WO1993013417A1 - Receptor/oncogene growth factor system in breast cancer - Google Patents

Abstract

An antibody which is immunoreactive against mammary-derived growth factor 1 (MDGF1) and related growth factors. A new growth factor/growth factor receptor-oncogene family. New therapeutic agents based on this system for treatment of disease.

Description

TITLE OF THE INVENTION

RECEPTOR/ONCOGENE GROWTH FACTOR SYSTEM IN BREAST CANCER

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a new receptor/oncogene growth factor system and its use in treatment of disease and in detecting other related growth factor systems.

Description of the Background

Tyrosine kinase activities are now recognized as being central to the development and progression of cancer. Two general areas of study are relevant. First, many kinase families exist and can be used to characterize the biological diagnosis and prognosis of cancer and other diseases. See Hunter et al, Ann. Rev. Biochem.. 54:197-205 (1985). Second, many of these tyrosine kinases are associated with different growth factor receptors such as the EGF receptor - erb B tyrosine kinase association. See Carpenter et al, J. Biol. Chem.. 265:7709-7712 (1976). Growth factors may stimulate early progression of cancer through such receptors independent of the genetic changes of amplification, translocation and mutation of their receptors which may make them oncogenic or cancerous. Genes encoding growth factors themselves may also become oncogenes. Better methods of breast cancer detection and sraging have recently come from the area of molecular biology. For example, the discovery of Her-2/neu tyrosine kinase oncogene has led to new prognostic test for breast cancer. Potentially even more importantly, knowledge of this oncogene has led to antibodies and other molecular probes to aid in elucidation of a larger family of related oncogenes with implications for better understanding of a wide range of human cancers. See Dickson RB and Lippman ME (Eds) Seminars in Cancer Biology: EGF family of receptors and ligands parts I and II. Vol. 1:4 and 1:5, W.B. Saunders, 1990.

However, at present, the knowledge of growth regulatory fac ors and their receptors is quite incomplete. Further developments are necessary to uncover new families of such activities which may provide added insight into the regulation of disease development, cancer, aging, cell function and tissue interaction.

SUMMARY OF THE INVENTION

Accordingly it is the object of the present invention to utilize the MDGFl receptor system for several purposes. The growth factor itself, its related analogues, or an antibody directed against the factor may be useful as therapeutic agents for a variety of pathologic conditions.

It is also an object of the present invention to provide various methods for the detection of MDGFl receptors and related molecules, and for determining the structure thereof and range of expression and function by stimulation of phosphotyrosine as determined by western Blot.

Finally, it is an object of the present invention to provide a means for utilizing structural information of a new growth factor - MDGFl, antibodies to the factor, and the radiolabelled and nonlabelled factor to characterize new growth factor-receptor-oncogenic molecules.

BRIEF DESCRIPTION OF THE DRAWINGS Figure IA illustrates a western blot analysis of MDGFl; Figure IB illustrates the identification of 62 kDa band by immunoprecipitation;

Figure 1C illustrates an autoradiogram of a gel of immunoprecipitates of [¹²⁵I]-MDGFl from milk in the presence of unlabeled factor;

Figure 2A illustrates metabolic labeling and immunoprecipitation of condition media;

Figure 2B illustrates a western blot analysis of cell lysates from MDA-MB 231, HLB-100 and 184 cells;

Figure 3A illustrates in vitro translation of mRNA and immunoprecipitation;

Figure 3B illustrates the tunicamycin treatment of HBL-100 cells;

Figure 3C illustrates N-linked glycosylation of the 62kDa MDGFl; Figure 4A illustrates western blot of phosphotyrosine in MDA-MB 468 cells;

Figure 4B illustrates the stimulation of tyrosine phosphorylation of MCF-7 cells by MDGFl; and

Figures 4C and 4D illustrate an autoradiogram of phosphorylated proteins in MDA-MB 468 (4C) and 184A1N4 cell (4D) lysates.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A mammary-derived growth factor, MDGFl, which stimulates mammary epithelial cells, was previously detected and purified from human milk and primary human mammary tumors. MDGFl binds to putative cell surface receptors of 120-140 kDa and stimulates proliferation of normal and malignant human mammary epithelial cells. In addition, MDGFl is known to stimulate collagen synthesis in fibroblasts and mammary epithelial cells. Bano et al, J. Biol. Chem. 260:5745-5752 (1985) . Bano et al, J. Biol. Chem. 265:1874-1880 (1990).

MDGFl is a high molecular weight (62 kDa) , acidic growth factor (pi 4.8) which can be distinguished from epidermal growth factor (EGF) which accounts for about 75% of the total growth factor activity in milk. MDGFl is readily distinguishable from EGF on the basis of size, pi and disulfide bond reducing agent sensitivity.

In accordance with the present invention, it has now been surprisingly discovered that MDGFl is a secreted mitogen detected in the conditioned media of human MDA-MB 231 breast carcinoma and immortalized, but non-malignant, HBL-100 cell lines and the normal 184 mammary epithelial cell strains. In accordance with the present invention, it has also been discovered that both EGF and MDGFl bind with comparable affinity to different receptors, respectively, 170 and 120-140 kDa size, and lead to an accumulation of phosphotyrosine, on a different peptide of 180-185 kDa in size.

Thus, in one aspect thereof, the present invention provides antibodies directed against MDGFl for the detection thereof by western blot, radioimmunoassay (RIA) and immunoprecipitation.

In another aspect thereof, the present invention provides a method for the determination of functional MDGFl receptors by stimulation of phosphotyrosine as determined by western blot.

The present inventors have found that human breast epithelial cells in culture produce MDGFl. In particular, a protein of the size of MDGFl was immunologically detected in the concentrated, conditioned medium prepared from human breast cancer cell line MDA-MB 231, the mammary-derived but non-tumorigenic HBL-100 line and the normal non-immortalized 184 cell strains. Then, a competitive radioreceptor assay (RRA) was used to estimate level of MDGFl in the conditioned medium. It was found that MDGFl was present in nanogram quantities per ml of media. _ -3.

Further, a 62 kDa protein was detected in the above cell lysates by western i munoblotting or by immunoprecipitation of etabolically labeled cell-conditioned media. In vitro translation of cell mRNA yielded a protein of 55 kDa, which was immunoprecipitated by anti-MDGFl antibody. Also, glycosylation of MDGFl appears indicated using tunicamycin treatment of cells.

Moreover, the present inventors have discovered that MDGFl induces accumulation of phosphotyrosine in a 130-185 kDa protein. This is significant inasmuch as tyrosine phosphorylation is known to play a critical role in the cell proliferation and cell transformation. Further, it was found that phosphorylation was not blocked by an antibody directed against the binding site of the EGF receptor. Thus, it is concluded that primary binding of MDGFl to a 120-140 kDa receptor occurs, and that phosphorylation on tyrosine of a 180-185 kDa protein is stimulated.

In view of the above discoveries, the present invention provides methods for detection of MDGFl and related molecules by antibody and oligonucleotide probe methodologies for the detection and cloning of MDGFl and related CDNAs. In addition, the present invention provides methods for the detection and study of MDGFl receptor/oncogenes by binding and crosslinking with radioiodinated MDGFl and by simulation of phosphotyrosine accumulation by western blot and other related methodologies. By the term "antibody methodologies'* is meant any immunoassay known to those skilled in the art which are capable of quantitatively detecting MDGFl and phosphotyrosine. For example, western blot, RIA and immunoprecipitation may be used.

A more detailed description of the drawings will now be provided.

Figure IA illustrates a western blot analysis of MDGFl. About 20 ng of the purified factor from milk was run on a 10% SDS-PAGE, transferred to nitrocellulose filter as described below in Experimental Procedures. The blots were visualized following incubation with preimmune serum (lane 1) or with polyclonal antiserum to the synthetic peptide raised in rabbits at a dilution of 1:500 (lane 2). Molecular markers are indicated at left in kilodaltons. Phosphorylase b, 130; bovine serum albumin, 75; and ovalbumin, 50.

Figure IB illustrates the identification of 62 kDa band by immunoprecipitation. [¹²⁵1]-MDGFl from milk was immunoprecipitated using MDGFl antisera or preimmune sera at two dilutions. The samples were analyzed by SDS gel electrophoresis with a 10% gel and autoradiography. Lanes 1 and 2 represent samples treated with prebleed and lanes 3 and 4, samples treated with anti-MDGFl sera (1:100 and 1:250 dilutions) .

Figure 1C illustrates an autoradiogram of a gel of immunoprecipitates of [¹²⁵1]-MDGFl from milk in the presence of unlabeled factor. Immunoprecipitation was carried out using anti-MDGFl and prebleed sera at a dilution of 1:100 as explained earlier. Unlabeled MDGFl was used at various concentrations ranging from 0 (lane 3) , 2.5 ng (lane 4) , 5 ng (lane 5) , 10 ng (lane 6) and 25 ng (lane 7) . Lanes 1 and 2 denote preimmune sera treated samples in the presence of 5 and 10 ng of unlabeled factor.

Figure 2A illustrates the metabolic labeling and immunoprecipitation of conditioned media. HBL-100 cells were labeled with [³⁵S]-methionine and [³⁵S]-cysteine, and conditioned medium was immunoprecipitated with either prebleed sera or with MDGFl antisera at a dilution of 1:250. Unlabeled factor was added at concentrations 0 (lane 1), 2.5 ng (lane 2), 5 ng (lane 3) and 10 ng (lane 4). The immunoprecipitates were resolved by electrophoresis on a 12.5% polyacrylamide gel and autoradiographed.

Figure 2B illustrates the western blot analysis of cell lysates from MDA-MB 231, HBL-100 and 184 cells. About 30 μg samples of lysates from cells were run on a 12.5% gel, transferred to nitrocellulose membrane and processed. Lanes 1-3 denote lysates from MDA-MB 231, HBL-100 and 184 cells. The filters were treated with immune and pre-immune sera, used at a dilution of 1:500.

Figure 3A illustrates the in vitro translation of mRNA and immunoprecipitation. Total RNA from two cell lines (HBL-100 and MDA-MB 231) were translated in vitro in a wheat -9-

germ system as described earlier and immunoprecipitated using MDGFl antisera. Figure depicts the result for HBL-100 cell line. Lane l denotes results with preimmune sera and lane 2 with immune sera, both used at 1:250 dilution. Arrow at right indicates the position of 62 kDa MDGFl.

Figure 3B illustrates tunicamycin treatment. HBL-100 cells were grown to confluency and were treated with 20 μg/ml of tunicamycin for 4 hrs at 37°C. Metabolic labeling with [³⁵S1-methionine and [³⁵S]-cysteine was performed as described in Experimental Procedures. Samples of conditioned medium were immunoprecipitated using anti-MDGFl or prebleed sera at a dilution of 1:250. After solubilization the immunoprecipitates were analyzed by 12.5% SDS-PAGE and subsequent fluorography. Lanes 3 and 4 denote tunicamycin treatment and lanes 1 and 2 in the absence of tunicamycin. Results for anti-MDGFl sera are shown in lanes 2 and 3 and that for prebleed sera in lanes 1 and 4.

Figure 3C illustrates the N-linked glycosylation of the 62 kDa MDGFl. Purified sample (0.5-1 μg) was incubated with 10 units per ml of N-glycanase (lane 2) or buffer only (lane 1) for 16 hrs at 37°C and subjected to western blot analysis and silver stained.

Figure 4A illustrates the western blot of phosphotyrosine in MDA-MB 468 cells. Adequate amount of cells (100,000) were incubated with no addition (lane 1) , 10 ng of purified MDGFl (lane 2), MDGFl in the presence of 100 ng of anti-EGF receptor monoclonal antibody #528 (lane 3), 10 ng EGF (lane 6), EGF in the presence of 100 ng of anti-EGF receptor monoclonal antibody 528 (lane 5) and 100 ng of^' monoclonal antibody 528 alone (lane 4) . Cells were solubilized in sample buffer and subjected to gel electrophoresis (7.5% gels) and western blot analysis using a commercially available monoclonal anti- phosphotyrosine antibody (2.5 μg/ml). Antibody reaction was visualized as noted in Experimental Procedures. Arrow at left indicates 180-185 kDa size band.

Figure 4B illustrates the stimulation of tyrosine phosphorylation of MCF-7 cells by MDGFl. Confluent MCF-7 cells were treated with 0 (lane 1), 10 nM of EGF (lane 2) or 10 nM of MDGFl (lane 3) for 20 min at 37°C, and then the lysates were subjected to SDS-PAGE, western blotting and incubated with monoclonal anti-phosphotyrosine. The blots were detected as explained above. Arrow at right indicates 180-185 kDa size band.

Figs. 4C and 4D illustrate an autoradiogram of phosphorylated proteins in MDA-MB 468 (4C) and 184A1N4 (4D) cell lysates. Total cell lysates were analyzed by 7.5% SDS- PAGE gels, followed by immunoblotting with anti- phosphotyrosine antibody as explained above. The blots were then processed with [¹²⁵1]-Protein A, dried and autoradiographed. Lane 1 denotes control lysate and lane 2, cells treated with 10 ng of MDGFl. The present invention will now be further illustrated by certain examples which are provided solely for purposes of illustration and are not intended to be limitative.

EXPERIMENTAL PROCEDURES Cell Cultures: MDA-MB 231, MDA-MB 468 and HBL-100 cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were maintained in improved minimal essential medium (IMEM, Biofluids, Rockville, MD) in the presence of 5% fetal calf serum (FCS) . The normal 184 cell strain and 184A1N4 (a carcinogen immortalized subclone) were obtained from Dr. Martha Starapfer (Berkeley, CA) and maintained in IMEM in 0.5% FCS in the presence of EGF (10 ng/ml. Collaborative Research), Insulin (10 μg/ml, Sigma, St. Louis, MO), transferrin (10 μg/ml, Sigma) and hydrocortisone (0.5 μg/ml, Sigma) according to the known protocol.

Preparation of Conditioned Medium: Cells were plated in 175-cm² T flasks and grown to confluence in IMEM supplemented with 5% fetal calf serum. Once confluent, the monolayers were switched to serum-free medium. After 24 to 48 hrs, the culture media were collected and concentrated 100-fold in an Amicon Ultra-filtration cell (YM 5 membrane, Amicon, Danvers, MA) . The concentrated media were dialyzed against 100 volumes of distilled water at 4°C. The material that precipitated during dialysis was removed by centrifugation. Protease inhibitors were added as described previously. The clarified sample was then lyophilized and stored.

Protein Sequencing: MDGFl sequencing was accomplished by electroblotting onto PVDF (polyvinylidene difluoride) membranes (Immunobilon Transfer, 0.45-μm pore size, Millipore) as described earlier. Approximately 100 pmol of purified factor was loaded on to a 12% polyacrylamide inigel. After electrophoresis, electroblotting and Coomassie Blue staining, the band corresponding to 62 kDa was cut out and used for amino acid sequencing in a gas-phase sequenator (Model 470A, Applied Biosystems, Foster City, Ca) . Sequence analysis was performed 3 times according to the protocol described by the manufacturer. Sequencing grade reagents were used throughout. The analysis of primary amino acid sequence was performed on an IBM AT personal computer utilizing the "PC GENE" software (IntelliGenetics, Mountain View, CA) .

Predicted location of the major antigenic sites of MDGFl: The method of Hopp and Woods was used to determine an average hydrophilicity value for a series of adjacent amino acids. It is reported that there is a strong correlation between the highest average hydrophilicity value and an antigenic region of the molecule. The analysis was performed with an averaging group length of 6 amino acids which provide the most reliable predictions. The peptide was made using t-Boc chemistry on an automated peptide synthesizer (Bio Research, San Rafael, CA) . Peptide synthesis reagents were purchased from Biosearch. Antibodies: Antisera to MDGFl were prepared by immunizing rabbits with the synthetic peptide corresponding to the N-terminal 18 amino acid residues. The immunogen was first conjugated to keyhole limpet hemocyanin (KLH) (20) and was emulsified in Complete Freund's adjuvant. Intradermal injections were given at multiple sites. Booster injections were given in incomplete Freund's adjuvant. Antibody titer was assayed by enzyme-linked immunosorbent assay (ELISA) . Measurement of MDGFl antibody titer by ELISA: Microensa plates (Dynatechimmunolon 11, Dynatech Laboratories, Inc, Chantilly, VA) were coated overnight at 37°C with the growth factor (200 ng) or synthetic peptide (100 ng) in 100 μl of phosphate-buffered saline (PBS) . The plates were washed three times with PBS containing 0.5% Tween-20 and blocked for 60 min with 1% BSA in PBS. The plates were washed again and 100 μl of serial dilutions (1-100 to 1-6400) of rabbit serum (preimmune and immune) in PBS containing 1% BSA was added for 120 min at 4°C. After the plate was thoroughly washed- with PBS-Tween, incubation was carried out with goat anti-rabbit immunoglobulin conjugated to horseradish peroxidase (Sigma, St. Louis, MO) for an hour at 4°C. The plates were then washed four times with PBS-Tween and incubated with ABTS (2,2' azino-bis (3 ethyl benzthiazoline-6-sulfonic acid) diazonium salt in citric acid solution containing 0.012% hydrogen peroxide. The color absorbance was measured at 405 nm using a UR raicroplate Reader (MR 700 Reader, Dynatech Labs) . Radioreceotor Assay: Radioreceptor assay was performed by simultaneous incubation of test samples with [¹²⁵I]-MDGFl using membranes from A431 cells. About 5 μg of purified factor was iodinated using lodogen method (Specific activity 62 μCi/μg) . Aliquots of membranes (2.5 μg protein) were incubated with [¹²⁵I]-MDGFl (40,000 cpm/well) in the presence of increasing dilutions of conditioned medium in a total volume of 150 μl of binding buffer (IMEM with 50 mM HEPES pH 7.4 containing 0.1% BSA). Following a two hr incubation at room temperature, the wells were washed extensively, bottoms were cut and counted in a gamma counter (Model B 5002, Packard Instrument Co.). Triplicate assays were done on each sample.

Radioreceptor assay and receptor crosslinking to reveal a 120-140 KDa binding site has been previously reported in the literature. See Bano et al, J. Biol. Chem. 265:1874-1880 (1990) .

Preparation of lysates and western immunoblotting: Cells were washed twice with PBS and then solubilized in SDS sample buffer (67 mM Tris-HCI, pH 6.8, 2% SDS, 10% glycerol, 2% β-mercaptoethanol, 0.3% bromophenol blue) at 100°C for 5 min. Proteins in the cell lysates were loaded on the stacking gel (3.2%) and separated in the resolving gel with 12.5% acrylamide, under conditions of constant current and running at 30 mA per gel. The proteins were then transferred from SDS-PAGE onto 0.45-μm nitrocellulose filters (Schleicher & Schuell, Inc., Keene, NH) using electroblotting techniques. The transfer buffer contained 150 mM glycine, 20 mM Tris in 20% methanol and the transfer was performed for one hour at 150 EiA constant current per gel at room temperature using a Hoefer trans-blot apparatus. After the transfer, filters were preincubated with 3% BSA in tris-buffered saline containing Tween (TBST, 10 mM Tris-HCI, pH 7.5, 150 mM NaCl, 0.05% Tween- 20) overnight at 40°C. A rabbit polyclonal antiserum raised against the synthetic peptide was used as the first antibody and a goat anti-rabbit antiserum (IgG) linked to alkaline phosphatase (Promega) was used as the second antibody. The blots were then transferred to color developing solution containing NBT (nitro blue tetrazolium 50 mg/ml in 70% dimethyl formamide) and BCIP (5-bromo-4-chloro-3-indolyl phosphate, 50 mg/ml in dimethyl formamide) in alkaline phosphatase buffer (Promega) (100 mM Tris-HCI, pH 9.5, 100 mM NaCl, 5 mM MgCl₂) . Positive reaction appears as purple bands on the nitrocellulose membrane.

Pulse labeling with r³⁵S~| Methionine and f³⁵S] Cysteine followed by immunoprecipitation: Cells, growing in IMEM to 80% confluency, were washed twice with PBS and were incubated for two hrs at 37°C in IMEM free of methionine and cysteine. The medium was replaced with fresh serum-free IMEM (lacking in methionine and cysteine), containing 200 μCi/ml [³⁵S]-cysteine and [³⁵S]-methionine (Amersham, Arlington Heights, IL, 1175 Ci/mmol) . The labeled cells were collected and washed twice with PBS and lysed in one ml of radioimmune precipitation assay buffer (RIPA-buffer, 100 mM Tris-HCI, pH 7.4, 300 mM NaCl, 2% Triton xlOO, 2% Na-deoxycholate, 0.2% SDS, 0.4% BSA and 2 mM PMSF) . The lysate was cleared by centrifugation and stored at -70°C ³⁵S-labeled proteins, released into the conditioned medium were harvested after 16-24 hrs and clarified by dialysis and centrifugation.

Immunoprecipitation of cell lysates or conditioned media was performed after incubation with antibodies (specific or nonspecific) for two hrs at 37°C. Ten mg of inactivated S.aureus were added and the immunoprecipitates were washed extensively with RIPA buffer. Finally the immunoprecipitates were solubilized in sample buffer at 100°C for 10 min and were electrophoresed on denaturing polyacrylamide gels (12.5%). Gels were fixed with 10% acetic acid, treated with Enlightening (Dupont) , dried and exposed to X-ray film at -70°C.

In vitro translation of mRNA and immunoprecipitation: Total RNA from MDA-MB 231 and HBL-100 cells was subjected to in vitro translation using wheat germ extract. Approximately 10 μg of each RNA were translated for 90 min at 25°C in a 50 μl reaction volume containing 100 μCi of [³⁵S]-methionine (1200 Ci/mmol) . The translated products were immunoprecipitated in the presence of specific and non-specific antisera. The precipitates were analyzed by SDS-PAGE as described above and were detected by autoradiography. Tunicamvcin treatment: Tunicamycin (Sigma, St.Louis, MO) was prepared by dissolving in 50 mM sodium carbonate buffer (pH 10.0) and filter-sterilized with 0.22 μm filter. Confluent monolayers of HBL-100 and MDA-MB 231 cells were incubated in IMEM in the presence or absence of 20 μg/ml of tunicamycin for 4 hrs prior to metabolic labeling, which was performed as described above. Immunoprecipitation was carried out using MDGFl antisera or prebleed sera and the immunoprecipitated products were run on a 12.5% SDS gels as described earlier and autoradiographed.

N-Glycanase digestion: About 0.5-1.0 μg of purified 62 kDa MDGFl was dried in a Speed-Vac Concentrator (Savant) , redissolved in 0.5% SDS and 1.0% 0-mercaptoethanol and diluted 5 fold into N-glycanase incubation buffer (0.2 M NaP0₄, pH 8.6, 1.25% Nonidet P-40) and treated with 10 units/mi of N-glycanase (Boehringer Mannheim, W. Germany) for 16 hrs at 37°C. Samples were run on a 12.5% SDS-PAGE gel, subjected to western blotting analysis and silver-stained.

Phosphotyrosine analysis: Monoclonal anti- phosphotyrosine was obtained from Amersham, UK. EGF receptor monoclonal antibody (mouse IgG, # 528) was purchased from Oncogene Science (Manhasset, N.Y.). Cell lysates from MCF-7, MDA-MB 468 or 184A1N4 were prepared after 20 min incubation with the growth factors (MDGFl and EGF) . Cells were lysed in 20 mM Tris-HCI (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA and 2 mM PMSF and leupeptin. Lysate samples (50 μg) were run on 7.5%. SDS-PAGE and transferred to nitrocellulose filters as described earlier. After blocking with 3% BSA, blots were incubated with 2 μg/ml of monoclonal anti-phosphotyrosine for 3 hrs and with anti-mouse IgG conjugated to alkaline phosphatase (Promega) for one hour, with extensive washings in between. The blots were developed with NBT and BCIP (Promega). Alternatively, [¹²⁵I]- Protein A (150,000 cpm/ml, NEN Products, Dupont) was used in the place of second antibody and autoradiography was performed on the dried blots.

RESULTS MDGFl partial seguencing and antibody preparation: The N-terminal 18 amino acid sequence of MDGFl was determined as indicated in Experimental Procedures. A search of the protein sequence data banks with FASTP program revealed that MDGFl was not closely related to any other growth factor (or protein) . The 18 amino acid partial sequence was analyzed for hydrophilicity and flexibility to attempt to predict antigenic sites. As shown in Table 1, residues 13-17 scored high for both parameters, indicating a high probability of an antigenic site in this region. A synthetic peptide corresponding to the sequence was prepared and coupled to KLH to be used as an antigen to raise MDGFl antisera in rabbits. ELISA results indicated that the synthetic peptide generated antisera with moderately high titer, as shown in Table 2, which could be used for further assays. Table 1. N-terminal partial amino acid sequence

I-P-V-K-Q-A-V-H-G-Q-F-L-L-P-K-Q-E-K

Ah = 0.42 (2-7)

Ah = 0.58 (4-9)

Ah = 1.23 (13-18)

B[Norm] = 0.977 (6-12)

B[Norm] = 0.994 (2-8)

3[Norml = 1.043 (11-17)

Amino acid sequencing was carried out for the first 18 N-terminal residues of MDGFl. Using the Geneprobe program, the method of Hopp and Woods was used to predict average hydrophilicity (Ah), and chain flexibility (B[Normj). The residues 13-17 represent overlap of the highest score for both parameters, indicating a high likelihood of an antigenic site localized here.

Further, in accordance with the present invention, it has been discovered that position 10, residue Q, may be eliminated without abolishing the ability of the peptide to elicit anitserum which is reactive to authentic MDGF-1. Instead of the sequence:

I-P-V-K-Q-A-V-H-G-Q-F-L-L-P-K-Q-E-K

the following sequence may be used for antiserum generation: I-p-V-K-Q-A-V-H-G-F-L-L-P-K-Q-E-K

In this application the standard one letter-abbreviation system is utilized. See Organic Chemistry of Biological Compounds. Barker (Prentice Hall 1971) .

SA test of ol clonal anti-MDGFl

About 200 ng of MDGFl was adsorbed to ELISA wells and an ELISA assay was carried out as described in Experimental Procedures. MDGFl peptide antisera or preimmune sera from rabbit was used at different dilutions and the color absorbance was read at 405 nm using a microplate Reader.

The factor purified from milk was subjected to SDS-PAGE and western blotting using the rabbit polyclonal antisera at a dilution of 1-500. The antibody detected a major MDGFl cross reactive species as expected at 62 kDa (Fig. IA, lane 2) . [¹²⁵I]-MDGFl was subjected to immunoprecipitation using MDGFl antisera at two dilutions (1-100 and 1-250) . As shown in Fig. IB, a major 62 kDa band was detected (lanes 3 and 4) which was not present when preimmune sera was used (lanes 1 and 2) . The band diminished in the presence of increasing concentrations of unlabeled peptide (Fig. 1C, lanes 6 and 7) . 10 ng of unlabeled MDGFl gave complete inhibition of the antibody reaction to [¹²⁵I]-MDGFl.

MDGFl in normal and malignant breast cell lines: To determine whether various human mammary cell lines produce MDGFl, conditioned medium from the cell lines was analyzed by radioreceptor assay using the purified, labeled factor. In these experiments, normal 184 mammary epithelial cells, the non-malignant HBL-100 breast cell line and the highly malignant MDA-MB 231 hormone independent lines were used. As shown in Table 3 (panel A) , undiluted, 100-fold concentrated medium conditioned by HBL-100, MDA-MB 231 and 184 showed maximum competition for the iodinated factor. The undiluted conditioned medium contained approximately 2-5 ng of MDGFl receptor binding activity per ml of medium. In contrast, the three other cell lines MCF-7 (estrogen receptor positive) , MDA-MB 468 (estrogen receptor negative) or 184A1N4 (carcinogen immortalized) did not show significant competing activity (Table 3, panel A). In order to test whether the factor made by the above cell lines is biologically active, the conditioned media were subjected to partial purification on isoelectric focusing column (IEF) and the fractions with a pi of 4.8 were tested for biological activity. Normal rat kidney (NRK) cells were used in accordance with a well-known procedure to determine the optimal cell growth and collagen synthesis stimulation of the IEF fractions. The results shown in Table 3 (panel B) suggest that the activity present in the conditioned media are biologically active in stimulating the growth of NRK cells and also in stimulating the synthesis of collagen. Purification by IEF and assay for collagen synthesis were carried out according to the previously published methods.

The polyclonal MDGFl antibody was also used to detect the 62 kDa growth factor in cell lysates and conditioned media from the two human mammary cell lines HBL-100 and MDA-MB 231. Metabolically labeled media from HBL-100 cell cultures were immunoprecipitated with antiserum as described earlier. Fig. 2A shows the presence of a 62 kDa immunoreactive band, (lanes 5 and 6) which disappeared in the presence of increasing concentrations of unlabeled factor (lanes 7 and 8) . Ten nanogram per ml of unlabeled MDGFl competed maximally for antibody reaction. Similar results were obtained with [³⁵S]-labeled MDA-MB 231 conditioned media.

Western blotting of cell lysates is depicted in Fig. 2B. MDA-MB 231, HBL-100 and 184 cell lysates showed an immunoreactive band at 62 kDa. Immunoprecipitated product derived from in vitro translation of MRNA from an immortalized, but non-malignant, HBL-100 cell line yielded a protein band with a molecular weight around 55 kDa (Fig. 3A, lane 2) . The same result was observed with MDA-MB 231 cell MRNA. In order to establish that MDGFl might be a glycoprotein, tunicamycin treatment, metabolic labeling and immunoprecipitation were done and the result is illustrated in Fig. 3B. Lane 2 depicts the 62 kDa band in the absence of tunicamycin and lane 3 shows a lower molecular weight band (55 kDa) after tunicamycin treatment. Lanes 1 and 4 are immunoprecipitates with prebleed sera in the presence (lane 4) or absence (lane 1) of tunicamycin. The result presented in Fig. 3C demonstrates that treatment of purified growth factor with N-glycanase leads to a reduction in molecular weight, again to 55 kDa (lane 2) indicating that the mature 62 kDa MDGFl contains N-linked carbohydrates.

Interaction of MDGFl with its receptor: The mechanism of MDGFl was studied to see whether it stimulates the accumulation of phosphotyrosine on cellular proteins as determined by western blot with an anti-phosphotyrosine antibody, as detected by an immune peroxidase reaction product. For these studies, cell lines (MCF-7, MDA-MB 468 or 184A1N4) were initially chosen which responded to both EGF and MDGFl, so that direct comparison could be made. As depicted in Fig. 4A, treatment of MDA-MB 468 cells with 10 ng of EGF stimulated phosphorylation on tyrosine of the 170 kDa EGF receptor as expected (lane 6) . This phosphorylation was blocked by preincubating the cells with 100 ng of a monoclonal antibody (# 528 IgG) directed against the binding site of the EGF receptor (lane 5) . Treatment of the cells with MDGFl stimulated appearance of a phosphotyrosine band at 180-185 kDa (lane 3) . In contrast to EGF receptor phosphorylation, MDGFl induced phosphorylation was not blocked by 528 IgG monoclonal antibody. A similar result was obtained with 184A1N4 cell lysates. Fig. 4B illustrates the western blot for MCF-7 cell lysates treated in the absence (lane 1) or presence of 10 nM of EGF (lane 2) and MDGFl (lane 3). While EGF only slightly stimulated autophosphorylation of its receptor, MDGFl again induced strong tyrosine phosphorylation of a 180-185 kDa protein. Some of these data were confirmed by detection of the phosphotyrosine-containing band by an alternate means. Figs. 4C and 4D show the result of the autoradiogram of the western im unoblot of MDA-MB 468 and 184A1N4 cell lysates after detection of anti-phosphotyrosine and antibody reaction with [¹²⁵I]-protein A. Lane 2 shows cell lysate stimulated with 10 ng of MDGFl and lane 1 is control lysate. The same 180-185 kDa phosphoprotein is detected.

In order to show that EGF and MDGFl are phosphorylating different receptors, an experiment was conducted by incubating A431 and MDA-MB 468 cells with or without growth factors (25 ng of EGF or MDGFl) . Then the EGF receptor was immunoprecipitated with EGF receptor antibody-1 (clone 528, Oncogene Science) . The washed immunoprecipitates were subjected, for l min at room temperature to a phosphorylation reaction mixture containing [γ-³²P] ] ATP, leading to self- phosphorylation. The immunoprecipitates were electrophoresed on 7.5% PAGE followed by autoradiography. Results indicated that self-phosphorylation of EGF receptor was enhanced by EGF but not by MDGFl.

DISCUSSION Human milk is probably one of the richest sources of growth factors known. The presence of growth factors in milk indicates that these activities may be produced by normal mammary epithelial cells. A series of other observations suggest that growth factors, such as TGF-α or EGF may also perform normal physiological functions during the growth and development of the mammary gland during pregnancy and pathophysiologic functions in development of breast cancer. Growth factors could have autocrine, paracrine and possibly even endocrine functions in proliferation of both normal and malignant breast tissue.

We have already detected an acidic growth factor with an apparent molecular mass of 62 Kda. The activity named mammary-derived growth factor 1 or MDGFl was purified to homogeneity. MDGFl differentially stimulated the synthesis of collagen IV and it binds to high affinity binding sites on normal rat, mouse and human mammary epithelial cells and A431 human epidermoid carcinoma membranes.

N-terminal amino acid sequence of MDGFl illustrated that it is not related to any other growth factor or protein. When the sequence was analyzed for hydrophilicity and flexibility, the result indicated that there was a high probability of an antigenic site in the region between 13-17 residues (Table l) , indicating that the synthetic peptide can be employed to induce antibodies that are reactive with native protein. Hence, a synthetic peptide was prepared and used to immunize rabbits to elicit MDGFl antiserum. ELISA assays showed that a 1:1000 dilution of the antisera could be used to detect MDGFl (Table 2) .

The polyclonal antibody was used to detect MDGFl in human milk and breast cancer cell line extracts by western blot. HBL-100 cell line which is initially derived from a spontaneously immortalized, milk-derived mammary epithelial cell, and MDA-MB 231 which is a hormone independent breast cancer cell line, were used in the i munoblot analysis. A major specific band was detected as expected with a molecular weight of 62 kDa (Fig. IA) . Immunoprecipitation of iodinated MDGFl using the antiserum was shown to recognize an immunoreactive MDGFl (Fig. IB) which diminished in the presence of unlabeled growth factor (Fig. 1C) . The antisera has also been used to detect MDGFl in primary human breast cancer biopsies by ELISA and immunostaining procedures. When HBL-100 and MDA-MB 231 cells were metabolically labeled and the conditioned media were subjected to immunoprecipitation, or the cell lysates were used in western blot analysis, a major specific band was detected at 62 kDa area (Figs. 2A and 2B) . Similar results were obtained with milk and partially purified milk fractions on western blot analysis using the polyclonal antiserum.

In vitro translation of mRNA from these two cell lines followed by immunoprecipitation using MDGFl antiserum indicated the molecular weight of the putative precursor of the MDGFl to be slightly lower than that of the native protein (Fig. 3A) . This shift in the molecular weight might be due to the glycosylated nature of the factor. Experiments with tunicamycin (Fig. 3B) and N-glycanase (Fig. 3C) strongly suggest this possibility.

Tyrosine phosphorylation appears to be closely involved in the regulation of cell growth or transformation. We have further characterized the mechanism of MDGFl action to evaluate whether MDGFl can stimulate the accumulation of phosphotyrosine on cellular proteins in breast epithelial cells. For this purpose cell lines were initially chosen which respond to both EGF and MDGFl so that direct comparisons could be made. Treatment of 184A1N4 or MDA-MB 468 cells with MDGFl or EGF stimulated tyrosine phosphorylation of proteins of 180-185 kDa and 170 kDa respectively (Fig. 4A, lanes 2 and 6) . Preincubating the cells with excess of blocking raonoclonal antibody to EGF receptor did not cause any change in the phosphor action of MDGFl treated cell lysate (lane 3). This indicates that although the two growth factors induced phosphorylation of similar size (but non-identical) proteins, that indeed the proteins were uniquely associated with their respective receptors. This was confirmed with MCF-7 cells, a cell line responsive to both growth factors, but with only a few thousand EGF receptors per cell. Addition of EGF to MCF-7 cell lysates failed to significantly stimulate the tyrosine phosphorylation of the EGF receptor (Fig. 4B, lane 2) whereas, MDGFl again strongly enhanced phosphorylation of 180-185 kDa band (lane 3). Further experiments with immunoprecipitated EGF receptor from A431 and MDA-MB 468 cell lysates revealed that autophosphorylation of EGF receptor was enhanced in the presence of EGF but not by the addition of MDGFl. These results strongly indicate that the phosphotyrosine-modified protein detected in response to EGF or MDGFl stimulation are different entities.

In accordance with one aspect of the present invention, polyclonal or monoclonal antibodies may be generally prepared against MDGFl and/or MDGFl receptors using methodologies as described, for example, in U.S. Patents 4,151,268, 4,197,237 and 4,123,431. Each and all of these patents are hereby incorporated in the entirety herein. The antibodies provided by the present invention are immunoreactive against MDGFl and/or MDGFl receptors. Generally, in accordance with the present invention, the antibodies produced are immunoreactive against a partial N-terminal amino acid sequence of the formulas:

j-p-V-K-Q-A-V-H-G-Q-F-L-L-P-K-Q-E-K

or other closely related formulas.

For example, as noted above the sequence:

I-P-V-K-Q-A-V-H-G-Q-F-L-L-P-K-Q-E-K

may be replaced with the sequence:

I-P-V-K-Q-A-V-H-G-F-L-L-P-K- -E-K

wherein I is isoleucine, P is proline, V is valine, K is lysine, E is glutamic acid, A is alanine, H is histidine, G is glycine, Q is glutamine, F is phenylamine and L is leucine.

Thus, the present antibodies are prepared against any growth factor which is related to MDGFl. By the term "related" is meant that the growth factor, at least, have the above amino acid sequence as an N-terminal sequence.

Further, monoclonal antibodies against MDGFl peptides can be prepared using conventional methodologies. In turn, these monoclonal antibodies may be used to identify MDGFl to detect and quantify MDGFl, or even to purify MDGFl by immunoaffinity chromatography. MDGFl may be used for a variety of purposes including treatment of a variety of pathologic conditions.

Additionally, in accordance with the present invention, antibodies, either polyclonal or monoclonal, may be used to attenuate or otherwise modulate the effect of MDGFl in a host. Any number of techniques may be used, in accordance with the present invention, for rendering MDFG1 peptides immunogenic.

For example, MDGFl peptides may be rendered immunogenic by conjugation with muramyl peptides as described in U.S. Patents 4,639,512 and 4,461,761. Also, MDGFl peptides may be rendered immunogenic by conjugation with other polypeptides as described in U.S. Patent 4,812,554. Each of U.S. Patents 4,639,512, 4,461,761 and 4,812,554 are incorporated herein in the entirety.

In more detail, the monoclonal and/or polyclonal antibodies of the present invention may be used to therapeutically detect and monitor the amount of MDGFl in mammalian serum. Generally, any suitable means of detection may be used. However, it is preferred that a two-site im unoassay be used. A two-site, two-step immunoradiometric procedure is generally described in U.S. 4,353,982, which is incorporated herein in the entirety.

In such a technique, an insoluble support is provided having a primary antibody attached thereto. The primary antibody may be bonded to the insoluble support by conventional processes.

The insoluble support having a primary antibody attached thereto is contacted with mixture containing serum sample in an aqueous dilution buffer for a time adequate to permit conjugation of the primary antibody with the peptide analyte. The insoluble support is then separated from the primary reaction mixture.

Then, the insoluble support is contacted with an aqueous conjugate dilution buffer solution containing a secondary antibody which binds selectively with the peptide analyte for a time adequate to permit conjugation of the secondary antibody to the peptide analyte. The secondary antibody is coupled to a label. But for the label, however, the secondary antibody is preferably the same as the first antibody.

Thereafter, the label attached to the insoluble support is detected and the amount of peptide analyte present is determined. The label may be a physically detectable label such as a radiolabel, fluorophore or chromophore or an enzyme, which is subsequently reacted with a substrate which yields a physically detectable reaction product.

As noted above, primary antibody can be bonded to the insoluble support by conventional processes. Procedures for binding of antibodies to insoluble supports are described in U.S. Patents 3,551,555, 3,553,310, 4,048,298 and RE-29,474, for example, all of which are incorporated herein in the antirety. 3inding of antibodies to polystyrene by adsorption has been described in U.S. Patents 3,646,346 and 4,092,408, for example, which are incorporated herein in the entirety.

A variety of materials can be used as the insoluble support, the most important factor being the binding of the primary antibody to the surface, the absence of interference with the conjugation reactions or with other reactions which can be employed to determine the presence and extent of the conjugating reaction. Organic and inorganic polymers, both natural and synthetic, can be used as the insoluble support. Examples of suitable polymers include polyethylene, polypropylene, polybutylene, poly(4-methylbutylene) , butyl rubber, silastic polymers, polyesters, polyamides, cellulose and cellulose derivatives, such as cellulose acetate, nitrocellulose and the like, acrylates, methacrylates, vinyl polymers, such as polyvinyl acetate, polyvinyl chloride, polyvinylidene chloride, polyvinyl fluoride, and the like, polystyrene and styrene graft copolymers, rayon, nylon, polyvinylbutyrate, polyformaldehyde, for example. Other materials which can be used as the insoluble support can be the latexes of the above polymers, silica gel, silicon wafers, glass, paper, insoluble protein, metals, metalloids, metal oxides, magnetic materials, semi-conductive materials, cermets and the like. Further, substances which form gels, such as proteins such as gelatins, lipopolysaccharides, silicates, agarose, polyacrylamides or polymers which form several aqueous phases such as dextrans, polyalkylene glycols (alkylene with 2 to 3 carbon atoms) or surfactants, e.g. amphophilico pounds such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like may also be used.

A preferred diagnostic support entails a polystyrene or styrene copolymers such as styrene-acrylonitrile copolymers, or polyolefins such as polyethylene or polypropylene, and acrylate and methacrylate polymers and copolymers. The primary antibody can be coordinated or bound to the insoluble support by adsorption, ionic bonding, van der Waals adsorption, electrostatic bonding, or other non-covalent bonding, or it can be bound to the insoluble support by covalent bonding. Preferred forms of insoluble supports are beads, microwells and dipsticks.

The aqueous dilution buffer may be a conventional buffer such as one containing a phosphate buffer, pH base 7.5 having a 0.1% sodium azide preservative. Such a buffer is described in U.S. 4,267,271, which is incorporated herein in the entirety. Additionally, the buffer may also contain a sulfhydryl group protecting agent such as is described in U.S. 4,267,271, which is incorporated herein in the entirety.

Generally, the buffer solution contains one or more buffering agents which are compatible with the other components of the solution and with the sulfhydryl group protecting agent, such as mercaptoethanol. Examples of buffers which are suitable for use with sulfhydryl group stability agents are tromethamine (Tris) , triethanolamine, imidazole acetate, imidazole and bis-(2-hydroxyethyl)-imino- tris-(hydroxymethyl)methane (bis-Tris) , for example. Generally, the buffer solution has a molar concentration of from 0.01 to 0.5 M and preferably from 0.05 to 0.2 M. In general, the buffer solution has a pH of from 6 to 9, preferably form 7 to 8.5.

The sample dilution buffer solution preferably contains multivalent cation inhibiting amount of chelating agent. The amount depends upon the choice of chelating agent. Suitable chelating agents include ethylenediamine tetracetic acid and the alkali metal and ammonium salts thereof (EDTA) . When the chelating agent is EDTA, the preferred amounts in the solution is within the range of from 0.01 to 0.1 (w/v)%.

The sample dilution buffer solution also preferably contains an antimicrobial agent such as sodium azide and the like which does not interfere with the immunochemical reactions. The preferred concentrations of sodium azide in the solution are from 0.017 to 0.3 wt.%.

The sample dilution buffer solution also preferably contains a quantity of accelerating agent sufficient to increase the analyte with primary antibody conjugation rate. Preferred accelerating agents are polyethylene glycols having molecular weights within the range of from 1000 to 8000 and optimally from 7000 to 8000. The accelerating agent concentration is preferably within the range of from 0 up to 2 wt.% in the solution.

The incubation between the primary antibody and sample is continued until substantial conjugation of the primary antibody with the analyte, if any is present, can occur. The time is dependent upon the size of the analyte and the temperature of the solution. Suitable incubation times are from about 2 to 240 minutes at temperatures within the range of from 15 to 45°C, the preferred incubation or contact time being within the range of from 5 to 45 minutes at the preferred temperatures within the range of from 18 to 30°C.

The aqueous dilution buffer solution is then rinsed from the insoluble support. Suitable rinse solutions are buffered solutions which do not leave a residue which will interfere with the second incubation step in the method of this invention. Conventional buffer solutions such as buffered salines, phosphate buffer solutions and borate buffer solutions can be used. A preferred rinse buffer solution is a phosphate buffer solution having a phosphate molarity of from 0.02 to 0.2 M and a pH of from 7 to 8.7.

In the second step, the insoluble support is contacted with an aqueous conjugate dilution buffer solution containing a secondary antibody which binds selectively with the analyte for a time sufficient to permit secondary antibody conjugation with analyte. The antibody is coupled with a label, but is otherwise preferably the same as the primary antibody. The aqueous conjugate dilution buffer solution can contain agents which reduce non-specific binding, accelerate the secondary conjugation reaction, and inhibit microbial growth. The buffering agents in the aqueous conjugate dilution buffer solution can be any conventional buffering agents which provide the desired pH range and do not interfere with the secondary conjugation reaction. Conventional buffer solutions such as buffered salines, phosphate buffer solutions and borate buffer solutions can be used. A preferred rinse buffer solution is a phosphate buffer solution having a phosphate molarity of from 0.02 to 0.2 M and a pH of from 7 to 8.7.

The aqueous conjugate dilution buffer solution is mixed with the secondary antibody for use in the second step of the immunoassay. While the secondary antibody is preferably the same as the primary antibody, the secondary antibody can be any monoclonal antibody, mixture of monoclonal antibodies, polyclonal antibodies or mixtures thereof which bind selectively with the analyte being determined in the assay. These antibodies are preferably coupled to a label. However, any physically detectable label or moiety which can be further treated to yield a physically detectable label can be used as the label.

The labels can be coupled with the secondary antibody by conventional procedures for attaching labels to proteins. The labels can be bonded or coupled to the protein reagents by che ical or physical bonding. Ligands and groups which can be conjugated to the secondary antibodies of this invention include elements, compounds or biological materials which have physical or chemical characteristics which can be used to distinguish the reagents to which they are bonded from compounds and materials in the sample being tested. A wide variety of such labels and methods for coupling them to antibodies are well-known and conventional in the art.

Radiolabeled secondary antibodies can be used in the method of this invention. The specific activity of a tagged antibody depends upon the half-life, isotopic purity of the radioactive label and how the label is incorporated into the antibody. Table 3 lists several commonly used isotopes, their specific activities and half-lives. In immunoassay tests, the higher the specific activity, in general, the better the sensitivity.

-38-

TABLE 3 Specific Activity of Pure

Isotope Isotope (Curies/mole) Half-Life

14, 6.25 X 10¹ 5720 years 2.91 X 10⁴ 12.5 years

35c 1.50 X 10⁵ 87 days

125_χ 2.18 X 10⁶ 60 days

32_? 3.16 X 10⁶ 14.3 days

131_τ 1.62 X 10⁷ 8.1 days

The antibodies of the present invention may be labeled with radioisotopes using conventional and well known processes. For example, U.S. 4,302,438 describes tritium labeling procedures, and is incorporated herein in the entirety. Also, U.S. 3,867,517 and 4,376,110 describe procedures for iodinating antibodies, and are both incorporated herein in the entirety.

Antibodies labeled with enzymes are particularly useful in the method of this invention. Examples of suitable systems, coupling procedures and substrate reactions therewith are disclosed in U.S. Patents Re. 31,006, 4,214,048, 4,289,747, 4,302,438, 4,312,943, 4,376,110 and the references cited therein, for example. Each of these patents are -39-

incorporated herein in the entirety. Examples of other suitable systems are described by Pesce et al, Clin. Chem. 20(3) :353-359 (1974) and Wisdom, G. , Clin. Chem. 22:1243 (1976) .

A list of suitable enzyme classes and specific examples therefor are recited hereinbelow in Table 4:

TABLE 4

Class Enzyme Example

Hydrolases Carbohydroases Amylases

Nucleases Polynucleotidase

Amidases Arginase

Purine deaminases Adenase

Peptidases Aminopolypeptidase

Proteinases Pepsin

Esterases Lipases

Iron Enzymes Catalase

Copper Enzymes Tyrosinases

Enzymes containing Coenzymes Alcohol dehydrogenase

Enzymes reducing cytochrome Succinic dehydrogenase

Yellow enzymes Diaphorase

Mutases Glyoxalase

Demolases Aldolase

Oxidases Glucose oxidase Horse radish peroxidase

Phosphatases Alkaline phosphatase Other enzymes 0-galactosidase Phosphorylases Hexokinases

A list of suitable enzymes are described in Hawk, et al. PRACTICAL PHYSIOLOGICAL CHEMISTRY, New York: McGraw-Hill pp. 306-397 (1954) .

Fluorogenic enzymes (enzymes in the presence of which a selected substrate will produce a fluorescent product) are useful labeling moieties. Methods for selectively conjugating enzymes to antibodies without impairing the ability of the antibody to bind with antigen are well known in the art. Suitable enzymes and procedures for coupling them to antibodies are described by Wilson, M. et al. Recent developments in the periodate method for conjugating horseradish peroxidase (HRPO) to antibodies. INTERNATIONAL CONFERENCE IN IMMUNOFLUORESCENCE AND RELATED STAINING TECHNIQUES. W. Knapp et al, editors. Amsterdam: Elsevier pp. 215-244 (1978), Sullivan, M. et al. Enzyme immunoassay: a review. Annals of Clinical Biochemistry. 16:221-240 (1979) and in U.S. Patent 4,190,496, for example. The preferred fluorogenic enzymes and suitable substrates corresponding thereto include horseradish peroxidase for which a suitable substrate is homovanillic acid or 4-hydroxy-3-methoxy- phenylacetic acid, 8-galactosidase for which a suitable substrate is 4-methylumbelliferyl-/3-D-galactoside, alkaline phosphatase for which a suitable substrate is 4-methylumbelliferyl phosphate and other umbelliferyl phosphates such as 4-carboxyumbelliferyl phosphate and umbelliferyl phosphate 4-carboxyalkyl-esters, etc.

Fluorescent labeled antibodies can be prepared from standard fluorescent moieties known in the art. Since antibodies and other proteins absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm and preferably above 400 nm. A variety of suitable fluorescers are described by Stryer, Science. 162:526 (1968) and Brand, L. et al, "Fluorescent probes for structure," Annual Review of Biochemistry. 41:843-868 (1972). The antibodies can be labeled with fluorescent groups by conventional procedures such as those disclosed in U.S. Patents 3,940,475, 4,289,747 and 4,376,110, for example, which are each incorporated herein in the entirety.

The aqueous conjugate dilution buffer solution can contain from 0.5 to 10 wt.% polyethylene glycol and optimally contains from 1 to 5 wt.% polyethylene glycol. The polyethylene glycol can have a molecular weight within the range from 1,000 to 8,000 and preferably within the range from 2,000 to 4,000. This reagent accelerates the rate of secondary conjugation. Excess amounts of polyethylene glycol may cause a precipitation of protein or flatten the response curve.

The conjugate dilution buffer solution can contain a non¬ specific binding inhibiting amount of a conventional non- immune serum or other water-soluble animal protein or water- soluble amino acid polymer. It preferably contains from 3 to 10 (v/v)% of non-immune protein or from 3 to 10 wt.% of the animal protein or amino acid polymer. Examples of suitable conventional and well-known non-immune serums include bovine serum albumins (BSA) , human (HSA) , rabbit (RSA) , goat (GSA) , sheep (SHA) , horse (HOSA) , etc. ; serum gamma globulin, of the previously described animals and other animal proteins such as ovalbumin, fibrinogen, thrombin, transferrin, glycoproteins, etc. Examples of water-soluble amino acid polymers include polylysine, polyglutamic acid, polyalanine, polyhistidine, polymethionine, and polyproline, for example.

The conjugation dilution buffer solution also preferably contains an amount of antimicrobial agent sufficient to inhibit microbial growth in the solution. The solution preferably contains from 0.01 to 0.2 wt.% of antimicrobial agent such as sodium ethylmercuric thiosalicylate or one or more of other antimicrobial agents which do not interfere with the secondary conjugation reaction. The incubation between the secondary antibody and the insoluble support is continued until substantial conjugation of the secondary antibody with the analyte, if any, present on the insoluble support can occur. The time is dependent upon the temperature of the solution. Suitable incubation times are from about 2 to 240 minutes at temperatures within the range of from 15 to 45°C, the preferred incubation or contact time being within the range of from 5 to 45 minutes at the preferred temperatures within the range of from 18 to 30°C.

The insoluble support is then rinsed to remove residual conjugate dilution buffer solution. The rinse solution should be free of any materials which would interfere with the subsequent label determination procedure. The rinse solution described above is suitable.

The label adhering to the insoluble support is then determined. If the label is a physically detectable moiety such as a radiolabel, chromophore or fluorophore, for example, it can be measured directly using suitable, conventional methods and devices.

If the secondary antibody label is an enzyme, the enzyme adhering to the insoluble support is determined by reacting it with a substrate which undergoes a chemical reaction in the presence of the enzyme to yield a physically detectable reaction product. The physically detectable reaction product produced by the reaction is then determined. The present invention also provides a test kit which may be used diagnostically in the detection of the above-disclosed peptides. In essence, the test kit may be used to detect the presence of MDGFl or may also be used to determine the amount of MDGFl present to ascertain whether antibodies should be administered to attenuate the effect of MDGFl.

Generally, the test kit of the present invention is for the detection of receptor blocking peptides of fibroblast growth factor receptor. Further, it is preferred that a two- site, two-step assay be conducted.

Generally, a mobile particulate solid phase having bound polyclonal or monoclonal antibodies thereto against the MDGFl is contacted with mammalian serum, particularly human serum.

Then, the solid-phase portion of each is removed and contacted with a tracer solution including a labelled second antibody which allows for the detection of the presence of peptide.

For example, by using the antibodies of the present invention, a test kit may be constructed analogous to that disclosed in U.S. Patent 4,624,916, which is incorporated herein in the entirety.

In more detail, the present test kit is for the detection of MDGFl and contains:

1) a solid-phase matrix containing an insoluble support having attached thereto a primary antibody against MDGFl, and having a liquid phase surrounding the solid-phase matrix; 2) a series of calibrating liquids each liquid containing a different known amount of MDGFl;

3) a wash buffer; and

4) a tracer solution containing a labeled secondary antibody against the peptide of interest.

Typically, five differing calibrations are included to provide an accurate calibration curve against the serum samples tested. However, as few as two differing calibrations or more than five of the same may be used.

In accordance with the present invention, both the primary and secondary antibodies are raised against the MDGFl, and are preferably the same but for the detectability of the second antibody.

cDNA Methodology: Molecular Cloning of cDNA Encoding MDGFl and Potentially Related Genes The availability of the amino terminal sequence of MDGFl as well as a high affinity rabbit antibody to the peptide makes two alternative approaches for cloning the cDNA for MDGFl practical. A lambda gtll cDNA expression library has been prepared by using poly A+ RNA from the human breast cancer cell line MDA MB 231. As previously noted, MDGFl is detected in the media conditioned by these cells by immunoprecipitation with the peptide antibody and western blotting of cell extracts from this cell line detects four specific bands including the major, expected 60 kDa species. The approach of screening for recombinant plaques expressing a betagalactosidase-MDGFl fusion protein using the peptide antibody may be used. Three hundred thousand plaques are plated at a density of 30,000 plaques per 150 mm petri dish. For screening, the procedure described by Mierendorf et al using Y1090(r-) as a host bacteria and the Protoblot Immunoscreening System of Promega is used. This system uses an anti-rabbit IgG alkaline phosphatase conjugate as a second antibody and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in combination with nitro blue tetrazolium (NBT) as color developing substrates.

Duplicate nitrocellulose lifts are processed with either the peptide antibody or the pre-immune rabbit serum. Areas containing plaques that give a positive signal only with the peptide antibody are picked and placed into lambda diluent [lOmM Tris-HCI (pH 7.5) , 10 mM MgCl₂] . The phage lysate is used to reinfect Y1090(r-) cells and then plated at a density that allows the subsequent isolation of a single positive plaque and the detection procedure repeated. Plating and detection are repeated a third time to insure that 100% of the plaques are now positive only for the peptide antibody. Analysis of individually isolated positive plaques involves first, the preparation of minipreps of phage DNA followed digestion with Eco RI to thereby obtain an estimate of the size of the cDNA insert. Initial sequencing reactions are performed using miniprep phage DNA with lambda gtll forward and reverse sequencing primers available from New England Biolabs. To facilitate subsequent sequencing and expression studies, the complete cDNA inserts are also amplified using the polymerase chain reaction technique with lambda gtll forward and reverse primers that also contain sequences for the generation of Not I restriction cleavage sites near the termini. This enzyme has an eight base pair GC rich recognition sequence and is therefore unlikely to occur within the cDNA. Subsequent amplification generates the complete cDNA insert flanked by lambda gtll sequences flanked by Not I sites. The PCR amplified material is digested with Not I and the size of the PCR amplified inserts is compared with the sum of the Eco RI generated fragments. The Not I digested material is then subcloned into the Not I site of the sequencing vector pGEM7zf+ and sequenced using oligonucleotides complementary to the SP6 and T7 promoter binding sites as primers. Double stranded DNA sequencing with oligonucleotides synthesized by the Lombardi Cancer Center Recombinant DNA core facility as primers is used.

The amplified cDNA contains lambda gtll sequences and from the results of the sequencing reactions, the junction of the phage and cDNA, the sense strand of the cDNA and the reading frame of the fusion protein may be determined. Since the peptide antibody was prepared against the amino terminus of the secreted peptide, it is likely that deduced amino acid sequence encoded by the cDNA contains these amino acids in the proximity of the junction of the phage and cDNA sequences. In instances where this is indeed the case, sequencing both strands of the rest of the CDNA insert with synthesized primers is continued and then the deduced amino acid sequence is compared with actual sequence information derived from V8 protease digests, or other protease successfully used to generate internal fragments, of the purified MDGFl protein. To determine if the cDNA is near full length, antisense riboprobes are synthesized for use in Northern analyses with RHA from HBL 100 and MDA MB231 RNA to determine if the size of the cDNA corresponds to the size of the mRNA. The deduced amino acid sequence is searched for the presence of a eukaryotic secretory signal sequence and the presence of an initial methionine codon that has upstream sequences that conform to the Kozak consensus sequence for initiation codons. The 3' end of the cDNA is searched for the presence of a polyadenylation addition signal. If indicated, the library may be rescreened using nucleic acid hybridization with probes taken from the extreme 5' and 3 * ends of the cDNA to obtain a full length cDNA.

It is possible that the MDGFl protein is derived from the post translational cleavage of another higher molecular weight polypeptide or that the secretory signal that is likely to be part of the protein is much further into the body of the protein than is usually encountered. In these cases, the amino terminal sequence of the MDGFl may not be in close proximity to the phage cDNA junction and additional sequencing of the cDNA inserts may be necessary.

If no positive plaques are detected with the MDA MB 231 library, an alternative strategy is employed. Two possible reasons for the failure to obtain a immunoreactive plaque are a) the cDNA is extremely rare and b) the peptide is encoded by the 5' end of an extremely large mRNA that is underrepresented in the library due the 3 ' end bias created by the use of oligo dT as a primer for reverse transcriptase in constructing the library. In such a case, two new libraries are constructed in lambda gtll using either oligo dT and randomly synthesized hexamers as primers. In this case, poly A+ mRMA from the HBL-100 cell line is used since immunoprecipitation experiments suggest that this cell line secretes more material into the media than does MDA MB 231 and would therefore be expected to contain a larger amount of the MDGFl mRNA. cDNA libraries are constructed using the method of Gubler and Hoffman with an RMA-cDNA synthesis kit. The cDNA is treated with Ecos RI methylase. Then, Eco RI linkers are then added and after digestion with ECO RI, excess linkers are removed by A-50m column chromatography according to Huynh et al. The pooled cDNA is then ligated to Eco RI digested and dephosphorylated phage arms (Promega) and packaged using a Stragene's Gigapack gold II packaging extracts.

If on the other hand, immunoreactive plaques are obtained with the MDA MB 231 library but sequencing of the inserts from these immunoreactive plaques fails to show any CDNA inserts that contain within the deduced amino acid sequence the sequence that corresponds to the amino terminal peptide, this indicates that the peptide antibody lacks the specificity that is required. In that case, the alternative approach of screening random primed and oligo dT primed HBL-100 lambda gtlO libraries with P-32 5' end-labeled oligonucleotide probes may be used. As suggested by the PROBE program of the PCGENE package of sequence analyses programs, two 26 bp probes are synthesized based on the amino acid sequence Val-Lys-Gln-Ala-Val-His-Gly-Gln. One probe contains all possible codon combinations for the amino acids whereas the second probe contains inosine at the degenerate third positions for the codons for Val, Ala, and Gly to reduce the number of possible combinations from 4096 to 16. End-labeling of pooled deoxyoligonucleotides and low stringency hybridizations and washes of replicate nitrocellulose filters containing recombinant phages are performed as described by Finch et al. Analysis of purified positive plaques are performed as described above.

Once cDNA clones are obtained and verified, by sequence analysis, for MDGFl, the cDNA clones may be used for library screening for potentially related molecules using similar procedures. For example, if an activity were obtained from a colon cancer cell line which induced MDGFl receptor phosphorylation, and there is a reason to believe that it was not MDGFl itself, then the MDGFl antibody could be used to purify and characterize the protein sequence or the cDNA could be used to clone the new factor by standard methods. The relationship of the new protein or cDNA may then be verified by standard sequence analysis. cDNA may used with cDNA libraries to get a full length cDNA and with genomic libraries to get a full length genomic clone of MDGFl by standard methodologies. Likewise, once a cDNA clone of a related MDGF- 1-like molecule is obtained, full length cDNA and genomic clones could be obtained in a straightforward manner.

Receptor Methodology

The nature of the MDGFl receptor binding site and the protein it phosphorylates on a tyrosine residue has been described above. These observations may lead to discovery of a new oncogene family, since growth factor receptor tyrosine kinase molecules are known to possess oncogenic activity in many cases. The 120-140 kDa binding component and the- 180-185 kDa phosphoprotein could be purified and sequenced by standard protein methodologies. Thus, the cDNA and genomic clones may be obtained. Antibodies may be made against both components. Thus these antibodies, cDNA and genomic probes for the receptor may be used to search for other related but non- identical receptor-tyrosine kinase-phosphoprotein molecules. Oncogenic activities of these cloned molecules may be assessed by gene transfection strategies as the normal or cancer cells. Since MDGFl protein is not related to other growth factors and since the MDGFl receptor is not known to bind other growth factors and since other growth factors are not known to induce the 180-185 kDa tyrosine-phosphoprotein it appears that the MDGFl receptor is a candidate oncogene and related to other unknown activities, thus entailing a new family.

MDGFl As A Therapeutic Agent

Since MDGFl is known to stimulate collagen synthesis selectively and potently, a therapeutic use is indicated in this respect. Further, MDGFl may be purified in large amounts using the antibody described. In addition, antibodies to block its activity may also be prepared using well known procedures.

MDGFl or its blocking antibody may be advantageously used in the treatment of inflammation, skin repair in wounding and burn healing, or in cancer, specifically using the antibody to block tumor stromal interactions or the growth factor to interfere with metastases. A blocking antibody may also interfere with tumor growth since the growth factor stimulates proliferation. In addition, MDGFl or antibody may be used in prevention of pulmonary fibrosis, hepatic or biliary cirrhosis, breast fibrocystic disease or fibroadenoma, or Ehlers-Danlos syndrome. MDGFl may also be used advantageously in the treatment of the gastro-intestinal tract to induce maturation in premature infants. Other uses of MDGFl or its antibody could be in treating bone fractures, osteoporosis, osteomalacia, bone changes due to hyperparathyroidism, Paget's disease, hypertrophic osteoarthropathy, fibrous dysplasia, fibrous cortical defect, congenital or hereditary disorders of the tendon or ligament, osteogenesis imperfecta, achondroplasia, osteopetrosis, multiple osteocartilaginous exostoses, endocondromatosis, and other bone injury due to infection, corneal problems due to diabetes mellitus, ventrolental fibroplasia, arthritis, villonodular synovitis, tenosynovitis and bursitis.

The present invention also provides a variety of other inventive aspects. For example, in accordance with the present invention, various pharmaceutical compounds may be conjugated to MDGFl and targeted to the MDGFl receptor. These compounds may include cytotoxins, chemotherapeutic drugs and radiopharmaceuticals well known to those skilled in the art. Also, these compounds may be conjugated to peptides and fragments that bind to the receptor.

Any pharmaceutical compound may be conjugated to MDGFl in accordance with the present invention. Further, any pharmaceutical compound may be conjugated to peptides and fragments capable of binding to MDGFl receptor. Coupled with the present disclosure, the artisan may use the techniques described in any or all of U.S. Patents 4,587,046; 5,034,223; 4,859,765; 4,707,356; 4,894,443; and 5,037,883; all of which are incorporated herein in the entirety. For example, anti-neoplastic agents such as vincristine, vinblastine, methotrexate, adriamycine, BCNU or CCNU may be used. However, any pharmaceutical compound may be used which is intended for delivery to the MDGFl receptor.

Moreover, the present invention also provides a methodology for blocking the MDGFl receptor using blocking peptides. For example, one skilled in the art would be able, in view of the above description and U.S. Patents 4,618,598; 4,716,147 and 4,753,927, each incorporated herein in the entirety, to prepare blocking peptides for the MDGFl receptor. Thus, the present invention specifically provides a peptide which is capable of blocking the MDGFl receptor, either partially or totally with respect to the availability of binding sites for MDGFl.

In another aspect, and as mentioned above, the present invention provides a method of stimulating collagen synthesis in a mammal, which entails administering an amount of MDGFl to the mammal effective for stimulating collagen synthesis.

Generally, from about 10^"4 mg to 10^~2 mg/kg of body weight is administered to the mammal. While any mammal, such as a horse, cow, pig, dog or cat may be treated in accordance with any aspect of the present invention, it is particularly preferred that the mammal treated be a human.

Furthermore, in accordance with the present invention, a method is provided for phosphorylating a receptor or a membrane bound protein. More particularly, this method entails inducing tyrosine phosphorylation on a 180-185 kDa protein in receptor-containing cells by contacting MDGFl with cells containing MDGFl receptors, thereby stimulating accumulation of phosphotyrosine on the receptor or a membrane bound protein.

As noted above, the present invention also provides antibodies against MDGFl and MDGFl receptors. These antibodies may be prepared using procedures, such as those found in U.S. Patents 5,015,571 and 5,034,515, both of which are incorporated herein in the entirety.

Additionally, the present invention also provides conjugates of pharmaceutical compounds to antibodies which are, in turn, conjugated to MDGFl receptors. These conjugates may be prepared using procedures such as those described in U.S. Patents 4,886,780; 4,981,979; 4,749,570; and 4,469,681, all of which are incorporated herein in the entirety. The present invention also provides conjugates of pharmaceutical compounds with antibodies, which are, in turn, conjugated to a protein. These conjugates may be prepared using procedures such as those described in U.S. Patents 4,867,973; 4,975,278; and 5,013,547, all of which are incorporated herein in the entirety. Any pharmaceutical compound may be so conjugated. The present invention also provides conjugates of MDGFl and MDGFl receptors to various contrast agents for imaging. In preparing and using such conjugates, procedures such as those described in U.S. Patent 4,824,986 may be used. U.S. Patent 4,824,986 is incorporated herein in the entirety. Such conjugates are useful in the diagnostic imaging of tumors and in tumor therapy.

Moreover, the present invention provides a pharmaceutical compound which is conjugated to MDGFl antibodies. This conjugate may be prepared in accordance with the present specification and in view of the various U.S. patents incorporated herein by reference.

Although the various aspects of the present invention will have significant application in the diagnosis and treatment of breast cancer, the present invention may also be used to advantage in conjunction with other types of cancer, particularly for solid tumors of other tissues, such as stomach, colon, uterine, ovarian and lung cancer.

Additionally, the present invention may also be used in conjunction with tissues other than cancer. As noted above, MDGFl or its blocking antibody may be used advantageously in the treatment of inflammation or in skin repair in wound and burn healing.

Finally, the present invention may be used to detect MDGFl in tissues other than tumor tissues, and also in bodily fluids. The term "bodily fluids" is intended to be all encompassing and includes, but is not limited to , fluids such as blood, urine and semen. In accordance with this aspect of the present invention, the procedures for detecting MDGFl are as escribed above, except that modifications using conventional techniques must be used for the collection of the bodily fluids and their appropriate storage conditions as they await analysis for MDGFl.

It is also noted that it is within the skill of one of the art, in view of the above disclosure, to find other related growth factors which are also detected by the present antibodies, which growth factors have different N-terminal sequences which, nevertheless, are substantially homologous to the present N-terminal sequences.

Further, the present method may be applied to any receptor involved in growth control which is susceptible to phosphorylation.

Having described the present invention, it will now be apparent to one skilled in the art that many changes and modifications can be made to the above-described embodiments without departing from the spirit and the scope of the present invention.

Claims

WHAT IS CLAIMED AS NEW AND DESIRED TO BE SECURED BY LETTERS PATENT OF THE UNITED STATES IS:

1. An antibody which is immunoreactive against mammary- derived growth factor 1 (MDGFl) and related growth factors.

2. The antibody of Claim 1, which is immunoreactive against an N-terminal amino acid sequence of the formula:

I-P-V-K-Q-A-V-H-G-Q-F-L-L-P-K-Q-E-K

or another N-terminal amino acid sequence having up to any two of the above-recited amino acid residues replaced by any other L-amino acid, wherein I is isoleucine, P is proline, V is valine, K is lysine, E is glutamic acid, A is alanine, H is histidine, G is glycine, Q is glutamine, F is phenylalanine, and L is leucine.

3. The antibody of Claim 2, wherein said other N-terminal amino acid sequence is:

I-P-V-K-Q-A-V-H-G-F-L-L-P-K-Q-E-K

wherein I is isoleucine, P is proline, V is valine, K is lysine, E is glutamic acid, A is alanine, H is histidine, G is glycine, Q is glutamine, F is phenylamine and L is leucine.

4. The antibody of Claim 1, which is a monoclonal antibody.

5. A method for detecting MDGFl receptors induced tyrosine phosphorylation on a 180-185 kDa protein in cells containing said MDGFl receptors, which comprises contacting MDGFl with cells containing said receptors, thereby stimulating accumulation of phosphotyrosine, and detecting said MDGFl receptors by quantitative assay.

6. A method for detecting one or more cDNA and genomic DNA fragments from MDGFl and related growth factors, which comprises contacting said cDNA fragments with an amino acid sequence having the N-terminal sequence:

I-P-V-K-Q-A-V-H-G-Q-F-L-L-P-K-Q-E-K

wherein I is isoleucine, P is proline, V is valine, K is lysine, E is glutamic acid, A is alanine, H is histidine, G is glycine, Q is glutamine, F is phenylalanine, and L is leucine. 7. A method for purifying MDGFl for use as a therapeutic agent for diverse pathologic conditions involving dysregulated collagen production, which comprises: a) contacting unpurified MDGFl with cells containing MDGFl receptors, or with anti MDGFl antibody thereby binding said MDGFl to said MDGFl receptors or antibodies, b) causing MDGFl bound to said receptors or antibodies to be released therefrom, and c) synthesizing MDGFl artificially from its cloned gene. 3. A complex which comprises a substance which is physiologically active in a mammal, which is conjugated to MDGFl.

9. The complex of Claim 7, wherein said physiologically active substance is a pharmaceutical compound.

10. The complex of Claim 7, wherein said physiologically active substance is a toxin.

11. A complex, which comprises a substance which is physiologically active in a mammal, which is conjugated to a peptide or peptide fragment which binds to MDGFl receptor.

12. The complex of Claim 10, wherein said physiologically active substance is a pharmaceutical compound.

13. The complex of Claim 10, wherein said physiologically active substance is a toxin.

14. A complex, which comprises a substance which is physiologically active in a mammal, which substance is conjugated to an antibody against MDGFl, which antibody is conjugated to a protein.

15. The complex of Claim 12, wherein said physiologically active substance is a pharmaceutical compound.

16. The complex of Claim 12, wherein said physiologically active substance is a toxin.

17. A complex, which comprises MDGFl or MDGFl receptors conjugated to a contrast agent for imaging.

18. A method of phosphorylating MDGFl receptors or a membrane-bound protein, which comprises inducing tyrosine phosphorylation on a protein in MDGFl receptor-containing cells by contacting MDGFl with cells containing MDGFl receptors, thereby stimulating accumulation of phosphotyrosine or the receptor or a membrane-bound protein.

19. A peptide which is capable of blocking MDGFl receptor either partially or totally.