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WO1989011492A1 - Inhibiteur de croissance de cellules mammaires humaines et procedes de production et d'utilisation - Google Patents

Inhibiteur de croissance de cellules mammaires humaines et procedes de production et d'utilisation Download PDF

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Publication number
WO1989011492A1
WO1989011492A1 PCT/US1989/002214 US8902214W WO8911492A1 WO 1989011492 A1 WO1989011492 A1 WO 1989011492A1 US 8902214 W US8902214 W US 8902214W WO 8911492 A1 WO8911492 A1 WO 8911492A1
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Prior art keywords
inhibitor
growth
human mammary
cells
cell
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PCT/US1989/002214
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English (en)
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Paul R. Ervin, Jr.
Max S. Wicha
Robert L. Cody
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The Regents Of The University Of Michigan
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Priority to JP89506373A priority Critical patent/JPH05506423A/ja
Publication of WO1989011492A1 publication Critical patent/WO1989011492A1/fr

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    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation

Definitions

  • the present invention relates generally to cell growth regulation and, more particularly, to human mammary cell growth inhibitor and methods of its production and use.
  • mammary cell growth factors which are not hormonal in nature have also been described. See, for example, Dickson, R. B. et al., Science. 232:1540-1543 (1986).
  • Specific growth factors which have been shown to have a stimulating effect on mammary tissue growth include platelet-derived growth factor, insulin-like growth factor (IGF-1) and transforming growth factor (TGF) alpha.
  • IGF-1 insulin-like growth factor
  • TGF transforming growth factor
  • TGF-beta has been shown to suppress mammary tissue growth. See, for example, Robert, A. B. et al. PNAS US, 82:119-123 (1985).
  • the present invention relates to a new cell growth inhibitor specific for human mammary cells and methods of its production and use.
  • the cell growth inhibitor of the present invention comprises a thermolabile protein which, when isolated from
  • NHMC-conditioned medium by ion exchange chromatography exhibits peaks of activity at molecular weights in the range of from about 40,000 to about 45,000 daltons and from about 50,000 to about 60,000 daltons as measured by MCF-7 growth assays of molecular sieve column fractions.
  • Inhibitor purified by antibody affinity chromatography exhibits peaks of activity in the range of from about
  • the inhibitory activity of cell growth inhibitor of the present invention is calcium-dependent, dose-dependent and its inhibitory effect is reversible.
  • the cell growth inhibitor is mammary tissue or cell specific, having an inhibitory effect on the growth of mammary cells, but having a lesser or no inhibitory effect on the growth of non-mammary cells.
  • the inhibitor of the present invention can thus be used prophylactically to decrease the risk of breast cancer, to screen for the risk or presence of breast cancer and monitor treatment, and as a therapeutic agent for the treatment of breast cancer.
  • Figure 1 is a bar graph of inhibitory activity of NHMC-conditioned media measured in MCF-7 cultures grown in low and high calcium, illustrating the calcium-dependence of inhibitor activity.
  • Figure 2 is a dose response curve of growth inhibition of transformed human mammary cells by NHMC-conditioned media.
  • Figure 3 is a graph of inhibitory activity of molecular sieve column fractions of NHMC-conditioned media on transformed human mammary cells, illustrating peak inhibitory activity of fractions having molecular weights of about 40,000-45,000 and about
  • Figure 4 is a silver stain of various samples separated on a hydrophobic interaction column and electrophoresed on a 10% polyacrylamide gel.
  • Figure 5 is a dose response curve illustrating the increased inhibition of growth of transformed human mammary cells with increasing volumes of monoclonal antibody affinity selected human mammary cell growth inhibitor.
  • Figure 6 is a fluorogram of affinity selected 35 S-methionine labeled human mammary cell growth inhibitor produced by normal human mammary cells.
  • Figure 7 is a Western blot using monoclonal antibody to the human mammary cell growth inhibitor to detect the presence of human mammary cell growth inhibitor in human cell lysates and sera.
  • Figure 8 is a graph of the effects of conditioned medium on MCF-7 growth.
  • Figure 9 is a bar graph illustrating the inhibitory effect of affinity purified human mammary growth inhibitor and abrogation of inhibitory activity by 3C6 antibody.
  • Figure 10 is a silver stain of affinity purified inhibitor and a fluorogram of 35 S-methionine labeled affinity selected human mammary growth inhibitor.
  • Figure 11 is a graph illustrating the heat and trypsin sensitivity of affinity purified inhibitor.
  • Figure 12 are immunoperoxidase stained normal human mammary cells and MCF-7 cells using 3C6 antibody.
  • the present invention generally relates to a human mammary cell growth inhibitor, to purified and enhanced concentrations of the inhibitor, and compositions thereof, for regulating cell growth, and to methods for its increased production and use.
  • enhanced concentration is meant a concentration of inhibitor greater than normal physiologic levels of inhibitor.
  • human mammary cell growth inhibitor is meant an inhibitor having the characteristics of the inhibitor of the present invention which in its active form is capable of inhibiting the growth of human mammary cells. It should be appreciated that the term "human” is not intended to limit the source of the inhibitor to humans or to necessarily limit its inhibitory effect to only humans.
  • a previously unknown mammary cell growth inhibitor has been found to be secreted by actively growing human mammary cells.
  • Functional mammary cell growth inhibitor is secreted by "normal", i.e. untransformed, human mammary cells in culture and is present in the sera of "normal” females, i.e. those free of breast cancer.
  • levels of mammary cell growth inhibitor produced by normal human mammary cells under standard culture conditions and the levels present in human sera were originally inadequate to isolate the inhibitor using standard techniques.
  • Increased production of functional inhibitor was, however, achieved by providing normal human mammary cells with a low calcium environment.
  • low calcium environment is meant an environment having a concentration of calcium below physiologic levels.
  • such environments include calcium concentrations below about 0.1 mM calcium, and preferably about 0.04 mM calcium.
  • Mammary cell growth inhibitor obtained from normal human mammary cells (NHMC) has been isolated and purified by ion exchange chromatography, hydrophobic interaction chromatography, and molecular sieve chromatography to a purity of about 90% . Further purification to about 95% purity has been achieved through ion exchange chromatography followed by monoclonal antibody affinity chromatography.
  • the human mammary cell growth inhibitor has been found to comprise a thermolabile protein, and inhibitor isolated from NHMC-conditioned media by DEAE ion exchange chromatography exhibits peaks of inhibitory activity in the range of from about 40,000 to about 45,000 MW and from about 50,000 to 60,000 MW as measured by MCF-7 growth assays of molecular sieve column fractions. All molecular weights referred to herein are given in daltons.
  • Inhibitor purified from NHMC-conditioned media by monoclonal antibody affinity chromatography exhibits peaks of inhibitory activity in the range of 45,000 to about 50,000 MW and in the range of from about 60,000 to 70,000 MW, determined by SDS polyacrylamide gel electrophoresis. More particularly, constant bands of about 47,000 and about 65,000 MW have been observed in silver stains of inhibitor purified from NHMC-conditioned media by DEAE Sephacryl ion exchange chromatography followed by monoclonal antibody affinity chromatography. Inhibitor so purified is hereinafter referred to as "affinity purified inhibitor". Fluorograms of inhibitor purified from NHMC-conditioned media directly by monoclonal antibody affinity chromatography (i.e.
  • the mammary cell growth inhibitor of the present invention is thus distinguishable from other cell growth inhibitors such as TGF-beta, which also has an inhibitory effect on mammary tissue growth, by its molecular weight, heat lability and cell specificity.
  • Polyclonal and monoclonal antibodies have been raised to the mammary cell growth inhibitor and used to further characterize and purify the inhibitor. As noted above, inhibitor purity of up to at least about 95% has been achieved using a monoclonal antibody affinity column. Both polyclonal and monoclonal antibodies to the inhibitor have also been shown to abrogate the inhibitor's activity. In the presence of complement, BT-20 and MCF-7 transformed human mammary cell lines, which apparently produce non-functional inhibitor, are lysed by animal antiserum to inhibitor, whereas non-mammary cell lines, which do not produce inhibitor are unaffected. This suggests the presence of inhibitor on the cell surface, a finding also confirmed by indirect immunofluoresence, and the presence of cell receptor sites for the inhibitor on the cell surface.
  • the inhibitory activity of crude and purified preparations of media conditioned by normal human mammary cells (NHMC) and containing mammary cell growth inhibitor has also been found to be mammary cell specific.
  • cell specific activity is meant that the inhibitor generally exhibits a greater inhibitory effect on the growth of the types of cells for which it is specific than on other cell types.
  • the mammary cell growth inhibitor of the present invention inhibits growth of NHMC and transformed mammary cell lines
  • the mammary cell growth inhibitor of the present invention is present in normal human mammary cells (NHMC), certain transformed mammary cell lines, and the sera of normal females, but its presence was not detected or was detected at low levels in other transformed mammary cell lines, non-mammary cell lines, male sera and sera of female breast cancer patients.
  • NHMC normal human mammary cells
  • the human mammary cell growth inhibitor of the present invention can be administered to patients in pharmaceutically acceptable delivery vehicles or systems, either alone or in combition with other forms of therapy, to treat benign and malignant mammary cell-proliferating conditions, or to increase inhibitor levels in the bloodstream or mammary tissue to decrease the risk of breast cancer.
  • low levels or absence of funtional human mammary cell growth inhibitor in the bloodstream or in mammary tissue may be indicative of breast cancer or a predisposition toward breast cancer and can be used as a diagnostic tool or to screen the population for those at high risk.
  • the regulatory effect exerted by the mammary cell growth inhibitor of the present invention is not via cell kill of the treated cells. Visual observation of normal human mammary cells treated by the inhibitor indicates that inhibited cells appear healthy, but that their nuclei decrease in size and the cells assume a characteristic flattened dormant morphology typical of differentiated cells. However, the precise mechanism of action of the mammary cell growth inhibitor of the present invention is not yet known.
  • NHMC-conditioned media used for treatment were obtained by growing normal human mammary cells (NHMC) for five days in the respective media shown in Table 1 below. The media was then removed, spun at 8,000 rpm for fifteen minutes, lyophilized and reconstituted in water at one-tenth the original concentration. Reconstituted media was filter sterilized and used to treat cultured cells.
  • Cells treated were normal human mammary cells (NHMC) obtained from reduction mammaplasty grown in primary culture for two weeks in DMEM/F12 supplemented with 10% fetal calf serum (FCS).
  • the cells were plated at 4 ⁇ 10 5 cells/T25 flask, allowed to attach overnight, then grown in IMDM defined media supplemented with 10% by volume conditioned media of the type noted in Table 1. Cells were followed until no further growth was observed, then trypsinized to remove them from the plates and counted by hemacytometer.
  • NHMC-conditioned approx. 2 cells were flat low calcium a media with small nuclei
  • NHMC-conditioned , 4 - 6 cells were of Defined MCDB media b normal shape, but nuclei appeared slightly shrunken
  • MCF-7 transformed human mammary cells were grown in media containing 0.04 mM calcium (low) and 24 mM calcium (high) alpha MEM supplemented with column fractions of NHMC-conditioned low calcium media.
  • the column fractions were obtained from a PBS elution of a G-100 Sephadex column onto which 2 ml of ultrafiltration concentrated (10x) conditioned media had been loaded. The fractions were used as a 10% by volume supplement to the MCF-7 cells in low and high calcium growth media.
  • the MCF-7 cells were grown in the supplemented media for four days, after which inhibitory activity was measured by decrease in cell numbers counted on a Coulter counter. As shown in Figure 1, the inhibitory activity of human mammary cell growth inhibitor was greater in media containing high concentrations of calcium, indicating that such activity is calcium-dependent.
  • EGF EGF
  • 98 ng/ml cholera toxin 10 ug/ml insulin
  • 1 ug/ml hydrocortisone 98 ng/ml cholera toxin
  • Cells were incubated at 37oC in a humidified CO 2 incubator.
  • MCF-7 cells were maintained in MEM supplemented with 10% fetal calf serum (FCS) in 1 ug/ml of insulin at 37oC in a humidified
  • MCF-7 cells were plated at 1 to 2 ⁇ 10 4 cells/ml,
  • % inhibition [1-(cell number of treated sample/cell number of untreated sample)] ⁇ 100
  • % recovery [1-(percent inhibition of treated sample with conditioned media removed/percent inhibition of treated sample with conditioned media)] ⁇ 100
  • % disinhibition [1-(percent inhibition of the sample/percent inhibition of conditioned media)] ⁇ 100.
  • MCF-7 cells were plated at 10 4 cells/ml in MEM supplemented with 10% FCS . The cells were grown for seven days after the addition of a range of volumes of conditioned media as indicated in Figure 2. The conditioned media used was low calcium
  • SPECIFIC EXAMPLE 4 Heat Denaturization and Trypsin Inactivation of Human Mammary Cell Growth Inhibitor MCF-7 cells were plated at 10 4 cells/ml in MEM supplemented with 10% by volume FCS. The cells were treated after
  • the inhibitory effect of the inhibitor was tested on both mammary and non-mammary human cells.
  • the different human cell lines and types listed below in Table 3 were plated at 10 4 cells/ml in Costar plates and then treated with unconditioned low calcium (0.04 mM) medium or low calcium medium which had been previously conditioned by NHMC. The cells were allowed to grow for one week, and then counted on a Coulter counter and percent inhibition calculated.
  • Human mammary cell growth inhibitor from low calcium NHMC-conditioned media was purified using the following protein purification technique.
  • NHMC-conditioned media with highest activity, as measured by MCF-7 inhibition assay were first concentrated by ultrafiltration through an Amicon filter with a 10,000 molecular weight cutoff and then fractionated by passage over a Sephadex G-100 column with molecular weight standards in PBS buffer. Fractions of various molecular weights from this separation were sterilized using 0.22 um Gelman acrodiscs and were used to treat MCF-7 cells at 10% by volume growing in culture. Treated MCF-7 cells were allowed to grow for seven days and then the growth was measured by counting cell density on a Coulter counter. The fractions exhibiting inhibitory activity were those of an apparent molecular weight of 40,000.
  • DEAE fractions prepared as described above which contained activity were loaded on a preparative polyacrylamide gel. Because of the superior purity of the region of the gel corresponding to the 35,000 to 50,000 molecular weight range surrounding the 40,000 to 45,000 inhibitory peak, this section of the gel was excized and the proteins electroeluted from this complete gel fragment. 20-50 ug/ml of the eluted proteins were then mixed with Freuds complete adjuvant 50% by volume and injected into two New Zealand white rabbits and a Balb/c mouse. The rabbits were immunized with 1.5 ml of the solution subcutaneously. The mouse was immunized with 0.2 ml of the solution injected into the peritoneal cavity.
  • mice were boosted with the same protein concentrations mixed in Freuds incomplete adjuvant, 50% by volume two weeks later.
  • the sera of these animals was tested one week after boosting for recognition of the inoculating solution using an ELISA assay.
  • Two weeks after boosting the mouse was tail vein injected with 20-50 ug/ml of the protein solution. Three days later, the mouse was sacrificed and the spleen removed.
  • Spleen cells removed from the mouse spleen and myeloma SP2/0 cells were fused in the presence of polyethylene glycol to produce hybridomas using standard immunological techniques as described in Selected Methods in Cellular Immunology by Michelle and Shiigi, W.H. Freeman and Co., San Francisco, California (1980).
  • the hybridomas from the cell fusion were grown in RPMI with 10% FCS and azoserine and hypoxanthine at appropriate dilutions.
  • Cell cultures were grown in 96-well micro-titer plates for two weeks in the presence of azoserine to select against unfused parental cells. The supernatants were then tested from individual cultures with the following standard ELISA.
  • 96-well Immulon 2 microtiter plates from Dynatech Laboratories, Inc., Chantilly, Virginia, were coated with 5 ug/ml of partially purified growth inhibitor in PBS which was obtained from the same electroeluted polyacrylamide gel slices used for the animal immunizations above. After incubation overnight at 4oC, the plates were then washed with a washing solution of 0.9% NaCl and 0.05% Triton X-100. 50 ul of the supernatants of individual hybridoma cultures were then added to the wells and incubated for 2 hours at room temperature.
  • the plates were washed with washing solution and then incubated for 2 hours at room temperature with goat anti-mouse IgG and IgM antibody conjugated to alkaline phosphatase from Southern Biotechnology Associates, Birmingham, Alabama, at a dilution of 1:1000 in 2% BSA in PBS.
  • the plates were then again washed with washing solution and 100 ul of a solution of 10 mg/ml colorometric substrate para-nitrophenyl phosphate, disodium salt from Sigma, St. Louis, Missouri was added. Color was allowed to develop for 1 to 20 hours.
  • a Dynatech MR700 microplate reader was then used to determine optical density at 410 nm wavelength. Cultures were selected on the basis of activity at least 4x the background, with 150 cultures screening positive on that basis.
  • Immune serum from rabbits which were immunized with the 35,000-50,000 molecular weight region was shown to contain a monospecific antibody to the protein when conditioned media was analyzed by Western blotting.
  • the immune serum also recognized the purified inhibitor eluted at 1 M NaCl from the hydrophobic interaction column described in Specific Example 6 as shown in Figure 4 at F.
  • Both rabbit immune sera containing polyclonal antibody and monoclonal antibody derived from the mouse were tested for their ability to block the inhibition which was observed in conditioned media.
  • MCF-7 cells were plated and then treated with 2% rabbit immune serum or 5% partially purified by ammonium sulfate precipitation 3C6 monoclonal antibody from ascites. These cells were also then treated with conditioned media and allowed to grow for seven days.
  • Disinhibition was determined by comparing the cell number for the samples treated with immune serum or 3C6 antibody to cell numbers which were treated with conditioned media alone. As shown by the data of Table 5 below, both the immune serum containing polyclonal antibody and 3C6 monoclonal antibody blocked the inhibitory activity of the inhibitor. It was also shown that immune sera blocked inhibitory response in a dose-dependent manner. TABLE 5 Effects of Antibodies on Inhibition Treatment % Disinhibition conditioned media 0 immune serum 42
  • 3C6 monoclonal antibody partially purified by ammonium sulfate precipitation from mouse ascites fluid were used to make a monoclonal antibody affinity column on CNBr activated sepharose using standard techniques. The column was used to remove the inhibitory activity from conditioned media passed over it and the inhibitory activity was eluted from this column. MCF-7 cells were plated with increasing volumes of low calcium NHMC-conditioned media purified on the 3C6 monoclonal affinity column, i.e. affinity selected inhibitor, and allowed to grow for seven days after which they were then counted.
  • SPECIFIC EXAMPLE 10 Effect of Conditioned Medium on MCF-7 Growth
  • Normal human mammary cells NHMC
  • NHMC characterized as epithelial by keratin expression
  • medium was conditioned by incubating confluent cultures of NHMC for 4 days in DMEM/F-12, at 40 nM CaCl 2 with 5% chelex treated bovine serum.
  • MCF-7 cells were plated at 2 ⁇ 10 5 cells /T25 flask in MEM with 10% fetal calf serum and 10 ug/ml insulin and allowed to attach overnight. Medium was then replaced with indicated concentration of medium conditioned by the NHMC cells. Conditioned or non-conditioned medium was concentrated 10x by amicon ultrafiltration. Cell number was determined at indicated times by Coulter counting. As shown in Figure 8, media conditioned by these cells was found to inhibit the growth of the transformed human mammary carcinoma cell line, MCF-7, in a time and dose-dependent manner, and the addition of 10% conditioned medium to these cultures resulted in a 75% inhibition of cell growth at 10 days compared to cultures supplemented with 10% non-conditioned medium.
  • the inhibitory activity in normal human mammary cell conditioned medium was purified by ion exchange chromatography on DEAE Sephacryl followed by affinity chromatography on a 3C6 monoclonal antibody affinity column.
  • One liter of conditioned medium was loaded on a DEAE Sephacryl column in 50 mM NaCl and eluted with a step gradient between 0.05 and 1.0 M NaCl. Dialysed fractions were assayed for MCF-7 growth inhibitory activity.
  • the inhibitory activity from conditioned medium eluted at 0.25 M NaCl.
  • Inhibitory activity which eluted between 0.1 and 0.5M NaCl was then loaded on a monoclonal antibody affinity column produced by binding 22 mg of 3C6 monoclonal antibody to 1.5 ml Biorad Affigel Beads.
  • the affinity column was next washed with 500 column volumes of PBS to remove unbound protein and bound protein was eluted with 0.1 M glycine pH 2.35.
  • the eluent was immediately neutralized with Tris base and dialysed against PBS.
  • Hybridoma supernatants were screened for their ability to recognize immunizing proteins and abrogate the inhibitory activity of conditioned medium on MCF-7 cells.
  • MCF-7 cells were plated as in Specific Example 10 and at day 1 the following supplements were added in fresh medium: (a) 10% NHMC-conditioned medium: (b) 10% NHMC-conditioned medium + 10% hybridoma supernatant from DF1/3C6; (c) 10 ng/ml affinity purified inhibitorr (d) 20 ng/ml affinity purified inhibitor; (e) 10 ng/ml affinity purified inhibitor + 5 ug/ml monoclonal antibody 3C6.
  • affinity purified inhibitor Is meant inhibitor purified from NHMC-conditioned media by a combination of DEAE Sephacryl ion exchange and 3C6 monoclonal antibody affinity chromatography as described in Specific Example 11.
  • Lanes (a) and (b) of Figure 10 show the respective results of silver staining of (a) molecular weights standards and (b) affinity purified inhibitor prepared as described previously.
  • inhibitor is synthesized by NHMC
  • cultures were labeled with 35 S-methionine in 5 ml of methionine-free DMEM medium supplemented with 200 uCi/ml of 35 S-methionine by incubating confluent T-75 flask of NHMC for 24 hours.
  • Affinity selected inhibitor was then prepared by subjecting the labeled culture supernatants to affinity chromatography on a 3C6 monoclonal antibody affinity column, and molecular weights determined by
  • Lane (c) is the fluorogram of
  • 35S-methionine labeled NHMC-conditioned medium eluted from the 3C6 affinity column.
  • lane B two prominent bands at approximately 47,000 and 65,000 appeared by silver staining of the affinity purified inhibitor.
  • a prominent band at about 47,000 and a doublet of a constant band at about 67,000 and an inconsistent minor band at about 63,000 were observed. This suggests that either the 47,000 and 63,000-67,000 species represent differentially processed or cleaved forms of the same protein or that these represent two distinct inhibitory factors that share a common epitope.
  • Affinity purified inhibitor (100 ng/ml) was heated to 37, 55, 70, or 100°C for 60 minutes. Inhibitory activity of these samples were then determined as previously described. Heating of the human mammary growth inhibitor to 70oC for 60 minutes, resulted in a 50% decrease in activity. Heating to 100oC for 60 minutes completely abrogated the inhibitory activity.
  • This pattern of heat sensitivity shown in Figure 11 distinguishes the human mammary growth inhibitor from TGF-beta which is stable to 100oC heating as described in Sporn, M.D., et al., Science 233:532 (1986).
  • the sensitivity of affinity purified inhibitor to trypsin was also tested. 5 ug/ml of trypsin was added to 1 ml of 100 ng/ml affinity purified inhibitor and incubated for one hour at 37oC.
  • trypsin inhibitor was added to stop trypsin degradation.
  • a control experiment of 50 ug of trypsin inhibitor added to 1 ml of 100 ng/ml affinity purified inhibitor was performed. 5 ug of trypsin was then added to the control mixture and the entire mixture incubated for one hour at
  • human mammary growth inhibitor on the growth of a variety of transformed mammary and non-mammary human cell lines was tested.
  • Cell lines were cultured in triplicate in RPMI + 10% fetal calf serum in 12 well plates at 2 ⁇ 10 4 cells per well and allowed to attach overnight. Cell number was determined at day 1 and cultures were then treated with a final concentration of either 10 ng/ml of inhibitor or 10 ng/ml BSA. Percent inhibition was calculated as previously described. As shown below in Table 7, human mammary growth inhibitor
  • HBL-100 a non-transformed mammary cell line, described by Gaffney, E.W., et al., J. Natl. Cancer. Inst., 63:913 (1979) showed inconsistent inhibition. Growth inhibition was seen in both estrogen responsive (MCF-7, ZR-75-1) and estrogen non-responsive mammary cell lines (BT-20, MDA-MB-231, evejos (established from a primary breast cancer at the University of Michigan and available on request from Paul Ervin, Jr. of the Dept. of Internal Medicine)).
  • the inhibitor had no effect on the growth of the eleven transformed human cell lines derived from non-mammary tissues shown in Table 7.
  • This tissue specificity of inhibitor further distinguishes it from TGF-beta which inhibits the growth of a wide variety of epithelial cell lines as described by Tucker, R.F., et al., Science, 226:205 (1984).
  • Western blot analysis of human mammary growth inhibitor and TGF-beta was performed with 3C6 and anti-TGF-beta antibody (R&D Products). 3C6 did not detect TGF-beta but did recognize the inhibitor. Conversely, anti-TGF-beta did not detect the inhibitor but did recognize TGF-beta.
  • the inhibitor of the present invention is distinct from
  • TGF-beta The tissue specificity of inhibition further distinguishes human mammary growth inhibitor from other smaller molecular weight inhibitory proteins previously isolated from mammary cells.
  • NHMC or MCF-7 cells were cultured for 3 days on chamber slides, rinsed with PBS and fixed in 2% paraformaldehyde in PBS for 10 minutes at 4oC. Subsequent steps were done at room temperature. Cells were permeableized with 0.1% Triton X-100 in PBS for 10 minutes and blocked with 1% BSA in PBS for 30 minutes and then incubated for 60 minutes with a 1:100 dilution of monoclonal antibody 3C6 in PBS + 1% BSA. Irrelevant IgM antibodies at equal concentrations were used as controls.
  • the cells were then washed and incubated in a 1:250 solution of peroxidase conjugated goat anti-mouse IgG antibody in 2.5% non-fat dry milk in PBS for 60 minutes and developed for 10 minutes with AEC solution. Slides were mounted with aquamount and photographed.
  • Figure 12 shows the staining results of (A) normal human mammary cells (X100), (B) MCF-7 cells (X100).
  • X100 normal human mammary cells
  • Figure 12A immunoreactive inhibitor was present in greater than 90% of the normal human mammary cells.
  • Figure 12B only 10-15% of MCF-7 cells showed weak staining. Samples that stained with 3C6 were negative with irrelevant antibody. Immunoperoxidase staining of the other transformed cell lines described in Table 7 was similarly performed.
  • BT-20, MDA-MB-231 and ZR-75-1, evejos and HBL-100 showed less than 10% weekly staining cells.
  • Non-mammary cells lines were negative.
  • conditioned media of the three transformed mammary cell lines tested were obtained from MCF-7, BT-20 and
  • MDA-MD-231 cell lines as described for NHMC and tested for MCF-7 growth inhibitor activity as previously described. These media were also analysed by Western Blot. Neither inhibitory activity or immunoreactive human mammary growth inhibitor was detected.
  • the bound protein was recognized on the plate by incubating for one hour at room temperature with 100 ul/well of a 2 ug/ml solution of biotin conjugated 3C6 antibody in PBS + 1% BSA.
  • the unbound antibody was removed from the plate by again flooding the plate with PBS + 0.1% Triton X-100 several times.
  • the bound antibody was then incubated with 100 ul/well alkaline phosphatase conjugated strept-avidin from Southern Biotechnology, Inc. at 0.4 ng/ml in PBS + 1% BSA for one hour at room temperature.
  • the Strept-avidin solution was removed from the plate by shaking and again washing with PBS + Triton X-100 twice.
  • Captured protein was then recognized by a colorimetric procedure using the alkaline phosphatase bound to the second antibody through the biotin conjugate. This was accomplished by incubating the plate with 100 ul/well of a solution of 1 (5 mg) p-nitrophenylphosphate tablet (Sigma 104-105 phosphatase substrate) per 11 ml of alkaline phosphate substrate buffer (400 ml dH 2 O, 24.5 mgs MgCl, 48 ml diethanolamine, brought to a pH of 9.8 with HCl and brought up to a total volume of 500 ml) until a yellow color was observed. Readings were taken at 30 minutes and one hour using a Dynatech Micro ELISA plate reader and the quantity of protein present was measured by determining the intensity of the yellow color which developed.
  • 1 5 mg
  • p-nitrophenylphosphate tablet Sigma 104-105 phosphatase substrate
  • alkaline phosphate substrate buffer 400 ml dH 2 O, 24.5 mgs

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  • Pathology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Un inhibiteur de croissance de cellules mammaires humaines, ainsi que de nouvelles compositions comprenant des concentrations améliorées et purifiées dudit inhibiteur, empêche la croissance de cellules mammaires humaines. L'inhibiteur de la présente invention purifié par chromatographie par affinité d'anticorps monoclonaux, comprend une protéine thermolabile et a des maximums d'activité inhibitrice à des poids moléculaires d'environ 47 000 et d'environ 63 000 à 67 000 déterminés par SDS-PAGE. L'activité inhibitrice dudit inhibiteur est spécifique aux cellules mammaires et dépend à la fois du calcium et de la dose. On peut induire une production accrue de l'inhibiteur par la croissance de cellules mammaires humaines sécrétrices d'inhibiteurs dans de faibles concentrations de calcium. On peut utiliser ledit inhibiteur de manière prophylactique afin de diminuer le risque de cancer du sein, afin de dépister le risque ou la présence de cancer de la poitrine, et comme agent thérapeutique pour le traitement du cancer du sein.
PCT/US1989/002214 1988-05-20 1989-05-22 Inhibiteur de croissance de cellules mammaires humaines et procedes de production et d'utilisation WO1989011492A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP89506373A JPH05506423A (ja) 1988-05-20 1989-05-22 ヒト乳房細胞成長インヒビターならびにその製造方法および使用

Applications Claiming Priority (2)

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US19665788A 1988-05-20 1988-05-20
US196,657 1988-05-20

Publications (1)

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WO1989011492A1 true WO1989011492A1 (fr) 1989-11-30

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EP (1) EP0414807A4 (fr)
JP (1) JPH05506423A (fr)
WO (1) WO1989011492A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998014577A3 (fr) * 1996-10-03 1998-04-09 Biotherapies Inc Sequence nucleotique et de proteique de mammastatine et procedes d'utilisation
WO1999032625A3 (fr) * 1997-12-19 1999-09-16 Univ Michigan Sequence nucleotidique et proteinique de la mammastatine et procedes d'utilisation
US6156732A (en) * 1998-02-19 2000-12-05 Ortho-Mcneil Pharmaceuticals, Inc. Azole peptidomimetics as thrombin receptor antagonists

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Biochemical and Biophysical Research Communications, Volume 122, issued 16 August 1984, SOTO et al, "Mechanism of Estrogen Action on Cellular Proliferation: Evidence for Indirect and Negative Control on Cloned Breast Tumor Cells", pages 1097-1103, see entire document. *
Biochimica et Biophysica Acta, Volume 846, issued 1985, BOHMER et al, "Specific Neutralizing Antiserum against a Polypeptide Growth Inhibitor for Mammary Cells Purified from Bovine Mammary Gland" pages 145-154, see entire document. *
Cancer Research, Volume 47, issued 1 March 1987, BRONZERT et al, "Estrogen Inhibition of a Mr 39,000 Glycoprotein Secreted by Human Breast Cancer Cells", pages 1234-1238, see entire document. *
Experimental Cell Research, Volume 150, issued 1984, BOHMER et al, "Purification of a Growth Inhibitor for Ehrlich Ascites Mammary Carcinoma Cells from Bovine Mammary Gland", pages 466-476. see entire document. *
In Vitro, Volume 14, issued 1978, PIGOTT et al, "Inhibition of Growth of a Human Mammary Cell Line by Normal Human Serum", see Abstract #109. *
In Vitro, Volume 20, issued August 1984, McGRATH et al, "Calcium Regulation of Normal Human Mammary Epithelial Cell Growth in Culture", pages 652-662, see entire document. *
Journal of Biological Chemistry, Volume 262, issued 5 November 1987, BOHMER et al, "Indentification of a Polypeptide Growth Inhibitor from Bovine Mammary Gland", pages 15137-15143, see entire document. *
See also references of EP0414807A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998014577A3 (fr) * 1996-10-03 1998-04-09 Biotherapies Inc Sequence nucleotique et de proteique de mammastatine et procedes d'utilisation
US6500937B1 (en) 1996-10-03 2002-12-31 University Of Michigan Nucleotide sequence encoding a mammary cell growth inhibitor
US6599495B1 (en) 1996-10-03 2003-07-29 Regents Of The University Of Michigan Nucleotide and protein sequence of mammastatin and methods of use
US7323173B2 (en) 1996-10-03 2008-01-29 The Regents Of The University Of Michigan Methods for treating breast cancer using a mammary cell growth inhibitor
US7332287B2 (en) 1996-10-03 2008-02-19 The Regents Of The University Of Michigan Methods and compositions for diagnosing breast cancer
EP1935899A1 (fr) * 1996-10-03 2008-06-25 The Regents Of The University Of Michigan Nucléotide et séquence de protéine de mammastatine et procédés d'utilisation
US7816097B2 (en) 1996-10-03 2010-10-19 The Regents Of The University Of Michigan Nucleotide and protein sequence of Mammastatin and methods of use
WO1999032625A3 (fr) * 1997-12-19 1999-09-16 Univ Michigan Sequence nucleotidique et proteinique de la mammastatine et procedes d'utilisation
US6156732A (en) * 1998-02-19 2000-12-05 Ortho-Mcneil Pharmaceuticals, Inc. Azole peptidomimetics as thrombin receptor antagonists

Also Published As

Publication number Publication date
EP0414807A1 (fr) 1991-03-06
JPH05506423A (ja) 1993-09-22
EP0414807A4 (en) 1991-07-03

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