US20030186382A1 - Novel antiangiogenic peptide agents and their therapeutic and diagnostic use - Google Patents
Novel antiangiogenic peptide agents and their therapeutic and diagnostic use Download PDFInfo
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- US20030186382A1 US20030186382A1 US09/819,094 US81909401A US2003186382A1 US 20030186382 A1 US20030186382 A1 US 20030186382A1 US 81909401 A US81909401 A US 81909401A US 2003186382 A1 US2003186382 A1 US 2003186382A1
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- 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
-
- 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
- C07K14/57518—Placental lactogen; Chorionic somatomammotropin
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- angiogenesis A growing number of serious, debilitating and often fatal diseases are associated with angiogenesis. These diseases are cumulatively called angiogenic diseases. Under normal physiological conditions, angiogenesis in mammals is endogenously controlled throughout the lifetime and neovascularization rarely occurs except during embryonic development, the reproductive cycle, and wound healing.
- Examples of the pathological conditions leading to development of angiogenic diseases are, among others, arthritis, rheumatoid arthritis, atherosclerotic plaques, corneal graft neovascularization, wound healing, hypertrophic or keloid scars, proliferative retinopathy, diabetic retinopathy, macular degeneration, granulations, neovascular glaucoma and uveitis.
- angiogenesis accompanies the growth of the placenta and solid tumors.
- Many tumors seem to produce factors which increase cell division of vascular endothelial cells and stimulate the migration and organization of endothelial cells into vessels resulting in neovascularization.
- factors which inhibit angiogenesis may be turned off.
- Activation of angiogenesis therefore, appears to be an essential stage in tumor progression.
- Various mammalian endogenous molecules have been identified as angiogenic factors stimulating angiogenesis directly or indirectly. These stimulators of angiogenesis include basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor (VEGF), tumor necrosis factor- ⁇ (TNF- ⁇ ), and angiogenin.
- bFGF basic fibroblast growth factor
- VEGF vascular endothelial cell growth factor
- TNF- ⁇ tumor necrosis factor- ⁇
- angiogenin angiogenin
- Human hormones such as growth hormone (hGH), prolactin (hPRL), placental lactogen (hPL) or growth hormone variant (hGH-V) are homologous protein hormones which are potent endogenous chemical substances asserting specific biological activities on their respective target organs. The biological activities of these hormones are not the same and they differ depending on the hormone and/or the hormone target organ.
- growth hormone which is a protein of the anterior lobe of the pituitary gland, promotes and regulates body growth and morphogenesis, fat mobilization and inhibitions of glucose utilization ( Trends Endocrinol. Metab., 3:117(1992)).
- Prolactin is a known hormone produced by the pituitary gland in all mammals.
- the normal biological function and activity of prolactin in mammals include regulation of reproduction, osmoregulation, the stimulation of milk production by the mammary gland, the modulation of steroidogenesis in the gonads, the stimulation of maternal behavior, and the modulation of immune function ( Life Sci., 57:1 (1995)).
- the human placenta expresses two proteins with significant structural homology to human growth hormone, namely human placental lactogen and a variant of human growth hormone, hGH-V, differing by 13 amino acid substitutions ( Hormonal Proteins Peptides, 4:61 (1977)).
- Human placental lactogen is somatotropic in fetal tissues and aids in stimulating mammary cell proliferation ( Endocrin. Rev., 12:316 (1991)). Human growth hormone variant rather than pituitary growth hormone regulates maternal metabolism during the second half of the pregnancy ( Endocrinology, 133:1292 (1993)). As described in Endocrinology, 121:2055 (1987) rodent placentas express and secrete several proteins such as proliferin and proliferin-related peptide possessing biological actions similar to prolactin rather than growth hormone.
- the human placenta serves as the major respiratory, nutritional and endocrine organ throughout fetal life and is critical to the survival and healthy development of the fetus.
- the placenta provides an intimate interface between the maternal and fetal blood supplies. Pathological impairment of placental invasion and poor vascular development has been associated with both fetal and maternal complications.
- Intrauterine fetal growth restriction is a condition that affects approximately 500,000 pregnancies annually in the United States. As a result of poor placental blood supply, and decreased fetal oxygenation and nutrition, this condition results in small-for-dates infants. These infants have a neonatal mortality rate 6-10 times higher than that of normal infants and are at risk for pulmonary and neurological problems at birth.
- Preeclampsia a condition that affects approximately 250,000 American pregnancies each year, puts mothers and their fetuses at high risk. Maternal hypertension, renal failure, hepatic failure, coagulopathy, cerebral edema seizures and stroke are the potential consequences of this syndrome. Fetal morbidity is 5 times higher than normal in preeclampsia.
- One aspect of the invention is an anti-angiogenic peptide substantially identical to about 10 to about 150 consecutive amino acids selected from the N-terminal end of human placental lactogen, human growth hormone, growth hormone variant hGH-V, or human prolactin, wherein the peptide
- (iii) binds to at least one specific receptor which does not bind an intact full length growth hormone, placental lactogen, prolactin, or growth hormone variant hGH-V.
- a further aspect of the invention is an isolated nucleic acid encoding the anti-angiogenic peptide of the invention.
- the nucleic acid may be DNA or RNA, and may comprise cDNA sequences.
- a further aspect of the invention is a host cell comprising the nucleic acid encoding the anti-angiogenic peptide of the invention.
- a further aspect of the invention is a method of producing the peptide of the invention, comprising expressing the nucleic acid in the host cell, and recovering the peptide.
- a further aspect of the invention is a method of treating an angiogenic disease in a subject, the method comprising administering to a subject in need of such treatment an angiogenesis inhibitory effective amount of the antiangiogenic peptide of the invention.
- a further aspect of the invention is a method of inhibiting tumor formation or growth in a patient, the method comprising administering to the patient an angiogenesis inhibitory effective amount of the antiangiogenic peptide of the invention.
- a further aspect of the invention is a method for diagnosing a probable abnormality of placental vascularization during pregnancy comprising assaying a level of at least one of endogenous N-terminal fragments of growth hormone, prolactin, growth hormone variant hGH-V, and placental lactogen in a tissue sample from a patient; and
- a level of the at least one of endogenous N-terminal fragments higher than the average level is a probable abnormality of placental vascularization during pregnancy.
- a further aspect of the invention is a method of modulating vascularization of a patient's placenta, the method comprising administering to the patient an angiogenesis inhibitory effective amount of the anti-angiogenic peptide of the invention.
- FIG. 1 is a graph showing in vitro antiangiogenic effect of peptides of the invention detected as an inhibition of bovine brain capillary endothelial cell proliferation.
- FIG. 2 is a graph of relative antiangiogenic activity in the chick chorioallantoic membrane of 16K N-terminal fragment peptides of human growth hormone, growth hormone variant hGH-V, human placental lactogen and human prolactin compared to their-respective intact 22 or 23K polypeptides and to a bovine serum albumin control.
- FIG. 3 comprises FIGS. 3 A-H, which are photographs of a subset of data from FIG. 2 comparing in vivo neovascularization of the chick chorioallantoic membrane following the treatment with the peptides of the invention.
- FIG. 4 comprises FIGS. 4 A-C.
- FIG. 4A is a Western blot analysis of tyrosine phosphorylation of mitogen-activated protein kinase.
- FIG. 4B is a graph depicting the percent level of inhibition of the bFGF stimulation of MAPK enzymatic activity by 16K N-terminal fragments.
- FIG. 3C is a graph depicting percent of stimulation over control by intact hormones without the presence of 16K fragment.
- FIG. 5 comprises FIGS. 5 A- 5 B.
- FIG. 5A depicts Western blot analysis of plasminogen activator inhibitor-1 protein levels and their quantitation following treatment with 16K N-terminal fragments or intact hormones.
- FIG. 5B is a Western blot analysis of plasminogen activator inhibitor (PAI-1) in untreated cells and cells treated with 16K N-terminal hGH, hGH, or a combination thereof.
- PAI-1 plasminogen activator inhibitor
- FIG. 6 depicts a silver stained SDS-polyacrylamide gels showing cleavage of human prolactin by cathepsin D in vitro.
- FIG. 7 is Western blot of human amniotic fluid prolactin showing cleavage of human prolactin in amniotic fluid into fragments of 18 kD and 13 kD.
- FIG. 8 comprises FIGS. 8A and 8B and depicts HPLC fractions of human prolactin cleaved with cathepsin D ( 8 A) and assayed for their ability to inhibit bFGF-induced bovine brain capillary endothelial (BBCE) cell 3 H-thymidine incorporation ( 8 B).
- BBCE bovine brain capillary endothelial
- FIG. 9 is a graph depicting increases in the number of 16K N-terminal hPRL binding sites in the human placenta throughout pregnancy expressed as specific binding.
- the invention provides novel antiangiogenic peptides which are potent inhibitors of angiogenesis.
- These peptides in general correspond to about 10 to about 150 consecutive amino acids, more preferably about 10 to about 100, most preferably about 10 to about 50 consecutive amino acids selected from the amino acid sequences of N-terminal fragments of about 13-16 kD (“16K”) derived from full length human growth hormone, the growth hormone variant hGH-V, human placental lactogen or human prolactin.
- 16K 13-16 kD
- peptides “substantially identical” in amino acid sequence are also included in the scope in the invention.
- the about 13-16K N-terminal fragments of the full length hormones are also referred to herein as “16K N-terminal human hormone” (16K hGH), “16K N-terminal human placental lactogen” (16K hPL), “16K N-terminal growth hormone variant hGH-V” (16K hGHV), and “16K N-terminal human prolactin” (16K hPRL).
- the peptides of the invention have the properties of (i) inhibiting capillary endothelial cell proliferation and organization; (ii) inhibiting angiogenesis in chick chorioallantoic membrane; and (iii) binding to at least one specific receptor which does not bind an intact full length growth hormone, placental lactogen or growth hormone variant hGH-V.
- 16K N-terminal fragments cleaved from this group of hormones bind to at least one new, previously undisclosed receptor or receptors other than their own specific receptors known in the art and have specific and distinguishable antiangiogenic activity is surprising and unexpected.
- the full length intact hormones have no antiangiogenic activity but are angiogenic in some settings.
- the antiangiogenic activity of the 16K N-fragments is not mediated via the intact hormones' own specific receptors.
- the peptides of the invention have similar biological potencies acting in the nM concentrations. Typically, one-half of the inhibitory action IA 50 is achieved at a very low concentration of about 0.8-1 nM.
- the peptides bind to the new receptor(s) with high affinity and in a saturable manner. Intact full length growth hormone, placental lactogen, growth hormone variant hGH-V, and bFGF do not compete for the same receptor site(s) and have different activities as well as levels of activities.
- the peptides of the invention inhibit the growth of bovine brain capillary endothelial (BBCE) cell proliferation confirming their antiangiogenic activity. Additionally, the peptides inhibit the mitogenic activity of bFGF and VEGF. While 16K N-terminal human growth hormone has this activity, rat 16K N-terminal growth hormone is not antiangiogenic.
- BBCE bovine brain capillary endothelial
- the peptides of the invention and their respective pharmaceutical compositions and preparations which are capable of inhibiting angiogenesis are useful for preventing or treating any disease or condition which is associated with or results in or from angiogenesis.
- diseases include formation of malignant tumors, angiofibroma, arteriovenous malformations, arthritis, such as rheumatoid arthritis, atherosclerotic plaques, corneal graft neovascularization, delayed wound healing, proliferative retinopathy such as diabetic retinopathy, macular degeneration, granulations such as those occurring in hemophilic joints, inappropriate vascularization in wound healing such as hypertrophic scars or keloid scars, neovascular glaucoma, ocular tumor, uveitis, non-union fractures, Osler-Weber syndrome, psoriasis, pyogenic glaucoma, retrolental fibroplasia, scleroderma, solid tumors, Kaposi's sar
- the peptides of the invention are also suitable for use in a method of inhibiting mammalian cell proliferation and organization that depends on vascularization, including the selective inhibition of vascularization of tumors, tumor size reduction and elimination.
- tumors undergoing angiogenesis include but are not limited to angiofibroma, arteriovenous malformations, ocular tumors, all solid tumors, Kaposi's sarcoma, trachoma and choriocarcinoma.
- the peptides of the invention may be used to assess and/or modulate the development of the vasculature of the placenta. Regulation of placental vascularization has important clinical implications, since two disorders of pregnancy, preeclampsia and intrauterine growth retardation, are associated with impairment of vascular development. No clinical tests exist to predict the occurrence of these disorders until pregnancy is seriously compromised.
- the peptides of the invention can also be used as contraceptive agents.
- Radioimmunoassay provides a diagnostic assay for the probable impairment of vascular development associated with preeclampsia, intrauterine growth retardation, and placental dysfunction.
- the activity level of enzymes which generate such fragments can also be measured as an indication of probable vascular abnormalities in the placenta. Measurements are typically done in the first trimester, but may be performed in the second and/or third trimesters.
- the diagnostic assay would comprise assaying a level of at least one of endogenous N-terminal fragments of prolactin, growth hormone, growth hormone variant hGH-V, and placental lactogen in a tissue sample from a patient; and comparing the level of the at least one of endogenous N-terminal fragments to an average level of the at least one of endogenous N-terminal fragments in a normal patient population; wherein a level of the at least one of endogenous N-terminal fragments higher than the average level is a probable abnormality of placental vascularization during pregnancy.
- the specified antibodies bind to a particular protein and do not bind in a significant amont to other proteins present in the sample.
- Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
- a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
- solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
- the peptides of the current invention can, for example, be synthesized, prepared from purified full-length hormones, or produced using recombinant methods and techniques known in the art. Although specific techniques for their preparation are described herein, it is to be understood that all appropriate techniques suitable for production of these peptides are intended to be within the scope of this invention.
- these techniques include DNA and protein sequencing, cloning, expression and other recombinant engineering techniques permitting the construction of prokaryotic and eukaryotic vectors encoding and expressing each of the peptides of the invention.
- the peptides of this invention are conveniently obtained by isolation of intact growth hormone from the human pituitary gland or plasma and isolation of lactogen and growth hormone variant hGH-V.
- the isolated intact hormones may be glycosylated and cleaved to varying degrees.
- the peptides may be prepared by peptide synthesis according to method described in Biotechnology and Applied Biochem., 12:436 (1990) or by methods described in Current Protocols in Molecular Biology, Eds. Ausubel, F. M., et al, John Wiley & Sons, N.Y. (1987).
- the peptides of the invention may be produced by expression of a nucleic acid encoding a peptide of interest, or by cleavage from a longer length polypeptide encoded by the nucleic acid. Expression of the encoded polypeptides may be done in bacterial, yeast, plant, insect, or mammalian hosts by techniques well known in the art. As for example, 16K hPRL has been produced in eukaryotic HCT 116 cells. The cDNA coding for the 16K hPRL (stop 140) has been cloned into the mammalian expression vector pRC-CMV.
- the construct was transferred into the HCT116 human colon cancer cells and stably transfected cells expressing 16K hPRL (stop 140) were obtained. Either conditioned media or purified 16K hPRL (stop 140) from these cells were able to inhibit bFGF-induced BBCE proliferation.
- a peptide of interest of the invention is obtained by cloning the DNA sequence encoding an intact full length human hormone into a vector; modifying the DNA codon corresponding to the last amino acid of a desired 16K N-terminal hormone fragment to a stop codon by mutagenesis techniques known in the art; and transforming a host cell with the modified nucleic acid to allow expression of the encoded peptide.
- the cloned hormone DNA is engineered to create a proteolytic cleavage site within the hormone polypeptide. The polypeptide is then cleaved after production in the host to generate the peptide of interest. Examples of mutagenesis techniques include, for example, methods described in Promega Protocols and Applications Guide, Promega Corp, Madison, Wis., p. 98 (1891) or according to Current Protocols in Molecular Biology, supra.
- the DNA sequence encoding human hormone preferably does not contain a signal peptide sequence.
- a DNA codon for methionine (Met) is typically inserted upstream of 5′ to the first DNA codon of the coding sequence.
- reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence.
- Optimal alignment of sequences for aligning a comparison window may, for example, be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. U.S.A. 85:2444 (1988), or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).
- the terms “substantial identity” or “substantial sequence identity” mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity or more.
- Percentage amino acid identity or “percentage amino acid sequence identity” refers to a comparison of the amino acids of two polypeptides which, when optimally aligned, have approximately the designated percentage of the same amino acids.
- “95% amino acid identity” refers to a comparison of the amino acids of two polypeptides which when optimally aligned have 95% amino acid identity.
- residue positions which are not identical differ by conservative amino acid substitutions.
- the substitution of amino acids having similar chemical properties such as charge or polarity are not likely to effect the properties of a protein. Examples include glutamine for asparagine or glutamic acid for aspartic acid.
- substantially purified when referring to a peptide or protein, means a chemical composition which is essentially free of other cellular components. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
- a protein which is the predominant species present in a preparation is substantially purified. Generally, a substantially purified or isolated protein will comprise more than 80% of all macromolecular species present in the preparation.
- the protein is purified to represent greater than 90% of all macromolecular species present. More preferably the protein is purified to greater than 95%, and most preferably the protein is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional techniques.
- nucleic acids that comprise DNA or RNA sequences encoding the peptides of the invention.
- The. nucleic acids of the invention may further comprise vectors for expression of the peptides of the invention.
- the DNA may comprise cDNA sequences encoding full-length hormones or sequences encoding N-terminal regions of the hormones. It is understood by one of ordinary skill in the art that because of degeneracy in the genetic code, substitutions in the nucleotide sequence may be made which do not result in changes in the encoded amino acid sequence. Thus, “substantially identical” sequences as defined herein are included in the scope of the invention. It is further understood by one of ordinary skill in the art that both complementary strands of any DNA molecule described herein are included within the scope of the invention.
- substantially identical or “substantial sequence identity” as applied to nucleic acid sequences and as used herein and denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, and more preferably at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
- the reference sequence may be a subset of a larger sequence.
- agonists of the receptor(s) for the anti-angiogenic peptides are provided.
- Such agonists include but are not limited to mutants of the peptides of the invention and peptide, non-peptide, and peptidomimetic analogs of the peptides of the invention.
- the method for treatment of angiogenic diseases comprises administering to a patient an angiogenesis inhibitory amount of one or more peptides of the invention.
- treatment is intended to refer to the prevention, amelioration, or reduction in severity of a symptom of angiogenesis.
- an angiogenic-inhibitory effective dose of a peptide of the invention is a dose sufficient to prevent, ameliorate, or reduce the severity of a symptom of angiogenesis.
- the peptides of the invention may be administered singly or in combination with each other or other angiogenesis inhibitory agents.
- the peptides of the invention are administered in an amount of about 8 micrograms to 3,000 ⁇ g/kg per day, and more preferably about 20 to 1,500 ⁇ g/kg per day preferably once or twice daily. However, other amounts, including substantially lower or higher amounts, may also be administered.
- the peptides of the invention are administered to a human subject in need of antiangiogenic treatment intramuscularly, subcutaneously, intravenously, intratumorally, by any other acceptable route of administration. In the case of ocular angiogenic diseases, the peptide may also be administered topically to the eye.
- the present peptide may be suitably utilized for the inhibition or retardation of this process.
- the composition of the invention to be administered may comprise an amount of the peptide about 12 to 3,500 ⁇ g/kg per day, and preferably about 25 to 2,700 ⁇ g/kg per day.
- different amounts of the peptide may also be administered as seen suitable by a practitioner for specific cases. Smaller amounts may be administered by injection into the anterior chamber of the eye.
- the peptide of this invention may be administered in an amount of about 10 to 3,750 ⁇ g/kg, and more preferably about 15 to 1,600 ⁇ g/kg. Any means of administration is suitable.
- Gene therapy techniques have the potential for limiting the exposure of a subject to a gene product, such as polypeptide, by targeting the expression of the therapeutic gene to a tissue of interest, such as skeletal muscle, myocardium, vascular endothelium or smooth muscle, or solid or circulating tumor cells.
- a tissue of interest such as skeletal muscle, myocardium, vascular endothelium or smooth muscle, or solid or circulating tumor cells.
- WIPO Patent Application Publication No. WO 93/15609 discloses the delivery of interferon genes to vascular tissue by administration of such genes to areas of vessel wall injury using a catheter system.
- an adenoviral vector encoding a protein capable of enzymatically converting a prodrug, a “suicide gene”, and a gene encoding a cytokine are administered directly into a solid tumor.
- Transductional targeting refers to the selective entry into specific cells, achieved primarily by selection of a receptor ligand.
- Positional targeting within the genome refers to integration into desirable loci, such as active regions of chromatin, or through homologous recombination with an endogenous nucleotide sequence such as a target gene.
- Transcriptional targeting refers to selective expression attained by the incorporation of transcriptional promoters with highly specific regulation of gene expression tailored to the cells of interest.
- tissue-specific promoters include a liver-specific promoter (Zou et al., Endocrinology 138:1771-1774 (1997)); a small intestine-specific promoter (Olivera et al., J. Biol. Chem. 271:31831-31838 (1996)); the promoter for creatine kinase, which has been used to direct of dystrophin cDNA expression in muscle and cardiac tissue (Cox et al., Nature 364:725-729 (1993)); and immunoglobulin heavy or light chain promoters for the expression of suicide genes in B cells a (Maxwell et al., Cancer Res. 51:4299-4304 (1991)).
- Amphotrophic retroviral vectors have been constructed carrying a herpes simplex virus thymidine kinase gene under the control of either the albumin or alpha-fetoprotein promoters (Huber et al., Proc. Natl. Acad. Sci. U.S.A. 88:8039-8043 (1991)) to target cells of liver lineage and hepatoma cells, respectively.
- tissue specific promoters can be used in retroviral vectors (Hartzoglou et al., J. Biol. Chem. 265:17285-17293 (1990)) and adenovirus vectors (Friedman et al., Mol. Cell. Biol. 6:3791-3797 (1986)) and still retain their tissue specificity.
- Other elements aiding specificity of expression in a tissue of interest can include secretion leader sequences, enhancers, nuclear localization signals, endosmolytic peptides, etc.
- these elements are derived from the tissue of interest to aid specificity.
- Viral vector systems useful in the practice of the instant invention include but are not limited to adenovirus, herpesvirus, adeno-associated virus, minute virus of mice (MVM), HIV, Sindbis virus, and retroviruses such as Rous sarcoma virus, and MoMLV.
- the nucleic acid encoding the therapeutic polypeptide or peptide of interest is inserted into such vectors to allow packaging of the nucleic acid, typically with accompanying viral DNA, infection of a sensitive host cell, and expression of the polypeptide or peptide of interest.
- nucleic acid encoding a therapeutic polypeptide or peptide of interest is conjugated to a cell receptor ligand for facilitated uptake (e.g., invagination of coated pits and internalization of the endosome) through a DNA linking moiety (Wu et al., J. Biol. Chem. 263:14621-14624 (1988); WO 92/06180).
- a DNA linking moiety Wang et al., J. Biol. Chem. 263:14621-14624 (1988); WO 92/06180.
- the DNA constructs of the invention can be linked through a polylysine moiety to asialo-oromucoid, which is a ligand for the asialoglycoprotein receptor of hepatocytes.
- viral envelopes used for packaging the recombinant constructs of the invention can be modified by the addition of receptor ligands or antibodies specific for a receptor to permit receptor-mediated endocytosis into specific cells (e.g., WO 93/20221, WO 93/14188; WO 94/06923).
- the DNA constructs of the invention are linked to viral proteins, such as adenovirus particles, to facilitate endocytosis (Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991)).
- molecular conjugates of the instant invention can include microtubule inhibitors (WO 94/06922); synthetic peptides mimicking influenza virus hemagglutinin (Plank et al., J. Biol. Chem. 269:12918-12924 (1994)); and nuclear localization signals such as SV40 T antigen (WO 93/19768).
- the nucleic acid can be introduced into the tissue of interest in vivo or ex vivo by a variety of methods.
- the nucleic acid is introduced into cells by such methods as microinjection, calcium phosphate precipitation, liposome fusion, or biolistics.
- the nucleic acid is taken up directly by the tissue of interest.
- nucleic acid is packaged into a viral vector system to facilitate introduction into cells.
- compositions of the invention are administered ex vivo to cells or tissues explanted from a patient, then returned to the patient.
- ex vivo administration of gene therapy constructs include Arteaga et al., Cancer Research 56(5):1098-1103 (1996); Nolta et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23 (1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); al., J. Thorac. Cardi. Surg. 11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).
- compositions of the invention will be formulated for administration by manners known in the art acceptable for administration to a mammalian subject, preferably a human.
- the compositions of the invention can be administered directly into a tissue by injection or into a blood vessel supplying the tissue of interest.
- the compositions of the invention are administered “locoregionally”, i.e., intravesically, intralesionally, and/or topically.
- the compositions of the invention are administered systemically by injection, inhalation, suppository, transdermal delivery, etc.
- compositions are administered through catheters or other devices to allow access to a remote tissue of interest, such as an internal organ.
- the compositions of the invention can also be administered in depot type devices, implants, or encapsulated formulations to allow slow or sustained release of the compositions.
- formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, (15th Edition, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87, by Blaug, Seymour, therein.
- These formulations include for example, powders, pastes, ointments, jelly, waxes, oils, lipids, anhydrous absorption bases, oil-in-water or water-in-oil emulsions, emulsions carbowax (polyethylene glycols of a variety of molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
- any of the foregoing formulations may be appropriate in treatments and therapies in accordance with the present invention, provided that the active agent in the formulation is not inactivated by the formulation and the formulation is physiologically compatible.
- the quantities of active ingredient necessary for effective therapy will depend on many different factors, including means of administration, target site, physiological state of the patient, and other medicaments administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the active ingredients. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, for example, in Goodman and Gilman's the Pharmacological Basis of Therapeutics, 7th Edition (1985), MacMillan Publishing Company, New York, and Remington's Pharmaceutical Sciences 18 th Edition, (1990) Mack Publishing Co, Easton Pa. Methods for administration are discussed therein, including oral, intravenous, intraperitoneal, intramuscular, transdermal, nasal, iontophoretic administration, and the like.
- compositions of the invention may be administered in a variety of unit dosage forms depending on the method of administration.
- unit dosage forms suitable for oral administration include solid dosage forms such as powder, tablets, pills, capsules, and dragees, and liquid dosage forms, such as elixirs, syrups, and suspensions.
- the active ingredients may also be administered parenterally in sterile liquid dosage forms.
- Gelatin capsules contain the active ingredient and as inactive ingredients powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.
- inactive ingredients examples include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like.
- Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract.
- Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
- compositions of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
- compositions of the invention may also be administered via liposomes.
- Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
- the composition of the invention to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a desired target, such as antibody, or with other therapeutic or immunogenic compositions.
- liposomes either filled or decorated with a desired composition of the invention can delivered systemically, or can be directed to a tissue of interest, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions.
- Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
- the selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
- a liposome suspension containing a composition of the invention may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the composition of the invention being delivered, and the stage of the disease being treated.
- nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
- a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more compositions of the invention, and more preferably at a concentration of 25%-75%.
- compositions of the invention are preferably supplied in finely divided form along with a surfactant and propellant.
- Typical percentages of compositions of the invention are 0.01%-20% by weight, preferably 1%-10%.
- the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
- Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
- Mixed esters such as mixed or natural glycerides may be employed.
- the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
- the balance of the composition is ordinarily propellant.
- a carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
- compositions of the invention can additionally be delivered in a depot-type system, an encapsulated form, or an implant by techniques well-known in the art. Similarly, the compositions can be delivered via a pump to a tissue of interest.
- compositions of the invention are typically administered to patients after the onset of symptoms, although treatment can also be prophylactic in some embodiments.
- treatment with direct administration of polypeptides is done daily, weekly, or monthly, for a period of time sufficient to reduce, prevent, or ameliorate symptoms.
- Treatment with the nucleic acids of the invention is typically done at intervals of several months. In some embodiments, administration of the compositions of the invention is done in utero.
- composition of the invention may also be provided in the kit as a slow-release composition such as a daily, weekly, monthly unit provided as a sponge, dermal patch, subcutaneous implant and the like in a wrapping or container as described above.
- a slow-release composition such as a daily, weekly, monthly unit provided as a sponge, dermal patch, subcutaneous implant and the like in a wrapping or container as described above.
- the patient may release a unit of the composition from the container and applies it as indicated in the kit instructions.
- the composition may then be replaced at the end of the specified period by a fresh unit, and so on.
- the present composition may also be administered by means of injection, as indicated above.
- the peptide may be administered by itself, or, for instance, in the case of a diabetic, in a composition also comprising insulin. The same is true for the slow-release forms of the composition.
- the peptide of the invention may be administered in a composition that also comprises another drug.
- One such case is that of cancer patients, where different anti-cancer drugs such as chemotherapeutic or contrast agents and target-specific antibodies, among others, may be provided in a composition also comprising the peptide of the invention.
- the proportion of peptides to the other drug(s) and carrier may be adjusted accordingly.
- the levels of the delivered peptide to a patient may be monitored by immunoassay.
- an antibody assay may be performed with antibodies specific to the peptide sequence by any of the protocols known in the art. Polyclonal or monoclonal antibodies or the 16K N-terminal fragment receptor may be utilized.
- the level of the peptide in blood may then be correlated with the progress of the inhibition of any of the diseases the patient is afflicted with.
- 16K N-terminal antiangiogenic agents were derived from full length human growth hormone, human growth hormone variant hGH-V, human placental lactogen and prolactin. These peptides were tested in vivo and in vitro for their binding and antiangiogenic activity in assays described in below. Results of these testings are illustrated in FIGS. 1 - 5 .
- the peptides were tested for their ability to inhibit the basal or stimulated growth of bovine brain capillary endothelial cells. Fibroblast growth factors were used to stimulate endothelial cell growth. Experimental procedures for these tests are described in below.
- FIG. 1 In vitro inhibition of bFGF stimulation of bovine brain capillary endothelial (BBCE) cell proliferation is shown in FIG. 1.
- FIG. 1 is a graph showing inhibition of bovine brain capillary endothelial cell proliferation expressed as the percent of inhibition of the stimulation by bFGF.
- bFGF (1 ng/ml) and 100, 50, 10, 5, 2, 1, 0.5, 0.2, or 0.1 nM purified protein were added once again to the dishes.
- the cells were incubated with 500,000 cpm of ( 3 H) thymidine for 4 hours, washed in 5% trichloroacetic acid, solubilized in NaOH, and counted as previously described in Endocrinology, 133: 1292 (1993).
- Each point seen in FIG. 1 represents the mean of triplicate wells. The experiments were repeated at least three times. Similar results were obtained in each experiment.
- FIG. 1 therefore, clearly shows that purified 16K hormone fragments of the invention are able to substantially inhibit the cell proliferation even in concentrations as low as 1 nM. At concentrations from 10-50 nM, these peptides completely inhibit endothelial cell proliferation in vitro.
- Symbols used for individual 16K peptides are: ( ⁇ ) 16K hPRL; ( ⁇ )16K hPL; ( ⁇ ) 16K hGH; ( ⁇ ) 16K hGH-V.
- the peptides of the invention were tested for their ability to inhibit basal or stimulated capillary formation in developing chick embryos, using the chick chorioallantoic membrane assay (CAM) described below.
- the ability to inhibit the development of capillaries in the chick chorioallantoic membrane is a measure of in vivo antiangiogenic activity.
- the peptides were applied to the chorioallantoic membrane by local placement of methylcellulose disks containing the peptide.
- FIGS. 2 and 3 In vivo studies using chick chorioallantoic membrane (CAM) assay are shown in FIGS. 2 and 3.
- FIG. 2 shows the antiangiogenic effect of the peptides of the invention calculated as the percentage of eggs showing anti-angiogenic response in the CAM assay. The procedure is described below.
- FIG. 2 compares antiangiogenic activity in the chick chorioallantoic membrane assay of the intact full length hPRL, hGH, hGH-V and hPL to the antiangiogenic activity of their respective 16K N-terminal fragment peptides.
- the antiangiogenic activity is described as antiangiogenic index expressed in percent.
- Bovine serum albumin (BSA) was used as a protein control.
- FIG. 3 shows photographs of results of the chick chorioallantoic membrane (CAM) assay following the treatment with 16K hGH, 16K hGH-V, 16K hPL and 16K hPRL compared to their respective full length hormones and to a bovine serum albumin control.
- FIG. 3 describes the inhibition of angiogenesis in early stage CAM assay.
- FIG. 3 shows photographs of neovascularization of chicken embryo subjected to treatment with BSA, (upper panel 1) serving as a protein control, to treatment with 16K peptides of the invention (upper panels 2-5) and to the treatment with intact full length hormones (lower panels 6-9).
- the sites of the disks, identified as black arrow, show clearly that at sites of treatment with 16K N-terminal fragments, the neovascularization is almost completely or substantially suppressed.
- high neovascularization is clearly visible. Such high neovascularization is completely unobservable in embryos treated with the 16K peptides of the invention.
- MAPK mitogen-activated protein kinase
- FIG. 4 represents a Western blot analysis of tyrosine phosphorylation of MAPK (FIG. 4A), and the percentage level of inhibition of the bFGF stimulation of MAPK enzymatic activity by 16K fragments (FIG. 4B) compared to % of stimulation over control without 16K fragment (FIG. 4C). Stimulation of the tyrosine phosphorylation of MAPK by bFGF was blocked by the addition of 16K hGH, 16K hGH-V and 16K hPL as well as 16K hPRL (FIG. 4C).
- 16K fragments inhibit bFGF-dependent MAPK tyrosine phosphorylation and activity, while full-length hormones stimulate these processes in the absence of bFGF.
- cell lysates and phosphotyrosine Western blots were prepared and in-gel MAPK activity was measured as previously described in EMBO J., 2:493 (1983).
- BBCE cells (105) were plated in 60-mm plates in 1 ml Dulbecco's modified Eagle's medium (DMEM) Gibco, containing 10% calf serum. Twenty-four hours after plating, the cells were transferred to DMEM containing 0.5% calf serum and incubated for 48 hours to induce quiescence.
- DMEM Dulbecco's modified Eagle's medium
- the cells were treated for 5 min with 10 nM of the purified 16K hGH, 16K hGH-V, 16K hPL or 16K hPRL and with 250 pM bFGF, or for 10 minutes with 10 nM of the intact full length corresponding hormone without bFGF. Control was left untreated. To terminate the incubation, the medium was removed by suction and the cells were washed twice with phosphate buffer solution (PBS).
- PBS phosphate buffer solution
- FIG. 4A P-Tyr panel—left side panel shows the inhibition of the bFGF-induced phosphorylation of the p42 and p44 MAPK which are 42 and 44K mitogen-activated kinases, respectively, also called erk-1 and erk-2.
- Right side panel shows the stimulation of the MAPK by the full length hormones in the absence of bFGF.
- Cell lysate proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with an anti-phosphotyrosine antibody (4G10, UBI, 1/2000 dilution).
- an anti-phosphotyrosine antibody (4G10, UBI, 1/2000 dilution).
- FIG. 4B Level of inhibition of the stimulation of MAPK enzymatic activity by 16K N-terminal fragments of the invention is seen in FIG. 4B. Results are compared to their respective intact hormone controls (FIG. 4C).
- FIGS. 4B and 4C show in-gel MAPK activity.
- cell lysates were resolved by SDS-PAGE in gels containing 0.5 mg/ml myelin basic protein (MBP) as discussed below.
- MBP myelin basic protein
- the gel proteins were denatured, renatured, and subjected to an in situ kinase reaction with 100 ⁇ Ci of ( ⁇ -32) ATP.
- the gels were then dried and autoradiographed.
- 32P incorporated into MBP was quantified by a phosphorimager. Numbers at the bottom of the histograms represent the number of experiments performed.
- FIGS. 4B and 4C show comparative results of MAPK activity, expressed as percent of bFGF stimulation.
- the bFGF stimulation of MAPK activity was 100%.
- the bFGF-induced MAPK activity was inhibited in all cases to less than 25%, with greatest suppression of MAPK activity observed after treatment with the 16K hGH suppressing the MAPK activity to about 10%.
- FIG. 4C shows the stimulation of MAPK activity with intact full length hormone in the absence of bFGF.
- the intact molecules seen in FIG. 4C actually stimulated the tyrosine phosphorylation of MAPK.
- the inhibition of bFGF-stimulated MAPK enzymatic activity by the 16K N-terminal fragments ranged between 75 and 85% (FIG. 4B), while the intact molecules actually stimulated MAPK activity.
- PAI-1 plasminogen activator inhibitor
- FIG. 5 is a Western blot analysis of the effect of the peptides of the invention on PAI-1 protein levels and their quantitation (FIG. 5A) and Western blot analysis of PAI-1 in untreated cells and cells treated with hGH, 16K hGH, or both (FIG. 5B).
- cell lysates were prepared as described in EMBO J., 2: 493 (1985).
- BBCE (10 5 ) cells were plated in 24-well plates in 1 ml Dulbecco's modified Eagle's medium (DMEM) containing lot calf serum. Twenty-four hours after plating, the cells were treated for 16 hours with purified 16K fragments or full-length hormone (10 nM) in serum-free DMEM. Untreated well was used as a control. 20 ⁇ l cell lysate was resolved by SDS-PAGE (10-4%) and the separated proteins transferred to a nitrocellulose membrane.
- DMEM Dulbecco's modified Eagle's medium
- the blots were blocked for 1 hour with 5% milk in Tris-buffered saline with 0.1% Tween 20 and probed for 2 hours with mouse anti-bovine PAI-1 monoclonal antibody obtained from Gibco, at 1:2000 dilution.
- the antigen-antibody complex was detected with horseradish-peroxidase-conjugated secondary antibody and an enhanced chemiluminescence system (ECL, Amersham).
- FIG. 5A shows the effect of 16K N-terminal peptides of the invention on the expression of PAI-1 compared to the effect of intact hormones.
- FIG. 5A shows that 16K fragments stimulate PAI-1 expression, where as the full length hormones have no effect.
- placental vascularization is critical to the survival of the developing conceptus, and requires extensive proliferation and remodeling of the decidual vascular tree. If vasculogenic mechanisms of placentation are similar to those reported for tumor invasion there are at least two forces driving the acquisition of the new blood supply. Maternal endothelial cells are recruited toward the placenta and concomitantly, placental cells migrate toward proliferating maternal vessels.
- Attachment of the implanting human placenta to the maternal decidua is achieved by contact between anchoring villi of the chorion frondosum and the decidua basalis.
- the characteristic hemochorial vascular anatomy of the human placenta provides a unique environment for oxygen, nutrient and waste product exchange. Irregular lobes or cotyledons separated by septa demarcate the maternal surface of the developing placenta. Normally, each cotyledon is supplied by one of approximately 120 maternal uterine spiral arteries.
- antiangiogenic proteins due to premature or excessive production of antiangiogenic proteins relative to angiogenic factors could lead to poor maternal vascularization of the placental bed and result in fetoplacental ischemia as observed in preeclampsia and IUGR ( Clin. Perinatol., 18: 661-82 (1994)).
- antiangiogenic peptides of the invention seem also to play a critical role in the human placenta, serving to regulate placental vascularization during normal pregnancy.
- VEGF vascular endometrium
- the first question in developing the diagnostic assay was to determine whether full length hPRL could be enzymatically cleaved to generate a biologically active 16K fragment.
- Cathepsin-like enzymes which have been shown to cleave rat PRL to the 16K isoform ( Endocrinology, 133:935-38 (1993)), e.g., cathepsin D, are expressed in several cells and tissues at the maternal-fetal interface. As shown herein, this enzyme can cleave human decidual PRL and can generate bioactive antiangiogenic fragments of purified hPRL.
- Transferred protein was stained with 0.1% Coomassie blue, the two bands of interest, namely 18K and 13K, excised from the PVDF membrane and the peptides were sequenced directly on a gas phase Beckman-Porton PI2090 protein sequencer by Edman degradation sequencing to analyze the NH 2 -termini of the cleaved fragments.
- the necessary molecular components required to generate 16K PRL are available at the site of placental vascularization.
- the activity level of cathepsin D and/or concentrations of 16K fragments of hPRL, hPL and hGH-V are thus advantageously utilized for detection of placental function, whether normal or abnormal.
- cathepsin D site in hPRL several other cleavage sites exist within the sequences of hGH, hGH-V and hPL that generate a N-terminal fragment of around 16 kDa.
- hGH can be cleaved by plasmin, thrombin and subtilisin ( EMBO J., 2:493 (1983)).
- hGH-V The consensus cleavage sequence for these enzymes is also present in hGH-V and we have shown herein that hGH-V is cleaved by thrombin. Furthermore, preeclampsia has been characterized as a disease of an excess in thrombin activity. Finally, hPL can be cleaved by plasmin to yield a 16K N-terminal fragment ( J. Biol. Chem. 254:2296 (1979)).
- Specimens of human placental tissues of first, second, and third trimester pregnancies were obtained from consenting women either undergoing elective pregnancy terminations (first and second trimester) or at the end of pregnancy (third trimester). The protocols were approved by the UCSF committee of human research. Tissues were kept at ⁇ 80° C. until further processing. Rinsed tissues were homogenized in 1:1 volume of 20 mM Tris, pH 7.4, 250 mM sucrose, 2 mM EDTA, and 0.2 mM Pefablock and centrifuged at 10,000 g for 30 min. The pellet-was resuspended in 5 ml of buffer and recentrifuged at 10,000 g for 30 min.
- Both supernatants were brought to 100 mM NaCl and 0.2 mM MgCl 2 to precipitate microsomal membranes.
- the pellet from a subsequent centrifugation at 30,000 g for 45 min was resuspended in 20 mM Hepes, 10% glycerol, 1 mM EDTA, 1 mM Pefablock, and 0.141U of aprotinin.
- Intact full length human prolactin (hPRL), human growth hormone (hGH), human growth hormone variant (hGH-V), human placental lactogen (hPL) and 16K N-terminal fragment of human prolactin (16K hPRL), growth hormone (16K hGH), growth hormone variant (16K hGH-V) and placental lactogen (16K hPL) were produced as described below.
- 16K hPRL (Met ⁇ 1 Pro 139 ) (SEQ ID NO:3) ATGTTGCCCA TCTGTCCCGG CGGGGCTGCC CGATGCCAGG TGACCCTTCG AGACCTGTTT 60 GACCGCGCCG TCGTCCTGTC CCACTACATC CATAACCTCT CCTCAGAAAT GTTCAGCGAA 120 TTCGATAAAC GGTATACCCA TGGCCGGGGG TTCATTACCA AGGCCATCAA CAGCTCCCAC 180 ACTTCTTCCC TTGCCACCCC CGAAGACAAG GAGCAAGCCC AACAGATGAA TCAAAAAGAC 240 TTTCTGAGCC TGATAGTCAG CATATTGCGA TCCTGGAATG AGCCTCTGTA TCATCTGGTC 300 ACGGAAGTAC GTGGTATGCA AGAAGCCCCG GAGGCTATCC TATCCAAAGC TGTAGAGATT 360 GAGGAGCAAA CCAAACGGCT TCTAGAGGGC ATGGAGCTGA TAGTCAGC
- hPRL (Met ⁇ 1 Cys 199 )antisense (SEQ ID NO:5) TACAACGGGT AGACAGGGCC GCCCCGACGG GCTACGGTCC ACTGGGAAGC TCTGGACAAA 60 CTGGCGCGGC AGCAGGACAG GGTGATGTAG GTATTGGAGA GGAGTCTTTA CAAGTCGCTT 120 AAGCTATTTG CCATATGGGT ACCGGCCCCC AAGTAATGGT TCCGGTAGTT GTCGACGGTG 180 TGAAGAAGGG AACGGTGGGG GCTTCTGTTC CTCGTTCGGG TTGTCTACTT AGTTTTTCTG 240 AAAGACTCGG ACTATCAGTC GTATAACGCT AGGACCTTAC TCGGAGACAT AGTAGACCAG 300 TGCCTTCATG CACCATACGT TCTTCGGGGC CTCCGATAGG ATACQTTTCG ACATCTCTAA 360 CTCCTCGTTT GGTTTGCCGA AGATCTCCCG TACCTCGACT ATCA
- 16K hPRL (MeT ⁇ 1 Thr 123 ) antisense (SEQ ID NO:6) TACAACGGGT AGACAGGQCC GCCCCGACGG GCTACGGTCC ACTGGGAAGC TCTGGACAAA 60 CTGGCGCGGC AGCAGGACAG GGTGATGTAG GTATTGGAGA GGAGTCTTTA CAAGTCGCTT 120 AAGCTATTTG CCATATGGGT ACCGGCCCCC AAGTAATGGT TCCGGTAGTT GTCGAGGGTG 180 TGAAGAAGGG AACGGTGGGG GCTTCTGTTC CTCGTTCGGG TTGTCTACTT AGTTTTTCTG 240 APAGACTCGG ACTATCAGTC GTATAACGCT AGGACCTTAC TCGGAGACAT AGTAGACCAG 300 TGCCTTCATG CACCATACGT TCTTCGGGGC CTCCGATAGG ATAGGTTTCG ACATCTCTAA 360 CTCCTCGTTT GGATT;
- 16K hPRL (Met ⁇ 1 Pro 139 ) antisense (SEQ ID NO:7) TACAACGGGT AGACAGGGCC GCCCCGACGG GCTACGGTCC ACTGGGAAGC TCTGGACAAA 60 CTGGCGCGGC AGCAGGACAG GGTGATGTAG GTATTGGACA GGAGTCTTTA CAAGTCGCTT 120 AAGCTATTTG CCATATGGGT ACCGGCCCCC AAGTAATGGT TCCCGTAGTT GTCGAGGGTG 180 TGAAGAAGGG AACGGTGGGG GCTTCTGTTC CTCGTTCGGG TTGTCTACTT AGTTTTTCTG 240 AAAGACTCGG ACTATCAGTC GTATAACGCT AGGACCTTAC TCGGAGACAT AGTAGACCAG 300 TGCCTTCATG CACCATACGT TCTTCGGGGC CTCCGATAGG ATAGGTTTCG ACATCTCTAA 360 CTCCTCGTTT GGTTTGCCGA AGATCTCCCG TACCTCGACT ATCA
- hPRL (Met ⁇ 1 Cys 199 ) MetLeuProIleCysProGlyGlyAlaAlaArgCysGlnValThrLseuArgAspLeuPhe (SEQ ID NO:9) ⁇ 1 5 10 15 AspArgAlaValValLeuSerHisTyrIleHisAsnLeuSerSerGluMetPheSerGlu 20 25 30 35 PheAspLysArgTyrThrHisGlyArgGlyPheIleThrLysAlaIleAsnSerCysHis 40 45 50 55 ThrSerSerLeuAlaThrProGluAspLysGluGlnAlaGlnGlnMetAsnGlnLysAsp 60 65 70 75 PheLeuSerLeuIleValSerIleLeuArgSerTrpAsnGluProLeuTyrHisLeuVal 80 85 90 95 ThrGluValArgGlyMet
- hRL (Met ⁇ 1 Phe 191 ) ATGGTCCAAA CCGTTCCGTT ATCCAGGCTT TTTGACCACG CTATGCTCCA AGCCCATCGC 60 (SEQ ID NO:13) GCGCACCAGC TGGCCATTGA CACCTACCAG GAGTTTGAAG AAACCTATAT CCCAAAGGAC 120 CAGAAGTATT CGTTCCTGCA TGACTCCCAG ACCTCCTTCT GCTTCTCAGA CTCTATTCCG 180 ACACCCTCCA ACATGGAGGA AACGCAACAG AAATCCAATC TAGAGCTGCT CCGCATCTCC 240 CTGCTGCTCA TCGAGTCGTG GCTGGAGCCC GTGCGGTTCC TCAGGAGTAT GTTCGCCAAC 300 AACCTGGTGT ATGACACCTC GGACAGCGAT GACTATCACC TCCTAAAGGA CCTAGAGGAA 360 GGCATCCAAA CGCTGATGGG GAGGCTGGAA GACGGCAGCC GCCGGACTATCACC
- hPL (MeT ⁇ 1 Phe 191 ) antisense TACCAGGTTT GGCAAGGCAA TAGGTCCGAA AAACTGGTGC GATACGAGGT TCGGGTAGCG 60 (SEQ ID NO:15) CGCGTGGTCG ACCGGTAACT GTGGATGGTC CTCAAACTTC TTTGGATATA GGGTTTCCTG 120 GTCTTCATAA GCAAGGACGT ACTGAGGGTC TGGAGGAAGA CGAAGAGTCT GAGATAAGGC 180 TGTGGGAGGT TGTACCTCCT TTGCGTTGTC TTTAGGTTAG ATCTCGACGA GGCGTAGAGG 240 GACGACGAGT AGCTCAGCAC CGACCTCGGG CACGCCAAGG AGTCCTCATA CAAGCGGTGG 300 TTGGACCACA TACTGTGGAG CCTGTCGCTA CTGATAGTGG AGGATTTCCT GGATCTCCTT 360 CCGTAGGTTT GCGACTACCC CTCCGACCTT CTGCCGTCGG
- hPL (Met ⁇ 1 Phe 191 ) MetValGlnThrValProLeuSerArgLeuPheAspHisAlaMetLeuGlnAlaHisArg (SEQ ID NO:17) ⁇ 1 5 10 15 AlaHisGlnLeuAlaIleAspThrTyrGlnGluPheGluGluThrTyrIleProLysAsp 20 25 30 35 GlnLysTyrSerPheLeuHisAspSerGlnThrSerPheCysPheSerAspSerIlePro 40 45 50 55 ThrProSerAsriMetGluGluThrGlnGlnLysSerAsnLeuGluLeuLeuArgIleSer 60 65 70 75 LeuLeuLeuIleGluSerTrpLeuGluProValArgPheLeuArgSerMetPheAlaAsn 80 85 90 95 AsnLeuValTyrAspThr
- hGH (Met ⁇ 1 Phe 191 ) ATGTTCCCAA CCATTCCCTT ATCCAGGCTT TTTGACAACG CTATGCTCCG 50 (SEQ lD NO:19) CGCCCATCGT CTGCACCAGC TGGCCTTTGA CACCTACCAG GAGTTTGAAG 100 AAGCCTATAT CCCAAAGGAA CAGAAGTATT CATTCCTGCA GAACCCCCAG 150 ACCTCCCTCT GTTTCTCAGA GTCTATTCCG ACACCCTCCA ACAGGGAGGA 200 AACACAACAG AAATCCAACC TAGAGCTGCT CCGCATCTCC CTGCTGCTCA 250 TCCAGTCGTG GCTGGAGCCC GTGCAGTTCC TCAGGAGTGT CTTCGCCAAC 300 AGCCTGGTGT ACGGCGCCTC TGACAGCAAC GTCTATGACC TCCTAAAGGA 350 CCTAGAGGAA GGCATCCAAA CGCTGATGGG GAGGCTGGAA GATGGCAGCC 400 CCCGGACTGG
- hGH (Met ⁇ 1 Phe 191 ) antisense TACAAGGGTT GGTAAGGGAA TAGGTCCGAA AAACTGTTGC GATACGAGGC 50 (SEQ ID NO:21) GCGGGTAGCA GACGTGGTCG ACCGGAAACT GTGGATGGTC CTCAAACTTC 100 TTCGGATATA GGGTTTCCTT GTCTTCATAA GTAAGGACGT CTTGGGGGTC 150 TGGAGGGAGA CAAAGAGTCT CAGATAAGGC TGTGGGAGGT TGTCCCTCCT 200 TTGTGTTGTC TTTAGGTTGG ATCTCGACGA GGCGTAGAGG GACGACGAGT 250 AGGTCAGCAC CGACCTCGGG CACGTCAAGG AGTCCTCACA GAAGCGGTTG 300 TCGGACCACA TGCCGCGGAG ACTGTCGTTG CAGATACTGG AGGATTTCCT 350 GGATCTCCTT CCGTAGGTTT GCGACTACCC CTCCGACCTT CTACCGTCGG 400 GGGCC
- hGH (Met ⁇ 1 Phe 191 ) MetPheProThrIleProLeuSerArgLeuPheAspAsnALaMetLeuArgAlaHisArg (SEQ ID NO:23) ⁇ 1 5 10 15 LeuHisGlnLeuAlaPheAspThrTyrGlnGluPheGluGluAlaTyrIleProLysGlu 20 25 30 35 GlnLysTyrSerPheLeuGlnAsnProGlnThrSerLeuCysPheSerGluSerIlePro 40 45 50 55 ThrProSerAsriArgGluGluThrGlnGlnLysSerAsnLeuGluLeuLeuArgIleSer 60 65 70 75 LeuLeuLeuIleGlnSerTrpLeuGluProValGlnPheLeuArgSerValPheAlaAsn 80 85 90 95 SerLeuValTyrGlyAlaS
- hGH-V (Met ⁇ 1 Phe 191 ) ATGTTCCCAA CCATTCCCTT ATCCAGGCTT TTTGACAACG CTATGCTCCG 50 (SEQ ID NO:25) CGCCCGTCGC CTGTACCAGC TGGCATATGA CACCTATCAG GAGTTTGAAG 100 AAGCCTATAT CCTGAAGGAG CAGAAGTATT CATTCCTGCA GAACCCCCAG 150 ACCTCCCTCT GCTTCTCAGA GTCTATTCCA ACACCTTCCA ACAGQGTGAA 200 AACGCAGCAG AAATCTAACC TAGAGCTGCT CCGCATCTCC CTGCTGCTCA 250 TCCAGTCATG GCTGGAGCCC GTGCAGCTCC TCAGGAGCGT CTTCGCCAAC 300 AGCCTGGTGT ATGGCGCCTC GGACAGCAAC GTCTATCGCC ACCTGAAGGA 350 CCTAGAGGAA GGCATCCAAA CGCTGATGTG GAGGCTGGAA GATGGCAGCC 400 CCCGGACT
- hGH-V (Met ⁇ 1 Phe 191 ) antisense TACAAGGGTT GGTAAGGGAA TAGGTCCGAA AAACTGTTGC GATACGAGGC 50 (SEQ ID NO:27) GCGGGCAGCG GACATGGTCG ACCGTATACT GTGGATAGTC CTCAAACTTC 100 TTCGGATATA GGACTTCCTC GTCTTCATAA GTAAGGACGT CTTGGGGGTC 150 TGGAGGGAGA CGAAGAGTCT CAGATAAGGT TGTGGAAGGT TGTCCCACTT 200 TTGCGTCGTC TTTAGATTGG ATCTCGACGA GGCGTAGAGG GACGACGAGT 250 AGGTCAGTAC CGACCTCGGG CACGTCGAGG AGTCCTCGCA GAAGCGGTTG 300 TCGGACCACA TACCGCGGAG CCTGTCGTTG CAGATAGCGG TGGACTTCCT 350 GGATCTCCTT CCGTAGGTTT GCGACTACAC CTCCGACCTT CTACCGTCGG 400 GGG
- hGH-V (Met ⁇ 1 Phe 191 ) MetPheProThrIleProLeuSerArgLeuPheAspAsnAlaMetLeuArgAlaArgArg (SEQ ID NO:29) ⁇ 1 5 10 15 LeuTyrGlnLeuAlaTyrAspThrTyrGlnGluPheGluGluAlaTyrIleLeuLysGlu 20 25 30 35 GlnLysTyrSerPheLeuGlnAsnProGlnThrSerLeuCysPheSerGluSerIlePro 40 45 50 55 ThrProSerAsnArgValLysThrGlnGlnLysSerAsnLeuGluLeuLeuArgIleSer 60 65 70 75 LeuLeuLeuIleGlnSerTrpLeuGluProValGlnLeuLeuArgSerValPheAlaAsn 80 85 90 95 SerLeuValTyr
- sequences of the 16K N-terminal fragments were obtained by mutagenesis of the sequences of the intact hormones. Mutagenesis was performed using the oligonucleotide-directed mutagenesis kit purchased from either Amersham International (Buckinghamshire, UK) or Boehringer Mannheim (Mannheim, Germany). Oligonucleotides were purchased from Eurogentec S.A. (Seraing, Belgium).
- a 100 ml culture of E. coli BL21(DE3) carrying the pT7L-hPRL plasmid was grown overnight at 37° C. in LB, medium-ampicillin 100 ⁇ g/ml. Twenty ml of this culture were used to inoculate 1 liter of LB medium-ampicillin 100 ⁇ g/ml. When the O.D. (600 nm) reached 0.9, 1 mm isopropyl ⁇ -D thiogalactopyranoside (IPTG) was added to the culture. The induced culture was grown for an additional 4 hour period.
- O.D. 600 nm
- IPTG isopropyl ⁇ -D thiogalactopyranoside
- Cells were collected by centrifugation at 5000 g for 15 min (4° C.) and resuspended in 50 mM Tris-HCl, 0.5 mM EDTA, (T 50 E 0.5 ), 0.1 mM PMSF, pH 8. Cells were broken in a cell disrupter. After centrifugation of cell lysates (12,000 g, 15 min, 4° C.), hPRL was found in the insoluble fraction (inclusion bodies).
- the inclusion bodies recovered in the pellet were washed 2 times in 250 ml of T 50 E 0.5 , 0.1 mM PMSF, pH 8. Inclusions bodies were solubilized in 200 mM phosphate buffer (pH 7) containing 8 M of deionized urea, 1% ⁇ -mercaptoethanol ( ⁇ -ME), 0.5 mM PMSF at a concentration of 100 ⁇ g of protein per ml of buffer.
- the protein solution was heated at 55° C. for 5 min and then incubated overnight at room temperature.
- the renaturation was performed by a continued dialysis (72 hours) against 500 volumes of 50 mM NH 4 HCO 3 , pH 7.5.
- the renatured protein was concentrated to 2 mg/ml and loaded on a Sephadex G100 column (50 mM NH 4 HCO 3 , pH 7.5) Fractions containing hPRL were pooled, dialyzed against 50 volumes of 20 mM Tris-HCl, pH 8 and loaded on a HitrapQ anion exchange column. Chromatography was performed in 20 mM Tris-HCl, pH 8 and hPRL was eluted within a gradient 0 to 1 M NaCl. Fractions containing purified hPRL were pooled, dialyzed against 20 mM NH 4 HCO 3 , pH 7.5 and lyophilized. The protein was purified to 95% homogeneity.
- This example describes the construction of plasmids coding for 3 different forms of 16K hPRL.
- the 16K N-terminal fragment of human prolactin was produced by three different methods.
- the DNA sequences coding for the three different forms of 16K hPRL have been obtained by mutating the DNA of hPRL (cloned in the laboratory) (SEQ ID NO: 1).
- the three different forms of 16K hPRL are called 16K hPRL (stop 124), 16K hPRL (stop 140) and 16K hPRL (IgA). All of the forms exhibit the same antiangiogenic activity.
- 16K hPRL (stop124)
- two mutations were introduced in the hPRL coding sequence (16K hPRL (Met ⁇ 1 Thr 123 )) (SEQ ID NO: 2).
- the Lys124 codon (AAA) was mutated to a stop codon (TAA); the Cys58 (TGC) into a Ser (TCC).
- TAA stop codon
- TGC Cys58
- TCC Ser
- the resulting plasmid was called pT7L-16K hPRL (stop124). It encodes a protein called 16K hPRL (stop 124) which is composed of the 123 N-terminal amino acids of hPRL with the substitution of a Ser at position 58 instead of a Cys (SEQ ID NO: 10).
- hPRL (IgA) which differs from hPRL at position 58 (Ser) and 140-144 (IgA specific cleavage site) (16K hPRL (Met ⁇ 1 Pro 142 ))
- inclusion bodies were recovered in the pellet and washed 2 times in 250 ml of T 50 E 0.5 , 0.1 mM PMSF, pH 8. Inclusions bodies were solubilized in 200 mM phosphate buffer (pH 7) containing 8 M of deionized urea, 1% 0-ME, 0.5 mM PMSF at a concentration of 100 ⁇ g of protein per ml of buffer. The protein solution was heated at 55° C. for 5 min and then incubated overnight at room temperature. The renaturation was performed by a continued dialysis (72 hours) against 500 volumes of 50 mM NH 4 HCO 3 , pH 7.5.
- the renatured protein was finally dialyzed against of 20 mM Tris-HCl, pH 8. After centrifugation at 5000 g to remove any precipitate, the supernatant was loaded on a MonoQ anion exchange column. Chromatography was performed in 20 mM Tris-HCl, pH 8 and 16K hPRL (stop124) was eluted within a gradient 0 to 1 M NaCl. Fractions containing 16K hPRL (stop124) were pooled and dialyzed against 50 mM NH 4 HCO 3 , pH 7.5. After centrifugation, supernatant was loaded on a Sephadex G75 column (50 mM NH 4 HCO 3 , pH 7.5). Fractions containing 16K hPRL (stop124) were pooled, dialyzed against 20 mM NH 4 HCO 3 , pH 7.5 and lyophilized.
- the 16K hPRL (stop124) was dialyzed against 50 mM NH 4 HCO 3 , pH 7.5. The protein was then concentrated to 2 mg/ml. The 16K hPRL (stop124) was denatured in 200 mM phosphate buffer, pH 7, containing 8 M of deionized urea, 1% ⁇ -ME, 0.5 mm PMSF.
- the molecular mass of the 16K hPRL (stop124) was calculated from its amino acid sequence to be 13.9 kD.
- the solution was heated at 55° C. for 5 min and then incubated overnight at RT.
- the denatured proteins were dialyzed against 20 mM ethanolamine-HCl, pH 9, 6 M deionized urea. After centrifugation (5000 g; 15 min.), the proteins were applied for purification onto an anion exchange Hitrap Q column. Chromatography was performed in 20 mm ethanolamine-HCl, pH 9, 6 M deionized urea and 16K hPRL (stop140) was eluted within a gradient 0 to 1 M NaCl.
- Cells were collected by centrifugation at 5000 g for 15 min (4° C.) and resuspended in 50 mM Tris-HCl, 0.5 mM EDTA, (T 50 E 0.5 ), 0.1 mM PMSF, pH 8. Cells were broken in a cell disrupter. After centrifugation of cell lysates (12,000 g, 15 min, (4° C.), 23K hPRL (IgA) was found in the insoluble fraction (inclusion bodies).
- the inclusion bodies recovered in the pellet were washed 2 times in 250 ml of T 50 E 0.5 0.1 mM PMSF, pH 8. Inclusions bodies were solubilized in 200 mM phosphate buffer (pH 7) containing 8 M of deionized urea, 1% ⁇ -ME, 0.5 mM PMSF at a concentration of 100 gg of protein per ml of buffer. The protein solution was heated at 55° C. for 5 min and then incubated overnight at room temperature. The renaturation was performed by a continued dialysis (72 hours) against 500 volumes of 50 mM NH 4 HCO 3 , pH 7.5.
- the 23K hPRL (IgA) was dialyzed against 50 mM NH 4 HCO 3 , pH 7.5.
- IgA protease the protein solution was concentrated to 1 mg/ml.
- the proteins were incubated 24 hours (25° C.) with 0.05% (w/w) of IgA protease (Boehringer Mannheim, Mannheim, Germany).
- the cleaved proteins were incubated for one hour with 1% ⁇ -ME before loading on a Sephadex G100 column (50 mM NH 4 HCO 3 , pH 7.5, 100 mM NaCl, 5 mM ⁇ -ME.
- the molecular mass of the 16K hPRL (IgA) calculated from its amino acid sequence was determined to be 16.1 kD.
- This example describes a procedure for preparation of intact human growth hormone (hGH (Met ⁇ 1 Phe 191 )) (SEQ ID NO: 23).
- hGH human growth hormone
- pT7L The coding region for human growth hormone (hGH) minus the signal peptide was inserted into plasmid pT7L.
- An initiation codon ATG was added to the 5′ end of the coding sequence of the hGH ( Science, 205:602 (1979) (SEQ ID NO: 19), and inserted into the pT7L E. coli expression vector.
- This plasmid was called pT7L-hGH.
- a 100 ml culture of E. coli BL21(DE3) carrying the pT7L-hGH plasmid was grown at 37° C. in LB medium-ampicillin 100 ⁇ g/ml. Twenty ml of this culture were used to inoculate 1 liter of LB medium-ampicillin 100 ⁇ g/ml. When the O.D. (600 nm) reached at 0.9, 1 mM isopropyl ⁇ -D thiogalactopyranoside (IPTG) was added to the culture. The induced culture was grown for an additional 4 hour period.
- O.D. 600 nm
- IPTG isopropyl ⁇ -D thiogalactopyranoside
- Cells were collected by centrifugation at 5000 g for 15 min (4° C.) and resuspended in 50 mM Tris-HCl, 0.5 mM EDTA, (T 50 E 0.5 ), 0.1 mM PMSF, pH 8. Cells were broken in a cell disrupter. After centrifugation of cell lysates (12,000 g, 15 min, 4° C.), hGH was found in insoluble fractions (inclusion bodies).
- the inclusion bodies recovered in the pellet were washed 2 times in 250 ml of T 50 E 0.5 , 0.1 mM PMSF, pH 8 and stored at ⁇ 20° C.
- Inclusion bodies were solubilized in 20 mM ethanolamine, pH 9 containing 8 M of deionized urea, it ⁇ -ME, 0.5 mM PMSF at a concentration of 100 gg of protein per ml of buffer.
- the protein solution was heated at 55° C. for 5 min and then incubated overnight at room temperature. The renaturation was performed by a continued dialysis (72 hours) against 500 volumes of 20 mM ethanolamine, pH 9.
- the renatured protein was loaded on a HitrapQ anion exchange column. Chromatography was performed in 20 mm ethanolamine, pH 9 and hGH was eluted within a gradient 0 to 1 M NaCl. Fractions containing hGH were pooled, concentrated to 2 mg/ml and loaded on a Sephadex G100 column (50 mM NH 4 HCO 3 , pH 7.5). Fractions containing purified hGH were pooled, dialyzed against 20 mM NH 4 HCO 3 , pH 7.5 and lyophilized. proteins were purified to 90% homogeneity.
- This example describes the procedure for preparation of the 16K N-terminal fragment of human growth hormone (16K hGH (Met ⁇ 1 Pro 133 )) (SEQ ID 24).
- a E. coli BL21(DE3) culture (100 ml) carrying the plasmid pT7L-16K hGH was grown overnight at 37° C. into TB medium-ampicillin 100 ⁇ g/ml. Twenty ml of this culture was used to inoculate 1 liter of TB medium-ampicillin 100 ⁇ g/ml. When the O.D.600 reached at 0.6, 1 mM isopropyl ⁇ -D thiogalactopyranoside (IPTG) was added to the culture. The induced culture was grown for an additional 4 hour period.
- IPTG isopropyl ⁇ -D thiogalactopyranoside
- 16K hGH was produced as an insoluble form (inclusion bodies).
- the latter were solubilized in 20 mM ethanolamine-HCl, pH 9, containing 8 M deionized urea, 1% ⁇ -ME.
- 0.5 mM PMSF (Buffer A) at a ratio of 100 ⁇ g of protein per ml of buffer.
- the solution was heated at 55° C. for 5 min and then incubated overnight at RT.
- the denatured proteins were dialyzed against 20 mM ethanolamine-HCl, pH 9, 6 M deionized urea. After centrifugation (5000 g; 15 min.), the proteins were applied for purification onto an anion exchange Hitrap Q column.
- the renaturation was performed by a continued dialysis for 72 hours.
- a continued dialysis was performed within a bath of 10 volumes of 20 mM ethanolamine-HCl, pH 9, 6 M deionized urea.
- the urea was progressively removed by buffer exchange against 500 volumes of 20 mM ethanolamine, pH 9 for 72 hours.
- the renatured proteins were then dialyzed against 50 mM NH 4 HCO 3 , pH 7.5, 0.1 M NaCl. After concentration to 2 mg/ml, the proteins were loaded a Sephadex G100 column (50 mM NH 4 HCO 3 , pH 7.5, 0.1 M NaCl). Fractions containing the 16K hGH were pooled, dialyzed against 20 mM NH 4 HCO 3 , pH 7.5 and lyophilized.
- the molecular mass of the 16K hGH calculated from its amino acid sequence was determined to be 15.5 kD. Proteins were purified to 95% homogeneity.
- This example describes a procedure for preparation of intact human growth hormone variant hGH-V (hGH-V(Met ⁇ 1Phe 191 )))(SEQ ID NO: 2.9)
- the coding region for hGH-V minus the signal peptide was inserted into a plasmid.
- the coding sequence for hGH-V was cloned by screening a placental cDNA library (Clonetech, HL 1008). An initiation codon ATG was added to the 51 end of the coding sequence of the hGH-V (SEQ ID NO: 25) and inserted into the pT7L E. coli expression vector. This plasmid was called pT7L-hGH-V.
- a 100 ml culture of E. coli BL21 (DE3) carrying the pT7L-hGH-V plasmid was grown overnight at 37° C. in LB medium-ampicillin 100 ⁇ g/ml. Twenty ml of this culture were used to inoculate 1 liter of LB medium-ampicillin 100 ⁇ g/ml. When the O.D. (600 nm) reached at 0.9, 1 mM isopropyl ⁇ -D thiogalactopyranoside (IPTG) was added to the culture. The induced culture was grown for an additional 4 hour period.
- O.D. 600 nm
- IPTG isopropyl ⁇ -D thiogalactopyranoside
- Cells were collected by centrifugation at 5000 g for 15 min (4° C.) and resuspended in 50 mM Tris-HCl, 0.5 mM EDTA, (T 50 E 0.5 ), 0.1 mM PMSF, pH 8. Cells were broken in a cell disrupter. After centrifugation of cell lysates (12,000 g, 15 min, 4° C.), hGH-V was found in the insoluble fraction (inclusion bodies).
- the inclusion bodies recovered in the pellet were washed 2 times in 250 ml of T 50 E 0.5 0.1 mM PMSF, pH 8. Inclusion bodies were solubilized in 20 mM ethanolamine (pH 10) containing 8 M of deionized urea, 1% ⁇ -ME. 0.5 mM PMSF at a concentration of 100 ⁇ g of protein per ml of buffer. The protein solution was heated at 55° C. for 5 min and then incubated overnight at room temperature. The renaturation was performed by a continued dialysis (72 hours) against 500 volumes of 20 mM ethanolamine (pH 10).
- the renatured protein was loaded on a HitrapQ anion exchange column. Chromatography was performed in 20 mM ethanolamine, pH 10 and hGH-V was eluted within a gradient of 0 to 1 M NaCl. Fractions containing hGH-V were pooled, concentrated to 2 mg/ml and loaded on a Sephadex G100 column (50 mM NH 4 HCO 3 , pH 9). Fractions containing purified hGH-V were pooled, dialyzed against 20 mM NH 4 HCO 3 , pH 9 and lyophilized. Proteins were purified to 95% homogeneity.
- This example describes the procedure for preparation of the 16K N-terminal fragment of human growth hormone variant hGH-V (16K hGH-V (Met 1 Arg 134 )) (SEQ ID NO: 30).
- hGH-V 16K hGH-V
- a natural cleavage site specific to thrombin is present at position Pro133-Arg134. Cleavage occurs after arginine 134.
- Human GH-V was produced and inclusion bodies denatured and renatured as described is Example 8.
- hGH-V was concentrated to 1 mg/ml and then enzymatically cleaved with thrombin (0.3%, 25° C. overnight, Sigma).
- the renaturation was performed by a continued dialysis for 72 hours. In order to remove the urea carefully, a first dialysis against 10 volumes of 20 mM ethanolamine-HCl, pH 9, 6 M deionized urea was performed. Second, the urea from this bath was removed by a continued dialysis against 500 volumes of 20 mM ethanolamine, pH 9. For the renaturation, a continued dialysis was performed within a bath of 10 volumes of 20 mM ethanolamine-HCl, pH 9, 6 M deionized urea whose the urea was progressively removed by buffer exchange against 500 volumes of 20 mM ethanolamine, pH 9 for 72 hours.
- the renatured proteins were then dialyzed against 50 mM NH 4 HCO 3 , pH 9, 0.1 M NaCl. After concentration to 2 mg/ml, the proteins were loaded a Sephadex G100 column (50 mM NH 4 HCO 3 , pH 9, 0.1 M NaCl). Fractions containing the 16K hGH-V were pooled, dialyzed against 20 mM NH 4 HCO 3 , pH 9 and lyophilized.
- the molecular mass of the 16K hGH-V calculated from its amino acid sequence was determined to be 15.7 kD.
- This example describes a procedure preparation of intact human placental lactogen (hPL (Met ⁇ 1 Phe 191 )) (SEQ ID NO: 17).
- the coding sequence of the hPL was cloned using RT-PCR experiments performed on syncytiotrophoblastic cells. An initiation codon ATG was added to the 51 end of the coding sequence of the hPL (SEQ ID NO: 13) and inserted into the pT7L E. coli expression vector. This recombinant plasmid was called pT7L-hPL.
- a 100 ml culture of E. coli BL21 (DE3) carrying the pT7L-hPL plasmid was grown overnight at 37° C. in LB medium-ampicillin 100 ⁇ g/ml. Twenty ml of this culture were used to inoculate 1 liter of LB medium-ampicillin 100 ⁇ g/ml. When the O.D. (600 nm) reached at 0.9, 1 mM isopropyl ⁇ -D thiogalactopyranoside (IPTG) was added to the culture. The induced culture was grown for an additional 4 hour period.
- O.D. 600 nm
- IPTG isopropyl ⁇ -D thiogalactopyranoside
- Cells were collected by centrifugation at 5000 g for 15 min (4° C.) and resuspended in 50 mM Tris-HCl, 0.5 mM EDTA, (T 50 E 0.5 ), 0.1 mM PMSF, pH 8. Cells were broken in a cell disrupter. After centrifugation of cell lysates (12,000 g, 15 min, 4° C.), hPL was found in insoluble fraction (inclusion bodies).
- the inclusion bodies recovered in the pellet were washed 2 times in 250 ml of T 50 E 0.5 , 0.1 mM PMSF, pH 8. Inclusions bodies were solubilized in 20 mM ethanolamine, pH 9 containing 8 M of deionized urea, 1% ⁇ -ME. 0.5 mM PMSF at a concentration of 100 ⁇ g of protein per ml of buffer. The protein solution was heated at 55° C. for 5 min and then incubated overnight at room temperature. The renaturation was performed by a continued dialysis (72 hours) against 500 volumes of 20 mM ethanolamine, pH 9.
- the renatured protein was loaded on a HitrapQ anion exchange column. Chromatography was performed in 20 mM ethanolamine, pH 9 and hPL was eluted within a gradient of 0 to 1 M NaCl. Fractions containing hPL were pooled, concentrated to 2 mg/ml and loaded on a Sephadex G100 column (50 mM NH 4 HCO 3 , pH 7.5)
- TGC Cys 53
- CGC Arg 134
- CCC Pro
- E. coli BL21 (DE3) culture (100 ml) carrying the plasmid pT7L-16K hPL was grown overnight at 37° C. in LE medium-ampicillin 100 ⁇ g/ml. Twenty ml of this culture was used to inoculate 1 liter of LB medium-ampicillin 100 ⁇ g/ml. When the O.D. (600 nm) reached at 0.9, 1 mM isopropyl ⁇ -D thiogalactopyranoside (IPTG) was added to the culture. The induced culture was grown for an additional 4 hour period.
- O.D. 600 nm
- IPTG isopropyl ⁇ -D thiogalactopyranoside
- Cells were collected by centrifugation at 5000 g for 15 min (4° C.) and resuspended in 50 mM Tris-HCl, 0.5 mM EDTA, (T 50 E 0.5 ), 0.1 mM PMSF, pH 3. Cells were broken in a cell disrupter. After centrifugation of cell lysates (12,000 g, 15 min, (4° C.), mutated hPL was found in the insoluble fraction (inclusion bodies).
- the inclusion bodies recovered in the pellet were washed 2 times in 250 ml of T 50 E 0.5 , 0.1 mM PMSF, pH 8 and stored at ⁇ 20° C. Inclusions bodies were solubilized in 20 mm ethanolamine, pH 9 containing 8 M of deionized urea, it ⁇ -ME. 0.5 mM PMSF at a concentration of 100 gg of protein per ml of buffer. The protein solution was heated at 55° C. for 5 min and then incubated overnight at room temperature (RT). The renaturation was performed by a continued dialysis (72 hours) against 500 volumes of 20 mM ethanolamine, pH 9. The protein was then concentrated to 2 mg/ml and cleaved with thrombin overnight (0.3%, 25° C.).
- the cleaved proteins were incubated for one hour with 1% ⁇ -ME. before loading on a Sephadex G100 column (50 mM NH 4 HCO 3 , pH 7.5, 100 mM NaCl, 5 mM ⁇ -ME.
- Fractions containing 16K hPL were pooled, dialyzed against 50 mM Tris-HCl, pH 8, 20% NH 2 SO4 and loaded on a phenyl sepharose 6 fast flow hydrophobic column. Chromatography was performed in 50 mM Tris-HCl, pH 8 and 16K hPL was eluted within a gradient 20 to 0% of NH 2 SO4.
- Fractions containing purified 16K hPL were pooled, dialyzed against 20 mM NH 4 HCO 3 , pH 7.5 and lyophilized. Proteins were purified to 95% homogeneity.
- the molecular mass of the 16K hPL calculated from its amino acid sequence was determined to be 15.6 kD.
- This example describes bovine brain capillary endothelial cell proliferation assay used for testing in vitro angiogenic activity of the peptides of the invention.
- This example illustrates preparation of bovine brain capillary endothelial cells for study of inhibitory effect of human 16K N-terminal fragments of lactogenic hormones.
- Bovine brain capillary endothelial cells were isolated according to J. Cell. Physiol, 127:121 (1986), and grown in low glucose Dulbecco's modified Eagle's medium containing 10 wt % calf serum, 2 mM glutamine, and 100 U/ml penicillin/streptomycin and 2.5 ⁇ g/ml fungizone.
- bFGF Basic fibroblast growth factor
- This example describes in gel mitogen activated protein kinase MAPK assay.
- Renatured MBP kinase activity was detected by incubating the gel for 60 min at room temperature in a reaction buffer containing 40 mM HEPES (pH 7.4) 2 mM DTT, 15 mM MgCl 2 , 300 ⁇ M sodium orthovanadate, 100 mM EGTA, 25 ⁇ M ATP and 100 ⁇ Ci of ( ⁇ - 32 P)ATP. Unincorporated radioactivity was removed by extensive washing in 5% trichloroacetic acid containing 1% tetrasodium pyrophosphate PPi. The gel was dried and exposed to X-ray film for 24 hours.
- This example describes Western blot for detection of mitogen activated protein kinase (MAPK) used for determination of specificity of the receptors bind peptides of the invention.
- MAPK mitogen activated protein kinase
- Antigen-antibody complexes were detected with horseradish peroxidase-coupled secondary antibodies using the Enhanced Chemiluminescence system (ECL, Amersham). The blots were exposed to Reflection NEF films (NEN). Western blots were stripped for reprobing with other primary antibodies by incubation for 30 min at 22° C. in a buffer containing 0.2 M glycine (pH 2.5) followed by two washes in PBS.
- This example describes the assay used for detection of inhibition of bovine plasminogen activator inhibitor-1 by peptides of the invention.
- the antigen-antibody complexes were detected with horseradish peroxidase conjugated secondary antibody and the Enhanced Chemiluminescence system.
- the blots were exposed to reflection EF films (NEN) to visualize the bands.
- This example illustrates chick chorioallantoic membrane assay used for testing of in vivo inhibitory activity of peptides of the invention.
- Chick embryos were placed in a Petri dish on day 3 of their development and cultured in a humidified incubator in an atmosphere of oxygen containing 2.5% CO 2 as described in Nature, 297:307 (1982).
- 5 mm disk of methylcellulose (0.5%, Sigma M0512) containing 20 ⁇ g of peptides of the invention and 2 ⁇ g bovine serum albumin (BSA) were laid on the advancing edge of the chick CAM as previously described in Nature, 297:307 (1982).
- BSA bovine serum albumin
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US10/714,067 US7300920B2 (en) | 1997-05-13 | 2003-11-14 | Antiangiogenic peptide agents |
US11/875,323 US20080096795A1 (en) | 1997-05-13 | 2007-10-19 | Novel Antiangiogenic Peptide Agents and Their Therapeutic and Diagnostic Use |
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US4949314A (en) * | 1966-08-16 | 1990-08-14 | The United States Of America As Represented By The Secretary Of The Navy | Method and means for increasing echo-ranging-search rate |
US4189426A (en) * | 1976-04-12 | 1980-02-19 | Hoffmann-La Roche, Inc. | Recombinant hormonal compositions and method |
US4725549A (en) * | 1980-09-22 | 1988-02-16 | The Regents Of The University Of California | Human and rat prolactin and preprolactin cloned genes |
US4853332A (en) * | 1982-10-19 | 1989-08-01 | Cetus Corporation | Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of biologically active proteins |
US4959314A (en) * | 1984-11-09 | 1990-09-25 | Cetus Corporation | Cysteine-depleted muteins of biologically active proteins |
US5217867A (en) * | 1988-11-30 | 1993-06-08 | The Salk Institute For Biological Studies | Receptors: their identification, characterization, preparation and use |
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- 1998-05-12 AU AU73832/98A patent/AU7383298A/en not_active Abandoned
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2001
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2007
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2511301A2 (de) | 2006-08-04 | 2012-10-17 | Medimmune Limited | Menschliche Antikörper gegen ERBB2 |
US20100113286A1 (en) * | 2007-05-05 | 2010-05-06 | Gilles Andre Lajoie | Methods for detection of preeclampsia |
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EP1173191A1 (de) | 2002-01-23 |
WO1998051323A1 (en) | 1998-11-19 |
US20080090750A1 (en) | 2008-04-17 |
US7300920B2 (en) | 2007-11-27 |
AU7383298A (en) | 1998-12-08 |
US20080096795A1 (en) | 2008-04-24 |
US20040077054A1 (en) | 2004-04-22 |
EP1173191A4 (de) | 2004-12-01 |
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