EP0416029A1 - Procalcitonin peptides - Google Patents

Procalcitonin peptides

Info

Publication number
EP0416029A1
EP0416029A1 EP89907003A EP89907003A EP0416029A1 EP 0416029 A1 EP0416029 A1 EP 0416029A1 EP 89907003 A EP89907003 A EP 89907003A EP 89907003 A EP89907003 A EP 89907003A EP 0416029 A1 EP0416029 A1 EP 0416029A1
Authority
EP
European Patent Office
Prior art keywords
peptide
proct
peptides
promoter
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP89907003A
Other languages
German (de)
French (fr)
Inventor
Bernard A. Roos
Douglas M. Burns
Howard A. Guy
Gary L. Mcknight
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Washington
Zymogenetics Inc
Original Assignee
University of Washington
Zymogenetics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Washington, Zymogenetics Inc filed Critical University of Washington
Publication of EP0416029A1 publication Critical patent/EP0416029A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • C07K14/575—Hormones
    • C07K14/585—Calcitonins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is related to peptides that stimulate the proliferation of osteoblasts and preosteoblasts, methods of making the peptides, therapeu- tic compositions containing the peptides, and methods of promoting bone growth in mammals.
  • Bone growth, maintenance and repair involve a balance between rates of bone formation and resorption. These two processes (together referred to as "remodel ⁇ ing”) are regulated by several hormones and growth factors, including parathyroid hormone, calcitonin, insulin, somatomedins, thyroid hormone, glucocorticoids, vitamin D, androgens, estrogens, epidermal growth factor, transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor. These chemical messengers influence the development and activity of osteoblasts and preosteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells) .
  • osteoblasts and preosteoblasts bone-forming cells
  • osteoclasts bone-resorbing cells
  • Bone remodeling is related to the role of the skeleton as a mineral reser ⁇ voir and serves to maintain cell viability, fluid exchange and bone strength. Imbalances in bone metabo ⁇ lism may result in osteoporosis and other diseases.
  • the bone-remodeling process is reviewed by Raisz and Kream (New Engl. J. Med. 309:29-35, 83-89, 1983) and Raisz (New Engl. J. Med. 318:818-827, 1988).
  • bones may be weakened or may fail to heal properly.
  • healing may proceed slowly in the elderly and in patients undergoing treatment with corticosteroids, such as transplant patients and those being treated for chronic lung disease.
  • corticosteroids such as transplant patients and those being treated for chronic lung disease.
  • victims of osteopor ⁇ osis suffer from an imbalance in bone metabolism which often leads to fractures and deformity.
  • Osteoporosis is currently treated with estro ⁇ gen, calcitonin, calcium, sodium fluoride, bisphosphon- ates, or a combination of these. These treatments probably do not correct the underlying cause of the disease and have only limited e fectiveness.
  • Estrogen treatment has been linked to an increased risk of endometrial cancer. Estrogen is of limited usefulness because only a fraction of women under age 55 respond; with further aging, estrogen responsiveness of bone diminishes.
  • the use of sodium fluoride is under ban ⁇ gation but ha.s not yet been approved. Further, bone formed under fluoride stimulation may be abnormal. In addition, significant toxicity is associated with fluoride administration. Calcium alone is ineffective except in cases of gross dietary calcium deficiency.
  • the present invention discloses isolated peptides and DNA sequences encoding peptides. that stimulate the proliferation of osteoblasts and preosteoblasts.
  • the peptides generally have the follow ⁇ ing characteristics: (a) they are at least 12 amino acids in length; (b) they are substantially homologous to at least a portion of rat N-procalcitonin (N-proCT) ; and
  • the peptide has an amino acid sequence selected from the group consisting of rat, human, chick and salmon N-proCT and rat, human and chick N-procalcitonin gene-related peptide (N-proCGRP) sequences, as shown in Figure 1.
  • N-proCGRP chick N-procalcitonin gene-related peptide
  • the present invention includes peptides derived from other species as well as derivatives of these peptides.
  • the peptide comprises an amino-terminal sequence selected from the group consisting of (a) V P L R S T L E S S P G; (b) A P F R S A L E S S P A; and (c) A P V R P G L E S I T D; and
  • the peptide is less than 32 amino acids in length.
  • a method of isolating a peptide that stimulates cell division in osteoblasts and preosteoblasts is disclosed. The method generally comprises (a) preparing an aqueous extract of cells capable of expressing a calcitonin or calcitonin-related gene; (b) fractionating the aqueous extract to enrich for peptides having a molecular weight less than approximately 15,000; and (c) fractionating the enriched fraction by hydrophobic chromatography and/or anion exchange chromatography to separate the peptide from the enriched fraction.
  • the step of fractionating the aqueous extract comprises reversed-phase HPLC and gel filtration of -the aqueous extract. It will be evident to one skilled in the art that the order of the steps may be modified without substantially affecting the desired result.
  • Host cells transfected or transformed with an expression vector comprising a transcriptional promoter operably linked to a DNA sequence encoding a peptide as described above are also disclosed. Suitable host cells in this regard include yeast host cells.
  • a method for producing a peptide that stimulates cell division in osteoblasts and preosteoblasts is disclosed.
  • the method generally comprises (a) introducing into a host cell an expression vector comprising a transcrip- tional promoter operably linked to a DNA sequence encod ⁇ ing a peptide having the characteristics described above; (b) culturing the host cell under suitable conditions; and (c) isolating the peptide from the host cell.
  • compositions comprising a peptide as described above in combination with a physio ⁇ logically acceptable carrier or diluent.
  • the therapeutic composition may be used within a method of promoting bone growth in a patient.
  • the composi- tion may be administered intranasally or by injection.
  • the composition may further include an effective amount of a substance such as estrogen, sodium fluoride, calcitonin or a bisphosphonate.
  • the composition may further include a growth factor such as insulin, an insulin-like growth factor, platelet-derived growth factor, transforming growth fac- tor alpha, transforming growth factor beta or epidermal growth factor in an amount sufficient to further increase DNA synthesis in osteoblasts and preosteoblasts.
  • a growth factor such as insulin, an insulin-like growth factor, platelet-derived growth factor, transforming growth fac- tor alpha, transforming growth factor beta or epidermal growth factor in an amount sufficient to further increase DNA synthesis in osteoblasts and preosteoblasts.
  • Figure 1 illustrates the amino acid sequences of representative peptides that stimulate the growth of osteoblasts and preosteoblasts. Boxes indicate blocks of identical amino acids. Asterisks indicate gaps intro ⁇ quiz ology. Numbers refer to the rat sequence.
  • N-proCGRP sequences correspond to the respec ⁇ tive N-proCT sequences, except where indicated. No sequence has been reported for salmon calcitonin gene- related peptide (CGRP). Amino acids are designated by the standard one-letter code.
  • Figure 2 illustrates the results of an immuno- assay on serially diluted synthetic peptides and extracts of thyroid cells (Ext) using an antiserum directed against the last six residues of rat N-proCT (NCAP) .
  • Figure 3 illustrates the results of an immuno- assay on serially diluted peptides and rat tumor cell extract (sample) using an antiserum directed against the first twelve amino acid residues of rat procalcitonin (NTP) .
  • NTP rat procalcitonin
  • Inverted triangles designate calcitonin, C-proCT, NCAP, CGRP and somatostatin.
  • Figure 4 shows a reversed-phase HPLC fractiona- tion profile of peptides from a thyroid tumor cell extract.
  • a 300 A/octyl reversed-phase HPLC column was used; one-milliliter fractions were collected, dried and assayed for immunoreactive NCAP (closed circles) and immunoreactive NTP (open squares) .
  • the dotted line shows the acetonitrile gradient.
  • FIG. 5 shows the results of gel filtration of partially purified N-proCT from rat medullary thyroid carcinoma (MTC) . Arrows indicate molecular weight markers.
  • N-proCT is the 7.4-KDa peak which possesses coincident NTP (open squares) and NCAP (closed circles) immunoreactivities.
  • Figure 6 illustrates the mitogenic effect of a representative procalcitonin-derived peptide on chick osteoblasts and preosteoblasts. Closed circles indicate synthetic NTP-Tyr [proCT(l-12)-Tyr] ; inverted triangles indicate synthetic calcitonin.
  • Figure 7 illustrates the mitogenic effect of partially purified rat N-proCT on chick (A) and rat (B) osteoblasts and preosteoblasts.
  • Figure 8 illustrates the results of a mitogene- sis assay on rat skin cells and chick bone cells using partially purified N-proCT and calcitonin.
  • Figure 9 illustrates the mitogenic effect of purified, synthetic human N-proCT on cultures of chick osteoblasts and preosteoblasts.
  • Figure 10 illustrates the mitogenic effects of synthetic human N-proCT on human osteoblasts prepared from patients with osteoarthritis (a) and human osteosar- coma cells (b) .
  • Figure 11 illustrates the coding sequence for rat N-proCT as constructed from oligonucleotides contain ⁇ ing yeast-preferred codons.
  • Figure 12 is a diagram of the assembly of the rat N-proCT coding sequence.
  • Figure 13 illustrates the construction of a yeast expression vector for N-proCT.
  • Figure 14 illustrates the plasmids pCPOT and pDPOT.
  • Figure 15 illustrates several yeast expression vectors used to produce procalcitonin-derived peptides.
  • Figure 17 illustrates the subcloning of the
  • Figure 18 illustrates the construction of plasmid pMVRl.
  • Figure 19 illustrates the insertion of MAT ⁇ 2 operator sequences into the TPI1 promoter.
  • Figure 20 illustrates the construction of plasmids pSXR109, pSXRHO, pSXRlll and pSXR112.
  • Figure 21 illustrates the results of experi ⁇ ments comparing bone growth in mice treated with N-proCT and untreated control mice.
  • the present invention provides a variety of peptides that specifically stimulate the proliferation of osteoblasts and preosteoblasts and are therefore useful in promoting bone growth in mammals.
  • these peptides may be derived from the amino-terminal region of procalcitonin or proCGRP. They may be synthesized de novo, isolated from suitable cells or tissues that naturally produce them, or produced through the use of recombinant DNA techniques.
  • Calcitonin is a 32-residue peptide hormone produced by C-cells of the thyroid gland and in large amounts by certain thyroid tumors. The hormone inhibits the activity of osteoclasts and by this mechanism reduces calcium levels in the blood. Calcitonin is secreted in response to high calcium and other nutritional signals. Rats fed a high-fat diet show increased levels of calci- tonin production, suggesting a role in fat metabolism. Injection of large doses of calcitonin can inhibit appe ⁇ tite and may also have analgesic effects. As is the case in the biosynthesis of other small peptide hormones, calcitonin is generated from a larger prohormone (Roos et al., Biochem. Biophys. Res.
  • Procalcitonin was first characterized in rats (Jacobs et al. , Science- 213 : 457- 459- 1981; A ara et al. , J. Biol. Chem. 257:2129-2132, 1S82); the rat sequence is now known to be structurally sr-imilar to the sequences of the human and chicken precursors (Gkonos et al. , J. Biol. Chem. 261: 14386- 14391, 1986; Lasmoles et al., EMBO J. 10:2603-2607, 1985).
  • Rat procalcitonin has 111 residues (Birnbaum et al., J. Biol. Chem. 259:2870-2874, 1984); the calcitonin sequence (proCTgo-91* 1 i* 3 nestled within the precursor, separated from amino- and carboxyl-terminal regions by flanking polybasic cleavage sites.
  • Calcitonin-rich cell lines developed for studies of calcitonin biosynthesis and secretion demonstrate a major pathway of procalci ⁇ tonin processing which yields calcitonin and the C- terminal hexadecapeptide, C-proCT (Birnbaum et al., J. Biol. Chem.
  • CGRP arises from differential RNA splicing. Mature
  • CGRP consists of 37 amino acids.
  • the precursor forms of the peptides (procalcitonin and proCGRP) are identical over the first 51 amino acid residues.
  • procalcitonin Like calcitonin,
  • CGRP is separated from its amino- and carboxyl-terminal flanking sequences by polybasic cleavage sites. CGRP can also reduce blood calcium and inhibit bone resorption, at least at high doses. Calcitonin and CGRP have separate cellular receptors, but each can bind to the other's receptor (Roos et al., Endocrinology 118:46-51, 1986).
  • N-terminal peptides designated N-proCT and N-proCGRP, respectively
  • These peptides have been found to have specific cell proliferative activity for osteoblasts and preosteoblasts when tested in a standard mitogenesis assay.
  • peptides derived from one species have been found to have specific cell proliferative activity for osteoblasts and preosteoblasts from another species.
  • calcitonin and CGRP from a number of species, including humans, rats and chickens, as well as salmon calcitonin, have been isolated and characterized.
  • the genes and cDNA sequences encoding these polypeptides have also been studied. These studies have shown that calcitonin and CGRP are encoded by the same gene and result from alternative RNA splicing.
  • a calcitonin pseudogene only known to be expressed in tumors, and a salmon calcitonin-like peptide have also been identified in humans.
  • the peptides of the present invention may correspond to amino-terminal sequences derived from precursors for any of these calcitonin gene products.
  • Exemplary peptides include human, chick, rat and salmon N-proCT (54 or 57 amino acids) and human, chick and rat N-proCGRP (52 or 55 amino acids), the sequences of which are shown in Figure 1.
  • the peptides of the present invention also include fragments of the amino-terminal portions of procalcitonin and proCGRP.
  • a 12-amino-acid fragment from the amino terminus of procalcitonin has been found to have unexpected, specific mitogenic activity for osteo ⁇ blasts and preosteoblasts.
  • the invention includes this 12-amino-acid fragment and larger procalcitonin-derived peptides having this amino-terminal sequence of 12 residues.
  • Preferred peptides in this regard include 31- amino-acid peptides from the amino termini of human, chick, rat and salmon N-proCT and human, chick and rat N- proCGRP. Additional bioactive peptides may be generated by cleavage of N-proCT or N-proCGRP with proteolytic enzymes or cyanogen bromide (CNBr) .
  • CNBr cyanogen bromide
  • CNBr cleavage of human N-proCT produces peptides of 36 and 21 amino acids.
  • minor changes may be made in amino acid sequen- ces without altering the useful properties of these peptides.
  • Such changes may result from genetic polymorphism or species diversity, or may be introduced into the peptides by genetic engineering. When introducing such changes, it is generally preferred to maintain those sequences that show high levels of interspecies homology (as shown in Figure 1) and to avoid making major changes in the chemical nature (e.g., charge, hydrophobicity, etc.) of the peptides.
  • the peptides of the present invention are commonly defined in the context of rat N-proCT. In general, the peptides of the present invention will be substantially homologous to a portion of rat N-proCT, generally at least about 40% homologous, reflecting the levels of interspecies homology shown in Figure 1.
  • the peptides will be substantially homologous to a portion of human N-proCT, which is about 60% homologous to rat N-proCT.
  • the peptides be substantially homologous to a portion of at least about 10 contiguous amino acids of the a ino-terminal-most 32 amino acids of rat N-proCT.
  • the peptides of the present invention may be isolated from tissues or cultured cells that naturally produce them, synthesized by conventional chemical proce ⁇ dures, or produced through the use of recombinant DNA techniques.
  • Cells and tissues known to produce calcitonin, CGRP or related gene products in recoverable amounts may be used as sources of the peptides. Suitable cell types include normal and neoplastic C-cells and brain cells. A preferred source of neoplastic C-cells is the 1-2-4 medullary thyroid carcinoma, disclosed by Roos et al. (Endocrinology 105: 26-32, 1979) .
  • the peptides are extracted from the cells or tissue using an aqueous buffer.
  • the tissue is minced and the peptides are extracted with a hot solution of an organic acid, using an acid concentration of about 1-2 N.
  • the extraction temperature is preferably at least 70°C, most preferably boiling.
  • minced tissue is added to about 10 to 20 volumes of 1.5 N acetic acid and boiled for 20 to 30 minutes.
  • the resulting ho ogenate is then cooled, and insoluble material and lipids are separated from the aqueous extract, preferably by centrifugation.
  • the aqueous extract is then size-fractionated, preferably in a two- stage process, combining a batch reversed-phase chroma ⁇ tography procedure with a gel-filtration procedure.
  • a preferred chromatographic medium is C-18 silica.
  • the peptides are then eluted from the medium with a suitable organic solvent.
  • the fractionation is performed on a 5-gram column of Vydac C-18, wide-pore, 30-micron silica that has been activated in 90% acetonitrile/0.1% trifluoroacetic acid (TFA) and equilib ⁇ rated in 0.1% TFA.
  • TFA trifluoroacetic acid
  • the column is washed with 25 ml of 0.1% TFA, followed by 50 ml of 20% acetonitrile/0.1% TFA.
  • N-proCT is eluted with 45%-90% acetonitrile/0.1% TFA.
  • the partially purified peptide is then dried and dissolved in 6 M guanidine and further purified by gel filtration.
  • a preferred gel- filtration medium is Sephadex G-50 (Pharmacia, Piscata- way, N.J.). Fractions containing the peptides of the present invention are identified by immunoassay and/or activity assay using cultured bone cells. The gel- filtration peak is further fractionated by hydrophobic chromatography, such as reversed-phase HPLC. C-8 silica, 10-micron, reversed-phase resin (Whatman) is a particu ⁇ larly preferred chromatographic medium in this regard. N-proCT elutes from C-8 silica at 37% acetonitrile and can be separated from other proteins by fractionation in a 35%-40% acetonitrile/0.1% TFA gradient run over 80 minutes.
  • Peak fractions are preferably identified by immunoassay. If desired, additional purification may be achieved through conventional methods, such as anion- exchange chromatography, HPLC and immunoaffinity chromato- graphy. Alternatively, the peptides may be purified by immunoaffinity chromatography followed by HPLC.
  • Peptides shorter than about 20 to 32 amino acids may be synthesized by conventional chemical tech ⁇ niques. Methods of peptide synthesis are disclosed by, for example, Merrifield (J. Am. Chem. Soc. 85:2149-2154, 1963) and Houghten (Proc. Natl. Acad. Sci. USA 82:5131- 5135, 1985). Peptides may also be obtained from various commercial suppliers, including Peninsula Laboratories (Belmont, Calif.), Bachem Bioscience, Inc. (Philadelphia, Pa.), Biosearch, Inc. (San Rafael, Calif.), and Applied Biosystems (Foster City, Calif. ) . The peptides of the present invention are preferably prepared using genetically engineered cells.
  • DNA sequence encoding the peptide of interest is joined to a suitable transcriptional promoter and inserted into a vector along with other elements (transcriptional terminators, poly- adenylation signals, enhancers, selectable markers, etc.) , which are selected according to the type of host cell chosen. Selection of the proper elements, construc ⁇ tion of expression vectors, and transformation or trans- fection of host cells are within the level of ordinary skill in the art.
  • DNA sequences encoding the peptides of the present invention are preferably synthesized.
  • oligonucleotides Use of synthesized oligonucleotides allows selection of codons in accordance with host cell preference. Methods for synthesizing DNA are disclosed by, for example, Caruthers et al. (U.S. Patent No. 4,458,066) and Itakura et al. (Science 198:1056-1063, 1977), although automated synthe ⁇ sis is generally preferred. In general, it is convenient to prepare oligonucleotides up to about 50 to 60 bases in length. Assembly of longer sequences is facilitated by designing the oligonucleotide pairs so that when annealed, overlapping, complementary ends are produced. The overlapping fragments are then ligated to produce longer sequences.
  • Suitable DNA sequences may also be obtained by enzymatic digestion of cDNA or genomic clones encoding calcitonin or CGRP precursors. Such clones are disclosed by Amara et al. (Nature 298:240-244, 1982) , Amara. et ai. (J. Biol. Chem. 257:2129-2132, 1982) , and Birnbaum et al. (J. Biol. Chem. 258:5463-5466, 1983) .
  • Preferred prokaryotic host cells for use in carrying out the present invention are strains of the bacteria Escherichia coli , although Bacillus and other genera are also useful.
  • Vectors used for expressing cloned DNA sequences in bacterial hosts will generally contain a selectable marker, such as a gene for antibiotic resistance, and a promoter that functions in the host cell.
  • Appropriate promoters include the trp DJ (Nichols and Yanofsky, Meth. Enzymol. 101:155-164, 1983), lac (Casadaban et al. , J. Bacteriol.
  • Plasmids useful for transforming bracteria include pBR322 (Bolivar et al., Gene 2_:95-113, 5:* 1977), the pUC plasmids (Messing, Meth. Enzymol. 101:20- 78, 1983; Vieira and Messing, Gene 19_:259-268, 1982), pCQV2 (Queen, ibid.) , and derivatives thereof. Plasmids may contain both viral and bacterial elements.
  • Eukaryotic microorganisms such as the yeasts ⁇ Saccharomyces cerevisiae and Schizosaccharomyces pombe or filamentous fungi (e.g., Aspergi llus spp. , ' Neurospora spp.), may also be used as host cells within the present invention.
  • S ⁇ cerevisiae is a particularly preferred host.
  • Techniques for transforming yeast are well known 5 in the literature, and have been described by, for instance, Beggs (Nature 275:104-108, 1978) and MacKay (Meth. Enzymol. 101:325-343, 1983).
  • Asperqillus species may be transformed according to known procedures, for example, that of Yelton et al. (Proc. Natl. Acad. Sci.
  • Suitable yeast expression vectors include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1979) , YEpl3 (Broach et al., Gene 8 ⁇ :121-133, 1979), pJDB249 and pJDB219 (Beggs, ibid.), and derivatives thereof.
  • Such vectors will generally include
  • a selectable marker such as the nutritional marker LEU2, which allows selection in a host strain carrying a leu2 mutation or, preferably, an "essential gene,” as described by Kawasaki and Bell (EP 171,142) .
  • a preferred such essential gene marker is the triose phosphate isomerase gene (POT1 gene) of Schizosaccharomyces pombe, which provides for stable plasmid maintenance in a triose phosphate isomerase-deficient host cell cultured in rich glucose medium.
  • Expression vectors containing the POT1 selectable marker include pCPOT (ATCC 39685) , pMPOT2 (ATCC 67788) and derivatives thereof.
  • promoters useful in yeast expression vectors include promoters from yeast glyco- lytic genes (Hitze ⁇ ran et al., J. Biol. Chem. 255 : 12073- 12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982; Kawasaki, U.S. Patent No. 4,599,311) or alcohol dehydrogenase (ADH) genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al. (Eds.) , New York: Plenum, 1982, p.
  • Derivatives and variants include naturally occurring mutant promoters (e.g., ADH2-4 C ) , engineered hybrid promoters (see, e.g., Bitter, WO 86/06077; Rosenberg et al. , EP 164,556) and other engineered derivatives. In general, such deriva ⁇ tives will provide enhanced promoter strength or altered regulatability as compared to the parent promoters.
  • Particularly preferred constitutive promoters are the triose phosphate isomerase (TPI1) promoter and the ADH2- 4 C promoter.
  • Particularly preferred regulated promoters include the wild-type ADH2 promoter and temperature- regulated hybrid promoters. Temperature-regulated hybrid promoters are constructed as described in U.S. Patent Applications Serial Nos. 889,100 and 036,823 by inserting one or more, preferably two or more, copies of a yeast mating-type regulatory element into a promoter, such as the TPI1 promoter. The resulting hybrid promoter is used in a host cell that is capable of expressing mating type elements in a temperature-sensitive manner. Suitable yeast host cells in this regard include temperature- sensitive sir and ste mutants.
  • Promoter strength is regulated by varying the growth temperature of the transformed cells, generally between 23°C and 36°C.
  • a transcriptional termination signal such as the TPI1 terminator
  • a signal sequence preferably from a yeast gene encoding a secreted protein, may be joined in the correct reading frame to the coding sequence for the peptide of interest. Suitable signal sequences include the pre-pro region of the MF ⁇ l gene (Kurjan and Herskowitz, Cell 30:933-943 , 1982; Kurjan et al. , U.S. Patent No.
  • Yeast host strains are widely available, for example, from American Type Culture Collection, Rockville, Md. , or the Yeast Genetic Stock Center, Berkeley, Calif. It is preferred that the host strain carries a pep4 mutation to reduce proteolytic degradation of the peptide of interest.
  • Expression vectors for use in mammalian cells will comprise a promoter capable of directing the transcription of a cloned DNA sequence introduced into a mammalian cell.
  • Particularly preferred promoters are the mouse metallothionein-1 (MT- 1) promoter (Palmiter et al., Science 222:809-814, 1983) or the major late promoter of adenovirus 2 (Berkner and Sharp, Nuc. Acids Res. 12:1925-1941, 1984) .
  • MT-1 mouse metallothionein-1
  • adenovirus 2 also included in such expression vectors is a polyadenylation signal, located downstream of the DNA sequence insertion site.
  • sequences such as enhancers and RNA splicing signals, may also be included.
  • the coding sequence for the peptide of incerest is joined in the correct reading frame to a mammalian secretory signal sequence so that the peptide will be secreted from the cell.
  • Cloned DNA sequences are introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al. , Cell 14: 725, 1978; Corsaro and Pearson, Somat. Cell Genet. 7: 603, 1981; Graham and Van der Eb, Virol. 5_2:456, 1973) or electroporation (Neumann et al. , EMBO J. 1:841-845, 1982) .
  • a selectable marker is generally introduced into the cells along with the sequence of interest in order to identify transfectants that have integrated the cloned DNA into the genome.
  • Preferred selectable markers include genes that confer resistance to drugs, such as neomycin, hygromycin and methotrexate.
  • Selectable markers may be introduced into the cell on a separate expression vector at the same time as the sequence of interest, or they may be introduced on the same expres- sion vector.
  • the copy number of the integrated DNA sequence may be increased through amplification by drug selection when using certain selectable markers, such as DHFR, which confers resistance to methotrexate.
  • the drug concentration is increased in a stepwise manner, with selection of resistant cells at each step. By selecting for increased copy number of cloned sequences, expression levels may be substantially elevated.
  • Host cells expressing the peptides are grown in a culture medium appropriate to the particular host.
  • a variety of media are available and a suitable medium is chosen on the basis of host cell nutrient requirements, plasmid selection, etc. (For media recipes, see, for example, catalogs of the American Type Culture Collec ⁇ tion, Rockville, Md.)
  • the recombinant peptides are purified from cell media or cleared cell lysates generally as previously described for extraction from tissue.
  • a sample containing a peptide of interest is fractionated by a combination of high-performance liquid chromatog- raphy and gel filtration. Peak fractions are identified by immunoassay or biological activity assay.
  • the peptides of the present invention are useful as therapeutic agents for the promotion of bone growth in warm-blooded animals. These peptides may find use in the prevention and treatment of osteoporosis, Paget's disease, and periodontal disease and in promoting the healing of fractures, particularly in patients in whom normal healing does not occur.
  • the peptides will be mixed with a physiologically acceptable carrier or diluent, such as sterile water or sterile saline.
  • the peptides may be packaged in lyophilized form and combined with the carrier or diluent prior to administration.
  • Peptides shorter than about 32 amino acids in length may be suitable for administration intranasally.
  • Methods for administering compositions intranasally are well known. Briefly, the peptide of interest is dissolved in a physiologically tolerable buffer and sprayed onto the nasal membrane. Longer peptides and shorter peptides not amenable to intranasal administration are administered by injection, preferably subcutaneous injection. The peptides may also be administered in suppositories. Suitable dosages will generally be in the range of about 0.2 ⁇ g to 2 mg per day per kg patient weight, preferably about 2 ⁇ g to 2 mg/kg/day, most preferably about 20 to 200 ⁇ g/kg/day, depending on the precise nature of the condition to be treated.
  • the peptides When administering the peptides intranasally, it is preferable to utilize divided doses over the course of the day. Substantially higher dosages may result in reduced proliferative activity.
  • the peptides may also be administered in combination with other therapeutic agents.
  • Preferred additives in this regard include estrogen, calcitonin, and bisphosphonates, which are known to reduce bone resorption and may therefore complement the action of the peptides of the present invention.
  • the activity of the peptides can be enhanced by combining them with growth factors. Notably, this enhancement is observed at growth factor doses at or below those reported to have general ⁇ ized growth-promoting effects.
  • Growth factors useful in this regard include insulin, insulin-like growth factors (somatomedins) , platelet-derived growth factor (PDGF) , transforming growth factors (TGF ⁇ and TGF ⁇ ) and epidermal growth factor (EGF) , with insulin-like growth factor I (IGF-I; also known as somato edin C) , with or without the IGF-I carrier protein, particularly preferred.
  • IGF-I insulin-like growth factor I
  • Methods for preparing these growth factors are known in the art. See, for example, Murray et al. , U.S. Patent 4,766,073; Derynck et al., EP 200,341; Derynck et al., U.S. Patent 4,742,003; Gregory et al. , U.S.
  • a peptide of the present invention is administered in combination with IGF-I at an IGF-I dose of 0.02-2 g per day per kg patient weight.
  • N-proCT was detected in normal and neoplastic tissue by immunoassay using antisera prepared against synthetic peptides (obtained from Peninsula Laboratories) corresponding to sequences in the amino-terminal and carboxyl-terminal regions of procalcitonin (Table 1) .
  • An NCAP antiserum was raised by immunizing rabbits with NCAP conjugated to keyhole limpet hemocyanin with l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (Birnbaum et al. , 1982, ibid.; Goodfriend et al. , Science 144:1344-1346, 1964) .
  • Two NTP antisera were raised by immunizing rabbits with synthetic NTP-Tyr conjugated to keyhole limpet hemocyanin (Goodfriend et al., ibid.) .
  • the NCAP radioimmunoassay was developed using radioiodinated synthetic [Tyr ⁇ ]-NCAP as tracer and standard. All iodinations were by the chloramine-T method and purified by Quso-32 silica adsorption (Roos and Deftos, in Methods of Hormone Radioassay, 2d ed. , Jaffe and Behr an (Eds.), New York:Academic Press, 1978, pp. 401-418) . The assay was developed to be specific for the free carboxyl-terminal portion of the N-proCT sequence.
  • radiolabeled NCAP analogs containing a modified amino terminus By screening antisera with radiolabeled NCAP analogs containing a modified amino terminus (tyrosine or Bolton-Hunter adduct) , antisera directed toward the carboxyl-terminal portion of NCAP were selected. All radioim unoassays were performed in 0.020 M sodium phosphate buffer (pH 7.5), containing 1 mM disodium EDTA, 0.005% sodium merthiolate, 0.1% bovine serum albumin (Pentex fraction V) , and 0.03% Brij-35 detergent. Standards ranging from 10 to 10,000 pg of [Tyr°]-NCAP were included in each assay.
  • NCAP antiserum from rabbit "Odessa" at a 1:3,500 final dilution, tracer, and standard or sample were added to a final volume of 500 ⁇ l per tube and incubated overnight at 4°C. Phase separa ⁇ tion was achieved by adding 1 ml/tube of 0.1% (w/v) Iggsorb (The Enzyme Center). The assay's limit of detec- tion for synthetic Tyr-NCAP and NCAP was about 0.20 pmol/tube, with 50% displacement of bound tracer occur ⁇ ring at roughly 1.8 pmol/tube. The Bolton-Hunter deriva- tive of NCAP had a displacement curve very similar to those of Tyr-NCAP and the unmodified NCAP peptide.
  • proCT4g_55 which lacks the last 2 amino acids of 5 NCAP (and N-proCT) .
  • the carboxyl-terminally extended NCAP-containing peptide, proCT46_59 was 1,000-fold less immunoreactiv ⁇ than NCAP, indicating that a free carboxyl-terminal serine is needed for an NCAP-containing peptide to cross-react in this NCAP assay. While the
  • NCAP immunoassay should recognize N-proCT (proCT ⁇ _57) , it should not detect procalcitonin or other NCAP-containing forms possessing a carboxyl-terminal extension.
  • NCAP did not cross-react in this NTP assay. Because 10 to 20 ⁇ l/tube amounts of 6 M guanidine-HCl interferred with the Vanessa radioimmunoassay, another NTP antiserum (from rabbit "Peggy") was used to analyze gel-filtration fractions containing guanidine. The Peggy antiserum was
  • Rat calcitonin radioimmunoassays were performed with calcitonin antiserum R-2, which is specific for the amidated carboxyl terminus, and with an antiserum that recognizes calcitonin's midregion (Roos et al. , 1979, ibid.; Birnbaum et al. , 1984, ibid.) .
  • Rat CGRP was measured with an immunoassay based on human CGRP anti ⁇ serum RB-2035, as previously described (Gkonos et al. , ibid.; Haller-Bre et al. , Endocrinology 121 : 1272-1277, 1987) .
  • WAG/Rij rats were maintained in an AAALAC- accredited animal facility.
  • the standard ad libitum diet was rodent laboratory chow #5001 (TekLad Co., Harlan Sprague Dawley, Inc.) . This diet contained 4.5% fat, 23% protein, and 6% fiber, with all the normal vitamin and mineral supplements.
  • To obtain normal thyroid tissue six- to nine-month-old female rats were sacrificed and their thyroids excised.
  • C-cell hyperplasia Massiller et al., Program of the Seventh Annual Meeting of the American Society for Bone and Mineral Research, Abstr.
  • Calcitonin-rich series 1-2-4 medullary thyroid carcinomas were generated by serial subcaps ⁇ lar renal transplantation into homologous weanlings as previously reported (Roos et al., 1979, ibid.) . Tumors of 10 to 40 g were removed surgically and extracted as described below.
  • tissue extracts 10 volumes (ml/g) of 2 N acetic acid/1% TFA was added to finely minced tissue, and the suspension was then boiled for 20 minutes. The sample was exhaustively homogenized using a Brinkmann Polytron at setting 10 and then cooled on ice. The resulting homogenat ⁇ was centrifuged at 15,000 x g for 15 minutes, and the supernatant was aspirated from the pellet.
  • peptides were extracted from the supernatant by a batch reversed-phase procedure in which the supernatant was applied to a 2- to 5-gram column (0.5 x 2 cm) of acetonitrile-activated, Vydac 218TPB30 C-18, large-pore silica beads.
  • the column was rinsed first with 0.1% TFA, then with 20% acetonitrile/ 0.1% TFA.
  • the sample was then eluted in a small volume of 90% acetonitrile/0.1% TFA and dried in a Savant SpeedVac system.
  • Monolayer cultures of the established CA-77 medullary thyroid carcinoma cell line were routinely grown in serum-free medium consisting of DMEM/nutrient mixture F-10 (Ham's) (1:1) supplemented with 1.28 g/liter aHC03, 5 ug/ml transferrin, 30 nM selenous acid, and 5 ug/ml insulin. The cultures were maintained in a humidified air-8% CO2 atmosphere.
  • CA-77 cells from passages 150-155 were subcultured at a density of 5 x 10 ⁇ cells per 25-cm2 f ask using previously described methods (Muszynski et al., J. Biol. Chem. 258:11678-11683, 1983); thereafter, medium was changed every 48 hours.
  • tissue culture media were clarified by centrifugation at 16,000 x g for 10 minutes, made 0.3 mg/ml in PMSF, and stored, frozen for analysis.
  • Cells were scraped from flasks into 1% TFA and homogenized by sonication.
  • Cellu ⁇ lar debris was pelleted by centrifugation and discarded; the supernatant was made 0.3 mg/ml in PMSF and stored frozen for analysis.
  • Culture medium and clarified cell extracts were passed over Vydac CI Q columns in the same manner as tissue extracts.
  • Tumor peptides were separated by reversed-phase HPLC.
  • Dried hot-acid extracts from 1-2-4 series medul ⁇ lary thyroid carcinomas were resuspended in 1% TFA (200- 600 ul) and were injected onto a Whatman Protesil 300 Octyl-25 analytical column using a Waters U6K injector and a modular Spectra-Physics HPLC system (Birnbaum et al., 1984, ibid.) .
  • Peptide separation was done with an acetonitrile gradient in 0.1% aqueous TFA.
  • the HPLC elution position of immunoreactive NCAP and immunoreac- tive NTP from normal rat thyroid was identical to that found in the 1-2-4 tumor tissue.
  • the apparent molar ratio of immunoreactive NCAP to immunoreactive NTP in the HPLC peak from normal thyroids was 1.0, the same as observed for the 1-2-4 tumor HPLC peak.
  • Guanidine gel filtration of an extract of normal-rat thyroids demon ⁇ strated a predominant 7.4-KDa immunoreactive NCAP/NTP peak, indistinguishable by size from the immunoreactive NCAP/NTP peak observed in the 1-2-4 tumor.
  • NCAP antiserum indicates that the carboxyi terminus of the 7.4-KDa peptide is the serine immediately preceding the first dibasic cleavage site in procalcitonin (serine-57) .
  • NTP-immunoreactivity and partial microsequencing of radiolabeled peptides from CA-77 cells indicate an amino terminus identical to pro ⁇ calcitonin' s. Therefore, the biochemical properties of the immunoreactive NTP- and immunoreactive NCAP-contain ⁇ ing peptide identify it as a 57-residue N-proCT species.
  • Rat medullary thyroid carcinoma tissue is weighed, minced, and added to 10 volumes (per gram wet weight) of 1.5 N acetic acid. This mixture is heated to boiling and boiled for an additional 20 minutes. The hot mixture is then homogenized in a Brink ann polytron (setting 10) . The homogenate is then rapidly cooled with ice water and maintained at roughly 4°C for 20 minutes, then centrifuged at 20,000 X g for 20 minutes. The aqueous supernatant is carefully aspirated so as not to disturb the pellet or floating lipid layer.
  • the supernatant is applied to a 5-gram column of Vydac C-18, wide-pore, 30-micron silica equilibrated in 0.1% TFA that has been previously activated in 90% acetonitrile/0.1% TFA.
  • the column is washed with 25 ml of 0.1% TFA, followed by 50 ml of 20% acetonitrile/ 0.1% TFA.
  • N-proCT is then eluted with 45% acetonitrile/ 0.1% TFA and dried in an unheated Savant SpeedVac.
  • the dried N-proCT is dissolved in 6 M guanidine and applied to a column of Sephadex G-50 equilibrated in 6 M guanidine-HCl (pH 5.5) .
  • the 7.4-kna NTP- and NCAP- containing material ( Figure 5) is pooled.
  • the peptide is then recovered from the pooled guanidine fractions by adsorption to and elution from a 1-gram column of Vydac C-18 and dried in the SpeedVac as described above.
  • the dried peak of N-proCT from the gel filtra ⁇ tion is resuspended in 300 ul of 0.1% TFA and injected onto a 0.6 X 25-cm column of Whatman C-8 silica, 10- micron (300 A pore size), reversed-phase resin.
  • N-proCT peptide elutes from the column at 37% acetonitrile and can be separated from other proteins by fractionation in a 35%-40% acetonitrile/0.1% TFA gradient run over 80 minutes ( Figure 4) .
  • a peak of constant specific immuno- reactivity is selected for amino acid analysis and sequencing.
  • the NTP-Tyr peptide (representing the initial dodecapeptide fragment of the 57-residue rat N-proCT) used for generation of RIAs (Example 1) was used to assess possible bone resorption and/or bone-cell- proliferative activity.
  • Calvaria were taken from 16-day embryonic chicks and neonatal rats and treated with collagenase to prepare bone cells.
  • the cells were suspended in serum-free BGJ ⁇ medium (Fitton-Jackson Modification from Gibco, Grand Island, N.Y.) and plated over 24 hours. The plates were rinsed to remove fibro- blasts and other nonosteoblastic cel ls.
  • Synthetic NTP- Tyr was added at concentrations between 0.001 and 100 uM for 22 hours, at which time the cells were labeled for 4 hours with tritiated thymidine. DNA synthesis was measured as TCA-precipitable tritiated thymidine and used as an index of cell proliferation. DNA synthesis was expressed relative to that of untreated cultures. With the synthetic N-proCT fragment, a strong mitogenic response was seen at 10 uM concentration, and significant effects were observable at 1 uM. Neither calcitonin nor CGRP had any mitogenic effect (Figure 6) .
  • N-proCT was partially purified from 1-2-4 tumor tissue by enriching roughly 100-fold over the starting tissue homogenate through the use of the boiling acid extraction method coupled with reversed-phase C-18 silica purification. This N-proCT was resuspended in BGJ D medium, neutralized, and added to rat or chick calvarial osteoblast cultures essentially as described above. Thymidine incorporation was increased in both the chick ( Figure 7A) and rat ( Figure 7B) bone-cell cultures.
  • the partially purified N-proCT preparation evokes a two- to threefold mitogenic response with a maximal response observed at 10 " ⁇ to lO- ⁇ M.
  • This sensitivity for intact N-proCT appears to be 100- to 1000-fold greater than that for the synthetic peptide fragment (NTP-Tyr).
  • the rat osteoblasts and preosteo- blasts appear to be somewhat more sensitive to the rat peptide than are the chick cells ( Figure 7); doses as low as lO-- ⁇ M had a stimulatory effect in the rat osteoblast cultures.
  • calcitonin nor CGRP which are active in hypocalcemia assays and bone-resorption assays, had effects in parallel chick osteoblast mitogenic assays at concentrations of 10 " ⁇ to 10 ⁇ 6 M.
  • Human N-proCT was synthesized by Applied Biosystems (Foster City, Calif.) and supplied as an impure preparation.
  • the human peptide was purified by high- performance liquid chromatography on a Vydac C-4 reversed-phase column followed by chromatography of the o major HPLC peak on a Lichroprep RP-18 (C-18 reversed- phase silica) column.
  • the Lichroprep column (0.5 g) was activated with 10 ml of 90% CH3CN containing 0.1% trifluoroacetic acid (TFA) .
  • the column was then washed with 20 ml of 0.1% TFA, and the N-proCT sample (0.5 ml) 5 was applied to the column.
  • the column was washed with 10 ml of 0.1% TFA followed by 10 ml of 20% CH3CN containing 0.1% TFA.
  • the N-proCT was eluted with 40% CH3CN/0.1% TFA. Greater than 90% of the N-proCT eluted at 40% CH3CN.
  • the sequence of the synthetic peptide was 0 analyzed and found to be identical to that of native human N-proCT ( Figure 1) .
  • Human osteoblast-enriched cultures were prepared from surgical femur fragments of one older man and two older women. The bone fragments were digested in collagenase. Cells were collected from the digests, rinsed in PBS, and plated into tissue culture flasks in calcium-free MEM containing 15% fetal calf serum. The resulting osteo ⁇ blast-enriched cultures were trypsinized from the dishes, rinsed in PBS, resuspended in BGJ ⁇ medium containing 5% fetal calf serum, and plated into 48-well microtiter plates (Costar, Inc.) at a density of 50,000 cells per well.
  • the swabs were washed twice for 10 minutes each in 12.5% TCA and once for 10 minutes in 95% ethanol, dried, placed in 4 ml of Ecolume (ICN, Irvine, Calif.) , and counted in a scintillation counter. Results showed that 10 nM human N-proCT produced a maximal stimulation (approximately a doubling) of thymidine incorporation into DNA, with a half-maximal effect at about 1 nM ( Figure I0a) .
  • the maxi ⁇ mally effective dose of insulin 10 ⁇ g J or 2 ⁇ M also doubled thymidine incorporation rates in these cultures.
  • Human N-proCT was tested in combination with insulin on human U-2 OS cells.
  • the cells were plated in 48-well microtiter plates as previously described, then Incubated for 48 hours in the presence of N-proCT, insulin, or N-proCT plus insulin.
  • trypsin and EDTA were added to final concentrations of 25 mg/ml and 1 mM, respectively, and the cultures were incubated at 37°C until the cells were visibly detached.
  • Fetal calf serum was then added to a final concentration of 15% to inactivate the trypsin.
  • Clumped cells were dispersed by repeated pipetting, and aliquots were taken for direct counting in a hemocytometer . Results, shown in Table 3, indicate a synergistic effect between N-proCT and insulin.
  • Synthetic human N-proCT was digested with lysyl 5 endopeptidase (Wako Chemicals USA, Inc., Dallas, Tex.). 345 ⁇ g of lyophilized N-proCT was dissolved in 110 ⁇ l of 8 M urea. Ten ⁇ l of 500 mM Tris pH 9 was added, and the mixture was incubated for 30 minutes at 37°C. 150 ⁇ l of 50 M Tris pH 9 was added to the mixture. Lysyl endopep- 0 tidase (3 mg/ml stock solution) was added to give a 1:100 enzyme:substrate ratio. The mixture was incubated at 37°C overnight, then stored frozen. The resultant peptides, N-proCT (1-37) and N-proCT (38-57) were recovered by chromatography on a Vydac C-4 column using a
  • Example 7 5 ⁇ M of N-proCT (1-37) was found to be at least- as effective as the same concentration of intact human N-proCT in competing for binding. Fragment (38-57) showed little, if any, ability to compete for binding.
  • N-proCT was constructed from the 6 synthesized oligonucle ⁇ otides shown in Table 4.
  • the oligonucleotides were JO
  • oligo ⁇ nucleotides were synthesized on an Applied Biosystems model 380A DNA synthesizer and purified by electrophor- esis on denaturing gels. The oligonucleotides were kinased, mixed in equimolar proportions and annealed.
  • the N-proCT fragment was linked to the ADH2-4 C promoter and TPI1 terminator.
  • the resulting expression unit was then inserted into a vector containing the POT1 selectable marker.
  • An ADH2-4 C promoter was constructed by joining the downstream portion of the wild-type ADH2 (alcohol dehydrogenase II) promoter to the upstream portion of the ADH2-4 C promoter described by Russell et al. (Nature 304:652-654, 1983) , The upstream sequences of the ADH2- 4 C promoter are responsible for its enhanced function. Construction of this promoter is illustrated in Figure 12.
  • site-specific _in vitro mutagenesis (Zoller et al., DNA 3 ⁇ :479-488, 1984) was done on the ADH2 insert in M13mpl9 using ZC237 as the mutagenic primer and ZC87 (5 1 TCC CAG TCA CGA CGT 3 1 ) as the second primer.
  • ZC237 As the mutagenic primer
  • ZC87 5 1 TCC CAG TCA CGA CGT 3 1
  • the oligonucleotide ZC237 looped out the structural portion of the ADH2 gene, fusing the 5' flank ⁇ ing sequence, including the translation start signal, with the Eco RI site of the Ml3mpl9 polylinker.
  • the replicative form of the mutagenized phage was made and cut with Bam HI and Eco RI to isolate the 1.2-kb promoter fragment. This fragment was ligated into pUC13 which had been linearized with Bam HI and Eco RI to generate plas- id p237-Wt.
  • p237-Wt promoter to the "promoter-up" mutant ADH2-4 C promoter, a 1.1-kb Bam HI- Sph I fragment from YRp7-ADR3-4c (Russell et al.
  • Plasmid pAT-1 comprises the expression unit of the ADH2 promoter from p237-Wt and an ⁇ -l-anti- trypsin cDNA-TPIl terminator sequence. These sequences were inserted into a portion of the vector pCPOT ( Figure 14). (Plasmid pCPOT has been deposited with ATCC as an E.
  • Plasmid pCPOT was cut with Bam HI and Sal I to isolate the approximately 10-kb linear vector fragment.
  • the 1.2-kb ADH2 promoter fragment was isolated from p237-Wt as a Bam HI-Eco RI fragment and ligated with the 1.5-kb ⁇ -1-antitrypsin cDNA-TPIl terminator fragment (Eco Rl-Xho I) and the linearized pCPOT in a three-part ligation to yield a plasmid designated pAT-1.
  • Plasmid pAT-1 contained three extra amino acid codons between the ADH2 translation start codon and the first amino acid codon for the mature form of AAT. These three codons were removed by site-specific iri vitro mutagenesis.
  • Plasmid pAT-1 was cut with Sph I and Bam HI to isolate the 190 bp ADH2 promoter fragment. This fragment was ligated into M13mpl8 which had been linear ⁇ ized with Bam HI and Sph I. The resulting construction was subjected to in vitro mutagenesis using ZC411 ( 5 'TAATACACAATAGGAGGA TCCC 3 ' ) as the mutagenic primer and ZC87 as the second primer to fuse the ADH2 translation start signal to the first codon of mature ⁇ -l-antitrypsin . Positive clones were confirmed by dideoxy sequencing from -170 bp from the ATG through the fusion point.
  • the 175-bp Sph I-Eco RI mutagenized promoter fragment was ligated into pUC19 which had been linearized with Sph I and Eco RI.
  • the resultant plasmid comprising the 3 '-most 170 bp of the ADH2 promoter and the ADH2 translation start codon fused to the first amino acid codon of the mature form of AAT in vector pUC19, was designated p411.
  • the 5'-most sequence of the ADH2-4 C promoter containing the alterations found by Russell et al. (ibid.) to influence promoter function, was added to the promoter fragment present in plasmid p411.
  • Plasmid p411 was digested with Sph I and Eco RI to isolate the 175-bp promoter fragment.
  • Plasmid p237-4 c was cut with Eco RI and Sph I to isolate the 3.71-kb fragment comprising pUC vector sequences and the 5 '-most promoter sequence that confers the "promoter- up" phenotype.
  • the 175-bp promoter fragment from p411 was ligated Into the p237-4 c vector fragment.
  • the ADH2 promoter from plasmid pAT-1 was modified to create a "universal" promoter by removing the ADH2 translation start site and the pUC18 polylinker sequences found in pAT-1. Plasmid pAT-1 was cut with Sph I and Bam HI to isolate the 190-bp partial ADH2 promoter fragment. This fragment was ligated into M13mpl8 which had been linearized with Bam HI and Sph I.
  • the resulting construction was subjected to in vitro mutagenesis using ZC410 ( 5 'CGTAATACAGAATTCCCGGG 3 ' ) as the mutagenic primer and ZC87 as the second primer to replace the ADH2 translation start signal and pUCl ⁇ polylinker sequences with a single Eco RI site fused to the M13mpl8 polylinker at the Sma I site. Positive clones were confirmed by dideoxy sequencing through the fusion point. For ease of manipulation, the mutagenized partial ADH2 promoter fragment was subcloned as a 175-bp Sph I-Eco RI fragment into pUC19 which had been linearized with Sph I and Eco RI.
  • the resulting plasmid contained the 3 '-most 175 bp of the ADH2 promoter.
  • the ADH2-4 C promoter was then modified to contain this 3' sequence by combining the p410ES promoter fragment (Sph I-Eco RI) with the 1.1-kb Bam Hl-Sph I ADH2-4 C pro ⁇ moter fragment from p237-4c.
  • the two promoter fragments were joined with Bam HI, Eco Rl-cut pUC13 in a three-part ligation.
  • the resultant plasmid confirmed by restric ⁇ tion analysis, contained the complete ADH2-4 C promoter mutagenized at the 3' end to place an Eco RI site in place of the translation start codon. This plasmid was designated p410-4c ( Figure 13) .
  • the Bam HI-Eco RI ADH2-4 C promoter fragment from p410-4c and the Eco Rl-Xba I N-proCT fragment were ligated to Bam HI, Xba I-digested M13mpl8.
  • M13 clones containing the insert were sequenced, and replicative form (RF) DNA was prepared from a clone containing the full insert.
  • the phage vector was digested with Bam HI and Xba I and the promote —N-proCT fragment was isolated.
  • This fragment was then joined to an Xba I-Bam HI TPIl terminator fragment from pKPIO (a plasmid comprising the TPIl promoter-alpha factor-VSB-TPII terminator expression unit from pSBl [Murray et al., U.S. Patent 4,766,073] inserted into a pBR322 vector lacking an EcoRI site) , and Bam Hi-digested pUC19. A clone containing the correct insert was then digested with Bam HI and the expression unit was isolated.
  • Plasmid pCPOT was cleaved with Sph I and Bam HI to remove 750 bp of 2 micron and pBR322 sequences.
  • the linearized vector was then joined to a 186-bp Sph I- Bam HI fragment derived from the pBR322 tetracycline resistance gene.
  • the resulting plasmid designated pDPOT ( Figure 12) was then digested with Bam HI and ligated to the Bam HI expression unit fragment, and the insert orien ⁇ tation of the resultant clones was determined.
  • a plasmid containing the ADH2-4 C promoter adjacent to the POTl gene was selected and designated pM271-9.
  • Expression vector pM271-9 was used to transform S. cerevisiae strains ZM118 (a MATa/MAT ⁇ diploid ho o- zygous for leu2-3,112 ura3 tpil: :URA3 + barl pep4: :URA3 + tcir° ] ) , ZM134 (MA a ⁇ tpi:URA3 pep4: :URA3 ieu2 ura3 sir3- _8 [cir + ] ) , and XB13-5B (MAT ⁇ ura3 leu2-3,112 barl ga!2 tpil: :URA3) .
  • TPI + colonies were selected and grown in glucose-containing rich media for two days at 30°C. Crude lysates were prepared and stored at -80°C. The lysates were thawed and cleared, and N-proCT was prepared from the cleared cell lysates essentially as described in
  • Example 2 N-proCT levels in cell lysates were assayed by NCAP radioimmunoassay. Results are shown in Table 5.
  • a second set of expression vectors was constructed by inserting the N-proCT expression unit into the vector pMP0T2, a vector containing the REP1, REP2, REP3 and ori sequences from the yeast 2-micron plasmid, an ampicillin resistance marker and the Schizosaccharo ⁇ myces pombe triose phosphate isomerase (POTl) gene.
  • pMP0T2 has been deposited with the American Type Culture Collection as an E ⁇ coli HB101 transformant under acces ⁇ sion number 67788. Plasmid p271-9 was digested with Bam HI and the N-proCT expression unit was isolated.
  • Plasmids pM274-4 and pM274-15 were transformed into S_ ; _ cerevisiae strain ZM118. ' Transformants were grown overnight at 30°C in 20 ml or 200 ml of yeast medium I (2% yeast extract, 6% glucose, 0.5% ammonium sulfate) containing 28 ⁇ g/ml leucine. Cells were sepa- rated from the medium by centrifugation, resuspended in PBS, and lysed by vortexing in the presence of glass beads. The resulting crude lysates were frozen and stored for later assay.
  • yeast medium I 2% yeast extract, 6% glucose, 0.5% ammonium sulfate
  • N-proCT activity was assayed in a 22-hour chick calvarial cell proliferation assay using native N-proCT (100 nM, from rat medullary thyroid carcinoma) and insulin (10 ⁇ g/ml) as positive controls.
  • native N-proCT 100 nM, from rat medullary thyroid carcinoma
  • insulin 10 ⁇ g/ml
  • Thawed yeast cell lysates were centrifuged at 16,800 x g for 15 minutes. The cleared lysates were then concen ⁇ trated by C-18 silica reversed-phase batch chromatography using Vydac wide-pore beads.
  • Samples are as follows: A, p274-4 clone 1, 16 nM; B, p274-4 clone 2, 3 nM; C, pMPOT2 control; D, p274-15 clone 1, 135 nM: E, p274-15, clone 2, >5 nM.
  • TPIl promoter fragment was obtained from plasmid pTPICIO (Alber and Kawasaki, J. Mol. Appl. Genet. l_:410-434, 1982) .
  • Plasmid pTPICIO was cut at the unique Kpn I site, the TPIl coding region was removed with Bal31 exonuclease, and an Eco RI linker (sequence: GGAATTCC) was added to the 3' end of the promoter. Digestion with Bgl II and Eco RI yielded a TPIl promoter fragment having Bgl II and Eco RI sticky ends.
  • Plasmid TEA32 was digested with Bgl II and Eco RI, and the " 900 bp partial TPIl promoter fragment was gel-purified.
  • Plasmid pIC19H (Marsh et al., Gene 32: 481-486, 1984) was cut with Bgl II and Eco RI and the vector fragment was gel-purified. The TPIl promoter fragment was then ligated to the linearized pIC19H and the mixture was used to transform E ⁇ coli RR1. Plasmid DNA was prepared and screened for the presence of a ⁇ 900- bp Bgl II-Eco RI fragment. A correct plasmid was selected and designated pICTPIP ( Figure 17). The complete TPIl promoter was then assembled.
  • Plasmid pIC7 (Marsh et al., ibid.) was digested with Eco RI, the fragment ends blunted with DNA polymerase I (Klenow fragment) , and the linear DNA recircularized using T4 DNA ligase. The resulting plasmid was used to transform E ⁇ _ coli RR1. Plasmid DNA was prepared from the transformants and screened for the loss of the Eco RI site. A plasmid having the correct restriction pattern was designated pIC7RI*. Plasmid pIC7RI* was digested with Hind III and Nar I, and the 2500-bp fragment was gel-purified. The partial TPIl promoter fragment (ca.
  • Plasmid pSXRIOl was constructed by ligating the 2.7-kb Sal I-Bam HI fragment of pUC9 with a 0.9-kb Xho I-Bam HI fragment of the TPIl promoter derived from plasmid pMVRl.
  • the Sph I site of the TPIl promoter in plasmid pSXRlOl was then changed to a unique Xho I site.
  • pSXRlOl DNA was cleaved with Sph I and dephosphorylated according to standard procedure (Maniatis et al., ibid.).
  • Sph I-Xho I adapter (GCTCGAGCCATG) was kinased in a reaction mixture containing 20 p oles of the adapter, 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidine, 1 mM ATP, and 5 units of polynucleotide kinase in a volume of 20 ul for 30 minutes at 37°C.
  • the kinased Sph I-Xho I adapter was ligated with Sph I-cut pSXRlOl, and the ligation mixture was used to transform E ⁇ coli RR1.
  • Plasmids with inserted adapter were identified by restriction analysis and named pSXR102 ( Figure 18) .
  • Oligonucleotides specify ⁇ ing the MAT ⁇ 2 operator (5' TCGAG TCA TGT ACT T.AC CCA ATT AGG AAA TTT ACA TGG 3 ' and 3 ' C AGT ACA TGA ATG GGT TAA TCC TTT AAA TGT ACC AGCT 5') were synthesized and kinased as described above.
  • Plasmid pSXR102 was cut with Xho I and dephosphorylated according to standard procedures. Three independent ligations were set up, with molar ratios of plasmid DNA to oligonucleotide of 1:1, 1:3 and 1:6, respectively.
  • Plasmids with inserted oligonucleotide(s) were identified by colony hybridiza ⁇ tion and restriction analysis. Subsequent DNA sequencing showed that pSXR103 contained one copy of the MAT0.2 operator, pSXR104 contained two copies, and pSXR108 contained four copies ( Figure 19) .
  • plasmids pSXR102, pSXR103, pSXR104 and pSXR108 were cut with Bam HI, dephosphor- ylated, and ligated with a 3.2-kb Bam HI-Bam HI fragment from plasmid plac7 containing the E_-_ coli lacZ gene.
  • the ligation mixtures were used to transform E ⁇ coli RR1.
  • Plasmids containing appropriate TPIl-lacZ fusions were identified by restriction analysis and named as follows: pSXR109, no MAT ⁇ 2 operator; pSXRHO, one MAT ⁇ 2 operator; pSXRlll, two copies of the MAT ⁇ 2 operator; and pSXR112, four copies of the MAT ⁇ .2 operator sequence ( Figure 20) .
  • An expression unit consisting of the temperature-regulated TPIl promote , N-proCT sequence and TPIl terminator was then constructed.
  • Plasmid pM271-9 was digested with Eco RI and Bam HI to remove the ADH2-4 C 5 promoter.
  • Plasmid pSXRlll was digested with Bgl II and Eco RI, and the SXRlll promoter fragment was isolated and joined to the linearized pM271-9.
  • the resultant plasmids were designated pM275-4 and pM275-5, depending on insert orientation ( Figure 15).
  • Q Plasmids pM275-4 and pM275-5 were transformed into S ⁇ cerevisiae strain ZM134. Transformants were grown in 20- ⁇ tI cultures of YEPD (1% yeast extract, 2% peptone, -2% glucose, 40 mg/1 adenine) at 30°C overnight to a cell density of approximately 8 x lO "7 cells/ml.
  • the 5 temperature was reduced to 23°-25°C, and the cultures were incubated for an additional two hours.
  • the cells were then pelleted, resuspended in 20 ml of fresh YEPD, and grown for 24 hours at 23°-25°C. Crude lysates were then prepared as described above and frozen at -80°C. 0
  • the wild-type ADH2 promoter was regenerated using the partial ADH2 promoter fragment from p410ES.
  • 5 Plasmid p410ES was digested with Sph I and Eco RI to isolate the 175-bp partial ADH2 promoter fragment. This fragment was joined with a 1-kb Bam Hl-Sph I fragment derived from pBR322-ADR2-BSa in a three-part ligation into pUC13 which had been linearized by digestion with 0' Ba HI and Eco RI.
  • the 1-kb fragment derived from pBR322-ADR2-BSa contained sequences that are homologous with the wild-type ADH2 promoter sequence.
  • the plasmid that resulted from the three-part ligation was confirmed by restriction analysis and designated ⁇ 410-Wt.
  • 5 Plasmid pM271-9 was digested with Bam HI and
  • Plasmids pM277-15 and pM277-16 were used to transform S ⁇ cerevisiae strain ZM118. Twenty-ml cultures in YEPD were grown at 30°C for approximately 48 hours. Crude lysates were prepared and frozen at -80°C.
  • N-proCT levels in cell lysates were assayed by NCAP radioimmunoassay using synthetic NCAP as a standard. Results of duplicate determinations are shown in Table 5.
  • a coding sequence for rat N-proCT was assembled essentially as described above, with the substitution of oligonucleotides ZC 2041 (5' AGC TTG GAC AAG AGA GTT CCC TTA AGA TCT ACC TTG GAA TCT TCT CCA GGT ATG GCT ACC TTG T 3') and ZC 2042 (5* CTT CAG ACA AGG TAG CCA TAC CTG GAG AAG ATT CCA AGG TAG ATC TTA AGG GAA CTC TCT TGT CCA 3' ) for ZC 1791 and ZC 1792, respectively.
  • the assembled coding sequence thus includes a Hind III "sticky end" at its 5' end.
  • the assembled coding sequence was joined to the TPIl terminator fragment (Xba I-Bam HI) from pKPlO.
  • the resulting Hind III-Bam HI fragment was joined to the ADH2-4 C promoter (3am HI-Eco RI from pM271-9), the ⁇ - factor pre-pro sequence (Eco RI-Hind III) and Bam Hi-cut pDPOT.
  • the resultant expression vectors were designated OM294-I (NproCT expression unit in opposite orientation 5 to the POTl marker) and ⁇ M294-4 (N-proCT expression unit in the same orientation as POTl) .
  • the vectors pM294-l and pM294-4 were trans ⁇ formed into S ⁇ cerevisiae strain XB13-5B. Duplicate 20- ml cultures were grown for 48 hours, and media samples 10 were diluted 1:1000 and assayed for N-proCT immuno ⁇ reactive material. Results (adjusted to full-strength media) are shown in Table 6.
  • the human N-proCT sequence was constructed from oligonucleotides (Table 7) that were designed to encode the peptide with yeast-optimized codons, a 5'-terminal Eco RI site and a 3'-terminal Xba I site.
  • the encoded sequence includes an initiator methionine residue that is
  • oligonucleotides A through F were synthesized, purified, kinased, mixed in equal proportions and annealed.
  • the resulting oligo ⁇ nucleotide pairs (A+F, B+E, C+D) were ligated and the mixture was digested with Eco RI and Xba I.
  • the coding sequence was isolated by electrophoresis on a native polyacrylamide slab gel and extracted from the gel.
  • Expression vectors were then constructed essentially as described in Example 4.
  • the ADH2-4 C promoter (Bam. HI-Eco RI fragment) , wild-type ADH2 promoter (3am HI-Eco RI fragment) or SXRlll promoter (Bgl II-Eco RI fragment) was joined to the N-proCT sequence together with the TPIl terminator from pKPlO.
  • the resulting expression units were then inserted into pMPOT2 or pDPOT, and the insert orientation was determined.
  • the human N-proCT expression vectors were transformed into appropriate yeast host strains.
  • Vectors containing the ADH2-4 C or wild-type ADH2 promoter were transformed into strains ZM118, XB13- 5B and ZM134.
  • Vectors containing the SXRlll promoter were transformed into ZM134.
  • Transformants were selected by growth on glucose-containing media. Cells were grown under appropriate conditions for regulated or constitu ⁇ tive expression of the peptide. The cells were then lysed, and cell-free lysates were prepared for assay.
  • Plasmid pM286-7 a pDPOT-based plasmid contain ⁇ ing the ADH2-4 C promoter with the N-proCT expression unit in the same orientation as the POTl gene, was transformed into strain ZM134. Transformed cells were cultured essen ⁇ tially as described above. The cells were harvested, lysed in 1 M acetic acid, and cent ifuged. The resulting cleared lysate was assayed for N-proCT production by radioimmunoassay using anti-NCAP antiserum. Samples of the cleared lysate were diluted in assay buffer (0.02 M NaP04, pH 7.4, 0.05% Na 3 , 0.05% NP-40, 1 M EDTA) .
  • Duplicate samples (100 ⁇ l) were added to tubes containing 200 ⁇ l assay buffer. To each tube was added 100 ⁇ l of antibody diluted 1:7 (1:3500 total dilution) in assay buffer. 100 ⁇ l of assay buffer containing 20,000 com of 125 ⁇ _ ⁇ a beled human N-proCT was then added to each tube and the tubes were incubated at room temperature for four hours. Staphylococcus a ⁇ reus cells (Pansorbin; Sigma Chemical Co.) were diluted 1:200 in assay buffer, 1 ml of the resulting suspension was added to each tube, and the tubes were mixed and incubated at room temperature for 20 minutes.
  • N-proCT levels were determined by comparison with a standard curve generated with synthetic human N-proCT diluted to 500,
  • oligonucleotides B, C, D, E, G and H were annealed as described above to give pairs G+H, B+E and C+D.
  • the pairs were ligated, the mixture was digested with Hind III and Xba I, and the fragment was gel-purified.
  • Vectors for secretory expression were then assembled.
  • the N-proCT fragment (Hind III-Xba I) and the ⁇ -factor pre-pro fragment (Eco RI-Hind III) from pKPlO were joined and inserted into expression vectors contain ⁇ ing the TPIl promoter and terminator as described above to construct plasmids pM285-13 (expression unit in same orientation as POTl ) and pM285-15 (expression unit in opposite orientation) .
  • a second set of secretory expres ⁇ sion vectors was constructed using the ADH2-4 C promoter from p410-4c and the TPIl terminator from pKPlO . These vectors were designated pM284-3 (expression unit in same orientation as POTl) and pM284-4 (expression unit in opposite orientation) .
  • Plasmid pM284-3 was transformed into S ⁇ cerevisiae strain XB13-5B. Plasmids pM285-13 and pM285- 15 were transformed into strains XB13-5B and ZMI34, Cells were cultured as previously described. Culture media were sterile filtered through 0.45-micron filters and assayed for N-proCT by radioimmunoassay using anti- NCAP antiserum as described above. Results of two sets of experiments are shown in Table 8.
  • Recombinant N-proCT was purified from yeast culture media by adding to the media 1% (by volume) acetic acid and 30% (by volume) acetonitrile. The mixture was passed over a Vydac C-4 reversed-phase column and eluted with a gradient of 30%-50% acetonitrile in H2O. The eluate was monitored for absorbance at 215 nm, and the peak fractions, containing N-proCT, were collected.
  • the animals were injected with tetracycline on day 2 and day 13 of the experiment to label the bones for histo- morphometry. The animals were sacrificed on day 14, and the bones were dissected free of soft tissue and fixed in neutral buffered formalin.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Obesity (AREA)
  • Diabetes (AREA)
  • Nutrition Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des peptides qui stimulent la prolifération d'ostéoblastes et de préostéoblastes. Des procédés permettant d'isoler les peptides ou de produire les peptides en utilisant des techniques d'ADN recombinant sont également décrits. Les peptides sont utiles dans des compositions thérapeutiques et sont particulièrement utiles pour favoriser la croissance osseuse chez les patients.The present invention relates to peptides that stimulate the proliferation of osteoblasts and preosteoblasts. Methods for isolating the peptides or producing the peptides using recombinant DNA techniques are also described. The peptides are useful in therapeutic compositions and are particularly useful for promoting bone growth in patients.

Description

Description
PROCALCITONIN PEPTIDES
Technical Field
The present invention is related to peptides that stimulate the proliferation of osteoblasts and preosteoblasts, methods of making the peptides, therapeu- tic compositions containing the peptides, and methods of promoting bone growth in mammals.
Background of the Invention
Bone growth, maintenance and repair involve a balance between rates of bone formation and resorption. These two processes (together referred to as "remodel¬ ing") are regulated by several hormones and growth factors, including parathyroid hormone, calcitonin, insulin, somatomedins, thyroid hormone, glucocorticoids, vitamin D, androgens, estrogens, epidermal growth factor, transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor. These chemical messengers influence the development and activity of osteoblasts and preosteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells) . Bone remodeling is related to the role of the skeleton as a mineral reser¬ voir and serves to maintain cell viability, fluid exchange and bone strength. Imbalances in bone metabo¬ lism may result in osteoporosis and other diseases. The bone-remodeling process is reviewed by Raisz and Kream (New Engl. J. Med. 309:29-35, 83-89, 1983) and Raisz (New Engl. J. Med. 318:818-827, 1988).
Although in healthy individuals bone growth proceeds normally and fractures heal without the need for pharmacologic intervention, in certain instances bones may be weakened or may fail to heal properly. For example, healing may proceed slowly in the elderly and in patients undergoing treatment with corticosteroids, such as transplant patients and those being treated for chronic lung disease. In addition, victims of osteopor¬ osis suffer from an imbalance in bone metabolism which often leads to fractures and deformity.
Osteoporosis is currently treated with estro¬ gen, calcitonin, calcium, sodium fluoride, bisphosphon- ates, or a combination of these. These treatments probably do not correct the underlying cause of the disease and have only limited e fectiveness. Estrogen treatment has been linked to an increased risk of endometrial cancer. Estrogen is of limited usefulness because only a fraction of women under age 55 respond; with further aging, estrogen responsiveness of bone diminishes. The use of sodium fluoride is under investi¬ gation but ha.s not yet been approved. Further, bone formed under fluoride stimulation may be abnormal. In addition, significant toxicity is associated with fluoride administration. Calcium alone is ineffective except in cases of gross dietary calcium deficiency. The benefits of calcium treatment, even in cases of defi¬ ciency, are limited in the absence of supplemental vitamin D. Treatment with calcitonin appears to reduce bone resorption, but any increase in bone mass is probably temporary. The eventual ineffectiveness of calcitonin is believed to result from diminished osteo- blast activity that occurs as a consequence of the coupling of bone formation and resorption. Calcitonin has not been shown to reduce the rate of bone fractures in osteoporosis victims. Side effects, including gastro¬ intestinal problems and nasal bleeding, may limit the use of calcitonin. Finally, while somatomedins (insulin-like growth factors) are now being evaluated for treatment of osteopenic states, complications and side effects seem likely since these growth factors exert effects on cells of soft tissue. There remains a need in the art for substances that promote bone formation and/or reverse bone loss. The present invention provides peptides having such activity and also provides other related advantages.
Disclosure of the Invention
Briefly stated, the present invention discloses isolated peptides and DNA sequences encoding peptides. that stimulate the proliferation of osteoblasts and preosteoblasts. The peptides generally have the follow¬ ing characteristics: (a) they are at least 12 amino acids in length; (b) they are substantially homologous to at least a portion of rat N-procalcitonin (N-proCT) ; and
(c) they increase DNA synthesis in osteoblasts and preosteoblasts by a factor of at least two, as compared to fibroblasts, at a concentration that is maximally stimulatory for osteoblasts and preosteoblasts. Within one aspect of the present invention, the peptide has an amino acid sequence selected from the group consisting of rat, human, chick and salmon N-proCT and rat, human and chick N-procalcitonin gene-related peptide (N-proCGRP) sequences, as shown in Figure 1. For purposes of convenience and clarity within the present invention, the peptides are commonly defined in terms of homology to rat N-proCT. However, it will be understood that the present invention includes peptides derived from other species as well as derivatives of these peptides. Within a related aspect of the present invention, the peptide comprises an amino-terminal sequence selected from the group consisting of (a) V P L R S T L E S S P G; (b) A P F R S A L E S S P A; and (c) A P V R P G L E S I T D; and
(d) A P A R T G L E S M T D. Within another aspect of the present invention, the peptide is less than 32 amino acids in length. Within a related aspect of the present inven¬ tion, a method of isolating a peptide that stimulates cell division in osteoblasts and preosteoblasts is disclosed. The method generally comprises (a) preparing an aqueous extract of cells capable of expressing a calcitonin or calcitonin-related gene; (b) fractionating the aqueous extract to enrich for peptides having a molecular weight less than approximately 15,000; and (c) fractionating the enriched fraction by hydrophobic chromatography and/or anion exchange chromatography to separate the peptide from the enriched fraction. Within a preferred embodiment, the step of fractionating the aqueous extract comprises reversed-phase HPLC and gel filtration of -the aqueous extract. It will be evident to one skilled in the art that the order of the steps may be modified without substantially affecting the desired result. Host cells transfected or transformed with an expression vector comprising a transcriptional promoter operably linked to a DNA sequence encoding a peptide as described above are also disclosed. Suitable host cells in this regard include yeast host cells. Within another aspect of the present invention, a method for producing a peptide that stimulates cell division in osteoblasts and preosteoblasts is disclosed. The method generally comprises (a) introducing into a host cell an expression vector comprising a transcrip- tional promoter operably linked to a DNA sequence encod¬ ing a peptide having the characteristics described above; (b) culturing the host cell under suitable conditions; and (c) isolating the peptide from the host cell.
Yet another aspect of the present invention is directed toward therapeutic compositions comprising a peptide as described above in combination with a physio¬ logically acceptable carrier or diluent. The therapeutic composition may be used within a method of promoting bone growth in a patient. Within such a method, the composi- tion may be administered intranasally or by injection. In one embodiment, the composition may further include an effective amount of a substance such as estrogen, sodium fluoride, calcitonin or a bisphosphonate. In another embodiment, the composition may further include a growth factor such as insulin, an insulin-like growth factor, platelet-derived growth factor, transforming growth fac- tor alpha, transforming growth factor beta or epidermal growth factor in an amount sufficient to further increase DNA synthesis in osteoblasts and preosteoblasts.
These and other aspects of the present inven¬ tion will become evident upon reference to the following detailed description and attached drawings.
Brief Description of the Drawings
Figure 1 illustrates the amino acid sequences of representative peptides that stimulate the growth of osteoblasts and preosteoblasts. Boxes indicate blocks of identical amino acids. Asterisks indicate gaps intro¬ duced to maximize ho ology. Numbers refer to the rat sequence. N-proCGRP sequences correspond to the respec¬ tive N-proCT sequences, except where indicated. No sequence has been reported for salmon calcitonin gene- related peptide (CGRP). Amino acids are designated by the standard one-letter code.
Figure 2 illustrates the results of an immuno- assay on serially diluted synthetic peptides and extracts of thyroid cells (Ext) using an antiserum directed against the last six residues of rat N-proCT (NCAP) .
Figure 3 illustrates the results of an immuno- assay on serially diluted peptides and rat tumor cell extract (sample) using an antiserum directed against the first twelve amino acid residues of rat procalcitonin (NTP) . Inverted triangles designate calcitonin, C-proCT, NCAP, CGRP and somatostatin.
Figure 4 shows a reversed-phase HPLC fractiona- tion profile of peptides from a thyroid tumor cell extract. A 300 A/octyl reversed-phase HPLC column was used; one-milliliter fractions were collected, dried and assayed for immunoreactive NCAP (closed circles) and immunoreactive NTP (open squares) . The dotted line shows the acetonitrile gradient.
Figure 5 shows the results of gel filtration of partially purified N-proCT from rat medullary thyroid carcinoma (MTC) . Arrows indicate molecular weight markers. N-proCT is the 7.4-KDa peak which possesses coincident NTP (open squares) and NCAP (closed circles) immunoreactivities.
Figure 6 illustrates the mitogenic effect of a representative procalcitonin-derived peptide on chick osteoblasts and preosteoblasts. Closed circles indicate synthetic NTP-Tyr [proCT(l-12)-Tyr] ; inverted triangles indicate synthetic calcitonin.
Figure 7 illustrates the mitogenic effect of partially purified rat N-proCT on chick (A) and rat (B) osteoblasts and preosteoblasts.
Figure 8 illustrates the results of a mitogene- sis assay on rat skin cells and chick bone cells using partially purified N-proCT and calcitonin. Figure 9 illustrates the mitogenic effect of purified, synthetic human N-proCT on cultures of chick osteoblasts and preosteoblasts.
Figure 10 illustrates the mitogenic effects of synthetic human N-proCT on human osteoblasts prepared from patients with osteoarthritis (a) and human osteosar- coma cells (b) .
Figure 11 illustrates the coding sequence for rat N-proCT as constructed from oligonucleotides contain¬ ing yeast-preferred codons. Figure 12 is a diagram of the assembly of the rat N-proCT coding sequence.
Figure 13 illustrates the construction of a yeast expression vector for N-proCT.
Figure 14 illustrates the plasmids pCPOT and pDPOT.
Figure 15 illustrates several yeast expression vectors used to produce procalcitonin-derived peptides. Figure 16 illustrates the results of a chick bone cell mitogenesis assay using recombinant N-proCT. The results are shown as mean + s.e.m. (n=4-6) for each test condition. Figure 17 illustrates the subcloning of the
S. cerevisiae TPI1 promoter.
Figure 18 illustrates the construction of plasmid pMVRl.
Figure 19 illustrates the insertion of MATα2 operator sequences into the TPI1 promoter.
Figure 20 illustrates the construction of plasmids pSXR109, pSXRHO, pSXRlll and pSXR112.
Figure 21 illustrates the results of experi¬ ments comparing bone growth in mice treated with N-proCT and untreated control mice.
Best Mode for Carrying Out the Invention
The present invention provides a variety of peptides that specifically stimulate the proliferation of osteoblasts and preosteoblasts and are therefore useful in promoting bone growth in mammals. Within a preferred aspect of the present invention, these peptides may be derived from the amino-terminal region of procalcitonin or proCGRP. They may be synthesized de novo, isolated from suitable cells or tissues that naturally produce them, or produced through the use of recombinant DNA techniques.
Calcitonin is a 32-residue peptide hormone produced by C-cells of the thyroid gland and in large amounts by certain thyroid tumors. The hormone inhibits the activity of osteoclasts and by this mechanism reduces calcium levels in the blood. Calcitonin is secreted in response to high calcium and other nutritional signals. Rats fed a high-fat diet show increased levels of calci- tonin production, suggesting a role in fat metabolism. Injection of large doses of calcitonin can inhibit appe¬ tite and may also have analgesic effects. As is the case in the biosynthesis of other small peptide hormones, calcitonin is generated from a larger prohormone (Roos et al., Biochem. Biophys. Res. Commun. jH): 1134-1140, 1974). Procalcitonin was first characterized in rats (Jacobs et al. , Science- 213 : 457- 459- 1981; A ara et al. , J. Biol. Chem. 257:2129-2132, 1S82); the rat sequence is now known to be structurally sr-imilar to the sequences of the human and chicken precursors (Gkonos et al. , J. Biol. Chem. 261: 14386- 14391, 1986; Lasmoles et al., EMBO J. 10:2603-2607, 1985). Rat procalcitonin has 111 residues (Birnbaum et al., J. Biol. Chem. 259:2870-2874, 1984); the calcitonin sequence (proCTgo-91*1 i*3 nestled within the precursor, separated from amino- and carboxyl-terminal regions by flanking polybasic cleavage sites. Calcitonin-rich cell lines developed for studies of calcitonin biosynthesis and secretion demonstrate a major pathway of procalci¬ tonin processing which yields calcitonin and the C- terminal hexadecapeptide, C-proCT (Birnbaum et al., J. Biol. Chem. 261:699-703, 1986; Birnbaum et al. , J. Biol. Chem. 257:241-244, 1982; Muszynski et al., J. Biol. Chem. 258:11678-11683, 1983).
Studies of calcitonin gene expression (Amara et al., Nature 298:240-244, 1982) have shown that the gene encodes two distinct secretory peptides. The second peptide, termed "calcitonin gene-related peptide," or
"CGRP," arises from differential RNA splicing. Mature
CGRP consists of 37 amino acids. The precursor forms of the peptides (procalcitonin and proCGRP) are identical over the first 51 amino acid residues. Like calcitonin,
CGRP is separated from its amino- and carboxyl-terminal flanking sequences by polybasic cleavage sites. CGRP can also reduce blood calcium and inhibit bone resorption, at least at high doses. Calcitonin and CGRP have separate cellular receptors, but each can bind to the other's receptor (Roos et al., Endocrinology 118:46-51, 1986).
The use of CGRP and CGRP analogs has been proposed for lowering blood pressure and gastric acid secretion (Evans et al. , U.S. Patent Nos. 4,530,838 and 4,549,986) .
There are at least three additional calcitonin- related human genes or pseudogenes for which direct or indirect evidence has emerged. Fischer et al. (J. Clin.
Endocrinol. Metab. 52:1314-1316, 1983) identified i munoreactive salmon calcitonin-like material in human thyroid and brain. Steenbergh et al. (in A. Pecile
(Ed.) , Calcitonin 1984, Elsevier, 1985, p. 23) suggest the existence of a human calcitonin pseudogene.
Steenbergh et al. (FEBS Lett. 209:97, 1986) isolated and characterized a gene for human CGRP-II which does not appear to undergo alternative expression to produce a calcitonin-like peptide. There are also now indications for a third human calcitonin-related gene (Thesis of
J.W.M. Hoppener, University of Utrecht, "The Human
Calcitonin/CGRP Genes," April 12, 1988) .
The inventors have found that processing of procalcitonin and proCGRP produces stable, N-terminal peptides (designated N-proCT and N-proCGRP, respectively) of about 52-57 amino acids in length, depending on the precursor protein. These peptides have been found to have specific cell proliferative activity for osteoblasts and preosteoblasts when tested in a standard mitogenesis assay. Furthermore, peptides derived from one species have been found to have specific cell proliferative activity for osteoblasts and preosteoblasts from another species.
As noted above, calcitonin and CGRP from a number of species, including humans, rats and chickens, as well as salmon calcitonin, have been isolated and characterized. The genes and cDNA sequences encoding these polypeptides have also been studied. These studies have shown that calcitonin and CGRP are encoded by the same gene and result from alternative RNA splicing. In addition, a calcitonin pseudogene, only known to be expressed in tumors, and a salmon calcitonin-like peptide have also been identified in humans. The peptides of the present invention may correspond to amino-terminal sequences derived from precursors for any of these calcitonin gene products. Exemplary peptides include human, chick, rat and salmon N-proCT (54 or 57 amino acids) and human, chick and rat N-proCGRP (52 or 55 amino acids), the sequences of which are shown in Figure 1. The peptides of the present invention also include fragments of the amino-terminal portions of procalcitonin and proCGRP. For example, a 12-amino-acid fragment from the amino terminus of procalcitonin has been found to have unexpected, specific mitogenic activity for osteo¬ blasts and preosteoblasts. The invention includes this 12-amino-acid fragment and larger procalcitonin-derived peptides having this amino-terminal sequence of 12 residues. Preferred peptides in this regard include 31- amino-acid peptides from the amino termini of human, chick, rat and salmon N-proCT and human, chick and rat N- proCGRP. Additional bioactive peptides may be generated by cleavage of N-proCT or N-proCGRP with proteolytic enzymes or cyanogen bromide (CNBr) . For example, CNBr cleavage of human N-proCT produces peptides of 36 and 21 amino acids. As will be appreciated by those skilled in the art, minor changes may be made in amino acid sequen- ces without altering the useful properties of these peptides. Such changes, including amino acid substitu¬ tions and deletions, may result from genetic polymorphism or species diversity, or may be introduced into the peptides by genetic engineering. When introducing such changes, it is generally preferred to maintain those sequences that show high levels of interspecies homology (as shown in Figure 1) and to avoid making major changes in the chemical nature (e.g., charge, hydrophobicity, etc.) of the peptides. As noted above, the peptides of the present invention are commonly defined in the context of rat N-proCT. In general, the peptides of the present invention will be substantially homologous to a portion of rat N-proCT, generally at least about 40% homologous, reflecting the levels of interspecies homology shown in Figure 1. Preferably the peptides will be substantially homologous to a portion of human N-proCT, which is about 60% homologous to rat N-proCT. In this regard, it is preferred that the peptides be substantially homologous to a portion of at least about 10 contiguous amino acids of the a ino-terminal-most 32 amino acids of rat N-proCT. The peptides of the present invention may be isolated from tissues or cultured cells that naturally produce them, synthesized by conventional chemical proce¬ dures, or produced through the use of recombinant DNA techniques.
Cells and tissues known to produce calcitonin, CGRP or related gene products in recoverable amounts may be used as sources of the peptides. Suitable cell types include normal and neoplastic C-cells and brain cells. A preferred source of neoplastic C-cells is the 1-2-4 medullary thyroid carcinoma, disclosed by Roos et al. (Endocrinology 105: 26-32, 1979) . The peptides are extracted from the cells or tissue using an aqueous buffer. Preferably, the tissue is minced and the peptides are extracted with a hot solution of an organic acid, using an acid concentration of about 1-2 N. The extraction temperature is preferably at least 70°C, most preferably boiling. In a particularly preferred embodi¬ ment, minced tissue is added to about 10 to 20 volumes of 1.5 N acetic acid and boiled for 20 to 30 minutes. The resulting ho ogenate is then cooled, and insoluble material and lipids are separated from the aqueous extract, preferably by centrifugation. The aqueous extract is then size-fractionated, preferably in a two- stage process, combining a batch reversed-phase chroma¬ tography procedure with a gel-filtration procedure. A preferred chromatographic medium is C-18 silica. The peptides are then eluted from the medium with a suitable organic solvent. Typically, the fractionation is performed on a 5-gram column of Vydac C-18, wide-pore, 30-micron silica that has been activated in 90% acetonitrile/0.1% trifluoroacetic acid (TFA) and equilib¬ rated in 0.1% TFA. After application of the sample, the column is washed with 25 ml of 0.1% TFA, followed by 50 ml of 20% acetonitrile/0.1% TFA. N-proCT is eluted with 45%-90% acetonitrile/0.1% TFA. The partially purified peptide is then dried and dissolved in 6 M guanidine and further purified by gel filtration. A preferred gel- filtration medium is Sephadex G-50 (Pharmacia, Piscata- way, N.J.). Fractions containing the peptides of the present invention are identified by immunoassay and/or activity assay using cultured bone cells. The gel- filtration peak is further fractionated by hydrophobic chromatography, such as reversed-phase HPLC. C-8 silica, 10-micron, reversed-phase resin (Whatman) is a particu¬ larly preferred chromatographic medium in this regard. N-proCT elutes from C-8 silica at 37% acetonitrile and can be separated from other proteins by fractionation in a 35%-40% acetonitrile/0.1% TFA gradient run over 80 minutes. Peak fractions are preferably identified by immunoassay. If desired, additional purification may be achieved through conventional methods, such as anion- exchange chromatography, HPLC and immunoaffinity chromato- graphy. Alternatively, the peptides may be purified by immunoaffinity chromatography followed by HPLC.
Peptides shorter than about 20 to 32 amino acids may be synthesized by conventional chemical tech¬ niques. Methods of peptide synthesis are disclosed by, for example, Merrifield (J. Am. Chem. Soc. 85:2149-2154, 1963) and Houghten (Proc. Natl. Acad. Sci. USA 82:5131- 5135, 1985). Peptides may also be obtained from various commercial suppliers, including Peninsula Laboratories (Belmont, Calif.), Bachem Bioscience, Inc. (Philadelphia, Pa.), Biosearch, Inc. (San Rafael, Calif.), and Applied Biosystems (Foster City, Calif. ) . The peptides of the present invention are preferably prepared using genetically engineered cells. Methods for producing recombinant proteins and peptides in bacteria, fungal cells and cultured higher eukaryotic cells are well known in the art. Briefly, a DNA sequence encoding the peptide of interest is joined to a suitable transcriptional promoter and inserted into a vector along with other elements (transcriptional terminators, poly- adenylation signals, enhancers, selectable markers, etc.) , which are selected according to the type of host cell chosen. Selection of the proper elements, construc¬ tion of expression vectors, and transformation or trans- fection of host cells are within the level of ordinary skill in the art. DNA sequences encoding the peptides of the present invention are preferably synthesized. Use of synthesized oligonucleotides allows selection of codons in accordance with host cell preference. Methods for synthesizing DNA are disclosed by, for example, Caruthers et al. (U.S. Patent No. 4,458,066) and Itakura et al. (Science 198:1056-1063, 1977), although automated synthe¬ sis is generally preferred. In general, it is convenient to prepare oligonucleotides up to about 50 to 60 bases in length. Assembly of longer sequences is facilitated by designing the oligonucleotide pairs so that when annealed, overlapping, complementary ends are produced. The overlapping fragments are then ligated to produce longer sequences.
Suitable DNA sequences may also be obtained by enzymatic digestion of cDNA or genomic clones encoding calcitonin or CGRP precursors. Such clones are disclosed by Amara et al. (Nature 298:240-244, 1982) , Amara. et ai. (J. Biol. Chem. 257:2129-2132, 1982) , and Birnbaum et al. (J. Biol. Chem. 258:5463-5466, 1983) . Preferred prokaryotic host cells for use in carrying out the present invention are strains of the bacteria Escherichia coli , although Bacillus and other genera are also useful. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold 5 Spring Harbor Laboratory, 1982). Vectors used for expressing cloned DNA sequences in bacterial hosts will generally contain a selectable marker, such as a gene for antibiotic resistance, and a promoter that functions in the host cell. Appropriate promoters include the trp DJ (Nichols and Yanofsky, Meth. Enzymol. 101:155-164, 1983), lac (Casadaban et al. , J. Bacteriol. 143:971-980, 1980), and phage λ (Queen, J. Mol. Appl. Genet. _2:l-~j_u. 1983) promoter systems- Plasmids useful for transforming bracteria include pBR322 (Bolivar et al., Gene 2_:95-113, 5:* 1977), the pUC plasmids (Messing, Meth. Enzymol. 101:20- 78, 1983; Vieira and Messing, Gene 19_:259-268, 1982), pCQV2 (Queen, ibid.) , and derivatives thereof. Plasmids may contain both viral and bacterial elements.
Eukaryotic microorganisms, such as the yeasts τχ Saccharomyces cerevisiae and Schizosaccharomyces pombe or filamentous fungi (e.g., Aspergi llus spp. , ' Neurospora spp.), may also be used as host cells within the present invention. S^ cerevisiae is a particularly preferred host. Techniques for transforming yeast are well known 5 in the literature, and have been described by, for instance, Beggs (Nature 275:104-108, 1978) and MacKay (Meth. Enzymol. 101:325-343, 1983). Asperqillus species may be transformed according to known procedures, for example, that of Yelton et al. (Proc. Natl. Acad. Sci.
30 USA 8^:1740-1747, 1984). Suitable yeast expression vectors include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1979) , YEpl3 (Broach et al., Gene 8^:121-133, 1979), pJDB249 and pJDB219 (Beggs, ibid.), and derivatives thereof. Such vectors will generally include
35 a selectable marker, such as the nutritional marker LEU2, which allows selection in a host strain carrying a leu2 mutation or, preferably, an "essential gene," as described by Kawasaki and Bell (EP 171,142) . A preferred such essential gene marker is the triose phosphate isomerase gene (POT1 gene) of Schizosaccharomyces pombe, which provides for stable plasmid maintenance in a triose phosphate isomerase-deficient host cell cultured in rich glucose medium. Expression vectors containing the POT1 selectable marker include pCPOT (ATCC 39685) , pMPOT2 (ATCC 67788) and derivatives thereof. In these vectors, it may be advantageous to insert the expression unit so that the direction of transcription is opposite to that of the POT1 gene. Preferred promoters useful in yeast expression vectors include promoters from yeast glyco- lytic genes (Hitzeπran et al., J. Biol. Chem. 255 : 12073- 12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982; Kawasaki, U.S. Patent No. 4,599,311) or alcohol dehydrogenase (ADH) genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al. (Eds.) , New York: Plenum, 1982, p. 335; Ammerer, Meth. Enzymol. 101: 192-201, 1983; Russell et al., Nature 304:652-654 , 1983) as well as derivatives and variants of these promoters. Derivatives and variants include naturally occurring mutant promoters (e.g., ADH2-4C) , engineered hybrid promoters (see, e.g., Bitter, WO 86/06077; Rosenberg et al. , EP 164,556) and other engineered derivatives. In general, such deriva¬ tives will provide enhanced promoter strength or altered regulatability as compared to the parent promoters. Particularly preferred constitutive promoters are the triose phosphate isomerase (TPI1) promoter and the ADH2- 4C promoter. Particularly preferred regulated promoters include the wild-type ADH2 promoter and temperature- regulated hybrid promoters. Temperature-regulated hybrid promoters are constructed as described in U.S. Patent Applications Serial Nos. 889,100 and 036,823 by inserting one or more, preferably two or more, copies of a yeast mating-type regulatory element into a promoter, such as the TPI1 promoter. The resulting hybrid promoter is used in a host cell that is capable of expressing mating type elements in a temperature-sensitive manner. Suitable yeast host cells in this regard include temperature- sensitive sir and ste mutants. Promoter strength is regulated by varying the growth temperature of the transformed cells, generally between 23°C and 36°C. In addition, it is preferable to include a transcriptional termination signal, such as the TPI1 terminator, within the expression vector. To facilitate purification of peptides produced in yeast transformants, a signal sequence, preferably from a yeast gene encoding a secreted protein, may be joined in the correct reading frame to the coding sequence for the peptide of interest. Suitable signal sequences include the pre-pro region of the MFαl gene (Kurjan and Herskowitz, Cell 30:933-943 , 1982; Kurjan et al. , U.S. Patent No. 4,546,082) or the BAR1 gene (MacKay et al., U.S. Patent 4,613,572). Yeast host strains are widely available, for example, from American Type Culture Collection, Rockville, Md. , or the Yeast Genetic Stock Center, Berkeley, Calif. It is preferred that the host strain carries a pep4 mutation to reduce proteolytic degradation of the peptide of interest.
Higher eukaryotic cells may also serve as suitable host cells within the present invention, with cultured mammalian cells preferred. Expression vectors for use in mammalian cells will comprise a promoter capable of directing the transcription of a cloned DNA sequence introduced into a mammalian cell. Particularly preferred promoters are the mouse metallothionein-1 (MT- 1) promoter (Palmiter et al., Science 222:809-814, 1983) or the major late promoter of adenovirus 2 (Berkner and Sharp, Nuc. Acids Res. 12:1925-1941, 1984) . Also included in such expression vectors is a polyadenylation signal, located downstream of the DNA sequence insertion site. Other sequences, such as enhancers and RNA splicing signals, may also be included. Generally the coding sequence for the peptide of incerest is joined in the correct reading frame to a mammalian secretory signal sequence so that the peptide will be secreted from the cell. Cloned DNA sequences are introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al. , Cell 14: 725, 1978; Corsaro and Pearson, Somat. Cell Genet. 7: 603, 1981; Graham and Van der Eb, Virol. 5_2:456, 1973) or electroporation (Neumann et al. , EMBO J. 1:841-845, 1982) . A selectable marker is generally introduced into the cells along with the sequence of interest in order to identify transfectants that have integrated the cloned DNA into the genome. Preferred selectable markers include genes that confer resistance to drugs, such as neomycin, hygromycin and methotrexate. Selectable markers may be introduced into the cell on a separate expression vector at the same time as the sequence of interest, or they may be introduced on the same expres- sion vector.
The copy number of the integrated DNA sequence may be increased through amplification by drug selection when using certain selectable markers, such as DHFR, which confers resistance to methotrexate. The drug concentration is increased in a stepwise manner, with selection of resistant cells at each step. By selecting for increased copy number of cloned sequences, expression levels may be substantially elevated.
Methods for expression of cloned DNA sequences in cells derived from other higher eukaryotes are disclosed by, for example, Miyajima et al. (Gene 58: 273- 282, 1987), Isa and Shima (J. Cell Sci. 8J3: 219-224, 1987), and Kretsovali et al. (Gene 5_8:167-176, 1987).
Host cells expressing the peptides are grown in a culture medium appropriate to the particular host. As will be evident to one of ordinary skill in the art, a variety of media are available and a suitable medium is chosen on the basis of host cell nutrient requirements, plasmid selection, etc. (For media recipes, see, for example, catalogs of the American Type Culture Collec¬ tion, Rockville, Md.) The recombinant peptides are purified from cell media or cleared cell lysates generally as previously described for extraction from tissue. Typically, a sample containing a peptide of interest is fractionated by a combination of high-performance liquid chromatog- raphy and gel filtration. Peak fractions are identified by immunoassay or biological activity assay. Other con¬ ventional separation techniques, including immunoaffinity chromatography and ion-exchange chromatography, may also be employed. The peptides of the present invention are useful as therapeutic agents for the promotion of bone growth in warm-blooded animals. These peptides may find use in the prevention and treatment of osteoporosis, Paget's disease, and periodontal disease and in promoting the healing of fractures, particularly in patients in whom normal healing does not occur. In general, the peptides will be mixed with a physiologically acceptable carrier or diluent, such as sterile water or sterile saline. Alternatively, the peptides may be packaged in lyophilized form and combined with the carrier or diluent prior to administration. Peptides shorter than about 32 amino acids in length may be suitable for administration intranasally. Methods for administering compositions intranasally are well known. Briefly, the peptide of interest is dissolved in a physiologically tolerable buffer and sprayed onto the nasal membrane. Longer peptides and shorter peptides not amenable to intranasal administration are administered by injection, preferably subcutaneous injection. The peptides may also be administered in suppositories. Suitable dosages will generally be in the range of about 0.2 μg to 2 mg per day per kg patient weight, preferably about 2 μg to 2 mg/kg/day, most preferably about 20 to 200 μg/kg/day, depending on the precise nature of the condition to be treated. When administering the peptides intranasally, it is preferable to utilize divided doses over the course of the day. Substantially higher dosages may result in reduced proliferative activity. The peptides may also be administered in combination with other therapeutic agents. Preferred additives in this regard include estrogen, calcitonin, and bisphosphonates, which are known to reduce bone resorption and may therefore complement the action of the peptides of the present invention. In addition, it has been found that the activity of the peptides can be enhanced by combining them with growth factors. Notably, this enhancement is observed at growth factor doses at or below those reported to have general¬ ized growth-promoting effects. Growth factors useful in this regard include insulin, insulin-like growth factors (somatomedins) , platelet-derived growth factor (PDGF) , transforming growth factors (TGFα and TGFβ) and epidermal growth factor (EGF) , with insulin-like growth factor I (IGF-I; also known as somato edin C) , with or without the IGF-I carrier protein, particularly preferred. Methods for preparing these growth factors are known in the art. See, for example, Murray et al. , U.S. Patent 4,766,073; Derynck et al., EP 200,341; Derynck et al., U.S. Patent 4,742,003; Gregory et al. , U.S. Patent 3,883,497; Eaton et al., EP 46,039; Jansen et al., Nature 306:609-611, 1983; and Bell et al. , Nature 3_1_0: 775-777 , 1984. Growth factors are combined with the peptides of the present invention in amounts that are experimentally demonstrated to potentiate the proliferative activity of the peptides with minimal effects on soft tissue. The growth factors will generally be used at or below the levels at which they have been clinically demonstrated to be effective individually. In a preferred embodiment, a peptide of the present invention is administered in combination with IGF-I at an IGF-I dose of 0.02-2 g per day per kg patient weight.
The following examples are offered by way of illustration and not by way of limitation.
Experimental
Example 1 - Identification of Rat N-proCT
A. Immunoassay Methods
N-proCT was detected in normal and neoplastic tissue by immunoassay using antisera prepared against synthetic peptides (obtained from Peninsula Laboratories) corresponding to sequences in the amino-terminal and carboxyl-terminal regions of procalcitonin (Table 1) . An NCAP antiserum was raised by immunizing rabbits with NCAP conjugated to keyhole limpet hemocyanin with l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (Birnbaum et al. , 1982, ibid.; Goodfriend et al. , Science 144:1344-1346, 1964) . Two NTP antisera were raised by immunizing rabbits with synthetic NTP-Tyr conjugated to keyhole limpet hemocyanin (Goodfriend et al., ibid.) .
TABLE 1
Peptide Sequence
52 57 NCAP (proCT52-57) Leu-Asp-Ser-Pro-Arg-Ser
[Tyr°]-NCAP (Ty ) -Leu-Asp-Ser-Pro-Arg-Ser
Bolton-Hunter-NCAP * (HPPA)-Leu-Asp-Ser-Pro-Arg-Ser
52 55 proCT45_55 Glu-Ala-Glu-Gly-Ser-Ser-Leu-Asp-Ser-Pro proCT46_59 Glu-Ala-Glu-Gly-Ser-Ser-Leu-Asp-Ser-Pro-
Arg-Ser-Lys-Arg
1
NTP-Tyr Val-Pro-Leu-Arg-Ser-Thr-Leu-Glu-Ser-Ser-Pro-
[proCTι_12] 12
Gly-(Tyr)
*HPPA, 3- (4-hydroxylphenyl) propionamide
The NCAP radioimmunoassay was developed using radioiodinated synthetic [Tyrυ]-NCAP as tracer and standard. All iodinations were by the chloramine-T method and purified by Quso-32 silica adsorption (Roos and Deftos, in Methods of Hormone Radioassay, 2d ed. , Jaffe and Behr an (Eds.), New York:Academic Press, 1978, pp. 401-418) . The assay was developed to be specific for the free carboxyl-terminal portion of the N-proCT sequence. By screening antisera with radiolabeled NCAP analogs containing a modified amino terminus (tyrosine or Bolton-Hunter adduct) , antisera directed toward the carboxyl-terminal portion of NCAP were selected. All radioim unoassays were performed in 0.020 M sodium phosphate buffer (pH 7.5), containing 1 mM disodium EDTA, 0.005% sodium merthiolate, 0.1% bovine serum albumin (Pentex fraction V) , and 0.03% Brij-35 detergent. Standards ranging from 10 to 10,000 pg of [Tyr°]-NCAP were included in each assay. NCAP antiserum (from rabbit "Odessa") at a 1:3,500 final dilution, tracer, and standard or sample were added to a final volume of 500 μl per tube and incubated overnight at 4°C. Phase separa¬ tion was achieved by adding 1 ml/tube of 0.1% (w/v) Iggsorb (The Enzyme Center). The assay's limit of detec- tion for synthetic Tyr-NCAP and NCAP was about 0.20 pmol/tube, with 50% displacement of bound tracer occur¬ ring at roughly 1.8 pmol/tube. The Bolton-Hunter deriva- tive of NCAP had a displacement curve very similar to those of Tyr-NCAP and the unmodified NCAP peptide. By contrast, virtually no tracer displacement was observed with proCT4g_55, which lacks the last 2 amino acids of 5 NCAP (and N-proCT) . The carboxyl-terminally extended NCAP-containing peptide, proCT46_59, was 1,000-fold less immunoreactivε than NCAP, indicating that a free carboxyl-terminal serine is needed for an NCAP-containing peptide to cross-react in this NCAP assay. While the
10 NCAP immunoassay should recognize N-proCT (proCTι_57) , it should not detect procalcitonin or other NCAP-containing forms possessing a carboxyl-terminal extension.
Antiserum to NTP-Ty , a segment also found at the amino terminus of the precursor to CGRP (Amara
15 et al., J. Biol. Chem. 257:2129-2132, 1982; Gkonos et al. , ibid.), was used for detection of the amino- terminal region of N-proCT. Antiserum from rabbit "Vanessa" was used at a final dilution of 1:1000. Antiserum, tracer, standard or sample, and buffer were
20 added to a final volume of 500 μl per tube and incubated overnight at 4°C The limit of detection of NTP-Tyr standard using antiserum Vanessa was typically 150 pg (0.08 pmol)/tube, with 50% displacement at 1.2 ng (0.74 pmol)/tube. Calcitonin, C-proCT, CGRP, somatostatin and
25 NCAP did not cross-react in this NTP assay. Because 10 to 20 μl/tube amounts of 6 M guanidine-HCl interferred with the Vanessa radioimmunoassay, another NTP antiserum (from rabbit "Peggy") was used to analyze gel-filtration fractions containing guanidine. The Peggy antiserum was
30 selected since it can be used with up to 40 μl/tube aliquots of 6 M guanidine-HCl. Peggy NTP antiserum was used at a final dilution of 1:20,000 in the standard 500 μl assay as described above. The limit of detection of NTP-Tyr standard was 12 pg (7 fmol)/tube, and 50%
35. displacement was at 185 pg (0.11 pmol)/tube.
Rat calcitonin radioimmunoassays were performed with calcitonin antiserum R-2, which is specific for the amidated carboxyl terminus, and with an antiserum that recognizes calcitonin's midregion (Roos et al. , 1979, ibid.; Birnbaum et al. , 1984, ibid.) . Rat CGRP was measured with an immunoassay based on human CGRP anti¬ serum RB-2035, as previously described (Gkonos et al. , ibid.; Haller-Bre et al. , Endocrinology 121 : 1272-1277, 1987) .
B. Preparation of Cell Extracts
WAG/Rij rats were maintained in an AAALAC- accredited animal facility. The standard ad libitum diet was rodent laboratory chow #5001 (TekLad Co., Harlan Sprague Dawley, Inc.) . This diet contained 4.5% fat, 23% protein, and 6% fiber, with all the normal vitamin and mineral supplements. To obtain normal thyroid tissue, six- to nine-month-old female rats were sacrificed and their thyroids excised. To induce C-cell hyperplasia (Miller et al., Program of the Seventh Annual Meeting of the American Society for Bone and Mineral Research, Abstr. 366, Washington, D.C., 1985), groups of female WAG/Rij rats were selected at 4 to 7 weeks after birth and thereafter maintained on a specific high-fat diet (TekLad custom high-fat diet #82376) . This diet con- tained 45% fat, 23% protein, and 5% fiber, with all the normal levels of vitamin and mineral supplements; the only significant difference between this high-fat diet and the normal diet (#5001) is the 10 times higher concen¬ tration of fat. At 7 to 10 months of age, groups of these rats were sacrificed and their thyroids taken for use in the assays. Tissue extracts were prepared as described below.
Calcitonin-rich series 1-2-4 medullary thyroid carcinomas were generated by serial subcapsύlar renal transplantation into homologous weanlings as previously reported (Roos et al., 1979, ibid.) . Tumors of 10 to 40 g were removed surgically and extracted as described below.
To prepare tissue extracts, 10 volumes (ml/g) of 2 N acetic acid/1% TFA was added to finely minced tissue, and the suspension was then boiled for 20 minutes. The sample was exhaustively homogenized using a Brinkmann Polytron at setting 10 and then cooled on ice. The resulting homogenatε was centrifuged at 15,000 x g for 15 minutes, and the supernatant was aspirated from the pellet. To minimize degradation and obtain reproducible chromatographic profiles, peptides were extracted from the supernatant by a batch reversed-phase procedure in which the supernatant was applied to a 2- to 5-gram column (0.5 x 2 cm) of acetonitrile-activated, Vydac 218TPB30 C-18, large-pore silica beads. The column was rinsed first with 0.1% TFA, then with 20% acetonitrile/ 0.1% TFA. The sample was then eluted in a small volume of 90% acetonitrile/0.1% TFA and dried in a Savant SpeedVac system. Monolayer cultures of the established CA-77 medullary thyroid carcinoma cell line were routinely grown in serum-free medium consisting of DMEM/nutrient mixture F-10 (Ham's) (1:1) supplemented with 1.28 g/liter aHC03, 5 ug/ml transferrin, 30 nM selenous acid, and 5 ug/ml insulin. The cultures were maintained in a humidified air-8% CO2 atmosphere. CA-77 cells from passages 150-155 were subcultured at a density of 5 x 10^ cells per 25-cm2 f ask using previously described methods (Muszynski et al., J. Biol. Chem. 258:11678-11683, 1983); thereafter, medium was changed every 48 hours. For secretion experiments, cells were cultured in DMEM alone, and the calcium concentration of the medium was adjusted with 100 mM CaCl2- Experiments began on day 10 after plating so that each flask contained roughly 5 x 10^ cells. At the end of the test and control incubations, tissue culture media were clarified by centrifugation at 16,000 x g for 10 minutes, made 0.3 mg/ml in PMSF, and stored, frozen for analysis. Cells were scraped from flasks into 1% TFA and homogenized by sonication. Cellu¬ lar debris was pelleted by centrifugation and discarded; the supernatant was made 0.3 mg/ml in PMSF and stored frozen for analysis. Culture medium and clarified cell extracts were passed over Vydac CI Q columns in the same manner as tissue extracts.
C. Identification of N-ProCT in C-Cell Extracts
The tracer competition curves with serially diluted extracts from thyroid and neoplastic C-cells were parallel to those generated with NCAP and [Tyr°]-NCAP (Figure 2) . No cross-reactivity was observed with puri- fied procalcitonin, NTP-Tyr, and a variety of other peptides, including C-cell products such as calcitonin, C-proCT, CGRP and somatostatin.
In the NTP immunoassay, tracer displacement curves with serial dilutions of C-cell extracts paral¬ leled that of the synthetic NTP-Tyr standard (Figure 3) .
D. Identification of N-ProCT in Tumor Cell Extracts
Tumor peptides were separated by reversed-phase HPLC. Dried hot-acid extracts from 1-2-4 series medul¬ lary thyroid carcinomas were resuspended in 1% TFA (200- 600 ul) and were injected onto a Whatman Protesil 300 Octyl-25 analytical column using a Waters U6K injector and a modular Spectra-Physics HPLC system (Birnbaum et al., 1984, ibid.) . Peptide separation was done with an acetonitrile gradient in 0.1% aqueous TFA. During the first 10 minutes, acetonitrile concentration was increased from 30% to 35%; thereafter, the acetonitrile concentration was increased linearly over the next 70 minutes, from 35% to 40%. A major coincident peak of immunoreactive NCAP and NTP eluted at 48 to 49 minutes (Figure 4) . These fractions lacked calcitonin im unore- activity. The immunoreactive NCAP/immunoreactive NTP form eluted earlier than procalcitonin (60 minutes) and later than calcitonin (18 minutes). The molar ratio of immunoreactive NCAP to immunoreactive NTP in the major immunoreactive peak was 1.0, as expected for N-proCT.
Gel filtration in 6 M guanidine HC1 was used to estimate the size of the immunoreactive peptides. A 0.9 x 60-cm column of Sephadex G-50 (superfine) was equil¬ ibrated in 6 M guanidine-HCl/O.01% bovine serum albumin (Birnbaum et al. , 1984, ibid.). Before chromatography, lyophilized samples were dissolved in column buffer supplemented with 5% 2-mercaptoethanol and heated to 70°C for 1 hour. Blue dextran and phenol red markers were added to the sample just before chromatography. 460-ul fractions were routinely collected. Size calibration was done with blue dextran (V0) , cytochrome C (12,800), rat CGRP dimer (7,600), human adrenocorticotropin hormone (4,500), rat CGRP (3,800), rat calcitonin (3,450), gamma- endorphin (1,860) and phenol red (Vs) . The equivalence of the major HPLC and gel- filtration peaks was confirmed by HPLC of the gel- filtration peak and gel filtration of the HPLC peak.
In 1-2-4 tumors, which have 30-fold more calcitonin than CGRP, a predominant peak containing both immunoreactive NCAP and NTP eluted with an apparent size of 7.4 KDa, the size predicted for N-proCT (Figure 5). No other significant peak of immunoreactive NCAP was observed, but minor peaks of immunoreactive NTP eluted at positions corresponding to sizes of 13 KDa and 4 KDa. The high molar ratio of immunoreactive NCAP to immuno¬ reactive NTP in the 7.4-KDa peak was due to guanidine interference with the radioi munoassays. While the procalcitonin (13 KDa) peak cross-reacted with calcitonin in the midregion calcitonin radioimmunoassay, neither the 7.4- nor 4-KDa immunoreactive NTP peak did. The 4-KDa immunoreactive NTP was probably a fragment of the larger immunoreactive NTP forms, since storage of partially purified 7.4-KDa immunoreactive NTP (in 1% TFA at -20°C) generated small amounts of the 4-KDa form. Generation of small immunoreactive NTP forms could be reduced by storage in 0.3 mg/ l PMSF and 6 M guanidine-HCl. Also, extracting tissue at 70°C with 6 M guanidine-HCl instead of cold 1% TFA reduced the levels of 4-KDa immunoreactive NTP.
CA-77 medullary thyroid carcinoma cells secreted a 7.4-KDa NCAP-containing peptide whose HPLC profile was indistinguishable from the cellular N-proCT observed in 1-2-4 tumors and thyroid. This peptide was present in basal culture medium in near equimolar ratio with calcitonin. Raising extracellular calcium, a known calcitonin secretagogue, from 0.5 mM to 1.8 or 4 M stimu- lated the secretion of immunoreactive NCAP equivalentiy with stimulation of calcitonin secretion. Treatment of these cultures with 4x10"^ M dexa ethasone for 4 days increased both the cellular levels of NCAP and calcitonin and the basal rates of NCAP and calcitonin secretion. As the untreated C-cell cultures, calcium stimulation of steroid-treated cultures increased the secretion of NCAP and calcitonin in parallel.
E. Immunoreactive Peptides in Normal Thyroids and Thyroids with C-Cell Hyperplasia
Evidence for j.rι vivo coordinate regulation of N-proCT and calcitonin was obtained by studying rats with diet-induced C-cell hyperplasia. The physiological increases in thyroidal calcitonin content induced by this chronic high-fat diet were mirrored by corresponding increases in immunoreactive NCAP and NTP content (Table 2). The immunoreactive NCAP and NTP forms in these thyroids were found to have gel-filtration and HPLC mobilities indistinguishable from those found in normal thyroid and the 1-2-4 tumor tissue. The molar ratio of immunoreactive NCAP to NTP within this HPLC peak was 1.0. The molar amounts of immunoreactive NCAP, NTP and calcitonin, ascertained with immunoassays, were nearly equivalent in normal thyroid (Table 2) . The HPLC elution position of immunoreactive NCAP and immunoreac- tive NTP from normal rat thyroid was identical to that found in the 1-2-4 tumor tissue. The apparent molar ratio of immunoreactive NCAP to immunoreactive NTP in the HPLC peak from normal thyroids was 1.0, the same as observed for the 1-2-4 tumor HPLC peak. Guanidine gel filtration of an extract of normal-rat thyroids demon¬ strated a predominant 7.4-KDa immunoreactive NCAP/NTP peak, indistinguishable by size from the immunoreactive NCAP/NTP peak observed in the 1-2-4 tumor.
TABLE 2
TISSUE CONCENTRATION OF PROCALCITONIN-DERIVED IMMUNOREACTIVITY*
Immunoreactive Immunoreactive Immunoreactive Calcitonin NCAP NTP
1-2-4 Tumor 4.50 + 0.27 4.75 + 0.21 3.25 + 0-45
Normal
Thyroid 0.98 + 0.29 0.96 + 0.32 0.88 + 0.29
High-fat-fed Rat Thyroid 4.63 + 0.39 4.25 + 0.53 4.25 + 0.57
Liver and
Muscle < 0.01 ' 0.01 <" 0.01
*The values (-H SEM) are expressed in pmol-equivalents per mg of tissue and are the means of four experimental determinations. The specificity of NCAP antiserum indicates that the carboxyi terminus of the 7.4-KDa peptide is the serine immediately preceding the first dibasic cleavage site in procalcitonin (serine-57) . NTP-immunoreactivity and partial microsequencing of radiolabeled peptides from CA-77 cells indicate an amino terminus identical to pro¬ calcitonin' s. Therefore, the biochemical properties of the immunoreactive NTP- and immunoreactive NCAP-contain¬ ing peptide identify it as a 57-residue N-proCT species.
Example 2 - Isolation of N-ProCT
Rat medullary thyroid carcinoma tissue is weighed, minced, and added to 10 volumes (per gram wet weight) of 1.5 N acetic acid. This mixture is heated to boiling and boiled for an additional 20 minutes. The hot mixture is then homogenized in a Brink ann polytron (setting 10) . The homogenate is then rapidly cooled with ice water and maintained at roughly 4°C for 20 minutes, then centrifuged at 20,000 X g for 20 minutes. The aqueous supernatant is carefully aspirated so as not to disturb the pellet or floating lipid layer.
The supernatant is applied to a 5-gram column of Vydac C-18, wide-pore, 30-micron silica equilibrated in 0.1% TFA that has been previously activated in 90% acetonitrile/0.1% TFA. Next, the column is washed with 25 ml of 0.1% TFA, followed by 50 ml of 20% acetonitrile/ 0.1% TFA. N-proCT is then eluted with 45% acetonitrile/ 0.1% TFA and dried in an unheated Savant SpeedVac. The dried N-proCT is dissolved in 6 M guanidine and applied to a column of Sephadex G-50 equilibrated in 6 M guanidine-HCl (pH 5.5) . The 7.4-kna NTP- and NCAP- containing material (Figure 5) is pooled. The peptide is then recovered from the pooled guanidine fractions by adsorption to and elution from a 1-gram column of Vydac C-18 and dried in the SpeedVac as described above. The dried peak of N-proCT from the gel filtra¬ tion is resuspended in 300 ul of 0.1% TFA and injected onto a 0.6 X 25-cm column of Whatman C-8 silica, 10- micron (300 A pore size), reversed-phase resin. N-proCT peptide elutes from the column at 37% acetonitrile and can be separated from other proteins by fractionation in a 35%-40% acetonitrile/0.1% TFA gradient run over 80 minutes (Figure 4) . A peak of constant specific immuno- reactivity is selected for amino acid analysis and sequencing.
Additional purification is achieved using ion- exchange chromatography, further HPLC purifications using different ion-pairing agents and HPLC matrices, and/or immunoadsorbent chromatography with a suitable high-titer antiserum.
Example 3 - Biological Activity of Amino-Terminal Procalcitonin Peptides
A. Rat N-ProCT
The NTP-Tyr peptide (representing the initial dodecapeptide fragment of the 57-residue rat N-proCT) used for generation of RIAs (Example 1) was used to assess possible bone resorption and/or bone-cell- proliferative activity. Calvaria were taken from 16-day embryonic chicks and neonatal rats and treated with collagenase to prepare bone cells. The cells were suspended in serum-free BGJ^ medium (Fitton-Jackson Modification from Gibco, Grand Island, N.Y.) and plated over 24 hours. The plates were rinsed to remove fibro- blasts and other nonosteoblastic cel ls. Synthetic NTP- Tyr was added at concentrations between 0.001 and 100 uM for 22 hours, at which time the cells were labeled for 4 hours with tritiated thymidine. DNA synthesis was measured as TCA-precipitable tritiated thymidine and used as an index of cell proliferation. DNA synthesis was expressed relative to that of untreated cultures. With the synthetic N-proCT fragment, a strong mitogenic response was seen at 10 uM concentration, and significant effects were observable at 1 uM. Neither calcitonin nor CGRP had any mitogenic effect (Figure 6) . Important to the interpretation of these experiments is the fact that the bone cells that are plated by this particular culture method are almost solely bone-forming cells (osteoblasts) and their precursors (preosteoblasts) . N-proCT was partially purified from 1-2-4 tumor tissue by enriching roughly 100-fold over the starting tissue homogenate through the use of the boiling acid extraction method coupled with reversed-phase C-18 silica purification. This N-proCT was resuspended in BGJD medium, neutralized, and added to rat or chick calvarial osteoblast cultures essentially as described above. Thymidine incorporation was increased in both the chick (Figure 7A) and rat (Figure 7B) bone-cell cultures. In both cases, the partially purified N-proCT preparation evokes a two- to threefold mitogenic response with a maximal response observed at 10"^ to lO-^ M. This sensitivity for intact N-proCT appears to be 100- to 1000-fold greater than that for the synthetic peptide fragment (NTP-Tyr). The rat osteoblasts and preosteo- blasts appear to be somewhat more sensitive to the rat peptide than are the chick cells (Figure 7); doses as low as lO--^ M had a stimulatory effect in the rat osteoblast cultures. Neither calcitonin nor CGRP, which are active in hypocalcemia assays and bone-resorption assays, had effects in parallel chick osteoblast mitogenic assays at concentrations of 10"^ to 10~6 M.
As control cultures for Iho bone cells, skin cell cultures were prepared from rat pups and plated over 24 hours in the absence of serum. When N-proCT was added to rat skin cells, there was only a slight mitogenic effect, even at the highest concentration (Figure 8) , despite a large effect of the same preparation on osteo- blasts and preosteoblasts. The mitogenic effect of N- proCT on osteoblasts and preosteoblasts was consistently two to three times that observed for fibroblasts at the concentration that was found to be maximally stimulatory for osteoblasts and preosteoblasts. Thus, the mitogenic effect of N-proCT would appear to be cell-specific
(osteoblasts and preosteoblasts but not fibroblasts) and peptide-specific (native peptides and synthetic NTP but not calcitonin or CGRP) . The potency of this mitogenic effect is similar to that of insulin and somatomedin.
B. Human N-proCT
Human N-proCT was synthesized by Applied Biosystems (Foster City, Calif.) and supplied as an impure preparation.
The human peptide was purified by high- performance liquid chromatography on a Vydac C-4 reversed-phase column followed by chromatography of the o major HPLC peak on a Lichroprep RP-18 (C-18 reversed- phase silica) column. The Lichroprep column (0.5 g) was activated with 10 ml of 90% CH3CN containing 0.1% trifluoroacetic acid (TFA) . The column was then washed with 20 ml of 0.1% TFA, and the N-proCT sample (0.5 ml) 5 was applied to the column. The column was washed with 10 ml of 0.1% TFA followed by 10 ml of 20% CH3CN containing 0.1% TFA. The N-proCT was eluted with 40% CH3CN/0.1% TFA. Greater than 90% of the N-proCT eluted at 40% CH3CN.
The sequence of the synthetic peptide was 0 analyzed and found to be identical to that of native human N-proCT (Figure 1) .
The mitogenic activity of the synthetic human N-proCT on chick osteoblasts and preosteoblasts was assayed as described above. Results of the assays are 5 shown in Figure 9 as the mean ±SEM (n=6) for each experi¬ mental group and are expressed relative to the mean of the control group. "Control" cultures received only BGJj-**, medium; "insulin" cultures contained 10 ug/ml insulin (approximately 1.5 uM) for the positive control.
Synthetic human N-proCT was also tested for its effects on human osteoblasts and osteosarcoma cells. Human osteoblast-enriched cultures were prepared from surgical femur fragments of one older man and two older women. The bone fragments were digested in collagenase. Cells were collected from the digests, rinsed in PBS, and plated into tissue culture flasks in calcium-free MEM containing 15% fetal calf serum. The resulting osteo¬ blast-enriched cultures were trypsinized from the dishes, rinsed in PBS, resuspended in BGJβ medium containing 5% fetal calf serum, and plated into 48-well microtiter plates (Costar, Inc.) at a density of 50,000 cells per well. After 16 hours, the medium was removed, the cultures were rinsed twice with PBS, and serum-free BGJj-, medium was added. Cultures were grown for six hours, after which the medium was removed and 200 μl of BGJj-, medium containing the test agent was added to each well. After 16-18 hours, 50 μl of BGJb medium containing 1 or 2 μCi of ^H-thymidine was added to each we] 1. After four hours, the medium was removed, and the wells were rinsed twice with PBS and allowed to dry. Each well was then scraped with a cotton swab moistened with 12.5% TCA. The swabs were washed twice for 10 minutes each in 12.5% TCA and once for 10 minutes in 95% ethanol, dried, placed in 4 ml of Ecolume (ICN, Irvine, Calif.) , and counted in a scintillation counter. Results showed that 10 nM human N-proCT produced a maximal stimulation (approximately a doubling) of thymidine incorporation into DNA, with a half-maximal effect at about 1 nM (Figure I0a) . The maxi¬ mally effective dose of insulin ( 10 μg J or 2 μM) also doubled thymidine incorporation rates in these cultures. Similar experiments were performed on human U-2 OS osteo- sarcoma cells (ATCC HTB 96) . Cells were maintained in BGJ5 medium, harvested by trypsinization from confluent flasks, washed twice in PBS, plated into 48-well micro- 1
titer plates in BGJt, medium containing 1% fetal calf serum, and cultured overnight. The cells were trans¬ ferred to serum-free BGJ5 medium, and after 6 hours ^H- thymidine incorporation was measured as described above. Human N-proCT stimulated thymidine incorporation into DNA with a maximal (approximately twofold) effect at >10 nM and a half-maximal effect at 5 nM (Figure 10b) . Compar¬ able maximal effects were observed with 30 ng/ml IGF-I (purified from human serum) or 10 μg/ml insulin. Human N-proCT also had a stimulatory effect on rat osteoblastic cells, but not on rat skin cells.
Human N-proCT was tested in combination with insulin on human U-2 OS cells. The cells were plated in 48-well microtiter plates as previously described, then Incubated for 48 hours in the presence of N-proCT, insulin, or N-proCT plus insulin. At the end of this time, trypsin and EDTA were added to final concentrations of 25 mg/ml and 1 mM, respectively, and the cultures were incubated at 37°C until the cells were visibly detached. Fetal calf serum was then added to a final concentration of 15% to inactivate the trypsin. Clumped cells were dispersed by repeated pipetting, and aliquots were taken for direct counting in a hemocytometer . Results, shown in Table 3, indicate a synergistic effect between N-proCT and insulin.
TABLE 3
Fold Increase Treatment Cells per Well Relative to Control
BGJb (control) 4,838 (± 796) 1 (± 0-12)
1 μM N-proCT 13,201 (± 1,462) 2.72 (± 0.30)
10 μg/ml insulin 12,900 (± 796) 2.62 (± 0.16) 5 μg/ml insulin 8,235 (± 667) 1.70 (± 0.14) 5 μg/ml insulin
+ 1 μM N-proCT 15,400 (± 1,054) 3.20 (± 0.22) C. Human N-ProCT Peptides
Synthetic human N-proCT was digested with lysyl 5 endopeptidase (Wako Chemicals USA, Inc., Dallas, Tex.). 345 μg of lyophilized N-proCT was dissolved in 110 μl of 8 M urea. Ten μl of 500 mM Tris pH 9 was added, and the mixture was incubated for 30 minutes at 37°C. 150 μl of 50 M Tris pH 9 was added to the mixture. Lysyl endopep- 0 tidase (3 mg/ml stock solution) was added to give a 1:100 enzyme:substrate ratio. The mixture was incubated at 37°C overnight, then stored frozen. The resultant peptides, N-proCT (1-37) and N-proCT (38-57) were recovered by chromatography on a Vydac C-4 column using a
■jr gradient of 0%-70% acetonitrile in 0.1% TFA.
Human N-proCT (1-37) and N-proCT (38-57) were tested for binding to U-2 OS cells in a competition binding assay using iodinated human N-proCT as tracer. Binding assays were performed essentially as described in
20 Example 7. 5 μM of N-proCT (1-37) was found to be at least- as effective as the same concentration of intact human N-proCT in competing for binding. Fragment (38-57) showed little, if any, ability to compete for binding.
Synthetic NCAP (Table 1) was assayed for
2 mitogenic activity on chick and rat osteoblastic cells as described above. The peptide exhibited no mitogenic activity at levels ranging from 0.1 μM to 0.1 M. Also, NCAP did not compete with intact human N-proCT binding to osteosarcoma cells.
30
Example 4 - Expression of Rat N-ProCT in Yeast
A. Synthesis of N-ProCT Coding Sequence
5 The coding sequence for the 57-amino-acid rat
N-proCT was constructed from the 6 synthesized oligonucle¬ otides shown in Table 4. The oligonucleotides were JO
designed to provide: Eco RI and Xba I restriction sites at the 5' and 3' termini, respectively, to facilitate expression vector construction; a yeas -optimized translation initiation site; an ATG initiation codon; codons selected on the basis of yeast codon preference, elimination of redundant restriction sites and introduc¬ tion of restriction sites to facilitate future sequence manipulations; and a TAA termination codon. The oligo¬ nucleotides were synthesized on an Applied Biosystems model 380A DNA synthesizer and purified by electrophor- esis on denaturing gels. The oligonucleotides were kinased, mixed in equimolar proportions and annealed. The annealed pairs were then ligated, and the resulting mixture was digested with Eco RI and Xba I. The 190-bp coding sequence (Figure 11) was then isolated by electro- phoresis on a native polyacryla ide slab gel and extracted from the gel. Assembly of the coding sequence is illustrated in Figure 12.
TABLE 4
ZC 1794
27 CTA GAT TTA AGA TCT TGG AGA GTC CAA ACT CGA GCC TTC AGC TTC
54 TTG TTC TTC TTC TTG TT
ZC 1793
27 CCA ATT CTC TAA CCT TCA TTT GCA TGT ΛGT TTT GAA CTA GTG CAG
54 CCA ACA ATC TAG CTT CTT ZC 1792
27
CTT CAG ACA AGG TAG CCA T.AC CTG GAG AAG ATT CCA AGG TAG ATC
54 TTA AGG GAA CCA TTT TTT AG
ZC 1791
27
AAT TCT AAA AAA TGG TTC CCT TAA GAT CTA CCT TGG AAT CTT CTC
54 CAG GTA TGG CTA CCT TGT
ZC 1790
27
CTG AAG AAG AAG CTA GAT TGT TGG CTG CAC TAG TTC AAA ACT ACA
54 TGC AAA TGA AGG TTA GAG
ZC 1789
27
AAT TGG AAC AAG AAG AAG AAC AAG AAG CTG AAG GCT CGA GTT TGG
54 ACT CTC CAA GAT CTT AAA T
B. Expression Using the AD1I2-4C Promoter
For expression in yeast, the N-proCT fragment was linked to the ADH2-4C promoter and TPI1 terminator. The resulting expression unit was then inserted into a vector containing the POT1 selectable marker. An ADH2-4C promoter was constructed by joining the downstream portion of the wild-type ADH2 (alcohol dehydrogenase II) promoter to the upstream portion of the ADH2-4C promoter described by Russell et al. (Nature 304:652-654, 1983) , The upstream sequences of the ADH2- 4C promoter are responsible for its enhanced function. Construction of this promoter is illustrated in Figure 12. The 2.2-kb Bam HI fragment containing the wild-type ADH2 structural gene and the 5 ' flanking sequences from pBR322-ADR2-BSa (Williamson et al., Cell 23:605-614, 1981) was ligated with M13mpl9 , which had been linearized with Bam HI. The orientation of the insert was deter¬ mined by restriction analysis. Oligonucleotide ZC237 (5* GCC AGT GAA TTC CAT TGT GTA TTA 3 ' ) was synthesized on an Applied Biosystems model 380A DNA synthesizer and puri¬ fied by polyacrylamide gel electrophoresis. To isolate the promoter, site-specific _in vitro mutagenesis (Zoller et al., DNA 3^:479-488, 1984) was done on the ADH2 insert in M13mpl9 using ZC237 as the mutagenic primer and ZC87 (51 TCC CAG TCA CGA CGT 31) as the second primer. In positive clones, the oligonucleotide ZC237 looped out the structural portion of the ADH2 gene, fusing the 5' flank¬ ing sequence, including the translation start signal, with the Eco RI site of the Ml3mpl9 polylinker. The replicative form of the mutagenized phage was made and cut with Bam HI and Eco RI to isolate the 1.2-kb promoter fragment. This fragment was ligated into pUC13 which had been linearized with Bam HI and Eco RI to generate plas- id p237-Wt. To change the p237-Wt promoter to the "promoter-up" mutant ADH2-4C promoter, a 1.1-kb Bam HI- Sph I fragment from YRp7-ADR3-4c (Russell et al. , ibid.) containing the alterations found to influence promoter function was subcloned into the vector fragment of p237- Wt which had been cut with Bam HI and Sph I. The result- ing plasmid was designated p237-4c (Figure 13).
The cloned ADH2-4C promoter was then modified by the addition of terminal restriction endonuclease cleavage sites. For convenience, this was done by fusing the promoter to the codon for the first amino acid of the mature form of human alpha-1-antitrypsin (AAT) in the plasmid pAT-1. Plasmid pAT-1 comprises the expression unit of the ADH2 promoter from p237-Wt and an α-l-anti- trypsin cDNA-TPIl terminator sequence. These sequences were inserted into a portion of the vector pCPOT (Figure 14). (Plasmid pCPOT has been deposited with ATCC as an E. coli strain HB101 transformant and has been assigned accession number 39685. It comprises the entire 2-micron plasmid DNA, the leu2-d gene, pBR322 sequences and the Schizosaccharomyces pombe POT1 gene. ) Plasmid pCPOT was cut with Bam HI and Sal I to isolate the approximately 10-kb linear vector fragment. The 1.2-kb ADH2 promoter fragment was isolated from p237-Wt as a Bam HI-Eco RI fragment and ligated with the 1.5-kb α-1-antitrypsin cDNA-TPIl terminator fragment (Eco Rl-Xho I) and the linearized pCPOT in a three-part ligation to yield a plasmid designated pAT-1. Plasmid pAT-1 contained three extra amino acid codons between the ADH2 translation start codon and the first amino acid codon for the mature form of AAT. These three codons were removed by site-specific iri vitro mutagenesis. Plasmid pAT-1 was cut with Sph I and Bam HI to isolate the 190 bp ADH2 promoter fragment. This fragment was ligated into M13mpl8 which had been linear¬ ized with Bam HI and Sph I. The resulting construction was subjected to in vitro mutagenesis using ZC411 (5 'TAATACACAATAGGAGGA TCCC3 ' ) as the mutagenic primer and ZC87 as the second primer to fuse the ADH2 translation start signal to the first codon of mature α-l-antitrypsin . Positive clones were confirmed by dideoxy sequencing from -170 bp from the ATG through the fusion point. For ease of manipulation, the 175-bp Sph I-Eco RI mutagenized promoter fragment was ligated into pUC19 which had been linearized with Sph I and Eco RI. The resultant plasmid, comprising the 3 '-most 170 bp of the ADH2 promoter and the ADH2 translation start codon fused to the first amino acid codon of the mature form of AAT in vector pUC19, was designated p411.
To generate the complete ADH2-4C promoter fused to the codon for the first amino acid of mature AAT, the 5'-most sequence of the ADH2-4C promoter, containing the alterations found by Russell et al. (ibid.) to influence promoter function, was added to the promoter fragment present in plasmid p411. Plasmid p411 was digested with Sph I and Eco RI to isolate the 175-bp promoter fragment. Plasmid p237-4c was cut with Eco RI and Sph I to isolate the 3.71-kb fragment comprising pUC vector sequences and the 5 '-most promoter sequence that confers the "promoter- up" phenotype. The 175-bp promoter fragment from p411 was ligated Into the p237-4c vector fragment. The result¬ ing plasmid, containing the complete ADH2-4C promoter fused to the first amino acid codon of the mature AAT sequence, was designated p237-4cM.
The ADH2 promoter from plasmid pAT-1 was modified to create a "universal" promoter by removing the ADH2 translation start site and the pUC18 polylinker sequences found in pAT-1. Plasmid pAT-1 was cut with Sph I and Bam HI to isolate the 190-bp partial ADH2 promoter fragment. This fragment was ligated into M13mpl8 which had been linearized with Bam HI and Sph I. The resulting construction was subjected to in vitro mutagenesis using ZC410 (5'CGTAATACAGAATTCCCGGG3 ' ) as the mutagenic primer and ZC87 as the second primer to replace the ADH2 translation start signal and pUClδ polylinker sequences with a single Eco RI site fused to the M13mpl8 polylinker at the Sma I site. Positive clones were confirmed by dideoxy sequencing through the fusion point. For ease of manipulation, the mutagenized partial ADH2 promoter fragment was subcloned as a 175-bp Sph I-Eco RI fragment into pUC19 which had been linearized with Sph I and Eco RI. The resulting plasmid, designated p410ES, contained the 3 '-most 175 bp of the ADH2 promoter. The ADH2-4C promoter was then modified to contain this 3' sequence by combining the p410ES promoter fragment (Sph I-Eco RI) with the 1.1-kb Bam Hl-Sph I ADH2-4C pro¬ moter fragment from p237-4c. The two promoter fragments were joined with Bam HI, Eco Rl-cut pUC13 in a three-part ligation. The resultant plasmid, confirmed by restric¬ tion analysis, contained the complete ADH2-4C promoter mutagenized at the 3' end to place an Eco RI site in place of the translation start codon. This plasmid was designated p410-4c (Figure 13) .
The Bam HI-Eco RI ADH2-4C promoter fragment from p410-4c and the Eco Rl-Xba I N-proCT fragment were ligated to Bam HI, Xba I-digested M13mpl8. M13 clones containing the insert were sequenced, and replicative form (RF) DNA was prepared from a clone containing the full insert. The phage vector was digested with Bam HI and Xba I and the promote —N-proCT fragment was isolated. This fragment was then joined to an Xba I-Bam HI TPIl terminator fragment from pKPIO (a plasmid comprising the TPIl promoter-alpha factor-VSB-TPII terminator expression unit from pSBl [Murray et al., U.S. Patent 4,766,073] inserted into a pBR322 vector lacking an EcoRI site) , and Bam Hi-digested pUC19. A clone containing the correct insert was then digested with Bam HI and the expression unit was isolated.
The final expression vector was then con¬ structed. Plasmid pCPOT was cleaved with Sph I and Bam HI to remove 750 bp of 2 micron and pBR322 sequences. The linearized vector was then joined to a 186-bp Sph I- Bam HI fragment derived from the pBR322 tetracycline resistance gene. The resulting plasmid, designated pDPOT (Figure 12), was then digested with Bam HI and ligated to the Bam HI expression unit fragment, and the insert orien¬ tation of the resultant clones was determined. A plasmid containing the ADH2-4C promoter adjacent to the POTl gene was selected and designated pM271-9. Expression vector pM271-9 was used to transform S. cerevisiae strains ZM118 (a MATa/MATα diploid ho o- zygous for leu2-3,112 ura3 tpil: :URA3+ barl pep4: :URA3+ tcir° ] ) , ZM134 (MA a Δtpi:URA3 pep4: :URA3 ieu2 ura3 sir3- _8 [cir+] ) , and XB13-5B (MATα ura3 leu2-3,112 barl ga!2 tpil: :URA3) . TPI+ colonies were selected and grown in glucose-containing rich media for two days at 30°C. Crude lysates were prepared and stored at -80°C. The lysates were thawed and cleared, and N-proCT was prepared from the cleared cell lysates essentially as described in
Example 2. N-proCT levels in cell lysates were assayed by NCAP radioimmunoassay. Results are shown in Table 5.
A second set of expression vectors was constructed by inserting the N-proCT expression unit into the vector pMP0T2, a vector containing the REP1, REP2, REP3 and ori sequences from the yeast 2-micron plasmid, an ampicillin resistance marker and the Schizosaccharo¬ myces pombe triose phosphate isomerase (POTl) gene. pMP0T2 has been deposited with the American Type Culture Collection as an E^ coli HB101 transformant under acces¬ sion number 67788. Plasmid p271-9 was digested with Bam HI and the N-proCT expression unit was isolated. This fragment was joined to Bam Hl-digested pMPOT2 to construct the vectors pM274-4 and pM274-15 (Figure 20). Plasmids pM274-4 and pM274-15 were transformed into S_;_ cerevisiae strain ZM118.' Transformants were grown overnight at 30°C in 20 ml or 200 ml of yeast medium I (2% yeast extract, 6% glucose, 0.5% ammonium sulfate) containing 28 μg/ml leucine. Cells were sepa- rated from the medium by centrifugation, resuspended in PBS, and lysed by vortexing in the presence of glass beads. The resulting crude lysates were frozen and stored for later assay.
Recombinant N-proCT activity was assayed in a 22-hour chick calvarial cell proliferation assay using native N-proCT (100 nM, from rat medullary thyroid carcinoma) and insulin (10 μg/ml) as positive controls. Thawed yeast cell lysates were centrifuged at 16,800 x g for 15 minutes. The cleared lysates were then concen¬ trated by C-18 silica reversed-phase batch chromatography using Vydac wide-pore beads. The fractions that eluted from the Vydac beads with 30%-50% acetonitrile/0.1% trifluoroacetic acid were dried in a SpeedVac and resuspended in 1 ml of BGJfc, medium. N-proCT concentra¬ tion in each sample was determined by NCAP RIA. The peptide preparations were applied to the cell cultures. After 18 hours, ^H-thymidine was added to each culture, and after an additional four hours TCA-precipitable tritium relative to untreated (control) cultures was determined for each test condition. Results are shown in Figure 15. Samples (clones and N-proCT concentrations) are as follows: A, p274-4 clone 1, 16 nM; B, p274-4 clone 2, 3 nM; C, pMPOT2 control; D, p274-15 clone 1, 135 nM: E, p274-15, clone 2, >5 nM.
C. Expression Using a Temperature-Regulated Hybrid Promoter
A partial TPIl promoter fragment was obtained from plasmid pTPICIO (Alber and Kawasaki, J. Mol. Appl. Genet. l_:410-434, 1982) . Plasmid pTPICIO was cut at the unique Kpn I site, the TPIl coding region was removed with Bal31 exonuclease, and an Eco RI linker (sequence: GGAATTCC) was added to the 3' end of the promoter. Digestion with Bgl II and Eco RI yielded a TPIl promoter fragment having Bgl II and Eco RI sticky ends. This fragment was then joined to plasmid YRp7' (Stinchcomb et al., Nature 282:39-43, 1979) which had been cut with Bgl II and Eco RI (partial) . The resulting plasmid, TE32, was cleaved with Eco RI (partial) and Bam HI to remove a portion of the tetracycline resistance gene. The linearized plasmid was then recircularized by the addition of an Eco RI-Bam HI linker to produce plasmid TEA32. Plasmid TEA32 was digested with Bgl II and Eco RI, and the "900 bp partial TPIl promoter fragment was gel-purified. Plasmid pIC19H (Marsh et al., Gene 32: 481-486, 1984) was cut with Bgl II and Eco RI and the vector fragment was gel-purified. The TPIl promoter fragment was then ligated to the linearized pIC19H and the mixture was used to transform E^ coli RR1. Plasmid DNA was prepared and screened for the presence of a ~900- bp Bgl II-Eco RI fragment. A correct plasmid was selected and designated pICTPIP (Figure 17). The complete TPIl promoter was then assembled.
Plasmid pIC7 (Marsh et al., ibid.) was digested with Eco RI, the fragment ends blunted with DNA polymerase I (Klenow fragment) , and the linear DNA recircularized using T4 DNA ligase. The resulting plasmid was used to transform E^_ coli RR1. Plasmid DNA was prepared from the transformants and screened for the loss of the Eco RI site. A plasmid having the correct restriction pattern was designated pIC7RI*. Plasmid pIC7RI* was digested with Hind III and Nar I, and the 2500-bp fragment was gel-purified. The partial TPIl promoter fragment (ca. 900 bp) was removed from pICTPIP using Nar I and Sph I and was gel-purified. The remainder of the TPIl promoter was obtained from plasmid pFATPOT (Kawasaki and Bell, EP 171,142). S^ cerevisiae strain E18 transformed with pFATPOT (designated ZYM-3) has been deposited with American Type Culture Collection under Accession No. 20699. pFATPOT was digested with Sph I and Hind III, and a 1750-bp fragment, which included a portion of the TPIl promoter, was gel-purified. The pIC7RI* fragment, the partial TPIl promoter fragment from pICTPIP, and the fragment from pFATPOT were then combined in a triple ligation to produce pMVRl (Figure 18).
The MATα2 operator sequence was then inserted into the TPIl promoter. Plasmid pSXRIOl was constructed by ligating the 2.7-kb Sal I-Bam HI fragment of pUC9 with a 0.9-kb Xho I-Bam HI fragment of the TPIl promoter derived from plasmid pMVRl. The Sph I site of the TPIl promoter in plasmid pSXRlOl was then changed to a unique Xho I site. pSXRlOl DNA was cleaved with Sph I and dephosphorylated according to standard procedure (Maniatis et al., ibid.). An Sph I-Xho I adapter (GCTCGAGCCATG) was kinased in a reaction mixture containing 20 p oles of the adapter, 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidine, 1 mM ATP, and 5 units of polynucleotide kinase in a volume of 20 ul for 30 minutes at 37°C. The kinased Sph I-Xho I adapter was ligated with Sph I-cut pSXRlOl, and the ligation mixture was used to transform E^ coli RR1. Plasmids with inserted adapter were identified by restriction analysis and named pSXR102 (Figure 18) . Oligonucleotides specify¬ ing the MATα2 operator (5' TCGAG TCA TGT ACT T.AC CCA ATT AGG AAA TTT ACA TGG 3 ' and 3 ' C AGT ACA TGA ATG GGT TAA TCC TTT AAA TGT ACC AGCT 5') were synthesized and kinased as described above. Plasmid pSXR102 was cut with Xho I and dephosphorylated according to standard procedures. Three independent ligations were set up, with molar ratios of plasmid DNA to oligonucleotide of 1:1, 1:3 and 1:6, respectively. The resultant ligation mixtures were used to transform E^ coli RR1. Plasmids with inserted oligonucleotide(s) were identified by colony hybridiza¬ tion and restriction analysis. Subsequent DNA sequencing showed that pSXR103 contained one copy of the MAT0.2 operator, pSXR104 contained two copies, and pSXR108 contained four copies (Figure 19) .
In the next step, plasmids pSXR102, pSXR103, pSXR104 and pSXR108 were cut with Bam HI, dephosphor- ylated, and ligated with a 3.2-kb Bam HI-Bam HI fragment from plasmid plac7 containing the E_-_ coli lacZ gene. The ligation mixtures were used to transform E^ coli RR1. Plasmids containing appropriate TPIl-lacZ fusions were identified by restriction analysis and named as follows: pSXR109, no MATα2 operator; pSXRHO, one MATα2 operator; pSXRlll, two copies of the MATα2 operator; and pSXR112, four copies of the MATα.2 operator sequence (Figure 20) . An expression unit consisting of the temperature-regulated TPIl promote , N-proCT sequence and TPIl terminator was then constructed. Plasmid pM271-9 was digested with Eco RI and Bam HI to remove the ADH2-4C 5 promoter. Plasmid pSXRlll was digested with Bgl II and Eco RI, and the SXRlll promoter fragment was isolated and joined to the linearized pM271-9. The resultant plasmids were designated pM275-4 and pM275-5, depending on insert orientation (Figure 15). Q Plasmids pM275-4 and pM275-5 were transformed into S^ cerevisiae strain ZM134. Transformants were grown in 20-πtI cultures of YEPD (1% yeast extract, 2% peptone, -2% glucose, 40 mg/1 adenine) at 30°C overnight to a cell density of approximately 8 x lO"7 cells/ml. The 5 temperature was reduced to 23°-25°C, and the cultures were incubated for an additional two hours. The cells were then pelleted, resuspended in 20 ml of fresh YEPD, and grown for 24 hours at 23°-25°C. Crude lysates were then prepared as described above and frozen at -80°C. 0
D. Expression Using Wild-Type ADH2 Promoter
The wild-type ADH2 promoter was regenerated using the partial ADH2 promoter fragment from p410ES. 5 Plasmid p410ES was digested with Sph I and Eco RI to isolate the 175-bp partial ADH2 promoter fragment. This fragment was joined with a 1-kb Bam Hl-Sph I fragment derived from pBR322-ADR2-BSa in a three-part ligation into pUC13 which had been linearized by digestion with 0' Ba HI and Eco RI. The 1-kb fragment derived from pBR322-ADR2-BSa contained sequences that are homologous with the wild-type ADH2 promoter sequence. The plasmid that resulted from the three-part ligation was confirmed by restriction analysis and designated ρ410-Wt. 5 Plasmid pM271-9 was digested with Bam HI and
Eco RI, and the Bam HI-Eco RI ADH2 promoter fragment from p410wt was inserted in place of the ADH2-4C promoter. The resultant plasmids were designated pM277-15 and pM277-16, depending on insert orientation (Figure 14).
Plasmids pM277-15 and pM277-16 were used to transform S^ cerevisiae strain ZM118. Twenty-ml cultures in YEPD were grown at 30°C for approximately 48 hours. Crude lysates were prepared and frozen at -80°C.
N-proCT levels in cell lysates were assayed by NCAP radioimmunoassay using synthetic NCAP as a standard. Results of duplicate determinations are shown in Table 5.
TABLE 5
% of cytoplasmic protein
Plasmid Strain 1 2 Average ρM271-9 ZM134 2.07 2.52 2.29
ZM118 0.54 0.69 0.62
XB13-5B 0.19 0.64 0.41
PM277-15 ZM118 0.48 0.41 0.44 pM277-16 ZM118 0 0.18 0.09 pDPOT ZM134 0 0 0
ZM118 0 0
Secretory Expression of Rat N-ProCT
A coding sequence for rat N-proCT was assembled essentially as described above, with the substitution of oligonucleotides ZC 2041 (5' AGC TTG GAC AAG AGA GTT CCC TTA AGA TCT ACC TTG GAA TCT TCT CCA GGT ATG GCT ACC TTG T 3') and ZC 2042 (5* CTT CAG ACA AGG TAG CCA TAC CTG GAG AAG ATT CCA AGG TAG ATC TTA AGG GAA CTC TCT TGT CCA 3' ) for ZC 1791 and ZC 1792, respectively. The assembled coding sequence thus includes a Hind III "sticky end" at its 5' end.
For expression vector construction, the assembled coding sequence was joined to the TPIl terminator fragment (Xba I-Bam HI) from pKPlO. The resulting Hind III-Bam HI fragment was joined to the ADH2-4C promoter (3am HI-Eco RI from pM271-9), the α- factor pre-pro sequence (Eco RI-Hind III) and Bam Hi-cut pDPOT. The resultant expression vectors were designated OM294-I (NproCT expression unit in opposite orientation 5 to the POTl marker) and ρM294-4 (N-proCT expression unit in the same orientation as POTl) .
The vectors pM294-l and pM294-4 were trans¬ formed into S^ cerevisiae strain XB13-5B. Duplicate 20- ml cultures were grown for 48 hours, and media samples 10 were diluted 1:1000 and assayed for N-proCT immuno¬ reactive material. Results (adjusted to full-strength media) are shown in Table 6.
TABLE 6 ι ς
Plasmid N-ProCT (μg/ml)
pM294-l 38
44 20 pM294-4 70
46
Example 5 - Expression of Human N-ProCT
25 The human N-proCT sequence was constructed from oligonucleotides (Table 7) that were designed to encode the peptide with yeast-optimized codons, a 5'-terminal Eco RI site and a 3'-terminal Xba I site. The encoded sequence includes an initiator methionine residue that is
30 excised by a methionine aminopeptidase _in vivo to yield the correct peptide.
35 TABLE 7
ZC1946
27
AAT TCT AAA AAA TGG CTC CAT TCA GAT CTG CTT TGG AAT
54 CTT CTC CAG CTG ACC CAG CTA CCT TGT
ZC1947
27 CTG AAG ACG AAG CTA GAT TGT TGT TGG CTG CAC TAG TTC
54 AAG ACT ACG TTC AAA TGA AGG CTT CTG
ZC1948
27 AAT TGG AAC AAG AAC AAG AAA GAG AAG GCT CGA GTT TGG
54 ACT CTC CAA GGT CTT AAA T
ZCI949
27 CTA GAT TTA AGA CCT TGG AGA GTC CAA ACT CGA GCC TTC 54
TCT TTC TTG TTC TTG TT
ZC1950
27 CCA ATT CAG AAG CCT TCA TTT GAA CGT AGT CTT GAA CTA
54 GTG CAG CCA ACA ACA ATC TAG CTT CGT
ZC1951
27 CTT CAG ACA AGG TAG CTG GGT CAG CTG GAG AAG ATT CCA
54 AAG CAG ATC TGA ATG GAG CCA TTT TTT AG
ZC1955
27 AGC TTG GAC AAG AGA GCT CCA TTC AGA TCT GCT TTG GAA
54 TCT TCT CCA GCT GAC CCA GCT ACC TTG T ZC1956
27 CTT CAG ACA AGG TAG CTG GGT CAG CTG GAG AAG ATT CCA
54 AAG CAG ATC TGA ATG GAG CTC TCT TGT CCA
For cytoplasmic expression, oligonucleotides A through F were synthesized, purified, kinased, mixed in equal proportions and annealed. The resulting oligo¬ nucleotide pairs (A+F, B+E, C+D) were ligated and the mixture was digested with Eco RI and Xba I. The coding sequence was isolated by electrophoresis on a native polyacrylamide slab gel and extracted from the gel.
Expression vectors were then constructed essentially as described in Example 4. The ADH2-4C promoter (Bam. HI-Eco RI fragment) , wild-type ADH2 promoter (3am HI-Eco RI fragment) or SXRlll promoter (Bgl II-Eco RI fragment) was joined to the N-proCT sequence together with the TPIl terminator from pKPlO. The resulting expression units were then inserted into pMPOT2 or pDPOT, and the insert orientation was determined.
For expression, the human N-proCT expression vectors were transformed into appropriate yeast host strains. Vectors containing the ADH2-4C or wild-type ADH2 promoter were transformed into strains ZM118, XB13- 5B and ZM134. Vectors containing the SXRlll promoter were transformed into ZM134. Transformants were selected by growth on glucose-containing media. Cells were grown under appropriate conditions for regulated or constitu¬ tive expression of the peptide. The cells were then lysed, and cell-free lysates were prepared for assay.
Plasmid pM286-7, a pDPOT-based plasmid contain¬ ing the ADH2-4C promoter with the N-proCT expression unit in the same orientation as the POTl gene, was transformed into strain ZM134. Transformed cells were cultured essen¬ tially as described above. The cells were harvested, lysed in 1 M acetic acid, and cent ifuged. The resulting cleared lysate was assayed for N-proCT production by radioimmunoassay using anti-NCAP antiserum. Samples of the cleared lysate were diluted in assay buffer (0.02 M NaP04, pH 7.4, 0.05% Na 3 , 0.05% NP-40, 1 M EDTA) . Duplicate samples (100 μl) were added to tubes containing 200 μl assay buffer. To each tube was added 100 μl of antibody diluted 1:7 (1:3500 total dilution) in assay buffer. 100 μl of assay buffer containing 20,000 com of 125ι_ιabeled human N-proCT was then added to each tube and the tubes were incubated at room temperature for four hours. Staphylococcus aυreus cells (Pansorbin; Sigma Chemical Co.) were diluted 1:200 in assay buffer, 1 ml of the resulting suspension was added to each tube, and the tubes were mixed and incubated at room temperature for 20 minutes. The tubes were centrifuged at 3000 rp for 15 minutes, the supernatants were poured off, and the radioactivity in the pellets was counted. N-proCT levels were determined by comparison with a standard curve generated with synthetic human N-proCT diluted to 500,
250, 125, 62.5, 31.25, 15.6 and 7.8 ng/ml. Approximately
0.025% of total soluble protein was found to be N-proCT.
For secretion of human N-proCT by yeast, oligonucleotides B, C, D, E, G and H were annealed as described above to give pairs G+H, B+E and C+D. The pairs were ligated, the mixture was digested with Hind III and Xba I, and the fragment was gel-purified.
Vectors for secretory expression were then assembled. The N-proCT fragment (Hind III-Xba I) and the α-factor pre-pro fragment (Eco RI-Hind III) from pKPlO were joined and inserted into expression vectors contain¬ ing the TPIl promoter and terminator as described above to construct plasmids pM285-13 (expression unit in same orientation as POTl ) and pM285-15 (expression unit in opposite orientation) . A second set of secretory expres¬ sion vectors was constructed using the ADH2-4C promoter from p410-4c and the TPIl terminator from pKPlO . These vectors were designated pM284-3 (expression unit in same orientation as POTl) and pM284-4 (expression unit in opposite orientation) .
Plasmid pM284-3 was transformed into S^ cerevisiae strain XB13-5B. Plasmids pM285-13 and pM285- 15 were transformed into strains XB13-5B and ZMI34, Cells were cultured as previously described. Culture media were sterile filtered through 0.45-micron filters and assayed for N-proCT by radioimmunoassay using anti- NCAP antiserum as described above. Results of two sets of experiments are shown in Table 8.
TABLE 8
Plasmid Host Strain Exported N-proCT (μg/ml)
1 2 avg. pM284-3 XB13-5B 6.71 5.97 6.34
PM285-13 XB13-5B 5.19 3.63 4.41 pM285-15 XB13-5B 2.96 4.80 3.88 pM285-15 ZM134 3.51 3.33 3.42
Recombinant N-proCT was purified from yeast culture media by adding to the media 1% (by volume) acetic acid and 30% (by volume) acetonitrile. The mixture was passed over a Vydac C-4 reversed-phase column and eluted with a gradient of 30%-50% acetonitrile in H2O. The eluate was monitored for absorbance at 215 nm, and the peak fractions, containing N-proCT, were collected.
Example 6 - In Vivo Activity of N-ProCT
Weanling Swiss Webster mice (21 days old; average body weight, 16 g; n=8) were injected subcutan- eously twice daily with 10 μg synthetic human N-proCT (dissolved in 0.01 M acetic acid) for 14 days. Control mice (n=7) were injected with vehicle. No significant difference in body weight occurred between treated and control mice. Thus, N-proCT was not toxic to the mice. The animals were injected with tetracycline on day 2 and day 13 of the experiment to label the bones for histo- morphometry. The animals were sacrificed on day 14, and the bones were dissected free of soft tissue and fixed in neutral buffered formalin. Tibia cross sections (50 μ ) at the tibiofibular junction were prepared from each animal, then quantified for tetracycline labeling with an image analysis program. The mean endosteal tetracycline- labeled area was significantly greater (+46%, p<0.02) in the N-proCT-treated animals than in the controls (Figure 21) . There was a significant reduction (-34.4%, p<0.001) in the number of osteσclasts/mm surface in the secondary spongiosa region in mice treated with N-proCT vs. controls. These data thus provide evidence that N-proCT affects bone formation in vivo and suggest that this C- cell product is an important regulator of bone growth.
Example 7 - Evidence for N-ProCT Receptors on Osteoblastic Cells
To elucidate the mechanism of action of N-proCT on osteoblast-like cells, radioiodinated human N-proCT was tested for high-affinity binding to cultured U-2 OS cells, which are derived from a human osteosarcoma . Synthetic human N-proCT (purified by reversed-phase HPLC) was radioiodinated by the chloramine T method and purified by elution from Quso G-32 silica/Dowex AG1-X8 (Bio-Rad Laboratories, Richmond, Calif.) . This labeled human N-proCT (60,000 cpm/ng) was used as tracer in these studies. Human U-2 OS cells were plated at 20,000 cells/well into a 48-well icrotiter plate. Six hours before the beginning of an experiment, and during all binding studies, the cells were maintained in serum-free BGJb tissue culture medium. Roughly 0.5% of added tracer is specifically bound to these cells. (Specific binding is defined as the binding in the absence of synthetic N- proCT less the amount of radioactivity remaining after incubation in the presence of high concentrations [5-8 μM] of unlabeied N-proCT. ) 5 μM human calcitonin and insulin did not compete for 125χ_ _pror;rrι binding to cells. Competitive binding with unlabeied human N-proCT has an apparent Kςj (half-maximal binding) of 10 nM, and competi¬ tion with synthetic human NTP-Tyr has a Kςj of 200 nM. These estimated dissociation constants agree very well with the concentrations of each peptide necessary to elicit half-maximal stimulation of U-2 OS cell prolifera¬ tion. Assuming equilibrium conditions and roughly 20,000 cells/well at the end of the binding experiments, there are at least 3000 binding sites per cell. These results provide further evidence that N-proCT is a C-cell peptide hormone that promotes bond formation via direct action on osteoblasts.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

ClaimsWe claim:
1. An isolated peptide having the following charac¬ teristics: is at least 12 amino acids in length; is substantially homologous to at least a portion of rat N-proCT; and increases DNA synthesis in osteoblasts and preosteo¬ blasts by a factor of at least two, as compared to fibro¬ blasts, at a concentration that is maximally stimulatory for osteoblasts and preosteoblasts.
2. The isolated peptide of claim 1 wherein said peptide consists of about 52 to 57 amino acids.
3. The isolated peptide of claim 1 wherein said peptide comprises an amino terminal sequence selected from the group consisting of (a) V P L R S T L E S S P G; (b) A P F R S A L E S S P A; (c) A P V R P G L E S I T D? and (d) A P A R T G L E S M T D.
4. The isolated peptide of claim 1 wherein said peptide has an amino acid sequence selected from the group consisting of rat, human, chick and salmon N-proCT and rat, human and chick N-proCGRP sequences, as shown in Figure 1.
5. The isolated peptide of claim 1 wherein said peptide is less than 32 amino acids in length.
6. An isolated peptide consisting of a sequence selected from the group consisting of (a) V P L R S T L E S S P G; (b) A P F R S A L E S S P A; (c) A P V R P G L E S I T D; and (d) A P A R T G L E S M T D.
7. A method of isolating a peptide that stimulates cell division in osteoblasts and preosteoblasts, comprising: preparing an aqueous extract of cells capable of expressing calcitonin or a calcitonin-related gene; fractionating said aqueous extract to enrich for peptides having a molecular weight less than approximately 15,000; and fractionating the enriched fraction by hydrophobic chromatography and/or anion exchange chromatography to separate the peptide from the enriched fraction.
8. The method of claim 7 wherein the step of fractionating the aqueous extract comprises reversed-phase HPLC and gel filtration of the aqueous extract.
9- An isolated DNA sequence having the following characteristics: is between 36 and 180 base pairs; and encodes a peptide according to any of claims 1-6.
10. A host cell transfected or transformed with an expression vector comprising a transcriptional promoter oper¬ ably linked to a DNA sequence encoding a peptide according to any of claims 1-6.
11. The host cell of claim 10 wherein said host cell is a yeast cell.
12. A method for producing a peptide that stimu¬ lates cell division in osteoblasts and preosteoblasts, comprising: culturing a host cell according to claims .10 or 11 under suitable conditions; and isolating the peptide from the host cell.
13. A therapeutic composition comprising a peptide according to any of claims 1-6, in combination with a physiologically acceptable carrier or diluent.
14. The therapeutic composition of claim 13, further comprising a growth factor in an amount sufficient to further increase DNA synthesis in osteoblasts and preosteo¬ blasts, wherein said growth factor is selected from the group consisting of insulin, insulin-like growth factors, platelet- derived growth factor, transforming growth factor alpha, transforming growth factor beta and epidermal growth factor.
15. The therapeutic composition of claim 13, further comprising insulin-like growth factor I in an amount sufficient to further increase DNA synthesis in osteoblasts and preosteoblasts.
EP89907003A 1988-05-03 1989-05-02 Procalcitonin peptides Withdrawn EP0416029A1 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US18991388A 1988-05-03 1988-05-03
US189913 1988-05-03
US27013788A 1988-11-14 1988-11-14
US270137 1988-11-14
US29571989A 1989-01-10 1989-01-10
US295719 1999-04-21

Publications (1)

Publication Number Publication Date
EP0416029A1 true EP0416029A1 (en) 1991-03-13

Family

ID=27392651

Family Applications (1)

Application Number Title Priority Date Filing Date
EP89907003A Withdrawn EP0416029A1 (en) 1988-05-03 1989-05-02 Procalcitonin peptides

Country Status (7)

Country Link
EP (1) EP0416029A1 (en)
JP (1) JPH03505327A (en)
KR (1) KR900700504A (en)
AU (1) AU3754889A (en)
DK (1) DK261990A (en)
FI (1) FI905453A0 (en)
WO (1) WO1989010934A1 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5693615A (en) * 1991-06-05 1997-12-02 The Procter & Gamble Company Therapeutic compositions for osteoinduction
ES2215359T3 (en) * 1991-12-18 2004-10-01 Icu Medical, Inc. FLUID TRANSFER PROCEDURE.
US5962270A (en) 1996-02-06 1999-10-05 Bionebraska, Inc. Recombinant preparation of calcitonin fragments and use thereof in the preparation of calcitonin and related analogs
BR0309877A (en) * 2002-05-03 2005-04-26 Millenium Biologix Inc Connective tissue stimulating peptides
CN114478745B (en) * 2022-03-30 2023-12-15 南京京达生物技术有限公司 Separation and purification method of recombinant procalcitonin PCT

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8910934A1 *

Also Published As

Publication number Publication date
JPH03505327A (en) 1991-11-21
AU3754889A (en) 1989-11-29
DK261990D0 (en) 1990-10-31
DK261990A (en) 1991-01-03
KR900700504A (en) 1990-08-13
WO1989010934A1 (en) 1989-11-16
FI905453A0 (en) 1990-11-02

Similar Documents

Publication Publication Date Title
US5128321A (en) PDGF analogs and methods of use
Okamura et al. A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol
US5827734A (en) Materials and methods for determining ob protein in a biological sample
Sara et al. Characterization of somatomedins from human fetal brain: identification of a variant form of insulin-like growth factor I.
Carlsson-Skwirut et al. Isolation and characterization of variant IGF-1 as well as IGF-2 from adult human brain
AU615654B2 (en) Expression of human proapolipoprotein a-i
Sugiyama et al. Synthesis of atrial natriuretic peptides and studies on structural factors in tissue specificity
Mizuno et al. Cloning and sequence of cDNA encoding a peptide C-terminal α-amidating enzyme from Xenopuslaevis
EP0289314B1 (en) Use of IGF-II in the treatment of bone disorders
Kuo et al. Amino acid sequence and characterization of a heparin-binding neurite-promoting factor (p18) from bovine brain.
Arimura et al. Isolation and identification of C-type natriuretic peptide in chicken brain
JPH0829097B2 (en) Fibroblast growth factor
CA2071538C (en) Hpth (1-37) fragment, its production, drug containing it and its use
JPH085797B2 (en) Cell growth stimulant
JP2823690B2 (en) Protease resistant PDGF and methods of use
US5359033A (en) Cleaved dimers of mullerian inhibiting substance-like polypeptides
Agerberth et al. Isolation and characterization of a 60-residue intestinal peptide structurally related to the pancreatic secretory type of trypsin inhibitor: influence on insulin secretion.
AU665034B2 (en) Production of human parathyroid hormone from microorganisms
Bajaj et al. Coypu insulin. Primary structure, conformation and biological properties of a hystricomorph rodent insulin
US5744444A (en) HPTH-fragment-(1-37), the preparation thereof, medicaments containing same and the use thereof
Matsuo et al. Molecular biology of atrial natriuretic peptides
EP0416029A1 (en) Procalcitonin peptides
WO1993023068A1 (en) Platelet derived growth factor antagonists
US5474982A (en) PDGF analogs and methods of use
EP0384731B1 (en) Osteogenic growth polypeptides identified from regenerating bone marrow

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19901030

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

17Q First examination report despatched

Effective date: 19930716

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19931127