WO1992019736A1 - Antagonistes de l'hormone de croissance - Google Patents

Antagonistes de l'hormone de croissance Download PDF

Info

Publication number
WO1992019736A1
WO1992019736A1 PCT/US1992/003532 US9203532W WO9219736A1 WO 1992019736 A1 WO1992019736 A1 WO 1992019736A1 US 9203532 W US9203532 W US 9203532W WO 9219736 A1 WO9219736 A1 WO 9219736A1
Authority
WO
WIPO (PCT)
Prior art keywords
bgh
growth
protein
hgh
growth hormone
Prior art date
Application number
PCT/US1992/003532
Other languages
English (en)
Inventor
John J. Kopchick
Wen Y. Chen
Original Assignee
Ohio University
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 Ohio University filed Critical Ohio University
Priority to EP92911122A priority Critical patent/EP0659213A1/fr
Priority to CA002102129A priority patent/CA2102129C/fr
Priority to JP4510937A priority patent/JPH06507632A/ja
Priority to AU18930/92A priority patent/AU666117B2/en
Publication of WO1992019736A1 publication Critical patent/WO1992019736A1/fr
Priority to NO19933909A priority patent/NO314309B1/no
Priority to FI934829A priority patent/FI934829A0/fi

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel growth hormo especially bovine growth hormone, muteins which inhibit growth of animals or otherwise antagonize the effects endogenous growth hormone.
  • These analogues may be expressed transgenic animals which thereby acquire a "reduced grow phenotype.
  • Bovine growth hormone is a protein of 191 am acids that is naturally synthesized in the anterior pituita The molecular weight of the mature protein is about 22, daltons, but it is initially made as a pre-growth hormone w an extra 26 amino acids on the amino terminal. This leader signal peptide) is normally cleaved during secretion of hormone by bovine pituitary cells.
  • the N-terminal can v due to variation in the site of cleavage during secretion) that the mature protein begins with either NH 2 -Ala-Phe-Pro NH 2 -Phe-Pro.
  • the amino acid at position 126 be either leucine or valine, apparently as a result of alle variation in the bovine population.
  • bGH has been produced by recombinant DNA techniqu see e.g., Fraser, U.S. 4,443,539 (yeast); Buell, EP Ap 103,395 (bacteria); Krivl, EP Appl. 193,515 (bacteri Kopchick, EP Appl. 161,640 (encapsulated mouse cells implan into animals); DeBoer, EP Appl. 75,444 (bacteria; gene modif to eliminate harmful secondary structure) and this facilitated the production of analogues of bGH by site-speci mutagenesis.
  • GB 2,073,245 describes production Met Pro (des Ala) bGH, Met Arg (des Ala) bGH, Met-Glu-Gly ( Ala) bGH, and des (Ala 1 Phe 2 -Pro 3 -Ala*) bGH i coli.
  • Bre et al., PNAS (USA) 85:3367-71 (1988) reported preparation the bGH mutant K112L, which extended the hydrophobic face the third alpha helix of bGH.
  • the 96-133 fragment of t mutant was also prepared.
  • Analogues of bGH have varied in growth-promoti activity, as have the known analogues of other growth hormone However, a growth hormone analogue having growth-inhibito activity has not previously been reported.
  • a variety of transgenic animals have been produ Hammer, et al., Nature, 315:680-683 (1985) (rabbits, sheep pigs) . Certain of these animals have been caused to expres growth hormone, and increased growth of such transgenic ani has been reported. Palmiter, et al., Nature 300:611 (19 microinjected the male pronucleus of fertilized mouse eggs a DNA fragment containing the promoter of the mo metallothionein-I gene fused to the structural gene of growth hormone.
  • transgenic mice developed f the genetically modified zygote exhibited a growth r substantially higher than that of control mice.
  • the genetically modified mouse serves as a test environment determining the effect of the hor one on animal growt Later, Palmiter, et al., Science, 222:809 (1983) demonstra that a similar enhancement of growth could be obtained transgenic mice bearing an expressible human growth horm gene. A like effect is observed when human growth horm releasing factor is expressed in transgenic mice. Hammer, al., Nature, 315:413 (1985).
  • Bovine growth hormone has also been expressed transgenic animals. McGrane, et al. J. Biol. Chem., 263:1144
  • the present invention relates to proteins which substantially homologous with a vertebrate growth hormone have growth-inhibitory activity.
  • G119R bGH Arg
  • G119P Pro
  • G119K Lys
  • Trp G119W
  • G119L Trp
  • This novel hormone may be administered mammals (or other vertebrates) , in particular humans bovines, when growth inhibition is desirable.
  • the hormone produced exogenously and administered to the subject is preferably produced expression in a suitable host of a gene coding for it.
  • a gene coding for it is most readily prepared by site-specific mutagenesis o bGH gene.
  • the hormone may also be produced by ot techniques, such as by condensation of fragments of native with a synthetic peptide carrying the replacement amino aci If a peptide fragment has the desired growth-inhibito activity, it may be prepared in toto by a Merrifield-ty synthesis.
  • this gene introduced into a prenatal form of a mammal by kno techniques, and the prenatal form is developed into transgenic mammal which expresses a reduced growth phenotyp Conceivably, a mammal could be genetically modified aft birt , i.e., "gene therapy”.
  • growth-inhibited animals may be produced eith by administration of the growth inhibitory hormone of th invention in pharmaceutical form, or by genetic transformati of a prenatal or postnatal form of the animal.
  • the growth-inhibitory hormone or the gene encodi it, is useful in the production of small animals for use research facilities where space is restricted, as pets for p lovers with limited quarters, and as livestock for farme having small tracts.
  • the hormone may also be useful in t treatment of human gigantism, and in research on gigantism a dwarfism, in the treatment of diabetes and its sequelae, in control of cholesterol, and in the prevention and treatment certain cancers.
  • Diabetic retinopathy is belie to arise as a result of the proliferation of microvascu endothelial cells in the retina.
  • Human growth hormone is kn to stimulate proliferation of microvascular endothelial cel See Rymaszewski, et al., Proc. Nat. Acad. Sci. USA 88:617 (1991) .
  • the growth hormone antagonists of the pres invention may therefore be useful in countering the adve effects of elevated levels of endogenous growth hormone microvascular tissues, such as the retina, in diabetics, or other individuals experiencing excessive growth horm levels.
  • Glomerulosclerosis occurs in a variety of glomeru diseases, including diabetic nephropathy. The cause unknown, but mesangial cell proliferation precedes accompanies mesangial sclerosis. Thus, dysregulation resident glomerular cells may be an important issue in development of glomerulosclerosis (Doi, et al., Am. J. Patho 137:541, 1990) . Transgenic mice which express bGH have been shown have enlarged glomeruli which progressed to a state glomerulosclerosis. Thus, GH has been implicated in development of diabetic glomerulosclerosis (Doi, et al., 1 and Bell, Am. J. Med. Sci., 301:195, 1991). A GH antagon could . alleviate the GH-dependent effect on the cells of diabetic kidney, and thereby be useful in the prevention treatment of glomerulosclerosis.
  • antagonists are used to reduce serum cholesterol levels.
  • these antagonists are therapeutically prophylactically useful in countering the adverse effects growth hormones, both endogenous hormones and hormon administered clinically.
  • Figure 1 Amino acid sequence of bGH (G119R) nucleotide sequence of the gene encoding this analogue. alpha helices are marked and the amino acids are numbered, w number 1 being the first amino acid of the mature protein. boldfaced bases and amino acids are those mutagenized in G119R mutant.
  • FIG. 2 General strategy of oligonucleotide direc mutagenesis.
  • pBGHIO 6 was used as the parental vector. contains mouse metallothionein I transcriptional regulat sequences (MT-1) fused to the bGH gene (BamHI joined w Bglll) which contains five exons (shaded boxes I-V) and int A. This fusion gene was incorporated into pBR322 at the Ec site. The pBR322 origin of replication (ORI) , ampicill resistant gene (Amp) , as well as the bGH translation st (ATG) and stop (TAG) codons are indicated. 5 1 and 3' no translated regions are shown in hatching.
  • ORI origin of replication
  • Amp ampicill resistant gene
  • ATG bGH translation st
  • TAG stop codons
  • Figure 3 is an idealized surface net (cylindric plot) representa ion of most of the third alpha helix of bovi growth hormone.
  • the surface net is produced by projection the helix onto a coaxial cylindrical sheet of paper, cutti this paper parallel to the helical axis and flattening it. T volumes of the amino acids are given in parentheses.
  • a dash line indicates the cleft or depression formed by Alal22-Glyll Aspll5.
  • Figure 4 is a plot of the secondary struc prediction (alpha-helix, beta-sheet, reverse turn, random c for amino acids 108-127 of bovine growth hormone (a) wild- (b) the mutant G119R and (c) the mutant A122D. These p were generated by the "Micro-Genie" program.
  • Figure 5 Scatchard plots of data from competi binding experiments for wild type bGH and bGH-M8 using m liver membrane preparations. The ordinate represents the r of bound to free bGH and the abscissa the concentration total bGH bound. Each point represents the mean of f experiments which were carried out in triplicate.
  • Figure 6 provides a growth rate comparison am control (non-transgenic) , G119R, G119L, G119K and G119P mi illustrating the growth-inhibitory effect of these mutants.
  • Figure 7 presents an axial view of the third al helix (109-126) of bGH, showing its a phipathic tendenci Hydrophobic amino acid sectors are shaded by dots; hydrophi amino acids are indicated by open sectors; the glycine sect a neutral amino acid, by slanted lines. The residue numb and hydrophilicity values (Hopp and Wood scale) are given.
  • Figure 8 presents side views of the third alpha he of wild type (left) and G119R mutant (right) bGHs projected the plane in which the side chain of the Arginine-119 of mutant G119R lies.
  • the glycine 119 residue found at the bot of the cleft is indicated by an arrow.
  • Figure 9 compares serum glucose, urea/nitrogen, and triglyceride levels of control mice, transgenic bGH-M8 (E117L G119R, A122D) -producing mice, and transgenic wtbGH-producing mice, of both sexes.
  • Fi ⁇ ure 10 compares serum cholesterol for transge wtbGH-producing mice, control mice, and transgenic bGH- producing mice, of both sexes.
  • Fi ⁇ ure 11 plots GPDH activity against bGH-M8 dos in a competitive inhibition assay for the antagonism of ability of GH (here, wild-type bGH) to promote differentiation of preadipocytes (NIH 3T3-F442A cells) .
  • Fi ⁇ ure 12 compares of the effect of bGH and bGH-M8 the differentiation of 3T3-F442A cells. At confluence, ce were incubated with increasing concentrations of bGH or bGH- Cells were harvested on day 8 for determination of G activity. The experiment was repeated twice with simi results. Each bar represents the mean value obtained f triplicate assays. The error bar represents the stand division.
  • Figure 13 shows the relationship between serum analog concentrations and the growth ratio of transgenic m (TG)/nontransgenic (NTG) .
  • the ordinate represents bGH anal concentrations in serum.
  • the abscissa represents the gro ratio of TG/NTG mice.
  • Fi ⁇ ure 14 shows the relationship between serum h analog concentrations and the growth ratio of transgenic mi (TG)/nontransgenic (NTG) .
  • the ordinate represents bGH anal concentrations in serum.
  • the abscissa represents the grow ratio of TG/NTG mice.
  • the present invention relates to growth hormo antagonists, especially growth inhibitors, which are peptid or proteins having a similarity in sequence and seconda structure to a vertebrate growth hormone, including but limited to mammalian growth hormones, especially human bovine growth hormones.
  • the compound comprises alpha helix having an amino acid sequence homology of at le about 50% with the third alpha helix of a vertebrate gro hormone, especially bovine or human growth hormone. Ot alpha helices of the native hormone may be omitted if this be done without loss of growth-inhibitory and/or other gro hormone antagonist activity.
  • the use of the term "antagoni is in a functional sense and is not intended to limit invention to compounds having a particular mechanism action.
  • the overall percentage homology of bovine gro hormone with other mammalian growth hormones is high: porc (92%) , ovine (99%) , human (66%) , and rat (87%) .
  • porc (92%) The overall percentage homology of bovine gro hormone with other mammalian growth hormones is high: porc (92%) , ovine (99%) , human (66%) , and rat (87%) .
  • porcine 97%
  • ovine ovine
  • human 66% and rat (94%)
  • rat rat
  • the secondary structure of a polypeptide is a regul arrangement of a linear segment of the polypeptide chain.
  • T most commonly encountered secondary structures are the bet sheets and the alpha-helices. See Schulz and Schime Principles of Protein Structure 69 (Springer-Verlag: 1979 The alpha helix is stabilized by hydrogen bonding betwe peptide amide and carbonyl groups of residues separated by single turn of the helix. Secondary structure predictions a based on observation of the frequency of occurrence of t amino acid in a beta-sheet, alpha-helix, etc. in a prote having a known three dimensional structure.
  • porcine grow hormone has been determined by X-ray diffraction and compar to that of other growth hormones.
  • Abdel-Meguid et al., Pro Nat. Acad. Sci., £4:6434 (1987).
  • it is a single domain protein arran as a four helix bundle with the helices in an antiparal relationship. Its four helixes are made up of residues 7- 75-87, 106-127 and 152-183.
  • Bovine growth hormone is 93% homologous at the am acid sequence level with porcine growth hormone, and bG structure has been deduced by study of the two sequences and the structure of porcine growth hormone. Its four al helixes have been reported to be assumed by amino acids 4- 66-80, 108-127 and 150-179.
  • the third alpha helix of bGH defined as amino acids 106-129. However, it will be noted t the ends of this helix have a less marked alpha heli secondary structure than does the central region, which is 1 126. The exact bounds of the third alpha helix may differ other GH's, depending on the alpha helical tendencies of "and" amino acids.
  • Watahik conserved region GD4 comprises the stretch LKDLEEGIIJALMRELED bovine growth hormone, i.e., residues 113 to 129.
  • Watahik Figure 3 identifies residues conserved among the GHs residues predicted to be important for the manifestation growth-promoting activity. Studying Watahiki's GD4 consensus region, seve families of growth hormones may be discerned.
  • the first fam (I) comprises cGH, rGH, pGH oGH, bGH, and hGH. These be with LKDLEEGI. They then continue with IQA (cGH, rGH, pG ILA (oGH, bGH) or IQT (hGH) . All members of family I t conclude GD4 with LMRELED (except for rGH, LMQELED, and h LMGRLED) .
  • the second family (II) comprises fGH, yGH, tGH sGH. These have the consensus sequence LS (E/D) LK (M G(L/I) (L/G/H/N) (K/L) LI (E/T/R/I) (A/G) (N/S) QD.
  • the present invention is not limited to the mutat of the third alpha helix of bovine or human growth hormo Rather, it encompasses the mutation of the third alpha helix any mammalian or other vertebrate growth hormone, includi but not limited to, the growth hormones whose sequences given in Watahiki (1989) : flounder, yellowtail, tuna, salm chicken, rat, porcine, ovine, bovine and human growth hormon Expression of mutants of other growth hormones is facilita by the availability of genes encoding the latter. See, e. Goeddel, Nature, 281:544-683 (1979) (hGH).
  • polypeptide which is substantia homologous to bovine growth hormone is deemed to include ( is not limited to) any polypeptide which differs from bovine human growth hormone by (a) a substitution at an amino a corresponding to amino acids 115 or 119 of bovine gro hormone, (b) a substitution at an amino acid corresponding an amino acid of bovine or human growth hormone which is conserved among the vertebrate growth hormones, especially t replacement of that amino acid by one found at the site i different growth hormone, and/or (c) truncation of amino aci 1- 95 and/or 134-191.
  • non-bovine vertebra growth hormones are "substantially homologous" with bovi and/or human growth hormone.
  • the polypeptide is least about 50% homologous, more preferably at least 8 homologous, with bovine or human growth hormone in subsequence substantially corresponding to the third al helix (approximately, residues 106-129) of bGH, and m preferably over the entire length of the polypeptide (ignor extraneous non-bGH-related fusions to the amino- or carbo terminal) .
  • the compound is considered to be growth-inhibitory the growth of test animals of at least one vertebrate spec which are treated with the compound (or which have b genetically engineered to express it themselves) significantly (at a 0.95 confidence level) slower than growth of control animals (the term "significant” being used its statistical sense) .
  • it is growth-inhibitory a plurality of species, or at least in humans and/or bovin Growth hormones have considerable interspecies cros reactivity.
  • Gill, et al., Biotechnology, 3.:643 (1985) report that recombinant chicken and bovine growth hormones accelera growth in juvenile pacific salmon.
  • the growth-inhibitory peptides (the term is used hereafter include proteins) of the present invention need not be as lar as bGH.
  • the peptides are at least 11 amino aci long (three turns of an alpha helix) and more preferably least 50 amino acids long. These peptides may retain t growth inhibiting action of, e.g., bGH (G119R) , yet lack othe undesirable biological activities of the native size mutan They may also have more desirable pharmacokinet characteristics.
  • the growth inhibitory peptides of the prese invention may also be larger than bGH, provided that t additional amino acids do not result in the compound bei unable to reduce the growth rate of a vertebrate. While the mechanism of action of applicant's gro inhibitory peptides is not known, it is believed that t function as antagonists to wild-type growth hormo endogenously produced by the target animal. We have sh that, e.g., bGH (G119R) and bGH (G119R, E117L, A122D) , b competitively inhibit the binding of wild type bGH to li membrane preparations.
  • the compo has a net result of inhibiting growth because its grow promoting activity is substantially less than that of wild t growth hormones (and perhaps is negligible) yet it can displ from growth hormone receptor (GHR) sites the endogenous nat growth hormone (whose stimulation of growth would have b more pronounced) .
  • GHR growth hormone receptor
  • the first receptor-binding reg of hGH is concave and is formed mainly by residues on expo faces of helix 4, but also by exposed residues of helix 1 residues in the region connecting helices 1 and 2.
  • the sec receptor-binding region comprises the exposed sides of heli 1 and 3 and is relatively flat. The role of the helix 3 shown best in DeVos 1 Fig.
  • the compounds of the present inventi have an ED50 which is less than about 10 times the ED50 of wi type bGH in an assay of the ability of the compound to displa radiolabeled wild type bGH from a liver membrane preparati made as described below. More preferably, the compounds ha an ED50 at least comparable to that of wild type bGH. Mo preferably, the compounds have a higher affinity for grow hormone receptors than does the growth hormone native to animal receiving the compound. For purification characterization of a human growth hormone receptor, see Leu et al.. Nature, 330:537-43 (1987).
  • a GH mutein may be considered an antagonist, even it lacks growth-inhibitory activity, if it antagonizes anot GH-mediated activity, e.g., its diabetogen glomerulosclerotic, hypercholesterolemic, or tumorige activities.
  • the preferred growth-inhibitory peptides characterized by a modification of the surface topography the third alpha helix. It will be seen from Figure 3 that the third alpha helix of "wild-type" bovine growth hormo there is a surface cleft or depression beginning, at Aspartate-115, deepening at the Glycine-119, and ending w the Alanine-122. All of the mutants prepared so far, b those which retain the wild-type growth-promoting activity those which do not, are consistent with the theory that grow promoting activity requires the presence of this cleft depression and that, if the center of this cleft is "filled by substitution of amino acids with bulkier side chains, mutein inhibits the growth of the subject.
  • alpha helix formers and breaker see Chou and Fasman, supra.
  • Glu, Ala and Leu are the preferr alpha helix formers while Pro and Gly are characterized strong helix breakers.
  • Substitutions which introduce stro alpha helix breakers are less desirable, but may be tolerat in a particular case, such as the end of the helix.
  • T secondary structures of our analogues have been predicted us the "Micro Genie" computer program, which uses the algorithm Gamier, et al., J. Biol. Chem., 120:97-120 (1978).
  • amino acid 119 glycine is both smallest amino acid residue and the one least favorable alpha-helix formation.
  • any ot amino acid may be substituted for it without destabilizing alpha helix, while at the same time filling in aforementioned cleft.
  • All of the G 119 bGH substitutions tes resulted in a "small animal" phenotype. These substituti were arginine (a large, positively charged AA) , proline cyclic aliphatic AA) , lysine (a large, positively charged A tryptophan (a large aromatic AA) and leucine (a larg nonpolar, aliphatic AA) .
  • hGH the homologous glycine is position 119.
  • Substitution of arginine or tryptophan result in an antagonist, however, hGH G120A retained growth-promoti activity. Consequently, it is presently believed that th glycine, which is conserved in all vertebrate GHs, may replaced by any amino acid other than alanine (the seco smallest amino acid) , and more preferably by any amino ac which is at least as large as proline (the smallest replaceme amino acid known to result in a "small" animal phenotype) . T deletion of G 119 is also known to result in a "small" anim phenotype.
  • the aspartate at position 115 may be replac by a bulkier amino acid which does not destroy the alpha heli
  • the replacement amino acid has a size greater th that of glutamate.
  • the amino acids histidine, methionin isoleucine, leucine, lysine, arginine, phenylalanine, tyrosi and tryptophan are substantially larger than glutamate. These, His, Met, Leu and Trp are more preferred because th combine the advantages of bulk with a reasonably stro alphahelical propensity.
  • the wild- ype G is the strongest alpha-helix former of all of the amino acid
  • the D115A mutant of bGH is not a GH antagonist, but Alanine smaller than Aspartic Acid, so this is not probative of value of replacing Aspll5 with a bulkier amino acid.
  • G119A might lead to a "sma phenotype if coupled with other mutations, e.g., at 115 122.
  • DNA which encodes bGH and is degenerate these positions, so as to there encode all possible ami acids, or only those with acceptable alpha-helic propensities, is prepared, e.g., by a "dirty bottle” synthesi Phage are prepared, as taught by Ladner, et al PCT/US89/03731, W090/02809, which display the mutant bGHs as domain of a chimeric coat protein.
  • the phage are incubat with a chromatographic support bearing a growth hormo receptor.
  • Nati bGH is also incubated with the support, before, during or aft the phage incubation. Bound phage are recovered, amplified a examined to determine the sequence of the mutant bGH (usual by sequencing the corresponding gene in the phage genome) These mutants have demonstrated the ability to compete wi wild type bGH for a growth hormone receptor. Their ability antagonize GH activity in vivo is then confirmed by, e.g.
  • phage bearing Wt-hGH from phage bea the low affinity hGH mutant R64A by means of affi chromatography (using the extracellular domain of the receptor bound to nonporous oxirane beads) .
  • mutations at position 119 which deemed necessary to impart the desired growth-inhibi activity
  • additional mutations are possible which will l the growth-inhibitory activity or other antagonist acti intact.
  • These mutations may take the form of single multiple substitutions, deletions, or insertions, nonessential regions of the polypeptide. For example, i possible to alter another amino acid in the alpha h provided that the substitution does not destroy the a helix. Preferably, such alterations replace an amino acid one of similar size and polarity. It may be advantageou modify amino acids flanking the primary mutation site 119 order to increase the alpha-helical propensities of sequence, particularly if the mutation at 119 is one expe to destabilize the helix.
  • Cunningham, et al. (1989) used homolog-scannin mutagenesis to identify the epitopes of hGH for its clone liver receptor. Only variant hormones having mutation regions C(54-74), F(164-190), and, to a lesser extent, A(ll exhibited reduced binding affinity.
  • Cunningham and We Science, 244:1081 (1989) used a related technique, alan scanning mutagenesis, to further study these regions. N however, that binding to the receptor utilized by Cunningha not necessarily critical to the growth-promoting or gro inhibitory activity of the mutant.
  • Truncations may be generated by gene modification or exopeptidase treatment.
  • the present invention is not limited to a particular method of producing the desired GH antagonists
  • these antagonists are produced by first altering gene encoding a vertebrate GH (e.g., bGH or hGH) having th "native" third alpha helix by site-specific mutagenesis, an then cloning and expressing the altered gene in a suitabl host.
  • a vertebrate GH e.g., bGH or hGH
  • the gene may b genomic origin, it may be cDNA prepared from bGH messenger it may be synthetic, or it may be a combination thereof.
  • the host may be any convenient organism, includin bacterial, yeast, or mammalian cell.
  • the gene is opera linked to a promoter functional in the host.
  • a constitut promoter would activate gene expression in a general mann i.e., in many tissue and at all times during development.
  • regulatable promoter may be activated in a tissue or c specific manner, at precise time during development, or response to changes in the environment.
  • a constitut promoter is' usually employed when larger amounts of g product (usually protein) is required or when the gene prod is required in many cells of many tissues.
  • a regulata promoter is utilized when one gene product is required in small number of cells of a particular tissue or at a given t during development.
  • the expression system may be engineered so that antagonist is secreted into the culture medium, or the h cells may be grown to a high cell density and then lysed release the compound.
  • t procedure involves purification by (a) ammonium sulf precipitation, (b) fractionation on DEAE-cellulose (or equivalent ion-exchange column), and (c) gel filtration (e. on a Sephadex G-25 and/or Sephacryl S-200 column) .
  • Ot . procedures applicable to purification of growth hormone-rela compounds are set forth in Reichert, Jr., "Purification Anterior Pituitary Hormones: Bovine, Rat and Rabbit," Me Enzymol., 37:360 et seq. (Academic Press, N.Y.:197 Polyclonal or monoclonal antibodies which specifica recognize the protein of interest may also be used in t purification process.
  • the purified antagonist may then be combined wi compatible, nontoxic pharmaceutical excipients a administered to an animal, e.g. to treat a conditi characterized by an excessive growth rate.
  • an animal e.g. to treat a conditi characterized by an excessive growth rate.
  • the antagonist may be administered orally or parenteral (including intravenously, subcutaneously and intramuscularl to humans, in any suitable pharmaceutical dosage form. In t case. of treatment of retinopathy, it may be administer directly to the eye by means of a conventional ocul pharmaceutical form.
  • An effective dosage and treatme protocol may be determined by conventional means, starting wi a low dose in laboratory animals and then increasing the dosa while monitoring the effects, and systematically varying t dosage regimen as well.
  • the trial dosages would be chos after consideration of the clinical literature with respect administration of growth hormones, and of somatostatin ( growth hormone release inhibitor) .
  • the gene is introduced into host cell which is developed into genetically transformed cell of a transgenic animal.
  • Linearized DNA bearing the growt hormone antagonist gene may be introduced into a gamete, o microinjected into the pronuclei of fertilized eggs, into cytoplasm, into the nuclei of two-cell embryos, into indivi cells of a blastocyst, or into the blastocoel cavity. (Som these targets may be reached by electroporation instea microinjection.)
  • a retrovirus bearing the may be constructed and used to infect preimplantation emb or tissue culture cells (e.g., embryonic stem cells) which be aggregated with such embryos.
  • genetically modified zygote after a brief in v cultivation, is implanted into a foster mother and carried term.
  • gene therapy post partum, see Cline, et Nature, 284:422-425 (1980); Williamson, Nature, 298:416 (1982) .
  • the gene is operably linked to a prom functional in the host, and the promoter may be constitutiv regulatable.
  • expression is regulated so abnor embryonic or fetal development is avoided.
  • the plasmid, pBGH-10delta6 was derived from pBGH and contains the complete coding region of bGH and intron Bovine growth hormone introns B, C and D are absent ( Figure This plasmid encodes "wild type" bGH, and its expression controlled by a 1700 base pair segment of the mo metallothionein I transcriptional regulatory sequence.
  • G 119 R, A 122 D were derived from pBGH-10delta6 and were genera by segment-directed mutagenesis using complement oligonucleotides to replace the DNA between the Tthllll s
  • the complementary oligonucleotides used for pBGHIO delta 6-G: 119 R were:
  • oligonucleotides encode DNA changes whi result in the substitutions of arginine for glycine at positi 119 in pBGH-10delta6-G119R; and leucine for glutamate position 117, arginine for glycine at position 119 aspartate for alanine at position 122 in pBGH-lOdelta ⁇ -E 11 G 119 R, and A 122 D.
  • amino acids were chosen because t have hydrophilic (arginine and aspartic acid) or hydropho (leucine) character [See Hopp and Woods, PNAS (USA), 78:3824 (1981)], positively (arginine) or negatively (aspartic ac charged side chains [See Kaiser and Kezdy, Science ac 223:249-55 (1984)], and high c-helical-forming potential [ Chou and Fasman, Ann. Rev. Biochem., 47:251-76 (197 furthering generation of an idealized amphiphilic a-helix [ Margalit, et al., J.
  • Mutant plasmid DNA's were identif by digestion with BamHI restriction site which simplif screening procedures.
  • the oligonucleotides were annealed subcloned between the Tthllll and Xmal sites using stand procedures (Maniatis et al. , Molecular Cloning (Cold Spr Harbor: 1982). Mutant plasmid DNA's were identified digestion with BamHI.
  • the nucleotide sequence of the mutated bovine gro hormone target regions were determined by using the dideoxy chain-termination method with modified T7 DNA polymer
  • DNA sequencing were synthesized using the DuPont Coder #300 synthesizer and purified by denaturing polyacrylami.de electrophoresis, passive elution and concentration by etha precipitation.
  • the oligonucleotide primers used for the dir sequencing analysis of the two mutants was the following: 18
  • mutant bGH genes were generated initially: o converts glycine 119 to arginine ("G119R") and the seco converts glutamate 117 to leucine, glycine 119 to arginine, a alanine 122 to aspartate (E117L, G119R, A122D) .
  • the plasmids encoding these mutations as well as wi type bGH DNA were transiently introduced in cultured mouse L cells, which were subsequently analyzed f bGH expression. Following "western analysis", protein bands approximately 22,000 daltons were observed for wild type b and bGH derived from the two mutant genes.
  • transgenic mouse lines which contain wi type and mutant bGH genes were produced by standa microinjection techniques (McGrane et al. , 1988) .
  • D extraction from mouse tails, dot blots, and ser determinations was as described (McGrane et al. , 1988) .
  • the genes contain the transcriptional regulato sequences of the mouse metallothionein I promoter which h been shown to be active in liver tissue as well as oth tissues of the transgenic mouse (Palmiter et al.. Natur 300:611-615 (1982)). Offspring generated by the microinjecti procedure were assayed for bGH DNA by slot blot hybridizati analysis. Mouse lines were generated which con approximately one copy of the recombinant bGH DNA seque derived from pBGH-10delta6, (wild type), pBGH-lOdelta ⁇ -G 1 and pBGH10delta6-E 117 L, G 119 R, A 1 2 D.
  • Serum from transg animals were assayed for bGH levels by the Western techni All mice which expressed the wild type bGH transgene in s also possessed a corresponding enhanced growth rate. which expressed mutant bGH (G 119 R or E 117 L, G 119 R, A 122 D) serum were dramatically and significantly smaller.
  • the growth ratio for wild type bGH transg mice relative to control littermates was 1.5 while the r for the two bGH mutant mice to control littermates was -
  • the growth ratio bet the transgenic and nontransgenic littermates ranged from to 1.00.
  • the degree of suppression of growth was dire related to the serum levels of the mutated bGH.
  • Three foun have been bred that pass the trait to offspring; ⁇ 50% of t offspring are positive for the gene and possess corresponding small phenotype.
  • IGF insulin- growth factors
  • bGH-M8 is active as an in vivo antagonist to mouse GH (mG thereby suppressing mouse IGF-1 production. If this is t then one would expect not only a reduction in serum mouse IG levels in bGH M8 transgenic mice but also an increase in production in the pituitary.
  • the IG levels in the serum of the "small" transgenic mice are ⁇ those of normal non-transgenic mice while mice containing w type bGH (large mice) have approximately 2x the IGF-1 levels non-transgenic mice.
  • muteins of bGH and hGH wi alterations in the third alpha helix have been prepared a tested for secretion in L cells, and, in selected cases, the effect on the growth of transgenic mice, with the followi results.
  • mutants are described by giving the origin amino acid, its position in the amino acid sequence of bGH, a the replacement amino acid, with the amino acids set for according to the internationally accepted single letter cod George, et al., Protein Seq. Data Anal., 1:27-39 (1987).
  • a first set of mutated bGH genes when expressed transgenic mice, resulted in animals with a growth rati similar to that of mice which express wild type bGH (i.e., 1.59 - 1.72) .
  • wild type bGH i.e., 1.59 - 1.72
  • a second set of mutated bGH genes when expressed transgenic mice, resulted in mice with a growth ratio smal than those animals which express wild type bGH (i.e., bet 1.29 -1.35) .
  • We refer to these bGH analogs as "part functional agonists" and have listed them in Table II.
  • a fourth set of mutated bGH genes when expressed transgenic mice, resulted in mice with a growth ratio between 0.57 and 1.0 (Table IV).
  • the growth ratio of the m was negatively correlated with the serum level of the analog, i.e., as the serum level of the bgh analog increas the growth ratio of the animals decreased. This correlation shown graphically in Figure 13.
  • mice which expr either wild type hGH, hGH G120A, hGH G120R and hGH G120W (Ta V .
  • Mice which express hGH G120A show a growth enhan phenotype similar to mice which express wild type hGH (Ta V) .
  • this hGH analog a "functional agonist.”
  • substitution of R or W for G at position 120 in h and subsequent expression in transgenic mice results animals with a growth ratio between 0.73 and 0.96 (Table and whose level of serum hGH analogs is negatively correla with the growth phenotype; i.e., as the serum levels of t hGH 120 analogs increase, the growth ratios decrease.
  • T correlation is shown in Figure 14.
  • th hGH 120 analogs as “functional antagonist.” It is important note that the glycine residue in bGH at position 119 is homologue of the glycine residue in hGH at position 120. T are both located in the central portion of the third a-helix
  • the mutant K112L, K114W shows the effect of expand the hydrophobic face of the helix. This mutant affects ani growth much as does wild type growth hormone.
  • the mutations K114P, E118P and L121P apparently destroy the alpha hel (Proline is a strong alpha helix breaker.)
  • the growth-relat biological activity is abolished.
  • the mutation E126G is special case; glycine is a helix breaker, but position 126 at the end of the helix so the normal biological activity retained. With G119P, however, one strong helix breaker w substituted for an even stronger one; the alpha helix w apparently preserved.
  • the third alpha helix of wild type growth hormo diverges from a perfect amphiphilic alpha helix at thr positions.
  • Glu is a hydrophilic amino acid the hydrophobic face.
  • Gly is a neutral ami acid in the hydrophilic face.
  • Ala is hydrophobic amino acid in the hydrophilic face.
  • Mutant G119P produced the small animal phenot even though proline is as . hydrophilic glycine.
  • Mutant G119L produced the small animal phenot even though leucine is hydrophobic and theref disrupts the hydrophilic face of the helix.
  • the present invent relates to mutations of the third alpha helix which result growth-inhibitory activity yet reduce or leave unchanged amphiphilic character of the helix.
  • Additional growth hormone antagonists may identified by systematically varying the codon corresponding G119 in bGH, so as to express the 18 other mutants having single amino acid change at this position. This is read accomplished by synthesizing oligonucleotides differing f those set forth in Example 1 at codon 119 so as to encode desired alternative amino acid. Similarly, one may alter homologous glycine reside in the third alpha helix of ot GHs, e.g., the G 120 of hGH. By similar means, variations the codons corresponding to other amino acids of the th alpha helix of a GH are investigated.
  • Glycerophosphate dehydrogena has been used as a differentiation marker for th GHinduced adipose conversion. Wise and Green, J. Biol. Chem. 254:273-75 (1979); Nixon and Green, Endocrinology, 114:52 (1984); Pairault and Green, Proc. Nat. Acad. Sci. (US 76:5138 (1979).
  • This assay is a convenient screening tool identifying potential GH antagonists.
  • mice transgenic for the wild type bGH gene are kn to develop progressive severe glomerulosclerosis and increa glomerular size.
  • bGH-Mll mice i.e., L121P, E1 mutants
  • bGH-M8 mice howev which had reduced serum IGF-1, body size, and glomerular s relative to nontransgenic mice, glomerulosclerosis was absen
  • the level of mouse growth suppression is correlated w serum levels of analogs (see Fig. 13) .
  • These mutated bGH ge are expressed in and secreted mouse L cells.
  • the secret pattern is similar to wild type bGH.
  • bGH Gly 119 is in a position equivalent to hGH Gly 1 Therefore, we refer to hGH Gly 120 consistently with literature.
  • Table VI Summary of mutated bGH genes expressed in mous cell without transgenic mice dats.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Endocrinology (AREA)
  • Environmental Sciences (AREA)
  • Biotechnology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Biochemistry (AREA)
  • Diabetes (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Emergency Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Cardiology (AREA)
  • Hematology (AREA)
  • Obesity (AREA)

Abstract

Antagonistes, inhibiteurs de croissance, de l'hormone de croissance bovine, obtenus par mutation de la troisième hélice alpha de cette protéine. Ces nouvelles hormones peuvent être administrées de manière exogène à des animaux. On peut également produire des animaux transgéniques qui expriment ledit antagoniste et présentent par conséquent un phénotype de croissance réduite.
PCT/US1992/003532 1991-05-01 1992-05-01 Antagonistes de l'hormone de croissance WO1992019736A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP92911122A EP0659213A1 (fr) 1991-05-01 1992-05-01 Antagonistes de l'hormone de croissance
CA002102129A CA2102129C (fr) 1991-05-01 1992-05-01 Antagonistes de l'hormone de croissance
JP4510937A JPH06507632A (ja) 1991-05-01 1992-05-01 成長ホルモンアンタゴニスト
AU18930/92A AU666117B2 (en) 1991-05-01 1992-05-01 Growth hormone antagonists
NO19933909A NO314309B1 (no) 1991-05-01 1993-10-28 Veksthormonantagonistpeptid, vektor og vertcelle inneholdende en DNA-sekvens som koder for peptider, fremgangsmåte for fremstilling,farmasöytisk sammensetning inneholdende veksthormonantagonistpeptidet og anvendelse avveksthormonantagonistpept
FI934829A FI934829A0 (fi) 1991-05-01 1993-11-01 Antagonister foer tillvaexthormon

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US69330591A 1991-05-01 1991-05-01
US693,305 1991-05-01

Publications (1)

Publication Number Publication Date
WO1992019736A1 true WO1992019736A1 (fr) 1992-11-12

Family

ID=24784129

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/003532 WO1992019736A1 (fr) 1991-05-01 1992-05-01 Antagonistes de l'hormone de croissance

Country Status (7)

Country Link
EP (1) EP0659213A1 (fr)
JP (1) JPH06507632A (fr)
AU (1) AU666117B2 (fr)
CA (1) CA2102129C (fr)
FI (1) FI934829A0 (fr)
NO (1) NO314309B1 (fr)
WO (1) WO1992019736A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994019004A1 (fr) * 1993-02-19 1994-09-01 Genentech, Inc. Antagonistes de ligands utilises dans le traitement du cancer du sein
US5506107A (en) * 1991-05-10 1996-04-09 Genentech, Inc. Selecting ligand agonists and antagonists
WO1996040203A1 (fr) * 1995-06-07 1996-12-19 Ohio University/Edison Biotechnology Institute Methodes de traitement utilisant des antagonistes de l'hormone de croissance
WO1997011178A1 (fr) * 1995-09-21 1997-03-27 Genentech, Inc. Variants de l'hormone de croissance humaine
EP0790305A1 (fr) * 1996-02-13 1997-08-20 JCR PHARMACEUTICALS Co., LTD. Mutante menschlichen Wachstumhormone und deren Verwendung
US5854026A (en) * 1988-10-28 1998-12-29 Genentech, Inc. Human growth hormone variant having enhanced affinity for human growth hormone receptor at site 1
US5958879A (en) * 1989-10-12 1999-09-28 Ohio University/Edison Biotechnology Institute Growth hormone receptor antagonists and methods of reducing growth hormone activity in a mammal
US6429186B1 (en) 1991-05-10 2002-08-06 Genentech, Inc. Ligand antagonists for treatment of breast cancer
US6583115B1 (en) 1989-10-12 2003-06-24 Ohio University/Edison Biotechnology Institute Methods for treating acromegaly and giantism with growth hormone antagonists

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3820497A4 (fr) * 2018-07-11 2022-03-23 Ohio University Inhibiteurs peptidiques de l'action de l'hormone de croissance et leurs procédés d'utilisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991005853A1 (fr) * 1989-10-12 1991-05-02 Ohio University Antagonistes d'hormones de croissance

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991005853A1 (fr) * 1989-10-12 1991-05-02 Ohio University Antagonistes d'hormones de croissance

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ENDOCRINOLOGY vol. 129, no. 3, September 1991, BALTIMORE, US pages 1402 - 1408; CHEN, W.Y. ET AL.: 'Functional antagonism between endogenous mouse growth hormone (GH) and a GH analog results in dwarf transgenic mice' *
ENDOCRINOLOGY vol. 130, no. 4, April 1992, BALTIMORE, US pages 2284 - 2290; OKADA, S. ET AL.: 'A growth hormone (GH) analog can antagonize the ability of native GH to promote differentiation of 3T3-F442A preadipocytes and stimulate insulin-like and lipolytic activities in primary rat adipocytes' *
JOURNAL OF BIOLOGICAL CHEMISTRY. vol. 266, no. 4, 5 February 1991, BALTIMORE US pages 2252 - 2258; CHEN, W.Y. ET AL.: 'Mutations in the third alpha-helix of bovine growth hormone dramatically affect its intracellular distribution in vitro and growth enhancement in transgenic mice' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 87, July 1990, WASHINGTON US pages 5061 - 5065; CHEN, W.Y. ET AL.: 'Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice' *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6143523A (en) * 1988-10-28 2000-11-07 Genentech, Inc. Human growth hormone variants
US6022711A (en) * 1988-10-28 2000-02-08 Genentech, Inc. Human growth hormone variants having enhanced affinity for human growth hormone receptor at site 1
US5854026A (en) * 1988-10-28 1998-12-29 Genentech, Inc. Human growth hormone variant having enhanced affinity for human growth hormone receptor at site 1
US6583115B1 (en) 1989-10-12 2003-06-24 Ohio University/Edison Biotechnology Institute Methods for treating acromegaly and giantism with growth hormone antagonists
US5958879A (en) * 1989-10-12 1999-09-28 Ohio University/Edison Biotechnology Institute Growth hormone receptor antagonists and methods of reducing growth hormone activity in a mammal
US5506107A (en) * 1991-05-10 1996-04-09 Genentech, Inc. Selecting ligand agonists and antagonists
US6429186B1 (en) 1991-05-10 2002-08-06 Genentech, Inc. Ligand antagonists for treatment of breast cancer
US6800740B1 (en) 1991-05-10 2004-10-05 Genentech, Inc. Variants of native growth hormones having non naturally occurring amino acid sequences or covalent modifications
US6936440B1 (en) 1991-05-10 2005-08-30 Genentech, Inc. Selecting ligand agonists and antagonists
AU685434B2 (en) * 1993-02-19 1998-01-22 Genentech Inc. Ligand antagonists for treatment of breast cancer
WO1994019004A1 (fr) * 1993-02-19 1994-09-01 Genentech, Inc. Antagonistes de ligands utilises dans le traitement du cancer du sein
US6004931A (en) * 1993-05-25 1999-12-21 Genentech, Inc. Method for inhibiting growth hormone action
US6136563A (en) * 1993-05-25 2000-10-24 Genentech, Inc. Human growth hormone variants comprising amino acid substitutions
WO1996040203A1 (fr) * 1995-06-07 1996-12-19 Ohio University/Edison Biotechnology Institute Methodes de traitement utilisant des antagonistes de l'hormone de croissance
WO1997011178A1 (fr) * 1995-09-21 1997-03-27 Genentech, Inc. Variants de l'hormone de croissance humaine
US5849535A (en) * 1995-09-21 1998-12-15 Genentech, Inc. Human growth hormone variants
AU718439B2 (en) * 1995-09-21 2000-04-13 Genentech Inc. Human growth hormone variants
US6057292A (en) * 1995-09-21 2000-05-02 Genentech, Inc. Method for inhibiting growth hormone action
EP0790305A1 (fr) * 1996-02-13 1997-08-20 JCR PHARMACEUTICALS Co., LTD. Mutante menschlichen Wachstumhormone und deren Verwendung
US6238915B1 (en) 1996-02-13 2001-05-29 Jcr Pharmaceuticals Co., Ltd. Mutant human growth hormones and their uses
AU723494B2 (en) * 1996-02-13 2000-08-31 Jcr Pharmaceuticals Co., Ltd. Mutant human growth hormones and their uses

Also Published As

Publication number Publication date
JPH06507632A (ja) 1994-09-01
NO314309B1 (no) 2003-03-03
NO933909L (no) 1993-12-20
AU666117B2 (en) 1996-02-01
AU1893092A (en) 1992-12-21
NO933909D0 (no) 1993-10-28
FI934829A (fi) 1993-11-01
FI934829A0 (fi) 1993-11-01
CA2102129C (fr) 2003-04-01
CA2102129A1 (fr) 1992-11-02
EP0659213A1 (fr) 1995-06-28

Similar Documents

Publication Publication Date Title
US5681809A (en) Growth hormone antagonists
US6583115B1 (en) Methods for treating acromegaly and giantism with growth hormone antagonists
US7101971B2 (en) Erythropoietin analog-human serum albumin fusion
Pursel et al. Genetic engineering of livestock
US6897290B1 (en) Modified RCA proteins
US20050181482A1 (en) Method for the production of an erythropoietin analog-human IgG fusion proteins in transgenic mammal milk
AU652545B2 (en) Growth hormone antagonists
US5958879A (en) Growth hormone receptor antagonists and methods of reducing growth hormone activity in a mammal
AU782925B2 (en) Myostatin and mimetics thereof
US6787336B1 (en) DNA encoding growth hormone antagonists
AU666117B2 (en) Growth hormone antagonists
CA2475273A1 (fr) Developpement modifie de l'hypophyse chez la progeniture de femelles animales portantes traitees par la therapie hormonale de liberation de l'hormone de croissance
RU2668174C2 (ru) Суперагонисты гликопротеинового гормона длительного действия
EP0893950A1 (fr) Activation selective de cellules cibles par expression d'un recepteur superieurement active par un ligand synthetique et couple a une proteine g
AU691525B2 (en) Modified truncated complement system regulators
JP2004500890A (ja) 遺伝子導入によって作成された血小板由来増殖因子
KR20070007894A (ko) 항비만약
WO1996040203A1 (fr) Methodes de traitement utilisant des antagonistes de l'hormone de croissance
WO2001072119A2 (fr) Modele animal non-humain permettant d'etudier la croissance insuffisante et le traitement de l'information ou les anomalies de la fonction cognitive et utilisation de ce modele
Kopchick et al. Structure/function studies of the bovine growth hormone third α-helical region employing transgenic mice
EP1493446A1 (fr) Régulateurs modifiés tronqués du système du complement
Majumdar Structural and functional divergence of the transcription factor Pit-1: analysis of the pou and transactivation domains.
NZ521152A (en) Assays for detecting the presence of myostatin and antagonists or agonists thereof using isolated myoblasts
NZ521151A (en) Truncated mature myostatin proteins and Piedmontese alleles and uses thereof in promoting muscle growth

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA FI JP KR NO

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

COP Corrected version of pamphlet

Free format text: PAGES 1/14,2/14,5/14,9/14 AND 10/14,DRAWINGS,REPLACED BY NEW PAGES BEARING THE SAME NUMBER

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2102129

Country of ref document: CA

Ref document number: 1992911122

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 934829

Country of ref document: FI

WWP Wipo information: published in national office

Ref document number: 1992911122

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1992911122

Country of ref document: EP