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WO1992009294A1 - Proteines purifiees tirees de tissu gestationnel mammifere regulant la proliferation cellulaire - Google Patents

Proteines purifiees tirees de tissu gestationnel mammifere regulant la proliferation cellulaire Download PDF

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Publication number
WO1992009294A1
WO1992009294A1 PCT/US1991/008046 US9108046W WO9209294A1 WO 1992009294 A1 WO1992009294 A1 WO 1992009294A1 US 9108046 W US9108046 W US 9108046W WO 9209294 A1 WO9209294 A1 WO 9209294A1
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Prior art keywords
substantially purified
purified protein
cells
protein
molecular weight
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PCT/US1991/008046
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English (en)
Inventor
Eytan R. Barnea
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Barnea Eytan R
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Application filed by Barnea Eytan R filed Critical Barnea Eytan R
Priority to JP4501045A priority Critical patent/JPH05508419A/ja
Publication of WO1992009294A1 publication Critical patent/WO1992009294A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to substantially purified agents normally expressed during mammalian pregnancy that may be used to control the proliferation of cells, and, in particular, provides for proliferative agents as well as antiproliferative agents.
  • the antiproliferative agents may be used to limit undesirable proliferation of cells, for example, in the treatment of cancer.
  • the proliferative agents may be utilized to increase cell proliferation and may be used, for example, in the treatment of infertility.
  • the fertilized egg, or zygote undergoes a number of cell divisions to form a ball of about 15 small cells, called the morula.
  • the morula enters the uterus and develops an inner cavity, thereby becoming a blastocyst consisting of (1) an inner cell mass which gives rise to the embryo; (2) a blastocyst cavity; and (3) an outer layer of cells, called the trophoblast.
  • the blastocyst attaches to the endometrial epithelium of the uterus and the trophoblastic cells invade the uterine wall.
  • the trophoblast differentiates into two types: cytotrophoblast and syncytiotrophoblast.
  • the syncytiotrophoblast is adjacent to the developing embryo and becomes a multinucleated protoplasmic mass in which no cell boundaries are discernible (Id., p. 34) .
  • This fluid, or embryolymph provides nutrition to the developing embryo and marks the beginning of the uteroplacental circulation.
  • the endometrium forms the maternal part
  • the trophoblast forms the fetal part, of the placenta (Id. , p. 36) .
  • hCG human chorionic gonadotrophin
  • hCS human chorionic somatomamotropin
  • hPL human placental lactogen
  • TGF-beta may be isolated from human placenta, among other tissues. It was observed that the response of cells to TGF-beta appeared to be bifunctional, in that TGF-beta was able to stimulate reversible transformation of murine fibroblasts, but was also able to inhibit anchorage- dependent growth of normal rat kidney fibroblasts and of human tumor cells by increasing cell cycle time.
  • etnansky (1987, Immunology 175:68) reports the isolation and characterization of a bovine placenta protein which specifically inhibits the proliferation of tumor cells. This protein, termed decidua inhibitory factor (DIF) , was estimated to have a molecular weight of about 60 kD by SDS-PAGE.
  • DIF decidua inhibitory factor
  • AR amphiregulin
  • TGF-alpha transforming growth factor alpha
  • the present invention provides for 5 gestational agents that may be used to control cell proliferation. It is based, at least in part, on the discovery that several agents produced by the . developing embryo appear to play an important role in achieving a system of checks and balances which regulate proliferation and differentiation of cells during pregnancy.
  • the present invention provides for a substantially purified protein having a molecular weight of less than about 10,000 daltons and preferably about 6,500-7,000 daltons that is expressed in mammalian embryos and that has an antiproliferative effect on certain cancer cells and on certain embryonal cells.
  • This protein termed JDK protein, was observed to exert an antiproliferative effect on all cancer cell lines tested, and was found to prevent tumor formation and to improve the survival of nude mice inoculated with fibrosarcoma cells.
  • the antiproliferative gestational protein of the invention may be utilized in the prevention and treatment of cancer. It may also be used as a contraceptive to prevent pregnancy.
  • the present invention provides for a substantially purified protein having a molecular weight of less than about 3,000 daltons which is expressed in mammalian embryonal tissue and has a proliferative effect on morular cells.
  • This gestational proliferative agent of the invention termed GP -1 has further been observed to increase the secretion of human chorionic gonadotrophin by placental explants, to promote the development of a morula into a blastocyst and to facilitate implantation.
  • the present invention provides for a second gestational proliferative agent, termed GPA-2, that has a molecular weight greater than 20,000 daltons.
  • GPA-2 gestational proliferative agent
  • FIGURE 1 Results of HPLC analysis of the ⁇ 8,000 MW fraction from spinal cord showing three dominant bands.
  • FIGURE 2. Results of PAGE analysis of the ⁇ 10,000 MW fraction from spinal cord.
  • FIGURE 3. Superfusion apparatus.
  • GROWTH CHAMBERS They contained the tissue being cultured.
  • TUBING SYSTEM Silicone permitted the gas to get in the tubing system.
  • HEAT/GAS EXCHANGER It maintains the thermal environment in and around the growth chambers.
  • GAS TRANSFER Its achieved by flooding the system with the desired gas.
  • MAXIMUM 3 ml/min. li. DIRECTION: The direction of the lighted arrow indicates direction of fluid through the pump tubing.
  • FLOW RATE From 00>99 represents a percentage of normal pumping rate. 13.
  • AT THIS POINT another peristaltic pump is added in order to add GnRH, or some other factors.
  • FIGURE 4 A. Suppressive effect of the ⁇ 8,000 MW fraction of extract on secretion of hCG by placental explants. Efferent sample fractions were collected every 2.4 minutes and were sequentially numbered.
  • FIGURE 5 Ability of various dilutions of total extract to suppress hCG secretion by placental explants. The following dilutions of extract in 0.05 M Tris-HCl PMSF-DTT (pH 7.4) were used: 1/20, 1/200, and 1/2000. Buffer alone was used as a control.
  • FIGURE 6 Suppressive activity of the ⁇ 8,000 MW fraction of extract on the proliferation of cancer cell lines. The number of cells in buffer-treated cultures serves as a control.
  • FIGURE 7. Percent suppression of MCF-7 proliferation as a function of increasing dosage of ⁇ 8,000 MW fraction protein.
  • FIGURE 8. Suppressive effect of embryo extract on mouse embryonal development in vitro. A. Percentage of untreated control embryos attaining the morular stage. B. Percentage of untreated control embryos exhibiting hatching/adhesion.
  • FIGURE 9 The effect of various molecular weight fractions of extract on nude mice survival following inoculation with fibrosarcoma. Fraction 1 corresponds to MW ⁇ 3,000; Fraction 2 corresponds to MW ⁇ 30,000 MW;
  • Fraction 3 corresponds to MW ⁇ 8,000
  • FIGURE 10 A ⁇ 3,000 MW fraction of embryo extract increased secretion of hCG by placental explants.
  • FIGURE 11 Results of HPLC analysis of the ⁇ 3,000 D molecular weight fraction associated with proliferative activity.
  • the present invention provides for gestational agents that may be used to control cell proliferation. It is based, at least in part, on the theory that pregnancy operates, figuratively speaking, like a reversible cancer.
  • the products of conception similar to cancer, are invasive, penetrate the circulation and metastasize, express similar surface antigens, and secrete certain cancer-related compounds, such as alphafetoprotein and carcinoembryonic antigen.
  • the conceptus like a tumor, is not rejected by the body, but rather harnesses maternal resources to secure its well-being. Unlike cancer, however, the invasiveness and tolerance associated with pregnancy are reversible at almost any time.
  • the various embodiments of the invention relate to such agents, and their use, for example, in the treatment of cancer and infertility, or as a contraceptive.
  • the present invention provides for a gestational antiproliferative agent that is comprised in an extract of mammalian embryonal tissue and that has a molecular weight less than about 10,000 daltons.
  • the agent has a molecular weight between about 6,500 and 7,000 daltons, and is termed JDK.
  • the gestational antiproliferative agent JDK may be prepared from any suitable mammalian embryo.
  • the size of the embryo will affect the ease with which individual organs are identified and isolated, it is desirable that the embryo be obtained from a relatively large mammal, such as, for example, but not by way of limitation, a human, non-human primate, horse, cow, sheep, or pig, etc. If obtained from a human, the embryo may be obtained, after proper consent, from the products of an elective or spontaneous abortion.
  • the embryo be of a developmental stage in which rapid development is occurring or has just begun to taper off.
  • embryos at a stage comparable to weeks six through nine of human gestation may preferably be used.
  • any organ of the embryo may be utilized.
  • the brain and spinal cord, (i.e. neural elements) or adrenal gland are preferably used.
  • Visceral organs, such as lung, liver, or kidney may also desirably be used.
  • the gestational antiproliferative agent, JDK, of the invention may be prepared as follows according to a specific, nonlimiting embodiment of the present invention.
  • Embryonal tissue may be minced and placed in cold Tris-HCl at pH 7.4 containing 10 mM dithiothreitol (DTT) and 2 mM phenyl-methyl-sulfonyl-fluoride (PMSF) , then sonicated on ice for about 60 seconds and then centrifuged at about 2400 rpm for about ten minutes to remove cellular debris.
  • An initial amount of embryonal tissue weighing about 50 mg may, for example, be processed in about 1 ml of buffer and may yield about 1 ml of supernatant.
  • the supernatant from the centrifuged material may then be separated into fractions based on molecular weight, using, for example, the Filtron System Omega Cell (Biolab) , 30 mm diameter, under nitrogen, and the fractions containing molecular weights less than about 10,000 daltons may desirably be obtained.
  • This fraction may, preferably, be further purified by polyacrylamide gel electrophoresis (PAGE) . That portion of the resulting gel corresponding to molecular weights between about 3,000 and 8,000 daltons, and preferably between about 6,500 and 7,000 daltons may then be separated from the rest of the gel and eluted to produce the gestational antiproliferative agent JDK.
  • the fraction may be further purified by HPLC as described in Section 6.1.3., infra, in which case three major bands may be expected to be produced, as set forth in Figure 1.
  • the present invention provides for a substantially purified protein having a molecular weight less than about 10,000 daltons, and preferably between about 6,500 and 7,000 daltons, that is normally expressed in mammalian embryonal tissue and that has an antiproliferative effect on certain cancer cells and certain embryonal cells.
  • a substantially purified protein having a molecular weight less than about 10,000 daltons, and preferably between about 6,500 and 7,000 daltons, that is normally expressed in mammalian embryonal tissue and that has an antiproliferative effect on certain cancer cells and certain embryonal cells.
  • the fact that it is "normally expressed" in a mammalian embryo is distinct from the substantially purified form of the protein recited, as the protein does not normally, (i.e. naturally) occur in substantially purified form.
  • the present invention further provides for a substantially purified protein, as described supra, that corresponds to the protein comprised in peak I, peak II, or peak III of the chromatogram depicted in Figure 1.
  • the gestational proliferative agent, or JDK, of the invention may also be prepared by any other method known in the art, including, but not limited to, chemical synthesis or recombinant DNA technology.
  • the present invention provides for the preparation and use of fragments or derivatives of JDK, including fragments produced chemically, by recombinant DNA, or by protease activity, and derivatives obtained by, for example, glycosylation. phosphorylation, dephosphorylation or chemical conjugation of any compound to JDK or a fragment thereof.
  • the present invention also provides for a protein related to JDK but normally expressed in a non-human embryo that is at least about 70 percent homologous to JDK.
  • JDK may be sequenced, and the amino acid sequence so obtained may be used to deduce oligonucleotide probes that may be used directly to screen recombinant DNA libraries, or in polymerase chain reaction, in order to obtain JDK- encoding nucleic acid sequences. These sequences may then be inserted into an appropriate expression vector system and then may be expressed in quantity using standard techniques. Alternatively, JDK may be produced by chemical synthesis or by any other method known in the art.
  • GPA-1 a gestational proliferative agent termed GPA-1 that is comprised in an embryonal tissue extract and that has a molecular weight less than about 3,000 daltons and preferably about 1,500-2,000 D.
  • the source of suitable embryonal tissue is as described in Section 5.1., supra. except that embryos at a developmental stage comparable to six weeks of human gestation may also be used.
  • GPA-1 may be prepared as follows according to a specific, nonlimiting embodiment of the present invention.
  • Embryonal tissue may be minced and placed in cold Tris-HCl at pH 7.4 containing 10 mM dithiothreitol (DTT) and 2 mM phenyl-methyl-sulfonyl- fluoride (PMSF) , then sonicated on ice for about 60 seconds, and then centrifuged at about 2400 rpm for about ten minutes to remove cellular debris.
  • An initial amount of embryonal tissue weighing about 50 mg may, for example, be processed in about 1 ml of buffer and may yield about 1 ml of supernatant.
  • the supernatant from the centrifuged material may then be separated into fractions based on molecular weight, using, for example, the Filtron System Omega Cell (Biolab) , 30 mm diameter, under nitrogen and the fraction containing molecular weights less than 3,000 D may desirably be obtained.
  • This fraction may, preferably, be further purified by polyacrylamide gel electrophoresis. That portion of the gel corresponding to a molecular weight less than about 3,000 D may then be separated from the rest of the gel and eluted to form the gestational proliferative agent GPA-1. See Section 8, infra.
  • the present invention provides for a substantially purified protein, GPA-1, having a molecular weight of less than about 3,000 daltons that is expressed in mammalian embryonal tissue and that has a proliferative effect on morular cells.
  • this protein has a molecular weight greater than 1,400 D, and in most preferred embodiments, the molecular weight is about 2,000 D.
  • GPA-1 also is capable of increasing the secretion of hCG by placental explants, of promoting the development of a morula into a blastocyst, and of facilitating implantation.
  • the present invention further provides for a second gestational proliferative agent termed GPA-2 that has a molecular weight above 20,000. 5.3 ANTIBODIES OF THE INVENTION
  • gestational proliferative agents i.e. GPA-1 and GPA-2
  • gestational antiproliferative agent i.e. JDK
  • active fragments or derivatives thereof may be used as immunogens to generate antibodies.
  • the amino acid sequence of the gestational agent may be analyzed in order to identify portions of the molecule which may be associated with increased immunogenicity.
  • the amino acid sequence may be subjected to computer analysis to identify surface epitopes.
  • the deduced amino acid sequences of gestational agent from different species could be compared, and relatively non-homologous regions identified; these non- homologous regions would be more likely to be immunogenic across various species.
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497) . as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , 1983, Immunology Today 4.:72), and the EBV-hybridoma technique to produce human monoclonal antibodies are within the scope of the present invention.
  • the monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g.. Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16).
  • Chimeric antibody molecules may be prepared containing a mouse antigen- binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and, Corynebacterium parvum.
  • Antibody molecules may be purified by known techniques, e.g. , immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography) , or a combination thereof, etc.
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab 1 fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the 2 Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • the gestational antiproliferative agent is a substantially purified protein as described in
  • the present invention provides for a method of treating a patient suffering from cancer comprising administering to the patient an effective amount of gestational antiproliferative agent JDK in a suitable pharmaceutical carrier.
  • the gestational antiproliferative agent is a substantially purified protein as described in
  • the present invention may be used to inhibit proliferation of a wide variety of cancer cells and to treat a wide variety of cancers. As exemplified in
  • the present invention may be used to inhibit proliferation of cancer cells arising from any tissue, including, but not limited to breast, bone marrow, lymphoreticular system, ovary, kidney, lung, brain, intestine, stomach, esophagus, pancreas, spinal cord, mucosa, germ cell, bone, muscle, skin (e.g. melanoma) etc.
  • the present invention may be used to treat cancer arising from any tissue in a patient, including, but not limited to, cancer of the breast, ovary, kidney,lung,brain, intestine, bone marrow, lymphoreticular system, stomach, esophagus, pancreas, spinal cord, mucosa, germ cell, bone, muscle, skin (e.g. melanoma), choriocarcinoma, etc.
  • An effective concentration of JDK may be defined as any concentration that is capable of inhibiting the proliferation of MCF-7 cells,under conditions as set forth in Section 7, infra , by at least 10 percent relative to untreated control.
  • An effective amount of JDK to be used in patient treatment may be defined as that amount that produces an effective concentration of JDK in the tissue of interest.
  • JDK may be administered by any route known in the art, including, but not limited to, intravenous, subcutaneous, intramuscular, by injection into a tumor or by other local injection, intranasal, intraocular, intraperitoneal, oral, etc.
  • JDK may be coupled to another molecule, such as an antibody, or to a magnetic bead in order to target delivery.
  • JDK may be administered via a solid or semisolid implant, such as a sustained-release implant.
  • JDK may be incorporated into microcapsules, microspheres, or liposomes.
  • JDK may be coupled to another molecule, such as an antibody, or to a magnetic bead in order to target delivery.
  • a suitable pharmaceutical carrier is any vehicle which is relatively nontoxic to humans and which preserves the activity of JDK.
  • Suitable carriers include, but are not limited to, water, saline, phosphate buffered saline, dextrose, etc.
  • gestational antiproliferative agent i.e. JDK
  • gestational proliferative agents i.e. GPA-1 and/or GPA-2
  • GPA-1 and/or GPA-2 gestational proliferative agents
  • the present invention provides for a method of contraception comprising administering to a female subject an effective amount of gestational antiproliferative agent JDK in a suitable pharmaceutical carrier.
  • the gestational antiproliferative agent JDK is a substantially purified protein.
  • the present invention further provides for a method of improving the fertility of a subject comprising administering to the subject an effective amount of gestational proliferative agent such as GPA- 1 and/or GPA-2 in a suitable pharmaceutical carrier.
  • the gestational proliferative agent(s) is (are) a substantially purified protein(s) .
  • a subject is any human or non-human mammalian subject.
  • the fertility promoting aspect of the invention may be particularly useful in improving the fertility of livestock.
  • An effective amount of gestational antiproliferative agent is construed, in embodiments relating to contraception, to refer to an amount that will result in a concentration in at least a portion of the reproductive organs of the subject that is the same as a concentration that will inhibit morular development of mouse embryos, under conditions as described in Section 7.1.3., by at least about 50 percent relative to untreated controls.
  • An effective amount of gestational proliferative agent is construed, in embodiments relating to fertility, to refer to an amount that will result in a concentration in at least a portion of the reproductive organs of the subject that is the same as a concentration that will increase the number of morula that progress to form blastocysts by at least 50 percent relative to untreated controls under conditions as described in Section 8, infra.
  • the fertility-controlling agents may be administered by any method known in the art, as described in Section 5.4., supra.
  • Suitable pharmaceutical carriers that may be used according to the invention are relatively nontoxic to the host and preserve the activity of the agent, and include those set forth in Section 5.4., supra.
  • antibody directed toward gestational proliferative agent may be used in methods of contraception.
  • the present invention provides for a method of contraception comprising administering to a female subject an effective amount of antibody that binds to a gestational proliferative agent.
  • the present invention provides for a method of contraception comprising immunizing a female subject with an immunogenic preparation comprising a gestational proliferative agent.
  • Such an immunogenic composition may comprise an adjuvant, such as Freund's complete adjuvant, and may desirably comprise gestational proliferative agent coupled to an immunogenic compound or a gestational proliferative agent obtained from a species that is different from the subject to be immunized.
  • an adjuvant such as Freund's complete adjuvant
  • gestational proliferative agent coupled to an immunogenic compound or a gestational proliferative agent obtained from a species that is different from the subject to be immunized.
  • immunization may result in irreversible contraception.
  • gestational antiproliferative agent may be used in the treatment of disorders of increased cell proliferation including, but not limited to, premalignant conditions, keloids. Von Recklinghausen disease, familial polyposis, benign neoplasms (e.g. breast adenomas) , autoimmune diseases such as rheumatoid arthritis, etc.
  • a gestational proliferative agent i.e. GPA-1 and/or GPA-2
  • GPA-1 and/or GPA-2 may be used in the treatment of disorders of decreased cell proliferation, such as burnt-out myeloproliferative disorders, pernicious anemia, Sjogren's syndrome, etc.
  • Gestational proliferative agent(s) may also be used to treat conditions in which increased cell proliferation is desirable, for example, to replace cells damaged by infarct, infection, injury, or exposure to a toxic agent (e.g. post-myocardial infarction myocardium, nervous system tissue that has been damaged or lost by infarction, infection, injury or exposure to toxic agent bone marrow that has been damaged by disease or by exposure to a toxic agent, skin that has been damaged or lost, e.g. in burn patients) .
  • a toxic agent e.g. post-myocardial infarction myocardium, nervous system tissue that has been damaged or lost by infarction, infection, injury or exposure to toxic agent bone marrow that has been damaged by disease or by exposure to a toxic agent, skin that has been damaged or lost, e.g. in burn patients.
  • gestational proliferative agent(s) may be used to promote liver regeneration.
  • gestational proliferative agent(s) may be used to facilitate the incorporation of grafted tissue into a subject.
  • gestational proliferative agent(s) may be used to promote wound healing and/or hair growth.
  • the gestational agents of the invention may be utilized in vivo or in vitro.
  • GPA-1 and/or GPA-2 may be used to promote the growth of cells or tissue in culture.
  • Visceral organs comprising lung, liver, kidney and adrenal, and/or neural organs such as spinal cord and brain, were extracted by sonication on ice followed by centrifugation (2,400 rpm x 10 in.) in 0.5 M Tris-HCl buffer at pH 7.4, dithiotrietol (DTT) 10 mM and phenyl-methyl-sulphonyl-fluoride (PMSF) 2 mM. Extraction with ethanol resulted in an approximately 3-fold lower activity product. 6.1.2. PREPARATION OF DIFFERENT MOLECULAR WEIGHT FRACTIONS
  • the extract obtained according to (A) above from embryonic spinal cord was filtered through different molecular weight (M.W.) filters using the PBS buffer with DTT and PMSF. Each 1 ml of extract was filtered with 30 ml of buffer. The collected extract was lyophylized and the sediment was suspended in distilled water and stored at -20°C.
  • fractions were prepared (the symbol " ⁇ ” being used, as conventional, to indicate fractions having "less than” the indicated M.W.): ⁇ 100,000, ⁇ 30,000, ⁇ 10,000, ⁇ 8,000 and ⁇ 3,000. All fractions were tested for the ability to modulate HCG production of placental explants by using the superfusion model described by Barnea and Kaplan (1989, J. Clin. Endocrinol. Metab. j59:215-217) (see Section 7, infra). Only fractions below about 10,000 were found to be biologically active.
  • High pressure liquid chromatography (HPLC) analysis was performed on the ⁇ 8,000 MW fraction of spinal cord extract using a Hitachi HPLC apparatus and a 25 cm RP-18 column at 25°C. The sample was applied in water and eluted using 40 percent H 2 0, 60 percent CH 3 OH at a flow rate of 1 ml/min.
  • tissue was sonicated in 0.5 ml of 0.2 M Tris buffer containing 10 '3 M PMSF and 2 mM DTT pH 7.4, centrifuged at 20,000 rpm and the supernatant was filtered through a 0.2 ⁇ m filter.
  • a sample (335 ⁇ l of 16 mg protein/ml) was applied on a HPLC column (Superdex 75HR 10/30, LKB, Pharmacia) which was previously equilibrated with the same buffer. Samples of 0.5 ml were collected at a flow rate of 0.5ml/min and frozen at -70°C until used. The molecular weights of the various fractions were estimated from the retention time of proteins with known molecular weights.
  • Extracts of various embryonic organs, prepared as described above, were subjected to fractionation by molecular weight. Only fractions having a molecular weight less than 10,000 daltons were found to be capable of altering HCG production of placental explants.
  • a superfusion apparatus (Accusyst, Endotronics, St. Paul, MN) ( Figure 3) with a multichannel peristaltic pump and fraction collector (model 272, ISCO, Durham,NC) was used to study the short term dynamics of human chorionic gonadotropin (hCG) secretion, as described in Barnea and Kaplan (1989, J. Clin. Endocrinol. .69:215-217).
  • Placental explants 200-300 mg wet weight) were placed into the culture chambers and a 18 mM HEPES/DMEM solution was washed through it in an atmosphere of 5% C0 2 and 95% air at 37°C.
  • Sample corresponding to a particular molecular weight fraction was applied by administering a one minute pulse such that all fractions could be analyzed in parallel.
  • Experiments were conducted for a 120-minute period. A 1 ml sample from the effluent of the apparatus was collected every 2.4 minutes for hCG measurements by radioimmunoassay. In such experiments, one channel served as control and four served as experimental channels. At given intervals a 1-minute pulse of an aqueous solution of the tested fraction was given through a peristaltic pump equipped with a digital flowmeter (Ismatech DD, Chicago, IL) . The fractions that caused a significant change in hCG secretion following administration of the pulses were considered as "active samples”. 7.1.2. TESTING OF JDK ACTIVITY ON CANCER CELL LINES
  • Cell lines for testing obtained from the American Type Culture Collection, Rockville, Maryland, were MCF-7, a breast cell line (accession number: HTB22) and HBL-40, a breast cell line exposed to SV40 virus, (accession number: HTB124) .
  • Other cell lines used included CRL, a kidney cell line, and GNL, an ovarian cell line.
  • Cells were cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml, and kept at 37°C in a 95 percent air/5 percent C0 2 atmosphere. 10-50 microliter aliquots of extract from ⁇ 8,000 and ⁇ 10,000 fractions, corresponding, respectively, to protein concentrations of 0.2-1.0 microgram per ml were added to identical cultures in order to evaluate the dose- response. Two control cultures were used for each cell line. The first was an untreated culture. The second control was a culture to which 50 microliters of Tris-HCl were added. Extract was added to cultures only at the beginning of the experiment. After 3-5 days, the number of cells was counted using an automatic cell counter.
  • the culture dishes were pre-coated with a 1 mm thickness of collagen.
  • mice (Balb/c) were treated by sublethal irradiation with x-rays to destroy their ability to form an immunological response.
  • mice were subcutaneously inoculated with 100,000 cells of a fibrosarcoma cell line. Five mice were used for each experiment, and five mice that were not inoculated served as controls. In addition, inoculated mice were treated by injecting 50 ⁇ l of about 1 ⁇ g/ml of protein-like material from different molecular weight fractions obtained as described supra immediately after tumor inoculation. Subsequently, the animals were kept in a germ free environment, fed ad libitum. and were examined weekly. After 3 weeks the mice were sacrificed and examined for the presence of tumors.
  • Figure 5 illustrates that even a 1/2000 dilution of extract, corresponding to a protein concentration of about 100 ng/ml, was observed to achieve about 50 percent of the suppressive effect associated with control.
  • Table II, infra, and Figure 7 illustrate the dose dependency of the antiproliferative effect of JDK in the ⁇ 8,000 MW fraction on MCF-7 cells in culture.
  • Cell count reduction became detectable when 10 ⁇ l of extract was used, corresponding to a protein concentration of 0.2 ⁇ g/ml. Maximal decrease was obtained with 50 ⁇ l of extract corresponding to 1.0 ⁇ g/ml of protein.
  • mice Thirty nude mice were inoculated with fibrosarcoma cells and divided into six groups of five mice each. Five uninoculated mice served as negative controls. One group of inoculated mice was treated only with Tris-HCl buffer. The remaining five groups were treated with 50 ⁇ l of about 1 ⁇ g/ml of one of the following extract fractions: ⁇ 3,000 MW; ⁇ 8,000 MW; ⁇ 8,000 MW; ⁇ 10,000 MW; and ⁇ 30,000 MW. As shown in Table III, all five mice in the group treated with the ⁇ 8,000 MW fraction were tumor-free. The only other group that contained tumor-free animals was the group receiving the ⁇ 8,000 molecular weight fraction (2 out of 5) . Of the remaining groups. Tumors were found to occur at the site of inoculation.
  • mice inoculated with fibrosarcoma and then either untreated (control) or treated with various molecular weight fractions of extract was evaluated over a three week period. Survival was measured as the number of mice surviving after the three week period.
  • the ⁇ 3,000 molecular weight fraction was obtained by mincing embryonal spinal cord in cold
  • Biolab Filtron System Omega Cell
  • ICR-strain mice were obtained from Charles River Laboratory. Females (between six and seven weeks old) were mated with ten to twelve-week old males. About 72 hours later, the females were sacrificed and morula-stage embryos were removed by flushing out the fallopian tubes with HAM-F1. The embryos were then placed in EBSS medium containing 10 percent newborn cord serum maintained in a humidified, 95 percent AIR/5 percent C0 2 atmosphere. For trophoblast development studies, the culture dishes were pre-coated with a 1 mm thickness of collagen. 8.1.3. ANALYSIS OF EFFECT OF EXTRACT ON HCG SECRETION BY PLACENTAL EXPLANTS
  • GPA-1 CAUSES INCREASED SECRETION OF HCG BY PLACENTAL EXPLANTS It was observed that the ⁇ 3,000 MW fraction, containing a protein termed GPA-1, of extract was able to increase hCG secretion by placental explants. Normally, hCG is secreted by placental extracts in a spontaneous pulsatile fashion with a relatively stable frequency. When a one minute pulse of the ⁇ 3,000 MW fraction was administered to explants via the superfusion apparatus, a major increase in hCG secretion was observed, as shown in Figure 10.
  • the number of morula treated with the ⁇ 3,000 MW fraction (GPA) that matured to form blastocysts was about three-fold greater than the number of blastocysts formed by morulas treated with buffer only. This observation occurred when morulas were treated with 10 microuters of GPA-1, which has a protein content of about 0.2 ⁇ g/ml. Exposure of the embryos to 50 microliters of GPA-1 containing fraction was found to increase the survival of the blastocysts by a factor of two relative to control, as measured over a 48 hour period.
  • GPA-1 PROMOTED TROPHOBLAST DEVELOPMENT When trophoblast development was evaluated using collagen-coated culture dishes, it was observed that the same level of development achieved by normal controls within 24 hours had been achieved by GPA-1- containing fraction-treated embryos within only 14 hours. These developmental changes included adhesion of the blastocyst to the collagen surface and penetration of the collagen by the differentiating trophoblastic cells. The process accelerated in vitro by GPA corresponds to the in vivo implantation process, and indicates that GPA-1 may be used to facilitate implantation, and, hence, increase fertility, in human beings.

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Abstract

L'invention concerne des agents purifiés exprimés normalement au cours d'une gestation de mammifères et pouvant être utilisé pour réguler la prolifération de cellules et, notamment, elle concerne des agents prolifératifs ainsi que des agents antiprolifératifs. Les agents antiprolifératifs peuvent être utilisés afin de limiter la prolifération indésirable de cellules, par exemple, dans le traitement du cancer. Les agents prolifératifs peuvent être utilisés afin d'accroître la prolifération cellulaire et, par exemple, dans le traitement de la stérilité.
PCT/US1991/008046 1990-11-28 1991-10-31 Proteines purifiees tirees de tissu gestationnel mammifere regulant la proliferation cellulaire WO1992009294A1 (fr)

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JP4501045A JPH05508419A (ja) 1990-11-28 1991-10-31 細胞増殖を制御する、哺乳動物妊娠期組織から精製されたタンパク質

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IL96499A IL96499A0 (en) 1990-11-28 1990-11-28 Anticancer protein-like extract
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4400640A1 (de) * 1994-01-12 1995-07-13 Eftag Entstaubung Foerdertech Verwendung von, Mittel mit und Verfahren zur Herstellung eines Arzneimittels mit Embryonalgewebe
WO1996018410A1 (fr) * 1994-12-13 1996-06-20 Alexandr Vasilievich Tikhonov Agent de traitement du cancer primitif du foie et procede de traitement du cancer primitif du foie
EP1091753A4 (fr) * 1998-07-02 2004-11-17 Envision Biomedical Consulting Proteines et peptides antiproliferants et antiviraux
EP1652529A3 (fr) * 1998-07-02 2007-11-21 Envision Biomedical Consulting Protéines et peptides antiprolifératifs et antiviraux
US7495071B2 (en) 2004-07-14 2009-02-24 Biospectrum, Inc. Antiproliferative peptides and antibodies for their detection
US9737585B2 (en) 2011-03-02 2017-08-22 Bioincept, Llc Compositions and methods for treatment of intracellular damage and bacterial infection
US10071131B2 (en) 2006-02-03 2018-09-11 Bioincept, Llc Compositions and methods for modulating the immune system
US11090355B2 (en) 2015-08-28 2021-08-17 Bioincept, Llc Compositions and methods for the treatment of neurodamage
US11096987B2 (en) 2015-08-28 2021-08-24 Bioincept, Llc Mutant peptides and methods of treating subjects using the same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4908206A (en) * 1986-09-16 1990-03-13 Amics Ag Extracts of embryonic organs, process for their preparation and pharmaceutical preparation containing them

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2451193A1 (fr) * 1979-03-13 1980-10-10 Brunel Henri Procede pour le traitement du cancer et preparation des produits employes pour ce traitement

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4908206A (en) * 1986-09-16 1990-03-13 Amics Ag Extracts of embryonic organs, process for their preparation and pharmaceutical preparation containing them

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4400640A1 (de) * 1994-01-12 1995-07-13 Eftag Entstaubung Foerdertech Verwendung von, Mittel mit und Verfahren zur Herstellung eines Arzneimittels mit Embryonalgewebe
WO1996018410A1 (fr) * 1994-12-13 1996-06-20 Alexandr Vasilievich Tikhonov Agent de traitement du cancer primitif du foie et procede de traitement du cancer primitif du foie
EP1091753A4 (fr) * 1998-07-02 2004-11-17 Envision Biomedical Consulting Proteines et peptides antiproliferants et antiviraux
EP1652529A3 (fr) * 1998-07-02 2007-11-21 Envision Biomedical Consulting Protéines et peptides antiprolifératifs et antiviraux
US7476649B2 (en) 1998-07-02 2009-01-13 Biospectrum, Inc. Antiproliferative and antiviral proteins and peptides
US7495071B2 (en) 2004-07-14 2009-02-24 Biospectrum, Inc. Antiproliferative peptides and antibodies for their detection
US10071131B2 (en) 2006-02-03 2018-09-11 Bioincept, Llc Compositions and methods for modulating the immune system
US10806771B2 (en) 2006-02-03 2020-10-20 Bioincept, Llc Compositions and methods for modulating the immune system
US9737585B2 (en) 2011-03-02 2017-08-22 Bioincept, Llc Compositions and methods for treatment of intracellular damage and bacterial infection
US10953071B2 (en) 2011-03-02 2021-03-23 Bioincept, Llc Compositions and methods for treatment of intracellular damage and bacterial infection
US11090355B2 (en) 2015-08-28 2021-08-17 Bioincept, Llc Compositions and methods for the treatment of neurodamage
US11096987B2 (en) 2015-08-28 2021-08-24 Bioincept, Llc Mutant peptides and methods of treating subjects using the same

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ZA919361B (en) 1992-09-30
JPH05508419A (ja) 1993-11-25
PT99622A (pt) 1992-10-30
EP0512102A4 (en) 1994-10-05
AU9085491A (en) 1992-06-25
IL96499A0 (en) 1991-08-16
EP0512102A1 (fr) 1992-11-11
IE914128A1 (en) 1992-06-03

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