WO2002000876A1 - Truncated egf receptor - Google Patents
Truncated egf receptor Download PDFInfo
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- WO2002000876A1 WO2002000876A1 PCT/AU2001/000782 AU0100782W WO0200876A1 WO 2002000876 A1 WO2002000876 A1 WO 2002000876A1 AU 0100782 W AU0100782 W AU 0100782W WO 0200876 A1 WO0200876 A1 WO 0200876A1
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- egfr ectodomain
- truncated egfr
- truncated
- ectodomain
- egf
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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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
Definitions
- the present invention relates to truncated EGF receptor molecules and to pharmaceutical compositions comprising these molecules.
- the present invention also relates to methods of screening for EGF receptor ligands and methods of treatment which involve the use of these molecules.
- the epidermal growth factor receptor (EGFR) family consists of four distinct tyrosine kinase receptors, EGFR/HER ErbBl, HER2/Neu/ErbB2, HER3/ErbB3 and HER4/ErbB4 (1). These receptors are widely expressed in epithelial, mesenchymal and neuronal tissues and play fundamental roles during development and differentiation. They are activated by a family of at least twelve ligands that induce either homo- or hetero-dimerisation of the EGFR homologues. ErbB2 is unable to bind ligand on its own but is a potent co-receptor for all ligands when co-expressed with other members of the EGFRHER/ErbB family.
- the EGFR is a large (1,186 residues), monomeric glycoprotein with a single transmembrane region and a cytoplasmic tyrosine kinase domain flanked by noncatalytic regulatory regions. Sequence analyses have shown that the ectodomain (residues 1-621) contains four sub-domains, here termed Ll, CR1, L2 and CR2, where L and CR are acronyms for large and Cys-rich respectively (2, 3). The Ll and L2 domains have also been referred to as domains I and III, respectively (4). The CR domains have been previously referred to as domains II and IV (4), or as Sl.l - Si.3 and S2.1 - S2.3 where S is an abbreviation for small (2).
- the present inventors have now made the surprising finding that the deletion of residues in the CR2 domain of the EGFR ectodomain gives rise to a truncated ectodomain with enhanced affinity for epidermal growth factors such as (EGF) and/or transforming growth factor- ⁇ (TGF- ⁇ ). This finding goes against recently reported results (8) showing that deletions or mutations in the CR2 region reduce EGFR binding affinity for EGF.
- EGF epidermal growth factors
- TGF- ⁇ transforming growth factor- ⁇
- the truncated EGFR ectodomains of the present invention may provide increased sensitivity in assays which screen for ligands of the EGF receptor. Furthermore, the truncated EGFR ectodomains of the present invention may have therapeutic potential given their high affinity for ligand and their ability to competitively inhibit EGF-induced proliferation responses in vitro.
- the present invention provides a truncated EGFR ectodomain, the truncated EGFR ectodomain lacking a substantial portion of the CR2 domain such that the truncated EGFR ectodomain has an increased binding affinity for at least one EGFR ligand when compared to the full length EGFR ectodomain.
- the EGFR ligand may be, for example, amphiregulin, heparin binding
- the truncated EGFR ectodomain has an increased binding affinity for EGF and/or TGF- ⁇ .
- the truncated EGFR ectodomain lacks at least the third to seventh modules of the CR2 domain. In a further preferred embodiment, the truncated EGFR ectodomain lacks at least the second to seventh modules of the CR2 domain. The truncated EGFR ectodomain may further lack a portion of the first module of the CR2 domain. In a further preferred embodiment, the truncated EGFR ectodomain lacks residues 514-621. In yet a further preferred embodiment, the truncated EGFR ectodomain lacks residues 502-621.
- the truncated EGFR ectodomain of the present invention comprises the Ll, CRl and L2 subdomains.
- the truncated EGFR ectodomain comprises residues 1-492 of the EGFR ectodomain. More preferably, the truncated EGFR ectodomain comprises residues 1-501 or residues 1-513 of the EGFR ectodomain.
- the truncated EGFR ectodomain has an affinity for EGF such that the K d is less than 30 nM, more preferably less than 25 nM. In a further preferred embodiment, the truncated EGFR ectodomain has an affinity for TGF- ⁇ such that the K d is less than 45 nM, more preferably less than 40 nM.
- the present invention provides a polynucleotide encoding a truncated EGFR ectodomain of the first aspect.
- the present invention provides an expression vector comprising a polynucleotide of the second aspect.
- the present invention provides a host cell comprising an expression vector of the third aspect.
- the present invention provides a method for producing a truncated EGFR ectodomain of the first aspect, the method comprising culturing a host cell of the fourth aspect under conditions which allow production of the truncated EGFR ectodomain and isolating the truncated EGFR ectodomain.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a truncated EGFR ectodomain according to the first aspect and a pharmaceutically acceptable carrier or diluent.
- the present invention provides a method of screening a putative compound for the ability to modulate the activity of the
- the method comprising exposing the putative compound to a truncated EGFR ectodomain according to the first aspect and monitoring the activity of the truncated EGFR ectodomain.
- a suitable assay procedure may involve a competition binding assay in a microplate format, where the putative compound is tested for its ability to inhibit the binding of labelled ligand such as IGF-1 or IGF-2 to the truncated EGF receptor ectodomain.
- the label may be a radiolabelled tag such as 125 I or a fluorescent tag such as fluorescein isothiocyanate or a lanthanide ion such as europium.
- the present invention provides a method of treating or preventing a disease associated with signalling by a molecule of the EGF receptor family in a subject, the method comprising administering to the subject a truncated EGFR ectodomain according to the first aspect.
- the disease associated with signalling by a molecule of the EGF receptor family is selected from psoriasis and tumour states comprising but not restricted to cancer of the breast, brain, ovary, cervix, pancreas, lung, head and neck, and melanoma, rhabdomyosarcoma, mesothelioma and glioblastoma.
- the method of the eighth aspect may be used alone or in combination with other therapeutic measures.
- the method of the fourth aspect may be used in combination with cytotoxic modalities, such as anti- EGFR antibodies, radiotherapy or chemotherapy, in the treatment of tumour states.
- Figure 1 BIAcore analysis of the interactions between sEGFR501 and
- SEGFR621 with immobilised hEGF or hTGF- ⁇ was passed over immobilised hEGF or hTGF- ⁇ .
- A sEGFR501 (140, 120, 100, 80, 60 and 40 nM) was passed over immobilised hEGF (160 RU immobilised). Samples (30 ⁇ l) were injected at a flow rate of 10 ⁇ l/min.
- B sEGFR501 was passed over immobilised hTGF- ⁇ (132 RU immobilised). Experimental details were as in panel A.
- C sEGFR621 (1000, 900, 800, 700, 600 and 500 nM) was passed over immobilised hEGF .
- FIG. 2 Scatchard analysis of equilibrium binding data.
- A sEGFR ⁇ Ol versus hEGF
- B sEGFR501 versus hTGF- ⁇
- C SEGFR621 versus hEGF
- D sEGFR621 versus hTGF ⁇ .
- FIG. 3 Inhibition of EGF-stimulated cell mitogenesis by sEGFR ⁇ Ol.
- A The stimulation of 3 H-thymidine incorporation by BaF/3ERX cells using serial dilutions of mEGF. The data was fitted by a sigmoidal function ( - ) to determine the EC 50 .
- B Inhibition of the mitogenic response of BaF/3ERX cells stimulated with mEGF (207 pM) by: sEGFR501 ( ⁇ - ⁇ ), sEGFR621 (• - •) or anti-EGFR antibody Mab528 (A - A). Each point was assayed in triplicate. Error bars are shown.
- FIG. 4 Covalent cross-linking of sEGFR501 dimers after incubation with mEGF.
- sEGFR ⁇ Ol 5 ⁇ M was incubated with (+) or without (-) mEGF (20 ⁇ M) in 20 mM HEPES (pH7.4) containing 150 mM NaCl for 1 h at room temperature followed by the addition of bis(sulfosuccinimidyl)suberate (BS3, Pierce, Rockford, IL, USA) to a final concentration of 0.5 mM and incubation for a further 30 min.
- BS3, Pierce, Rockford, IL, USA bis(sulfosuccinimidyl)suberate
- the reaction was terminated and the degree of dimer formation was monitored by SDS-PAGE and immunoblotting with anti-EGFR Mab528 (7) (0.5 ⁇ g/ml) and horseradish-peroxidase-labelled goat anti-mouse IgG (Bio-Rad) with detection by ECL (Amersham Pharmacia Biotech). Analysis by non-reducing SDS-PAGE was necessary since the antibody used to detect sEGFR ⁇ Ol (Mab528) is conformation-dependent.
- FIG. 5 Analysis of EGF/sEGFR501 interactions using the analytical ultracentrifuge.
- A Sedimentation equilibrium analysis of EGF, sEGFR501 and a mixture of EGF and sEGFR ⁇ Ol. The equilibrium distributions were obtained after centrifugation at 12,000 rpm at 20oC for 16 h.
- D 20 ⁇ M EGF;
- o 10 ⁇ M sEGFR ⁇ Ol,
- ⁇ 20 ⁇ M EGF + 10 ⁇ M sEGFR ⁇ Ol.
- the lines of best fit drawn though the data for EGF and sEGFR ⁇ Ol are for single species and for molecular weight values of 6,000 and 6 ⁇ ,600 respectively.
- the line drawn through the data for the EGF/sEGFR ⁇ Ol mixture is the line of best-fit calculated assuming two species with the molecular weight of the first species fixed at 6,000 and a fitted value of 106,400 for the molecular weight of the second species.
- FIG. 6 BIAcore analysis of the binding of the Gly44lLys sEGFR ⁇ Ol mutant to immobilised hEGF and hTGF- ⁇ .
- Purified Gly44lLyssEGFR ⁇ 01 24 - 38 ⁇ nM was passed over immobilised hTGF- ⁇ (Panel A) or hEGF (Panel B) using the experimental conditions described in Figure 1.
- EGF EGF receptor family
- EGF receptor family molecules show similar domain arrangements and share significant sequence identity, preferably at least 40% identity.
- full length EGFR ectodomain refers to the ectodomain consisting of residues 1-621 of the EGF receptor.
- the amino acid sequence of the full length ectodomain has been previously described (13).
- the full length ectodomain contains four sub-domains, referred to as Ll, CRl, L2 and CR2, where L and CR are acronyms for large and cys-rich respectively.
- the CR2 sub-domain consists of the following seven modules joined by linkers of 2 or 3 amino acid residues and bounded by cysteine residues as follows: First module: cys residues 482-499
- Second module cys residues ⁇ 02- ⁇ ll
- Third module cys residues ⁇ l ⁇ - ⁇ 31
- truncated ectodomains may be prepared by recombinant DNA technology as described herein or as described previously (8).
- truncated ectodomains may be prepared by subjecting the full length ectodomain or full length receptor to limited proteolysis as described previously (9).
- Binding affinity and inhibitor potency may be measured for candidate truncated ectodomains using biosensor technology.
- Truncated EGFR ectodomains of the invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
- a truncated ectodomain of the invention may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein of the invention.
- modified ectodomains may be constructed by making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for binding activity. Generally, substitutions should be made conservatively; i.e., the most preferred substitute amino acids are those having physiochemical characteristics resembling those of the residue to be replaced. Similarly, when a deletion or insertion strategy is adopted, the potential effect of the deletion or insertion on biological activity should be considered.
- deletions and substitutions will preferably result in homologous or conservatively substituted sequences, meaning that a given residue is replaced by a biologically similar residue.
- conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, Met or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn.
- Other such conservative substitutions for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.
- truncated EGFR ectodomains Modifications encompassed by the present invention also include various structural forms of the primary protein which retain binding affinities. Due to the presence of ionizable amino and carboxyl groups, for example, a truncated ectodomain may be in the form of acidic or basic salts, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction.
- the primary amino acid structure may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants.
- Other modifications within the scope of this invention include covalent or aggregative conjugates of the truncated ectodomain with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions.
- the conjugated polypeptide may be a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co-translationally or post- translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast ⁇ -factor leader).
- Truncated EGFR ectodomain fusions may comprise peptides added to facilitate purification or identification of the truncated ectodomain (e.g., poly-His) or to enhance stability or delivery of the ectodomain in vivo.
- the truncated EGFR ectodomains of the present invention may also be fused to the constant domain of an immunoglobulin molecule.
- a recombinant chimeric antibody molecule may be produced having truncated EGFR ectodomain sequences substituted for the variable domains of either or both of the immunoglubulin molecule heavy and light chains and having unmodified constant region domains. This may result in a single chimeric antibody molecule having truncated EGFR ectodomains displayed bivalently. Such polyvalent forms of the truncated EGFR ectodomain may have enhanced binding affinity for EGFR ligands. Details relating to the construction of such chimeric antibody molecules are disclosed in WO 89/09622 and EP 315062.
- truncated ectodomains of the present invention may be used to target compounds to cancer cells.
- truncated EGFR ectodomain fusions may comprise compounds useful in the diagnosis or treatment of cancer cells such as drugs, isotopes or toxins.
- Truncated EGFR ectodomain derivatives may also be obtained by cross-linking agents, such as M-maleimidobenzoyl succinimide ester and N- hydroxysuccinimide, at cysteine and lysine residues.
- the truncated ectodomains may also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen bromide-activated, bisoxirane- activated, carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-linking).
- substrates such as cyanogen bromide-activated, bisoxirane- activated, carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-linking).
- the truncated ectodomain may be used to selectively bind (for purpose of assay or purification) anti-EGFR antibodies or EGF. It may also be desirable to use derivatives of the ectodomains of the invention that are conformationally constrained.
- Conformational constraint refers to the stability and preferred conformation of the three-dimensional shape assumed by a peptide.
- Conformational constraints include local constraints, involving restricting the conformational mobility of a single residue in a peptide; regional constraints, involving restricting the conformational mobility of a group of residues, which residues may form some secondary structural unit; and global constraints, involving the entire peptide structure.
- truncated EGFR ectodomains of the present invention may be used as immunogens, reagents in receptor-based immunoassays, or as binding agents for affinity purification procedures of EGF or other binding ligands.
- Truncated EGFR ectodomains may be tested for their ability to modulate receptor activity using a cell-based assay incorporating a stably transfected, EGF-responsive reporter gene (10).
- the assay addresses the ability of EGF to activate the reporter gene in the presence of novel ligands. It offers a rapid (results within 6-8 hours of hormone exposure), high- throughput (assay can be conducted in a 96-well format for automated counting) analysis using an extremely sensitive detection system (chemiluminescence).
- chemiluminescence chemiluminescence
- Once candidate compounds have been identified, their ability to antagonise signal transduction via the EGF-R can be assessed using a number of routine in vitro cellular assays such as inhibition of EGF- mediated cell proliferation.
- the efficiency of truncated EGFR ectodomains as tumour therapeutics may be tested in vitro in animals bearing tumour isografts and xenografts as described (11, 12
- Truncated EGFR ectodomains of the invention may preferably be combined with various components to produce compositions of the invention.
- the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use) .
- Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
- the compositions may be formulated, for example, for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration.
- each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
- the truncated ectodomains of the present invention will be useful in diagnostic assays for EGFR ligands, as well as in raising antibodies to the EGFR for use in diagnosis and therapy.
- purified truncated EGFR ectodomains may be used directly in therapy to bind or scavenge EGFR ligands, thereby providing means for regulating the activities of these ligands.
- truncated EGFR ectodomains of the present invention may be administered for the purpose of inhibiting EGF-dependent responses.
- the plasmid pEGFR used in the construction of truncated hEGFR cDNAs, comprises nucleotides 167-3970 of hEGFR (13) in the multiple cloning site of plasmid pUCl ⁇ . Coding is in the opposite sense to the LacZ ⁇ peptide, and the insertion is downstream of the Xbal site of pUCl ⁇ . This plasmid was used later in excision of the truncated constructs for insertion into the mammalian expression vector pEEl4 (14).
- 167-3150 of hEGFR was constructed by ligation oi ⁇ iXb ⁇ l/Nsil fragment from pEGFR and Xb ⁇ l/Pstl-cnt pBluescript KS+ . From this plasmid, a 4 kbp fragment Bbs ⁇ /Bglll fragment (containing all of pBluescript KS+ and nucleotides 167-ll ⁇ O and 29 ⁇ l-31 ⁇ 0 of EGFR) and a ⁇ 28 bp BbsVPvu ⁇ .
- nucleotides 1151-1679 were ligated with a 70 bp PCR-derived PvuR and Bglil fragment, encoding amino acids 474-476 of hEGFR, an enterokinase cleavage site and a c-myc epitope tag to facilitate purification.
- the 70 bp PCR cassette was produced using a similar previous construct (15) as template.
- PCR was used with three oligonucleotides to produce a fragment of hEGFR cDNA (nucleotides 1121 to 1760 or 1121 to 1797 respectively), followed by a sequence encoding an enterokinase cleavage site, a c-myc epitope tag and a termination codon.
- the upstream primer in PCR corresponded to an arbitrary choice of nucleotides 1121-1140 of hEGFR cDNA, while two overlapping downstream primers were used to construct additional sequence adjacent to nucleotide 1760 or 1797 respectively.
- the PCR products were cloned using the pCR-Script vector (Stratagene).
- Clones incorporating the mutations were identified by colony hybridisation (17) using 32 P-labelled mutagenic oligonucleotide as a probe, and the mutations were confirmed by DNA sequence analysis (18). Vehicles for mammalian cell expression were generated for each mutant by excising the 1.7 kbp fragment harbouring the mutated sEGFR ⁇ Ol cDNA from M13 RF-DNA by Xbal digestion and inserting it into plasmid pEEl4 (16) at the Xbal site.
- Truncated hEGFR ectodomains and mutants were identified by probing membranes with horseradish peroxidase (HRP) -conjugated Mab9ElO (Roche), followed by chemilumiscent detection with Pierce Super Signal substrate.
- HRP horseradish peroxidase
- Stable cell lines expressing sEGFR ⁇ Ol were established in the Lec8 mutant cell line from CHOK (7) using glutamine synthetase as a selectable marker (l ⁇ ).
- Supematants from methionine sulfoximine (MSX)-resistant cell colonies were screened for secreted receptor by biosensor analysis (see below) or by dot blotting onto nitrocellulose and probing with HRP-Mab9ElO. A single cell line was selected for cloning by limiting dilution.
- Lec8 cells expressing sEGFR ⁇ Ol protein were cultured in a Celligen Plus bioreactor (New Brunswick Scientific, New Jersey, USA) using 70 g Fibra-Cell Disks carriers with 1.7 L working volume. Continuous perfusion culture using glutamine-free DMEM/F12 medium supplemented with non- essential amino acids, nucleosides and 10% FCS was maintained for 6 weeks. Selection pressure was maintained with 2 ⁇ ⁇ M MSX for the duration of the fermentation. Perfusion rate was adjusted as required to ensure a residual glucose level of 1.0-1.5 g/L, with a corresponding lactate concentration of 2.0- 2.3 g/L. Purification of Truncated EGFR Ectodomains.
- conditioned medium containing the sEGFR truncated proteins (4 L) was adjusted to pH 8.0 with Tris-HCl (Sigma) containing sodium azide (0.02% (w/v)) (TBSA), and particulates removed by centrifugation prior to recovery of c-myc-tagged protein by affinity purification at 4°C on a column of monoclonal antibody 9E10 covalently-bound to agarose, using peptide elution (15).
- Eluted protein was further purified by size exclusion chromatography on Superdex 200 (HRlO/30, Amersham Pharmacia Biotech) at room temperature using TBSA buffer at a flow rate of 0.8 ml/min. Protein was detected by absorbance at 280nm.
- BIAcore Binding Assays Protein-protein interactions were monitored in real time on an instrumental optical biosensor using surface plasmon resonance detection (BIAcore 2000 or 3000, BIAcore, Uppsala, Sweden). Recombinant hEGF or hTGF- ⁇ (Gropep, Sydney, Australia) were purified immediately prior to immobilisation by micropreparative RP-HPLC using a SMART system (Amersham Pharmacia Biotech) as described previously (20). The proteins were immobilised onto the biosensor surface using amine coupling chemistry (N-hydroxysuccinimide and N-ethyl-N'- dimethylaminopropyl-carbodiimide) at a flow rate of 4 ⁇ l/min. Typically 100 - 200 RU were immobilised equivalent to 0.1 - 0.2 ng/mm 2 (20). Automated targeting of immobilisation levels was achieved using the BIAcore 3.1 control software (21).
- sEGFR621 23
- sEGFR ⁇ Ol sEGFR ⁇ Ol mutant samples were characterised by micropreparative size exclusion chromatography (Superose 12 3.2/30, Amersham Pharmacia Biotech) to ensure size homogeneity (20) and pooled fractions were diluted in BIAcore buffer (HBS: 10 mM Hepes pH 7.4 containing 3.4 mM EDTA, O.l ⁇ mM NaCl and 0.00 ⁇ % (v/v) Tween 20) to the appropriate concentration.
- HBS 10 mM Hepes pH 7.4 containing 3.4 mM EDTA, O.l ⁇ mM NaCl and 0.00 ⁇ % (v/v) Tween 20
- samples (30 ⁇ l) at concentrations of 10-1000 nM were injected sequentially over the sensor surfaces at a flow rate of ⁇ or 10 ⁇ l/min.
- KD Kinetic rate constants
- Chemically cross-linked sEGFR ⁇ Ol dimers were generated by the incubation of sEGFR ⁇ Ol ( ⁇ ⁇ M) with mEGF (20 ⁇ M) in 20 mM HEPES pH7.4 containing l ⁇ O mM NaCl for lh at room temperature followed by the addition of bis(sulfosuccinimidyl)suberate (BS3, Pierce, Rockford, EL, USA) to a final concentration of O. ⁇ mM and incubation for a further 30 min. The reaction was terminated by the addition of Tris-HCl buffer (pH 7.5) to a final concentration of 10 mM. Monomer-dimer separation was achieved on Novex non-reducing SDS-PAGE gels (10%). Proteins were transferred onto poly (vinyl difluoride) (PVDF) membranes (Bio-Rad,
- BaF/3ERX cells a cell line derived from
- sEGFR ⁇ Ol or sEGFR621 or the anti-EGFR monoclonal antibody Mab ⁇ 28 were added to the first titration point and titrated in two-fold dilutions across the 96 well plate in duplicate with or without a constant amount of mEGF (207 pM).
- 3H-Thymidine O. ⁇ ⁇ Ci/well was added and the plates were incubated for 20 h at 37°C in 5% C0 2 .
- the cells were then lysed in O. ⁇ M NaOH at room temperature for 30 min before harvesting onto nitrocellulose filter mats using an automatic harvester (Tomtec, Connecticut, USA). The mats were dried in a microwave, placed in a plastic counting bag and scintillant (10 ml) was added.
- 3 H-Thymidine incorporation was determined using an automated beta counter (1205 Betaplate, Wallac, Finland).
- sEGFR501 purified from a Mab9E10 anti-c-myc peptide affinity column using peptide elution showed a single symmetrical peak on size exclusion chromatography (apparent molecular mass of —80 kDa) and migrated as a single band of —70 kDa on SDS-PAGE under reducing conditions (not shown).
- sEGFR ⁇ Ol gave a unique expected sequence, LEEKKVXQGT (13) on N-terminal amino acid sequence analysis, the X at cycle 7 being due to the presence of a disulphide-bonded cysteine residue at that position.
- the apparent molecular mass of approximately 70 kDa on SDS-PAGE is due to the residual glycosylation reported for the glycosylation defective Lee 8 cells (33) since the calculated mass of human sEGFR ⁇ Ol apo-protein is —57.5 kDa.
- the BIAcore biosensor was used to determine both the rate and equilibrium binding constants for the interaction between sEGFR ⁇ Ol and hEGF or hTGF- ⁇ .
- Full length ectodomain (sEGFR621) was used as a positive control for the surface reactivity, since this interaction has been studied in detail previously (23, 27).
- Example 3 Antagonist activity of sEGFR501.
- the competition assay ( Figure 3B) used a constant concentration of mEGF (207 pM), which causes maximal stimulation ( Figure 3A), and varying levels (0.0004 ⁇ -0. ⁇ ⁇ M) of sEGFR ⁇ Ol, sEGFR621 or the neutralising anti-EGFR monoclonal antibody Mab ⁇ 28 raised against epidermal growth factor receptors on a human epidermoid carcinoma cell line, A431 (19). This antibody has been shown to prevent the growth of A431 cell xenografts, bearing high numbers of EGF receptors, in nude mice.
- the molecular weight (65,600 Da) and partial specific volume (0.71 ml/g) determined for 10 ⁇ M sEGFR ⁇ Ol alone was calculated from the sedimentation equilibrium distribution (Figure 5A) and is based on the known amino acid composition and a calculated value of 12% (w/w) for the carbohydrate composition.
- Sedimentation equilibrium data for a mixture of EGF (20 ⁇ M) and sEGFR ⁇ Ol (10 ⁇ M) was analyzed assuming two species ( Figure ⁇ A).
- the molecular weight of the first species was fixed at the value obtained for free EGF (6,000 Da) with the molecular weight and weight fraction of the second species used as fitting parameters. Under these conditions the molecular weight of the second species provides a good approximation to the weight- average molecular weight of sEGFR ⁇ Ol and its complexes.
- Biosensor analysis was also used to analyse the binding of the transiently expressed sEGFR ⁇ Ol mutants to both immobilised hEGF and hTGF- ⁇ surfaces.
- the presence of the mutant proteins in culture supematants from transfected cell lines was demonstrated by both immunoblotting with the anti-EGFR monoclonal antibody, Mab ⁇ 28, and biosensor analysis using Mab 528 immobilised on the surface.
- the Glu367Lys mutant and the Glu472Lys mutant showed similar binding characteristics to sEGFR ⁇ Ol when passed over the hEGF sensor surface (data not shown).
- the Gly441Lys mutant showed much reduced binding, even though the Mab ⁇ 28 surface had indicated that the Gly44lLys mutant was present at higher concentrations than sEGFR ⁇ Ol.
- the Gly44lLys mutant now showed the highest binding, whilst the binding of the Glu367Lys mutant the Glu472Lys mutant and wild type sEGFR ⁇ Ol were again similar but lower.
- mutant proteins present in the conditioned media from transient transfected 293T fibroblasts were concentrated and purified by a combination of affinity purification using the 9E10 monoclonal antibody and size exclusion chromatography on Superdex 200 and Superose 12.
- the sensorgrams obtained with the immobilised hEGF and hTGF- ⁇ surfaces are shown in Figures 6A,B.
- sEGFR ⁇ Ol which lacks most of CR2, exhibits ligand-induced receptor dimerisation ( Figures 4 and ⁇ ) indicating that the regions responsible for dimerisation are unlikely to include CR2. It also confirms that membrane anchoring is not required for the generation of high affinity dimers in contrast to the situation with ErbB2/ErbB3 heterodimers and neuregulin.
- the ultracentrifugation analyses showed that the binding sites on sEGFR ⁇ Ol were saturated, and the extent of dimerisation began to plateau, at molar ratios greater than of 1:1 (Figure ⁇ C), even at the relatively low concentration of sEGFR ⁇ Ol of ⁇ M (320 ⁇ g /ml).
- sEGFR ⁇ Ol will have therapeutic potential given its high affinity for ligand and its ability to competitively inhibit EGF-induced proliferation responses in a model cell system (Figure 3). This inhibition was greater than that achieved in the same assay with a neutralising monoclonal antibody raised against the receptor (Mab ⁇ 28), chimeric forms of which (C22 ⁇ ) are currently in clinical trials.
- sEGFR ⁇ Ol was also employed to investigate the residue responsible for the differential binding between hTGF- ⁇ and hEGF observed with chicken EGFR (9).
Abstract
Description
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Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/070,007 US6946543B2 (en) | 2000-06-28 | 2001-06-28 | Truncated EGF receptor |
CA002381241A CA2381241A1 (en) | 2000-06-28 | 2001-06-28 | Truncated egf receptor |
EP01944755A EP1200581A4 (en) | 2000-06-28 | 2001-06-28 | Truncated egf receptor |
JP2002506192A JP2004500902A (en) | 2000-06-28 | 2001-06-28 | Truncated EGF receptor |
AU67156/01A AU784697B2 (en) | 2000-06-28 | 2001-06-28 | Truncated EGF receptor |
US11/209,187 US7449559B2 (en) | 2000-06-28 | 2005-08-22 | Truncated EGF receptor |
AU2006203619A AU2006203619A1 (en) | 2000-06-28 | 2006-08-22 | Truncated EGF receptor |
US12/254,655 US20090117134A1 (en) | 2000-06-28 | 2008-10-20 | Truncated EGF Receptor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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AUPQ8418 | 2000-06-28 | ||
AUPQ8418A AUPQ841800A0 (en) | 2000-06-28 | 2000-06-28 | Truncated egf receptor |
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US11/209,187 Continuation-In-Part US7449559B2 (en) | 2000-06-28 | 2005-08-22 | Truncated EGF receptor |
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EP (1) | EP1200581A4 (en) |
JP (1) | JP2004500902A (en) |
AU (1) | AUPQ841800A0 (en) |
CA (1) | CA2381241A1 (en) |
WO (1) | WO2002000876A1 (en) |
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HRP20030853B1 (en) * | 2001-04-25 | 2011-11-30 | Lek Pharmaceutical And Chemical Company D.D. Legal Affairs And Industrial Property Dept. | Crystalline form of omeprazole |
WO2019070856A1 (en) * | 2017-10-03 | 2019-04-11 | Precision Biosciences, Inc. | Modified epidermal growth factor receptor peptides for use in genetically-modified cells |
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- 2001-06-28 JP JP2002506192A patent/JP2004500902A/en active Pending
- 2001-06-28 US US10/070,007 patent/US6946543B2/en not_active Expired - Fee Related
- 2001-06-28 WO PCT/AU2001/000782 patent/WO2002000876A1/en active Application Filing
- 2001-06-28 EP EP01944755A patent/EP1200581A4/en not_active Withdrawn
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2005
- 2005-08-22 US US11/209,187 patent/US7449559B2/en not_active Expired - Fee Related
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
HRP20030853B1 (en) * | 2001-04-25 | 2011-11-30 | Lek Pharmaceutical And Chemical Company D.D. Legal Affairs And Industrial Property Dept. | Crystalline form of omeprazole |
EP1421113A1 (en) * | 2001-08-03 | 2004-05-26 | Commonwealth Scientific And Industrial Research Organisation | Methods of screening based on the egf receptor crystal structure |
EP1421113A4 (en) * | 2001-08-03 | 2005-04-13 | Commw Scient Ind Res Org | Methods of screening based on the egf receptor crystal structure |
WO2019070856A1 (en) * | 2017-10-03 | 2019-04-11 | Precision Biosciences, Inc. | Modified epidermal growth factor receptor peptides for use in genetically-modified cells |
EP4269560A3 (en) * | 2017-10-03 | 2024-01-17 | Precision Biosciences, Inc. | Modified epidermal growth factor receptor peptides for use in genetically-modified cells |
Also Published As
Publication number | Publication date |
---|---|
US20090117134A1 (en) | 2009-05-07 |
EP1200581A1 (en) | 2002-05-02 |
AUPQ841800A0 (en) | 2000-07-20 |
JP2004500902A (en) | 2004-01-15 |
US7449559B2 (en) | 2008-11-11 |
CA2381241A1 (en) | 2002-01-03 |
EP1200581A4 (en) | 2003-04-02 |
US6946543B2 (en) | 2005-09-20 |
US20060234343A1 (en) | 2006-10-19 |
US20030108953A1 (en) | 2003-06-12 |
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