineering, Principles & Methodε, 12:87-98 (1990). BMP-10 (95% pure) was eluted from the column in 6 molar GHCl pH 5.0. Protein renaturation out of GHCl can be accomplished by εeveral protocolε. (Jaenicke, R. and Rudolph R., Protein Structure - A Practical Approach, IRL Preεε, New York (1990). The pH 5.0 eluate waε diluted and reapplied to a εecond nickel-chelate column. Bound protein waε renatured by running a linear descending gradient of GnHCl. Thiε allowε for folding to occur on the column. The protein waε then eluted with 250 μm imidazole pH 5.0.
Example 2 Tiεεue Diεtribution of BMP-10
Northern blot analyεiε waε carried out to examine the levelε of expression of BMP-10 in human tisεueε. Total cellular RNA εampleε were iεolated with RNAzol™ B system (Biotecx Laboratories, Inc., 6023 South Loop Eaεt, Houston, TX 77033). About 10 ug of total RNA isolated from each human tisεue εpecified was separated on 1% agarose gel and blotted onto a nylon filter (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Preεε (1989)). The labeling reaction waε done according to the Stratagene Prime- It kit with 50 ng DNA fragment. The labeled DNA waε purified with a Select-G-50 column from 5' Prime — 3 Prime, Inc., 5603 Arapahoe Road, Boulder, CO 80303. The filter waε then hybridized with radioactive labeled full length BMP-10 gene at 1,000,000 cp /ml in 0.5 M NaPo4 and 7% SDS overnight at 65"C. The filterε were waεhed twice at room temperature and twice at 60*C with 0.5 x SSC, 0.1% SDS, the filterε were then exposed at -70*C overnight with intensifying screen. The meεεage RNA for BMP-10 iε abundant in lung. Figure 2.
Numerouε modifications and variations of the present invention are posεible in light of the above teachingε and, therefore, within the scope of the appended claimε, the invention may be practiced otherwise than as particularly deεcribed.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: HE, ET AL.
(ii) TITLE OF INVENTION: Bone Morphogenic Protein - 10
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) COUNTRY: USA
(F) ZIP: 07068
(V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/209,214
(B) FILING DATE: 10 MARCH 1994.
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER:
(B) FILING DATE:
(Viii) ATTORNEY/AGENT INFORMATION: (A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134
(C) REFERENCE/DOCKET NUMBER: 325800-140
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 360 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
AAGACGATTC AGAAAGCCCG GAGGAAGCAG TGGGATGAGC CGAGGGTGTT CTCCCGGAGG 60 TACCTGAAGG TGGACTTCGC AGACATCGGC TGGAATGAAT GGATAATCTC ACCGAAATCT 120 TTTGATGCCT ACTACTGCGC GGGAGCATGT GAGTTCCCCA TGCCTAAGAT CGTTCGTCCA 180 TCCAACCATC CCACCATCCA GAGCATTGTC AGGGCTGTGG GCATCATCCC TGGCATCCCA 240 GAGCCCTGCT GTGTTCCCGA TAAGATGAAC TCCCTTGGGG TCCTCTTCCT GGATGAGAAT 300 CGGAATGTGG TTCTGAAGGT GTACCCCAAC ATGTCCGTGG ACACCTGTGC CTGCCGGTGA 360
(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 119 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Lys Thr lie Gin Lyε Ala Arg Arg Lyε Gin Trp Aεp Glu Pro Arg
5 10 15
Val Phe Ser Arg Arg Tyr Leu Lyε Val Aεp Phe Ala Aεp lie Gly
20 25 30
Trp Asn Glu Trp lie lie Ser Pro Lyε Ser Phe Aεp Ala Tyr Tyr
35 40 45
Cyε Ala Gly Ala Cyε Glu Phe Pro Met Pro Lyε lie Val Arg Pro
50 55 60
Ser Aεn Hiε Ala Thr He Gin Ser He Val Arg Ala Val Gly He
65 70 75
He Pro Gly He Pro Glu Pro Cyε Cyε Val Pro Aεp Lyε Met Aεn
80 85 90
Ser Leu Gly Val Leu Phe Leu Aεp Glu Asn Arg Aεn Val Val Leu
95 100 105
Lyε Val Tyr Pro Aεn Met Ser Val Aεp Thr Cys Ala Cys Arg
110 115
-27-
SUBSTΠTUΓE SHEET (RULE 26)

Claims

WHAT IS CLAIMED IS:
1. An iεolated polynucleotide encoding for BMP-10, εaid polynucleotide εelected from the group conεiεting of
(a) a polynucleotide encoding for the BMP-10, polypeptide having the deduced amino acid εequence of Figure la or an fragment, analog or derivative of εaid polypeptide;
(b) a polynucleotide encoding for the BMP-10 polypeptide having the amino acid εequence encoded by the cDNA contained in ATCC Depoεit No. 75672 or an fragment, analog or derivative of εaid polypeptide.
2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.
3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.
4. The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.
5. The polynucleotide of Claim 2 wherein said polynucleotide encodeε for BMP-10 having the deduced amino acid sequence of Figure la.
6. The polynucleotide of Claim 2 wherein εaid polynucleotide encodeε for the BMP-10 polypeptide encoded by the cDNA of ATCC Depoεit No. 75672.
7. The polynucleotide of Claim 1 having the coding εequence for BMP-10 aε εhown in Figure la.
8. The polynucleotide of Claim 2 having the coding sequence for BMP-10 depoεited aε ATCC Depoεit No. 75672.
9. A vector containing the DNA of Claim 2.
10. A hoεt cell genetically engineered with the vector of Claim 9.
11. A proceεε for producing a polypeptide compriεing: expreεεing from the hoεt cell of Claim 10 the polypeptide encoded by εaid DNA.
12. A proceεε for producing cellε capable of expreεεing a polypeptide compriεing genetically engineering cellε with the vector of Claim 9.
13. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having BMP-10 activity.
14. A polypeptide selected from the group consisting of (i) a BMP-10 polypeptide having the deduced amino acid sequence of Figure la and fragments, analogs and derivativeε thereof and (ii) a BMP-10 polypeptide encoded by the cDNA of ATCC Deposit No. 75672 and fragmentε, analogs and derivatives of said polypeptide.
15. The polypeptide of Claim 14 wherein the polypeptide is BMP-10 having the deduced amino acid sequence of Figure la.
16. An antibody against the polypeptide of claim 14.
17. A method for the treatment of a patient having need of BMP-10 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 14.
18. A method for the detection of a lung abnormality in a patient comprising: drawing a blood sample from the patient; separating the blood serum from the blood sample; adding to the blood εerum an effective amount of the antibody of claim 16; performing an immunoaεεay; and determining the concentration of BMP-10 in the blood serum.
19. A pharmaceutical composition compriεing the polypeptide of Claim 14 and a pharmaceutically acceptable carrier.
20. The method of Claim 17 wherein εaid therapeutically effective amount of the polypeptide iε adminiεtered by providing to the patient DNA encoding εaid polypeptide and expreεεing εaid polypeptide in vivo .
PCT/US1994/005292 1994-03-10 1994-05-12 Bone morphogenic protein-10 WO1995024474A1 (en)

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EP1027430A1 (en) * 1997-07-16 2000-08-16 Human Genome Sciences 64 human secreted proteins
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Cited By (20)

* Cited by examiner, † Cited by third party
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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 (en) * 1997-07-16 2000-08-16 Human Genome Sciences 64 human secreted proteins
EP1027430A4 (en) * 1997-07-16 2001-09-19 Human Genome Sciences 64 human secreted proteins
EP1095159A1 (en) * 1998-07-15 2001-05-02 Human Genome Sciences, Inc. Bone morphogenic protein
EP1095159A4 (en) * 1998-07-15 2003-01-02 Human Genome Sciences Inc Bone morphogenic protein
US7625867B2 (en) 2004-03-26 2009-12-01 Acceleron Pharma Inc. BMP-3 propeptides and related methods
WO2005113590A3 (en) * 2004-05-12 2006-03-02 Acceleron Pharma Inc Bmp10 propeptides and related methods
WO2005113590A2 (en) * 2004-05-12 2005-12-01 Acceleron Pharma Inc. Bmp10 propeptides and related methods
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 (en) 2014-07-10 2020-06-24 Cambridge Enterprise, Ltd. Bone morphogenetic proteins
EP3906936A1 (en) 2014-07-10 2021-11-10 Cambridge Enterprise, Ltd. Bone morphogenetic proteins
US11572396B2 (en) 2014-07-10 2023-02-07 Cambridge Enterprise Limited Therapeutic use of bone morphogenetic proteins

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