JPH05506351A - Antibodies to human breast cell growth inhibitors and methods of production and use thereof - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Antibodies to human breast cell growth inhibitors and methods of production and use thereof 8 related petitions This application is filed by Ervin on July 61, 1986. Part of No. 891,135 entitled “Difactin” Continuing application by Irvine et al. published May 20, 1988 @Human Mammary Ce1l Growth Inhibitorand Met hods of Production and Use @(Human breast cell growth No. 196,657 entitled "Inhibitors and Methods of Their Preparation and Uses" Ant filed May 27, 1988 by Ardine et al., which is a continuation-in-part application. i bod i e s.

to Human Mammary Ce1l Growth Inhibit or and Metbo-ds of Production and Us e’ (Antibodies against human breast cell growth inhibitors and their production methods) This is a continuation-in-part application no.

field of invention FIELD OF THE INVENTION The present invention relates generally to cell growth regulation, and more particularly to human breast cell growth. Antibodies specific for inhibitors and methods for their production and use.

The following hyperdomas are American Type CulturaColl ection (12301Parklawn Drive, Rockvi-1 1e, Maryland 20825).

Hypuridoma Accession Number Deposit Date DF1/3C6HB9722 198 May 24, 1988 DF1/1 o4 Hs9723 May 24, 1988 DF1 /6E8 HBi01!52 May 22, 1989 Background of the invention Human breast tissue undergoes an explosion of proliferative activity at the onset of menarche and during each menstrual cycle. happen. Studies on the effects of estroden on breast tissue and tumors have shown that Strobin has been shown to be the primary growth initiation factor for breast tissue ( For example, Martin, L.

gstrogen in the gnvlronm8nt, 105-150 Page and 5onnenschein, C., et al., J., Mat., Canc. rnst,.

64: 211-214, see 198C1). Growth of estradiol sensitivity The existence of factors has also been established (e.g., 5oto, A, M, et al., Cancer Ras, 46: 2271-2275, 1.986 M). Furthermore, the book Breast cell growth factors that are not hormones have also been reported (e.g., Dicks on, R, B, et al., 5science, 232: 1540-1543, 1 986). It has been shown to have a stimulatory effect on breast tissue growth. Specific growth factors include platelet-derived growth factor and insulin-like growth factor (IGF). -1) and transforming growth factor (TGF) αβ can suppress breast tissue growth. (e.g., Robert, A. B., et al., PNAS US , 82: 119-125.1985).

Breast cell growth is of more than academic interest.

If unchecked, neoplastic growth in breast tissue can lead to malignant tumors that continue to grow uncontrollably. This has progressed to become the cause of death for as many as 1,000 women each year. Cell increase specifically in breast tissue If there is a growth inhibitory factor that can inhibit growth, it would be possible to prevent such malignant tumors and breast cancer. It is thought to be a powerful tool for treatment. Breast cell growth inhibitors such as - Parent Application No. 196,657 filed May 20, 1988 and No. 891.13i5. The descriptions of both applications are incorporated herein by reference. Introduce it into the book.

Immunological purification and concentrated N of breast cell growth inhibitors may be useful for prevention and protection against breast cancer. This greatly facilitates the use of this inhibitor as a therapeutic agent. breast Immunoassay for the detection of the presence and concentration of inhibitors in tissues and serum It also helps in the diagnosis of breast cancer, screening of subjects at high risk of breast cancer, and treatment. Monitoring a patient's inhibitor concentration is sometimes highly advantageous. body fluids or The EIJSA kit for the detection of breast growth inhibitors in tissue samples is also Convenient for diagnosis and screening.

Summary of the invention The present invention provides antibodies specific for human breast cell growth inhibitors and methods for producing the same. and regarding use. does not produce monoclonal antibodies specific to this inhibitor. A hybridoma cell line was created using cell fusion and selection techniques. child Polyclonal antibodies against inhibitors of and the recovery of antibodies from its serum. In accordance with the principles of the invention, Antibodies generated against the inhibitors can also be obtained by purifying and treating the inhibitors of the invention. and concentration in human breast and non-breast cells and human serum. used to test for the presence of inhibitors.

The human breast cell growth inhibitor against which the antibodies of the invention are made is thermolabile. Consists of protein. NHMC! IiJ! From media to ion exchange chromatography Therefore, the active peak of the isolated inhibitor is the MCF of the molecular sieve column fraction. 7 growth assay, the molecular weight is about 40,000 to about 45,000 f. Approximately 50. It was observed in the range of Q11 to about 60,000 Daltons. but However, inhibitors purified by antibody affinity chromatography When measured by 5DS-PAGE, the activity is about 45,000 to about 50,000 00 Dalton range and about 60,000 to about 70.00 Daltons. D CI O Dalton range Shown in the box.

Increased production of the functional inhibitors of the invention results from low calcium levels in inhibitor-secreting mammary cells. induced by growth in sium concentrations. Inhibitor activity also It is time-dependent, dose-dependent, and reversible. The inhibitor is present in the serum of normal women. However, it is absent or at low concentrations in the serum of normal men and breast cancer patients. There are only degrees. Therefore, inhibitors may be used to prevent breast cell expansion. Administered alone or in combination with other drugs as a therapeutic agent for the treatment of cancer or breast cancer be able to.

Based on the relationship between breast cell growth inhibitor concentration and breast cancer, The immunoassay of the present invention detects the presence or absence and concentration of inhibitors in breast cancer or breast cancer. diagnosis of or precancerous cell growth conditions, screening for predisposition to breast cancer, and treatment. It can be used to monitor a patient's inhibitor concentration during treatment.

The invention and its advantages may be further understood by reference to the drawings and specific examples, which describe preferred embodiments of the invention. I believe that you will be able to understand it better if you read it with reference to it.

Brief description of the drawing Figure 1 shows the measurements in MCF-7 cultures grown in low and high calcium. It is a bar graph of the inhibitory activity of the NHMCmM medium, which shows the calcium dependence of the inhibitory activity. It is an example of gender.

Figure 2 shows the dose response of NHMC-conditioned medium in inhibiting the growth of transformed human breast cells. It is a curve.

Figure 3 shows NHMCi! Transformed human mammary cells of molecular sieve column fraction of Il culture medium. This is a graph of inhibitory activity against cells with a molecular weight of about 40,000 to about 45,000. range and the peak of inhibitory activity of the fractions ranging from about 50,000 to about 60,00 CJ. Here are some examples.

Figure 4 shows separation on a hydrophobic interaction column and a 10-column polyacrylamide gel. The results of silver staining of each sample subjected to electrophoresis are shown.

Figure 5 shows human breast cell growth inhibitors selected for monoclonal antibody affinity. Doses illustrating enhanced growth inhibition of transformed human breast cells with increasing doses of This is a reaction curve.

Figure 6 shows 353-methionine labeled human milk produced by normal human breast cells. Fluorogram of tuft cell growth inhibitor.

Figure 7 shows the use of monoclonal antibodies against human breast cell growth inhibitors. Western Probe for Detecting Inhibitors in Human Cell Lysates and Serum It is.

FIG. 8 shows that using a monoclonal antibody against human breast cell growth inhibitor, Western for detecting inhibitors in the serum of normal subjects and breast cancer patients It's a plot.

Figure 9 shows the inhibition of breast cell growth inhibitor activity by purified monoclonal antibodies. Dose-response curve of inhibition.

FIG. 10 is a graph of the effect of conditioned medium on the growth of MCF-7.

Figure 11 shows the inhibitory activity and inhibition of affinity-purified human breast growth inhibitors. Figure 2 is a bar graph illustrating inhibition of activity by 3C6 antibody.

Figure 12 shows silver staining of affinity purified inhibitors and 358-methio Fluorogram of a nin-labeled affinity-selected human breast growth inhibitor. .

Figure 13 illustrates the heat and trypsin sensitivity of affinity purified inhibitors. This is a graph showing.

Figure 14 shows normal human breast cells and immunoperoxidase staining using 3C6 antibody. and MCF-7 cells.

Description of preferred embodiments of the invention The present invention generally relates to antibodies specific for human breast cell growth inhibitors; relating to its manufacturing method and use. What does the term “human breast cell growth inhibitor” mean? , the characteristics of the inhibitor of the present invention, the active form of which can suppress the growth of human breast cells. means an inhibitor that has The word “human” refers to the origin of inhibitors in humans. It is not intended that the limiting effect or the suppressive effect necessarily be limited to humans only. It should be understood that this is not the case.

Hybridoma cells that produce monoclonal antibodies specific for this inhibitor The system was created by cell fusion and selection techniques. Poly for inhibitors Clonal antibodies are produced by in vivo immunization of an animal and the production of antibodies from its serum. Created by the collection. Both polyclonal and monoclonal antibodies Blocks the inhibitory activity of the inhibitor and induces normal human breast cells to secrete the inhibitor. completely eliminates the inhibitory activity from the conditioned medium. High concentration, purified composition of inhibitor were also produced using antibodies of the invention. The term "high concentration" refers to the inhibitor refers to the concentration of an inhibitor that exceeds the normal physiological level of . In accordance with the principles of the invention Antibodies against the inhibitor can be used to detect the inhibitor in human cells and serum. was used to assay for the presence or absence of

Breast cell growth inhibitors, for which antibodies have been created, are actively proliferating. is secreted by human breast cells and is the parent application of the above-mentioned May 20, 1988 application. No. 196.657 and Original H. No. 891,135. Functionality Breast cell growth inhibitors are used to treat "normal" or non-transformed human breast cells in culture. secreted by breast cancer cells and present in the serum of “normal” women, i.e. women without breast cancer. Exists. However, the levels produced by normal human breast cells under standard culture conditions The levels present in human serum and the levels of inhibitors originally used in standard technology It was unsuitable for isolation. However, normal human breast cells are Increased production of functional inhibitors was achieved by placing the inhibitors in the “Low Cal The term ``calcium'' refers to any environment that has a calcium concentration below physiological levels. do.

More specifically, for at least one in vitro low calcium environment, , such an environment contains less than about [1,1mM calcium, preferably about Q,Q4 Includes calcium concentrations below mM calcium.

Breast cell growth inhibitors obtained from normal human breast cells (NHMC) are exchange chromatography, hydrophobic interaction chromatography, and molecular sieves It was isolated and purified to approximately 90% purity by chromatography. Approximately 5% more Purification to purity is achieved by ion exchange chromatography, followed by monoclonal antibody Achieved by affinity chromatography.

Human breast cell growth inhibitor was found to consist of a pentathermal protein, DEA NE prepared by E ion exchange chromatography (inhibitor isolated from MC) Bitter is approximately 40% as determined by MCF-7 growth assay of molecular sieve column fractionation. ,000 to about 45,000 gluton MW and about 50.0 [1C1-6G, It shows a peak of inhibitory activity in the range of 000 gluton Mw. Molecular weight herein are all displayed in Daltons.

However, monoclonal antibody affinity chromatography Inhibitors purified from NHMCW4 culture medium were purified using SDS polyacrylamide. Inhibitory activity peak 't measured by del electrophoresis, 45,000 to about 50. OOOM The W(7) range is approximately 60,000 to 70,000 MW. More details Alternatively, DEAE 5epharyL ion exchange chromatography can be used to NKMC-conditioned medium by clonal antibody affinity chromatography Silver staining of the inhibitor purified from A certain band was observed. Inhibitors purified in this way are referred to as ``affinity''. It is called "tea refining inhibitor". Direct monoclonal antibody affinity cloning NHMC-conditioned medium by matography (i.e. without any ion exchange) Inhibitors isolated and purified from The fluorogram of It showed a constant band and a small inconsistent band at about 63,000 MW. . That is, the breast cell growth inhibitor of the present invention also inhibits breast tissue growth. From other cell growth inhibitors, such as TGF-β, which have an inhibitory effect on identified by their molecular weight, thermolability and cell specificity.

Regulated by normal human breast cells (NHMCs) to inhibit breast cell growth inhibitors The inhibitory activity of the crude and purified prevolamination of the media contained also It was found to be different. The term "cell-specific" activity generally refers to It is a powerful inhibitor for the cell type for which it exhibits specificity, even if the target is active against other cell types. It means to exert an effect. Breast cell growth inhibitors of the invention are NHM C and transformed breast cell lines MCF-7, BT-20, ZR-75-1 .

@VejO8 and MDA-MB-231 growth is suppressed, but 11 species Little or no growth inhibition was observed with this inhibitor in non-mammary cell types. There wasn't. From the study of inhibitory activity at various calcium concentrations, the inhibitory activity also , is clearly calcium dependent and requires elevated calcium levels has been done.

Dose-response studies also show that inhibition of breast cell growth by inhibitors is dose-responsive. It is shown. Another study showed that inhibition of breast cell growth by inhibitors It has been shown that the inhibitor is reversible upon removal.

The regulatory effects of the breast cell growth inhibitors of the present invention are mediated by cell death of treated cells. It's not something you can do. Cells treated with inhibitors appear healthy to the naked eye However, the nuclear size decreases and the cells exhibit a stable dormant morphology typical of differentiated cells. It seems to show characteristics. However, the breast cell growth inhibitors of the present invention The exact mechanism of action is still unknown.

Polyclonal clones made for breast cell growth inhibitors in accordance with the principles of the present invention. monoclonal and monoclonal antibodies for further inhibitor characterization and purification. used to do. Monochrome inhibitors of at least about 95% purity Obtained using a lonal affinity column. Polik against inhibitors Both local and monoclonal antibodies are NHMC-modulation abolished the inhibitory activity or as evidenced by the depletion test. It was shown to completely remove the inhibitor from the culture medium. Created for inhibitors It was also revealed that the disinhibition by the antibody was dependent on the antibody dose. clearly BT-20 and MC'F-7 transformed human milk producing non-functional inhibitors The tuft cell line is lysed with animal antiserum against the inhibitor in the presence of complement and then incubated. Non-breast cell lines that do not produce the anti-inhibitor are unaffected and do not produce the inhibitor on the cell surface. suggests the presence of an inhibitor.

This finding has also been confirmed by indirect immunofluorescence. This data is suggests that cellular receptor sites for inhibitors exist on the cell surface .

Immunoassays utilizing antibodies of the invention also detect the presence of breast cell growth inhibitors. used for detection and quantification of The inhibitor is normal human breast cells (N) ( MC), as a non-functional form in certain transformed breast cell lines, and in serum from normal women. However, other transformed breast cell lines, non-breast cell lines, male serum and None or only low levels were detected in the serum of women with breast cancer.

Low levels of functional human breast cell growth inhibitors in the bloodstream or breast tissue or its absence is indicative of breast cancer or a predisposition to breast cancer. In other words, the above output Breast cell growth, as described in Application Nos. 196.657 and 891,135. Inhibitors are used to reduce the risk of breast cancer or to inhibit benign and malignant breast cells. For the treatment of proliferative conditions, administering to a patient to inhibit inhibitors in the blood or breast tissue. can increase the level of. Presence of inhibitors in human tissues and serum The immunoassay of the present invention, which detects breast cancer, can be used as a diagnostic method for breast cancer. For screening high population populations and for monitoring patient inhibitor levels during procedures. It is useful for Detecting binding of antibodies to breast cell growth inhibitors The EL and ISA kits of the present invention, which use inhibitors and reagents for facilitate diagnosis and examination.

specific example In Specific Examples 1-26, unless otherwise indicated, the following general experimental protocol is used: I used call. All percentages are by volume unless otherwise indicated.

Cell culture and media preparation: Normal human breast cells from reduction mammoplasty were obtained from Michigan Cancer F. Obtained from Foundation's Herb 5 Oule. cells (low calci 40-60 nM calcium chloride, 5% Sigma Chalex-treated Mass serum, 20n, 9/mCoLlaborative EGF, 98n& /+114! Cholera toxin, 10 μi/Ml insulin and 1 μg/IL In Gibco I) MEM/F12 medium containing t-hydrococcinin maintained. Cells were incubated at 37°C in a humidified CO2 incubator. did. If NHMC-conditioned medium is required, cells are incubated at low temperature for at least 96 hours. The cells were adapted to Le 7um medium.

McF-7m cells were prepared with 10% fetal bovine serum (Fe2) and 1μEl/Ill. 37° in a humidified co□ incubator in MEM supplemented with insulin. It was maintained at C.

Growth and inhibition assays: After removing trypsin from the stock culture medium, MCF-7 cells were treated with 10 volume% FC3 and Prepare 12 wells in MEM supplemented with 1 μg/1jLl insulin as desired. Using a tar culture plate, 1-2 x 10' cells/m/, 1 rttl/ It was triple plated with Uxul and allowed to adhere overnight at 37°C.

Fresh medium was added and the cultures were treated by adding the appropriate samples. 37 cells The cells were allowed to grow for 7 days at ℃ and then counted.

Cell counting: Cells were washed with room temperature phosphate-buffered saline (PBS) and then incubated with SigmaC The nuclei were released by lysis at 37°C with atrimide surfactant solution, and the core Counted with a router counter. The cell number deviates by more than 5% t from the average cell number. The values were averaged after excluding values showing inappropriateness.

Calculation (using all cell count average values): Inhibition rate = [number of cells in ic treated sample/number of cells in untreated sample] 3X100 Jin recovery rate - [1-(!ll) Jin suppression rate of conditioned medium removal treatment sample/conditioned medium treatment sample Qi suppression rate of sample)〕X1　Clf] Chi deinhibition rate-[1-(% inhibition of sample/depression inhibition rate of conditioned medium)]X100 Specific example 1 NHMC-conditioned medium-treated NHMC growth inhibition and cell morphology changes The NHMC-conditioned medium used for treatment was normal human breast cells (SUCC) below t- were obtained by growing for 5 days in each medium shown in Table 1. Then cultivate Remove the base, centrifuge at 8.00 rpm for 15 minutes, lyophilize, and reduce to the initial concentration. ↓The sea urchin was rebuilt underwater. Sterile filter the reconstituted medium; It was used to treat cultured cells. Treated cells were supplemented with 11:l fetal bovine serum (Fe2). Reduced breasts grown for 2 weeks in DMlffl/F12. Normal human mammary cells (NHMC) obtained from the tumor. - Birds from the next culture After removing psin, t4×105 cells/’r were plated with 257 rascos. After allowing the mixture to adhere overnight, supplement with a 10-width conditioned medium of the type listed in Table 1. Grown in rMDM specific medium. Cells are then no longer observed to grow. The cells are then trypsinized to remove them from the plate and counted in a hemocytometer. I counted.

As is evident from the data in Table 1, NHMC-conditioned media with low calcium concentrations High calcium concentration when added to NHMC in IMDM growth medium Unlike other cases, it exhibits non-toxic growth-inhibitory effects on normal human breast cells and is a low-cal NHMC cells grown at concentrations of Cium have higher amounts of functional breast cell growth inhibitors. It was suggested that it produced tar. Table 1 also shows the low calcium WC-conditioned medium The results show that the cells inhibited by this method also took on morphologically distinct characteristics.

F12 medium with small nuclei reduction ao, 04mM calcium b Physiological concentration of calcium Specific example 2 Calcium dependence of inhibitory activity To test whether the inhibitory activity of human breast cell growth inhibitors is calcium dependent. In order to 0, 04mM calcium (low) and 24mM supplemented column fraction of culture medium The cells were grown in DMEM/Fl2 medium containing calcium (high) αMEN. column The fraction is G-1 [1Q 5e loaded with 2 dt of ultrafiltration concentrated (10X) conditioned medium. Obtained from PBS elution of a phadax column. Fractionation, low and high calcium It was used as a 10 volume supplement for MCF-7 cells in growth medium. MCF '-7 cells were grown for 4 days in supplemented medium and then counted in a Coulter counter. Inhibitory activity was measured by the decrease in number of counted cells.

As shown in Figure 1, the inhibitory activity of the human breast cell growth inhibitor Tar higher in media containing lucium, indicating that this activity is calcium dependent. It shows.

Specific example 3 Dose response of MCF-7 cells treated with NHMC-pi culture medium MCF-7 cells were plated at 104 cells/go in MEM supplemented with 10% FC8. I clicked. After adding the volume of conditioned medium shown in Figure 2, cells were allowed to grow for 7 days. I let it grow. The conditioned medium used was previously conditioned for 96 hours by growth of NHMC. Low calcium DMEM/F12.

As is clear from the dose-response curve in Figure 2, MCF-7 is used to supplement the medium. Increasing the volume of conditioned medium increases the level of inhibitory activity; It has been shown to be dose dependent.

Specific example B Reversibility of the inhibitory activity of human breast cell growth inhibitors using 10 T25 flasks. MCF-7M cells t 1 x 10’m in 10% FC3 supplemented MEM 5m Cells/ml were teflated. 6 flasks contain 1 volume of low calcium N HMC-conditioned medium was also supplemented and the remaining four were used as untreated controls. cells add Grow for 5 days at 37°C in a humid CO□ incubator. After 5 days, untreated control The cell membranes of the cells in two flasks and two treated flasks were lysed with Csrtimida. , count with a free supranuclear Coulter counter and average the cell number as described above. The chi suppression rate was increased. Similarly, after 5 days, 10 μm of FC8 was added to the remaining flask medium. Add fresh MEM medium and add N)(MC-conditioned medium to the remaining two treated cultures. The two pre-treated cultures and the remaining two untreated cultures were not treated. There wasn't. Cells were grown for an additional 5 days, lysed with nails, and supranuclear coulter The cells were counted using a counter, the cell numbers were averaged, and the inhibition rate and recovery rate were determined.

As is clear from the data in Tables 2 and 3, the treatment was carried out on day 0 and from day 5 onwards. For cells that were not treated until day 10, a 40-fold recovery from inhibition was observed.

Table 2 Regret suppression rate 1chi CM” 2a person “CM = Low Calcium NHMC-conditioned medium Table 3 Reversibility of inhibition 1% CM 1chi CM 30% 1 CM None 18 regrets Heat-denatured human breast cell growth inhibitor/inhibitor and avian MCP-78 cells were added in 10 volumes. The cells were plated at 104 cells/go in MKM supplemented with FC'S. cells for 24 hours After that, add 2.5μg7rnt+)pusin-containing solution 5% in PBS and pre-incubate at 37°C. 10% low calcium NThC treated for 1 hour or heated to 70°C for 1 hour - treated with conditioned medium. However, the conditioned medium used for control supplementation was initially Treated with inhibitor and then incubated with trypsin. cells for 7 days The cells were allowed to grow and then counted using a Coulter counter to determine the rate of inhibition of detachment.

Human breast cell growth inhibitor activity as exemplified by the data in Table 4 below. is sensitive to heat and trypsin, and this inhibitor has proteinaceous properties. It shows.

Table 4 Inactivation of inhibitors Specificity of human breast cell growth inhibitor Specificity of human breast cell growth inhibitor and whether its activity is due to just that of a universal cytotoxic metabolic by-product. To demonstrate that the inhibitor's inhibitory activity was inhibited in mammary and non-mammary cells, Test both. The various human cell lines or types listed in Table 5 below are Plate at 104 cells/WLt in star plates and unadjusted low-cal with calcium (0.04mM) medium or low calcium medium adjusted in advance with NHMC. Please handle it. After the cells were grown for one week, they were counted using a Coulter counter and then quenched. Achieved the control rate.

The specificity of the inhibitor for human breast cells is demonstrated by the data in Table 5. As shown in the figure, growth inhibition of the mammary paper layer and lack or reduction of inhibition in the non-mammary paper layer. Therefore, it is exemplified. Breast cell types of two harvested animals, MDA-M B-231 and D The lack of inhibition in σ4475 makes these transformed cells resistant to the inhibitor. their response to or inhibitor recognition on their surface, e.g. Suppressive effect due to defects in the receptor site of the inhibitor or these cells by producing an appropriate amount of stimulator to eliminate This is thought to be due to the fact that it has an obstructive sound.

Table 5 Specificity of suppressive effect of HHMC conditioned medium Breast NHMC50 Breast BT-2050 Breast MCF-778 Breast HBL-10029 Milk N ZR-75-126 Breast MDA-MB-2! +1 < 5 Breast DU-4475 < 5 11,%H,m 5w948<! 5 Fibrous meat II HT1080 <5 Epithelial cancer HeLa <5 Melanoma wM164〈5 Lymphoma U937 <5 Myeloid leukemia K562 <5 Cell sphere HF (5 Promyelocytic leukemia J774-2<5T-cell Molt4<5 Purification and activity of human breast cell growth inhibitors Low calcium NHMC-conditioned media Human breast cell growth inhibitors from natural sources were purified using standard protein purification methods detailed below. It was purified using In other words, it shows the highest activity as measured by the MCF-7 inhibition assay. The NHMC-conditioned medium was first added to Amiconfil with a molecular weight cutoff of 10,000. Concentrated by ultrafiltration through a filter and then combined with molecular weight standards in PBS buffer. The mixture was fractionated by passing through an 8ephadex G-1[IQ column. This separation Various molecular weight fractions t-0, 22 μm from Ge1man Acrodisc Treatment of MCF-7 cells sterilized and then grown in culture in 10 volumes used for. After growing treated MCF-7 cells for 7 days, Coulter counter The inhibition rate was determined by counting using a micrometer. The fraction that showed inhibitory activity had an apparent molecular weight of approx. 4a, aa aO fraction.

NHMC-conditioned medium was passed through a DEAE ion exchange column at low salt concentration. A higher degree of purification was achieved. Then a step gradient of NaCl concentration or Na Protein was eluted from this column using a C1!1 linear ascending gradient. this The inhibitory activity of the fractions from the separation was determined by dialysis of the fractions against PBS and filtration of the fractions. by the use of the solution in the treatment of MCF-7 cells as described above in this example. It was measured. Considering the inhibitory activity, the protein activity is 3.5 M Na in a step gradient. In the C4 minute direct, the linear gradient reached a maximum of about 0.3 M in the fraction of NaCt. activity.

The fraction from the DEAg column containing inhibitory activity was further 5uperose i 2F Separated by molecular sieve chromatography on a PLC power ram and arranged in order of size, The activity of the fractions was measured. In this assay, inhibition occurs in fractions after a 24-hour labeling period. Measurement of 3H-thymidine uptake by MCF-7 cells after 48 hours of exposure to did. As shown in Figure 3, this separation yields two main inhibitory activity peaks, viz. The apparent molecular weight is about 40.000-45. [10[1 peak and further apparent A peak with a molecular weight of approximately 50,000 to 60,000 was observed. phenyl 5ep Further separation on a harO8e hydrophobic interaction column gave the inhibitors a silver stain. It has been purified to a level that cannot be detected by color, but it is clear from the data in Table 6 below. As such, peak suppression activity was present in the fraction eluted with 1M NaC2.

However, as shown in lane D of FIG. 0'l Silver staining of samples separated by electrophoresis on SDS polyacrylamide By color, no inhibitor protein was detected in this fraction.

Table 6 Hydrophobic interaction chromatography Chi inhibition rate of fractions separated by (Bl 2 M NaCt elution (C11,5M NaC6 elution fDl I M NaC6 elution (El O, 5M NaC6 elution (CM) Low calcium NHMC-conditioned medium specific example 8 Production of antibodies against human breast cell growth inhibitors and elimination of inhibitor activity loss The DEAE fraction containing activity was prepared as described above using a polyacrylamide gel. Loaded on top. Molecular weight range encompassing inhibition peaks from 40,000 to 45,000 35, DQ O~50. D01: lK equivalent -r rrk area (DflfLfc For purity reasons, we excised this portion of Del and extracted the protein from this complete Del 7 fragment. was electroeluted. Elute protein 20-50μI/1llfr - then 50 volumes 70 India Complete Adjuvant mixed with New Sealant White Rabbit and Ba1 b/C mice were injected. 0.2 d of this solution in the peritoneal cavity of a rabbit? injection were immunized.

Two weeks later, the animals were treated with 50 volumes of 70 indo-incomplete azojuvant at the same protein concentration. A booster was administered by mixing with One week after the booster, the serum of these animals was The recognition of electroconductive solutions was tested using the ELrSA assay. booster 2 weeks Afterwards, a protein solution of 20-50 μg/1116 was injected into the tail vein of the mouse. 3 days Afterwards, the mice were sacrificed and the entire spleen was removed.

Spleen cells extracted from mouse spleen and mouse myeloma Co, San Fran As described in Cisco, Ca1ifornia (j980) fused in the presence of polyethylene glycol using standard immunological techniques. Generated a predoma. Hybridomas from cell fusion were prepared in R containing 15% FC3. Cultured in PMI, to which hyboxanthin and azoserine were added at appropriate concentrations. The unfused parental cells were grown for 2 weeks in 96-well microtiter plates. selected against. The supernatant from each culture was then subjected to the following ELISA. , tested the ability of individual cultures to produce antibodies against the inhibitor.

Dynatsch Laboratories, Inc. (Chantil Ly, Vir-ginia) 96-well Immulon 2 microta Iterplate’l, the same electrophoretic polypeptide used to immunize the animals described above. 5 in PBS of partially purified growth inhibitor obtained from crylamide del slices. Coated with μl//1rLl. -After overnight incubation at 4°C, Wash the plate with 9% NaCl and 0.05% Triton X-100. Washed with cleaning solution. Then add 50 μt of clay tube solution from each hybridoma culture solution to the well. and incubated for 2 hours at room temperature. Clean with Platera wash solution and remove alkaline Yai anti-mouse IgG and IgMvL bodies conjugated to lyphosphatase (5ou thern Biotechnology Associates, Birmi ngham, Alabama) and PBS Middle School, BSA Middle School i:1ooo Nozomi. The cells were incubated for 2 hours at room temperature in a diluted solution. Then wash the plate again. The chromogenic substance p-nitrophenyl phosphate disodium salt (Sigma, St, Louis, Missouri) 10 @g/go solution 100 μt was added. Color was developed for 1 to 20 hours. Next, Dynatsch M R70 Measure the optical density at a wavelength of 410" using a microplate reader. Ta. Screen cultures based on activity at least 4 times that of PacGrande Based on this criterion, all 150 positive cultures were selected.

The cloned population of ELISA positive cells was then expanded. Wash the growth medium After removing azoserine and adjusting the medium, it was tested in a growth inhibition assay. test Cells from the culture that showed no suppression or had blocked suppressive activity were collected by limited dilution. subcloned into single cell clones and then retested for effects on suppressive activity. Ta.

Up to Sadeclonal t5X10' cells that do not show suppression or have abolished suppressive activity. Proliferated and pristane-stimulated C3H/HsN x Ba1b/C(Fl) It was injected intraperitoneally into mice to produce antibodies in ascites fluid. against inhibitors The noclonal antibody was given the generic name DFii.

Three types of monoclonal antibodies prepared according to this example and used in the following tests. and the hybridomas that produce these antibodies are DF1/3C6, DF1/ID4, and and DF1/688. These antibodies and hybridomas University of Michigan Medical Center (Ann Arbor, Michigan) 48109) from Dr. Max Wicha. Also, this Hypuri The doma cell line is American Type Culture Co11ec. tion (i 2501 Parklawn Drive, Rockville e, Maryland 2Q 352) with accession number HB9722 (DF1/3 C6), HB9723 (DF1/1 o4) and HB10152 (DF1/6 B8).

Immune serum from rabbits immunized with a molecular weight range of 35,000 to 50,000 was When conditioned media are analyzed by Western blotting, monospecificity for proteins It was revealed that it contains sexual antibodies. This immune serum is also described in specific case B. The purified inhibitor eluted with 1M NaC4 from the hydrophobic interaction column of It was recognized as shown in Figure 4F.

Rabbit immune serum containing polyclonal antibodies and monoclonal antibodies derived from mice Both local antibodies were prepared using MCF-7 with low calcium NHMC-conditioned medium. It was tested for its ability to block cellular inhibition. MCF-7 cells t1X10' cells/d and partially purified with 2eS rabbit immune serum or ammonium sulfate precipitation. The cells were treated with 5% 306 monoclonal antibody from ascitic fluid. These cells then Also treated with 10 volumes of low calcium NHMC-'tA culture medium for 7 days. I let it grow. Cells were then counted and inhibited with immune serum or 3C, IS antibody. All control rates were measured.

Immune serum containing polyclonal antibodies, as evidenced by the data in Table 7 below. Both the 3C6 monoclonal antibody and the 3C6 monoclonal antibody t-blocked the inhibitory activity. immunity Serum was also eluted with iM NaC2 from the hydrophobic interaction column described in Specific Example 6. The purified inhibitor was also recognized as shown in Figure 4F. Other tests have shown that It is also clear that immune serum blocks the inhibitory response in a dose-dependent manner. action Treatment-related disinhibition rate Conditioned medium 0 Immune serum 42 3C6 monoclonal antibody 100 Dose response of inhibition by purified inhibitors isolated from conditioned media Using 3C6 monoclonal antibody purified from mouse ascites by ammonium sulfate precipitation, CNBr-activated 5epharo by the method described in further detail in Specific Example 14 A monoclonal antibody affinity column on se was created.

A column is used to pass the yI culture medium through it to remove inhibitory activity, and this column The inhibitory activity was eluted from.

Grating MCF-7 cells and adding 3c6 monoclonal affinity low calcium NHMC-conditioned medium purified on After 7 days of growth and treatment with increasing amounts of "selective inhibitor", cells were I counted. Suppression by increasing the volume of purified conditioned medium was suppressed by 10% PBS-treated MCF. -7 cell samples compared to inhibition. As shown in Figure 5, inhibitor-containing inhibition until the solution is at a dose high enough to produce approximately 90 mm of growth inhibition. showed dose dependence.

The protein recognized by the monoclonal antibody is produced by NHMC cells. To ensure that the protein produced by NHMC was Metabolically labeled by the use of s-methionine. Cellular proteins are methinine-free G In ibco DMEM, add 353 to 200μci/mz of methionine, 37 Labeling was carried out for 20 hours at ℃. The labeled medium was then removed and dialyzed against PES.

The labeled v4S medium was then passed over a 30(5) monoclonal affinity column and Elute the retained proteins. Next, the eluted protein was subjected to polyacrylamide gel electrophoresis. It was fractionated by motion. As shown in the fluorogram in Figure 6, the 3C6 monochrome The null antibody affinity column allows 358-methionine-labeled proteins to be detected in normal human milk. Inhibitory activity produced by tuft cells (Nmc)? Holds in the same position as the molecular weight it contains did.

Specific example 10 Presence of human breast cell growth inhibitors in human cell lysates and serum The inhibitor is of human origin and is clearly excreted from breast cells. Togara, human breast cell lines and human serum were tested for the presence of inhibitor proteins. Tested by Western blotting. Cells were lysed with 216DS in PBS Human serum samples were individually mixed with reducing buffer and washed with 10% SDS polyacrylate. It was attached to an electric glue over an amidol. The protein from the protein was mixed with 10 liters of methanol, 0. 15M glycine in 25mM Tris at 180mA overnight on nitro. Transferred to cellulose. Filter paper in PB8 solution (2 bovine serum aldemin BSA) 10 I tried using CJml. 3C6 monoclonal antibody was first dissolved in PBS. Added with Yu BSA 10 Go.

The filter paper was washed twice with 100M PBS solution. 125I-labeled sheep anti-mouse The two antibodies were incubated at 1 μC’i/1 in PBS with 5 g of non-fat dry milk powder (NFDM). It was diluted to 0.0 g and added. The filter paper was washed 5 times with PBS solution for 30 minutes each time. vinegar All washes were performed with PBS Plus [ ], 1% Baker Tween 20. It was carried out.

As is clear from the Western plot shown in Figure 7, the inhibitor human breast cell lysate, MCF-7 and BT-20 human breast cell lysate, and Although present in normal human female serum, MDA-MB-231 and HBL-i Q: Absent or detectable in human breast cell lysate and male serum There was no such level.

Specific example 11 Absence or reduced levels of human breast cell growth inhibitors in breast cancer patients Presence or level of inhibitors in patient serum of the type described in the legend to Figure 8. was determined by Western blot m-. Sample 2 μt of each sample of human serum. Mix with pull loading buffer IQQttt, It was subjected to electrophoresis on midgel. Protein from Glue, 10% methanol% 0.1 overnight at 180 mA in 5M glycine and 2S mM Tris. and electroplotted onto nitrocellulose filter paper. Next, filter paper t2 ( l　Block with 100mf of BSA-containing pBs for 3 hours to prevent non-specific protein binding to the filter paper. reduced the 3C6 monoclonal partially purified from ascites by ammonium sulfate precipitation Add 100μt of antibody and 10d of PBS containing 2% BSA to the filter paper and incubate overnight at 4°C. The cells were incubated and washed three times with PBS. PBS containing 5% skim milk powder iQ d and 125 knee sheep anti-mouse IgG10μC1/1011Lt then filter paper and incubated for 2 hours at room temperature.

Next, take the filter paper in PBS and wash it 5 times for 10 minutes each time with 1% Tween. It was then dried on filter paper. When the filter paper is exposed to film for 4 days and developed, the result shown in Figure 8 is obtained. Western plots were obtained.

As shown in Figure 8, in 3 out of 4 breast cancer patients (lanes 3-4 and 7-10) ), no detectable levels of inhibitor were observed. 1 case (lane 5 and A decreased but detectable level of the inhibitor was observed in the serum of both patients and 6). . A second Western plot was also prepared according to the procedure above and obtained the same results. .

Specific example 12 Dose response of disinhibition of inhibitor activity by 3C6 monoclonal antibody MCF-7 cells were treated with RPMI containing 10 FC8 and 10 μg/goinsulin. Medium 1×104 cells/d were plated and allowed to attach overnight at 37°C. unintentionally cells on a 3C6 monoclonal antibody affinity column prepared using standard methods. 50 μt1 of inhibitor from the low calcium NHMC-N culture medium purified above. and ammonium sulfate precipitated partially purified 5C6 monoclonal antibody in various volumes shown in Table 8. Processed. After treatment, cells were incubated for 7 days at 37°C and then counted. .

As is clear from the data in Table 8 and Figure 9, which is shown as a dose-response curve, Increasing the amount of antibody added also increases disinhibition, and disinhibition by 3C6 antibody is dose-responsive. It turns out that it is sexual. The reluctance inhibition rate and the disengagement inhibition rate were calculated as described above. .

Table 8 Dose response for disinhibition of suppressive activity by 3C6 antibody 50 μt 100 μt 22 μt 50μt 5Qμt 1a chi 50μt 25μt 8% 50μtCJpl　O% 0 μt 5 [] μt − Complement lysis of MCF-7 cells with immune serum MCF-7 cells were treated with 10% FC8 and 1×104 cells/d in MEM containing 10 afi/rttl insulin. Grate tinbure, allowed to adhere overnight at 37°C. The cells are then treated with the following supplements ( The cells were treated in triplicate with medium supplemented with 100ml (100ml).

That is, 404 PBS; 10% low calcium NRMC-V@ low calcium 10% NFIMC-adjusted medium Immunization medium described in Guwosu Specific Example 8 10% immune serum obtained from sealant white rabbit; IC1 immune serum: and immunity 10 μm of New Sealant White rabbit serum obtained from the same animal before treatment (pre-immune blood Kiyoshi). Treated cells were grown for 7 days in supplemented medium, then lysed and supranuclear coated. Counts were made with a Luther counter and the counts were averaged for each.

As is clear from the data in Table 9 below, and by observation of the culture medium, immune serum It was shown that the inhibitor caused complement lysis in human breast cells, indicating that the inhibitor This suggests that it exists on the cell surface. However, present in MCF-7 cells The inhibitor did not inhibit the growth of MCF-7 cells, and Suppression of growth occurs upon treatment with inhibitors produced by normal human breast cells. , the inhibitor of MCF-7 cells is considered to be non-functional. You should understand. NH containing functional inhibitors as shown in Table 9 Addition of MC-conditioned medium increased complement lysis. The above test was conducted two more times. However, the same result was obtained.

Table 9 Complement lysis treatment of MCF-7 with immune serum; count after 7 days 10min PBC10204 10cm cv; &7386 10 CM + iQ Is, 997 10 regrets 1984 101psc11680 a Low calcium NHMC-conditioned medium, 6 Immune serum. preimmune serum Specific example 14 Depletion of suppressive activity from NHMC-conditioned media by immune serum. CNB r-activated 5apharose 4B serum-binding column was used with standard immunological Prepared by method. In detail, 2g of CNBr, 5epharose, 4 Bf Swell in 1mM HCl for 15 minutes and rinse with the same solution on the lath funnel until sintering is complete. dried rll/l for a total of 200 g/y - added in several portions, between successive additions. Supernatant liquid? I aspirated it. Distribute the solution into 3 tubes and add 0-5M Na Ct content 0. i M Coupling buffer of NaHCO3 (p) 18.3) I Washed with CI Go. From immune serum (Is), pre-immune serum (ps) and immune serum 5 taph A-purified IgG (SA-IgG) 11 dt each - 10 each +1/ml of coupling buffer, add this solution t'' to 3 2 tubes. and incubated for 2 hours at room temperature with constant mixing. Then ruru , poured into a syringe lined with lath cotton to elute unbound proteins, and then emptied into a syringe. Rinse with 1FC0,2M Glycino (p[48) 10%. Next, place the column in a cup. Pulling buffer, then 0.1M acetate buffer (pH 4) containing 0.5M NaC2 ) and washed again with coupling buffer. Store the column in a 4”C.

Before use, mix the Is, PS and SA-1gG columns with 2d/column of conditioned medium. and recirculated over the column 5 times. Next, remove the medium and replace the column with 211 Ll. Clove with PBS to a final volume of 4M per column passage medium sample. Kara The cells were washed once more with 20 g of PBS and stored again at 4°C. Next, the column gold For the passed v4m medium, removal of inhibitor by column was performed using MCF- 7 growth assay. To measure depletion, add MCF-7 to the growth medium. Add 10 volumes of conditioned medium or 5 volumes of non-depleted conditioned medium eluted from the column as an additive. A 7-day MCF-7 disinhibition test was conducted using In nine cases where depletion occurs, Elute the affinity column with 0.2 M glycyhydride (pH 3) 2rnt, Antibody/inhibitor was removed from the column. Antioxidant eluate’i Transparent to PBS The inhibitor activity was then examined by MCF-7 growth inhibition assay. Nine.

As is clear from the data in Table 10, the immune serum column and 5taph A- The purified IgG column removed some of the inhibitory activity, which is evident from the data in Table 11. As expected, it eluted from the column again.

Table 10 Inhibitor depletion by column Additives Cell number on day 7 Suppression'L % depletion rate C10 YuP B S 2607 1 0 regret -5%LcMa26135 0% 100%5%CMb12949 50% 0% 10 excellent CM from IS column 14839 43 liters 1 from 14% ps column 0 CM 11888 54 0% SA-IgG'''5ゝra') 16352 37 Regret 26 Yu 10chi CM 0 unadjusted, column not passed low calcium medium 5 low calcium vII 4 adjusted, column not passed Expressed as passage medium 0 derepression rate Table 11 Antigen eluate inhibitory activity Additive cell number suppression rate on day 7 10%LCM 41062 0min 10 ps column antigen eluate 1583 23% 10 ps column UXIW eluate 36993 10% SA-gG antigen eluate 3059625% inhibition Disinhibition by active hybridoma supernatant containing 54FC3 and hypoxanthine , - RPMI medium supplemented with indicator to 46 individual hybridoma subclones. F. The culture solution was adjusted until it became acidic, usually for 3 or 4 days. Then, Remove cells and medium from culture medium, centrifuge, and supernatant hybridoma conditioned medium. Make sure to collect the liquid. MCF-7 whole cells, 10 pieces FC8-containing M medium 2 x 10' thin Plate cells/ml in 12-well Costa plates at 37°C. I applied it at night. 50 μt/well of hybridoma supernatant and low Add calcium NHMC-UF4 JI medium 1'0[]μt/well and add cells. The cells were grown for 7 days, counted, and the rate of inhibition of detachment was determined. 46 Ibridomas tested In the supernatant M solution, the 13 hyperidoma supernatants listed in Table 12 below are 100% Disinhibition of hypohyridoma epitubular fluid/CM showing disinhibition of inhibitory activity in DF1/1405 50% DF1/15C562 width DF1/12C539 Jin DF1/9a8 100% DF’1/9A10 41st Section DF1/7G8 35% DF1/13D3 24% DF1/908 (subclone) 73% DFI/11D7 784 DF1/8A11 89% DF1/3c6 100% DF1/6c6 76% DPI/2D2 56 regret Specific example 16 Hyperdoma supernatant depletion assay 96-well microtiter plate t-100μ Otsu/sea urchin h 10Ag /mlyaeK-? Us' IgG! Coating was carried out overnight at 14°C. the next day, Plate washed twice with t-PBS and as described above for each of the 14 hybridoma clones. Add 75 μt/well of gold to the L. hybridoma supernatant prepared as described above and incubate at 4°C. Incubated overnight. Next, after washing well i twice with PBS, each well Add low calcium A) JHMC-prepared medium 50μt/In1t- and incubate at 4°C. ffl incubate friend. Then, remove the medium, sterilize the heel, and use CAE to remove lCI. MC plated for 7 days at 1 x 10 m cells/d in FC8tlrMgM. F-7 cells were treated to remove inhibitors from the conditioned medium with plate-bound antibodies. used in a disinhibition assay to measure depletion.

Among the 14 hybridomasade clones tested, as shown in Table 13 below: , seven hybridoma subclones named as follows, from conditioned media: Depletion of suppressive activity over 5G1 occurred by 100%1.

Table 13 ...Depleted DF1/9G8 55% with Ibrid-Masadecumin supernatant DF1/16G9 91% DF1/7C) 8 61% oy1/6B8 66% DFI/11D7 100% DF1/1D4 66% DF1/306 57% Displayed by a% disinhibition rate Isotype determination of monoclonal antibodies against human breast cell growth inhibitors Determining the 111 intib of monoclonal antibodies 3C6, ID4 and (SBB) To do this, EiLISA'i was used.

Dynatech Laboratories, Inc. (ChantiL 96-well Immulon 1 microtie (Ly, Virginia) Tago Co., Burlingame, Ca. 1ifornia) anti-mouse IgM and IgG antibodies, 50μ in PBS Coating was performed by incubating overnight at 4°C.

After incubation, plate 'tO, 9% NaCA and 0. Q5(+Tri It was washed with a washing solution of tonX-10[). Hypuridoma fine as described above. Add 50 μt of the N. iploidoma subclone supernatant prepared from the cell culture to each well. In addition, without dilution, 24 BSA-containing PBS from 1 = 1 to 1:128 It was added as a 2-fold dilution series. After incubating for 1 hour at room temperature, plate Washed with the washing solution described above. Labeled with alkaline phosphatase Southern Biotechnology A s5ociates, Birmingham, Alabama) t-2% Dilute 1:1000 in PBS containing BSA to 50μ.

added to each well. Pre-incubation with a dilution series of specific hybridoma supernatants Add individual concentrations of antibodies to specific antigens to the plated wells. Me, some wells contained antibodies against gM, and some wells contained rgG3. , IgG1. Antibodies against either IgG 2b or IgG 2a I made it possible to be given. After incubating for an additional hour at room temperature, the plate was washed with a washing solution and the chromogenic substance p-nitrophenyl lysate/disodium acid (Sig ma, St,, Louis, Missouri) 10q/rtt! melt 100 μt of liquid was added. Color was developed for 1 to 20 hours. Next, Dynatech Optical density at wavelength 410 nm using MR700 microplate reader The degree was measured. Monoclonal antibodies of known isotypes were used as positive and negative controls. I used it. This procedure confirms that the antibodies are 3C6, ID4 and 6B8iIgM. It has been decided that. No cross-reactivity with other isotypes was observed.

Specific example 18 Against the growth of MCF-7! Effect of culture medium: Improves epithelial properties and properties through keratin expression. Normal human breast cells (NHMC) were induced from reduced breasts and 5 oul e, H, S, et al.: In vitro, 22: 6 (1986). T serial passages were carried out in low calcium medium (<60 nM) according to the instructions. Medium is 5% Contains chelex-treated bovine serum, a OnM cact2oMz / F 12 A confluent culture of NHMC was incubated for 4 days to prepare a confluent culture.

MCF-7 cells were incubated with 10 ml of fetal calf serum and 10 μfi/III insulin. Contains M-medium, 2 x 105 cells/grating in T25 flask, - night I made him wear it.

The medium was then replaced with medium at the indicated concentrations adjusted with NHMC cells. adjustment or Unconditioned medium was concentrated 10-fold by Amicon ultrafiltration. Coulter at specified time - Cell number was determined by counter. As shown in Figure 10, these cells The medium conditioned by the transformed human breast cancer cell line, MCF-7'fc, It was found that the effect was suppressed in a time-dependent and dose-dependent manner. Add 10 microns to these culture solutions. Addition of conditioned medium increased cell growth on day 1a compared to culture medium supplemented with unconditioned medium. A 75% reduction in length occurred.

Specific example 19 Antibody affinity loading of human breast growth inhibitors Inhibitory activity t-DEAES in normal human breast cell conditioned medium By ion exchange chromatography on yl, then 5C6 monoclonal butt Purified by affinity chromatography on a body affinity column. . 1t of DEAE 5apha-cry in conditioned medium 50mM NaCt l　Loaded on the column and eluted with a stepwise gradient of 0.05-1.0M NaC2. I put it out. The dialyzed fraction was assayed for MCF-7 growth inhibitory activity. wRi medium? Their inhibitory activity was eluted at 0-25 M NaC2.

Next, the inhibitory activity eluted with 0, 1 to 0-5 M NaCt was determined by the 3C6 mono Clonal antibody t1.5M +7) to Biorad Affigel beads It was loaded onto a monoclonal antibody affinity column prepared by binding. next Wash the affinity column FF with 1500 column volumes of PBS to remove unbound proteins. The protein was removed and the bound protein was eluted with 0.1M glycine-2.35. The eluate is directly It was then neutralized with Tris base and dialyzed against PBS.

Specific activity is defined as the amount of protein required for 50% inhibition of MCF-7 cell growth. and expressed in units/■. The concentration of inhibitory protein was visualized by silver staining on SDS-P. Determined by comparison with albumin standards on AGE and Biorad protein assays. Established. The final purity of the inhibitory protein was determined to be over 50 min. The result is the 14th below. Shown in the table. A final purity of at least about 95% is achieved by repeating the purification process described above. was achieved.

Table 14 0.5M　OwaF,　~ Fraction 20.3 47.7 41x 40.3 MCF -7 Affinity for growth Effect of purified human breast growth inhibitor and 3C6 antibody on hybridoma Regarding the supernatant, the recognition ability of immune proteins and v4 regulation for MCF-7 cells were determined. The ability of the medium to inhibit suppressive activity was screened.

As in Specific Example 10, MCF-7 cells were grown on a grating plate, and after the first day. The supplements below were added in fresh medium. That is, (a110% normal human breast adjustment Medium; +b+io NHMC conditioned medium +DF1/! ;Hyperidoma from C6 Upper setting t104; (cl 10 nl/ld affinity purification inhibitor (d120 ng/d affinity purification inhibitor; (θ11 0nN/11t affinity purification inhibitor + 5μm1/goshi clonal The antibody is 3C6. “The term affinity purification inhibitor is NHMC-regulated. DEAR5ephacryL ion exchange solution was added from the culture medium as described in Specific Example 19. and 5C6 monoclonal antibody affinity chromatography. means 9-inhibitor.

Cell growth was measured on day 7 and expressed as percent inhibition compared to an equal volume of unconditioned medium. Ta. As shown in Figure 11, the purified substance inhibited the growth of MCF-7 cells in a dose-dependent manner. 50% growth inhibition at inhibitor concentration 10 ng/IL/, 20 ng/IL/ d showed more than 90% inhibition of cell growth. in conditioned media or purified inhibitors The inhibition of cell growth by monoclonal antibody 3C6 was abolished by monoclonal antibody 3C6. monochrome Null antibody alone had no significant effect on the growth of MCF-7 cells.

Specific example 21 SDS-PAGE and Fluorography of Purified Human Breast Growth Inhibitor analysis Lanes (a) and (bl) in Figure 12 indicate (a) the molecular weight standard and (bl) the Silver staining results for nine affinity-purified inhibitors prepared by shows. To also clarify that the inhibitor is synthesized by NEMC, Cultures were grown in methionine-free DEME supplemented with 200 μC1/d of 358-methionine. Confluence of NHMCs in medium 5g T-757 was incubated for 24 hours. Labeled with 353-methionine. Next, the culture supernatant was treated with 306 monoclonal anti- Affinity chromatography on a body affinity column Tea selection inhibitor t-preparation sheet, 10% 5DS 7” SDS-PA-G Molecular weight was measured by E. Lane (C) shows the elution from the 306 affinity column. This is a fluorogram of the 35B-methionine labeled NHMC-ill culture medium. Ru.

As shown in Figure 12, lane B, the affinity purified inhibitor was silver stained. Two prominent bands were observed at approximately 47,000 and 65,000.

The fluorogram of the affinity-selective inhibitor is shown in lane C. approximately 47,000 outstanding bands and approximately 67,000 constant doublet bands; and a minor inconsistent band was observed at approximately 63,000. This is 4 7.0 [10 and 63. OCJ O~67.000 harvests are different versions of the same protein grosetted or truncated, or two species with a common epitope This suggests a distinct inhibitory factor.

Specific example 22 Heat denaturation and tryptic sensitivity of affinity-purified human breast cell growth inhibitors receptivity Affinity purification inhibitor (10ng/d) 37.55.70 or 1 00°C for 60 minutes.

The inhibitory activity of these samples was determined as described above. human breast growth inhibitor Heating the bitters to 70° C. for 60 minutes resulted in a 50 million drop in activity. 6 to 100℃ After heating for 0 minutes, the inhibitory activity completely disappeared. This thermal sensitivity shown in Figure 13 The pattern is 5porn.

M.D. et al.: 5science, 233:532 (1986). Human breast growth stimulation from TGF-β, which is stable even when heated to 100°C, as shown in Different from inhibitors.

The sensitivity of the affinity purified inhibitors to trypsin was also tested. Af Infinity Purification Inhibitor 10 On,! i’ / ml, 5 in 1 rul All/ml of Trip'/7f was added and incubated at 37°C for 1 hour.

After incubation for 1 hour, trypsin inhibitor was added at 50 μg/Ill. Stopped Shin's decomposition. Affinity purification inhibitor 100n#/go, 1 A control experiment was also performed in which 50 μg of trypsin inhibitor was added to -. Then trypsin 5μyt was added to the control mixture and the entire mixture was incubated at 37°C for 1 hour. fruit Both test and control samples were then sterile filtered and the MCF in Specific Example 18 - 7 culture solution was added with gold, and the inhibitory activity was assayed. As shown in Figure 13, the affinity The inhibitory activity of the purified inhibitor was completely abolished by the addition of trypsin.

Specific example 23 Effects of human breast growth inhibitors on the growth of human cell lines Human breast growth inhibitors for the growth of various transformed breast and non-mammary human cell lines The effect of the tar was tested.

The cell line was prepared in RPMI + 10% fetal bovine serum using a 12-well plate. Each well was cultured in triplicate at 2×10′ cells/well and allowed to attach overnight.

On the first day, the cells were counted and then treated with the inhibitor at a final concentration of 1Qn, jil/atg. - The case was treated with BSA. The inhibition rate was calculated as described above.

As shown in Table 15, human breast growth inhibitor (10nJ7/m) was tested. A significant inhibition of the growth of five transformed human breast cell lines resulted. Gaffney , E, W.

Suppression of the growth of the non-transformed breast cell line, HBL-1Q, described in The specified result was not entered. Nisthrobin responsiveness (MCF-7, ZR-75- 1) and Est o fif 7 non-responsive CB T-2Q, MDA-M B-231, evejos (established from primary mammary cancer in Michigan canine science, available on request from the Department of Internal Medicine, Paul Ervin)] for any of the cell lines. Growth suppression was observed.

Unlike its action on breast cells, the inhibitors are effective against the non-breast tissues listed in Table 15. No effect was shown on the growth of the 11 derived human cell lines.

This tissue specificity of the inhibitor was determined by Tucker, R.F. A wide range of epithelial cells, as described in CE, 226: 115 (1984). This further allows for differentiation from TGF-β, which suppresses the growth of the system.

In addition, Western plot analysis of human breast growth inhibitor and T()F-β analysis, 3C6 and anti-TGF-β antibody (R-&D Products). It was implemented more. 3C6 did not detect TGF-β but recognized the inhibitor. Ta. Conversely, anti-TGF-β detected no inhibitor, but TGF-βt- I recognized it. That is, due to molecular weight, heat sensitivity, tissue specificity, and antibody reactivity, The inhibitor of the present invention is identified as TGF-β. The tissue specificity of inhibition is further Compare human breast growth inhibitors to other small molecular weight inhibitors previously isolated from breast cells. It is distinguished from protein.

Breast cancer MCF-76i-17/-g Breast cancer BT-2055+/-17 Breast cancer MDA-MB-23125+/-4 Breast cancer ZR-75-157+/-4 Breast cancer Evej as 21-+//-4 Breast epithelium HBL-10019+/- 15 Lung adenocarcinoma A 427 3+/-1 Colon cancer SW 94B -8+/-5 Fibrosarcoma HT 1080 8+/-16 Premonocytic leukemia HL 60-9+/ -3 Bladder cancer 1 (ill 4+/-7 Lymphoblastic leukemia Raji -2+/-11 Myeloid monocytic leukemia Weh↓- 51+/-6 Slave cell carcinoma A 253 0+/-6 Wired cell carcinoma UM-3CC-17a -12+/-11 Wired cell carcinoma 4-5ea -38-6+/-8 Cervical cancer He La -17+/-20 Negative numbers are successful. Indicates long stimulation Specific example 24 Immunoperoxidase staining of human breast cells by normal and transformed human cell lines To examine inhibitor production, 3C6 monoclonal antibody was used at the interface. Perform immunoperoxidase staining of active agent permeable normal and transformed cells.

NHMC or MCF-7 cells were cultured on chamber slides for 3 days, and PB The cells were washed with S and fixed in 2% paraformaldehyde in PBS at 4°C for 10 minutes. Below Subsequent steps were performed at room temperature. Cells were treated with 0.1% Triton X-100 in PBS. permeabilize for 10 minutes, soak for 30 minutes with l5ssA in PBS, and then 1:100 dilution of monoclonal antibody 3C6 in PBS containing % BSA for 60 min. Incubated. Equal concentrations of irrelevant IgM antibodies were used as controls. next Cells were washed with Yaza anti-mouse IgG antibody conjugated in PBS containing 2.5% nonfat dry milk. Incubate for 60 minutes in a 1:250 solution of oxidase and 1:1 in an AEC solution. Developed for 0 minutes. I attached an Aquamount to the slide and took pictures.

Figure 14 shows captive normal human breast cells (X100) and (BIMCF-7 cells). (Xl　00) staining results are shown. As shown in Figure 14A, the immunoreactive inhibitor Bitter was present in over 90% of normal human breast cells. On the other hand, as shown in Figure 14B, In contrast, only 10-15% of MCF-7 cells showed weak staining.

Samples stained with 3C6 were negative for irrelevant antibodies.

Immunoperoxidase staining of other transformed cell lines listed in Table 15 was performed in the same manner. did. BT-20, MDA-MB-231 and ZR-75-1, evej In os and HBL-100, weakly stained cells of less than 10 cells were observed. . Non-breast cell lines were negative.

Specific example 25 Three transformed humans tested for human breast growth inhibitors using transformed cell lines Cell line conditioned media contains immunoreactive or bioactive human breast cell growth inhibitors. - could not be detected.

Conditioned medium was prepared in the same manner as described for NHMC, MCF-7, BT -20 and MDA-MD-251 cell lines and as previously described. MCF-7 growth inhibitory activity was investigated. These media were analyzed by Western blot. I also analyzed it. Suppressive activity was also detected in immunoreactive human breast growth inhibitors. There wasn't.

Specific example 26 Sandwich ELISA and quantification of human breast growth inhibitor levels Please note that the Dynatech microtiter plate was mentioned above in Specific Example 7. from hybridoma clone oF1/6B8 produced in this manner. rgM monoclonal antibody, 10 ng/- solution in PBS of purified 688 in all wells. 100 μl was used and incubated overnight at 4°C. coated plastic Letra was followed by 300 μt of bovine serum aldemin (BSA) per well. , Sihiro Chemical)-containing PBS t- and incubation at room temperature for 1 hour. Bate and block to reduce total non-specific binding. plate? , well Atta! 1100 μt of affinity-purified inhibitor or inhibitor as standard. The cells were incubated with human serum for 2 hours at room temperature. Next plate t Q-1%’rr Rinse and wash with PBS containing ttQn　X　-100 to remove unbound proteins. , and then the entire solution was shaken off the plate. This operation sound was repeated four times.

The binding protein is on the plate and per well! 14 BSA-containing PB of 1100μ Incubate for 1 hour at room temperature with 2 μm 17d solution of biotin-conjugated 5C6 antibody in S. Recognized by bate. Unbound antibodies were plated again in O, 1% Clear the plate by rinsing it several times with PBS containing Triton X-100. It was removed. The binding protein was then incubated with alkaline phosphatase in PBS containing i% BsA. -se-conjugated streptavidin (5outhern Biotachnology) .

Inc.) 0.4 ng/’ solution, 100 μt per well for 1 hour at room temperature. Incubated. Shake the plate to remove the streptavidin solution from the grate. It was removed and washed twice again with PBS + Triton X-100.

The captured protein is then bound to a second antibody via a biotin conjugate. It was recognized by a colorimetric method using calyphosphatase. That is, the plate , Alka IJ IJ phosphate substrate buffer (400ml dH20.

24-5 rn9 Mget, 48 ml di x l / -h 7 mini y f HCL tep) Adjust to I9.8 and make total 500-) 1 tablet per 11d (5 curvature) p-nitrophenyl phosphorus all salt tablets (Sigma104-105 ho sphatase substrate) solution, 100 µt per well, and incubate until a yellow color is observed. Incubated. After 30 minutes and 1 hour, the Dynatec Micro Read using an IJ-der, the amount of protein present is determined by the generated yellow color. The strength was measured.

To quantify the actual nJil/ml of protein, this method is first applied to purified insulin. Bitter protein concentration f Biorad protein assay or similar assay For example, using 5DS-PAGE comparison, then pure protein t-PBS + This was done by serially diluting the sample in a 1% BSA solution and creating a standard curve. Not yet The known samples were then compared to this standard curve to determine protein levels.

The present invention may be embodied in several forms without departing from its spirit or essential characteristics. The above embodiments are merely illustrative and do not limit the present invention. It's not something you do. The scope of the invention is defined by the appended claims. Therefore, all changes that constitute operational equivalents within the scope of the claims are hereby included. within the scope of the claims.

Engraving (no changes to the content) G-100 force 5mu i1+'-yo & MCF'-, 7 cells' success! 1Wts ground use quantity response nm to 50 250 500 Concentration land addition capacity rμri Figure 2 Activity of molecular node column fractionation Transverse cleft inhibitor 1 of MCI”-7: Inhibition by C102550700200 Purified inhibitor addition volume rμ/) Figure 5 kdABCDE):” 4-・ Figure 4 ABC[) (D) 35s-methionine detection in human culture medium Western blot detection Presence of inhibitor in serum 1, normal female serum 2 Normal male serum J Breast cancer patients, serum of Mc 4. Mc serum for breast cancer patients 5 Serum from breast cancer patients 7.8 5. Serum of breast cancer patients T and Bk 2 Breast cancer patient A, new serum a Breast cancer patient A, new serum 9. Serum of breast cancer patients P and Do 10. Serum of breast cancer patients P and Do Figure 8 0 20 40 60 Bo 100 1200 J7 55 70 100 f+I　I4-/Figure 13 Time (number of culture days) 10% unconditioned medium (10 μ 5% conditioned medium 10 lengths 10% 401 medium 6 liters).

Values are the mean ± SEM of triplicate determinations repeated twice.

Amendment for blocking procedure due to monoclonal antibody 1 with inhibitory activity (voluntary) January 2Σ, 1991

Claims (1)

[Claims] 1. Antibodies consisting of monoclonal antibodies that bind to human breast cell growth inhibitors 2. 5. The antibody according to claim (1), wherein the antibody is chimeric. Antibodies are inhibitors The antibody according to claim (1), which binds to the active site of 4. The antibody according to claim (1), wherein the antibody binds to an inactive site of the inhibitor. 5. 6. The antibody according to claim (1), wherein the antibody is an IgQ antibody. The antibody is an IgM antibody. 7. Antibody according to claim (1) The monoclonal antibody was prepared using hybridoma cell line A. A claim that is the product of a hybridoma having the characteristics of TCC Accession No. HB9722 Antibody 8 described in (1). The monoclonal antibody was produced using a hybridoma cell line ATCC. The product according to claim (1) is a product of a hybridoma having the characteristics of serial number HB9723. Antibody 9. The monoclonal antibody is hybridoma cell line ATCC accession number H The antibody according to claim (1), which is a product of a hybridoma having the characteristics of B10152. Body 10. The following characteristics, namely (a) NHMC, MCF-7, BT-20 breast Inhibitory effect on cell growth, (b) Calcium dependence of inhibitory activity, (c) Inhibitory activity (d) reversibility of inhibitory activity upon decreasing inhibitor concentration; (e) a substantial decrease in inhibitory activity upon heating to 100°C; and (f) a binding to cell growth inhibitors with a substantial decrease in inhibitory activity upon treatment with Monoclonal antibodies that match 11. 12. Antibody according to claim (10), wherein the antibody is an IgM antibody. transformed cells and , a splenic gland that produces monoclonal antibodies against human breast cell growth inhibitors. Hybridoma generated by fusion with cysts 15. Hypuridoma is Hypuridoma The Doma cell line has the characteristics of ATCC Accession No. HB9722 as described in claim (12). Hypuridoma 14. Hypridoma is a characteristic of hyperdoma cell ATCC accession number HB9723. The hyperidoma according to claim (12), which has 15. The hybridoma is a hybridoma cell line ATCC accession number HB1015 The hyperidoma according to claim (12), which has the characteristics of 2. 16. (a) preparing a biological sample from a human subject; and (b) growing human breast cells. preparing an antibody specific for the inhibitor; and (c) testing the binding of the antibody to the inhibitor. (d) combining the antibody with a biological sample and human breast cell populations in the sample; If a long inhibitor is present, the inhibitor must be present for a sufficient time and in sufficient quantity for the antibody to bind to it. each step of contacting with an amount of antibody, and then (e) assaying the binding of the antibody to the inhibitor. 17. A method for screening breast cancer in a human subject, comprising: Antibodies are monoclonal The method according to claim (16), wherein the antibody is a polyantibody. 18. The method according to claim (16), wherein the antibody is a polyclonal antibody. 19. The method according to claim (16), wherein the biological sample is breast tissue. 20. The method according to claim (16), wherein the biological sample is serum. 21. (a) preparing a biological sample of body fluid or tissue of a human subject; (c) preparing an antibody specific for a breast cell growth inhibitor; preparing an assay for antibody binding; (d) combining the antibody with a biological sample and the sample; The presence of human breast cell growth inhibitor in the nine-fold antibody is sufficient for the antibody to bind to it. (e) contacting with a sufficient amount of antibody for a sufficient amount of time to inhibit binding of the antibody to the inhibitor; and (f) determine the amount of inhibitor present in the sample relative to standard levels. Human Breast Cell Growth Inhibitor in Human Subjects from Each Step Associated with Measuring How to monitor the levels of 22. (a) providing breast tissue with a therapeutically effective amount of a human breast cell growth inhibitor; (b) a human body consisting of each step of monitoring the level of breast cell growth inhibitor; How to prevent or treat breast cancer 25. and (c) monitoring the level of inhibitors present in vivo. The in vivo inhibitor level monitoring process The process involves (1) preparing a sample of body fluid or tissue of interest; and (ii) preparing a sample of human breast cell tissue. (iii) conjugation of the antibody to the inhibitor; (iv) combining the antibody with the body fluid or tissue present in the sample; for sufficient time and sufficient time for the antibody to bind to the human breast cell growth inhibitor (v) assaying binding of the antibody to the inhibitor, and then (v) assaying the binding of the antibody to the inhibitor. vi) the steps of determining the amount of inhibitor present in the sample; (22) Method described 24 The delivery process maintains therapeutically effective levels of the inhibitor in vlvo. The method according to claim (22), wherein the method is repeated to 25. Claim 22, wherein the inhibitor is supplied to the breast tissue via the circulatory system. the method of 26. The method of claim 22, wherein the inhibitor is injected into breast tissue. 27. The method according to claim (22), wherein the antibody is a monoclonal antibody. 28. Antibodies that bind predominantly to human breast cell growth inhibitors in biological fluids , and reagent means for detecting binding of the antibody to the inhibitor; and the reagent means is a human breast cell growth inhibitor present in sufficient quantity to perform the assay. kit for testing the presence of 29. Kit 30 according to claim (28), wherein the antibody is an IgQ antibody. Antibodies are IgM Kit 31 according to claim (28), which is an antibody. The assay is an enzyme-linked immunoassay. Kit according to claim (28) 32. The kit according to claim (28), wherein the assay is a radioimmunoassay. 33. The kit according to claim (28), wherein the assay is a hemagglutination assay. 34. The kit according to claim (28), wherein the assay is a cell inhibition assay. 35. The kit according to claim (28), wherein the antibody is a monoclonal antibody. 36. The kit according to claim (28), wherein the antibody is a polyclonal antibody. 37. A method of purifying human breast cell growth inhibitor in solution, comprising: (a) preparing a purified monoclonal antibody against the inhibitor; (b) combining the solution with the antibody; contacting the antibody for a sufficient amount of time and in a sufficient amount for the antibody to bind to the inhibitor; and (c) from each step in which the inhibitor is recovered from the antibody-inhibitor complex. Detailed description of the method invention