EP0597845A4 - 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.

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Publication number
EP0597845A4
EP0597845A4 EP19910910516 EP91910516A EP0597845A4 EP 0597845 A4 EP0597845 A4 EP 0597845A4 EP 19910910516 EP19910910516 EP 19910910516 EP 91910516 A EP91910516 A EP 91910516A EP 0597845 A4 EP0597845 A4 EP 0597845A4
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European Patent Office
Prior art keywords
cells
tgfα
glycoprotein
erbb
growth
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English (en)
French (fr)
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EP0597845A1 (de
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Marc Lippman
Ruth Lupu
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Georgetown University
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Georgetown University
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    • 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.
  • a variety of proto-oncogenes and oncogenes have been implicated in the activation of tumor cells as regulating factors.
  • oncogenic protein kinases are believed to induce cellular transformation through either inappropriate or excessive protein phosphorylation, resulting in the uncontrolled growth of malignant neoplasms. See Histopathology, Wrba, F. et al, 15, 71-76 (1989).
  • the c-erbB-l 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 stimulating factor.
  • a fourth member of this group of proto-oncogenes, called neu was identified in ethylnitrosourea-induced rat neuroblastomas.
  • 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 (September 15, 1989).
  • 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 pl85 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 approximately one-third of all cancers occurring in women and together are responsible for approximately one-fourth of cancer related deaths in females.
  • the c-erbB-2 oncogene has been found to be amplified in 25 to 30% of human primary breast cancers. See Science, Slamon, D. et al, 244, 707-712 (May 12, 1989).
  • the c-erbB-2 oncogene is known to express a 185Kd transmembrane glycoprotein (pl85 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 pl85 erb8 ⁇ 2 .
  • no ligand is known which binds to this protein.
  • a need continues to exist for a ligand for (pl85 erb8 ⁇ 2 ) .
  • a ligand for (pl85 erb8 ⁇ 2 ) might be used to counteract the effects of c-erbB-2 oncogene over expression in facilitating carcinogenesis.
  • Figure 1 illustrates the isolation of the present 30 Kd growth factor.
  • Portion A illustrates the use of low affinity heparin chromatography
  • 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.
  • Figure 4 illustrates the phosphorylation of pi85 erbB"2 protein in intact CHO/DHFR and CHO/erbB-2 cells.
  • Figure 5 illustrates a pl85 erbB ⁇ 2 receptor competition assay in SK-Br-3 cells.
  • Figure 6 illustrates the inhibition of pl85 erbB " 2 crosslinking with 4D5 antibody by gp30.
  • the human c-erbB-2 oncogene encodes a 185 Kd transmembrane glycoprotein having protein kinase activity.
  • This glycoprotein known as pl85 erbB"2 , shows extensive structural similarity with the pl70 epidermal growth factor receptor (EGFR) and is therefore thought to be growth factor receptor.
  • EGFR pl70 epidermal growth factor receptor
  • TGF ⁇ the normal ligands for the EGFR, interact directly with pl85 erbB"2 . In fact, no ligand for this glycoprotein has been described.
  • a 30 Kd growth factor which is secreted from the estrogen receptor negative cell line MDA-231 is effective as a ligand for pl85 erbB"2 glycoprotein.
  • the 30 Kd glycoprotein of the present invention also exhibits TGF ⁇ -like activity.
  • the present 30 Kd glycoprotein binds to EGFR, is capable of phosphorylating EGFR as well as inducing NRK colony formation. This is quite surprising inasmuch as the present 30 Kd growth factor is distinct from the normal precursor for TGF ⁇ or mature TGF ⁇ as shown by peptide mapping of the translated proteins.
  • the 30 Kd glycoprotein was immunoprecipitated by an anti-TGF ⁇ polyclonal antibody and exhibited TGF ⁇ -like biological activity as assayed by EGF radioreceptor assay and NRK and A1N4T cell colony formation assays.
  • the 30 Kd growth factor also stimulated autophosphorylation of the EGF receptor more efficiently than mature 6 Kd TFG ⁇ .
  • 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.
  • Tunicamycin treatment in vivo or N-glyconase deglycosylation in vitro revealed a precursor of 22 Kd in contrast to the 18 Kd precursor for mature TGF ⁇ .
  • the same is used to inhibit the growth of cells which overexpress the c-erbB-2 oncogene.
  • 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 American type Culture Collection (Rockville, MD) .
  • 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 improved 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 Industries,
  • 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 samples containing TGF ⁇ -like activity were incubated with 20 g porcine pancreatic elastase (Sigma) dissolved in 50 mM glycylglycine, pH 7.9, for 1 hour at 22°C. The samples were then subjected to immunoprecipitation and SDS-PAGE analysis.
  • Antiserum against human TGF ⁇ was obtained by immunization of a rabbit on day 0 with 400 g of recombinant TGF ⁇ synthesized in E. coli. provided by Genentech Corp.
  • 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 follows: day 60, 175 g TGF ⁇ and days 90, 150, 180, and 210, 100 g TGF ⁇ .
  • the booster injections were given subcutaneously at multiple sites in incomplete Freund's adjuvant.
  • the rabbit serum was assayed for antibody titer by ELISA at 10 to 14 days following each injection.
  • the samples to be assayed 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 anti-rabbit immunoglobulin in TBS-BSA-Tween.
  • the plates were then washed five times with PBS-Tween and incubated for 4 hrs at 22°C with 100 1 per well of 0.1 mg/ml o-phenylenediamine, 0.012% H 2 0 2 in 0.1 M Phosphate-citrate buffer (pH 5.0). The reaction was stopped by the addition of 50 1/well of 2.5 N H 2 S0 4 and the absorbance was measured at 492 nm using a UR 700 Microplate Reader (Dynatech Lab., Inc. Chantilly, VA) .
  • 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 5xl0 4 cpm of [ 125 I] TFG ⁇ or 2xl0 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) .
  • 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 Ki ball and Warner. A431 cells were disrupted under nitrogen and the nuclei and organelles pelleted by low speed centrifugation. The membranes were then pelleted by centrifugation at 35,000 rp for l hour and resuspended in - 20 mM HEPES buffer, pH 7.4. Membranes (2.5 g/ml) were plated into 96 well plates and allowed to dry overnight at 37°C before use. Standard binding competition studies were performed using [ 125 I]EGF (ICN, Costa Mesa, California, specific activity- 100 Ci/g, about 50,000 CPM/well) .
  • a standard curve was constructed with 0.075-10ng of unlabelled hEGF (receptor grade, Collaborative Research) .
  • the different fractions to be analyzed were lyophilized and reconstituted in PBS (0.5 ml/500 ml conditioned media). After incubation of the labelled EGF and 10 1 of the samples for 2 hours at 37°C in binding buffer (IMEM containing 50 mM HEPES and 0.1% BSA pH 7.7), the wells were washed, cut from the plate and counted. EGF-competing activity, was computed using a Hewlett Packard RIA Program.
  • 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 illipore filter before plating. Plates were incubated in a humidified, 5% C0 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) .
  • Cells were grown in IMEM containing 5% FCS. Upon confluence cells were detached using trypsin-versene (Biofluids, Rockville MD) and passed at 1:20 to 1:50 dilutions. Cells were seeded in 12-well plates at 4,000-10,000 cells/well, depending on the cell type (MDA-MB-231-8,000 cells/well in serum free IMEM). After 24 hours the media was changed and the cells were treated with EGF, TGF ⁇ or TGF ⁇ -like protein were harvested at 1, 2 and 4 days using trypsin-versene. The cells were counted using a coulter counter.
  • 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 rp 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 lyophilized and resuspended in 300 1 PBS for assay and served as a source for further purification.
  • 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 subsequently added to final concentrations of 20% and 2%, respectively. Proteolysis was stopped by boiling for 2 min. The samples were then injected on a C18 Reversed Phase HPLC column.
  • 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.
  • Total cellular RNA was extracted from cells by homogenizing in guanidine isothiocyanate followed by centrifugation over a cesium chloride cushion.
  • Poly (A) + mRNA was eluted in 10 mM Tris after passing total cellular RNA over an oligo (DT) cellulose column (Pharmacia, Piscataway, NJ) equilibrated with 10 mM Tris-0.5 M NaCl pH 8.0. After precipitation in ethanol (66% vol/vol) and 0.1 M acetic acid, both total and poly(A) + selected RNA were resuspended in 10 m Tris-1 mM EDTA buffer and separated on 1% agarose, 6% formaldehyde gels.
  • Electrophoresis was carried out at 20 volts over 14-16 hours in: 5 mm NaAc 1 mM EDTA, 20 mM 3-[N-morpholino] propane sulfonic acid pH 7.0 (MOPS-Sigma) .
  • the gels were stained with ethidium bromide 2.0g/ml to allow inspection of the quality and quantity of RNA () .
  • In vitro translation assays were performed using Weat Germ kit according to the manufacturer's instructions (Promega) .
  • MDA-MB-231 cells were metabolically labelled with [ 35 S] methionine and [ 35 S] cysteine.
  • Metabolically labelled conditioned media from MDA-MB-231, TGF ⁇ -transfected MCF-7 (H8) , and HS578T cells were tested by solid phase RIA for immunoreactivity with a polyclonal antibody (R399) and a monoclonal antibody raised against recombinant 6 kDa TGF ⁇ .
  • Metabolically labelled TGF ⁇ -like material from MDGA-mb-231 cells reacted only with the polyclonal antibody.
  • TGF ⁇ -like polypeptide secreted from MDA-MB-231 cells was a glycosylated form of TGF ⁇ .
  • MDA-MB-231 cells were incubated with tunicamycin, an inhibitor of co-translational N-linked glycosylation, and the media was immunoprecipitated with the anti-TGF ⁇ polyclonal antibody a species of 22 kDa substituted that previously observed of 30 kDa.
  • 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.
  • TGF ⁇ -like polypeptide 5 ml of 100-fold concentrated, dialyzed conditioned medium was chromatographed by gel filtration using Sephadex G-100. Elution was performed with 1.0 M acetic acid and fractions were characterized for protein content. TGF ⁇ -like activities were eluted from the column in a single broad peak. Maximal activity was observed at an apparent molecular weight of 30 kDa and was separated from the bulk of contaminating proteins present in the bed volume. All the fractions demonstrating TGF ⁇ immunoreactivity also contained EGF receptor binding activity. The relative amounts of receptor binding activity and immunoreactivity present in these fractions, however appeared to differ.
  • TGF ⁇ -like polypeptide from 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 recovered in the unabsorbed fractions. A sharp peak of 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 stimulate the growth of NRK cells in soft agar was determined.
  • a pool of the active fractions (indicated with an horizontal bar) was rechromatographed on the same column. Fractions were eluted with a 0-20% acetonitrile gradient in 0.05% TFA for 5 minutes followed by a linear 20-40% acetonitrile gradient. The TGF ⁇ -like polypeptide activity was eluted at 25-30% acetonitrile and effectively separated from other contaminant proteins.
  • TGF ⁇ -like polypeptide In order to achieve a complete separation of TGF ⁇ -like polypeptide from those impurities detected by silver staining (data not shown) we used size exclusion chromatography under acidic conditions. The active fractions for EGF receptor-competing activity were pooled and analyzed by SDS-PAGE. One single polypeptide band was observed after silver staining.
  • 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.
  • I t t a Total protein was determined using BSA as a standard. The iantitation of step 6 was based on I extrapolation from standard values. The absolute specific activity of a companion aliquot was found to be 1 million Units/mg.
  • One unit of EGF fiompeting activity is defined as the amount of protein that inhibits the binding of [ 125 1]EGF to the receptor by 50%.
  • the EGF receptor binding activity of the 30 kDa TGF ⁇ - like protein was compared with that of EGF in a radioreceptor assay. Both growth factors competed with [ 1 5 I]EGF for receptor sites on A431 membranes.
  • the specific EGF-competing activity of the purified TGF ⁇ -like polypeptide was found to be 1-1.5 x 10 6 units/mg; l.lng of TGF ⁇ -like polypeptide was required to inhibit EGF binding by 50%. TGF ⁇ -like polypeptide was as effective as EGF in EGF receptor binding.
  • the purified 30 kDa was found to be 1-1.5 x 10 6 units/mg; l.lng of TGF ⁇ -like polypeptide was required to inhibit EGF binding by 50%.
  • TGF ⁇ -like polypeptide was as effective as EGF in EGF receptor binding.
  • the purified 30 kDa was found to be 1-1.5 x 10 6 units/mg; l.lng of TGF ⁇ -like polypeptide was required
  • 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 epithelial cells 184A1N4 and anchorage-independent growth assays of 184AlN4-derived cells partially transformed by SV40 T antigen, 184A1N4T. Dose response curves of TGF ⁇ -like polypeptide on these cells were similar to those observed with EGF and TGFU.
  • the biological activity of the purified 30 kDa TGF ⁇ -like factor was further assessed by examining its ability to induce autophosphorylation of the EGF receptor.
  • 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 reversed phase chromatography. Three major peptide peaks eluted at different acetonitrile concentrations by reversed phase chromatography. However, the concentrations at which those peptides isolated from MDA-MB-231 cells eluted (16%, 18.7%, and 21.7%) were different from the peptides isolated from H8 and FeSrV cells (24%, 29%, and 32.6%).
  • the peptide elution pattern of the TGF ⁇ (6 Kd) derived from H8 cells and Rat-FeSrV cells was essential identical.
  • 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 MDA-468 cells. This antibody did not, however, completely block the phosphorylation induced by the present 30 Kd ligand and SK-Br-3 cells. However, it did block the phosphorylation induced by TGF ⁇ .
  • 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 phosphorylation was observed in untreated cells. Hence, from the above, a direct interaction between the 30 Kd ligand and the 185 Kd glycoprotein appears to occur.
  • Cells were treated with the present 30 Kd growth factor, EGF, TGF ⁇ and anti-erbB-2 antibody in order to inhibit the proliferation of SK-Br-3 cells.
  • the present 30 Kd glycoprotein exhibited no effect on MCF-7 cells, which have normal levels of EGFR and erbB-2. Additionally, EGF and TGF ⁇ inhibited the anchorage dependant growth of MDA-468 cells and SK-Br-3 cells, but not that of MDA-453 or MCF-7 cells. EGF-induced anchorage dependant growth inhibition of SK-Br-3 and MDA-468 cells was reversed by an anti-EGFR blocking antibody. In the presence of the 30 Kd glycoprotein, the growth inhibition of SK-Br-3, MDA-453 and MDA-468 cells was nearly complete.
  • the growth inhibitory property of the present 30 Kd ligand appears to be similar to that described for EGF on size whichever express EGFR such as A431 cells and MDA-468 cells.
  • 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.
  • IMEM Biofluids
  • MTX Methotrexate
  • 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 chromatography.
  • EGF/TFGA active fractions obtained after heparin-sepharose chromatography were chromatographed twice on a ⁇ Bondapak C 3 column in 0.05% TFA. Samples were eluted with a steep gradient of acetonitrile. Fractions that showed EGF receptor binding activity were then rechromatographed and eluted with a shallow acetonitrile gradient. EGF competing activity was constantly eluted at a 25-30T acetonitrile gradient. The resulting fraction was analyzed on a 15% SDS-PAGE followed by silver staining. Sizes are shown in kilodaltons.
  • SK-Br-3 cells were grown in 90% confluence in 24-2311 plates (Costar) .
  • Cells were treated at 30°C with IMEM (lanes 1 and 2) , IMEM containing 25 nb/ml recombinant TGF ⁇ (Genetech, CA) (lanes 3 and 4) , and IMEM containing 5 ng/ml of gp30 (lanes 5 and 6) , all of these in the presence (lanes 1, 4, 5) and the absence (lanes 2, 3, 6) of an anti-EGF receptor blocking antibody (Genetech, CA) .
  • 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 incubated with a color development substrate solution containing NBT and BCIP (Promega) .
  • 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 of gp30 (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.
  • FIG. 4 Phosphorylation of P185 Protein in Intact CHO/DHFR. and CHO/erbB-2 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. The EGFKR were saturated with 30 nM EGF for 2 hours at 4°C. pl85 binding studies were then performed for 3 hours at 4°C with 1 nM iodinated 4D5 in the presence of various concentrations of unlabeled gp30 for 4D5. After the incubation, cells were washed 3 times with binding buffer and then solubilized with 1% SDS. No specific binding was determined with excess (100 nM) of unlabeled antibody. Each group was assayed in triplicate. The experiments were performed five times and the results were reproducible.
  • binding buffer DMEM/F12 pH 7.4, containing 1 mg/ml BSA,
  • 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 NH4C1.
  • 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 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 pi85 erbB ⁇ 2 .
  • the present invention provides conjugates of the 30 Kd glycoprotein ligand with either EGFR or pl85 erbB"2 .
  • the present invention provides diagnostic and therapeutic methods using these conjugates.
  • 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 gp3o in patient sera.
  • the mere detection of either pl85 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 pl85 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 advantageously in a biochemical detection method in which the 30 Kd glycoprotein ligand is bound to a surface and put into contact with aqueous solution containing a tumor portion containing cells which are suspected of 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 pl85 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 non-specific binding. 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 containing reagent solution. The agent for limiting 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 gp3o.
  • 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 pl85 erbB"2 .
  • the present invention also provides an assay and a test kit for the detection of gp30 using monoclonal antibodies to gp30.
  • the monoclonal antibodies to gp30 are preferably bound to the microtiter or multi-well plate and exposed to patient sera suspected of containing gp30.
  • a conventional detecting means Upon detecting the presence of gp30 by a conventional detecting means, a conclusion of poor prognosis would be made necessitating the use of more aggressive treatment for the tumor.
  • the presence of the 30 Kd glycoprotein (gp30) in patient sera can be detected utilizing either monoclonal or polyclonal antibodies in virtually any type of immunoassay. This includes both single-site or twosite or "sandwich" assays of the non-competitive types, as well as in traditional competitive binding assays.
  • 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 pl85, EGFR or gp30.
  • the 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 gp30-EGFR and/or gp30-pl85 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 administered. It is preferred, however, if from about 5-500 ng per day are used.
  • the above amounts may vary on a case-by-case basis.
  • the present 30 Kd glyccoprotein may be administered by itself, as a therapeutic agent, it may be administered in combination with one or more other therapeutic agents.
  • the 30 Kd glycoprotein may be administered with any chemotherapeutic substance, growth inhibitor or immunestimulating substance.
  • the present invention specifically contemplates such combinations.

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US7135456B1 (en) 1991-04-10 2006-11-14 Acorda Therapeutics, Inc. Glial mitogenic factors, their preparation and use
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
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
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
US5834229A (en) * 1991-05-24 1998-11-10 Genentech, Inc. Nucleic acids vectors and host cells encoding and expressing heregulin 2-α
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US7037888B1 (en) 1992-04-03 2006-05-02 Acorda Therapeutics, Inc. Methods for treating muscle diseases and disorders
AU676476B2 (en) * 1992-04-29 1997-03-13 Georgetown University Binding peptides which interact with ligand growth factors of the epidermal growth factor receptor and erbB-2-receptor
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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' *
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