EP0597845A1 - Wachstumsfaktor der den wachstum von zellen, die das onkogen erbb-2 aus dem menschen überexpremieren, inhibiert - Google Patents

Wachstumsfaktor der den wachstum von zellen, die das onkogen erbb-2 aus dem menschen überexpremieren, inhibiert

Info

Publication number
EP0597845A1
EP0597845A1 EP91910516A EP91910516A EP0597845A1 EP 0597845 A1 EP0597845 A1 EP 0597845A1 EP 91910516 A EP91910516 A EP 91910516A EP 91910516 A EP91910516 A EP 91910516A EP 0597845 A1 EP0597845 A1 EP 0597845A1
Authority
EP
European Patent Office
Prior art keywords
cells
tgfα
glycoprotein
erbb
growth
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91910516A
Other languages
English (en)
French (fr)
Other versions
EP0597845A4 (de
Inventor
Marc Lippman
Ruth Lupu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Georgetown University
Original Assignee
Georgetown University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Georgetown University filed Critical Georgetown University
Publication of EP0597845A4 publication Critical patent/EP0597845A4/de
Publication of EP0597845A1 publication Critical patent/EP0597845A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a growth factor which interacts with the human oncogene erbB-2, and which inhibits the growth of cells overexpressing this oncogene.
  • Carcinogenesis is believed to be a multi-step process of alteration of genes which are involved in the growth control of cells.
  • oncogenes have been implicated in the activation of tumor cells as regulating factors. For example, oncogenic protein kinases are believed to induce cellular transformation through either inappropriate or excessive protein
  • the c-erbB-1 gene encodes the epidermal growth factor or their receptors.
  • the c-sis gene encodes the B-chain of the platelet-derived growth factor.
  • the c-fms gene encodes a related or identical molecule for the receptor of the granulocyte-macrophage colony
  • HER-2/neu or c-erbB-2 The human counterpart of neu, called HER-2/neu or c-erbB-2, has been sequenced and mapped to the chromosomal locus 17q21. See Cancer Research. Schneider, P.M. et al, 49, 4968-4971
  • the HER-2/NEU OR c-erbB-2 oncogene belongs to the erbB-like oncogene group, and is related to, but distinct from the epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • the oncogene has been demonstrated to be implicated in a number of human adenocarcinomas leading to elevated levels of expression of the p185 protein product. For example, the oncogene has been found to be amplified in breast, ovarian, gastric and even lung adenocarcinomas.
  • Carcinoma of the breast and ovary account for
  • the c-erbB-2 oncogene is known to express a 185Kd transmembrane glycoprotein (p185 erb8-2 ).
  • the expressed protein has been suggested to be a growth factor receptor due to its homology with EGFR.
  • known EGFR ligands such as EGF or TGF ⁇ , do not bind to p185 erb8-2 .
  • no ligand is known which binds to this protein.
  • FIG. 1 illustrates the isolation of the present 30 Kd growth factor.
  • Portion A illustrates the use of low
  • portion B illustrates the use of reversed-phase chromatography.
  • Figure 2 illustrates the detection of phosphorylated proteins in SK-Br-3 cells.
  • Figure 3 illustrates the detection of phosphorylated proteins in MDA-453 cells.
  • pi85 erbB-2 protein in intact CHO/DHFR and CHO/erbB-2 cells pi85 erbB-2 protein in intact CHO/DHFR and CHO/erbB-2 cells.
  • Figure 5 illustrates a p185 erbB-2 receptor competition assay in SK-Br-3 cells.
  • Figure 6 illustrates the inhibition of p185 erbB-2 crosslinking with 4D5 antibody by gp30. Best Mode for Carrying Out the Invention
  • the human c-erbB-2 oncogene encodes a 185 Kd
  • transmembrane glycoprotein having protein kinase activity This glycoprotein, known as p185 erbB-2 , shows extensive structural similarity with the p170 epidermal growth factor receptor (EGFR) and is therefore thought to be growth factor receptor.
  • EGFR epidermal growth factor receptor
  • TGF ⁇ the normal ligands for the EGFR
  • no ligand for this glycoprotein has been described. It would be extremely desirable to find a ligand for this 185 Kd
  • the 30 Kd glycoprotein of the present invention is the 30 Kd glycoprotein of the present invention.
  • the present 30 Kd glycoprotein binds to EGFR, is capable of phosphorylating EGFR as well as inducing NRK colony
  • the 30 Kd glycoprotein was observed, unlike EGF and TGF ⁇ , to bind to heparin-sepharose, and was purified to apparent homogeneity by heparin affinity chromatography and subsequent reversed phase chromatography.
  • MDA-MB-231 cells confirmed these observations.
  • Biochemical characterization of the 30Kd TGF ⁇ -like protein was obtained by V8-protease digestion of the de-glycosylated polypeptides and translated products. Peptide mapping of the
  • polypeptide while related to the EGF/TGF ⁇ family, is encoded by a different gene and is not a post-translation modification of mature TGF ⁇ .
  • the present 30 Kd glycoprotein may be used, by itself, or in conjunction with other medicinal substances to inhibit the growth of any cells which overexpress the c-erbB-2 oncogene.
  • the present 30 Kd glycoprotein may be used advantageously to inhibit the growth of adenocarcinoma cells, preferably those of breast, ovarian, gastric and lung tissue which overexpress the erbB-2 oncogene and EGFR.
  • MDA-MB-231 and NRK clone 49F fibroblasts were obtained from the following sources.
  • Hs578T cells, A431 cells, and H8 cells, a TGF ⁇ -transfected MCF-7 breast cancer cell line were available upon request from a variety of sources.
  • Carcinogen-immortalized normal mammary epithelial cell subline 184A1N4 and its SV40-transfected derivative 184A1N4T were also available on request.
  • Rat-FeSrV transfected cells were also provided upon request. All cell lines were propagated in improved modified Eagle's medium (IMEM, Gibco, Grand Island NY) supplemented with 10% fetal bovine serum (FBS, Gibco).
  • IMEM modified Eagle's medium
  • FBS fetal bovine serum
  • Conditioned media collections were carried using a well-known procedure.
  • the media were concentrated 100-fold in an Amicon ultra-filtration cell (YM5 membrane) (Amicon, Danvers, MA) . Once clarified and concentrated, the media were stored at -20°C while consecutive collections were made during the following days.
  • the concentrated media were dialyzed using Spectraphore 3 tubing (Spectral Medical
  • Tunicamycin (Sigma, St. Louis, MO) was dissolved in 50 mM sodium carbonate (pH 10.0) and filter-sterilized with a 0.22 m filter. Confluent monolayers of MDA-MB-231, MCF-7 and Hs578T cells were grown in IMEM in the presence of 20 g/ml tunicamycin (unless otherwise specified) for 4 hours prior to metabolic labelling. Metabolic labelling was then performed as described above with continued tunicamycin treatment.
  • the immunogen was first conjugated to keyhole limpet hemocyanin (KLH) and was emulsified in complete Freund's adjuvant and was injected intradermally at multiple sites. Additional injections were given as
  • the rabbit serum was assayed for antibody titer by ELISA at 10 to 14 days following each injection.
  • Monoclonal Antibodies A monoclonal antibody against recombinant TGF ⁇ was kindly provided by Genetech Corp. Measurement of Anti TGFU Antibody (R.399) Levels by ELISA Micro-Elisa plates (Dynatech-Immunolon II, Dynatech Laboratories, Inc. Chantilly VA.) were coated for 16 hours at 4°C with 500 ng/ml of recombinant TGF ⁇ in 50 mM sodium - carbonate buffer (pH 9.6). The samples to be assayed
  • antibody were serially diluted 1:1,000-1:64,000 with 0.15 M NaCl, 0.05 M Tris-HCl (pH 7.4), 2 mM EDTA, 5 mg/ml bovine serum albumin, 0.05% Tween 20 (TBS-BSA-Tween) and were incubated in the wells for 2 hours at 37°C. The plates were washed five times with PBS-Tween and then incubated for 1 hr at 37°C with horseradish peroxidase-conjugated goat
  • TGF ⁇ RIA The presence of peptides immunologically related to TGF ⁇ was determined using a RIA kit with a polyclonal anti-rat TGF ⁇ and rat [ 25 I]TGF ⁇ (Biotope, Inc., Seattle, WA). This antibody does not cross-react with human EGF. Aliquots of conditioned media were reduced with 40 mm dithiothreitol and denatured by immersion for 1 minute in a boiling water bath. Assays were done in duplicate according to the manufacturer's protocol and each collection of conditioned media was assayed at least twice.
  • Solid Phase RIA 96 well microtiter plates were coated with anti-TGF ⁇ antibody (R399 or monoclonal antibody) for 2 hours at 37°C. The wells were then filled with 100 1 of the column fraction to be assayed for TGF ⁇ activity. A standard curve was constructed using 0.075 to 15 ng unlabelled TGF ⁇ . After the 2 hours incubation 5x10 4 cpm of [ 125 I] TFG ⁇ or 2x10 5 cpm of metabolically labelled antigen was added per well. The plates were incubated further for 16 hours at 4°C. The wells were then washed and counted using a gamma counter (Model B5002, Packard Instruments Co., Sterling, VA). The EGF RIA was performed with an anti-EGF antibody (Oncogene Science clone 144-8, Manhasset, NY). A standard curve was constructed using human EGF (HEGF, receptor grade. Collaborative Research, Waltham, MA).
  • the purified 30 kDa TGF ⁇ -like protein was subjected to digestion with N-glyconase. Samples equivalent to 100 ng were incubated with 50 1 ot 0.2 M sodium phosphate (pH 8.6), 1.25% NP40 and 2-6 g N-Glycanase (Genzyme Corp., Boston, MA) were subsequently added to each sample and incubated at 37°C for 16 hours. 50 1 of 3-fold concentrated loading buffer was added before electrophoretic analysis, performed as outlined above. The gel was silver stained.
  • A431 membranes were prepared according to the method of Kimball and Warner. A431 cells were disrupted under
  • EGF-competing activity was computed using a Hewlett Packard RIA Program.
  • Anchorage-independent Growth Assay Soft agar cloning assays were carried out using a 1 ml bottom layer of IMEM containing 0.6% Bacto-agar (Difco, Detroit, MI), 10% FBS, and 2 mM glutamine in 35mm tissue dishes (Costar, Cambridge, MA). A 0.8 ml top layer of IMEM containing the test samples, 0.36% agar, 10% FBS, and 3 x 10 4 NRK cells was added after solidification of the bottom layer. Each sample was plated in triplicate. All samples were sterilized by filtration using a 0.22 m Millex CU millipore filter before plating. Plates were incubated in a humidified, 5% CO 2 atmosphere at 37°C and were counted after 12 days incubation with a Bausch and Lomb Stem Cell Colony Counter (Artex Systems Corp, Farmingdale, NY).
  • Lyophilized conditioned medium was dissolved in 1 M acetic acid to a final concentration of about 25 mg/ml total protein. Insoluble material was removed by centrifugation at 10,000 rpm for 15 minutes. The sample was then loaded onto a Sephadex G-100 column (XK 16, Pharmacia, Piscataway, NJ) , was equilibrated and was subjected to elution with 1 M acetic acid at 4°C with an upward flow of 30 ml/hr. 100 ng of protein was processed from 4 ml of 100-fold concentrated medium. Fractions containing 3 ml of eluate were
  • Electroeluted proteins were dissolved at approximately 0.5 mg/ml in loading buffer which contained 0.125 M Tris-HCl (pH 6.8), 0.5% SDS, 10% Glycerol and 0.001% Bromophenol Blue. The samples were then heated at 100°C for 5 minutes. Proteolytic digestion were carried out at 37°C for 30 minutes by the addition of Staphylococcus aureus Protease V8 (Sigma, St. Louis, MO) to a final concentration of 25 g/ml according to methods. P-mercaptoethanol and SDS were
  • EGF Receptor Subconfluent A431 cells were cultured in IMEM for 10-12 hours. The cells were treated with 10-30 nM TGF ⁇ , EGF or TFG ⁇ -like growth factor for 30 minutes at 37°C. Cells were lysed in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 1 MM EDTA, 2 mM PMSF, 42 mM leupeptin and immunoprecipitated as described above using monoclonal antibody 225 directed against the EGF receptor Oncogene Science, Manhasset, NY).
  • the immunoprecipitates were washed three times with RIPA buffer and resuspended in 40 1 TNE (0.01M Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM EDTA). Five Ci of [ ⁇ - 32 P]ATP was added to the immunoprecipitates and the total ATP concentration was adjusted to 15 mM (final) in a volume of 60 1. The reaction mixture was incubated for 5 minutes on ice before addition of 20 1 of 3x sample buffer. The samples were boiled for 5 minutes and analyzed by denaturing 7.5% SDS-PAGE.
  • MDA-MB-231 cells were metabolically labelled with
  • TGF ⁇ -like Polypeptide TGF ⁇ -like, material was isolated from serum-free conditioned media of MDA-MB-231 cells. Levels of TGF ⁇ -like polypeptide were quantified by three independent assays: capacity to induce anchorage-independent growth of NRK fibroblasts in soft agar, ability to compete with ( 125 I]EGF for EGF receptor binding on A431 human carcinoma cell membranes and cross-reactivity with polyclonal antibodies raised against mature TGF ⁇ . EGF receptor binding activity and TGF ⁇ immunoreactivity were detected using a RIA kit provided by Biotops. To determine the approximate molecular weight of the MDA-MB-231 derived TGF ⁇ -like polypeptide, 5 ml of 100-fold concentrated, dialyzed conditioned medium was chromatographed by gel filtration using Sephadex G-100.
  • MDA-MB-231 cells was carried out using heparin-sepharose affinity chromatography. Heparin-sepharose affinity chromatography was performed on unconcentrated conditioned media from MDA-MB-231 cells. In all experiments, less than 20% of the TGF ⁇ activity loaded onto the column was
  • EGF receptor binding activity was eluted by heparin-sepharose chromatography at a concentration of 0.4-0.5 M NaCl. This activity represented one major 30 kDa molecular weight protein, which retained 70%-80% of the load activity.
  • the TGF ⁇ -like polypeptide was further purified by reversed phase chromatography (HPLC) in two steps.
  • HPLC reversed phase chromatography
  • a pool of fractions containing EGF receptor-competing activity from heparin-sepharose chromatography was reconstituted in 0.05% TFA in water and then chromatographed on a Bondapak C 3 column. A steep acetonitrile gradient (0-100%) was used in this step.
  • TGF ⁇ -like polypeptide elutes as a sharp peak in 30% acetonitrile and is separated from the bulk of the contaminating proteins. The capacity of the individual fractions to compete for EGF receptor binding and to
  • TGF ⁇ -like polypeptide activity was eluted at 25-30% acetonitrile and effectively separated from other contaminant proteins.
  • TGF ⁇ -like polypeptide A summary of the steps leading to the isolation and purification of TGF ⁇ -like polypeptide is presented in Table 1. A 27% recovery of activity and approximate 5400 fold purification was achieved.
  • the EGF receptor binding activity of the 30 kDa TGF ⁇ - like protein was compared with that of EGF in a
  • TGF ⁇ -like polypeptide specific EGF-competing activity of the purified TGF ⁇ -like polypeptide was found to be 1-1.5 x 10 6 units/mg; 1.1ng of TGF ⁇ -like polypeptide was required to inhibit EGF binding by 50%. TGF ⁇ -like polypeptide was as effective as EGF in EGF receptor binding. Furthermore, the purified 30 kDa
  • TGF ⁇ -like polypeptide stimulated the growth of serum NRK fibroblasts and induced colony formation of these cells in soft agar.
  • the bioactivity of the purified TGF ⁇ -like polypeptide was also tested by anchorage-dependent growth assays of the carcinogen- immortalized human mammary
  • A431 cells which overexpress the EGF receptor, were incubated with various concentrations of EGF, TGFU or TGF ⁇ -like growth factors. Each of the three peptides similarly stimulated phosphorylation of the EGF receptor.
  • peptide mapping was performed using the method of Clevand. Immunoprecipitation of metabolically labelled conditioned media from MDA-MB-231, H8, and Rat-FeSrV cells was carried out with the R399 anti-TCF ⁇ polyclonal antibody.
  • Precipitates were analyzed on SDS-PAGE and the specific bands were electroeluted (30 Kd from MDA-MB-231 cells, 6 kDa from H8 cells, and 18 kDa from the Rat-FeSrV cells). These proteins were subjected to enzymatic treatment with
  • N-glyconase and elastase The precipitated bands sizes are summarized in Table 2.
  • the products were then subjected to a peptide digestion using 25 g/ml V8-protease. After complete digestion, the samples were analyzed by C18
  • MDA-468 and SK-Br-3 In order to characterize the cellular effects of the present 30 Kd glycoprotein ligand, its induction of tyrosine phosphorylation was assessed in the human breast cancer lines MDA-468 and SK-Br-3. Notably, MDA-46 8 cells have amplification and over expression of the EGFR gene and do not express erbB-2 receptor-like protein. SK-Br-3 cells have amplification and over expression of the erbB-2 gene as well as relatively elevated levels of EGFR.
  • the 30 Kd ligand, TGF ⁇ and EGF were found to induce tyrosine phosphorylation in both cell lines an EGFR blocking antibody abolished the phosphorylation induced by the three growth factors in
  • phosphorylation of a protein is different from EGFR occurs in SK-Br-3 cells treated with 30 Kd factor.
  • Example 2 In human mammary carcinoma cell line MDA-453, which over expresses erbB-2, but which has undetectable levels of the EGF receptor protein or mRNA, the 30 Kd ligand was observed to induce a significant increase in tyrosine phosphorylation in a dose dependent manner at concentrations ranging from 1.25 mg/ml to 50 mg/ml. By contrast, EGF and TGFU were unable to induce tyrosine phosphorylation in the 185 kDa range, at a concentration of 25 mg/ml. No
  • Example 4 Further, the growth of CHO/erbB-2 transfected cells was inhibited by 70-80% after treatment with the present 30 Kd glycoprotein. No effect was observed on the CHO/DHFR control transfectants and the parenteral CHO line. TGF ⁇ at the same molar concentration did not exhibit any effect on the proliferation of any of the three lines. Tyrosine phosphorylation and cell proliferation of the CHO/DHFR cells and the parenteral CHO cell line is not effected after treatments by the present 30 Kd ligand or TGF ⁇ .
  • Example 5 Cell Growth Inhibition by gp30
  • Sk-Br-3, MDA 453, MDA 468 and MCF-7 cells were plated in 24 well plates in IMEM (Biofluids) supplemented with 5% FCS.
  • Parental CHO cells, and CHO cells transfected with the DHFR gene or the erbB-2 gene were plated in 24 well plates (Costar) in ⁇ -MEM (Biofluids) supplemented by 10% dialyzed FCS, 0.75 mg/ml G418 and Methotrexate (MTX) 50 nM for the CHO parental and CHO-DHFR CELLS for 250 nM for the
  • CHO-erbB-2 After 24 hours media was removed and replaced with control serum free media (SFM) containing fibronectin, transferrin, hepes, glutamine, trace elements, and BSA, or SFM with the addition of 2.0 ng/mlgp30, 10 ng/ml
  • SFM serum free media
  • FIG. 1 Isolation of gp30 Part A illustrates the use of low affinity heparin chromatography.
  • affinity chromatography of conditioned media from MDA-231 cells was performed on a heparin-sepharose column. Fractions were analyzed for EGF receptor binding activity of A431 cell membranes. Aliquots from the input media and from the fractions containing activity were analyzed by a 15% SDS-PAGE, followed by silver staining. Lane 1 shows unconcentrated conditioned media. Lane 2 represents the active fraction.
  • Part B illustrates the use of reversed-phase
  • SK-Br-3 cells were grown in 90% confluence in 24-2311 plates (Costar). Cells were treated at 30°C with IMEM
  • Electrophoretic transfer was carried out at room temperature for one hour at 125 Ma in a buffer containing 25 Mm glycine, 129 Mm Tris (Ph 8.3) and 20% methanol. Following transfer, the filter was blocked with 5% BSA in Tris-Buffered Saline containing 0.5% Tween 20. An antiphosphotryosine antibody (Amersham) was reacted with the immobilized proteins in 5% BSA (Sigma RIA Grade). Immunecomplexes were detected by a goat anti-mouse antibody conjugated to alkaline phosphatase. Blots were then
  • MDA-45e cells were grown to 90% confluence in 24-2311 plates (Costar) and treated at 37°C with IMEM (lane 1), IMEM containing 25 ng/ml of recombinant TGF ⁇ (Genetech, CA) (lane 10), or IMEM containing 1.25-40 ng/ml ofgp30 (lanes 2-9). After 20 minutes media was removed and cells were lysed in 100 ⁇ l of sample buffer as described in Figure 2. After 5 minutes at 95°C, 50 ⁇ g of protein was loaded in a 7.5% SDS-PAGE. Proteins were then transferred to nitrocellulose membrane for immunoblotting with an antiphosphotryosine antibody (Amersham) as described in Figure 2.
  • CHO-DHFR Fig. 4A
  • CHO-erbB-2 Fig. 4B cells
  • SK-Br-3 cells were plated in 24 well plates in IMEM (Biofluids) supplemented with 5% FCS. After a wash with binding buffer (DMEM/F12 pH 7.4, containing 1 mg/ml BSA, 10 Mm hepes and 20 Mm glutamine) cells were incubated for 30 minutes at 37°C with binding buffer.
  • binding buffer DMEM/F12 pH 7.4, containing 1 mg/ml BSA, 10 Mm hepes and 20 Mm glutamine
  • FIG. 5 Binding was performed with iodinated 4D5 (1 nM) alone (lane 1), in the presence of 100 nFM unlabeled 4D5 (lane 2) and in the presence of 2nM gp30 (lane 3). 100 nM EGF were used as a control (lane 4). Cells were then treated with a cross-linking agent EGS for 45 minutes at 4°C, then quenched by adding 0.1 ml of 20 Mm NH4Cl. The solubilized cells were immunoprecipitated with a polyclonal antibody to the C-terminal domain of erbB-22 (Genetech, CA). The precipitates were analyzed on a 5% SDS-PAGE.
  • the 30 Kd glycoprotein of the present invention may thus be used advantageously to inhibit the growth of various types of adenocarcinoma cells which overexpress the erbB-2 oncogene and EGFR.
  • the present 30 Kd may thus be used advantageously to inhibit the growth of various types of
  • glycoprotein is used in inhibit the growth of adenocarcinoma cells of breast, ovarian, gastric and lung tissue which overexpress the erbB-2 oncogene and EGFR.
  • relatively low concentrations of the glycoprotein may be used.
  • an aqueous solution having a concentration of about 1-50 ng/ml may be conveniently administered to a patient such that a total of from about - 1-10,000 ng of glycoprotein are administered per day. It is preferred, however, if about 1-1,000 ng are administered per day.
  • the present invention thus relates to the use of the present 30 Kd TGF ⁇ -like glycoprotein in direct interactions with EGFR and p185 erbB-2 .
  • the present invention provides conjugates of the 30 Kd
  • the present invention provides diagnostic and therapeutic methods using these conjugates. Further, the present invention provides a diagnostic test kit using the present conjugates.
  • the present invention relates to the preparation of monoclonal antibodies of gp3o, and the use of these monoclonal antibodies to detect the presence of gp30 in patient sera.
  • the present invention also provides a method for detecting gp30 in patient sera.
  • the mere detection of either p185 or gp30 is a basis for concluding that the detected protein is being overexpressed. This conclusion, in turn, leads to a poor patient prognosis necessitating the use of more aggressive treatment of the tumor.
  • the present invention specifically first contemplates the use of conjugates of the 30 Kd glycoprotein and EGFR, and of the 30 Kd glycoprotein and p185 erbB-2 in detecting the presence of adenocarcinoma cells which overexpress either EGFR or erbB-2 oncogene.
  • the adenocarcinoma cells detected are of breast, ovarian, gastric and lung tissue.
  • the present conjugates may be used
  • EGFR or erbB-2 oncogene overexpressing either EGFR or erbB-2 oncogene. This is conveniently done as either EGFR or p185 may be found on the cell surfaces. If such cells are present, either the EGFR or p185 erbB-2 will become bound to the ligand. Thereafter, the aqueous solution is separated from the bound antiligand material, and the antiligand material may be conveniently detected with a known detection means associated therewith. For example, an amplified enzymelinked immunoassay may be used. The surface to which the ligand is bound is treated with one or more agents for limiting the amount of
  • Such agents reduce the "noise" arising due to non-specific binding when interpreting the assay.
  • a diagnostic test kit may be constructed in a variety of ways.
  • a test kit may be constructed to contain a vessel containing a test liquid having a surface to which gp30 ligand is bound. This is preferably a multi-well test plate. Also contained is at least one other vessel
  • non-specific binding may be incorporated within a solution of the kit or may have been used to treat the surface of the first vessel before it is supplied.
  • a portion of the tumor or a tumor sample may be worked up into an aqueous solution and put into contact with the bound gp30.
  • the present invention contemplates and is specifically directed to any diagnostic or therapeutic method for the detection of adenocarcinoma cells which overexpress EGFR or erbB-2 oncogene, which method uses the formation of a conjugate between the 30 Kd glycoprotein of the present invention and either EGFR or p185 erbB-2 .
  • the present invention also provides an assay and a test kit for the detection of gp30 using
  • monoclonal antibodies can be used for this purpose, it is preferred that monoclonal antibodies be used.
  • the monoclonal antibodies to gp30 are preferably bound to the microtiter or multi-well plate and exposed to patient sera suspected of containing gp30.
  • test kit is also provided.
  • the kit contains a first container containing an antibody having specificity for gp 30 and a second container containing a second antibody having specificity for gp30 and being labelled with a reporter molecule capable giving a detectable signal.
  • the first antibody is immobilized on a solid surface.
  • the 30 Kd glycoprotein of the present invention is wellcharacterized by: 1) being a heparin-binding protein;
  • polyclonal or monoclonal antibodies produced against gp30 may be produced in accordance with well-known techniques. For example, see Current Protocols in Molecular Biology, edited by F.M. Ausubel et al (Wiley 1987), in particular Chapter 11 on Immunology. Also, the immunoassays used in the assays and diagnostic test kits of the present invention are well known to the artisan as evidenced by the above treatise, and by the methods disclosed in U.S. Patent 4,921790 which patent has been specifically incorporated herein in the entirety.
  • the diagnostic aspects of the present invention relate to the use of methods and test kits for the detection of either p185, EGFR or gp30.
  • detection of any one of these proteins may form the basis for a poor prognosis necessitating the use of aggressive treatment of one or more adenocarcinomas.
  • the present invention also relates to gp30, itself, and conjugates of gp-0-EGFR and/or gp30-p185 erbB-2 .
  • the therapeutic aspects of the present invention relate to the use of gp30 to inhibit the growth of adenocarcinoma cells which overexpress EGFR and/or erbB-2 oncogene.
  • the amount of gp30 to be administered as a therapeutic agent will be determined on a case by case basis by the attending physician.
  • the extent of the adenocarcinoma, body weight and age of the patient are considered while up to about 10,000 ng per day may be used, generally not more than 1,000 ng per day of gp30 is
  • the 30 Kd glycoprotein may be administered with any chemotherapeutic substance, growth inhibitor or immunestimulating substance.
  • the present invention specifically contemplates such combinations.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP91910516A 1990-05-25 1991-05-22 Wachstumsfaktor der den wachstum von zellen, die das onkogen erbb-2 aus dem menschen überexpremieren, inhibiert Withdrawn EP0597845A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US52843890A 1990-05-25 1990-05-25
PCT/US1991/003443 WO1991018921A1 (en) 1990-05-25 1991-05-22 GROWTH FACTOR WHICH INHIBITS THE GROWTH OF CELLS OVEREXPRESSING THE HUMAN ONCOGENE erbB-2
US528438 1995-09-14

Publications (2)

Publication Number Publication Date
EP0597845A4 EP0597845A4 (de) 1993-06-18
EP0597845A1 true EP0597845A1 (de) 1994-05-25

Family

ID=24105687

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91910516A Withdrawn EP0597845A1 (de) 1990-05-25 1991-05-22 Wachstumsfaktor der den wachstum von zellen, die das onkogen erbb-2 aus dem menschen überexpremieren, inhibiert

Country Status (5)

Country Link
EP (1) EP0597845A1 (de)
JP (1) JPH05509079A (de)
AU (1) AU667026B2 (de)
CA (1) CA2083688A1 (de)
WO (1) WO1991018921A1 (de)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7135456B1 (en) 1991-04-10 2006-11-14 Acorda Therapeutics, Inc. Glial mitogenic factors, their preparation and use
US7115554B1 (en) 1993-05-06 2006-10-03 Acorda Therapeutics, Inc. Methods of increasing myotube formation or survival or muscle cell mitogenesis differentiation or survival using neuregulin GGF III
US7285531B1 (en) 1991-04-10 2007-10-23 Acorda Therapeutics, Inc. Method for prophylaxis or treatment of a nervous system, pathophysiological condition involving a glial growth factor sensitive cell by administration of a glial growth factor
US5530109A (en) * 1991-04-10 1996-06-25 Ludwig Institute For Cancer Research DNA encoding glial mitogenic factors
US7319019B1 (en) 1991-04-10 2008-01-15 Acorda Therapeutics, Inc. Glial mitogenic factors lacking an N-terminal signal sequence
US7094749B1 (en) 1991-04-10 2006-08-22 Acorda Therapeutics, Inc. Glial mitogenic factors, their preparation and use
US5834229A (en) * 1991-05-24 1998-11-10 Genentech, Inc. Nucleic acids vectors and host cells encoding and expressing heregulin 2-α
IL101943A0 (en) * 1991-05-24 1992-12-30 Genentech Inc Structure,production and use of heregulin
US7037888B1 (en) 1992-04-03 2006-05-02 Acorda Therapeutics, Inc. Methods for treating muscle diseases and disorders
CA2134544A1 (en) * 1992-04-29 1993-11-11 Ruth Lupu Binding peptides which interact with ligand growth factors of the epidermal growth factor receptor and erbb-2 receptor
GB9217316D0 (en) * 1992-08-14 1992-09-30 Ludwig Inst Cancer Res Schwann cell mitogenic factor,its preparation and use
US5770567A (en) * 1994-11-14 1998-06-23 Genentech, Inc. Sensory and motor neuron derived factor (SMDF)
US5912326A (en) * 1995-09-08 1999-06-15 President And Fellows Of Harvard College Cerebellum-derived growth factors
HRP970371A2 (en) * 1996-07-13 1998-08-31 Kathryn Jane Smith Heterocyclic compounds

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4705677A (en) * 1977-05-03 1987-11-10 Makari Jack G Immunization
US4746539A (en) * 1983-11-23 1988-05-24 The Ohio State University Research Foundation Purification of cancer-associated protein and preparation of antibody thereto

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
EMBO JOURNAL vol. 8, no. 1, 1989, OXFORD,UK pages 167 - 173 LEE ET AL 'HER2 CYTOPLASMIC DOMAIN GENERATES NORMAL MITOGENIC AND TRANSFORMING SIGNALS IN A CHIMERIC RECEPTOR' *
PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH vol. 31, March 1990, WASHINGTON D.C.,USA page 83 LUPU ET AL 'A NOVEL TGF-ALPHA RELATED GROWTH FACTOR INTERACTS DIRECTLY WITH EGF RECEPTOR AND ERBB-2' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES,USA vol. 85, 1988, WASHINGTON D.C.,USA pages 5389 - 5393 KOKAI ET AL 'PHOSPHORYLATION PROCESS INDUCED BY EPIDERMAL GROWTH FACTOR ALTERS THE ONCOGENIC AND CELLULAR NEU (NGL) GENE PRODUCTS' *
SCIENCE vol. 244, 1989, WASHINGTON D.C.,USA pages 707 - 712 SLAMON ET AL 'STUDIES OF THE HER-2/NEU PROTO-ONCOGENE IN HUMAN BREAST AND OVARIAN CANCER' *
SCIENCE vol. 249, September 1990, WASHINGTON D.C.,USA pages 1552 - 1555 LUPU ET AL 'DIRECT INTERACTION OF A LIGAND FOR THE ERBB2 ONCOGENE PRODUCT WITH THE THE EGF RECEPTOR AND P185ERBB2' *
See also references of WO9118921A1 *

Also Published As

Publication number Publication date
AU667026B2 (en) 1996-03-07
CA2083688A1 (en) 1991-11-26
JPH05509079A (ja) 1993-12-16
AU7960591A (en) 1991-12-31
EP0597845A4 (de) 1993-06-18
WO1991018921A1 (en) 1991-12-12

Similar Documents

Publication Publication Date Title
US5869618A (en) Antibodies to ligand growth factors
US5459061A (en) Hybridomas producing monoclonal antibodies which specifically bind to continuous epitope on the human EGF receptor and compete with EGF for binding to the EGF receptor
JP3293823B2 (ja) 肝細胞成長因子（ｈｇｆ）レセプターの部分切除形態
AU667026B2 (en) Growth factor which inhibits the growth of cells overexpressing the human oncogene erbB-2
US5362716A (en) Methods for stimulating hematopoietic progenitors using hepatocyte growth factor and lymphokines
US5656435A (en) Antibodies to peptides having NGF-like activity, said antibodies having no substantial cross-reactivity with NGF, and use thereof
Yarden et al. Biochemical analysis of the ligand for the neu oncogenic receptor
US5571895A (en) Modified GAF polypeptides
EP0641358B1 (de) Bindungspeptide zur interaktion mit wachstumsfaktoren als liganden des rezeptors des epidermiswachstumsfactor und des erbb-2-rezeptor
Mroczkowski et al. Identification of biologically active epidermal growth factor precursor in human fluids and secretions
JPH07224089A (ja) ｎｅｕレセプターの刺激因子
EP0574414B1 (de) Wachstumsfaktor-ligand, der an den erbb-2-rezeptor bindet und die zellulären reaktionen induziert
EP0377855B1 (de) Endothelialzellen-Wachstumsfaktor
Seno et al. Purification and characterization of a recombinant human cripto-1 protein
Lupu et al. The role of erbB-2 and its ligands in growth control of malignant breast epithelium
Lupu et al. Purification and characterization of a novel growth factor from human breast cancer cells
US6183971B1 (en) Human betacellulin-specific antibodies and uses thereof
US5644026A (en) Epitaxin, a cell motility factor
US5187062A (en) Method for detecting and measuring FGF
JP3232415B2 (ja) モノクローナル抗体，その製造法および用途
Kidwell et al. Growth factor production by mammary tumor cells
CA2008735C (en) Human epidermal growth factor (egf) receptor sequences and egf competitive monoclonal antibodies
Imam et al. Detection by a human monoclonal antibody of a glycoprotein associated with malignant proliferation of mammary epithelial cells
JPH07135994A (ja) 抗体、ポリペプチド、その製造法および用途
Ke et al. Development and characterization of a specific anti-caveolin-1 antibody for caveolin-1 functional study in human, goat and mouse

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19921222

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

17Q First examination report despatched

Effective date: 19950418

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 19961118