AU705793B2 - Assay, receptor proteins and ligands - Google Patents

Description

f 1A- ASSAY, RECEPTOR PROTEINS LIGANDS 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.

Background and Prior Art Cellular proliferation and differentiation in multicellular organisms requires precise coordination and regulation. Binding of extracellular signalling molecules, such as cytokines and growth factors, to specific cell surface receptors provides one major mechanism by which this is achieved. The receptors span the cell membrane, and are classified as to whether or not they possess an intrinsic protein tyrosine kinase domain in their intracellular region. The tyrosine kinase phosphorylates tyrosine residues in many intracellular proteins, providing an important step in signal transduction. Receptors which 20 have an intrinsic protein tyrosine kinase are known as receptor tyrosine kinases (RTK; growth factor receptor).

With the advent of the Polymerase Chain Reaction (PCR) many novel members of the family have been identified via the highly conserved structural elements within their 25 catalytic domains. Even though these receptors share a common cytoplasmic protein tyrosine kinase (PTK) domain, they have distinctive structural elements in their extracellular domains. These structural elements can be used to classify the RTKs into a given subfamily, and 30 demonstrate the structural diversity that exists within the RTK family (Ullrich and Schlesinger, 1990).

Three RTKs have been studied, named NYK (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

I

2 structural features found in RTKs, and are classified into three distinct sub-families. Neuroepithelial tyrosine kinase (NYK); also called Foetal liver kinase (flk-1) or vascular endothelial growth factor receptor 2 (VEGFR2), is a receptor for vascular endothelial growth factor (VEGF) and possibly for one other factor, and is a class II RTK 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 two Ig-like domains, three EGF repeats and three fibronectin type III repeats (Runting et al, 1993). RYK is an orphan receptor with so-called leucine-rich repeats, but lacks any 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. When a tissue or organ needs to provide itself with a blood supply, a new wave of blood vessel development (angiogenesis) must take place; this requires the division and growth of the endothelium. Of the known proteins capable of recruiting blood vessel development, one particular growth factor, vascular endothelial growth factor (VEGF), is demonstrably one of the most important stimuli to the division of endothelial cells during 25 angiogenesis.

Significant angiogenesis occurs in the adult only in certain situations, such as: a) the endometrium of the female uterus, shed and re-established during the menstrual cycle, 30 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 complication of diabetes which leads to blindness.

Since tumours are unable to establish themselves 3 without a substantial vasculature, the process of tumour angiogenesis has been a target for the development of anticancer drugs. Indeed, the process of tumour angiogenesis is the one thing that all metastatic 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.

It is thought that at least two receptors for VEGF are involved in angiogenesis, but that NYK is the major signalling receptor. Thus, compounds able to bind to the receptor so as to inhibit the activity of VEGF would have a wide range of therapeutic applications. Because angiogenesis is essentially only involved in the situations mentioned above, such inhibitors would be less likely to cause major side effects than conventional medications.

Although there are some agents which are known to inhibit angiogenesis, these known inhibitors act either on a different receptor or via a different pathway. In order 20 to discriminate between inhibitor and non-inhibitor compounds, and to demonstrate specificity of inhibition of the VEGF receptor, in vitro assay systems have been developed based on the growth factor VEGF and its receptor VEGFR2 (NYK), which are able to detect inhibitors of the 25 interaction of these two molecules. There are two uses for these systems; first, to establish the sites of interaction between VEGF and its receptor, the better to enable rational design of drugs which will act as either agonists or inhibitors of this process; second, as a natural product 30 screen, wherein the inhibition of the VEGF/VEGFR2 interaction will be evidence for the presence of an inhibitor in a particular natural source.

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. However, it will be clearly understood that the invention may use isolated NYK Slb -4 extracellular domain produced by other methods. The method described herein for producing the isolated extracellular domain is convenient and allows production of large quantities of the protein. The receptors are present only in very small quantities normally. Hence our method allows the production of recombinant proteins specifying the extracellular domains of these receptors, to act as affinity reagents for detecting the interactive ligand of interest and also to act as an immunogen for production of antibodies. One of the major considerations when expressing the extracellular domains of receptors is to ensure that the various structural motifs are folded in the correct conformation. Techniques such as simply introducing an in-frame stop codon at the extracellular domain/transmembrane boundary can be used effectively, if monoclonal antibodies which recognise the folded receptor are available for purification of the expressed protein.

Recently, however, the advent of PCR cloning strategies has meant that the complete cDNA sequence is 20 often obtained well before any protein data or monoclonal antibodies are available. This can be circumvented by the use of marker peptides or polypeptides which can be ligated to the protein of interest, and to which affinity reagents are available, such as alkaline phosphatase (Flanagan and 25 Leder, 1990), the Fc region of immunoglobulin (Fanslow et al, 1992), glutathione-S-transferase (Smith and Johnson, 1988), and synthetic peptides such as FLAG M Patent No. 4,703,004; Hopp et al, 1992) and His-Tag T7-Tag HSV-Tag" and S-Tag (Novagen). Some of these markers, 30 especially in applications such as screening for ligands on the biosensor, suffer from the disadvantage that they have their own binding specificity (eg. Fc regions), may interfere with the folding of the domain, or may sterically hinder possible ligand binding sites. In addition, some of these markers have only been developed in prokaryotic expression systems, which may be inappropriate for expression of complex receptors because post-translational 5 modification may be required for activity.

The FLAG" 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). To date the majority of studies with the FLAG M marker have used bacterial expression systems (Hopp et al, 1992; Li et al, 1992; Su et al, 1992; Zhang et al, 1991), although one recent study has employed a mammalian expression system (Gerard and Gerard, 1990).

Techniques are described herein which alleviate some of the problems outlined above. A site-directed mutagenesis approach is used to generate restriction sites at the junction of the extracellular and predicted 20 transmembrane domains to facilitate the in-frame ligation of a marker peptide to the ligand binding region of the receptor. In this study the small FLAG TM marker peptide and a CHO cell expression system have been employed for large scale protein production. Soluble fusion proteins were 25 purified from cell supernatants using the commercially available anti-FLAG M affinity gel, and mild elution with free peptide. This approach has produced pure, functional receptor extracellular domains from a mammalian source.

4 While FLAG" is specifically referred to herein, it will be 30 clearly understood that other small marker peptides may be employed, using the same site-directed mutagenesis and inframe ligation method. Monoclonal antibodies to some such peptides are commercially available.

By employing a single site-directed mutagenesis step, we have engineered a restriction enzyme site in the receptor cDNAs to enable the ligation of a pair of oligonucleotide linkers encoding the FLAG' octapeptide.

6 This avoids the use of PCR on large sections of cDNA (1-3 kb) which often possess regions of high GC content at the 5' end. The FLAG T octapeptide marker is positioned at the C-terminal end of the extracellular domain, thereby leaving the N-terminal region of the receptor, an area frequently involved in ligand binding, unmodified. This approach has been used to construct FLAGT"-fusion proteins with RTKsfrom three distinct subfamilies: VEGFR2, tie2 and RYK. Recombinant fusion proteins expressed in a CHO cell expression system have been purified using anti-FLAG" antibody affinity chromatography. The proteins were eluted from the affinity column by competition with free FLAG

T

peptide, which has minimised their exposure to potentially denaturing agents. This method is generally applicable to expressing and purifying functional extracellular domains of cell surface receptors using a well-characterised marker peptide. This method is particularly useful for studies such as monoclonal antibody production, assays such as immunoassays, biosensor experiments or ligand isolation 20 studies which require functional material.

Summary of the Invention In a first aspect the invention provides a method 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 with 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.

S. 30 The putative ligand may be any molecule suspected S" of being able to bind NYK whether from natural or synthetic origin. In the case of synthetic molecules these may be any synthetic molecule including those of a combinatorial library. In the case of natural molecules, the invention provides a particularly useful method for screening compounds present in rain forest plant extracts, and 7 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. Furthermore, 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. In such cases 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 25 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 8 well known to those skilled in the art.

Binding may be determined by any convenient means. For example, 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. Alternatively an in vitro bioassay may be used to monitor the function induced by binding. Many suitable detection systems are known in the art. Thus 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-activated cell sorting; biosensor assays; and bioassays. In addition to detecting binding, the degree of binding may be measured by one or more of the above techniques.

In a second aspect the invention provides a method of detecting a ligand of the receptor protein NYK in 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 25 suitable to allow binding of any of said ligand to said receptor and determining whether binding of said ligand has occurred.

The biological sample may be any biological sample suspected of containing a ligand of NYK, in 30 particular any samples suspected of containing VEGF such as ascites, serum, blood and solid tissues such as tumours and the like. Detection of VEGF is important as this molecule is a marker for tumours and diabetic retinopathy.

The terms "receptor protein NYK, derivative thereof or functional equivalent thereof" and "under conditions suitable to allow binding" have the same meanings as given above.

9 The method of the invention also extends to the detection of agents which are able to inhibit binding of NYK ligands to NYK.

Accordingly in a third aspect 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 NYK or a derivative thereof or functional equivalent thereof and (ii) a known ligand of NYK under conditions suitable to allow binding of said NYK or derivative thereof or functional equivalent to said known ligand, and determining whether binding has occurred.

Inhibition of interaction between NYK and its ligand in vivo may result in inhibition of angiogenesis or inhibition of vascular permeability.

The terms "receptor protein NYK, derivative thereof or functional equivalent thereof" and "under 20 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 .25 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 30 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 10 methods described above.

Preferably the NYK derivative used in the methods of the invention comprises the extracellular region of NYK, more preferably in the form of a fusion protein.

In a fourth aspect 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 comprising contacting a molecule which is suspected of having said increased ability with NYK, or a derivative thereof or a functional equivalent thereof under conditions sufficient to allow binding of said NYK or derivative or functional equivalent thereof with said molecule and determining whether binding has occurred, and (ii) comparing said determination with the amount of binding in a control which control comprises contacting and a native ligand under suitable conditions.

The term "molecule with an increased ability to stimulate activity of the receptor protein NYK compared to 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 effect 25 will usually be in terms of the ability of the molecule to bring about a better angiogenic response in a bioassay than the native ligand, VEGF. Alternatively the enhanced ability may be in respect of an ability to induce vascular permeability. Such molecules are expected to be useful in pharmaceutical applications where angiogenesis is required such as wound healing and the like. The molecule will generally be a protein. Preferably the molecule is a mutant of wild type VEGF with enhanced angiogenic activity.

The other terms have the same meanings as given above.

In a fifth aspect the invention provides a method of detecting the receptor protein NYK or a variant thereof 1 4 11 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.

The term "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. In a preferred embodiment the known ligand is coupled to a biosensor chip.

This provides a very sensitive assay for the receptor or variants thereof.

In a sixth aspect the invention relates to ligands, inhibitors and agonists identified by the methods of the invention. Preferably said 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.

In a particularly preferred aspect 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. Preferably 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 12 mutants. For example murine VEGF exists in different amino 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. In addition homologs of VEGF have been found in a pox virus "orf" (Lyttle et al, 1994). Such homologs could 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. Preferably the mutants comprise amino acid deletions, substitutions or insertions in the equivalent of the cysteine-knot motif of murine wild type VEGF. Such amino acid substitutions or insertions may be native amino acids or amino acids from synthetic sources.

Preferably 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. Still more preferably the polypeptides comprise mutations at one or more of the following positions corresponding to mouse VEGF: 73, 111, 117 and 109 to 115. Even more preferably the polypeptides are the mutants K73S, H111G, G117V or VEGFO described herein or variants thereof having substantially the same biological activity as said mutant. Variants of these 25 mutants may be produced by standard conservative amino acid substitutions. The methods of producing such variants will be known by those skilled in the art.

~Alternatively preferably the mutant is an agonist of the receptor protein NYK and comprises 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 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. More preferably the agonist comprises K106"2 as herein described or a variant thereof retaining substantially the same 13 biological activity as the mutant.

In a seventh aspect the invention provides 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. The appropriate pharmaceutically acceptable carrier or diluent will be known by those skilled in the art. Similarly 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.

In an eighth aspect 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.

In a particularly preferred aspect the invention 25 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. Preferably the polypeptide encoded is a mutant of a wild type VEGF as discussed above. More preferably 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.

Preferably 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 14 which corresponds to the V3 domain of wild type VEGF. Still more preferably the nucleic acid molecule encodes polypeptides with mutations at one or more of the following positions corresponding to mouse VEGF: 73, 111, 117 and 109 to 115. Even more preferably the nucleic acid molecule encodes K73S, H111G, G117V or VEGFO or variants thereof having substantially the same biological activity.

Alternatively preferably 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 corresponding to wild type mouse VEGF with an amino acid substitution at or around the same position. Still more preferably the nucleic acid molecule encodes K1062 as herein described or a variant thereof retaining substantially the same Sbiological activity.

20 One particularly rapid and convenient test system for the first, second, third, fourth and fifth aspects of the invention uses an optical biosensor, such as the BIAcore T (Pharmacia Biosensor AB, Uppsala, Sweden), which enables the use of proteins or peptides immobilised to a 25 sensor chip. The biosensor assay is very simple, reproducible and rapid, while having high specificity. The biosensor assay enables a very high throughput of samples, 4 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 agonists S" of VEGF. However, it is envisaged that test compounds of a wide variety of structures can be tested using this method.

In a particularly preferred embodiment, 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 ligand 15 compound to bind to the immobilised NYK protein or of a putative inhibitor to inhibit binding of a known ligand, respectively.

In an alternative preferred embodiment, 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.

According to a ninth aspect, 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, 25 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, se*e) subjecting the protein to a further purification step.

S Preferably 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.

Preferably the marker peptide is FLAG"; however, other small marker peptides are known in the art.

16 Preferably the host cells are Chinese hamster ovary (CHO) cells, but a variety of other convenient host cells is also known in the art. Preferably the affinity purification step is carried out using affinity chromatography, more preferably with anti-FLAG" affinity gel and mild elution with free FLAG" peptide.

Restriction sites at the junction of the domains 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 BglII or BamHI. For convenience in selection of recombinant proteins, a unique restriction site, such as Clal, may be inserted at the 3' end of the oligonucleotide encoding the marker protein.

According to a tenth aspect, the invention provides an extracellular domain of a receptor protein tyrosine kinase (extracellular RTK domain), produced using S. the method of the invention. Preferably the receptor 20 protein tyrosine kinase is selected from the group consisting of neuroepithelial tyrosine kinase (NYK), tie2, 0• and RYK. More preferably the receptor protein tyrosine kinase is neuroepithelial tyrosine kinase.

According to a eleventh aspect the invention 25 provides an isolated nucleic acid molecule generated by the method of the first aspect of the invention.

According to a twelfth aspect, the invention ,"provides a monoclonal antibody directed against an eo *extracellular RTK domain. Methods for producing monoclonal antibodies are routine in the art. Such monoclonal antibodies are useful for immunodiagnosis, immunotherapy, immunohistochemistry, and immunoassay.

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.

In the ninth to twelfth aspects of the invention 17 the RTK is preferably selected from the group consisting of NYK, tie2, and RYK, and is most preferably

NYK.

Detailed Description of the Invention flk-1 MAb

MSX

NYK

PCR

RTK

RYK

tie2

VEGF

VEGFR2 Abbreviations used herein are as follows: foetal liver kinase-1 monoclonal antibody methionine sulphoxide neuroepithelial tyrosine kinase polymerase chain reaction receptor tyrosine kinase related to tyrosine kinases tyrosine kinase with Ig-like domains and EGF repeats vascular endothelial growth factor vascular endothelial growth factor receptor 2.

*o a.

The invention will now be described in detail by way of reference only to the following examples, and to the drawings, in which Figure 1 shows the results of analysis of VEGF binding to immobilised

NYK-EX-FLAG

m using the BIAcore

T

Purified NYK-EX-FLAG M was coupled to a CM5 sensor chip as described herein. The following solutions were then analysed for their binding to NYK-EX-FLAG'"; CHO cell medium containing VEGF, CHO cell medium containing tie2-EX-FLAG

T

(Control 1) or buffer alone (Control 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 BIAcoreT buffer and point D, the overall change in baseline due to specific 18 binding of the VEGF.

Figure 2 illustrates the mechanism of signal transduction by cytokine receptors, such as those for IL-3 and erythropoietin (Epo), RTK and by an RTK-Epo chimera.

Figure 3 shows flow cytometry analysis of VEGFR2- EpoR expression on the surface of BA/F3 cells using monoclonal antibody 4H3 and control antibody 4g8. The traces are control cell line 115 stained with antibody 4H3 and the BAF/3-NYK-EpoR cell line stained with either 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 in the art.

Figure 5 shows the sequence of FLAG linker oligonucleotides. The linker sequence encodes the FLAG" octapeptide, including an in-frame stop codon. BglII compatible overhangs are present at both ends, to allow ligation into DNA digested with BglII, BamKI, Bcll, NdeII or XhoII. The internal Clal site allows for identification of recombinants in the ligation reaction.

Figure 6 shows a schematic representation of the methods used to make a growth factor receptor extracellular domain-FLAG fusion protein. After digestion of the mutant 25 receptor with either BglII or BamHI, the FLAG linker oligonucleotides were ligated together and recombinants containing the FLAG' sequence in frame with the extracellular domain sequence selected. The ability of the FLAG"-fusion protein to be expressed was first assessed by transient expression in COS cells and then the inserts were transferred to a CHO cell expression vector utilising Xbal.

Figure 7 shows the results of transient transfection of constructs into COS cells and detection by 35 immunoprecipitation. Panel shows NYK-EX-FLAG-CDM8; Panel shows tie2-EX-FLAG"-CDM8; in each case CDM8 alone is used as control. NYK-EX-FLAG'"-CDM8 *o 19 tie2-EX-FLAG"-CDM8 and CDM8 alone (A and B) were transfected into COS cells by the DEAE dextran method.

Cells were biosynthetically labelled with 3SS-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. Expended tissue culture supernatant from CHO cell lines expressing NYK-EX-FLAG' (lanes 1-5 elution with 25 gg/ml FLAG' peptide, lanes 6-8 elution with glycine-HCl, pH tie2-EX-FLAG" (lanes 1-3 elution with 25 pg/ml FLAG" peptide, lanes 4 and 5 with 50 gg/ml peptide and lanes 6-9 glycine-HCl pH 3.0 and (C) RYK-EX-FLAG (eluted with 25 Ag/ml free FLAGm peptide, lanes 1-4, lane l=void volume) were passed over independent M2-gel affinity columns and washed as described herein.

Elution was performed with free FLAG" peptide (25-50 g/ml), or 0.1 M glycine pH 3.0 containing 0.02% Tween 20. The proteins eluted were analysed by SDS-PAGE 25 under reducing conditions and detected by silver staining.

Molecular weight markers are indicated.

Figure 9 shows the results of analysis of binding of monoclonal antibodies to tie2-EX-FLAG" to native and denatured tie2-EX-FLAG", using the BIAcore".

The following description utilises the extracellular domain of the NYK protein, produced in CHO cells as a fusion protein with the marker peptide FLAG

M

(Hopp et al, 1992) linked at the C-terminus of the extracellular domain, and purified by affinity 35 chromatography. Production of this fusion protein, designated NYK-EX-FLAG', is described in Examples 7 to 9.

However, it is to be clearly understood that the invention o o 20 is not limited to NYK-EX-FLAG", and that NYK, and particularly the extracellular domain thereof, produced by other methods is within the scope of the invention.

Example 1 Binding Experiments The NYK-EX-FLAG' fusion protein was tested for its ability to bind a known ligand, vascular endothelial growth factor (VEGF) (Millauer et al, 1993).

Binding studies were performed on the optical biosensor (BIAcore", Pharmacia Biosensor AB, Uppsala, Sweden) using proteins immobilised to a CM5 sensor chip (Pharmacia). Immobilisation of NYK-EX-FLAG' was performed using standard NHS/EDC chemistry as previously described (Nice et al, 1994). Approximately 15 ng/mm 2 (15,000 RU) of NYK-EX-FLAG™ were coupled to the biosensor chip as determined by the BIAcore™ analysis. The integrity of immobilised protein was examined by comparing the BIAcore" response to medium conditioned with VEGF-pCDM8 transfected CHO cells tie2-EX-FLAG" transfected CHO cells and buffer alone. Between each cycle, the derivatised sensor surface was regenerated with 50 mM diethylamide, pH 12.0, 0.1% Triton X-100. In control experiments it was established that this treatment had no effect on the binding capacity of the immobilised receptor for VEGF.

The sensorgrams depicted in Figure 1 show that a 25 sensor chip derivatised with NYK-EX-FLAG" gave rise to a S.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 from cells expressing another RTK protein, tie2-EX-FLAG' (Control 1) or buffer alone (Control 2) were below 20 RU.

The rise in response units seen in the BIAcore™ sensorgram of VEGF binding to NYK-EX-FLAG™ (t=100-520s, B-C) is also consistent with the binding of a specific ligand. This contrasts with the sensorgrams seen for Controls 1 and 2, which show no increase over this period. A panel of monoclonal antibodies which recognise native NYK and tie2 21 (described in our International Patent Application No. PCT/US95/01743, filed 9 February 1995) also yielded specific responses to NYK-EX-FLAG" and tie2-EX-FLAG" 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-FLAG immobilised on the sensor chip. Conditioned media containing the monoclonal antibody at a concentration of 10-20 Ag/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.

Table 1 Binding of Anti-NYK Monoclonal Antibodies to NYK-EX-FLAG' Immobilised on a Sensor Chip

U

U

U

a. a.

Monoclonal antibody Relative Response (RU) 3B6 1194 3C8 875 4H3 1163 Buffer 139 Three different monoclonal antibodies directed to tie2-EX-FLAG", designated respectively lell,3gl, and 4g8 25 were tested for their ability to bind to tie2-EX-FLAG immobilised on the sensor chip.

In order to study the effect of denaturation of the tie2-EX-FLAG'" protein on antibody binding, tie2-EX-FLAG" was immobilised on the BIAcore'" chip. The results are shown in Figure 9. The response to the three purified antibodies, each at the same concentration, on 22 tie2-EX-FLAG 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-FLAG' 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-FLAG", as shown by the absence of increase in response units; see top right-hand trace. The other two antibodies, 3gl and 4g8, still show binding after denaturation of the tie2-EX-FLAG", albeit at a low level, showing that although the conformation of the tie2-EX-FLAGm is altered, it still remains attached to the chip.

The location of the FLAG' peptide at the C-terminus did not adversely affect either experiments on the BIAcore" or MAb production to the receptors. The purified NYK-EX-FLAG specifically bound the ligand VEGF.

Also, there was no apparent binding from serum containing media, in contrast to our observation with alkaline phosphatase-fusion proteins. The apparent inertness of the FLAG 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 25 applications, since it avoids false positives.

Example 2 Detection of VEGF in Serum Mice were immunised with 1 x 106 C6 glioma cells or 1 x 106 U937 cells subcutaneously and the tumours allowed to develop to approximately 0.5-1.0 cm 3 At this 30, point blood samples were drawn from the animals and serum subsequently collected. The serum was assayed for binding 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). Results are shown in Table 2 below.

e 23 Tabl e 2 Detection of VEGF in the Serum of Tumour Bearing Mice R. T-T.

VEGF

(na/ml) Control #1 Control #2 Tumour #1 Tumour #2 rVEGF Run #1 rVEGF Run #2 R.U. VEGF (na/mi) 2.0 1.0 61.0 63.0 1551 598 335 154 150 83 62 29 351 365 10,000 3,125 1,560 780 1,000 500 250 125 63 EXaLMPle 3 Detection of Rcombnan=t-

VEGF

Recombinant VEGF (5 ;11) was resuspended in 20 mM sodium acetate pH 4.5/0.02% Tween 20 and immobilised onto a standard BlAcore chip. Conditioned medium was flowed over the chip and bound material desorbed with 10 mM HCl. These results are shown below in Table 3. *e

S

S. S.

S

Set.

S

5 S. S S. a S S a.

24 Table 3 Binding of NYK Extracellular Domain Construct to Recombinant VEOF Coupled to the Biosensor Chip.

Sample Relative Respone Medium 53 NYK extracellular domain 1043 anti-VEGF antibody 2746 Example 4 Detection of Monoclonal Antibodies 4H3 and 3B6 Bindinq to immnobilised

NYK-EX-FLAG

Monoclonal antibodies directed to the extracellular domain of VEGFR2 were used to detect the iimobilised NYK-EX-FLAG on the BlAcore. Antibody (10 Al/ml) was applied to the chip and the relative response determined. The results are shown in Table 4 below.

Table 4 Binding of Anti-VEGFR2 Monoclonal Antibodies to immobilised

NYK-EX-FLAG

Antibody I Relative Response 4H3 1769 3B6 1194 4H3 1391 ,(+3B6 already bound) a.

a 25 Example 5 Bioassay for NYK Using An NYK- Erythropoietin Receptor Fusion Protein Recent studies by Pacifici and Thomason (1994) suggest that most RTKs when transfected into factordependent cell lines such as FDCP-1 and BA/F3 do not produce a significant proliferative response to ligand.

However, if a chimeric molecule comprising the extracellular domain of the RTK and the transmembrane and cytoplasmic domain of the cytokine receptor for erythropoietin receptor (EpoR) is used, the level of proliferation achieved is similar to that given by the wildtype cytokine receptor. This implies that aggregation of the receptor cytoplasmic domain is sufficient for some proliferative responses to be generated. The mechanism is illustrated in Figure 2.

Using site directed mutagenesis a silent mutation in the EpoR cytoplasmic domain was effected to remove a BglII restriction enzyme site at position 866 of the mouse Epo receptor published nucleotide sequence (D'Andrea et al, 1989). Another BglII site was then subsequently introduced at the junction of the extracellular domain and putative transmembrane domain of the EpoR. The cytoplasmic and transmembrane domain of the Epo receptor was then ligated in frame to the extracellular domain of the NYK receptor 25 via the BglII site described in Examples 7 to 9. The insert was then subcloned into the pBOS expression vector via XbaI. This construct is designated pBOS-NYK/Epo.

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 pg/ml gentamicin, 20 pg/ml L-glutamine, 1.2 mg/ml G418 growth medium.

35 Individual clones growing after 7-14 days were picked and expanded in liquid culture.

26 NYK/Epo expressing colonies were selected by two procedures: immunoprecipitation of 3 as-Met/Cys-labelled receptors with anti-NYK MAbs (prepared as described in our copending International Patent Application No.

PCT/US95/01743 filed 9 February 1995) and analysis of

SDS-PAGE;

(ii) stimulation of the cell line with VEGF.

1000 NYK/Epo-BA/F3 cells or non-expressing control BA/F3 cells were resuspended in a 15 pl volume of growth medium containing 10% medium conditioned by the growth of COS cells transfected with pCDM8-VEGF165.

Stimulation of cells was judged over a period of 2-14 days when compared to control populations.

Three cell lines were demonstrated to respond to VEGF and to express the NYK-Epo chimera, as summarised in Table 5 and demonstrated by flow cytometry (Fig. Flow cytometry analysis of VEGFR2-EpoR expression on the surface of BA/F3 cells was conducted as follows BA/F3 cells (5 x 10 s 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 receptor (4H3 described in PCT/US95/01727) or control antibodies 25 directed to the tie2 receptor (PCT/US95/01743). After washing the cells were incubated with a goat anti-rat-FITC antibody, subsequently washed and the cells analysed by flow cytometry using a FACScan" and CellQuest" software (Becton Dickinson). The results are shown in Fig. 3. The traces are control cell line 115 stained with antibody 4H3 and the BA/F3-NYK-EpoR cell line stained with either 4H3 or control antibody 4g8 These cell lines were also shown to be specifically stimulated to grow by incubation with 35 10-20 pg/ml of MAbs directed to the extracellular domain of the NYK receptor. This is the result of cross-linking of receptors at the cell surface, which mimics ligand-induced e* 27 dimerisation of the receptor.

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.

Table Stimulation of NYK-EpoR Transfected BA/F3 Cells Clone WEH13D 1VEGF165 Growth comdiumonI condiine BA/F3 BA! F3 BA/F3 vector only BA/F3 -NYK-EpoR#2 BA/F3-NYK-EpoR#18 BA/F3 -NYK-EpoR#23 Transfected or untransfected BA/F3 cells were removed from WEH13D conditioned medium, washed three to four times in PBS and resuspended in medium containing 20 5 ng/ml of VEGF165.

Growth was determined between 2 and 4 days in VEGF by visual inspection of cultures.

4* 4. 0 a 0 *4 28 Example 6 Detection of Mutant VEGF 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 at position 73. The wild type leader sequence is also included in the mutants shown in Figure 4. Mutations were confirmed by sequencing. The sequence of the mutants is shown in Table 6.

e *e a. *aa.

a a 0Oe a a.

a** a. a a a a a a a a a a a a. a a *aa a a..

a. a a a a a a a a a a a a. a a a. eta at a a.

Table 6 Sequ~ences of mutants produced Underlining indicates the amino acid substitution(s) which results in the mutant.

Mutant Amino acid sequence Change made Name and corresponding to create nucleotide sequence mutant 1(73S I E Y I F S P S C V P L K S ATA GAG TAC ATC TTC AGC CCC AGC TGT GTG CCG CTO (SEQ.ID.No.1) H111G R I K P G Q S Q H I G H G COG ATC AAA CCT GGC CAA AGC CAG CAC ATA GGA (SEQ.ID.NO.2) G117V Q S Q H I V E M S F L Q G V CAA AGC CAG CAC ATA GTA GAG ATG AGC TTC CTG CAG (SEQ.ID.NO.3) 1(106*2 L Q H S R C E C R P W "STOP" K(4W CTA CAG CAC AGC CGA TOT GAA TGC AGA CCA TGG TAA (SEQ.ID.NO.4) W -*STOP VEGFO R I R S G D R P S I G E K R CGG ATC AGA TCT GOC GAT AGA CCG TCC ATA GGA GAG (SEQ.ID.NO.5) P S Q D

S-*R

Q P H S 30 Constructs were transfected into COS cells by the 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 cells not expressing the receptor. After 48-72 hours the assay was assessed for the amount of proliferation and scored as 0 no viable cells, 1 less than 25% coverage of the well, 2 25-50% coverage of the well, 3 50-75% coverage of the well and 4 greater than 75% of the well covered.

NT=not tested. The expression levels of the VEGF mutants was assessed by immunoprecipitation of 35S-Met/Cys labelled COS cell conditioned media with an antisera to VEGF and analysis by SDS-PAGE. The results are shown in Table 7.

S

o o p t 60 *o 31 Table 7 Response of NYK-EpoR transfected BA/F3 cells to stimulation with conditioned medium from COS cells transfected with pCDM8 or PCDNAl-Amp containing VEGF wildtype or mutated forms generated by site-directed mutagenesis.

MUTANT

BIOASSAY

Q62D NT D66S 3 E69S

NT

2 E72T 3 K73S 1 N87G 2 E92H 2 H111G 1 I116Y 2 G117V 1 C2,4S 2 K106*2 4 VEGFO 1 PLGF WT 0 VEGF WT 3 VECTOR 0 a. *a 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.

The following examples relate to production of recombinant receptor for use in the above assays.

32 Reagents Purified M2 monoclonal antibody which recognises an internal epitope on the FLAG" octapeptide (Cat# IB13025), M2-gel (IB13021) and free FLAG" peptide (IB13070) were obtained from International Biotechnologies, New Haven, CT.

A cDNA clone encoding VEGF (165 amino acid form) was isolated by PCR from reverse transcribed mouse colon mRNA. The VEGF cDNA was sequenced and found to be identical to the known sequence (Breier et al, 1992). The 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-FLAG binding studies (see below). The bioactivity of VEGF produced in this manner was evaluated in a Miles vascular permeability assay (Miles and Miles, 1952) and the VEGF was found to be functional.

Oligonucleotides All oligonucleotides were synthesised by the Joint Protein Structure Laboratory of the Ludwig Institute for Cancer Research and The Walter and Eliza Hall Institute. The FLAG' linker oligonucleotides were synthesised as two 39mers which were complementary over 35 nucleotides, giving a four base overhang corresponding to BglII cohesive ends (Figure The oligonucleotides encoded the amino acid sequence of the FLAG' octapeptide, o' 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 0 C for 1 hour prior to ligation. Mutagenic oligonucleotides (27-35 mers) which introduced either BglII or BamHI sites were made to sequences at the receptor extracellular/transmembrane border, as defined by hydrophobicity plots.

33 Example 7 Site-directed Mutagenesis and Liqation of Linker Sequence The strategy developed for isolating the extracellular domain of NYK, tie2 and RYK is summarised in Figure 6. Site-directed mutagenesis was used to generate mutant forms of the cDNA to enable the FLAG" m sequence, encoded in a set of oligonucleotides (Figure 5) to be placed in-frame, thereby creating the appropriate extracellular domain-FLAG" fusion protein. Initially, site-directed mutagenesis was performed on single stranded DNA of the growth factor receptor to introduce either a BglII or BamHI site at the border of the extracellular and transmembrane regions using a mutagenic oligonucleotide.

BglII enzyme sites were introduced into the NYK and RYK cDNAs, whereas a BamHI site was introduced into the tie2 cDNA due to an existing BglII 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 pCDM8 (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 BglII or BamHI sites at S. their extracellular domain and the transmembrane domain boundaries (as determined by hydrophobicity plots (Kyte and Dolittle, 1982) using the DNA STAR" software) were digested o with the appropriate restriction enzyme, phosphatased and ligated with the oligonucleotide linker sequence encoding the FLAG" M marker peptide (IBI);

S

5

'GATCTGACTACAAGGACGACGACGATGACAAGTGAATCGATA',

(N)Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Term(C)).

A unique ClaI site was installed at the 3' end of the oligonucleotides, and recombinants were selected by Clal restriction enzyme digestion. All clones were sequenced to ensure that no other mutations had been introduced, and 34 that the FLAG' sequence was in the correct orientation.

The FLAG' fusion proteins were designated

NYK-EX-FLAG,

tie2-EX-FLAG" and RYK-EX-FLAG, respectively.

The small phosphorylated FLAG linker oligonucleotides (39 mers) ligated easily into the digedted vector fragments, and recombinants were accurately detected by use of the internal Clal site.

Example 8 Cell Culture COS cells were maintained in RPMI-1640 medium with 10% FCS and 50 ig/ml gentamicin at 37 0 C and 5% CO 2 COS cells were transfected by the DEAE dextran method as described previously (Aruffo and Seed, 1987).

pEE6-NYK-EX-FLAG, pEE6-tie2-EX-FLAG and pEE6-RYK-EX-FLAG constructs were transfected into CHO-K1 cells by calcium phosphate precipitation and selected in Glasgow Modified Eagle's Medium (without L-glutamine, without NaHCO 3 Cytosystems, Castle Hill, supplemented with fetal calf serum, 50 g/ml gentamicin, non-essential amino acids, sodium pyruvate and 25 mM methionine sulphoxide (MSX). Bulk production of the protein was achieved by culturing the cells on Cytodex-3 beads (Pharmacia, Uppsala, Sweden) in roller bottles in the presence of 2 mM sodium butyrate. Cells were grown until they reached about 80-90% confluence. Conditioned medium was collected every week by replacing with fresh medium. Supernatants were harvested from the roller bottles 16-20 days after seeding and the following cocktail of protease inhibitors was added: "0.02% NaN 3 1 mM PMSF, 0.2 TIU/ml aprotinin, g/ml pepstatin and 0.5 mM EDTA. The supernatants were either used directly or stored at -70C prior to use.

The constructs and controls were transfected into COS cells and supernatants were analysed for fusion protein three days post-transfection. Biosynthetic labelling of the cells, immunoprecipitation with M2-gel and SDS-PAGE analysis demonstrated products of the expected size, as shown in Figure 7. Diffuse bands in the range of 35 120-130 kD for NYK-EX-FLAG" (Figure 7A) and 100-110 kD for tie2-EX-FLAGm (Figure 7B) were specifically immunoprecipitated with the M2-gel. Predicted sizes for the NYK-EX-FLAGm, tie2-EX-FLAG and RYK-EX-FLAGm 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 Daltons, 95,000 Daltons and 38,000 Daltons respectively.

These initial studies in COS cells have shown that the FLAG™ peptide can be expressed at the C-terminus of the receptor extracellular domains, (ii) the FLAG"-fusion protein is secreted by the cell, and (iii) the FLAG'-fusion protein can be recognised effectively by the M2 antibody.

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 ig/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 ig of fusion protein per ml of expended tissue culture supernatant.

Example 9 Purification of Fusion Proteins NYK-EX-FLAGw, tie2-EX-FLAGm and RYK-EX-FLAG" were purified from medium conditioned by transfected CHO cells using affinity chromatography on M2 (anti-FLAG) gel (IBI).

A separate column was used for each receptor construct.

Supernatant (100-200 ml) was passed over a 1-2 ml column of M2-gel which was subsequently washed with 10 volumes of mM Tris-HCl pH 8.0, 150 mM NaC1, 0.02% Tween e 36 volumes of 50 mM triethylamine (TEA) pH 10.0, 150 mM NaC1, 0.02% Tween 20 and again with 10 volumes of mM Tris-HCl pH 8.0, 150 mM NaC1, 0.02% Tween 20. This step may not be necessary, depending on the yield and purity of protein which is required. Bound material was eluted with either 100 mM glycine-HCl pH 3.0, 0.02% Tween (neutralised in 1/10 volume 1 M Tris-HC1 pH 8.6) or 25-50 gg/ml FLAG" peptide (N-Asp-Tyr-Lys-Asp- Asp-Asp-Asp-Lys-C) in 10 mM Tris-HCl pH 8.0, 150 mM NaC1, 0.02% Tween 20. Columns were eluted in 1 ml fractions, and Al samples combined with 10 Al of reducing

SDS-PAGE

sample buffer were analysed by SDS-PAGE and proteins detected by silver staining. Following desorption with free peptide the affinity resin was treated with 0.1 M glycine pH 3.0 to remove bound peptide prior to the next purification cycle. For coupling to the sensor chip and depletion of free FLAG" peptide, fusion proteins were further purified by high-performance liquid chromatography in a Tris-free buffer system (Nice et al, 1994) on a AMonoQ PC 1.615 anion exchange column (Pharmacia) to give a single homogeneous species corresponding to either NYK-EX-FLAG, tie2-EX-FLAG or RYK-EX-FLAG"'.

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 eluted from the column with excess free FLAG" peptide, and further purification to homogeneity was achieved by microbore anion exchange HPLC. This also enabled the removal of excess free FLAG" peptide. Desorption of non-specific proteins was achieved by a two-step washing procedure involving Sbuffers of pH 8.0 and pH 10.0 respectively. This protocol appeared sufficient to remove the large majority of S..a contaminants, as judged by SDS-PAGE analysis of the purified fractions, which is illustrated in Figure 8.

Purification of NYK-EX-FLAG" and tie2-EX-FLAG gave a 37 predominant bands in the size range of 120-130 kD and 100-110 kD respectively (Figure 8A and 8B). Purification and analysis of RYK-EX-FLAG' gave in addition to the predicted 35-40 kD species a range of other higher molecular weight proteins (Figure 8C). These are consistently present in all RYK-EX-FLAG purifications, and may represent oligomerised forms of the fusion protein.

Competition with 25-50 gg/ml of FLAG' peptide was sufficient to remove the bound extracellular domain-FLAG", as further elution of the column with 0.1 M glycine-HC1 pH 3.0 gave little if any fusion protein (Figure 8A-C).

Interestingly, elution with low pH yielded more contaminating proteins in the samples than elution with FLAG peptide alone. Figure 8B (lanes 6-9) shows a range of contaminants in the low-pH eluate that were not seen with the peptide eluted material. We conclude that the FLAG' peptide is relatively specific in its ability to elute only FLAG-M2 interactions, and does not dissociate most other non-specific interactions. We detected a small amount of high molecular weight material, presumably immunoglobulin, eluting from the column after pH treatment; this was not eluted with the peptide at pH Fractionation of the M2-affinity eluate on a pmonoQ column effectively removed the free FLAG M peptide, which could be recycled for future use. Our overall yield of HPLC purified extracellular domain from the CHO cell conditioned medium was calculated to be in the order of 80 jg of protein per 100 ml.

To confirm that the material eluting from the M2 affinity column was in a native conformation, the NYK-EX-FLAG" fusion protein was tested for its ability to o bind a known ligand, vascular endothelial growth factor (VEGF) (Millauer et al, 1993) as described in Example 1.

Advantages of the Invention In this study we have described a protocol for isolating functional extracellular domains of receptor a 38 tyrosine kinases using a combination of site-directed mutagenesis, the FLAG' peptide system and CHO cell expression. Our need to develop this method was initially prompted by a complete lack of reagents for characterising the proteins encoded by three recently isolated cDNA clones. The advantage of our protocol is that it allows for the precise positioning of the FLAG' sequence at the junction of the extracellular and predicted transmembrane domain of a nominated receptor. BglII/BamHI restriction enzyme sites were used to introduce the FLAG" sequence because of their compatibility with each other and the absence of at least one of these in the cDNA clones being studied. In addition, the strategy provides a universal adaptor site whereby the receptor extracellular domain can be ligated to other proteins of interest. For example, the extracellular domain could be ligated into the vector AP-tag-l (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. For example, a fusion protein in which the extracellular domain 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 for our technique, as it can be used for generating single stranded DNA for site-directed mutagenesis, as well as for transient expression in COS cells. This allows the rapid testing of constructs for expression of the fusion protein before proceeding to large scale production. Subcloning of the final constructs into the CHO cell expression vector pEE6 is also simple, due to compatible XbaI sites present in the pCDM8 and pEE6 polylinkers. We have used sitedirected mutagenesis to introduce the necessary restriction enzyme sites at the junction of the extracellular and transmembrane domains. This technique has some advantages over PCR, especially when attempting to generate large 39 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).

Alternatively, PCR could be used to generate smaller fragments encoding the BglII/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 FLAG" 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 FLAG 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. We could not make use of the divalent cation-dependent nature of the Ml MAb for mild elution, as this MAb requires a free FLAGm N-terminus for recognition; therefore the M2 antibody was utilised. Mild elution is important when dealing with receptors which possess uncharacterised ligands, as the integrity of the ligand binding site cannot be readily assessed.

The location of the FLAG' peptide at the C-terminus did not adversely affect either experiments on the BIAcore™ or MAb production to the receptors. The purified NYK-EX-FLAG specifically bound the ligand VEGF.

Also, there was no apparent binding from serum containing media, in contrast to our observation with alkaline phosphatase-fusion proteins. The apparent inertness of the FLAG 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-FLAGM fusion proteins using a mammalian expression system. Utilising p.

40 such a system means that glycosylation and protein folding are most likely to resemble the native state. Linkage of a 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 FLAG' peptide means that the effects on the protein being studied are minimised. Other workers have successfully expressed a FLAG (N-terminus)-human platelet-activating receptor fusion protein in mammalian cells (COS-7) to demonstrate its topology at the membrane, although studies examining the function of the receptor were not performed (Gerard and Gerard, 1990). This is the first report of the FLAG" peptide as a C-terminal marker in a mammalian expression system. FLAG" was designed originally to be linked to the N-terminal of the desired protein, and has a structure such as to permit ready cleavage from this N-terminal

(U.S.

Patent No. 4,703,004).

This type of approach is generally applicable to the examination of cell surface receptors, in particular for workers intending to isolate the cognate ligands of these receptors. Although the FLAG" system itself is not inexpensive, it compares favourably with the development costs of a specific MAb affinity column, for which one would still have the problem of elution conditions to contend with. The time scale of the present technique is about two to four months from isolation of a clone encoding the extracellular domain to having microgram-milligram quantities of purified fusion protein.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

e 9 9 41 Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", means "including but not limited to" and is not intended to exclude other additives, components, integers or steps.

References cited herein are listed on the following pages, and are incorporated herein by this reference.

The entire disclosure in the complete specification of our Australian Patent Application No.

46061/96 is by this cross-reference incorporated into the present specification.

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REFERENCES

Aruffo A. and B. Seed.

"Molecular cloning of a CD28 cDNA by a high-efficiency

COS

cell expression system" Proc. Natl. Acad. Sci. 1987 84 8573'-8577 Brejer U. Albrecht, S. Sterrer and W. Risau.

"Expression of vascular endotheial growth factor during embryonic angiogenesis and endothelial. cell differentiation" Development, 1992 114 521-532 Fanslow D. Anderson, K.H. Grabstein, E.A. Clark, D. Cosman and R.J. Armitage.

"Soluble forms of CD40 inhibit biological responses of human B cells" J. Immunol., 1992 149 655-660.

Flanagan J.G. and P. Leder.

"The kit ligand: a cell surface molecule altered in steel mutant fibroblasts" Cell, 1990 63 185-194.

Gerard N.P. and C. Gerard.

n~ostuctonand exrsino a novel recombinant anaphylatoxin, C~a-N19, as a probe for the human receptor" Biochemistry, 1990 29 9274-9281.

D' Andrea, H.F. Lodisch and G.G. Wong "Expression cloning of the murine erythropoietin receptor" Cell, 1989 57 277-285 43 Hopp K.S. Prickett, V.L. Price, R.T. Libby, C.J. March, D.P. Cerretti, D.L. Urdal and P.J. Conlon.

"A short polypeptide marker sequence useful for recombinant protein identification and purification.

Biotechnology, 1992 6 1204-1210.

Hovens S.A. Stacker, A. Andres, A.G. Harpur, A. Ziemiecki and A.F. Wilks.

"RYK, a receptor tyrosine kinase-related molecule with unusual kinase domain motifs" Proc. Natl. Acad. Sci. 1992 89 11818-11822.

Kozak M.

"An Analysis of Vertebrate mRNA Sequences: Intimations of Translational Control" J. Cell Biol., 1991 115 887-903.

Kunkel T.A.

"Rapid and efficient site-specific mutagenesis without phenotypic selection" Proc. Natl. Acad. Sci. 1985 82 488-492.

Kyte J. and R.F. Doolittle.

"A simple method for displaying the hydropathic character of a protein" Journal of Molecular Biology, 1982 157 105-132.

Li Y.N. Jan and L.Y. Jan.

"Specification of subunit assembly by the hydrophobic amino-terminal domain of the shaker potassium channel" Science, 1992 257 1225-1230.

Miles A.A. and E.M. Miles.

"Vascular reactions to histamine, histamine-liberator and leukotaxine in the skin of guinea-pigs" Journal of Physiology (London), 1952 118 228-257.

-44 Millauer S. Wizigraman.Voo, H. Scbnurch, R. Martinez, N.P.H. Moller, W. Risau and A. Ulirich.

"High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenes js" Cell, 1993 72 835-846.

Nice M. Lackmann, F. Smyth, L. Fabri and A.W. Burgess.

"Synergies between micropreparative high-performance liquid chromatography and an instrumental optical biosensor" Journal of Chromatography, 1994 660 169-185.

Oelrichs H.H. Reid, 0. Bernard, A. Ziemiecki and A.F. Wilks.

"IJYK/FLK-1: A putative receptor protein tyrosine kinase isolated from E10 embryonic neuroepithejum is expressed in endothelial cells of the developing embryo" Oncogene, 1993 8 11-18.

Pacifici R.E. and A.R. Thomason "Hybrid tyrosine kinase/cytokine receptors transmit mitogenic signals in response to ligand" J. Biol. Chem., 1994 269 1571-1574.

Paul D. Merberg, H. Finnerty, G.E. Morris, J.C. Morris, 5.5. Jones, R. Kriz, K.J. Turner and C.R. Wood.

"Molecular cloning of the cDNA encoding a receptor tyrosine **kinase-related molecule with a catalytic region homologous to c-met. International Journal of Cell" Cloning, 1992 10 309-314.

Runting S.A. Stacker and A.F. Wilks.

"ltie2, a putative protein tyrosine kinase from a new class of cell surface receptor" Growth Factors, 1993 9 99-105.

Samibrook E.F. Fritsch and T. Maniatis.

"Molecular Cloning A Laboratory Maniual" Cold Spring Harbor Laboratory Press, 1989.

Smith D.B. and K.S. Johnson.

"Single-step purification of polypeptides expressed -in E.colj as fusions with glutathjone S-transferaseu Gene, 1988 67 31-40.

Stacker C.M. Hovens, A. Vitali, M.A. Pritchard, E. Baker, G.R. Sutherland and A.F. Wilks.

"Molecular cloning and chromosomal localisation of the human homologue of a receptor related to tyrosine kinases (ryk)" Oncogene, 1993 8 1347-1356.

Su A.K. Prestwood and R.A. McGraw.

"Production of recombinant porcine tumour necrosis factor alpha in a novel E.coli expression system" Biotechniques, 1992 13 756-762.

Terman M. Dougher Vermazen, M.E. Carrion, D. Dimitrov, D.C. Armellino, D. Gospodarowicz and P. Bohlen.

"Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor" Biochem. Biophys. Res. Coxnmun., 1992 187 1579-1586.

aUllrich A. and J. Schlessinger.

"Signal transduction by receptors with tyrosine kinase activity" o Cell, 1990 61 203-212.

**a.Wilks

A.F.

"Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction" aProc. Natl. Acad. Sci. 1989 86 1603-1607.

46 Zhang K.N. Wills, M. Husmann, T. Hermann and M. Pfahl.

"Novel pathway for thyroid hormone .receptor action through interaction with jun and fos oncogene activities" Mol. Cell Biol., 1991 11 6016-6025.

47 SEQUENCE LISTING GENERAL

INFORMATION:

APPLICANT: LUDWIG INSTITUTE FOR CANCER

RESEARCH

(ii) TITLE: ASSAY AND PEPTIDES FOR USE THEREIN (iii) NUMBER OF SEQUENCES: 12 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSESS: Felfe Lynch STREET: 805 Third Avenue CITY: New York STATE: New York COUNTRY:

U.S.A.

POSTCODE: 10022 COMPUTER READABLE

FORM:

MEDIUM TYPE: 3.5 1.44 Mb storage diskette COMPUTER: IBM PS/2 OPERATING SYSTEM:

PC-DOS

SOFTWARE: Wordperfect (vi) PRIOIR APPLICATION

DATE:

APPLICATION NO.: PN 0300, PN 0301 FILING DATE: 23 DECEMBER 1995 (vii) CURRENT APPLICATION

DATA:

APPLICATION NO.: Not yet assigned FILING DATE: Not yet assigned (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Patricia A. Pasqualini REFERENCE NO.: LUD-5401-PCT (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (212) 688-9200 TELEFAX: (212) 838-3881

S

St.

*et 48 INFORMATION FOR SEQ ID NO. 1: SEQUENCE

CHARACTERISTICS:

LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 Ile Glu Tyr Ile Phe Ser Pro Ser Cys Val Pro Leu ATA GAG TAC ATC TTC AGC CCC AGC TGT GTG CCG CTG INFORMATION FOR SEQ ID NO. 2: SEQUENCE

CHARACTERISTICS:

LENGTH: 11 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 Arg Ile Lys Pro Gly Gin Ser Gin His Ile Gly CGG ATC AAA CCT GGC CAA AGC CAG CAC ATA GGA INFORMATION FOR SEQ ID NO. 3: SEQUENCE

CHARACTERISTICS:

LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 *9 Gln Ser Gln His Ile Val Glu Met Ser Phe Leu Gin CAA AGC CAG CAC ATA GTA GAG ATG AGC TTC CTG CAG 49 INFORMATION FOR SEQ ID NO. 4: SEQUENCE

CHARACTERISTICS:

LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 Leu Gin His Ser Arg Cys Glu Cys Arg Pro Trp CTA CAG CAC AGC CGA TGT GAA TGC AGA CCA TGG TAA INFORMATION FOR SEQ ID NO. SEQUENCE

CHARACTERISTICS:

LENGTH: 12 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Arg Ile Arg Ser Gly Asp Arg Pro Ser Ile Gly Glu CGG ATC AGA TCT GGC GAT AGA CCG TCC ATA GGA GAG INFORMATION FOR SEQ ID NO. 6: SEQUENCE CHARACTERISTICS: LENGTH: 190 TYPE: amino acid S. STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 Met Asn Phe Leu Leu Ser Trp Val His Trp Thr Leu Ala Leu Leu Leu 10 Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala Pro Thr Thr Glu Gly 25 Glu Gin Lys Ser His Glu Val Ile Lys Phe Met Asp Val Tyr Gin Arg 40 Ser Tyr Cys Arg Pro Ile Glu Thr Leu Val Asp Ile Phe Gin Glu Tyr 50 55 Pro Asp Glu Ile Glu Tyr Ile Phe Ser Pro Ser Cys Val Pro Leu Met 65 70 75 Arg Cys Arg Gly Cys Cys Asn Asp Glu Ala Leu Glu Cys Val Pro Thr 85 90 Ser Ser Cus Cys 145 Lys Glu Glu Gin Arg 130 Ser Cys Leu 50 Ser Asn Ile Thr Met Gin le Met Arg Ile Lys Pro His Gin 100 105 110 His Ile Giy Giu Met Ser Phe Leu Gin His Ser Arg Cys Glu 115 120 125 Pro Lys Lys Asp Arg Thr Lys Prp Giu Ann His Cys Giu Pro 135 140 Giu Arg Arg Lys His Leu Phe Val Gin Asp Pro Gin Thr Cys 150 155 160 Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gin Leu 165 170 175 Ann Giu Arg Thr Cys Arg Cys Ann Leu Pro Arg Arg 180 185 190 INFORMATION FOR SEQ ID NO. 7: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 190 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: iinear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 Met Ann Phe Leu Leu Ser 5 Tyr Leu His His a a* a

S

a Glu Ser Pro Arg Ser Ser Cys Cys 145 Ly's Glu Gin Tyr Asp Cys Giu Gin Arg 130 Ser Cys Leu Lys Ser Cys Arg Glu Ile Ala Gly Ser Ann 100 His Ile 115 Pro Lys Giu Arg Ser Cys Asn Glu 180 Ala His Pro Giu Cys 85 Ile Gly Lys Arg Lys 165 Arg Lys Giu Ile Tyr 70 Cys Thr Giu Asp Lys 150 Ann Thr Trp Val His Trp Thr Leu 10 Trp Ser Gin Ala Ala Pro 25 Val Ile Lys Phe Met Asp 40 Giu Thr Leu Val Asp Ile 55 60 Ile Phe Lys Prp Ser Cys 75 Ann Asp Giu Ala Leu Giu 90 Met Gin Ile Met Arg Ile 105 Met Ser Phe Leu Gin His 120 Arg Thr Lys Pro Glu Asn 35 140 His Leu Phe Val Gin Asp 155 thr Asp Ser Arg Cys Lys 170 Cys Arg Cys Asn Leu Pro 185 Ala Thr Val Phe Val Cys Lys Ser 125 His Pro Ala Arg Leu Thr Tyr Gin Pro Val Pro 110 Arg Cys Gin Arg Axg 190 Leu Leu Giu Gly Gin Arg Glu Tyr Leu Met Pro Thr Gly Gin Cys Giu Giu Pro Thr Cys 160 Gln Leu 175 51 INFORMATION FOR SEQ ID NO. 8: Wi SEQUENCE CHARACTERISTICS: LENGTH: 190 TYPE: amino acid STPANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 Met Asn Phe Leu Leu Ser Trp, Val His Trp Thr Leu Ala 10 Tyr Leu His His Ala Lys Trp, Ser Gin Ala Ala Pro Thr 25 Glu Gin Lys Ser His Glu Val Ile Lys Phe Met Asp Val 40 45 Ser Tyr Cys Arg Pro Ile Glu Thr Leu Val Asp Ile Phe so 55 60 Pro Asp Giu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Vai 70 75 Arg Cys Ala Gly Cys Cys Asn Asp Glu Ala Leu Giu Cys 90 Ser Glu Ser Asn Ile Thr Met Gin Ile Met Axrg Ile Lys 100 105 Ser Gin His Ile Val Glu Met Ser Phe Leu Gin His Ser 115 120 125 Cys Arg Pro Lys Lys Asp Arg Thr Lys Pro Giu Asn His 130 135 140 Cys Ser Giu Arg Arg Lys His Leu Phe Vai Gin Asp Pro 145 150 155 Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala 165 170 Glu Leu Asn Giu Arg Thr Cys Arg Cys Asn Leu Pro Arg 180 185 Leu Leu Thr Giu Tyr Gin Gin Glu Pro Leu Val Pro Pro His 110 Arg Cys Cys Giu Gin Thr Arg Gin 175 Arg 190 Leu Gly Arg Tyr Met Thr Gin Glu Pro Cys 160 Leu *6

C.

C

C

*0 C.

C

CC..

C C

C

C INFORMATION FOR SEQ ID NO. 9: Wi SEQUENCE CHARACTERISTICS: LENGTH: 133 TYPE: amino acid STRAINDEDNESS: single TOPOLOGY: linear 52 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 Met Asn Phe Tyr Leu His Giu Gin Lys Ser Tyr Cys s0 Pro Asp Giu Arg Cys Ala Ser Giu Ser Ser Gin His 115 Cys Arg Pro 130 Leu Leu His Ala Ser His Arg Pro Ile Glu Gly Cys Asn Ile 100 Ile Gly Lys Trp Ser Trp Val Lys Trp Ser Glu Val Ile 40 le Glu Thr 55 Tyr Ile Phe 70 Cys Asn Asp Thr Met Gin Glu Met Ser 120 His Trp Thr Leu Ala Leu 10 Gin Ala Ala Pro Thr Thr 25 Lys Phe Met Asp Val Tyr Leu Val Asp Ile Phe Gin Lys Pro Ser Cys Val Pro 75 Giu Ala Leu Glu Cys Val 90 Ile Met Arg Ile Lys Pro 105 110 Phe Leu Gin His Ser Arg 125 Leu Leu Giu Gly Gin Arg Glu Tyr Leu Met Pro Thr His Gin Cys Glu INFORMATION FOR SEQ ID NO. Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 190 TYPE: amino acid STRA1NDEDNESS: single TOPOLOGY: linear ese.

*559

S

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Asn Phe Leu Leu Ser Trp Val His Trp, Thr Leu Ala Leu Leu Leu 5 10 Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala Pro Thr Thr Giu Gly 25 Giu Gin Lys Ser His Glu Val Ile Lys Phe Met Asp Val Tyr Gin Arg 35 40 Ser Tyr Cys Arg Pro Ile Giu Thr Leu Val Asp Ile Phe Gin Ghi Tyr 55 Pro Asp Giu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met 65 70 75 Arg Cys Ala Gly Cys Cys Asn Asp Giu Ala Leu Giu Cys Val Pro Thr 85 90 53 Ser Giu Ser Asn Ile Thr Met Gin Ile Met Arg le Arg Ser Giy Asp 100 105 110 Arg Pro Ser le Gly Giu Met Ser Phe Leu Gin His Ser Arg Cys Giu 115 120 125 Cys Arg Pro Lys Lys Asp Arg Thr Lys Pro Giu Asn His Cys Glu Pro 130 135 140 Cys Ser Giu Arg Arg Lys His Leu Phe Val Gin Asp Pro Gin Thr Cys 145 I50 155 Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Aia Arg Gin Leu 165 170 175 Giu Leu Asn Giu Arg Thr Cys Arg Cys Asn Leu Pro Arg Arg 180 185 190 INFORM~ATION FOR SEQ ID NO. 11: SEQUENCE

CHARACTERISTICS:

LENGTH: 8 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 Asp Tyr Lys Asp Asp Asp Asp Lys INFORMATION FOR SEQ ID NO. 12: SEQUENCE

CHARACTERISTICS:

LENGTH: 78 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 54 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 GATCTGACTA CAAGGACGAC GATGkCAAGT GAATCGATA~, CTGA.TGTTCC TGCTGCTACT GTTCACTTAG CTATCTAG 78 a. a a.

a a a.

a S a *ae.

a a. a a..

a. a S 55 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An isolated polypeptide which is a mutant of wild type VEGF and is capable of inhibiting or promoting the activity of the receptor protein NYK.

2. A polypeptide according to Claim 1, wherein said polypeptide comprises amino acid deletion or substitution in the equivalent of the cysteine-knot motif of wild type

VEGF.

3. A polypeptide according to Claim 1, wherein said polypeptide comprises one or more amino acid substitutions S* in the region which corresponds to the V3 domain of wild 15 type VEGF.

4. A polypeptide according to Claim 1, wherein said polypeptide comprises a mutation at least one position corresponding to mouse VEGF, said position selected from the group consisting of positions 73, 109, 110, 111, 112, 113, 114, 115 and 117.

0*SS 5. A polypeptide according to Claim 1, wherein said polypeptide is selected from the group consisting of K73S, 25 H111G, G117V, VEGFO and variants thereof having substantially the same biological activity.

6. A polypeptide according to Claim 1, comprising an agonist of the receptor protein NYK and 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.

7. A polypeptide according to Claim 6, wherein said S= 35 truncation is at or about residue 133 corresponding to wild H:\Bkrot\Ieep\speci\31583-97.doc 19/03/99

Claims (6)

1. 8. A pharmaceutical composition comprising a polypeptide according to any one of Claims 1 to 7 and a pharmaceutically acceptable carrier or diluent.
2. 9. An isolated nucleic acid molecule which encodes a polypeptide capable of inhibiting or promoting the activity of the receptor protein NYK. A molecule according to Claim 9, wherein said molecule has a nucleotide sequence which encodes a polypeptide with amino acid deletions or insertions in the 15 equivalent of the cysteine-knot motif of wild type VEGF.
3. 11. A molecule according to Claim 9, wherein said So molecule has a nucleotide sequence which encodes a polypeptide with one or more substitutions in the region which corresponds to the V3 domain of a wild type VEGF.
4. 12. A molecule according to Claim 11, wherein said molecule encodes a polypeptide with a mutation at least one position, said position selected from the group consisting S 25 of positions 109, 110, 111, 112, 113, 114, 115 and 117 corresponding to wild type mouse 3VEGF.
5. 13. A molecule according to Claim 12, wherein said •molecule encodes a polypeptide selected from the group consisting of K73S, H111G, G117V, VEGFO and variants thereof having substantially and same biological activity.
6. 14. A molecule according to Claim 9, wherein said molecule encodes a truncated protein terminating at or around residue 133 corresponding to wild type mouse VEGF H:\Bkrot\Keep\speci\31583-97.doc 19/03/99 57 and having an amino acid substitution at around the same position. An immunogenic composition comprising a polypeptide according to any one of Claims 1 to 7, wherein said polypeptide is specific for the NYK receptor extracellular domain and a pharmaceutically acceptable adjuvant. Dated this 16th day of March 1999 LUDWIG INSTITUTE FOR CANCER RESEARCH By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and U 15 Trade Mark Attorneys of Australia f S. S U H:\Bkrot\Keep\speci\31583-97.doc 19/03/99 F- 58 ABSTRACT This invention relates to growth factor receptors of the receptor protein tyrosine kinase family, preparation of the extracellular domain of th--e receptor in large amounts, ligands for the receptors, nucleic acids encoding the ligands, and the use of the receptors and ligands in assays. H:\Bkrot\Keep\speci\31583-97.doc 19/03/99