EP0828843A1 - Heregulines de recombinaison et leurs fonctions biologiques lors de l'activation d'un recepteur - Google Patents

Heregulines de recombinaison et leurs fonctions biologiques lors de l'activation d'un recepteur

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Publication number
EP0828843A1
EP0828843A1 EP96915791A EP96915791A EP0828843A1 EP 0828843 A1 EP0828843 A1 EP 0828843A1 EP 96915791 A EP96915791 A EP 96915791A EP 96915791 A EP96915791 A EP 96915791A EP 0828843 A1 EP0828843 A1 EP 0828843A1
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European Patent Office
Prior art keywords
growth factor
fusion protein
rhrg
cells
rehrg
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German (de)
English (en)
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Jean-Michel Culouscou
Gary W. Carlton
Alejandro A. Aruffo
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Bristol Myers Squibb Co
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Bristol Myers Squibb Co
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    • 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
    • C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to the generation of versatile recombinant heregulins (HRGs) and some of the biological functions and intracellular signaling pathways that these proteins trigger following receptor activation.
  • HRGs versatile recombinant heregulins
  • Heregulins are mosaic glycoproteins that bind to, and induce tyrosine phosphorylation of the HER4/P180 ⁇ rbB4 receptor.
  • Heregulins (HRGs), Holmes, W. E. et al.. (1992), Science 256. 1205-1210, neu differentiation factor (NDF), Peles, E. et al.. (1992), C 69, 205-216; Wen, D. et al.. (1992), Qg ⁇ 69, 559-572 and Wen, D. et al.. (1994), Mol. Cell Biol. 14, 1909-1919, glial growth factors (GGFs), Marchionni, M. A. et al..
  • HRG acetylcholine receptor-inducing activity
  • the HRG isoforms originate from a single gene by alternative RNA splicing.
  • HRG cDNAs encode large transmembrane precursors with multiple domains including an immunoglobulin-like domain, a spacer domain with several glycosylation sites, an EGF-like domain, a juxtamembrane domain of variable length, a transmembrane region, and a cytoplasmic domain.
  • the soluble mature HRGs are released from the cell surface by proteolytic cleavage.
  • GGFs Marchionni, M. A. et al.. (1993) Nature 362, 312-318, and ARIA, Falls, D. L et al.. (1993) Cell 72, 801 -815, which were isolated from brain tissues, also contain a kringle-like domain that is absent in HRGs and NDFs.
  • ⁇ and ⁇ HRG isoforms display sequence differences in the third loop of the EGF-like domain, and the juxtamembrane domain.
  • the EGF-like domain of HRGs contains six cysteine residues that are characteristic of the EGF family of growth factors including EGF, Carpenter, G. et al.. (1979) Ann. Rev. Biochem..
  • HRGs contain an EGF-like motif, they do not bind to EGFR/P170 erbB1 , Holmes, W. E. et aL (1992). Science 256. 1205-1210.
  • the HRGs bind to HER4/pl80 erbB4 , a recently isolated member of the epidermal growth factor receptor family, Plowman, G. D. et al.. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1746-1750; Culouscou, J. M. et al.. (1993) J. Biol. Chem. 268, 18407-18410 and Plowman, G. D. et al..
  • the HER3/P180 ⁇ rb ⁇ 3 receptor another member of this family has also been reported to be a receptor for HRGs, Carraway, K. L et al.. (1994) J. Biol. Chem. 269, 14303-14306. HRGs do not directly interact with HER2/p185 erbB2 receptor, as originally proposed, Holmes, W. E. et al.. (1992), Science 256, 1205-1210 and Peles, E. et al.. (1992), C_eJl 69, 205-216. The HER2/p185 erbB2 indirectly participates in HRG-mediated signaling through transphosphorylation or receptor heterodimerization with HER4 and/or HER3, Plowman, G. D. et al..
  • receptor tyrosine kinases such as the epidermal growth factor receptor, the platelet-derived growth factor receptor and the insulin receptor.
  • the studies carried out on receptor tyrosine kinases have demonstrated a crucial role for receptor autophosphorylation in intracellular signal transduction following ligand binding, Ullrich, A. et al.. (1990) C ⁇ H 61 , 203-212 and White, M. F. et al.. (1994) J. Biol. Chem. 269, 1 -4. It has been demonstrated that specific autophosphorylation sites on receptor tyrosine kinases serve as recognition structures for target molecules containing Src homology 2(SH2) domains.
  • SH2 Src homology 2
  • SH2 domains are conserved noncatalytic sequences of approximately 100 amino acid found in various signaling molecules and oncogenic proteins, Koch, C. A. et al.. (1991 ) Science 252. 668- 674 and Songyang, Z. et al.. (1993) C_eJi 72, 767-778.
  • SH2 domain-containing proteins bind with high affinity to phosphotyrosine residues in the context of specific flanking amino acids. For example, the p85 subunit of phosphatidylinositol (P
  • SHEET (RULE 2 ⁇ ) l)3 ' -kinase (P I 3-K), the p21 ras GTPase activating protein (GAP), and phospholipase C ⁇ (PLC- ⁇ ) have been shown to contain SH2 domains. More recently, SH2 domain-containing proteins that lack an apparent catalytic domain and seem to function as adaptors linking proteins involved in signal transduction have been described, Lowenstein, E. J. et al.. (1992) C 70, 431 -442; Pelicci, G. et al.. (1992) £eJJ 70, 93-104; Egan, S. E. et al..
  • She was identified and cloned based on its homology to SH2 sequences from the human c-fes gene, Pelicci, G. ⁇ LaL, (1992) £ 70, 93-104.
  • the She cDNA is predicted to encode two proteins of 46 and 52 kDa that contain a single C-terminal SH2 domain and a collagen-homologous region that is rich in glycine and proline. No catalytic domain was identified in She.
  • Anti-She antibodies have been shown to recognize three proteins of 46, 52, and 66 kDa in a wide range of mammalian cells.
  • She proteins are associated with a transformed phenotype in fibroblasts, Pelicci, G. et al.. (1992) Cell 70, 93-104, and neuronal differentiation of PC12 cells, Rozakis-
  • the present invention is directed to the generation of versatile recombinant heregulins (HRGs) and the biological functions and intracellular signaling pathways that these proteins trigger following receptor activation.
  • HRGs versatile recombinant heregulins
  • the present invention relates to the cloning of the cDNA fragments encoding the EGF-like domains of HRG- ⁇ ,- ⁇ 2, or -83 into an eukaryotic expression vector containing sequences encoding a thrombin cleavage site, followed by the Fc portion of a human IgGI.
  • the present invention also relates to the production of the recombinant fusion proteins rHRGs-T-Fc which can be used as chimeric proteins or as EGF-like domains (reHRGs) after thrombin cleavage and removal of the Fc portion of the molecule.
  • the present invention demonstrates that the recombinant HRGs, in either form bind to and activate the HER4 receptor and that the She proteins are tyrosine phosphorylated following HRG stimulation.
  • the present invention also demonstrates that rHRG- ⁇ -T-Fc bound to human breast cancer cells that express HER4 receptors and induced the expression of the intercellular adhesion molecule-1.
  • reHRG-82 markedly induced phosphorylation of She proteins on tyrosine, suggesting a role for these adaptor molecules in HRG-mediated signaling.
  • Figure 1 illustrates tyrosine autophosphorylation of the HER4 receptor following rHRGs-T-Fc stimulation.
  • Figure 2 illustrates the binding of rHRG- ⁇ -T-Fc to MDA- MB-453 cells.
  • Figure 3 illustrates the induction of ICAM-1 expression in response to rHRG- ⁇ -T-Fc.
  • Figure 4 illustrates thrombin cleavage of rHRG- ⁇ 3-T-Fc.
  • Figure 5 illustrates the stimulation of protein phosphorylation in response to reHRGs.
  • Figure 6 illustrates tyrosine phosphorylation of She proteins upon HER4 activation.
  • Figure 7 shows the nucleotide and amino acid sequences (SEQ. ID. NOS. 8 and 9) of a heregulin alpha fusion protein.
  • the nucleotide bases correspond to the following:
  • the present invention is directed to the generation of recombinant EGF-like domains of HRGs and the biological effects induced by them as well as identifying intracellular molecules involved in HER4 signaling.
  • the present invention relates to the cloning of the EGF-like domains of HRG- ⁇ ,- ⁇ 2 and - ⁇ 3 into an eukaryotic expression vector in frame with sequences encoding a thrombin cleavage site followed by the Fc portion of a human IgGI. More preferably, the vector further comprises the signal sequence of the CD5 protein which allows
  • the vector is a mammalian expression vector.
  • the present invention relates to the creation of chimeric genes which direct the expression of recombinant fusion proteins, rHRGs-T-Fc.
  • the recombinant fusion proteins are expressed in large amounts by transfecting the vector onto a suitable host.
  • the preferred host is mammalian COS cells. These proteins are shown to stimulate the phosphorylation of HER4/P180 ⁇ rbB4 .
  • the bivalent fusion proteins generated are useful as growth factors since they activate growth factor receptors. These fusion proteins are also useful in being detected like antibodies, via their Fc domain.
  • the recombinant fusion proteins can also be used in high throughput screening assay for identifying low molecular weight agonist or antagonist of HER3 and HER4 receptors.
  • the high throughput screening assay involves screening several hundreds of compounds in a short period of time in microliter well plates using reagents. The assay is carried out with the assistance of robotics and automation. The assay could be a binding assay or an enzyme assay, etc., depending on the compound being screened.
  • the present invention is also useful for the production of large amounts of of other recombinant growth factors, such as epidermal growth factor, transforming growth factor-alpha, amphiregulin, betacellulin, heparin-binding epidermal growth factor, vaccinia growth factor, cripto, insulin growth factor, insulin ⁇ like growth factor, transforming growth factor-beta, platelet-derived growth factor, fibroblast growth factor, and nerve growth factor.
  • growth factors such as epidermal growth factor, transforming growth factor-alpha, amphiregulin, betacellulin, heparin-binding epidermal growth factor, vaccinia growth factor, cripto, insulin growth factor, insulin ⁇ like growth factor, transforming growth factor-beta, platelet-derived growth factor, fibroblast growth factor, and nerve growth factor.
  • the present invention also relates to purified EGF-like domains (reHRGs) after thrombin protease cleavage of the rHRGs-T-Fc fusion proteins. These reHRGs are shown to stimulate protein phosphorylation in HER4 expressing cells.
  • the present invention is also directed to a method of purifying the fusion proteins rHRGs-T-Fc in a single step by protein A-Sepharose chromatography.
  • the present invention demonstrates that reHRG-B2 markedly induces phosphorylation of She proteins on tyrosine. This suggests a role for these adaptor molecules in HRG-mediated signaling.
  • NDF neu differentiation factor ARIA acetylcholine receptor inducing activity GGF glial growth factor p1 85 erbB2 H ER2 encoded protein
  • ICAM-1 intercellular adhesion molecule-1 SH2 Src homology 2 CHO Chinese hamster ovary
  • the EGF-like domain of HRG- ⁇ corresponds to residue 177 to 241 of the mature protein, while that of HRG- ⁇ 2 and - ⁇ 3 corresponds to residue 177 to 238 and residue 177 to 241 , respectively.
  • the three fusion proteins rHRGs-T-Fc are prepared by transient expression in COS cells, purified from
  • SUBSTITUTE SHEET (RULE 28) culture supernatants on protein A-Sepharose, and gave yields in the range of 350 to 1900 ⁇ g/l.
  • the CD5 signal peptide allowed efficient processing and secretion of the rHRGs-T-Fc. All three fusion proteins are secreted as disulfide-linked homodimers similar to immunoglobulins and therefore are each capable of presenting two HRG-EGF-like domains.
  • a study is preferably carried out of their potential to induce phosphorylation of HER4 as well as morphological changes and up-regulation of ICAM-1.
  • the chimeric genes of other growth factors such as, epidermal growth factor, transforming growth factor-alpha, amphiregulin, betacellulin, heparin-binding epidermal growth factor, vaccinia growth factor, cripto, insulin growth factor, insulin-like growth factor, transforming growth factor- beta, platelet-derived growth factor, fibroblast growth factor, and nerve growth factor, can be constructed and expressed in a similar manner as for rHRGs-T-Fc.
  • HER4 receptor The activation of the HER4 receptor by rHRGs-T-Fc is examined with CHO/HER4 cells that express high levels of recombinant human p180erbB4/HER4 and have previously been shown to respond to HRG, Plowman, G. D. et al.. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1746-1750 and Culouscou, J. M. et al.. (1993) J. Biol. Chem. 268. 18407-18410.
  • rHRG- ⁇ -T-Fc, - ⁇ 2-T-Fc, and - ⁇ 3-T-Fc are added to CHO/HER4 cells at 50 and 200 ng/ml for 10 minutes at 37°C. Cells are then lysed and then the pattern of tyrosine phosphorylated proteins are analyzed by anti-
  • SUBSTITUTE SHEET (RULE 28) phosphotyrosine Western blotting as compared to untreated cells. As shown in Fig. 1 A, all three rHRGs-T-Fc induced the hyper- phosphorylation of the HER4 receptor. Ligand activation not only resulted in receptor autophosphorylation, but also in the tyrosine phosphorylation of several substrates, including a Mr 100,000 band, not identified (Fig. 1A). When tested on CHO/EGFR cells that express high levels of recombinant human EGFR, rHRGs-T-Fc (200 ng/ml) failed to activate the EGFR (Fig. 1 B).
  • the EGF (200 ng/ml) markedly induced phosphorylation of the EGFR in CHO/EGFR cells (Fig. 1 B, lane 2). These demonstrate that the rHRGs-T-Fc are active molecules and are able to specifically induce HER4 tyrosine phosphorylation.
  • rHRG- ⁇ -T-Fc The binding of rHRG- ⁇ -T-Fc to HER4 expressing cells can be shown in the MDA-MB-453 cells that are known to express the HER4 receptor, Plowman, G. D. et al.. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1746-1750, as well as in the related receptors HER2 and HER3, Kraus, M. H.et aL (1987) EMBO J. 6, 605-610 and Kraus, M. H. et al.. 1989) Proc. Natl. Aca. Sci. U.S.A. 86, 9193- 9197.
  • rHRG-a- T-Fc Bound fusion proteins are detected by adding fluorescein- conjugated anti-human IgG antibodies which recognize the human Fc portion of rHRG- ⁇ -T-Fc.
  • rHRG- ⁇ -T- Fc bound to MDA-MB-453 cells-(Fig. 2B, 10 ⁇ g/mL; and 2D, 1 ⁇ g/mL).
  • the fluorescence as analyzed by confocal microscopy, is localized at the periphery of the cells which is consistent with the fact that the staining is performed on live cells kept on ice.
  • the fluorescein-conjugated anti-human IgG showed no detectable binding to the MDA-MB-453 cells (Fig.
  • NDF the rat homologue of HRG
  • HRG the rat homologue of HRG
  • rHRG- ⁇ -T-Fc the ability of rHRG- ⁇ -T-Fc to induce the expression of ICAM-1 at the surface of MDA-MB-453 cells
  • these cells are treated for 3 days with 50 ng/ml of the fusion protein and stained with an anti-ICAM-1 antibody. Bound anti-ICAM-1 antibodies are detected using a fluorescein-conjugated anti- mouse IgG antibody.
  • rHRG- ⁇ -T-Fc induced a clear up-regulation of ICAM-1 expression in MDA-MB-453 cells as compared to untreated cells (Fig. 3A) and cells treated with an irrelevant Tek-Fc fusion protein (Fig. 3C).
  • p45 a HRG isoform purified from conditioned medium from HepG2 cells, Culouscou, J. M. et al.. (1993) J. Biol. Chem. 268, 18407-18410, is used at 50 ng/ml as a positive control and induced up-regulation of ICAM-1 (Fig. 3D).
  • the result shows that the rHRGs-T-Fc elicited biological responses similar to those elicited by the natural HRGs in breast carcinoma cells expressing the HER4 receptor and can be used to study the biological consequences of HRG binding to such cells.
  • the DNA fragments encoding the EGF-like domains of HRGs are amplified by PCR, purified and inserted into a CDM7-
  • SUBSTITUTE SHEET (RULE 28) derived expression vector containing sequences encoding a thrombin cleavage site upstream and in frame with the Fc portion of a human IgGI antibody.
  • the addition of a thrombin cleavage site in the expression vector is based on a system developed by Hakes and Dixon, Hakes, D. J. et al.. (1992) Anal. Biochem. 202, 293-298, for recombinant protein expression in bacteria.
  • the presence of a thrombin cleavage site in the rHRGs-T-Fc allows for separation of the two functional domains of the fusion proteins.
  • the purified EGF-like domains are recovered as monomeric proteins since the thrombin site is located upstream of the hinge region of the Fc domain of the fusion proteins.
  • rHRG- ⁇ 2-T-Fc and - ⁇ 3-T-Fc are incubated with human thrombin.
  • the recombinant EGF-like domains of HRGs (reHRGs) are then separated from the Fc portion of the molecules by protein A-Sepharose chromatography. reHRGs are recovered in the column flow-throughs. Fc portions are recovered from protein A-Sepharose by acid elution. Fig.
  • FIG. 4 shows a silver stained polyacrylamide gel of the rHRG- ⁇ 3-T-Fc before thrombin cleavage (lane 1 ).
  • the intact fusion protein displays an apparent molecular mass of 40 kDa under reducing conditions, corresponding to its monomeric form.
  • lane 2 After cleavage but before protein A-Sepharose (lane 2), a 34 kDa band, corresponding to the Fc portion of the fusion protein, and a 6 kDa band, corresponding to the EGF-like domain of HRG- ⁇ 3, are identified.
  • the two fragments are separated by protein A-Sepharose chromatography.
  • the 6 kDa EGF-like domain of HRG- ⁇ 3 (reHRG- ⁇ 3) is recovered in the column flow-through (lane 3), and the 34 kDa Fc domain of the fusion protein is acid eluted from the column (lane 4).
  • SUBSTITUTE SHEET (RULE 28) The stimulation of protein phosphorylation in response to reHRGs are carried out on the purified reHRG- ⁇ 2, reHRG- ⁇ 3, and Fc domains of rHRG- ⁇ 2-T-Fc and rHRG- ⁇ 3-T-Fc in MDA-MB- 453 cells.
  • the intact rHRG- ⁇ 2-T-Fc and - ⁇ 3-T-Fc (Fig. 6B, lanes 3 and 6, respectively) are potent stimulators of tyrosine phosphorylation of a 180 kDa protein, as compared to background levels of phosphorylation observed in the absence of treatment (lane 1) or following EGF treatment (lane 2).
  • the region proximal to the EGF domain (referred to as the juxtamembrane region in the HRG/NDF precursor forms) can be absent, e.g.
  • HRG- ⁇ 2/NDF- ⁇ 2 or comprise up to 26 amino acids, e.g. NDF- ⁇ 4, Holmes, W. E. et al.. (1992), S_cjej ⁇ c_e_ 256, 1205-1210 and Wen, D. et al.. (1994), Mol. Cell Biol. 14, 1909-1919.
  • a truncated form of NDF- ⁇ that lacks the juxtamembrane region displays the same receptor binding affinity as the full-length NDF- ⁇ isoform, implying that this region proximal to the EGF domain is
  • SUBSTITUTE SHEET (RULE 28) The phosphorylation of She upon HER4 receptor activation is carried out as follows: the activation of receptor tyrosine kinases, such as the EGF receptor, the insulin receptor, and the PDGF receptor results in phosphorylation of a number of intracellular signaling molecules, Ullrich, A. et al.. (1990) Cell 61 , 203-212 and White, M. F. et al.. (1994) J. Biol. Chem. 269, 1 -4. For analyzing the molecules that are involved in HRG signaling, MDA- MB-453 cells are stimulated with or without 200 ng/ml reHRG- ⁇ 2.
  • CHO/HER4 cells and MDA-MB-453 cells are exposed to reHRG- ⁇ 2, and lysed.
  • Equivalent amounts of cell lysates are immunoprecipitated with an anti-She antibody, and blotted with either anti-She (Fig. 6A), or anti-phosphotyrosine antibodies (Fig. 6B).
  • Fig. 6A anti-She
  • Fig. 6B anti-phosphotyrosine antibodies
  • 6A shows that equal amounts of proteins from stimulated and unstimulated cell lysates are loaded per lane, and that MDA-MB-453 cells (lanes 1 and 2) express only p46Shc and p52Shc (p66Shc is not detected in the assay), whereas
  • SUBSTITUTE SHEET (RULE 28) CHO/HER4 cells (lanes 3 and 4) express all three She isoforms. p66Shc is translated from a different transcript than the other two She isoforms and is not expressed in every cell type, for example it is absent in human hematopoietic cell lines, Pelicci, G. et al.. (1992) C_eJi 70, 93 -104. As seen in Fig. 6B, reHRG- ⁇ 2 induced hyper-phosphorylation of She in both cell types. In MDA-MB-453 cells, reHRG- ⁇ 2 stimulation resulted in tyrosine phosphorylation of both p46Shc and p52Shc (lanes 1 and 2).
  • p46Shc appeared to display a relatively high endogenous level of phosphorylation in those cells, and is only marginally affected following HRG treatment.
  • results presented in the instant invention indicate recombinant EGF-like domains of HRG- ⁇ , - ⁇ 2, and - ⁇ 3 fused to a thrombin cleavage site followed by the Fc domain of a human IgGI.
  • These reagents are useful in in vitro assays as fusion proteins or as a source of truncated recombinant HRGs.
  • results also indicate that both forms in vitro can activate the HER4 receptor and elicit known HRG biological responses.
  • the present invention shows for the first time, that following HRG stimulation, She proteins which have been implicated in Ras activation pathway are phosphorylated on tyrosine.
  • the availability of the recombinant HRGs will allow further experiments to dissect the mechanism of HRG receptor signaling as well as compare the
  • SUBSTITUTE SHEET (RULE 28) HER4 substrates to those of other members of the EGFR family of tyrosine kinases.
  • Antibodies - RC20 recombinant antiphosphotyrosine antibody (Transduction Laboratories) and PY20 antiphosphotyrosine antibody (ICN Biomedicals, Inc.) used in Western blotting studies were purchased from Transduction Laboratories and ICN Biomedicals, Inc.
  • Polyclonal anti-She antibodies were purchased from Upstate Biotechnology Incorporated and the monoclonal anti-She antibody was purchased from Transduction Laboratories.
  • BBA 3 the anti- human ICAM-1 monoclonal antibody, was purchased from R & D Systems.
  • CHO/EGFR cells were generated by Dr. B. Thome (Bristol-Myers Squibb, Seattle, WA.) as follows: the complete recombinant human EGF receptor coding sequence was inserted into a CDM8 expression vector containing the neomycin resistance gene. The resulting construct was transfected into Chinese hamster ovary cells (CHO-KI). G418 resistant clones were analyzed for EGFR
  • SUBSTITUTE SHEET (RULE 28) expression.
  • Levels of expression of functional EGFR in CHO/EGFR stable cells were assessed by stimulating the cells with EGF, immunoprecipitating the EGFR and determining its phosphorylation level by phosphotyrosine Western blotting as reported, by Plowman, G. D. et al., (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1746-1750.
  • CHO/HER4 cells expressing high levels of recombinant human HER4 have previously been described, by Plowman, G. D. et al.. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1746-1750; Culouscou, J. M. et al.. (1993) J. Biol. Chem. 268, 18407-18410 and Plowman, G. D. et al.. (1993) Nature (London) 366, 473-475.
  • DNA fragments encoding part of the spacer domain of the human HRGs, the EGF-like domains, the transmembrane domain, and a few residues of the cytoplasmic domain were amplified by RT-PCR from total RNA isolated from HepG2 cells.
  • the oligonucleotide primers were designed based on the sequence of the human HRG- ⁇ , Holmes, W. E. et al.. (1992), Science 256. 1205-1210.
  • PCR primers used were synthesized and have the following sequences:
  • SEQ. I.D. NO.: 2 Amplification was performed with Taq DNA polymerase (Perkin-Elmer Roche) using 35 cycles, each cycle being composed of a 1 minute at 95°C denaturing step, 1 minute
  • the EGF-like domains of HRG- ⁇ , -B2, and -B3 were generated by PCR using HRG- ⁇ , or - ⁇ 2 template plasmids generated as described above.
  • the oligonucleotide primers described below were designed to place a Spel site at the 5 ' end and a BamH1 site at the 3 ' end of the amplified products for cloning purposes.
  • the epidermal growth factor-like domain of the human HRG-a was amplified using the following sequences: 5 ' -GAGACTAGTAGCCATCTTGTAAAATGTGCG-3 '
  • PCR conditions consisted of 40 cycles of 30 sec at 94°C, 1 minute at 55°C, and 2 minutes at 72°C, using Pfu polymerase and reagents recommended by the vendor
  • the PCR product encoded complementary sequences corresponding to residue 177 to 241 of HRG- ⁇ .
  • the epidermal growth factor-like domains of human HRG- ⁇ 2 and - ⁇ 3 were amplified using a HRG- ⁇ 2 clone as a template.
  • the forward primer is shown above (SEQ. I.D. No.: 3).
  • the HRG- ⁇ 2 reverse primer had the sequence:
  • SUBSTITUTE SHEET (RULE 28) polymerase using the same temperature conditions as that used for HRGa.
  • This PCR product encoded sequences corresponding to residue 177 to 238 of HRG- ⁇ 2.
  • the HRG- ⁇ 3 reverse primer contained a silent point mutation introducing a Hindlll site for diagnostic purposes and had the following sequence:
  • PCR conditions consisted of 40 cycles of 1 minute at 94°C, 2 minutes at 50°C, and 3 minutes at 72°C using Pfu polymerase.
  • the PCR product encoded sequences corresponding to residues 177 to 241 of HRG- ⁇ 3.
  • PCR products were digested with BamHI and Spel and ligated to a BamHI-Spel-cut CDM7-derived vector containing cDNA sequences coding for the CD5 signal peptide 5 ' of the cloning site for proper secretion of the expressed proteins, as well as cDNA sequences encoding a thrombin cleavage site (amino acid sequence DPGGGGGRLVPRGFGTG; Sequence I.D. No. 7) and cDNA sequences encoding the hinge and constant regions of a human IgGI, 3 ' of the cloning site.
  • THROMBIN CLEAVAGE OF FUSION PROTEINS Fusion proteins were incubated for 30 minutes at room temperature with human thrombin (purchased from Sigma, St.
  • CHO/HER4 cells (5x104), CHO/EGFR cells (2x104), and MDA-MB-453 cells (4x105) were seeded in 48-well plates. 24 hours later, cells were serum-starved for 8 hours and then
  • SUBSTITUTE SHEET (RULE 28) stimulated with various samples for 10 minutes at 37°C. Supematants were discarded, and cells were lysed by adding boiling electrophoresis sample buffer. Lysates were subjected to SDS-PAGE on 8% polyacrylamide gels (purchased from Novex) and then electroblotted onto nitrocellulose. PY20 monoclonal anti-phosphotyrosine antibody (purchased from ICN) and horseradish peroxidase-conjugated goat anti-mouse IgG F(ab ' )2 (purchased from Cappel) were used as primary and secondary probing reagents, respectively. Immunoreactive bands were visualized using enhanced chemiluminescence (purchased from Amersham Corp.). The results showed the pattern of tyrosine phosphorylated proteins in HER4 receptor which is illustrated in Fig. 1.
  • CHO/HER4 cells were seeded in 100-mm dish. 80-90% confluent monolayers were washed and incubated with the various recombinant HRGs for 10 minutes at 37°C. Monolayers were washed with ice-cold PBS, and solubilized for 10 minutes on ice in PBSTDS lysis buffer (10 mM sodium phosphate, pH 7.3, 150 mM NaCI, 1% Triton-X100, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 1 mM Na3 V04, 20 ⁇ g/ml aprotinin, 20 ⁇ g/ml leupeptin, 20 ⁇ g/ml pepstatin. The protein concentrations of the clarified extracts were determined using a BCA protein assay kit (purchased from Pierce). Lysates (1 mg per immunoprecipitation) were incubated overnight at 4°C with
  • MDA-MB-453 cells were plated in 8-well borosilicate chambered slides (purchased from Lab-Tek). For receptor binding visualization, after a 48 hour-culture period, the cells were placed on ice for 10 minutes, washed twice with ice-cold binding buffer (DMEM supplemented with 44 mM sodium bicarbonate, 50 mM Bes, pH 7.0,0.1% bovine serum albumin) and then incubated on ice for 2 hours with rHRG-a-T-Fc, or as a negative control, an irrelevant fusion protein consisting of the extracellular domain of Tek receptor, Dumont, D. J. et al..
  • DMEM ice-cold binding buffer
  • rHRG-a-T-Fc or as a negative control, an irrelevant fusion protein consisting of the extracellular domain of Tek receptor, Dumont, D. J. et al..
  • Oncogene 8, 1293-1301 fused to the thrombin cleavage site followed by the Fc region of an IgGI as in rHRGs-T-Fc.
  • SUBSTITUTE SHEET (RULE 28) mammalian cells used to construct and generate the Tek-Fc fusion protein were identical to the ones used to make the rHRGs-T-Fc.
  • the cells were washed twice and incubated for 45 minutes on ice with a fluorescein-conjugated goat anti-human IgG F(ab ' )2 (purchased from Tago).
  • the cells were rinsed twice with PBS and fixed for 20 minutes in PBS, 2% formaldehyde. The results showed that the recombinant fusion protein can be used to stain cells that express HER4 receptor.
  • the MDA-MB-453 cells were incubated for three days with 50 ng/ml rHRG- ⁇ -T-Fc, p45, Culouscou, J. M. et al.. (1993) J. Biol. Chem. 268, 18407-18410, Tek-Fc fusion protein as a negative control, or culture medium alone. Staining was then performed on live cells. The cells were washed and incubated for 1 hour on ice with an anti-ICAM-1 antibody (purchased from R & D Systems) diluted 1 :500 in binding buffer.
  • an anti-ICAM-1 antibody purchased from R & D Systems
  • the cells were washed and incubated for 45 minutes on ice with a fluorescein-conjugated goat anti-mouse IgG F(ab ' )2 (purchased from Tago). The cells were rinsed and fixed as described above. The levels of receptor staining and ICAM-1 expression were analyzed using a Leica confocal microscope. The results showed that fusion protein induced the expression of ICAM-1 in breast carcinoma cells that express HER4 receptor and is illustrated in Fig. 3.
  • CHO/EGFR cells were incubated in the absence (lane 1) or the presence of EGF (lane 2), rHRG- ⁇ -T-Fc (lane 3), rHRG- ⁇ 2-T-Fc (lane 4), and rHRG- ⁇ 3-T-Fc (lane 5) used at 200 ng/ml.
  • Cells lysates were processed as described in A. The positions of HER4 and EGFR are indicated. These results illustrated in Fig. 1 B showed that rHRGs-T-Fc failed to activate the EGFR.
  • Fig. 1 B (lane 2) showed that EG'F markedly induced phosphorylation of the EGFR in CHO/EGFR cells.
  • MDA-MB-453 cells were cultured for 24 hours in 8-well Lab-Tek (Nunc) chamber slides. Cells were treated with 50 ng/ml rHRG-a-T-Fc (panel B), irrelevant fusion protein (panel C), p45, Culouscou, J. M. et al.. (1993) J. Biol. Chem. 268, 18407-18410, (panel D), or left untreated (panel A). Following three additional days of incubation the cells were stained with an anti-ICAM-1
  • THROMBIN CLEAVAGE OF rHRG- ⁇ 3-T-FC - Figure 4 The fusion protein was incubated with human thrombin at room temperature for 30 minutes, and loaded on a protein A- Sepharose column. The EGF-like domain of HRG- ⁇ 3 (reHRG-B3) was recovered in the column flow-through while the Fc portion of the fusion protein was eluted from the column. The resulting products were analyzed by SDS-PAGE, and silver stained.
  • Lane 1 rHRG- ⁇ 3-T-Fc, untreated; lane 2, rHRG- ⁇ 3-T-Fc, after thrombin cleavage; lane 3, protein A-Sepharose column flow-through (reHRG- ⁇ 3); lane 4, protein A-Sepharose column eluate (Fc portion of the fusion protein).
  • Fig. 4 (lane 1 ) showed a silver stained polyacrylamide gel of the rHRG- ⁇ 3-T-FC before thrombin cleavage.
  • Fig. 4 (lane 2) showed a 34 kDa band corresponding to the Fc portion of the fusion protein and a 6 kDa band corresponding to the EGF-like domain of HRG- ⁇ 3. The 6 kDa
  • MDA-MB-453 cells were incubated in the absence (lane 1 ) or the presence of EGF (lane 2), rHRG- ⁇ 2-T-Fc (lane 3), reHRG- ⁇ 2 (lane 4), Fc portion of the rHRG- ⁇ 2-T-Fc fusion protein (lane 5), rHRG- ⁇ 3-T-Fc (lane 6), reHRG- ⁇ 3 (lane 7), Fc portion of the rHRG- ⁇ 3-T-Fc fusion protein (lane 8), at 200 ng/ml.
  • Cells were lysed, proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane and blotted with an anti-phosphotyrosine antibody.
  • MDA-MB-453 cells (lanes 1 and 2) and CHO/HER4 cells (lanes 3 to 6) were treated with (+) or without (-) 200 ng/ml of reHRG- ⁇ 2 for 10 minutes at 37°C and solubilized.
  • Cell lysates containing equal amounts of protein (1 mg) were precipitated with a polyclonal rabbit anti-She antibody. Immune complexes were washed, separated by SDS-PAGE, and transferred to nitrocellulose.
  • She proteins were detected by immnunoblot using a monoclonal anti-She antibody.
  • Fig. 6A The results illustrated in Fig. 6A showed that MDA-MB- 453 cells (lanes 1 and 2) express only p46Shc and p52Shc whereas CHO/HER4 cells (lanes 3 and 4) express all three She isoforms.
  • Fig. 6B showed that reHRG- ⁇ 2 induced hyper- phosphorylation of She in both cell types.
  • MDA-MB-453 cells reHRG- ⁇ 2 stimulation resulted in tyrosine phosphorylation of both p46Shc and p52Shc (lanes 1 and 2).
  • phosphorylation of'p66Shc was markedly increased in CHO/HER4 cells (lanes 3 and 4). Longer exposure time of the blot (Fig.
  • the expression plasmids of other growth factors can be constructed analogously from cDNA's of other growth factors, such as epidermal growth factor, transforming growth factor-alpha, amphiregulin, betacellulin, heparin-binding epidermal growth factor, vaccinia growth factor, cripto, insulin growth factor, insulin- like growth factor, transforming growth factor-beta, platelet-derived growth factor, fibroblast growth factor, or nerve growth factor, according to Example 1 and expressed according to Example 2.
  • Other growth factors such as epidermal growth factor, transforming growth factor-alpha, amphiregulin, betacellulin, heparin-binding epidermal growth factor, vaccinia growth factor, cripto, insulin growth factor, insulin- like growth factor, transforming growth factor-beta, platelet-derived growth factor, fibroblast growth factor, or nerve growth factor, according to Example 1 and expressed according to Example 2.

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Abstract

L'invention porte sur la production d'hérégulines de recombinaison polyvalentes ainsi que sur quelques fonctions biologiques et voies de signalisation intracellulaires déclenchées par ces protéines à la suite de l'activation d'un récepteur. L'invention a également trait au clonage de fragments d'ADN complémentaire codant le domaine du type EGF de l'héréguline-α, β2 ou β3 dans un vecteur d'expression eucaryote et à la transfection de ce vecteur dans des cellules mammaliennes. Ces hérégulines de recombinaison s'avèrent utiles dans le cadre de l'activation du récepteur HER4.
EP96915791A 1995-05-16 1996-05-14 Heregulines de recombinaison et leurs fonctions biologiques lors de l'activation d'un recepteur Withdrawn EP0828843A1 (fr)

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US5713848A (en) 1993-05-19 1998-02-03 Dubrul; Will R. Vibrating catheter
US6479254B2 (en) 1996-03-22 2002-11-12 Human Genome Sciences, Inc. Apoptosis inducing molecule II
US7964190B2 (en) 1996-03-22 2011-06-21 Human Genome Sciences, Inc. Methods and compositions for decreasing T-cell activity
US6635743B1 (en) 1996-03-22 2003-10-21 Human Genome Sciences, Inc. Apoptosis inducing molecule II and methods of use
US6495520B2 (en) 1996-03-22 2002-12-17 Human Genome Sciences, Inc. Apoptosis Inducing Molecule II and methods of use
US6136558A (en) * 1997-02-10 2000-10-24 Genentech, Inc. Heregulin variants
WO1998035036A1 (fr) * 1997-02-10 1998-08-13 Genentech, Inc. Variants d'hereguline
ZA986077B (en) 1997-07-09 2000-01-10 Genentech Inc ErbB4 receptor-specific neuregulin related ligands and uses therefor.
US6121415A (en) 1997-07-09 2000-09-19 Genentech, Inc. ErbB4 receptor-specific neuregolin related ligands and uses therefor
US6994856B1 (en) 1997-07-24 2006-02-07 Genentech, Inc. ErbB4 receptor-specific neuregulin related ligands and uses therefor
IL137419A0 (en) * 2000-07-20 2001-07-24 Yissum Res Dev Co Nk cells activating receptors and their therapeutic and diagnostic uses
CN102676571A (zh) * 2011-11-18 2012-09-19 北京市结核病胸部肿瘤研究所 一种真核蛋白表达载体及其构建和应用

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