EP0804728A1 - Assay, receptor proteins and ligands - Google Patents

Assay, receptor proteins and ligands

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Publication number
EP0804728A1
EP0804728A1 EP95944197A EP95944197A EP0804728A1 EP 0804728 A1 EP0804728 A1 EP 0804728A1 EP 95944197 A EP95944197 A EP 95944197A EP 95944197 A EP95944197 A EP 95944197A EP 0804728 A1 EP0804728 A1 EP 0804728A1
Authority
EP
European Patent Office
Prior art keywords
nyk
molecule
protein
receptor
ligand
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95944197A
Other languages
German (de)
English (en)
French (fr)
Inventor
Steven A. Ludwig Inst. Cancer Research STACKER
Andrew F. Ludwig Inst. Cancer Research WILKS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
Original Assignee
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AUPN0301A external-priority patent/AUPN030194A0/en
Priority claimed from AUPN0300A external-priority patent/AUPN030094A0/en
Application filed by Ludwig Institute for Cancer Research Ltd, Ludwig Institute for Cancer Research New York filed Critical Ludwig Institute for Cancer Research Ltd
Priority to EP97203361A priority Critical patent/EP0854185A3/en
Publication of EP0804728A1 publication Critical patent/EP0804728A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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

Definitions

  • This invention relates to growth factor receptors of the receptor protein tyrosine kinase family, preparation of the extracellular domain of the receptor in large amounts, ligands for the receptors, nucleic acids encoding the ligands, and the use of the receptor and ligands in assays.
  • tyrosine kinases receptor tyrosine kinases
  • RTKs Three RTKs have been studied, named ⁇ YK (VEGFR2) , tie2 (tek) and RYK, respectively, which were isolated using PCR-based technology (Wilks, 1989) .
  • the extracellular domains of these receptors display a diverse array of structural features found in RTKs, and are classified in three distinct sub-families.
  • Neur ⁇ epithelial tyrosine kinase (NYK) ; also called Foetal liver kinase (flk-1) or vascular endothelial growth factor receptor 2 (VEGFR2) , a receptor for vascular endothelial growth factor (VEGF) and possibly for one other factor, and is a class III RT with seven Ig-like extracellular domains (Oelrichs et al, 1993; Terman et al, 1992) .
  • tie2 belongs to a family of RTKs whose extracellular regions consist of an array of Ig-like domains, three EGF repeats and three fibronectin type III repeats (Runting et al , 1993) .
  • RYK is an orpha receptor with so-called leucine-rich repeats, but lacks other identifiable structural motifs (Hovens et al, 1992; Paul et al, 1992; Stacker et al, 1993) .
  • the cellular lining of blood vessels (the endothelium) is an important interface between the blood and the tissues.
  • angiogenesis vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • angiogenesis occurs in the adult on in certain situations, such as: a) the endometrium of the female uterus, she and re-established during the menstrual cycle, b) embryo implantation and development, c) tissue remodelling after wounding, d) the blood vessels recruited during the establishment of a tumour, a process known as tumour angiogenesis, and e) diabetic retinopathy, a major complicatio of diabetes which leads to blindness.
  • tumour angiogenesis Since tumours are unable to establish themselve without a substantial vasculature, the process of tumour angiogenesis has been a target for the development of anti- cancer drugs. Indeed, the process of tumour angiogenesis is the one thing that all meta ⁇ tatic tumours have in common. However, the turnover rate for normal endothelial cells in the adult is far shorter, and is 12-18 months. Thus an anti-angiogenesis drug would be an important adjunct to any tumour therapy.
  • the methods of the invention utilise the isolated extracellular domain of the NYK protein.
  • a particularly convenient method for production of the isolated extracellular domain is described.
  • the invention may use isolated NYK extracellular domain produced by other methods.
  • the met described herein for producing the isolated extracellula domain is convenient and allows production of large quantities of the protein.
  • the receptors are present on in very small quantities normally.
  • our method all the production of recombinant proteins specifying the extracellular domains of these receptors, to act as affinity reagents for detecting the interactive ligand o interest and also to act as an immunogen for production antibodies.
  • One of the major considerations when expressing the extracellular domains of receptors is to ensure that the various structural motifs are folded in correct conformation. Techniques such as simply introducing an in-frame stop codon at the extracellular domain/transmembrane boundary can be used effectively, i monoclonal antibodies which recognise the folded recepto are available for purification of the expressed protein.
  • marker peptides or polypeptides which can be liga to the protein of interest, and to which affinity reagen are available, such as alkaline phosphatase (Flanagan an Leder, 1990) , the Fc region of immunoglobulin (Fanslow e al, 1992), glutathione-S-transferase (Smith and Johnson, 1988), and synthetic peptides such as FLAGTM (U.S. Patent No.
  • markers especially in applications such as screening for ligands the biosensor, suffer from the disadvantage that they ha their own binding specificity (eg. Fc regions) , may interfere with the folding of the domain, or may sterica hinder possible ligand binding sites.
  • some these markers have only been developed in prokaryotic expression systems, which may be inappropriate for expression of complex receptors because post-translation modification may be required for activity.
  • the FLAGTM peptide system was originally described as an N-terminal marker for recombinant bacterial proteins. It is a small, eight-amino acid water soluble peptide, hydrophilic in nature, to which a series of monoclonal antibodies have been made, these are commercially available.
  • the peptide was designed to localise to the outside of fusion proteins and, due to its size, provide minimal interference to the folding of the protein being studied (Hopp et al, 1992) .
  • a site-directed mutagenesis approach is used to generate restriction sites at the junction of the extracellular and predicted transmembrane domains to facilitate the in-frame ligation of a marker peptide to the ligand binding region of the receptor.
  • the small FLAGTM marker peptide and a CHO cell expression system have been employed for large scale protein production. Soluble fusion proteins were purified from cell supernatants using the commercially available anti-FLAGTM affinity gel, and mild elution with free peptide. This approach has produced pure, functional receptor extracellular domains from a mammalian source.
  • FLAGTM is specifically referred to herein, it will be clearly understood that other small marker peptides may be employed, using the same site-directed mutagenesis and in- frame ligation method. Monoclonal antibodies to some such peptides are commercially available.
  • the invention provides a meth of detecting a ligand able to bind the receptor protein NYK, comprising contacting a receptor protein NYK or a derivative thereof or a functional equivalent thereof wit a putative ligand under conditions suitable to allow binding of said putative ligand to said receptor, derivative thereof or equivalent thereof and determining whether binding has occurred.
  • the putative ligand may be any molecule suspect of being able to bind NYK whether from natural or synthet origin. In the case of synthetic molecules these may be any synthetic molecule including those of a combinatorial library.
  • the invention provides a particularly useful method for screening compounds present in rain forest plant extracts, and extracts from marine life such as corals and other marine organisms.
  • the method may also be Used for screening compounds derived from plants and animals from other aquatic or terrestrial habitats.
  • the method of the invention may be used initially to screen relatively unpurified compositions or extracts and thus extends to a method of detecting one or more putative ligands in a sample.
  • the sample suspected of containing one or more ligands is contacted with the receptor protein NYK or derivative or functional equivalent thereof.
  • Said samples may be from the sources described above but also include samples from biological origin such as tissue samples from patients including tumour tissue samples, serum samples and the like.
  • the term "receptor protein NYK or a derivative thereof or a functional equivalent thereof refers to naturally occurring NYK proteins including allelic variants thereof, to native NYK which has been modified and to synthetic NYK.
  • the term functional equivalent specifically refers to proteins which retain NYK receptor function. Modification of native NYK includes cleaving the native molecule to obtain the extracellular portion which contains the receptor functions. Synthetically produced NYK is also contemplated and this covers NYK produced by peptide synthesis, for example. Similarly, recombinant NYK is included within the scope of the term and this refers to recombinantly produced NYK including fusion proteins which retain receptor activity. The term also extends to NYK and derivatives or functional equivalents thereof which are expressed on the surface of cells. Preferably such NYK and derivatives or functional equivalents thereof are encoded by a recombinant vector present in said cell.
  • the term "under conditions suitable to allow binding of said putative ligand to said receptor, derivative thereof or functional equivalent thereof” includes parameters such as a suitable period of time, suitable pH, temperature and other parameters which will be well known to those skilled in the art.
  • Binding may be determined by any convenient means.
  • either the NYK, the NYK derivative thereof or functional equivalent thereof, or the putative ligand to be tested for binding may be labelled with a detectable marker, such as a radioactive label, a fluorescent label, or a marker detectable by way of an enzyme reaction.
  • a detectable marker such as a radioactive label, a fluorescent label, or a marker detectable by way of an enzyme reaction.
  • an in vi tro bioassay may be used to monitor the function induced by binding. Many suitable detection systems are known in the art.
  • assay systems which are suitable for use in the invention include, but are not limited to, immunoassays such as enzyme linked immunosorbent assay (ELISA) ; affinity-type assays such as those using coated microtitre plates or slides, or affinity chromatography; fluorescence-activate cell sorting; biosensor assays; and bioassays.
  • immunoassays such as enzyme linked immunosorbent assay (ELISA)
  • affinity-type assays such as those using coated microtitre plates or slides, or affinity chromatography
  • fluorescence-activate cell sorting such as those using coated microtitre plates or slides, or affinity chromatography
  • biosensor assays include, but are not limited to, immunoassays such as enzyme linked immunosorbent assay (ELISA) ; affinity-type assays such as those using coated microtitre plates or slides, or affinity chromatography; fluorescence-activate cell sorting; biosensor assays; and bioassays.
  • the degree of binding may be me
  • the invention provides a method of detecting a ligand of the receptor protein NYK a biological sample said method comprising contacting a receptor protein NYK or a derivative thereof or a functional equivalent thereof with a biological sample suspected of containing said ligand under conditions suitable to allow binding of any of said ligand to said receptor and determining whether binding of said ligand h occurred.
  • the biological sample may be any biological sample suspected of containing a ligand of NYK, in particular any samples suspected of containing VEGF such ascites, serum, blood and solid tissues such as tumours a the like. Detection of VEGF is important as this molecul is a marker for tumours and diabetic retinopathy.
  • receptor protein NYK, derivative thereof or functional equivalent thereof and "under conditions suitable to allow binding” have the same meanings as given above.
  • the method of the invention also extends to the detection of agents which are able to inhibit binding of NYK ligands to NYK.
  • the present invention provides a method of detecting a molecule which is capable of inhibiting interaction of NYK and a ligand comprising contacting a molecule which is suspected of inhibiting said interaction with
  • receptor protein NYK derivative thereof or functional equivalent thereof
  • under conditions suitable to allow binding have the same meanings as given above.
  • the molecule suspected of being an inhibitor of the NYK ligand interaction may be any molecule which may be an antagonist of this interaction. Such molecules may be derived from natural or synthetic sources such as those described above. The method is particularly useful for screening plant and animal extracts for NYK antagonists. It is important to note that the molecule suspected of being an inhibitor may be a molecule which binds to any region of the NYK protein, not necessarily the receptor site of the protein which has the receptor functions. Any molecule which indirectly inhibits the receptor function may also be detected by the method of the invention. Thus the nature of the NYK protein, equivalent or derivative thereof will to some extent determine the type of inhibitor which is identified by the method of the invention.
  • Determination of binding may be performed by the methods described above.
  • the NYK derivative used in the metho of the invention comprises the extracellular region of NY more preferably in the form of a fusion protein.
  • the invention provides a method of detecting a molecule which has an increased ability to stimulate activity of the receptor protein NYK compared to a native ligand of NYK, said method comprisin contacting a molecule which is suspected of having said increased ability with
  • molecule with an increased ability to stimulate activity of the receptor protein NYK compared t a native ligand of NYK refers to any molecule which is capable of bringing about an enhanced effect on the receptor compared to a native ligand.
  • Such enhanced effe will usually be in terms of the ability of the molecule t bring about a better angiogenic response in a bioassay th the native ligand, VEGF.
  • the enhanced abili may be in respect of an ability to induce vascular permeability.
  • Such molecules are expected to be useful in pharmaceutical applications where angiogenesis is require such as wound healing and the like.
  • the molecule will generally be a protein.
  • the molecule is a mutant of wild type VEGF with enhanced angiogenic activit
  • the invention provides a meth of detecting the receptor protein NYK or a variant thereo in a sample comprising contacting a sample suspected of containing NYK or a variant thereof with a known ligand of NYK under conditions suitable to allow binding to occur between said NYK or variant thereof and said known ligand and determining whether binding has occurred.
  • a variant thereof refers to naturally occurring alleles and mutants of NYK and to synthetically produced variants of NYK. Such variants retain the functional activity of the native receptor.
  • the other terms have the same meanings as given above.
  • the sample may be any sample such as those mentioned above.
  • the known ligand may be any ligand of NYK and is preferably an antibody, more preferably a monoclonal antibody.
  • the method of determining binding may be any method such as those described above.
  • the known ligand is coupled to a biosensor chip. This provides a very sensitive assay for the receptor or variants thereof.
  • the invention relates to ligands, inhibitors and agonists identified by the methods of the invention.
  • ligands, inhibitors and agonist are in the form of an isolated preparation. This means that they have been purified or separated to at least some degree from the other compounds with which they naturally or usually occur.
  • the invention provides a polypeptide which is a mutant of wild type VEGF and is capable of inhibiting or promoting the activity of the receptor protein NYK by directly binding the receptor or by some other means.
  • the wild type VEGF on which the mutants are modelled may be VEGF of any origin.
  • the VEGF is of mammalian origin such as human, murine, bovine, ovine, porcine, equine, guinea pig, etc .
  • Different VEGF splice variants may be used as a basis for the mutants.
  • murine VEGF exists in different ami acids lengths such as 120, 164, 188 and 206 amino acids (Brier et al, 1992) .
  • Human VEGF also exists in different amino acid lengths such as 121, 165, 189 and 206 amino acids.
  • homologs of VEGF have been found in a pox virus "orf" (Lyttle et a , 1994) . Such homologs coul also be used as a basis for the mutants.
  • the mutants may be produced by recombinant DNA techniques, direct peptide synthesis or any other convenient technique.
  • the mutants comprise amino acid deletions, substitutions insertions in the equivalent of the cysteine-knot motif o murine wild type VEGF.
  • Such amino acid substitutions or insertions may be native amino acids or amino acids from synthetic sources.
  • the mutant is an inhibitor of the receptor protein NYK and comprises one or more amino acid substitutions in the region which corresponds to the V3 domain of wild type VEGF.
  • the polypeptides comprise mutations at one or more of the following positions corresponding to mouse VEGF: 73, 111, 117 and 109 to 115.
  • the polypeptides are the mutants K73S, H111G, G117V or VEGF0 described herein or variants thereof having substantially the same biological activity as said mutant. Variants of these mutants may be produced by standard conservative amino aci substitutions. The methods of producing such variants wil be known by those skilled in the art.
  • the mutant is an agonis of the receptor protein NYK and comprises a truncated for of wild type VEGF with one or more amino acid svibstitution in the region which corresponds to the V3 region of wild type VEGF.
  • the agonist comprises a truncated protein terminating at or about residue 133 corresponding to wild type mouse VEGF with an amino acid substitution at or around the same position.
  • the agonist comprises K106 * 2 as herein describe or a variant thereof retaining substantially the same - 13 - biological activity as the mutant.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a ligand, inhibitor or agonist identified by the methods of the invention described above, together with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent will be known by those skilled in the art.
  • methods of producing the pharmaceutical compositions will be known by those skilled in the art. Such compositions may be produced by reference to standard textbooks such as Remington Pharmaceutical Sciences, 17th Edition, Elsevier Publishing Co, Eton, Pennsylvania, USA.
  • the invention relates to an isolated nucleic acid encoding a protein which is a ligand, inhibitor or agonist of NYK, a NYK derivative or a NYK functional equivalent thereof.
  • the nucleic acid molecule may be DNA or RNA, single or double stranded, linear or covalently closed circular. It may be composed of natural or synthetic nucleotide bases. Those skilled in the art will appreciate that due to redundancy in the genetic code several different codons may be used to encode the same amino acid.
  • the invention provides a nucleic acid molecule which encodes a polypeptide capable of inhibiting or promoting the activity of the receptor protein NYK by directly binding the receptor or by some other means.
  • the polypeptide encoded is a mutant of a wild type VEGF as discussed above.
  • said nucleic acid molecule comprises a nucleotide sequence which comprises amino acid deletions, substitutions or insertions in the equivalent of the cysteine-knot motif of wild type VEGF.
  • said nucleic acid encodes a mutant which is an inhibitor of the receptor protein NYK and comprises a nucleotide sequence which encodes a polypeptide with one or more amino acid substitutions in the region which corresponds to the V3 domain of wild type VEGF.
  • the nucleic acid molecule encodes polypeptides with mutations at one or more of the followi positions corresponding to mouse VEGF: 73, 111, 117 and 1 to 115.
  • the nucleic acid molecule encodes K73S, H111G, G117V or VEGFO or variants thereof having substantially the same biological activity.
  • the nucleic acid molecule encodes an agonist of the receptor protein NYK which is a truncated form of wild type VEGF with one or more amino acid substitutions in the region which corresponds to the V3 region of wild type VEGF. More preferably the nucleic acid molecule encodes a truncated protein terminating at or around residue 133 correspondin to wild type mouse VEGF with an amino acid substitution a or around the same position. Still more preferably the nucleic acid molecule encodes K106 * 2 as herein described a variant thereof retaining substantially the same biological activity.
  • One particularly rapid and convenient test syst for the first, second, third, fourth and fifth aspects of the invention uses an optical biosensor, such as the BIAcoreTM (Pharmacia Biosensor AB, Uppsala, Sweden) , which enables the use of proteins or peptides immobilised to a sensor chip.
  • the biosensor assay is very simple, reproducible and rapid, while having high specificity.
  • T biosensor assay enables a very high throughput of samples and is amendable to automation. It is therefore suitable for screening of natural products for their ability to inhibit binding to NYK protein, or for activity as agonis of VEGF.
  • test compounds of wide variety of structures can be tested using this metho
  • the methods of the first, second, third and fourth aspects of the invention comprise the steps of immobilising the extracellular domain of NYK to an optical biosensor chip, and detecting binding by measuring the ability of a ligan compound to bind to the immobilised NYK protein or of a putative inhibitor to inhibit binding of a known ligand, respectively.
  • the methods of the first, second, third and fourth aspects of the invention uses a bioassay comprising the step of exposing a cell expressing an NYK extracellular domain as a fusion protein together with a cytokine receptor to a ligand capable of binding to the NYK extracellular domain and determining whether binding has occurred.
  • the invention provides a method of production of a recombinant protein having the biological activity of an extracellular domain of a receptor protein tyrosine kinase, comprising the steps of: a) generating in a nucleic acid encoding the protein a restriction site at the junction of the extracellular and predicted transmembrane domains of the receptor, wherein the restriction site is chosen so that it will be unique to a construct produced in step b, b) ligating to the nucleic acid produced in a) an oligonucleotide sequence encoding a marker peptide and an in-frame stop codon, optionally with a further unique restriction site at the 3' end of the nucleic acid, c) expressing the construct produced in b) in a mammalian host cell in culture, d) isolating said protein by using an affinity reagent specific for the marker peptide, and optionally, e) subjecting the protein to a further purification step.
  • step d) consists of subjecting the culture medium conditioned by host cells to purification although depending on the exact construct and host system there may be other ways of obtaining purification of the protein.
  • the marker peptide is FLAGTM; however, other small marker peptides are known in the art.
  • the host cells are Chinese hamster ovary (CHO) cells, but a variety of other convenient host cells is also known in the art.
  • the affinity purification step is carried out using affinity chromatography, more preferably with anti-FLAGTM affinity gel and mild elution with free FLAGTM peptide.
  • Restriction sites at the junction of the domain may be introduced using site-directed mutagenesis or via PCR reaction; the nature of the restriction sites will depend on the specific sequence of the receptor cDNA, but is conveniently a site for a restriction enzyme such as Bgrlll or Ba- ⁇ H .
  • a unique restriction site such as Clal, may be inserted at the 3' end of the oligonucleotid encoding the marker protein.
  • the invention provides an extracellular domain of a receptor protein tyrosine kinase (extracellular RTK domain) , produced usin the method of the invention.
  • a receptor protein tyrosine kinase is selected from the group consisting of neuroepithelial tyrosine kinase (NYK), tie2 and RYK. More preferably the receptor protein tyrosine kinase is neuroepithelial tyrosine kinase.
  • the invention provides an isolated nucleic acid molecule generated by t method of the first aspect of the invention.
  • the invention provides a monoclonal antibody directed against an extracellular RTK domain.
  • Methods for producing monoclon antibodies are routine in the art. Such monoclonal antibodies are useful for im unodiagnosis, i munotherapy, immunohistochemistry, and i munoassay.
  • compositions comprising an extracellular RTK domain of the invention, or a monoclonal antibody thereto together with a pharmaceutically acceptable carrier, are also within the scope of the invention.
  • the RTK is preferably selected from the group consisting of NYK, tie2, and RYK, and is most preferably NYK.
  • RYK related to tyrosine kinases tie2 tyrosine kinase with Ig-like domains and EGF repeats VEGF vascular endothelial growth factor VEGFR2 vascular endothelial growth factor receptor 2.
  • Figure 1 shows the results of analysis of VEGF binding to immobilised NYK-EX-FLAGTM using the BIAcoreTM.
  • CM5 sensor chip Purified NYK-EX-FLAGTM was coupled to a CM5 sensor chip as described herein. The following solutions were then analysed for their binding to NYK-EX-FLAGTM; CHO cell medium containing VEGF, CHO cell medium containing tie2-EX-FLAGTM (Control 1) or buffer alone (Control 2) .
  • the figure represents the overlay of three sensorgrams representing the relative response units (RU) seen after injection of the above samples over the time of the experiment, measured in seconds.
  • RU relates to the mass change at the surface of the biosensor chip associated with ligand binding.
  • Point A represents the baseline prior to addition of the test samples
  • B is the point at which the test samples are injected
  • point C represents the end of the injection phase where the samples are replaced with BIAcoreTM buffer
  • point D the overall change in baseline due to specific binding of the VEGF.
  • Figure 2 illustrates the mechanism of signal transduction by cytokine receptors, such as those for IL- and erythropoietin (Epo) , RTK and by an RTK-Epo chimera.
  • Figure 3 shows flow cytometry analysis of VEGFR
  • the traces are control cell line 115 stained with antibody 4H ( ) and the BAF/3-NYK-EpoR cell line stained with eith 4H3 (-) or control antibody 4g8 (-) .
  • Figure 4 shows the amino acid sequences of modulators of NYK receptor function.
  • the corresponding nucleotide sequences will be well known to those skilled the art.
  • Figure 5 shows the sequence of FLAGTM linker oligonucleotides.
  • the linker sequence encodes the FLAGTM octapeptide, including an in-frame stop codon.
  • Bglll compatible overhangs are present at both ends, to allow ligation into DNA digested with Bgl l , BamHI, Bell , Ndell or XhoII.
  • the internal Clal site allows for identificati of recombinants in the ligation reaction.
  • Figure 6 shows a schematic representation of th methods used to make a growth factor receptor extracellul domain-FLAGTM fusion protein.
  • the FLAGTM linker oligonucleotides were ligated together and recombinants containing the FLAGTM sequence in frame with the extracellular domain sequence selected.
  • the ability of t FLAGTM-fusion protein to be expressed was first assessed b transient expression in COS cells and then the inserts were transferred to a CHO cell expression vector utilisin Xbal .
  • Figure 7 shows the results of transient transfection of constructs into COS cells and detection b immunoprecipitation.
  • Panel (A) shows NYK-EX-FLAGTM-CDM8;
  • Panel (B) shows tie2-EX-FLAGTM-CDM8; in each case CDM8 alo is used as control.
  • NYK-EX-FLAGTM-CDM8 (A), tie2-EX-FLAGTM-CDM8 (B) and CDM8 alone (A and B) were transfected into COS cells by the DEAE dextran method.
  • Cells were biosynthetically labelled with 3s S-cysteine/methionine for 16 h and the supernatants immunoprecipitated with M2-gel. Washed beads were eluted with SDS-PAGE sample buffer and analysed by SDS-PAGE. The gels were dried and labelled proteins detected by exposing to a storage phosphor screen and analysing using a 400 series Phosphorimager and Imagequant v3.0 software (Molecular Dynamics, Sunnyvale, CA) .
  • Figure 8 shows the results of analysis of affinity-purified fusion proteins by SDS-PAGE and silver staining.
  • Figure 9 shows the results of analysis of binding of monoclonal antibodies to tie2-EX-FLAGTM to native and denatured tie2-EX-FLAGTM, using the BIAcoreTM.
  • the following description utilises the extracellular domain of the NYK protein, produced in CHO cells as a fusion protein with the marker peptide FLAGTM (Hopp et al , 1992) linked at the C-terminus of the extracellular domain, and purified by affinity chromatography. Production of this fusion protein, designated NYK-EX-FLAGTM, is described in Examples 7 to 9. However, it is to be clearly understood that the invention is not limited to NYK-EX-FLAGTM, and that NYK, and particularly the extracellular domain thereof, produced b other methods is within the scope of the invention.
  • VEGF vascular endothelial growth factor
  • Binding studies were performed on the optical biosensor (BIAcoreTM, Pharmacia Biosensor AB, Uppsala, Sweden) using proteins immobilised to a CM5 sensor chip
  • the sensorgrams depicted in Figure 1 show that sensor chip derivatised with NYK-EX-FLAGTM gave rise to a relative response of 146 RU upon injection of VEGF containing CHO cell supernatants; compare A to D. By comparison, signals from control CHO cell supernatant fro cells expressing another RTK protein, tie2-EX-FLAGTM (Control 1) or buffer alone (Control 2) were below 20 RU.
  • a panel of monoclonal antibodies which recognise native NYK and tie2 (described in our International Patent Application No. PCT/US95/01743, filed 9 February 1995) also yielded specific responses to NYK-EX-FLAGTM and tie2-EX-FLAGTM derivatised biosensor chips respectively.
  • Table 1 shows the results of binding of three different monoclonal antibodies, respectively designated 3B6, 3C8 and 4H3 (described in International application PCT/US95/01727 filed 9 February 1995) , to NYK-EX-FLAGTM immobilised on the sensor chip.
  • Conditioned media containing the monoclonal antibody at a concentration of 10-20 ⁇ g/ml were applied to the sensor chip, and the relative response compared to that shown using buffer alone.
  • the sensor chip was regenerated with a high pH wash between applications.
  • tie2-EX-FLAGTM was immobilised on the BIAcoreTM chip. The results are shown in Figure 9. The response to the three purified antibodies, each at the same concentration, on tie2-EX-FLAGTM in the native form is shown on the left. Bound antibody was removed by washing with a high pH wash as shown by the dip in response units following antibody binding. The tie2-EX-FLAGTM was then denatured using guanidium hydrochloride and 2-mercaptoethanol, and the ability of the antibodies to bind was again tested, as shown in the right column of Figure 9.
  • the lell antibody no longer binds after denaturation of the tie2-EX-FLAGTM, as shown by the absence of increase in response units; se top right-hand trace.
  • the other two antibodies, 3gl and 4g8, still show binding after denaturation of the tie2-EX-FLAGTM, albeit at a low level, showing that althou the conformation of the tie2-EX-FLAGTM is altered, it stil remains attached to the chip.
  • mice were immunised with 1 x 10 6 C6 glioma cells or 1 x 10 6 U937 cells subcutaneously and the tumours allowed to develop to approximately 0.5-1.0 cm 3 .
  • blood samples were drawn from the animals and serum subsequently collected.
  • the serum was assayed for bindin to the NYK-FLAG derivatised chip and the amount of VEGF present estimated by comparison to a standard curve generated using recombinant human VEGF (Preprogen) . Resul are shown in Table 2 below. Table 2
  • pBOS-NYK/Epo was then cotransfected into the factor-dependent pre-B cell line BA/F3 with the neomycin resistance plasmid.
  • the BA/F3 cell line is dependent on the presence of IL-3/GM-CSF for growth; removal of these factors results in rapid cell death within 24-48 h.
  • Cells were selected in DMEM, 10% HI FCS, 50 ⁇ g/ml gentamicin, 20 ⁇ g/ml L-glutamine, 1.2 mg/ml G418 growth medium. Individual clones growing after 7-14 days were picked and expanded in liquid culture. NYK/Epo expressing colonies were selected by tw procedures:
  • VEGF vascular endothelial growth factor
  • Fig. 3 flow cytometry analysis of VEGFR2-EpoR expression on the surfa of BA/F3 cells was conducted as follows BA/F3 cells (5 x 10 5 ) transfected with the NYK-
  • EpoR construct BA/F3-NYK-EpoR#18 or cells transfected with vector alone (115) were reacted with monoclonal antibodies to the extracellular domain of the NYK recepto (4H3 described in PCT/US95/01727) or control antibodies directed to the tie2 receptor (PCT/US95/01743) . After washing the cells were incubated with a goat anti-rat-FIT antibody, subsequently washed and the cells analysed by flow cytometry using a FACScanTM and CellQuestTM software (Beeton Dickinson) . The results are shown in Fig. 3. The traces are control cell line 115 stained with antibody 4H
  • the assay therefore specifically detects interactions of the NYK extracellular domain and its ligand VEGF, and is therefore useful for detection and evaluation of substances that may modulate this interaction, or detection and evaluation of inhibitors of such modulatory substances.
  • Transfected or untransfected BA/F3 cells were removed from WEHI3D conditioned medium, washed three to four times in PBS and resuspended in medium containing 5 ng/ml of VEGF165.
  • Mutants were generated in the pCDNA-1 Amp expression vector. These were generated by using oligonucleotide directed mutagenesis by the technique already described. The mutations were either single or multiple amino acid substitutions in the wild type VEGF.
  • the amino acid sequence of wild type VEGF used as a basis for these mutants is the same as the sequence for K73S shown in Figure 4 except that the wild type has a lysine (K) at position 73.
  • the wild type leader sequence is als included in the mutants shown in Figure 4. Mutations were confirmed by sequencing. The sequence of the mutants is shown in Table 6.
  • Constructs were transfected into COS cells by t DEAE-Dextran method and conditioned media collected for seven days post transfection.
  • the conditioned media was diluted to 10% in the bioassay buffer as described previously in the presence of BA/F3-NYK-EpoR or Ba/F3 cel not expressing the receptor.
  • NT not tested.
  • the expression levels of the VEGF mutants was assessed by immunoprecipitation of 35S-Met/Cys labell COS cell conditioned media with an anti ⁇ era to VEGF and analysis by SDS-PAGE. The results are shown in Table 7.
  • the above mutants were tested for their ability to induce or inhibit vascular permeability by the Miles Assay with the appropriate controls.
  • This assay involves administering a blue dye to an animal which is distributed in the animal's blood vessels then intradermally injecting the animal with the mutant and observing the effect on vascular permeability.
  • VEGF vascular endothelial growth factor
  • mRNA reverse transcribed mouse colon mRNA
  • the VEGF cDNA was sequenced and found to be identical to the known sequence (Breier et al, 1992) .
  • Th fragment encoding VEGF was subcloned into the pEE6 vector and transfected into CHO cells as described below.
  • Supernatants from stably-transfected CHO cells were collected and used for the NYK-EX-FLAGTM binding studies (see below) .
  • the bioactivity of VEGF produced in this manner was evaluated in a Miles vascular permeability ass (Miles and Miles, 1952) and the VEGF was found to be functional.
  • oligonucleotides were synthesised by the Joint Protein Structure Laboratory of the Ludwig Institut for Cancer Research and The Walter and Eliza Hall Institute.
  • the FLAGTM linker oligonucleotides were synthesised as two 39mers which were complementary over 35 nucleotides, giving a four base overhang corresponding to Bglll cohesive ends ( Figure 5) .
  • the oligonucleotides encoded the amino acid sequence of the FLAGTM octapeptide, an in-frame stop codon and a Clal restriction enzyme site to allow selection of recombinants.
  • Oligonucleotide linkers were phosphorylated with polynucleotide kinase using a conventional method, as previously described (Sambrook et al , 1989) and annealed at 37°C for 1 hour prior to ligation. Mutagenic oligonucleotides (27-35 mer which introduced either Bglll or BaznHI sites were made to sequences at the receptor extracellular/transmembrane border, as defined by hydrophobicity plots.
  • Example 7 Site-directed Mutagenesis and Ligation of
  • Site-directed mutagenesis was used to generate mutant forms of the cDNA to enable the FLAGTM sequence, encoded in a set of oligonucleotides ( Figure 5) to be placed in-frame, thereby creating the appropriate extracellular domai -FLAGTM fusion protein.
  • site-directed mutagenesis was performed on single stranded DNA of the growth factor receptor to introduce either a Bglll or BamHl site at the border of the extracellular and transmembrane regions using a mutagenic oligonucleotide.
  • Bglll enzyme sites were introduced into the NYK and RYK cDNAs, whereas a BamHI site was introduced into the tie2 cDNA due to an existing Bglll site.
  • Full length cDNA clones encoding the mouse NYK/FLK-1/VEGFR2 receptor, mouse tie2 receptor and the human RYK receptor were subcloned into the mammalian expression vector pCDM ⁇ (Invitrogen) using the BstXI restriction enzyme site and BstXI linkers. Single stranded UTP-containing DNA (Kunkel, 1985) was generated, and used as templates to make mutants of NYK, tie2 and RYK cDNA which encoded the required restriction enzyme sites.
  • Mutant receptor cDNAs containing Bglll or BamHI sites at their extracellular domain and the transmembrane domain boundaries were digested with the appropriate restriction enzyme, phosphatased and ligated with the oligonucleotide linker sequence encoding the FLAGTM marker peptide (IBI) ;
  • the small phosphorylated FLAGTM linker oligonucleotides (39 mers) ligated easily into the digest vector fragments, and recombinants were accurately detect by use of the internal Clal site.
  • COS cells were maintained in RPMI-1640 medium with 10% FCS and 50 ⁇ g/ml gentamicin at 37°C and 5% C0 2 . COS cells were transfected by the DEAE dextran method as described previously (Aruffo and Seed, 1987) .
  • pEE6-NYK-EX-FLAGTM, pEE6-tie2-EX-FLAGTM and pEE6-RYK-EX-FLA constructs were transfected into CHO-K1 cells by calcium phosphate precipitation and selected in Glasgow Modified Eagle's Medium (without L-glutamine, without NaHC0 3 , Cytosystems, Castle Hill, N.S.W.) supplemented with 10% fetal calf serum, 50 ⁇ g/ml gentamicin, non-essential amin acids, sodium pyruvate and 25 mM methionine sulphoxide (MSX) .
  • Glasgow Modified Eagle's Medium without L-glutamine, without NaHC0 3 , Cytosystems, Castle Hill, N.S.W.
  • MSX methionine sulphoxide
  • Predicted sizes for the NYK-EX-FLAGTM, tie2-EX-FLAGTM and RYK-EX-FLAGTM fusion proteins including the contribution of N-linked glycosylation, as predicted from immunoprecipitation of the mature cell surface receptors (Runting et al, 1993; Stacker et al, 1993), are 125,000 Dalton ⁇ , 95,000 Daltons and 38,000 Daltons respectively.
  • the constructs were then subcloned into the CHO cell expression vector pEE6 and transfected into CHO cells.
  • Supernatants of stably transfected CHO cells were screened for expression of fusion proteins by immunoprecipitation following biosynthetic labelling. Clones that produced fusion proteins of the predicted size were selected, and large scale production of the secreted protein undertaken. Expression levels of transfected CHO cells in 25 mM MSX were sufficient (0.5-1.0 ⁇ g/ml), so that selection in higher amounts of MSX was not attempted.
  • CHO cells were grown on Cytodex-3 beads in roller bottles and produced in the range of 1 ⁇ g of fusion protein per ml of expended tissue culture supernatant.
  • NYK-EX-FLAGTM, tie2-EX-FLAGTM and RYK-EX-FLAGTM were purified from medium conditioned by transfected CHO cells using affinity chromatography on M2 (anti-FLAGTM) gel (IBI) . A separate column was used for each receptor construct.
  • Bound material was eluted with either 100 mM glycine-HCl pH 3.0, 0.02% Twee (neutralised in 1/10 volume 1 M Tris-HCl pH 8.6) or 25-50 ⁇ g/ml FLAGTM peptide (N-Asp-Tyr-Lys-Asp- Asp-Asp-Asp-Lys-C) in 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% Tween 20. Columns were eluted in 1 ml fractions, 10 ⁇ l samples combined with 10 ⁇ l of reducing SDS-PAGE sample buffer were analysed by SDS-PAGE and proteins detected by silver staining.
  • fusion proteins were further purified by high-performance liquid chromatograph in a Tris-free buffer system (Nice et al, 1994) on a ⁇ MonoQ PC 1.615 anion exchange column (Pharmacia) to give single homogeneous species corresponding to either NYK-EX-FLAGTM, tie2-EX-FLAGTM or RYK-EX-FLAGTM.
  • the strategy developed for purification of the fusion proteins from medium conditioned with transfected CHO cells involved a single immunoaffinity chromatography step on an M2-antibody column (2 ml of packed beads per 100 ml of CHO cell supernatant) .
  • the proteins were elute from the column with excess free FLAGTM peptide, and furth purification to homogeneity was achieved by microbore ani exchange HPLC. This also enabled the removal of excess free FLAGTM peptide.
  • Desorption of non-specific proteins was achieved by a two-step washing procedure involving buffers of pH 8.0 and pH 10.0 respectively. This protoco appeared sufficient to remove the large majority of contaminants, as judged by SDS-PAGE analysis of the purified fractions, which is illustrated in Figure 8.
  • the NYK-EX-FLAGTM fusion protein was tested for its ability to bind a known ligand, vascular endothelial growth factor (VEGF) (Millauer et al, 1993) as described in Example 1.
  • VEGF vascular endothelial growth factor
  • the strategy provides a universal adaptor site whereby the receptor extracellular domain ca be ligated to other proteins of interest.
  • t extracellular domain could be ligated into the vector AP-tag-1 (Flanagan and Leder, 1990) for expression of fusion proteins with secreted alkaline phosphatase or any other protein domain to which an in-frame, compatible restriction enzyme site has been incorporated.
  • AP-tag-1 Felanagan and Leder, 1990
  • a fusion protein in which the extracellular doma of NYK protein is linked to the cytoplasmic and transmembrane domain of the erythropoietin receptor is described in a concurrently-filed application.
  • the pCDM8 vector was used as the base vector fo our technique, as it can be used for generating single stranded DNA for site-directed mutagenesis, as well as fo transient expression in COS cells. This allows the rapid testing of constructs for expression of the fusion protei before proceeding to large scale production. Subcloning the final constructs into the CHO cell expression vector pEE6 is also simple, due to compatible Xba.1 sites present in the pCDM8 and pEE6 polylinkers. We have used site- directed mutagenesis to introduce the necessary restricti enzyme sites at the junction of the extracellular and transmembrane domains.
  • PCR has some advantage over PCR, especially when attempting to generate large fragments, or when oligonucleotide primers are derived from sequences possessing GC-rich regions, as frequently occurs in the 5' regions of cDNAs encoding RTKs (Kozak, 1991) .
  • PCR could be used to generate smaller fragments encoding the Bglll/BamHI sites and then ligated to the extracellular domain cDNA via an appropriate restriction enzyme site.
  • a major advantage of this system is the commercial availability of the FLAGTM system components, and their effectiveness in yielding relatively pure fusion protein preparations with a one-step procedure from starting material containing 10% fetal calf serum.
  • the ability to elute with the free FLAGTM peptide is also a major advantage, maximising the likelihood of obtaining native receptor extracellular domain by avoiding the need for exposure to extreme pH or to chaotropic agents.
  • the location of the FLAGTM peptide at the C-terminus did not adversely affect either experiments on the BIAcoreTM or MAb production to the receptors.
  • the purified NYK-EX-FLAGTM specifically bound the ligand VEGF.
  • there was no apparent binding from serum containing media in contrast to our observation with alkaline phosphatase-fusion proteins.
  • the apparent inertness of the FLAGTM peptide in protein interactions compared with other markers such as alkaline phosphatase or the Fc portion of immunoglobulin, makes this system ideal for biosensor-based applications, since it avoids false positives.
  • Our method permits the isolation of purified, functional receptor extracellular domain-FLAGTM fusion proteins using a mammalian expression system.
  • utilising such a system means that glycosylation and protein foldi are most likely to resemble the native state.
  • Linkage of well-characterised marker peptide means that reagents are available to affinity purify reasonable quantities of the fusion protein, which will facilitate further study.
  • the size and C-terminal location of the FLAGTM peptide means that the effects on the protein being studied are minimised.
  • FLAGTM N-terminus
  • COS-7 mammalian cells
  • FLAGTM peptide was designed originally to be linked to th N-terminal of the desired protein, and has a structure su as to permit ready cleavage from this N-terminal (U.S. Patent No. 4,703,004) .
  • kit ligand a cell surface molecule altered in steel mutant fibroblasts
  • NYK/FLK-1 A putative receptor protein tyrosine kinase isolated from ElO embryonic neuroepitheium is expressed endothelial cells of the developing embryo
  • Hybrid tyrosine kinase/cytokine receptors transmit itogenic signals in response to ligand
  • tie2 a putative protein tyrosine kinase from a new clas of cell surface receptor
  • Arg lie Lys Pro Gly Gin Ser Gin His lie Gly CGG ATC AAA CCT GGC CAA AGC CAG CAC ATA GGA
  • Arg lie Arg Ser Gly Asp Arg Pro Ser lie Gly Glu CGG ATC AGA TCT GGC GAT AGA CCG TCC ATA GGA GAG

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US6872699B2 (en) 1992-11-13 2005-03-29 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften, E.V. Truncated Flk-1 receptor protein, methods of use and a recombinant vector containing a nucleotide encoding the truncated Flk-1 protein
US6177401B1 (en) 1992-11-13 2001-01-23 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften Use of organic compounds for the inhibition of Flk-1 mediated vasculogenesis and angiogenesis
US6846914B2 (en) * 1996-06-19 2005-01-25 Regeneron Pharmaceuticals, Inc. Tie-2 ligand-3
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US6365154B1 (en) * 1998-09-28 2002-04-02 Smithkline Beecham Corporation Tie2 agonist antibodies
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US6521424B2 (en) 1999-06-07 2003-02-18 Immunex Corporation Recombinant expression of Tek antagonists
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US7482116B2 (en) 2002-06-07 2009-01-27 Dna Genotek Inc. Compositions and methods for obtaining nucleic acids from sputum
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