WO1989011491A1 - Anticorps contre l'inhibiteur de croissance de cellules mammaires humaines et procedes de production et d'utilisation - Google Patents

Anticorps contre l'inhibiteur de croissance de cellules mammaires humaines et procedes de production et d'utilisation Download PDF

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WO1989011491A1
WO1989011491A1 PCT/US1989/002215 US8902215W WO8911491A1 WO 1989011491 A1 WO1989011491 A1 WO 1989011491A1 US 8902215 W US8902215 W US 8902215W WO 8911491 A1 WO8911491 A1 WO 8911491A1
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antibody
inhibitor
cell growth
hybridoma
human mammary
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PCT/US1989/002215
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English (en)
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Paul R. Ervin, Jr.
Max S. Wicha
Mark S. Kaminski
Robert L. Cody
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The Regents Of The University Of Michigan
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Priority to JP89506374A priority Critical patent/JPH05506351A/ja
Publication of WO1989011491A1 publication Critical patent/WO1989011491A1/fr

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    • 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
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to cell growth regulation and, more particularly, to antibodies specific for human mammary cell growth inhibitor and methods of their 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).
  • Immunological purification and concentration of mammary cell growth inhibitor would greatly facilitate the use of the inhibitor as a prophylactic and therapeutic agent against breast cancer.
  • Immunoassays for detecting the presence and levels of the inhibitor in mammary tissue and serum would also be highly advantageous in the diagnosis of breast cancer, in screening those at high risk of breast cancer, and in monitoring patient levels of inhibitor during treatment.
  • ELISA kits for detecting the mammary growth inhibitor in samples of body fluid or tissue would facilitate such diagnosis and screening.
  • SUMMARY OF THE INVENTION The present invention relates to antibodies specific for human mammary cell growth inhibitor and methods of their production and use. Hybridoma cell lines producing monoclonal antibodies specific for the inhibitor have been generated using cell fusion and selection techniques.
  • Polyclonal antibodies to the inhibitor have also been raised by in vivo immunization of animals and recovery of antibodies from their serum.
  • antibodies raised to the inhibitor have also. been used to purify and concentrate the inhibitor of the present invention and to assay for the inhibitor's presence in human mammary and non-mammary cells and human sera.
  • the human mammary cell growth inhibitor to which antibodies of the present invention have been raised comprises a thermolabile protein.
  • Peak activities of inhibitor isolated from NHMC- conditioned media by ion exchange chromatography have been observed at molecular weights of 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 seive column fractions.
  • Inhibitor purified by antibody affinity chromatography exhibits peaks of activity in the range of from about 45,000 to about 50,000 daltons and in the range of from about 60,000 to about 70,000 daltons as measured by SDS-PAGE.
  • Increased production of functional inhibitor of the present invention has been induced by growth of inhibitor-secreting human mammary cells in low concentrations of calcium.
  • the activity of the inhibitor is also calcium-dependent, dose-dependent and reversible.
  • the inhibitor is present in the sera of normal females, but absent or present in low levels in the sera of normal males and breast cancer patients.
  • the inhibitor is also 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 can thus be administered alone or in combination with other agents to prevent mammary cell proliferation and as a therapeutic agent for the treatment of breast cancer.
  • immunoassays of the present invention which detect the presence or absence and levels of the inhibitor in human tissue and serum can be used to diagnose breast cancer or precancerous cell-proliferating conditions, to screen for predisposition to breast cancer, and to monitor patient levels of inhibitor during treatment.
  • 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 inhibition 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 in the range of from about 40,000 to about 45,000 and in the range of from about 50,000 to about 60,000.
  • 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 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 inhibitor in human cell lysates and sera.
  • Figure 8 is a Western blot using monoclonal antibody to the human mammary cell growth inhibitor to detect the presence of inhibitor in sera of normal and breast cancer patients.
  • Figure 9 is a dose response curve of disinhibition of mammary cell growth inhibitor activity by purified monoclonal antibody.
  • Figure 10 is a graph of the effects of conditioned medium on MCF-7 growth.
  • Figure 11 is a bar graph illustrating the inhibitory effect of affinity purified human mammary growth inhibitor and abrogation of inhibitory activity by 3C6 antibody.
  • Figure 12 is a silver stain of affinity purified inhibitor and a fluorogram of 35 S-methionine labeled affinity selected human mammary growth inhibitor.
  • Figure 13 is a graph illustrating the heat and trypsin sensitivity of affinity purified inhibitor.
  • Figure 14 are immunoperoxidase stained normal human mammary cells and MCF-7 cells using 3C6 antibody.
  • the present invention generally relates to antibodies specific for a human mammary cell growth inhibitor, and to methods for their production and use.
  • 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.
  • Hybridoma cell lines producing monoclonal antibody specific for the inhibitor have been generated by cell fusion and selection techniques.
  • Polyclonal antibodies to the inhibitor have been raised by in vivo immunization of animals and recovery of antibody from their serum. Both polyclonal and monoclonal antibodies have been shown to block inhibitory activity of the inhibitor or to deplete inhibitory activity from media conditioned by normal human mammary cells which secrete the inhibitor.
  • Enhanced concentrations and purified compositions of the inhibitor have also been produced using antibodies of the present invention.
  • “enhanced concentration” is meant a concentration of inhibitor greater than normal physiologic levels of inhibitor.
  • antibodies to the inhibitor have also been used to assay for the presence or absence of inhibitor in human cells and serum.
  • Mammary cell growth inhibitor to which the antibodies have been raised is secreted by actively growing human mammary cells and has been described in the aforementioned parent Application Serial No. 196,657 filed May 20, 1988, and original Application Serial No. 891,135.
  • Functional mammary cell growth inhibitor is secreted by "normal”, i.e. untransformed, human mammary cells in culture and is present in the serum of "normal" females, i.e. those free of breast cancer.
  • the levels produced by normal human mammary cells under standard culture conditions and the levels present in human serum 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. More particularly, with respect to at least in vitro low calcium environments, 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 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 by DEAE ion exchange chromatography exhibits peaks of inhibitory activity in the range of from about 40,000 to about 45,000 dalton MW and from about 50,000 to 60,000 dalton MW as measured by MCF-7 growth assays of molecular sieve column fractions.
  • 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 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".
  • NHMC-conditioned media Fluorograms of inhibitor isolated or purified from NHMC-conditioned media directly by monoclonal antibody affinity chromatography (i.e. sans ion exchange), hereinafter referred to as "affinity selected inhibitor", revealed constant bands at about 47,000 and about 67,000 MW and an inconsistent minor band at about 63,000 MW.
  • 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.
  • TGF-beta which also has an inhibitory effect on mammary tissue growth, by its molecular weight, heat lability and cell specificity.
  • 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 MCF-7, BT-20, ZR-75-1, evejos and MDA-MB-231, but little or no growth inhibition of eleven non-mammary cell types by the inhibitor has been observed.
  • Studies of inhibitory activity in varying concentrations of calcium indicate that inhibitory activity is also calcium-dependent, and requires elevated levels of calcium. Dose response studies also indicate that the inhibition of mammary cell growth by the inhibitor is dose-responsive.
  • polyclonal and monoclonal antibodies raised against the mammary cell growth inhibitor have been used to further characterize and purify the inhibitor. Inhibitor purity of up to at least about 952 has been achieved using a monoclonal antibody affinity column. Both polyclonal and monoclonal antibodies to the inhibitor have also been shown to abrogate the inhibitory activity as shown by disinhibition studies, or to remove the inhibitor from NHMC-conditioned media as shown by depletion studies. Disinhibition by antibodies raised to the inhibitor has also been shown to be antibody dose-responsive.
  • 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 cells lines which do not produce inhibitor are unaffected, suggesting the presence of inhibitor on the cell surface, a finding also confirmed by indirect immunofluoresence. This data suggests the presence of cell receptor sites for the inhibitor on the cell surface.
  • Immunoassays utilizing antibodies of the present invention have also been used to detect the presence or absence of and quantify mammary cell growth inhibitor.
  • the inhibitor has been found in normal human mammary cells (NHMC), in certain transformed mammary cell lines in non-functional form, and in the sera of normal females, but its presence was not detected or was detected only in low levels in other transformed mammary cell lines, non-mammary cell lines, male sera and sera of female breast cancer patients. It appears that a low level or absence of functional human mammary cell growth inhibitor in the bloodstream or in mammary tissue may be indicative of breast cancer or a predisposition to breast cancer.
  • NHMC normal human mammary cells
  • the mammary cell growth inhibitor can be administered to patients to increase inhibitor levels in the bloodstream or in mammary tissue in order to decrease the risk of breast cancer or to treat benign and malignant mammary cell-proliferating conditions.
  • Immunoassays of the present invention for detecting the presence of inhibitor in human tissue and serum are thus useful as a diagnostic tool for breast cancer, for screening the population for those at high risk of breast cancer, or for monitoring patient levels of inhibitor during treatment.
  • ELISA kits of the present invention using the inhibitor and reagent to detect the binding of antibody to the mammary cell growth inhibitor facilitates such diagnosis and screening.
  • 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 CO 2 incubator.
  • FCS fetal calf serum
  • MCF-7 cells were plated in triplicate at 1 to 2 ⁇ 10 4 cells/ml, 1 ml/well in 12-well Costar culture plates in MEM supplemented with 10% by volume FCS, and, optionally, 1 ug/ml insulin, after trypsin removal from stock cultures and were allowed to attach overnight at 37oC. The media was refreshed and the cultures were treated by addition of the appropriate samples. Cells were allowed to grow undisturbed for seven days at 37oC and were then counted.
  • Cell Counts Cells were washed with room temperature phosphate-buffered saline (PBS) and then lysed with Sigma Cetrimide detergent solution at 37oC to liberate their nuclei for counting on a Coulter Counter. Cell counts were averaged, with a deviation from the mean cell number greater than 5 % being discarded as unacceptable.
  • PBS room temperature phosphate-buffered saline
  • % 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
  • 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% 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.
  • media which was NHMC-conditioned at low calcium concentrations, as opposed to higher concentrations was added to the NHMC in IMDM growth media, it had a non-toxic growth-inhibiting effect on normal human mammary cells, indicating that more functional mammary cell growth inhibitor is produced by NHMC cells grown at low calcium concentrations.
  • cells inhibited by the low calcium NHMC-conditioned media also took on a characteristic differentiated morphology.
  • NHMC-conditioned approx. 2 cells were flat low calcium a DMEM/F12 with small nuclei media
  • NHMC-conditioned cells were of Defined MCDB media b 4 - 6 normal shape, but nuclei appeared slightly shrunken a 0.04 mM calcium b physiologic levels of calcium
  • MCF-7 transformed human mammary cells were grown in DMEM/F12 media containing 0.04 mM calcium (low) and 24 mM calcium (high) alpha MEM supplemented with column fractions of low calcium NHMC-conditioned 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 the 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.
  • 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
  • DMEM/F12 which had been conditioned for 96 hours by the growth of NHMC.
  • Inhibitor MCF-7 cells were plated at 1 ⁇ 10 4 cells/ml in 5 ml MEM media with 10% FCS in ten T25 flasks. Six of the flasks were also treated with 1% by volume low calcium NHMC-conditioned media supplement and the remaining four were used as untreated controls.
  • Cells were grown for five days at 37oC in a humidified CO 2 incubator. After five days, cells in two of the untreated control flasks and two of the treated flasks were counted by lysing the membranes of the cell with Cetrimide and counting the liberated nuclei on a Coulter counter, the cell counts averaged, and percent inhibition determined as described previously. Also at five days, the media in the remaining flasks was refreshed with MEM media with
  • MCF-7 cells 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 24 hours with 10% low calcium NHMC-conditioned media which had been supplemented with 5% of a solution containing 2.5 ug/ml trypsin in
  • 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 5 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, then counted on a Coulter counter and percent inhibition calculated.
  • Specificity of the inhibitor for mammary cells is illustrated by the growth inhibition of mammary cells and lack of or decreased inhibition in non-mammary cells as shown by the data in Table 5.
  • the lack of inhibition in two mammary cell types, MDA-MB-231 and DU 4475 may be due to the fact that these transformed cells are in some way defective in their response to or recognition of the inhibitor, for example, by virtue of a defect in receptor sites for the inhibitor on their surface, or because stimulators are produced by these cells in adequate amounts to overcome the inhibitory effect.
  • Human mammary cell growth inhibitor from low calcium NHMC-conditioned media was purified using standard protein purification techniques more fully described as follows: 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 0.2% urn 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, then counted on a Coulter counter, and inhibition measured. The fractions exhibiting inhibitory activity were those of an apparent molecular weight of about 40,000.
  • 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 Freund's 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. Animals were boosted with the same protein concentrations mixed in Freund's incomplete adjuvant, 50% by volume, two weeks later.
  • Spleen cells removed from the mouse spleen and mouse 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 cultured in RPMI with 15% FCS, to which were added hypoxanthine and azoserine at the appropriate dilution in 96-well microtiter plates for two weeks to select against unfused parental cells.
  • the supernatants from individual cultures were then tested for ability to produce antibody to the inhibitor from individual cultures with the following 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 % hours at room temperature.
  • the plates were washed with washing solution and then incubated for % 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.
  • DF1/3C6, DF1/1D4 and DF1/6B8 Three of the monoclonal antibodies and hybridomas producing these antibodies made in accordance with this Example and used in further studies were designated DF1/3C6, DF1/1D4 and DF1/6B8. These antibodies and hybridomas are available from Dr. Max Wicha at the University of Michigan, Medical Center, Ann Arbor, Michigan 48109 and the hybridoma cell lines are also on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, Accession Nos. HB972% (DF1/3C6), HB9723 (DF1/1D4) and HB10152 (DF1/6B8).
  • 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.
  • MCF-7 cells were plated at 1 ⁇ 10 4 cells/ml treated with 2% rabbit immune serum or 5% 3C6 monoclonal antibody from ascites partially purified by ammonium sulfate precipitation. These cells were also then treated with 10% by volume low calcium NHMC-conditioned media and allowed to grow for seven days. The cells were then counted and percent disinhibition by immune serum or 3C6 antibody determined.
  • 3C6 monoclonal antibody from mouse ascites fluid purified by ammonium sulfate precipitation was used to make a monoclonal antibody affinity column on CNBr activated sepharose using the techniques which are described more fully in Specific Example 14.
  • 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. Inhibition by increasing volumes of purified conditioned media was compared to inhibition by a sample of MCF-7 cells that were treated with 10% PBS. As shown In Figure 5, inhibition appeared dose-dependent until a high enough volume of inhibitor-containing solution resulted in growth inhibition of nearly 90%.
  • the proteins being produced by NHMC were metabolically labeled using 3 5 S-methionine. Cell proteins were labeled with 35 S-methionine at
  • Labeled media was then drawn off and dialyzed against PBS.
  • the labeled conditioned media was then passed over the 3C6 monoclonal affinity column, and the retained proteins were eluted.
  • the eluted proteins were then fractionated by polyacrylimide gel electrophoresis.
  • the fluorogram depicted in Figure 6 shows that the 3C6 monoclonal antibody affinity column retained 35 S-methionine labeled proteins at the same molecular weight as those containing the inhibitory activity produced by normal human mammary cells
  • the inhibitor was present in normal human mammary cell lysates, MCF-7 and BT-20 human mammary cell lysates, and in normal human female sera, but absent or present in undetectable levels in MDA-MB-231 and
  • HBL-100 human mammary cell lysates, and male serum HBL-100 human mammary cell lysates, and male serum.
  • a Western blot was used to determine the presence or level of inhibitor in patient serum of the types denoted in the legend of Figure 8. 2 ul of each sample of human serum was mixed with 100 ul of sample loading buffer and electrophoresed on a 10% SDS-polyacrylamide gel. The proteins from the gel were electroblotted onto a nitrocellulose filter overnight in 10% methanol, 0.15 M glycine and 25 mM Tris at 180 milliamps. The filter was then blocked with 100 ml of PBS with 2% BSA for three hours to decrease non-specific protein binding to the filter.
  • SPECIFIC EXAMPLE 12 Dose Response of Disinhibition of Inhibitor Activity by 3C6 Monoclonal Antibody MCF-7 cells were plated at 1 ⁇ 10 4 cells/ml in RPMI with
  • CNBr-activated sepharose 4B serum coupled columns were prepared by standard immunological methods. More specifically, % g of CNBr sepharose 4B were swollen for 15 minutes in 1 mM HCl and washed on a scintered glass filter with the same solution, with a total of approximately 200 ml/g of dry gel being added in several aliquots, the supernatant being sucked up between successive additions. The gel solution was aliquoted into three tubes and washed with 10 ml of a coupling buffer (pH 8.3) of 0.1 M NaHCO 3 containing 0.5 M NaCl.
  • a coupling buffer pH 8.3
  • IgG immune serum
  • PS preimmune serum
  • SA-IgG Staph A-purified IgG from immune serum
  • the IS, PS and SA-IgG columns were washed with PBS, after which % ml/column of low calcium NHMC-conditioned media was added to each column and recirculated five times over the columns. The media was then drained and the columns rinsed with
  • the immune serum column and Staph A-purified IgG columns removed some inhibitory activity which, as shown by the data in Table 11, was also eluted from the columns.
  • RPMI media with 5% FCS and hypoxanthine which contained pH indicator was conditioned by 46 individual hybridoma subclones until acidic conditions of the culture were observed, usually about 3 or 4 days. The cells and media were then removed from culture and centrifuged, and the hybridoma conditioned media supernatant recovered. MCF-7 cells were plated in 12-well Costar plates at 2 ⁇ 10 4 cells/ml In MEM with 10% FCS and allowed to attach overnight at 37oC. 50 ul/well of the hybridoma supematants and 100 ul/well of low calcium NHMC-conditioned media were added to each test well, the cells grown for 7 days then counted and percent disinhibition calculated. Of the 46 hybridoma supematants tested, the 13 supematants of the hybridomas listed below in Table 1% showed disinhibition of inhibitor activity of up to 100%.
  • 96-well microtiter plates were coated with 100 ul/well of 10 ug/ml of goat anti-mouse IgG overnight at 4oC. The next day, the plates were washed two times with PBS and 75 ul/well hybridoma supernatant prepared as described above of fourteen individual hybridoma subclones were added to the wells (4 wells/sample) and incubated overnight at 4oC. The wells were then washed two times with PBS, after which 50 ul/ml of low calcium NHMC-conditioned media was added to each well and allowed to incubate overnight at 4oC .
  • the media were then removed, sterilized, and used to treat MCF-7 cells plated at 1 ⁇ 10 4 cells/ml in MEM and 10% FCS for 7 days in a disinhibition assay to determine depletion of inhibitor from the conditioned media by the plate-bound antibodies.
  • the 7 hybridoma subclones designated below caused over 50% and up to 100% depletion of the inhibitory activity from the conditioned media.
  • 50 ul of hybridoma subclone supematants prepared as described above from hybridoma cell cultures were then individually added to the wells without dilution and in serial two-fold dilutions from 1:1 to 1:128 in 2% BSA in PBS. After one hour of incubation at room temperature, the plate was washed with the above washing solution. 50 ul of goat anti-mouse isotype-specific antibodies labeled with alkaline phosphatase from Southern Biotechnology Associates, Birmingham, Alabama, at a 1:1000 dilution in 2% BSA in PBS was added to the wells.
  • NHMC normal human mammary cells
  • NHMC normal human mammary cells
  • ⁇ 60nM low calcium media
  • 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 10 ⁇ by amicon ultrafiltration. Cell number was determined at indicated times by Coulter counting. As shown in Figure 10, 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.
  • SPECIFIC EXAMPLE 19 Antibody Affinity Purification of Human Mammary Growth Inhibitor
  • 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 2% 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.
  • Specific activity was defined in units/rag where one unit is the amount of protein necessary to produce 50% inhibition of MCF-7 cell growth. Concentration of the inhibitory protein was determined by comparison with albumin standards on SDS-PAGE gels visualized by silver stain and Biorad protein assays. Final purity of the inhibitory proteins was determined to be greater than 50%. The results are summarized in Table 14 below. Upon repetition of the purification scheme set forth above, final purity of at least about 95% was achieved.
  • Hybridoma supematants 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% normal human mammary conditioned medium: (b) 10% NHMC conditioned medium + 10% hybridoma supernatant from DF1/3C6; (c) 10 ng/ml affinity purified inhibitor; (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 19.
  • Affinity selected inhibitor was then prepared by subjecting the culture supematants to affinity chromatography on a 3C6 monoclonal antibody affinity column, and molecular weights determined by SDS-PAGE on a 10% SDS gel. Lane (c) is the fluorogram of 35 S-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
  • Affinity purified inhibitor (100 ng/ml) was heated to 37,
  • affinity purified inhibitor 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. After the one hour incubation, 50 ug/ml of 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 37oC. Both experimental and control samples were then sterilized by filtration and added to MCF-7 cultures as in Specific Example 18 to assay for inhibitory activity. As shown in Figure 13, the inhibitory activity of the affinity purified inhibitor was completely abrogated by the addition of trypsin.
  • 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 1% 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.
  • human mammary growth inhibitor (10 ng/ml) produced significant inhibition of the growth of the five transformed human mammary cell lines tested.
  • 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 15. 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).
  • 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.
  • Figure 14 shows the staining results of (A) normal human mammary cells (X100), (B) MCF-7 cells (X100). As shown in Figure 14A, Immunoreactive inhibitor was present in greater than 90% of the normal human mammary cells. In contrast, as shown in Figure 14B, 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 15 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.
  • Neither immunoreactive or biologically active human mammary cell growth inhibitor could be detected in conditioned media of the three transformed mammary cell lines tested.
  • Conditioned medium was 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 colorometric 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 Dynatec 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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Abstract

Un anticorps monoclonal tiré d'antisérum animal et des anticorps monoclonaux produits par des lignées cellulaires d'hybridome se lient à un inhibiteur de croissance spécifique à des cellules mammaires sécrété par des NHMC (cellules mammaires humaines normales). Lorsqu'on le purifie par chromatographie des anticorps monoclonaux par affinité, l'inhibiteur de la présente invention présente des maximums d'activité d'environ 47000 et environ 63000 à 67000 mesurés par SDS-PAGE. L'inhibiteur de croissance est présente dans les sérums de femmes normales, mais est absent ou présent dans des niveaux moindres dans les sérums d'hommes normaux et de patientes atteintes du cancer du sein. On peut utiliser des analyses de liaison de l'anticorps à l'inhibiteur afin de diagnostiquer la présence du cancer du sein, afin de dépister les personnes présentant un risque élevé de cancer du sein, et afin de contrôler les niveaux d'inhibiteur des patients dans le tissu mammaire et le sérum pendant le traitement.
PCT/US1989/002215 1988-05-20 1989-05-22 Anticorps contre l'inhibiteur de croissance de cellules mammaires humaines et procedes de production et d'utilisation WO1989011491A1 (fr)

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Cited By (2)

* 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

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4379839A (en) * 1977-05-23 1983-04-12 The Trustees Of Columbia University In The City Of New York Method for detecting cancer
US4753894A (en) * 1984-02-08 1988-06-28 Cetus Corporation Monoclonal anti-human breast cancer antibodies

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4379839A (en) * 1977-05-23 1983-04-12 The Trustees Of Columbia University In The City Of New York Method for detecting cancer
US4753894A (en) * 1984-02-08 1988-06-28 Cetus Corporation Monoclonal anti-human breast cancer antibodies

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biochimica et Biophysica Acta, Volume 846, issued 1985, BOHMER et al, "Specific Neutralizing Antiserum against a Polypetide Growth Inhibitor for Mammary Cells Purified from Bovine Mammary Gland" pages 145-154, see entire document. *
See also references of EP0417168A4 *
The 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. *

Cited By (8)

* 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
WO1998014577A2 (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

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