WO1989011492A1

WO1989011492A1 - Human mammary cell growth inhibitor and methods of production and use

Info

Publication number: WO1989011492A1
Application number: PCT/US1989/002214
Authority: WO
Inventors: Paul R. Ervin, Jr.; Max S. Wicha; Robert L. Cody
Original assignee: The Regents Of The University Of Michigan
Priority date: 1988-05-20
Filing date: 1989-05-22
Publication date: 1989-11-30
Also published as: JPH05506423A; EP0414807A1; EP0414807A4

Abstract

A human mammary cell growth inhibitor, and novel compositions comprising enhanced and purified concentrations thereof, inhibits the growth of human mammary cells. Inhibitor of the present invention purified by monoclonal antibody affinity chromatography comprises a thermolabile protein and has peaks of inhibitory activity at molecular weights of about 47,000 and about 63,000-67,000, as determined by SDS-PAGE. The inhibitory activity of the inhibitor is mammary cell specific, and is both calcium and dose-dependent. Increased production of the inhibitor can be induced by growth of inhibitor-secreting human mammary cells in low concentrations of calcium. The inhibitor can be used prophylactically to decrease the risk of breast cancer, to screen for the risk or presence of breast cancer, and as a therapeutic agent for the treatment of breast cancer.

Description

HUMAN MAMMARY CELL GROWTH INHIBITOR AND METHODS OF PRODUCTION AND USE

RELATED APPLICATIONS

This is a continuation-in-part of Application Serial No. 196,657 entitled "Human Mammary Cell Growth Inhibitor and Methods of

Production and Use" filed on May 20, 1988 by Ervin et al. which is a continuation-in-part of Application Serial No. 891,135 entitled

"Difactin" filed on July 31, 1986 by Ervin.

FIELD OF THE INVENTION 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.

MICROORGANISM DEPOSITS

The following hybridomas have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville,

Maryland 20825:

Hybridoma Accession No. Date of Deposit

DF1/3C6 HB9722 May 24, 1988

DF1/1D4 HB9723 May 24, 1988 DF1/6B8 HB10152 May 22, 1989

BACKGROUND OF THE INVENTION Human mammary tissues undergo a burst of proliferative activities at the onset of menarch and during each menstrual cycle. Studies on the effects of estrogen on mammary tissues and tumors indicate that estrogen is a primary growth-initiating factor for mammary tissues. See, for example, Martin, L., Estrogens in the Environment, pp. 103-130 and Sonnenschein, C. et al., J . Nat . Cane . Inst.. 64:211-214 (1980). The existence of growth factors which are estradiol-sensitive has also been established. See, for example, Soto, A. M. et al., Cancer Res., 46:2271-2275 (1986). In addition, 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. See, for example, Huff, K. K. et al., Endoc. Soc. Abst.. 310 (1986). TGF-beta, on the other hand, has been shown to suppress mammary tissue growth. See, for example, Robert, A. B. et al. PNAS US, 82:119-123 (1985).

The interest in mammary cell growth regulation is of more than academic importance. Neoplastic growths of mammary tissues, if unchecked, develop into uncontrollably-proliferating malignant tumors, which are the cause of death of thousands of women yearly. Growth inhibition factors which could suppress cell growth in mammary tissue would provide a dynamic tool for preventing such malignancies and in treating breast cancer.

SUMMARY OF THE INVENTION

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, however, exhibits peaks of activity in the range of from about

45,000 to about 50,000 dalton MW and from about 60,000 to about 70,000 dalton MW as determined by SDS-PAGE. Increased production of functional inhibitor has also been induced by growth of inhibitor-secreting human mammary cells in low concentrations of calcium.

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.

A better understanding of the present invention and its advantages will be had from a reading of the Description of the Preferred Embodiments of the Invention taken in combination with the drawings and specific examples.

BRIEF DESCRIPTION OF THE DRAWINGS 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

55,000.

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 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 ³⁵S-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.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

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. By "enhanced concentration" is meant a concentration of inhibitor greater than normal physiologic levels of inhibitor. By "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. By "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 (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, however, 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. 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.

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. By "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. Additional tests have shown that the inhibitor's suppression of mammary cell growth is reversible upon removal of the inhibitor.

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. Thus 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. In addition, 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.

SPECIFIC EXAMPLE 1 Growth Inhibition and Changes in Cell Morphology of NHMC Treated with NHMC-Conditioned Media

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). After trypsin removal from primary culture, the cells were plated at 4 × 10⁵ 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.

As shown by the data in Table 1, when 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, suggesting that more functional mammary cell growth inhibitor is produced by NHMC cells grown at low calcium concentrations. As also shown in Table 1, cells inhibited by the low calcium NHMC-conditioned media also took on a characteristic differentiated morphology.

TABLE 1

Conditioned Media Number of Cell Divisions After Cell Supplement Treatment with Supplemented Media Morphology

NHMC-conditioned approx. 2 cells were flat low calcium^a media with small nuclei

NHMC-conditioned > 6 normal healthy DMEM/F12 media^b cells with a rounded shape

NHMC-conditioned , 4 - 6 cells were of Defined MCDB media^b normal shape, but nuclei appeared slightly shrunken

a 0.04 mM calcium b physiologic levels of calcium

SPECIFIC EXAMPLE 2 Calcium Dependence of Inhibitory Activity To test whether the inhibitory activity of the human mammary cell growth inhibitor was calcium-dependent, 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. SPECIFIC EXAMPLES 3 THROUGH 18

For Specific Examples 3 through 18, the following general experimental protocol was used, except where otherwise indicated:

Cell Culture and Conditioning of Media: Normal human mammary cells from reduction mammaplasty were obtained from Herb Soule of the Michigan Cancer Foundation. The cells were maintained in Gibco DMEM/F12 with 40-60 nM calcium chloride, 5% Sigma Chelex treated horse sera, 20 ng/ml Collaborative

EGF, 98 ng/ml cholera toxin, 10 ug/ml insulin and 1 ug/ml hydrocortisone. Cells were incubated at 37ºC in a humidified CO₂ 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

C0„ incubator. When NHMC-conditioned media was required, the cells were allowed to condition the low calcium medium for at least 96 hours.

Growth and Inhibition Assays: MCF-7 cells were plated at 1 to 2 × 10⁴ cells/ml,

1 ml/well in 12-well Costar culture plates in MEM supplemented with 10% FCS and, optionally, 1 ug/ml insulin, after trypsin removal from stock cultures and 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% were considered unacceptable and discarded. Calculations (using cell count averages):

% 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.

SPECIFIC EXAMPLE 3

Dose Response of MCF-7 Cells Treated with NHMC-Conditioned Media MCF-7 cells were plated at 10⁴ 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.

As shown by the dose response curve of Figure 2, when the volume percentage of conditioned media used to supplement the MCF-7 media was increased, the level of inhibitory activity increased, illustrating that the level of inhibitory activity is dose-dependent.

SPECIFIC EXAMPLE 4 Heat Denaturization and Trypsin Inactivation of Human Mammary Cell Growth Inhibitor MCF-7 cells were plated at 10⁴ cells/ml in MEM supplemented with 10% by volume FCS. The cells were treated after

24 hours with 10% by volume low calcium NHMC-conditioned media which had been treated with 5% by volume of a solution containing 2.5 ug/ml trypsin in PBS for one hour at 37°C or which had been heated to 70ºC for one hour. The conditioned media used as a supplement for the control was, however, treated first with trypsin inhibitor and then incubated with trypsin. The cells were allowed to grow for seven days and then counted. Loss of activity was measured by percent disinhibition. As illustrated by the data in Table 2 below, human mammary cell growth inhibitor activity is sensitive to heat and trypsin, indicating that the inhibitor is proteinaceous in nature.

TABLE 2 Inactivation of Inhibitor Treatment % Disinhibition

Control 5

Trypsin 31

Heat 53

SPECIFIC EXAMPLE 5 Specificity of Human Mammary Cell Growth Inhibitor

To test the specificity of the human mammary cell growth inhibitor and to show that its activity was not just that of a universally cell-toxic metabolic by-product, 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 10⁴ 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.

As shown by the data in Table 3, 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. 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. TABLE 3 Specificity of Inhibitory Effect of NHMC-Conditioned Media Cell Type Designation % Inhibition

Mammary NHMC 50

Mammary BT-20 50

Mammary MCF-7 78

Mammary HBL-100 29

Mammary ZR-75-1 26

Mammary MDA MB-231 <5 Mammary DU 4475 <5

Colon Adenocarcinoma SW948 <5

Fibrosarcoma HT1080 <5

Epiloid Carcinoma HeLa <5

Melanoma WM 164 <5

Lymphoma U 937 <5

Mylogeneous Leukemia K 562 <5

Fibroblasts HF <5

Promyeloid Leukemia J774-2 <5

T-Cell Molt 4 <5

SPECIFIC EXAMPLE 6

Purification of Human Mammary Cell Growth Inhibitor and Activity Isolated from Conditioned Media

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.

Greater purification was achieved by passing NHMC-conditioned media over a DEAE ion exchange column at low salt concentration. Proteins were then eluted from this column using either a step-gradient of NaCl concentrations or an increasing linear gradient of NaCl concentration. The inhibitory activity of the fractions from this separation was determined by dialyzing the fractions against PBS, sterilizing the fraction by filtration, and using the solutions to treat MCF-7 cells as previously described. When inhibitory activity was determined, the activity of the protein was greatest in the 0.5 M NaCl fraction from the step gradients and in the fractions of approximately 0.3 M NaCl for the linear gradient.

Fractions from the DEAE column which contained inhibitory activity were further separated and sized by molecular sieve chromatography on a Superose 12 FPLC column and the inhibitory activity of the fractions was measured. Inhibition in this assay was measured by ³H-thymidine incorporation of the MCF-7 cells after a 24 hour labeling period and 48 hours exposure to the fractions. From this separation, as shown in Figure 3, there were two major peaks of inhibitory activity: a peak at an apparent molecular weight of about 40,000 to 45,000 and an additional peak at an apparent molecular weight of about 50,000 to 60,000. Further separation over a phenyl sepharose hydrophobic interaction column purified the inhibitor to levels undetectable by silver stain, although, as shown by the data below in Table 4, peak inhibitory activity was present in the fraction eluted with 1 M NaCl. However, as shown in Figure 4 at D, a silver stain of samples separated on the hydrophobic interaction column then electrophoresed on a 10% SDS polyacrylamide gel failed to detect inhibitor protein in this fraction.

TABLE 4

Percentage Inhibition for Fractions Separated by Hydrophobic Interaction Chromatography

Sample % Inhibition

B 3 C 31 D 51 E 39 CM 76

(B) 2 M NaCl eluent

(C) 1.5 M NaCl eluent

(D) 1 M NaCl eluent

(E) 0.5 M NaCl eluent (CM) conditioned media

SPECIFIC EXAMPLE 7

Preparation of Antibodies to Human Mammary Cell Growth Inhibitor and Abrogation of Inhibitor Activity

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. Animals 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.

Cloned populations of ELISA-positive cell cultures were then expanded. Expanded cultures were washed free of the azoserine and allowed to condition the media before they were tested in the growth inhibition assays. Cells from tested cultures which were shown to disirihibit or block inhibitory activity were then subcloned to single cell clones by limiting dilution and then retested for ability to affect inhibitory activity. Subclones which were shown to disinhibit or block inhibitory activity were then expanded to 5 × 10⁶ cells and injected into the peritoneal cavity of pristane-primed C3H/HeN × Balb/c (F₁) mice for production of the antibody in ascites fluid. Monoclonal antibodies to the inhibitor were given the general designation DF1. 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. HB9722 (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. 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 100

SPECIFIC EXAMPLE 8

Dose Response of Inhibition by Purified Human Mammary Growth Inhibitor Isolated from Conditioned Media 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. 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%. To insure that the protein recognized by the monoclonal antibody was one that had been produced by the NHMC cells, the proteins being produced by NHMC were metabolically labeled using³⁵ S-methionine. Cell proteins were labeled with ³⁵S-methionine at

200 uCi/ml in methionine-free Gibco DMEM at 37°C for 20 hours. 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 ³⁵S-methionine labeled proteins at the same molecular weight as those containing the inhibitory activity produced by normal human mammary cells (NHMC).

SPECIFIC EXAMPLE 9 Presence of Human Mammary Cell Growth Inhibitor in Human Cell Lysates and Sera

Since the inhibitor was of human origin and obviously exported from the mammary cells, human mammary cell lines and human sera were tested for the presence of the protein by Western blotting. Cells were lysed with 2% SDS in PBS and human sera were mixed with reducing buffer and run on 10% SDS-PAGE. Proteins from the gels were transferred to nitrocellulose in 10% methanol, 0.15 M glycine, 25 mM Tris at 180 ma overnight. Filters were blocked with

2% BSA in PBS and the first 3C6 monoclonal antibody was added with 2% BSA In PBS. ¹²⁵I-labeled sheep anti-mouse second antibodies were diluted in 5% by weight NFDM in PBS at 10 uCi/10ml. All washes were performed with PBS plus 0.1% Baker Tween 20. As shown by the

Western blot depicted in Figure 7, 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.

SPECIFIC EXAMPLE 10 Effect of Conditioned Medium on MCF-7 Growth Normal human mammary cells (NHMC), characterized as epithelial by keratin expression, were derived from reduction mammaplasties and serially passaged in low calcium media (<60nM) as described by Soule, H.S., et al., In Vitro, 22:6 (1986). Medium was conditioned by incubating confluent cultures of NHMC for 4 days in DMEM/F-12, at 40 nM CaCl₂ with 5% chelex treated bovine serum.

MCF-7 cells were plated at 2 × 10⁵ 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.

SPECIFIC EXAMPLE 11 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 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.

Specific activity was defined in units/mg 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%. These results are summarized in Table 6 below. Upon repetition of the purification scheme set forth above final, purity of at least about 95% was achieved. TABLE 6

Total Specific

Sample Protein Activity Purification Yield (% .

Conditioned

Media 2150 mg 1.16 1-fold 100 0.5M DEAE

Fraction 20.3 mg 47.7 41-fold 40.3 Affinity

Purified 0.0012 mg 106,000.0 91, 380-fold 5.3

SPECIFIC EXAMPLE 12

Effect of Affinity Purified Human Mammary Growth Inhibitor and 3C6 Antibody on MCF-7 Growth

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. By "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.

Cell growth was determined at; day 7 and percent inhibition, compared to an equivalent volume of non-conditioned medium, was determined. As shown in Figure 9, the purified material inhibited the growth of MCF-7 cells in a dose dependent manner with 50% growth inhibition at an inhibitor concentration of 10 ng/ml and greater than a 90% Inhibition of cell growth at 20 ng/ml. Inhibition of cell growth by either conditioned medium or purified Inhibitor was abrogated by monoclonal antibody 3C6. Monoclonal antibody alone had no significant effect on MCF-7 cell growth. SPECIFIC EXAMPLE 13

SDS-PAGE and Fluorographic Analysis of Purified Human Mammary Growth Inhibitor

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. In order to also demonstrate that inhibitor is synthesized by NHMC, cultures were labeled with ³⁵S-methionine in 5 ml of methionine-free DMEM medium supplemented with 200 uCi/ml of ³⁵S-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

SDS-PAGE on a 10% SDS gel. Lane (c) is the fluorogram of

35S-methionine labeled NHMC-conditioned medium eluted from the 3C6 affinity column.

As shown in Figure 10 , lane B , two prominent bands at approximately 47,000 and 65,000 appeared by silver staining of the affinity purified inhibitor. As shown in the fluorogram of affinity selected inhibitor in lane c, 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.

SPECIFIC EXAMPLE 14

Heat Denaturization and Trypsin Sensitivity of Affinity Purified Human Mammary Cell Growth Inhibitor

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.

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 10 to assay for inhibitory activity. As shown in Figure 11, the inhibitory activity of the affinity purified inhibitor was completely abrogated by the addition of trypsin.

SPECIFIC EXAMPLE 15 Effects of Human Mammary Growth Inhibitor on Growth of Human Cell Lines

The effects of 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 × 10⁴ 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

(10 ng/ml) produced significant inhibition of the growth of the five transformed human mammary cell lines tested. The growth of 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)). In contrast to the effect on mammary cells, 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). In addition, 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. Thus, by molecular weight, heat sensitivity, tissue specificity and antibody reactivity, 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.

TABLE 7

Cell Type Designation Inhibition

Mammary Carcinoma MCF-7 61 +/- 3 Mammary Carcinoma BT-20 53 +/- 17 Mammary Carcinoma MDA-MB-231 25 +/- 4 Mammary Carcinoma ZR-75-1 37 +/- 4 Mammary Carcinoma Evej os 21 +/- 4 Mammary Epithelium HBL-100 19 +/- 15

Lung Adenocarcinoma A 427 3 +/- 1 Colon Carcinoma SW 948 -8 +/- 5 Fibrosarcoma HT 1080 8 +/- 16 Promonocytic Leukemia HL-60 -9 +/- 3 Bladder Carcinoma Hill 4 +/- 7 Lymphoblastic Leukemia Raji -2 +/- 11 Myelomonocytic Leukemia Wehi-3 1 +/- 6 Squamous Carcinoma A 253 o +/- 6 Squamous Carcinoma UM-SCC-17a -12 +/- 11 Squamous Carcinoma UM-SCC-38 -6 +/- 8 Cervical Carcinoma HeLa -17 +/- 20

Negative numbers indicate growth stimulation. SPECIFIC EXAMPLE 16 Immunoperoxldase Staining of Human Mammary Cells

In order to examine inhibitor production by normal and transformed human cell lines, immunoperoxidase staining of detergent permeabilized normal and transformed cells using the 3C6 monoclonal antibody was performed.

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). As shown in Figure 12A, immunoreactive inhibitor was present in greater than 90% of the normal human mammary cells. In contrast, as shown in 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.

SPECIFIC EXAMPLE 17 Lack of Secretion of Human Mammary Growth Inhibitor by Transformed Cell Lines

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.

SPECIFIC EXAMPLE 18 Sandwich ELISA and Quantification of Level of Human Mammary Growth Inhibitor

Dynatech microtiter plates were incubated with 100 ul/well of 10 ug/ml purified 6B8, an IgM monoclonal antibody from hybridoma clone DF1/6B8, produced as described previously in Specific Example

7, in PBS at 4ºC overnight. Coated plates were then blocked to reduce non-specific binding by incubating the coated wells with 300 ul of PBS + 1% bovine serum albumin (BSA) from Sigma Chemical for one hour at room temperature. Plates were incubated for two hours at room temperature with 100 ul/well of either affinity purified inhibitor as a standard or human serum. Plates were next washed with PBS + 0.1% Triton X-100 to remove unbound protein by flooding the plate with this wash solution and then shaking the solution off the plate. This procedure was repeated four times.

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 dH₂O, 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.

Actual ng/ml quantification of protein was accomplished by this method by determining protein concentration of the purified inhibitor first using a .Biorad protein assay or a similar assay such as SDS-PAGE comparison and then producing a standard curve of the pure protein by successive dilution in a solution of PBS + 1% BSA.

Unknown samples were then compared to this standard curve to determine protein level. As this invention may be embodied in several forms without departing from the spirit or essential characteristics thereof, the present embodiment is therefore illustrative and not restrictive, and since the scope of the invention is defined by the appended claims, all changes that fall within the metes and bounds of the claims or that form their functional as well as conjointly cooperative equivalents are therefore intended to be embraced by those claims.

Claims

What is Claimed is:

1. A cell growth inhibitor in substantially pure form, wherein functional inhibitor has the following characteristics:

(a) an inhibitory effect on the growth of female NHMC obtained from reduction mammoplasty, MCF-7, and BT-20 mammary cells;

(b) calcium-dependence of inhibitory activity;

(c) dose-dependence of inhibitory activity;

(d) reversibility of inhibitory activity;

(e) substantial decrease of inhibitory activity upon heating to 100°C; and

(f) substantial decrease of inhibitory activity upon treatment with trypsin.

2. The inhibitor of Claim 1, wherein the composition further comprises a physiologically compatible solution of the inhibitor.

3. The inhibitor of Claim 1, wherein the molecular weight of one form of affinity purified inhibitor is about 47,000 as determined by 10Z SDS-PAGE.

4. The inhibitor of Claim 1, wherein the molecular weight of one form of affinity purified inhibitor is about 65,000 as determined by 10Z SDS-PAGE.

5. .The inhibitor of Claim 1, wherein the molecular weight of one form of affinity selected inhibitor is about 47,000.

6. The composition of Claim 1, wherein the molecular weight of the one form of affinity selected inhibitor is in the range of from about 63,000 to about 67,000 as determined by 10% SDS-PAGE.

7. The inhibitor of Claim 1, wherein significant inhibitory activity as measured by MCF-7 growth assay is present in NHMC-conditioned media molecular sieve column fractions of a molecular weight of about 45,000.

8. The inhibitor of Claim 1, wherein significant inhibitory activity as measured by MCF-7 growth assay is present in NHMC-conditioned media molecular sieve column fractions of a molecular weight of about 55,000.

9. A composition of matter for regulating cell growth comprising an enhanced concentration of a growth inhibitor, the inhibitor comprising a thermolabile protein having a molecular weight in the range of from about 45,000 to about 70,000, wherein functional growth inhibitor is capable of inhibiting the growth of human mammary cells and is specific for mammary cells.

10. The composition of Claim 9, wherein the composition further comprises a pharmaceutically acceptable delivery vehicle.

11. The composition of Claim 9, wherein the enhanced concentration of growth inhibitor is substantially pure.

12. The composition of Claim 9, wherein the level of secretion of functional growth inhibitor by human mammary cells is calcium-dependent.

13. The composition of Claim 10, wherein the level of mammary cell growth inhibition is calcium-dependent.

14. The composition of Claim 10, wherein the human mammary cells comprise transformed female human mammary cells.

15. A method of increasing the level of functional human mammary cell growth inhibitor secreted by a human mammary cell comprising the steps of: (a) providing a human mammary cell capable of secreting functional human mammary cell growth inhibitor; and

(b) providing the secreting cell with a low calcium environment.

16. The method of Claim 15, wherein the human mammary cell provided is a cell in tissue culture and the low calcium environment comprises cell culture medium having a concentration of less than approximately 0.1 mM calcium.

17. The method of Claim 15, wherein the human mammary cell is a normal female human mammary cell.

IS. The method of Claim 16, wherein the calcium concentration is approximately 0.04 mM.

19. A method of decreasing the proliferation of a population of human mammary cells comprising the steps of:

(a) contacting at least a portion of the population with an effective amount of functional human mammary cell growth inhibitor; and

(b) monitoring the level of proliferation of the mammary cell population.

20. The method of Claim 19, further comprising the stepof Increasing the level of calcium of the cell population environment an amount sufficient to increase inhibitory activity of the mammary cell growth inhibitor.

21. A method of treating human breast cancer in a patient comprising the steps of:

(a) administering a therapeutically effective amount of human mammary cell growth inhibitor to the patient; and

(b) monitoring the level of inhibition of mammary cell growth.

22. The method of Claim 21, further comprising the step of regulating the calcium levels of the patient.

23. A mammary cell growth inhibitor in substantially pure form which is immunoreactive with monoclonal antibodies produced by a hybridoma selected from the group consisting of hybridoma DF1/3C6 assigned ATCC Accession No. HB9722, hybridoma DF1/1D4 assigned ATCC Accession No. HB9723, hybridoma DF1/6B8 assigned ATCC Accession No. HB10152.

24. The inhibitor of Claim 24, wherein the inhibitor is immunoreactive with monoclonal antibodies produced by hybridoma DF1/3C6.

25. The inhibitor of Claim 24, wherein the inhibitor is immunoreactive with monoclonal antibodies produced by hybridoma DF1/1D4.

26. The inhibitor of Claim 24, wherein the inhibitor is Immunoreactive with monoclonal antibodies produced by hybridoma DF1/6B8.

27. Substantially purified human mammary growth inhibitor.

28. The inhibitor of Claim 27 in an active form.

29. The inhibitor of Claim 27 in an inactive form.