ANTIBODIES TO HUMAN MAMMARY CELL GROWTH INHIBITOR AND METHODS OF PRODUCTION AND USE

Abstract: Polyclonal antibody from animal antiserum and monoclonal antibodies produced by hybridoma cell lines bind to a mammary cell specific growth inhibitor secreted by NHMC. When purified by affinity monoclonal antibody chromatography, the inhibitor of the present invention exhibits peaks of activity at about 47,000 and about 63,000-67,000, as measured by SDS-PAGE. The growth inhibitor is present in the sera of normal females, but is absent or present in decreased levels in the sera of normal males and female breast cancer patients. Binding assays of the antibody to the inhibitor can be used to diagnose the presence of breast cancer, to screen for those at high risk breast cancer, and to monitor patient levels of inhibitor in mammary tissue and serum during treatment.

Description: ANTIBODIES TO HUMAN MAMMARY CELL GROWTH INHIBITOR AND METHODS OF PRODUCTION AND USE RELATED APPLICATIONS This is a continuation-in-part of Application Serial No. 199,608 entitled "Antibodies to Human Mammary Cell Growth Inhibitor and Methods of Production and Use" filed on May 27, 1988 by Ervin et al. , which 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 antibodies specific for human mammary cell growth inhibitor and methods of their 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 is of more than academic interest. 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 specifically in mammary tissue would provide a dynamic tool for preventing such malignancies and in treating breast cancer. Such a mammary cell growth inhibitor has been described in aforementioned parent Application Serial No. 196,657 filed on May 20, 1988 and original Application Serial No. 891,135, both herein incorporated by reference. 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. In accordance with the principles of the present invention, 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, however, 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. Because of the relationship of mammary cell growth inhibitor levels to 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. 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 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 10Z 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. 35 Figure 6 is a fluorogram of 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 35 and a fluorogram of 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. DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION The present invention generally relates to antibodies specific for a human mammary cell growth inhibitor, and to methods for their production and use. 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. 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. By "enhanced concentration" is meant a concentration of inhibitor greater than normal physiologic levels of inhibitor. In accordance with the principles of the present invention 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. uπtransformed, human mammary cells in culture and is present in the serum of "normal" females, i.e. those free of breast cancer. However, 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. 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 902. Further purification to about 95X 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. All molecular weights referred to herein are give 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 polyacryla ide 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". Fluorogra s 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. T e 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 mammairy cell growth is reversible upon removal of the inhibitor. The regulatory effect exerted by the mammary cell growth inhibitor of the present invention is not via cell kill of the treated cells. Upon visual observation, normal human mammary cells treated by the inhibitor appear healthy, but 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. In accordance with the principles of the present invention, 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. 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 cells lines which do not produce inhibitor are unaffected, suggesting the presence of inhibitor on the cell surface, a finding also confirmed by indirect i munofluoresence. 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. Thus, as described in the aforementioned Applications Serial Nos. 196,657 and 891,135, 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. SPECIFIC EXAMPLES For Specific Examples 1 through 26, the following general experimental protocol was used, except where otherwise indicated and all percentages given are by volume unless otherwise indicated: Cell Culture and Conditioning of Media: Normal human maπππary cells from reduction mammaplasty were obtained from Herb Soule of the Michigan Cancer Foundation. The cells were maintained in Gibco DMEM/F12 media with (low calcium) 40-60 nM calcium chloride, 52 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 37 C in a humidified CO- incubator. When NHMC-conditioned media was required, the cells were allowed to condition the low calcium medium for at least 96 hours. MCF-7 cells were maintained in MEM supplemented with 102 fetal calf serum (FCS) in 1 ug/ml of insulin at 37 C in a humidified C0„ incubator. Growth and Inhibition Assays: 4 MCF-7 cells were plated in triplicate at 1 to 2 x 10 cells/ml, 1 ml/well in 12-well Costar culture plates in MEM supplemented with 102 by volume FCS, and, optionally, 1 ug/ml insulin, after trypsin removal from stock cultures and were allowed to attach overnight at 37 C. 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 37 C 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 37 C to liberate their nuclei for counting on a Coulter Counter. Cell counts were averaged, with a deviation from the mean cell number greater than 52 being discarded as unacceptable. Calculations (using cell count averages) : 2 inhibition - [l-(cell number of treated sample/cell number of untreated sample) ] x 100 2 recovery - [1-(percent inhibition of treated sample with conditioned media removed/percent inhibition of treated sample with conditioned media)] x 100 2 disinhibition - [1-(percent inhibition of the sample/percent inhibition of conditioned media)] x 100 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- enth 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 102 fetal calf serum (FCS) . After trypsin removal from primary culture, the cells were plated at 4 x 10 cells/T25 flask, allowed to attach overnight, then grown in IMDM defined media supplemented with 102 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 hemac tometer. 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, indicating 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 3 DMEM/F12 with small nuclei media NHMC-conditioned > 6 normal healthy DMEM/F12 medi cells with a rounded shape NHMC-conditioned , cells were of Defined MCDB media normal shape, but nuclei appeared slightly shrunken 0.04 mM calcium 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 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 (lOx) conditioned media had been loaded. The fractions were used as a 102 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. 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 EXAMPLE 3 Dose Response of MCF-7 Cells Treated with NHMC-Conditioned Media 4 MCF-7 cells were plated at 10 cells/ml in MEM supplemented with 102 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 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 Reversal of Inhibitory Activity of Human Mammary Cell Growth Inhibitor 4 MCF-7 cells were plated at 1 x 10 cells/ml in 5 ml MEM media with 102 FCS in ten T25 flasks. Six of the flasks were also treated with 12 by volume low calcium NHMC-conditioned media supplement and the remaining four were used as untreated controls. Cells were grown for five days at 37 C in a humidified C0- 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 102 FCS, with two of the remaining treated cultures again receiving treatment with NHMC-conditioned media supplement, and with two of the previously treated cultures and the remaining two untreated cultures receiving no treatment. The cells were allowed to grow for five additional days and were then lysed and the cell nuclei counted on a Coulter counter, cell counts averaged, and percent inhibition and recovery determined. As shown by the data in Tables 2 and 3, 402 recovery from inhibition was observed in the cells treated at Day 0 but untreated from Day 5 to Day 10. TABLE 2 Percent Inhibition Treatment Day 0 2 Inhibition Day 5 none 02 12 CM* 282 - CM - low calcium NHMC-conditioned media TABLE 3 Reversal of Inhibition Treatment Day 0 Treatment Day 5 2 Inhibition Day 10 none none 02 12 CM 12 CM 302 12 CM none 182 SPECIFIC EXAMPLE 5 Heat Denaturization and Trypsin Inactivation of Human Mammary Cell Growth Inhibitor 4 MCF-7 cells were plated at 10 cells/ml in MEM supplemented with 102 by volume FCS. The cells were treated after 24 hours with 102 low calcium NHMC-conditioned media which had been supplemented with 52 of a solution containing 2.5 ug/ml trypsin in PBS for one hour at 37 C or which had been heated to 70 C 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 on a Coulter counter and percent disinhibition determined. As illustrated by the data in Table 4 below, human mammary cell growth inhibitor activity is sensitive to heat and trypsin, indicating that the inhibitor is proteinaceous in nature. TABLE 4 Inactivation of Inhibitor Treatment 2 Disinhibition Control 5 Trypsin 31 Heat 53 SPECIFIC EXAMPLE 6 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 4 Table 5 were plated at 10 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. TABLE 5 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 M Maammmmaarryy D DUU 44447755 <5 Colon Adenocarcinoma SW948 <5 Fibrosarcoma HT1080 <5 Epiloid Carcinoma HeLa <5 Melanoma WM 164 <5 LLyymmpphhoommaa UU 993377 <5 Mylogeneous Leukemia K 562 <5 Fibroblasts HF <5 Promyeloid Leukemia J774-2 <5 T-Cell Molt 4 <5 SPECIFIC EXAMPLE 7 Purification of Human Mammary Cell Growth Inhibitor and Activity 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 0.22 urn Gelman acrodiscs and were used to treat MCF-7 cells at 102 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. Greater purification was achieved by passing NHMC-conditioned 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 in this Example. 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 3 was measured by H-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 6, peak inhibitory activity was present in the fraction eluted with 1 M NaCl. However, as shown in Figure 4 at lane D, a silver stain of samples separated on the hydrophobic interaction column then electrophoresed on a 102 SDS polyacrylamide gel failed to detect inhibitor protein in this fraction. TABLE 6 Percentage Inhibition for Fractions Separated by Hydrophobic Interaction Chromatography Sample 2 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) low calcium NHMC-conditioned media SPECIFIC EXAMPLE 8 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 e ' luted proteins were then mixed with Freund's complete adjuvant 502 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, 502 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 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 152 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 4 C, the plates were then washed with a washing solution of 0.92 NaCl and 0.052 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 22 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, disodiu 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 disinhibit 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 x 10 cells and injected into the peritoneal cavity of pristane-primed C3H/HeN x Balb/c (F- ) 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 of MCF-7 cells by low calcium 4 NHMC-conditioned media. MCF-7 cells were plated at 1 x 10 cells/ml treated with 22 rabbit immune serum or 52 3C6 monoclonal antibody from ascites partially purified by ammonium sulfate precipitation. These cells were also then treated with 102 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. As shown by the data of Table 7 below, both the immune serum containing polyclonal antibody and 3C6 monoclonal antibody blocked the inhibitory activity of the inhibitor. 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 lane F. It was also shown in other studies that immune serum blocked inhibitory response in a dose-dependent manner. TABLE 7 Effects of Antibodies on Inhibition Treatment 2 Disinhibition conditioned media 0 immune serum 42 3C6 monoclonal antibody 100 SPECIFIC EXAMPLE 9 Dose Response of Inhibition by Purified Inhibitor Isolated From Conditioned Media 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. 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 102 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 902. 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 using 35 35 S-methionine. Cell proteins were labeled with S-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 35 3C6 monoclonal antibody affinity column retained S-methionine labeled proteins at the same molecular weight as those containing the inhibitory activity produced by normal human mammary cells (NHMC). SPECIFIC EXAMPLE 10 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 serum were tested for the presence of inhibitor protein by Western blotting. Cells were lysed with 22 SDS in PBS and samples of human serum were individually mixed with reducing buffer and electrophoresed on a 102 SDS polyacrylamide gel. Proteins from the gels were transferred to nitrocellulose in 102 methanol, 0.15 S glycine, 25 mM Tris at 180 milliamps overnight. Filters were blocked with 100 ml of 22 bovine serum albumin (BSA) in PBS solution. 3C6 monoclonal antibody was first added with 10 ml of 22 BSA in PBS. Filters were washed two times with 100 ml of PBS 125 solution. I-labeled sheep anti-mouse second antibodies diluted in PBS with 52 by weight non-fat dry milk (NFDM) at 10 uCi/lOml were then added. Filters were washed five times at 30 minutes each with PBS solution. All washes were performed with PBS plus 0.12 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 11 Absence or Decreased Levels of Human Mammary Cell Growth Inhibitor in Breast Cancer Patients 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 102 SDS-polyacrylamide gel. The proteins from the gel were electroblotted onto a nitrocellulose filter overnight in 102 methanol, 0.15 M glycine and 25 mM Tris at 180 milliamps. The filter was then blocked with 100 ml of PBS with 22 BSA for three hours to decrease non-specific protein binding to the filter. 100 ul of 3C6 monoclonal antibody from ascites partially purified by ammonium sulfate precipitation and 10 ml PBS with 22 BSA were added to the filter, incubated overnight at 4 C, then washed three times 125 In PBS. 10 ml of PBS with 52 non-fat dry milk and I-sheep anti-mouse IgG at 10 uCi/10 ml was then added to the filter and incubated for 2 hours at room temperature. The filter was then washed five times for ten minutes each time in PBS and 0.12 Tween 20 after which the filter was dried. The filter was exposed to film for 4 days, then developed to give the Western blot of Figure 8. As shown in Figure 8, three of the four patients with breast cancer (lanes 3-4 and 7-10) had no detectable levels of inhibitor, whereas one patient (lanes 5 and 6) had a reduced but detectable level of inhibitor in her serum. A second Western blot was prepared following the above procedures and similar results were obtained. SPECIFIC EXAMPLE 12 Dose Response of Disinhibition of Inhibitor Activity by 3C6 Monoclonal Antibody 4 MCF-7 cells were plated at 1 x 10 cells/ml in RPMI with 102 FCS and 10 ug/ml insulin and allowed to attach overnight at 37 C. The cells were then treated with 50 ul of inhibitor from low calcium NHMC-conditioned media purified on a 3C6 monoclonal antibody affinity column prepared by standard techniques and various volumes shown in Table 8 of 3C6 monoclonal antibody partially purified by ammonium sulfate precipitation. The cells were incubated at 37 C for seven days after treatment then counted. As shown by the data in Table 8, which Is also shown as a dose response curve in Figure 9, addition of increasing amounts of antibody increased disinhibition, indicating that disinhibition by the 3C6 antibody is dose-responsive. Percent inhibition and disinhibition were calculated as described previously. TABLE 8 Dose Response of Disinhibition of Inhibitor Activity by 3C6 Antibody Antibody Purified Partially Purified Inhibitor from CM 3C6 Antibody % Disinhibition 50 ul 100 ul 222 50 ul 50 ul 102 50 ul 25 ul 82 50 ul 0 ul 02 0 ul 50 ul SPECIFIC EXAMPLE 13 Complement Lysis of MCF-7 Cells with Immune Serum 4 MCF-7 cells were plated at 1 x 10 cells/ml in MEM with 102 FCS and 10 ug/ml insulin and allowed to attach overnight at 37 C. Cells were then treated in triplicate with media with the following supplements by volume: 102 PBS; 102 low calcium NHMC-conditioned low calcium media; 102 NHMC-conditioned media plus 102 immune serum obtained from New Zealand white rabbits immunized as described in Specific Example 8; 102 immune serum; and 102 New Zealand white rabbit serum obtained from the same animals prior to immunization (preimmune serum) . The treated cells were allowed to grow seven days in the supplemented media, and were then lysed and their nuclei counted on a Coulter counter and the counts for each averaged. As shown by the data in Table 9 below, and by visual observation of the cultures, immune serum caused complement lyses of human mammary cells, indicating that inhibitor is present on the cell surface. It should be noted, however, that the inhibitor present in MCF-7 cells appears to be non-functional, since it does not inhibit the growth of the MCF-7 cells, yet inhibition of MCF-7 cell growth is observed upon treatment with inhibitor produced by normal human mammary cells. As also shown in Table 9, complement lysis increased with the addition of NHMC-conditioned media which contains functional inhibitor. The above study was conducted two additional times with similar results. TABLE 9 Complement Lysis of MCF-7 by Immune Serum Treatment Cell Count after 7 Days 102 PBS 10204 102 CM a 7386 102 CM + 102 IS 997 102 IS 1984 102 PS C 11680 , low calcium NHMC-conditioned media Immune serum c preimmune serum SPECIFIC EXAMPLE 14 Depletion of Inhibitory Activity from NHMC-conditioned Media by Immune Serum CNBr-activated sepharose 4B serum coupled columns were prepared by standard immunological methods. More specifically, 2 g of CNBr sepharose 4B were swollen for 15 minutes in 1 mM HC1 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 containing 0.5 M NaCl. One ml of immune serum (IS), preimmune serum (PS) and Staph A-purified IgG from immune serum (SA-IgG) were each dissolved in 10 ml of coupling buffer, the solution added to one of the three gel tubes and incubated for two hours at room temperature with constant mixing. The gel was then poured into a syringe blocked with glass wool, unbound proteins were eluted, and the columns were then rinsed with 10 ml of 0.2 M glycine at pH 8. The column was next rinsed with coupling buffer, then 0.1 M acetate buffer at pH 4 with 0.5 M NaCl, then again with coupling buffer. The columns were stored at 4 C. Prior to use, the IS, PS and SA-IgG columns were washed with PBS, after which 2 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 2 ml PBS resulting In a final volume of 4 ml per sample of column-passed media. The columns were then rinsed an additional time with 20 ml of PBS, and again stored at 4 C. The conditioned media passed through the columns was then tested for removal of inhibitor by the column with an MCF-7 growth assay. To determine depletion, MCF-7 disinhibition studies were conducted using 102 by volume of conditioned media eluted from the columns or 5% non-depleted conditioned media as a supplement to the MCF-7 growth media for 7 days. If depletion occurred, the immune affinity column was eluted with 2 ml of 0.2 M glycine at pH 3 to remove antigen/inhibitor from the column. The antigen eluents were dialyzed against PBS then tested in an MCF-7 growth inhibition assay for inhibitor activity. As shown by the data in Table 10, 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. TABLE 10 Depletion of Inhibitor by Columns Supplement Cell Count at 7 Days 2 Inhibition 2 Depletion 102 PBS 26071 52 LCM a 26135 02 1002 52 CM 12949 502 02 102 CM from IS column 14839 432 142 102 CM from PS column 11888 542 02 102 CM from SA-IgG column 16352 372 262 , low calcium media not conditioned and not passed over column low calcium conditioned media not passed over column measured as 2 disinhibition TABLE 11 Inhibitory Activity of Antigen Eluents Supplement Cell Count at 7 Days % Inhibition 102 LCM 41062 02 102 IS column antigen eluent 1583 232 102 PS column antigen eluent 36993 102 102 SA-IgG antigen eluent 30596 252 SPECIFIC EXAMPLE 15 Disinhibition of Inhibitor Activity by Hybridoma Supernatant RPMI media with 52 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 x 4 10 cells/ml In MEM with 102 FCS and allowed to attach overnight at 37 C. 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 12 showed disinhibition of inhibitor activity of up to 1002. TABLE 12 Disinhibition by Hybridoma Supernatant/CM Hybridoma Subclone 2 Disinhibition DF1/14D5 502 DF1/15C5 622 DF1/12C5 392 DF1/9G8 1002 DF1/9A10 412 DF1/7G8 352 DF1/13D3 242 DF1/9G8 (subclone) 732 DF1/11D7 782 DF1/8A11 892 DF1/3C6 1002 DF1/6C6 762 DF1/2D2 562 SPECIFIC EXAMPLE 16 Depletion Assay of Hybridoma Supernatant 96-well microtiter plates were coated with 100 ul/well of 10 ug/ml of goat anti-mouse IgG overnight at 4 C. 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 4 C. 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 4 C. The media were then removed, sterilized, and used to treat MCF-7 4 cells plated at 1 x 10 cells/ml in MEM and 102 FCS for 7 days in a disinhibition assay to determine depletion of inhibitor from the conditioned media by the plate-bound antibodies. As shown In Table 13 below, of the 14 hybridoma subclones tested, the 7 hybridoma subclones designated below caused over 502 and up to 1002 depletion of the inhibitory activity from the. conditioned media. TABLE 13 Depletion by Hybridoma Subclone Supernatant Hybridoma Subclone % Depletion DF1/9G8 552 DF1/16G9 912 DF1/7G8 612 DF1/6B8 662 DF1/11D7 1002 DF1/1D4 662 DF1/3C6 572 a measured by 2 disinhibition SPECIFIC EXAMPLE 17 Isotyping of Monoclonal Antibodies to Human Mammary Cell Growth Inhibitor An ELISA was used to determine the Ig isotype of the monoclonal antibodies 3C6, 1D4 and 6B8. The wells of a 96-well Immulon 1 microtiter plate from Dynatech Laboratories, Inc., Chantilly, Virginia, were coated with 50 ul of goat anti-mouse IgM and IgG antibodies from Tago Co., Burlingame, California at 10 ug/ml in PBS and incubated overnight at 4 C. After incubation, the plates were washed with a washing solution of 0.92 NaCl and 0.052 Triton X-100. 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 22 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 22 BSA in PBS was added to the wells. Individual preparations of antibodies to a specific isotype were added to wells previously incubated with the serial dilution of a specified hybridoma supernatant, so that some wells received antibodies against IgM and some against either IgG3, IgGl, IgG2b or IgG2a. After another hour of Incubation at room temperature, the plate was washed with washing solution and then 100 ul of solution of 10 mg/ml of a 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 a 410 n wavelength. Monoclonal antibodies of known isotypes were used as positive and negative controls. Using this procedure, 3C6, 1D4 and 6B8 were determined to be IgM antibodies. No cross-reactivity with other isotypes was observed. SPECIFIC EXAMPLE 18 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 CaCl with 52 chelex treated bovine serum. MCF-7 cells were plated at 2 x 10 5 cells /T25 flask in MEM with 102 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 x 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 102 conditioned medium to these cultures resulted in a 752 inhibition of cell growth at 10 days compared to cultures supplemented with 102 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 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/rag where one unit is the amount of protein necessary to produce 502 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 502. The results are summarized in Table 14 below. Upon repetition of the purification scheme set forth above, final purity of at least about 952 was achieved. TABLE 14 Total Specific Sample Protein Activity Purification Yield (2) 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 20 Effect of Affinity Purified Human Mammary Growth Inhibitor and 3C6 Antibody on MCF-7 Growth 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) 102 normal human mammary conditioned medium: (b) 102 NHMC conditioned medium +- 102 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. 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 19. 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 11, the purified material Inhibited the growth of MCF-7 cells in a dose dependent manner with 502 growth inhibition at an inhibitor concentration of 10 ng/ml and greater than a 902 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 21 SDS-PAGE and Fluorographic Analysis of Purified Human Mammary Growth Inhibitor Lanes (a) and (b) of Figure 12 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, 35 cultures were labeled with S-methionine in 5 ml of methionine-free 35 DMEM medium supplemented with 200 uCi/ l of S-methionine by incubating confluent T-75 flask of NHMC for 24 hours. Affinity selected inhibitor was then prepared by subjecting the culture supe atants to affinity chromatography on a 3C6 monoclonal antibody affinity column, and molecular weights determined by SDS-PAGE on a 35 102 SDS gel. Lane (c) is the fluorogram of S-methionine labeled NHMC-conditioned medium eluted from the 3C6 affinity column. As shown in Figure 12, 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 22 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 70 C for 60 minutes, resulted in a 502 decrease in activity. Heating to 100 C for 60 minutes completely abrogated the inhibitory activity. This pattern of heat sensitivity shown in Figure 13 distinguishes the human mammary growth inhibitor from TGF-beta which is stable to 100 C 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 37 C. 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 37 C. 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. SPECIFIC EXAMPLE 23 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 4 RPMI + 102 fetal calf serum in 12 well plates at 2 x 10 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 15, 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- ransformed mammary cell line, described by Gaffπey, 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 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). 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 15 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 Evejos 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 0 +/- 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 24 I munoperoxidase 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 22 paraformaldehyde in PBS for 10 minutes at 4 C. Subsequent steps were done at room temperature. Cells were permeableized with 0.12 Triton X-100 in PBS for 10 minutes and blocked with 12 BSA in PBS for 30 minutes and then incubated for 60 minutes with a 1:100 dilution of monoclonal antibody 3C6 in PBS + 12 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 2.52 non-fat dry milk in PBS for 60 minutes and developed for 10 minutes with AEC solution. Slides were mounted with aqua ount and photographed. 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 902 of the normal human mammary cells. In contrast, as shown in Figure 14B, only 10-152 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 102 weekly staining cells. Non-mammary cells lines were negative. SPECIFIC EXAMPLE 25 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 26 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 4 C overnight. Coated plates were then blocked to reduce non-specific binding by incubating the coated wells with 300 ul of PBS + 12 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 + 0.12 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 + 12 BSA. The unbound antibody was removed from the plate by again flooding the plate with PBS + 0.12 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 + 12 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„0, 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. 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 + 12 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. ANNEX H3 International Application No: PCT/ MICROORGANISMS OM« J inm to r aMfWw «ιtn ma ■ucrMfM'Uf ntarrM » an M — — ._— ** βw I loatmncATtOH or » funfit mum n imiuwn an ait Ml»n> E)' (1 of 3 sheets) American Type Culture Collection » ♦ 12301 Par la n Drive Rockville, Maryland 20852 United States of America 24 Mav 1 ( ?88 (24.05.88) HB 9722 t. AOømOMAk INDICATIONS ' Ikm MM H I I •). TMa iwlarw— «ιι M CWIIHII •« a i fvta aiBcn aMβ* e. DUICMATIO rrATta pea VMKM INDICATIONS AM HAOI >ιι<tMi D. ma AΛAn ramtSMwβ o* INDICATIONS • mm MM H MI I TM i ir«M« -mm mw iH M a— WIWM M tM lianiaiiial turmu M • (laaci+i tM nwil W M I I. Q Thii IMI w IWOTM mnti tM imatnatiaMi mwnn ιm M (ta M CMCM lAuUMfUM OfflMrt Q TM MM «i IHWI (tram tM Militant) kr tM limiimiiim IMH >• tana K mOIM IJUun 1H1) " ANNEX H3 Intamational Application No : PCT/ MICROORGANISMS OWMH *Mβι in canMcβan wtt t mtcrM —ai m rmt-r- t * *, -^^ . al oiβ Mni iMmnuTwi or DEPOSI « f-ftr-w iiaaaia an nanu ή M an an 'EJ* (2 of 3 sheets) American Tvpe Culture Collection ΛMIM m* wiiwi IIIMIMMII (H 12301 Parklnwn D ive Rockville, Maryland 20852 United States of America 24 May 1088 (24.05.88) HB 9723 B. ADDITIONAL INDICATIONS < t m Mm ri M< aaaiiratiil. That lalaiWMid » em C. DCSISNATtB STATVS POK WHICH IND4CATIONS AM NAO! • (H tM iMit ina an I tor al *aa*n ■. SVAftΛTI πiMOMM O* IND CATWNS • OMM MM rt Ml I TM i BwfMu m - (lueiti tM pmnl MOM «t tM MWauiu I. π Thl * aMβl ma mcanr-m ami tM mtamatiaMI Matacman mn MM (U M tlaOM -η 1 — (AwtMrtaM laWaai) Q TM MM al IM»« (Item tM MMcant) fcr tM IMMMMMI Im g <• lAunwuM Ofkwl Farm PCT/HO/M* Maimarr 1W1) ANNEX M3 Intamational Application No: PCT/ MICROORGANISMS . at tM MOKMK I A. IDCNTIPICATION OΨ OflPOSTT » M naM.ai . M*g]» (3 0 f 3 sheets) American Type Culture Collection AMfβM al lniiMry maaiM in (M 12301 Parklawn Drive Rockville, Maryland 20852 United States of America 22 May 1989 (22.05.89) HB 10152 B. ADOmONAl INDICATIONS ' (lamw M a). TMa laliiiaMin M < C. DBSWNATtD BTATtS PON WMCM INDICATIONS AM MAOI • (H βta I B. DS ANATI PU∞tSMmβ OP INDICATIONS • flam aMM M • TM BUTMM Mar • (taaill) IM pmnl i Q TMa aMai ma i MM (ta M c ca kr IM (Aι D TM < ■a* dram M Mainaatl kr ma IIWIMIIMI SMTMM >• (AvmaruM OStea Farm P T/KO IM (JM H IMI)

Claims: What is Claimed is: 1. An antibody comprising a monoclonal antibody which binds to human mammary cell growth inhibitor. 2. The antibody of Claim 1, wherein the antibody is chimearic. 3. The antibody of Claim 1, wherein the antibody binds an active site of the inhibitor. 4. The antibody of Claim 1, wherein the antibody binds an inactive site of the Inhibitor. 5. The antibody of Claim 1, wherein the antibody is an IgG antibody. 6. The antibody of Claim 1, wherein the antibody is an IgM antibody. 7. The antibody of Claim 1, wherein the monoclonal antibody is a product of a hybridoma having the characteristics of hybridoma cell line ATCC Accession No. HB9722. 8. The antibody of Claim 1, wherein the monoclonal antibody is a product of a hybridoma having the characteristics of hybridoma cell line ATCC Accession No. HB9723. 9. The antibody of claim 1, wherein the monoclonal antibody is a product of a hybridoma having the characteristics of hybridoma cell line ATCC Accession No. HB10152. 10. A monoclonal antibody which binds to a cell growth inhibitor having the following characteristics: (a) inhibitory effect on the growth of NHMC, MCF-7, BT-20 mammary cells; (b) calcium-dependence of inhibitory activity; (c) dose-dependence of inhibitory activity; (d) reversibility of inhibitory activity upon a decrease of inhibitor concentration; (e) substantial decrease of Inhibitory activity upon heating to 100°C; and (f) substantial decrease of inhibitory activity upon treatment with trypsin. 11. The antibody of Claim 10, wherein the antibody is an IgM antibody. 12. A hybridoma produced by the fusion of a transformed cell and a spleen cell which produces monoclonal antibody to human mammary cell growth inhibitor. 13. The hybridoma of Claim 12, wherein the hybridoma has the characteristics of hybridoma cell line ATCC Accession No. HB9722. 14. The hybridoma of Claim 12, wherein the hybridoma has the characteristics of hybridoma cell line ATCC Accession No. HB9723. 15. The hybridoma of Claim 12, wherein the hybridoma has the characteristics of hybridoma cell line ATCC Accession No. HB10152. 16. A screening procedure for breast cancer in a human subject comprising the steps of: (a) providing a biological sample from the subject; (b) providing an antibody specific for human mammary cell growth inhibitor; (c) providing an assay for the binding of the antibody to the inhibitor; (d) bringing the antibody into contact with the biological sample for a time sufficient and in an amount sufficient for the antibody to bind human mammary cell growth inhibitor if present in the sample; and (e) assaying for the binding of the antibody to the inhibitor. 17. The procedure of Claim 16, wherein the antibody is a monoclonal antibody. 18. The precedure of Claim 16, wherein the antibody Is a polyclonal antibody. 19. The procedure of Claim 16, wherein the biological sample is breast tissue. 20." The procedure of Claim 16, wherein the biological sample is serum. --- 21. A method of monitoring the level of human mammary cell growth inhibitor in a human subject comprising the steps of: (a) providing a biological sample of body fluid or tissue of the subject; (b) providing an antibody specific for human mammary cell growth inhibitor; (c) providing an assay for the binding of the antibody to the inhibitor; (d) bringing the antibody into contact with the biological sample for a time sufficient and in an amount sufficient for the antibody to bind to human mammary cell growth inhibitor present in the biological sample; (e) assaying for the binding of the antibody to the inhibitor; and (f) measuring the amount of inhibitor present in the sample relative to a standard level. 22. A method of preventing or treating human breast cancer comprising the steps of: (a) supplying the breast tissue with a therapeutically effective amount of human mammary cell growth inhibitor; and (b) monitoring the level of mammary cell growth inhibition. i Γ rtt «ι«ι 23. The method of Claim 22, further comprising the step of: (c) monitoring the level of inhibitor present in vivo, the step of monitoring in vivo inhibitor levels further comprising the steps of: (i) providing a sample of body fluid or tissue of the subject; (ii) providing an antibody specific for human mammary cell growth inhibitor; (ill) providing an assay for the binding of the antibody to the inhibitor; (iv) bringing the antibody into contact with the body fluid or tissue for a time sufficient and in an amount sufficient for the antibody to bind to human mammary cell growth Inhibitor present in the sample; (v) assaying for the binding of the antibody to the inhibitor; and (vi) measuring the amount of inhibitor present in the sample. 24. The method of Claim 22, wherein the step of supplying is repeated to maintain therapeutically effective levels of Inhibitor n vivo. 25. The method of Claim 22, wherein the inhibitor is supplied to the breast tissue through the circulatory system. 26. The method of Claim 22, wherein the inhibitor is injected into breast tissue. 27. The method of Claim 22, wherein the antibody is monoclonal antibody. 28. A kit to assay for the presence of human mammary cell growth inhibitor comprising: an antibody which preferentially binds to human mammary cell growth inhibitor in a biological fluid; and reagent means for detecting the binding of the antibody to the inhibitor, wherein the antibody and reagent means are present in amounts sufficient to perform the assay. 29. The kit of Claim 28, wherein the antibody is an IgG. antibody. 30. The kit of Claim 28, wherein the antibody is an IgM antibody. 31. The kit of Claim 28, wherein the assay is an enzyme-linked immunoassay. 32. The kit of Claim 28, wherein the assay is a radioimmunoassay. 33. The kit of Claim 28, wherein the assay is a hemagglutination assay. 34. The kit of Claim 28, wherein the assay is a cell inhibition assay. 35. The kit of Claim 28, wherein the antibody is a monoclonal antibody. 36. The kit of Claim 28, wherein the antibody is a polyclonal. 37. A method for purifying human mammary cell growth inhibitor in solution comprising the steps of: (a) providing purified monoclonal antibody to the inhibitor; (b) contacting the solution with antibody in an amount sufficient and for a time sufficient to bind the antibody to the inhibitor; and (c) recovering the inhibitor from the antibody-inhibitor complex.

Inventors: CODY ROBERT L, ERVIN PAUL R JR, KAMINSKI MARK S, WICHA MAX S, CODY, ROBERT, L., ERVIN, PAUL, R., JR., KAMINSKI, MARK, S., WICHA, MAX, S.
Applicants: UNIV MICHIGAN, THE REGENTS OF THE UNIVERSITY OF MICHIGAN
Application year: 1989
Main classification: C07K15
IPC codes: A61K38, A61K39, A61P35, C07K14, C07K16, C12N15, C12P21, C12R1, G01N33