HGF POLYPEPTIDES AND THEIR USE IN THERAPY

Abstract: A variant hepatocyte growth factor (HGF) which is substantially incapable of binding a heparan sulphate proteoglycan but which is capable of binding to the HGF receptor for use in medicine.

Description: HGF POLYPEPTIDES AND THEIR USE IN THERAPY The present invention relates to polypeptides and their use in therapy and in particular to the use of mutant hepatocyte growth factors in therapy. Hepatocyte growth factor (HGF), also known as scatter factor (SF) is a polypeptide growth factor which induces growth, movement and differentiation in target cells. HGF is synthesised as a single-chain precursor which is proteolytically cleaved to an active two-chain form. The active form of HGF is believed to mediate its effect by binding to a tyrosine kinase receptor (MET) encoded by the c-met oncogene. Wild-type HGF has a number of potential therapeutic uses. However, wild-type HGF is rapidly eliminated from the plasma (t l Λ approximately 4.5 min). The clearance rate can be reduced somewhat by complexing HGF to heparin or other polyanions prior to administration. Matsumoto et al (1991) Biochem. Biophys. Res. Comm. 181, 691-699 describes that the deletion of kringle domains or the N-terminal hairpin structure in HGF results in marked decreases in related biological activities. Miau et al (1996) Biochem. Biophys. Res. Comm. 223, 487-491 purports to describe the identification of a novel variant HGF secreted by spleen- derived stromal cells. Lokker et al (1994) Prot. Eng. 7, 895-903 describes the mutational analysis and molecular modelling of the N-terminal kringle-containing domain of HGF and identifies amino acid side chains important for interaction with the c-Met receptor. Mizuno et al (1994) . Biol. Chem. 269, 1131-1136 describes mutants in which the hairpin loop or a kringle domain have been deleted. Schwall et al (1996) J. Cell Biol. 133, 709-718 reports that heparin induces dimerization and confers proliferative activity onto the HGF antagonists NK1 and NK2 which are truncated variants of HGF. Lokker et al (1992) EMBO J. 11, 2503-2510 describes various mutants of HGF. WO 92/05184 and WO 96/40914 relate to a truncated form of HGF which purportedly antagonises the activity of HGF. WO 93/23541, US 5,547,856, US 5,316,921 and US 5,580,963 relate to HGF variants that are resistant to proteolytic cleavage. US 5,464,815 describes a method for extending the plasma half-life of heparin-binding proteins. WO 96/28475 describes HGF modified with polyethylene glycol. The present invention describes HGF variants which have significantly reduced ability to bind heparin or heparan sulphate proteoglycan (HSPG) but which are still able to bind to the HGF receptor (MET). At least some of these variants have a longer circulatory half-life in vivo and a greater mitogenic activity than wild-type HGF when administered to rats. Thus, one object of the invention is to overcome the need to complex HGF with heparin-like compounds or other polyanions in order to improve pharmacokinetics and in vivo activity. The variant HGF molecules of the invention may activate the HGF receptor in substantially the same way as wild-type HGF or they may act as antagonists of wild-type HGF or they may act in some other way. In any case the variant HGF molecules are useful in medicine. A first aspect of the invention provides a variant hepatocyte growth factor (HGF) which is substantially incapable of binding a heparan sulphate proteoglycan but which is capable of binding to the HGF receptor for use in medicine. By "variant hepatocyte growth factor" we mean a hepatocyte growth factor (HGF) which varies in primary amino acid structure from a wild- type form. For example, the amino acid sequence of a wild-type form of human HGF is given in Figure 7. It is well known that certain polypeptides, especially those from humans, are polymorphic and it will be appreciated that some natural variation of the human HGF sequence may occur and that such human HGF molecules will be considered to be wild-type provided that the HGF binds heparan sulphate proteoglycan (HSPG) and is able to bind the HGF receptor and give rise to the known biological effect. By "substantially incapable of binding HSPG" we include the meaning that the variant HGF binds HSPG significantly less well than wild-type HGF. It is preferred if the variant HGF binds HSPG at least 10-fold less well than wild-type HGF, preferably at least 20-fold less well, still more preferably 50-fold less well and most preferably at least 100-fold less well. Conveniently the HSPG-binding ability of wild-type HGF and variant HGF is measured with reference to the HSPGs produced by the cells in Example 1 described in Example 1. Preferably, the variant HGF has a decreased affinity for heparin. Typically, although not always, the variant HGF of the invention will have a decreased ability to bind both heparin and HSPG. Conveniently, the variant HGF herein disclosed may be one that binds heparin significantly less well than wild-type HGF. It is preferred if the variant HGF binds heparin at least 10-fold less well than wild-type HGF, preferably at least 20-fold less well, still more preferably at least 50-fold less well and most preferably at least 100-fold less well. Conveniently, the heparin-binding ability of wild-type HGF and variant HGF is measured using the methods described in Example 1. In any case, suitably, the variant HGF has substantially the same mitogenic and motogenic activities on target cells as wild-type HGF (at least those variants which are not antagonists of HGF). Preferably the NK2 fragments of wild-type HGF or variant HGF are used to determine the binding affinity for heparin. Using the method of Example 1 wild-type human HGF has a molar dissociation constant (Kj) of ~ 1 x 10 "9 M (1 nM) and the NK2 fragment of wild-type HGF has a molar dissociation constant (Kj) of around 2.3 nM with respect to heparin. The naturally occurring form of NK2 is a product of alternative splicing of the primary HGF transcript and encodes the leader sequence (residues 1-31), the N domain containing the hairpin loop (residues 70-96), kringle 1 (residues 128-206), kringle 2 (residues 211-288) and an extra three residues after kringle 2 (residues 289-291). By "capable of binding to the HGF receptor" we include the meaning that the variant HGF can bind with substantially the same affinity to a soluble form of the HGF receptor (MET) as wild-type HGF as described in Example 1. For the avoidance of doubt the variant HGFs of the invention include HGF variants that are capable of binding HGF receptor leading to either (a) activation of the receptor or (b) inhibition of receptor signalling by endogenous (or wild-type) HGF. Preferably the variant HGF binds to the HGF receptor with an affinity of between 0J- and 10-fold that of wild-type HGF. Preferably, in some cases, the variants of HGF are not antagonists of HGF and for some variants the effect of binding the variant HGF to MET in a cell that contains MET and can respond to binding of HGF to MET is substantially the same as the effect of binding wild-type HGF to MET. Thus, in these cases the activity of the variant HGF which is not an antagonist of HGF and the activity of wild-type HGF on target cells in vitro is substantially the same. Still preferably, as is described below, some variant HGF molecules are antagonists of HGF but these nevertheless fall within the scope of the invention. As is discussed in more detail below, all of these types of molecules are believed to be useful in medicine and so are prepared in a form packaged and presented for use in medicine. Before the present work it had not been shown that variants of HGF which are substantially incapable of binding heparin or HSPG but which are capable of binding to the HGF receptor have improved activities in vivo compared to wild-type or other variant forms of HGF. In particular at least some of the variants of the invention show unexpectedly slower clearance from serum than wild-type HGF and at least some of d e variant HPG molecules of the invention unexpectedly induce DNA synthesis in adult liver to a higher degree than wild-type HGF. By "HGF" we include single chain forms as well as forms which have been proteolysed to the two-chain form. Preferably the variant HGF is a variant HGF wherein a positively-charged amino acid residue in the hairpin loop structure of wild-type HGF has been replaced with an amino acid residue with no charge or a negative charge. The hairpin loop structure spans amino acid residues 70 to 96 of wild-type HGF. By a "positively-charged amino acid residue" we mean Arg (R), Lys (K) or His (H). Histidine has a partial positive charge at physiological pH. Arg and Lys are often called basic amino acid residues. By a "negatively-charged amino acid residue" we mean Glu (E) or Asp (D). Apart from the given positively- or negatively-charged amino acid residues all other naturally occurring amino acid residues which are directly encoded by the genetic code have no charge. Glu and Asp are often called acidic amino acid residues. It is particularly preferred if the variant is one in which a positively- charged residue in the hairpin loop structure is replaced by a negatively- charged residue. It is therefore preferred if a Lys or Arg or His residue is replaced by a Glu or Asp residue. It is particularly preferred if a Lys or Arg residue is replaced by a Glu residue. It will be appreciated that replacement of positively-charged residues by non-charged or negatively-charged residues is readily achieved by the well known methods of protein engineering as described below. However, variant HGF molecules which are substantially incapable of binding heparin or HSPG but which are capable of binding to the HGF receptor may be obtained by, for example, deleting positively charged amino acid residues present in the hairpin loop structure by protein engineering methods or, for example, by chemically modifying positively-charged residues in the hairpin loop structure. Protein engineering methods are preferred for replacement of amino acid residues; however, it may be possible to, for example, accomplish selective modification of arginine residues using cyclohexan-l,2-dione. Preferably the HGF is a human HGF. The amino acid sequence of a wild-type human HGF is given in Figure 7. It is particularly preferred if the variant HGF is a variant human HGF in which positively-charged amino acid residues of the hairpin loop region are replaced by negatively- charged amino acid residues or amino acid residues with no charge. However, it will be appreciated that HGF from other mammalian species have an analogous hairpin loop structure which contains positively-charged residues and variants of these non-human HGF molecules in which the positively-charged amino acid residues are replaced by negatively-charged amino acid residues or by non-charged amino acid residues are included within the scope of the invention provided that such molecules are substantially incapable of binding heparin or HSPG but which are capable of binding to the HGF receptor. In wild-type human HGF the positively-charged amino acid residues in the hairpin loop structure include R73, R76, K78, K85, K91, R93 and K94. It is particularly preferred if the variant human HGF is one wherein at least Arg73 (R73) has been replaced by an amino acid residue with no charge or with a negative charge. Preferably the variant human HGF includes the mutation Arg73 Glu (R73E) or Arg73 Asp (R73D); most preferably Arg73 Glu (R73E). It is also particularly preferred if the variant human HGF is one wherein at least Arg76 (R76) has been replaced by an amino acid residue with no charge or with a negative charge. Preferably the variant human HGF includes the mutation Arg76 Glu (R76E) or Arg76 Asp (R76D); most preferably Arg76 Glu (R76E). An especially preferred embodiment of the invention is a variant human HGF wherein both amino acid residues Arg73 and Arg76 (R73 and R76) have been replaced independently of each other with an amino acid residue with no charge or with a negative charge. It is preferred that both Arg73 and Arg76 are replaced with a negatively-charged amino acid residue such as Glu (E) or Asp (D); it is particularly preferred if the variant human HGF comprises amino acid replacements Arg73 Glu and Arg76 Glu (R73E and R76E). Conveniently, in addition to die replacement of Arg73 and Arg76 other positively charged amino acid residues in the hairpin loop structure are replaced with a negatively-charged amino acid residue or an amino acid residue with no charge. Suitably, in addition to d e replacement of Arg73 and Arg76 of human HGF, Arg93 (R93) and/or Lys78 (K78) are replaced witii a negatively-charged amino acid residue or an uncharged amino acid residue. Preferably Arg93 (R93) and/or Lys78 (K78) are replaced widi Glu (E) or Asp (D), most preferably Glu (E). Thus, a preferred embodiment of me invention is a variant human HGF comprising amino acid replacements R73E, R76E and R93E, and a variant human HGF comprising amino acid replacements R73E, R76E and K78E. The variant HGF molecules of the invention include HGF molecules which, in addition to containing the mutations referred to above, may also be modified further in order to modulate the biological activity of the molecule (for example, in order to make an HGF antagonist) or tiiey may be modified further in order to facilitate synthesis and/or purification of ύJe variant HGF molecule. However, these further modifications retain the given properties of the variant HGF molecule namely mat the variant is substantially incapable of binding HSPG or heparin as the case may be but is capable of binding to the HGF receptor. Suitably the variant HGF may comprise a "tag" peptide sequence which may facilitate its purification. Typically, the tag comprises a binding site for a component which may be immobilised in order to allow a solid phase purification of the variant HGF following binding of the variant HGF to the component. The tag may be, for example, the His n sequence (n>4) which binds to Ni 2+ ions, or it may be an epitope for a monoclonal antibody such as die well known Myc-tag epitope. Other possibilities are well known in the art. It is preferred, however, if die variant human HGF consists of a wild-type HGF wid amino acid replacements Arg73 Glu and Arg76 Glu (R73E, R76E) or a wild-type HGF with amino acid replacements Arg73 Glu, Arg76 Glu and Arg93 Glu (R73E, R76E, R93E) or a wild-type HGF with amino acid replacements Arg73 Glu, Arg76 Glu and Lys78 Glu (R73E, R76E, K78E). The variant human HGF with amino acid replacements R73E, R76E and R93E is called HP1 in Example 1 and the variant human HGF with amino acid replacements R73E, R76E and K78E is called HP2 in Example 1 (see legend to Figure 1). It is also preferred if me variant human HGF comprises mutations of L80 and/or F82. In particular a preferred variant human HGF comprises me mutations L80S and F82Q. The variant human HGF with amino acid replacements L80S and F82Q is called HP4 in Example 1 and is a variant HGF for use in medicine of the invention. It will be appreciated diat it may be beneficial to introduce one or more of these mutations into a variant HGF which also contains one or more of the mutations in the hairpin loop which remove the positive charge as herein described. In addition to die aforementioned mutations it may be advantageous to replace other positively-charged amino acid residues in the human HGF molecule with non-charged or negatively-charged amino acid residues. For example, it may be advantageous to introduce one or more replacements of K91 and K94 (in the hairpin structure) and H241, R242, K244 and R249 (in kringle 2 domain). A variant human HGF which consists of wild-type HGF in which Lys85 is replaced by Glu (K85E; HP5 - see legend to Figure 1) is not a variant of die invention since it behaves essentially like wild-type HGF. However, this mutation may be beneficially included witii the other mutations herein disclosed. The variant HGF is preferably one which, in relation to its MET binding ability and its ability to transduce signals into die cell, has substantially die same activity as wild-type HGF. However, it is also preferable that the variant HGF is one which antagonises the action of wild-type HGF. Variant HGF molecules which antagonise the action of wild-type HGF are known in the art. For example, WO 92/05184 and WO 96/40914 relate to truncated forms of HGF which specifically antagonise or partially antagonise die activity of wild-type HGF. WO 93/23541, US 5,547,856, US 5,316,921 and US 5,580,963 relate to variant HGF molecules which are resistant to proteolytic cleavage by enzymes capable of in vivo conversion of HGF into its two-chain form. All of these patent applications and patents are incorporated by reference. Thus, it is preferred if die variants described in these patent applications and patents are further modified in the way described herein by, for example, making additional mutations which make the variant HGF substantially incapable of binding heparin or HSPG but which is capable of binding to die HGF receptor. Preferably die mutations disclosed herein are combined with the mutations which confer resistance to proteolytic cleavage by enzymes capable of in vivo conversion of HGF into its two-chain form such as diose described in US 5,316,921. More preferably, these mutations, which may be combined with the mutations disclosed herein in relation to the hairpin loop structure, are ones in which an amino acid alteration at or adjacent to any of amino acid positions 493, 494, 495 and 496 of wild-type human HGF sequence. Thus, preferred variant HGF molecules of the invention which are believed to be antagonists of wild-type HGF are, for example, those comprising the amino acid replacements R73E, R76E and R93E, or R73E, R76E and K78E and any one or more of replacements of amino acid positions 493, 494, 495 and 496 of wild-type human HGF. As discussed above die aforementioned variant HGF molecules are useful in medicine. A further aspect of the invention provides a pharmaceutical composition comprising a variant HGF as defined in the first aspect and a pharmaceutically acceptable carrier. The aforementioned compounds of me invention or a formulation diereof may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time. Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of me invention and not deleterious to me recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free. The formulations may conveniently be presented in unit dosage form and may be prepared by any of me methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general me formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or bom, and men, if necessary, shaping the product. Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. A tablet may be made by compression or moulding, optionally wim one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of me powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of me active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile. Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacantii; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising me active ingredient in a suitable liquid carrier. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render die formulation isotonic with the blood of die intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and diickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of die sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction tiiereof, of an active ingredient. It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include odier agents conventional in die art having regard to die type of formulation in question, for example those suitable for oral administration may include flavouring agents. Localised delivery may be desirable where possible; for example when delivery is desired to die liver it may be via the hepatic artery. A furtiier aspect of the invention provides a method of treating a patient in need of or who would benefit from treatment wim a hepatocyte growth factor or an antagonist diereof the method comprising administering to the patient an effective amount of a variant HGF as defined in me first aspect of the invention. It will be appreciated diat widi the advances in gene dierapy it may be possible to administer the variant HGF via administration of a genetic construct which encodes die variant HGF. This is specifically included in me method of treatment of the invention. The merapeutic use of wild-type and particular variant HGF molecules distinct from those disclosed herein has been described previously. For example, studies in mice suggest mat HGF may function as a tumour suppressor in the early stages of liver carcinogenesis (Santoni et al (1996) Proc. Natl. Acad. Sci. USA 93, 9577-9582), and HGF is reported to inhibit the growtii of Hep G2, HCC, B6/F1 melanoma and KB squamous carcinoma cells in vitro (Tajima et al (1991) FEBS Lett. 291, 229-232). HGF prevents onset and progression of hepatic fibrosis/cirrhosis in rats and completely abrogates deadi caused by severe hepatic cirrhosis and dysfunction (EP 0 456 188 and Matsuda et al (1995) /. Biochem. 118, 643-649). HGF enhances die efficiency of retroviral transduction of primary hepatocytes (Pages et al (1996) Biochem. Biophys. Res. Comm. 222, 726-731). HGF stimulates liver regeneration in 70%-hepatectomised rats (Ishii et al (1995) J. Biochem. 117, 1105-1112). HGF has been proposed as a therapy against vascular smoodi muscle cell proliferation (Nakamura et al (1995) Biochem. Biophys. Res. Comm. 215, 483-488, and decrease in local HGF concentrations due to high D-glucose may be a trigger of endodielial injury in diabetes, potentially resulting in the progression of atherosclerosis (Nakamura et al (1997) Diabetes 46, 138-142). HGF is a potent angiogenesis factor in vivo (Rosen et al (1993) Symp. Soc. Exp. Biol. 47, 227-234) and thus may have utility in wound healing. HGF is reported to enhance repair of intestinal epithelial erosions/ulcerations (Nusrat et al (1994) J. Clin. Invest. 93, 2056-2065). HGF has been proposed to block side effects of radio/chemotherapy (WO 93/08821; WO 95/25537), as a remedy for cranial nerve disorders (WO 95/07709), as a remedy for cartilage disease (WO 96/05855), to relieve side effects caused by immunosuppressants (WO 95/25537) and as a dierapeutic agent for renal disease (EP 0 462 549). See also Matsumoto & Nakamura, Hepatocyte Growth Factor, pages 450-474 In: Acute Renal Failure, New Concepts and dierapeutic strategies, ed. M.S. Goligorsky & J.H. Stein, Churchill Livingstone, New York. All of these documents are incorporated herein by reference. HGF, or variants diereof, is reported to be useful in the treatment of various odier disorders. WO 97/09997 suggests diat HGF may be useful in treating cystic fibrosis. WO 97/12628 suggests diat HGF may enhance resurfacing of blood vessels damaged or traumatized, for instance, by vascular surgery or angioplasty, and WO 97/12629 suggests diat HGF may be useful in enhancing angiogenesis. The a fragment of HGF may act as an antagonist which specifically inhibits die ability of cancer cells to invade or metastasise by means of HGF (see WO 97/16205), whereas in WO 96/32960 HGF is employed as the active ingredient which exerts die effect of suppressing cytotoxicity in an ischaemic model and, dierefore, is efficacious as a preventive and/or remedy for ischaemic diseases. WO 95/29694 indicates that HGF can accelerate collagen hydrolysis to treat fibrosis and odier diseases caused by a lowered collagenase activity, and EP 0 661 995 suggests diat HGF may be used in the prevention of die establishment of, or progress of, liver damage in patients at risk of liver damage. HGF variants which prevent HGF from binding to MET are proposed for use in a method of preventing tumour cell metastasis (see, for example, EP 0 805 203). In addition, WO 98/00543 describes various HGF receptor agonists, and WO 97/07824 indicates mat the HGF gene may be employed, widi the use of liposomes, in gene therapy. The variant HGF molecules which bind MET and which transduce cellular signals in substantially die same way as wild-type HGF are believed to be dierapeutically useful in me same way as wild-type HGF but since die variant HGF molecules are substantially incapable of binding HSPG or heparin the variant molecules are believed to have a superior effect in vivo since, for example, the variant HGF molecules are believed to give greater tissue penetration, hence me ability to reach cell or tissue comparttnents which exogenous, wild-type HGF cannot reach effectively (or would require a much higher dose. Accordingly, it is believed diat die dierapeutic efficacy of at least some of the variant HGF molecules of the invention is higher man for wild-type HGF and diat a lower dierapeutic dose can be used which may result in lower side effects. Thus, diis class of variant HGF molecules are believed to be useful as a cancer dierapeutic, in treating human liver fibrosis/cirrhosis, as a dierapeutic agent in hepatectomy and liver injury, as an adjuvant for gene merapy of the liver, in therapy against vascular smoodi cell muscle cell proliferation, to enhance repair of intestinal epidielial erosions/ulcerations, as an angiogenesis factor, to block side effects of radio- or chemotherapy, as a remedy for cranial nerve disorder, as a remedy for cartilage disease, to relieve side effects caused by immunosuppressants and as a dierapeutic agent for renal diseases. These variant HGF molecules may also be useful in treating chronic skin wounds. The variant HGF molecules of the invention are believed to be useful in treating cancer in order to prevent or retard spreading and metastasis. A further aspect of die invention provides die use of a variant HGF as defined in the first aspect of the invention in the manufacture of a medicament for treating a patient in need of or who would benefit from treatment with a HGF or an antagonist. A further aspect of the invention provides a variant hepatocyte growdi factor (HGF) which is substantially incapable of binding heparin or HSPG but which is capable of binding to the HGF receptor provided tiiat die variant HGF is not a variant of human HGF in which die replacements (a) R73E, R76E and R93E or (b) R73E and R76E or (c) K91E, R93E and K94E have been made. The preferences for die variant HGF molecules of this aspect of the invention are die same as mose for the first aspect of the invention. A further aspect of the invention provides a polynucleotide encoding a variant hepatocyte growdi factor (HGF) which is substantially incapable of binding heparin or HSPG but which is capable of binding to die HGF receptor provided diat die variant HGF is not a variant of human HGF in which the replacements (a) R73E, R76E and R93E or (b) R73E and R76E or (c) K91E, R93E and K94E have been made. The polynucleotide of this aspect of the invention can be readily made, for example by site-directed mutagenesis using mismatched oligonucleotides or by using the polymerase chain reaction or by de novo polynucleotide syntiiesis or by any other methods. Such memods are well known in die art of molecular biology and at least some of die methods are described in detail in Sambrook et al (1989), Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. A further aspect of die invention provides a vector comprising a polynucleotide of die invention and a still furdier aspect of die invention provides a host cell comprising a polynucleotide or vector of die invention. The polynucleotide or vector may be DNA or RNA; preferably it is DNA. A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to die vector DNA. The vector and DNA segment are then joined by hydrogen bonding between die complementary homopolymeric tails to form recombinant DNA molecules. Syndietic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated wi i bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini widi dieir 3'-5'-exonucleolytic activities, and fdl in recessed 3 '-ends with their polymerizing activities. The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated widi a large molar excess of linker molecules in me presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of die reaction are DNA segments carrying polymeric linker sequences at meir ends. These DNA segments are men cleaved wi i the appropriate restriction enzyme and ligated to an expression vector diat has been cleaved widi an enzyme that produces termini compatible with diose of the DNA segment. Syndietic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA. A desirable way to modify die DNA encoding the polypeptide of the invention is to use me polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491. In this method die DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art. The DNA (or RNA) is then expressed in a suitable host to produce a variant HGF (polypeptide constituting die compound of die invention) of die invention. Thus, the DNA encoding die polypeptide constituting the compound of die invention may be used in accordance widi known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is dien used to transform an appropriate host cell for the expression and production of die polypeptide of die invention. Such techniques include diose disclosed in US Patent Nos. 4,440,859 issued 3 April 1984 to Rutter et al, 4,530,901 issued 23 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to Crowl, 4,677,063 issued 30 June 1987 to Mark et al, 4,678,751 issued 7 July 1987 to Goeddel, 4,704,362 issued 3 November 1987 to Itakura et al, 4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 July 1988 to Toole, Jr. et al, 4,766,075 issued 23 August 1988 to Goeddel et al and 4,810,648 issued 7 March 1989 to Stalker, all of which are incorporated herein by reference. The DNA encoding me polypeptide constituting die compound of die invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon die nature of die host, the manner of the introduction of die DNA into the host, and whedier episomal maintenance or integration is desired. Generally, die DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by die desired host, altiaough such controls are generally available in the expression vector. The vector is dien introduced into die host through standard techniques. Generally, not all of die hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into me expression vector a DNA sequence, widi any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, die gene for such selectable trait can be on anodier vector, which is used to co-transform the desired host cell. Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of die teachings disclosed herein to permit the expression of die polypeptide, which can dien be recovered. Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells. The vectors include a prokaryotic replicon, such as die ColEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in omer, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of die genes in a bacterial host cell, such as E. coli, transformed tiierewidi. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and p-7>r99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA. A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses die SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses me glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of die cloned gene. Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps). The present invention also relates to a host cell transformed widi a polynucleotide vector construct of the present invention. The host cell can be eidier prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bediesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343). Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney- derived COS-1 cells available from the ATCC as CRL 1650. Transformation of appropriate cell hosts with a DNA construct of die present invention is accomplished by well known methods that typically depend on die type of vector used. Widi regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are avadable from Stratagene Cloning Systems, or Life Technologies Inc. , Gaithersburg, MD 20877, USA. Electroporation is also useful for transforming cells and is well known in die art for transforming yeast cell, bacterial cells and vertebrate cells. For example, many bacterial species may be transformed by die methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5X PEB using 6250V per cm at 25μFD. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182. Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce die polypeptide of die invention. Cells can be harvested and lysed and dieir DNA content examined for the presence of the DNA using a method such as mat described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of die protein in the supernatant can be detected using antibodies as described below. In addition to directly assaying for die presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when die recombinant DNA is capable of directing die expression of die protein. For example, cells successfully transformed widi an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for die protein using suitable antibodies. Thus, in addition to the transformed host cells diemselves, me present invention also contemplates a cultore of diose cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. It is preferred if die host cells are insect cells and die expression is a baculovirus expression. It is also preferred if the host cells are Pichia pastoris. It will be appreciated diat, since it is useful to use the variant HGF molecules, or at least polynucleotides encoding diem, in gene dierapy, the invention also includes methods of introducing die said polynucleotides into the cells of the patient. The invention will now be described in detail wi i reference to die following Examples and Figures in which Fig. 1 shows the heparin binding and MDCK scatter activity of wt- and mutant forms of HGF/SF. The following mutants were generated eidier in e hairpin structure (HP) or kringle 2 (Kr2) of HGF/SF: HP1: R73E, R76E, R93E; HP2: R73E, R76E, K78E; HP3: K91D, R93E, K94D; HP4: L80S, F82Q; HP5: K85E; HP6: D90K; Kr2-1: H241S, R242E, K244E, R249G and Kr2-2: R281G, W282G, Y284A). a) Expression ofwt- and mutant forms of HGF/SF. The figure shows an SDS gel under reducing conditions of proteins present in die supernatant of Neuro 2a cells transiently transfected widi HGF/SF cDNAs and labelled widi 35 S-cysteine. The migration of the 60 kDa A chain and die 32/34 kDa B chain is indicated. Control lanes are supernatants of Neuro 2a cells transfected widi vector alone. The mutant HPΔ has the complete hairpin structure deleted (residues 68-100) and dierefore the mobility of the A chain is slightly increased compared to wt-HGF/SF. b) Binding of 20 ng/ml of wt- or mutant HGF/SF proteins to wells coated with 200 μg/ml of heparin. The data are expressed as percentage of wt-HGF/SF. Deletion of die hairpin structure (HPΔ) reduced heparin binding to < 5 % . Similarly, all mutants in which basic amino acid residues were exchanged (HP1 , HP2, HP3) showed strongly reduced heparin binding. Mutations outside die positively charged surface (HP4, HP5, HP6) either had a less pronounced effect (HP4) or no effect (HP5 and HP6). Substitution of positively charged amino acids on die surface of kringle 2 (Kr2-1) reduced heparin-binding to 18% of wt-HGF/SF, whereas deletion of die lysine-binding pocket (Kr2-2) had no effect. c) Scatter of MDCK colonies by wt- and mutant HGF/SFs expressed in Neuro 2a cells. The data are expressed as percentage of wt-HGF/SF. Deletion of die hairpin structure (HPΔ) abolished biological activity but point mutations of positively charged amino acids in die hairpin structure had variable effects on biological activity. Thus die HPl and HP2 mutants had biological activities comparable to wt-HGF/SF whereas the activity of mutant HP3 was only 17% of wt-HGF/SF. The kringle 2 mutants showed eidier a severe loss of biological activity (Kr2-1) or no change (Kr2-2). Fig. 2. shows a BIAcore analysis of wt-NK2 and HP1-NK2 binding to immobilised heparin Panel a shows a sensorgram overlay of wt-NK2 (10-300 nM) binding to a heparin surface (maximum binding capacity 340 RU). Panel b shows a corresponding set of sensorgrams for HP1-NK2 (10-300 nM) (surface capacity ca 170 RU). A lower surface capacity and longer injection time were used for HP1-NK2 to allow for the great differences in association rates. The spikes at the beginning and die end of die sample pulses are artefacts resulting from subtraction of the curves from the blank surface (not shown) and are due to small, irresolvable timing errors. Panel c shows a - tff vs concentration (C, nM) and panel d shows a plot of die observed rate constant k. vs concentration (C, nM) for wt-NK2 (solid squares) and HP1-NK2 (open squares). Fig. 3 shows the total binding of wt-HGF/SF and HPl to mink lung cells. The total binding of wt-HGF/SF and HPl to mink lung cells in a 96 well plate is shown. wt-HGF/SF showed strong concentration-dependent binding to the cell surface of mink lung cells (solid squares) which could be competed by addition of soluble heparin at 0J mg/ml (solid triangles). In contrast, die binding of HPl was negligible at concentrations up to 675 ng/ml (open squares) and was not affected by the addition of heparin (open triangles). Fig. 4 shows die binding of wt-HGF/SF and HPl to a soluble form of the MET-receptor (MET-Fc) and effect of wt-HGF/SF and HPl on DNA synthesis in mink lung cells. a) Binding of wt-HGF/SF (solid bars) and HPl (open bars) to a soluble form of the MET receptor (MET-Fc). Immobilised wt-HGF/SF or HPl were incubated with 200 ng/ml of MET-Fc and bound MET-Fc was detected with an anti-human IgG antibody. The binding of die HPl mutant was only slightly lower man for wt-HGF/SF. b) Effect of wt-HGF/SF (solid squares) and HPl (open squares) on DNA synthesis in mink lung cells. 10,000 serum-starved mink lung cells were incubated widi die indicated concentrations of wt-HGF/SF or HPl. After 24 hours, 3 H-thymidine was added and die amount of radioactivity incorporated into DNA was measured in duplicate samples. The figure shows DNA synmesis induced by wt-HGF/SF or HPl after subtraction of background counts ( 3 H-thymidine incorporated by cultures incubated in control medium). The stimulation induced by HPl was only slightly lower than for wt-HGF/SF. Fig. 5 shows die effect of wt-HGF/SF and HPl on die morphology of mink lung and MDCK cells. The figure compares mink lung cells (panels a, b, and c) and MDCK cells (panels d, e and f) cultured in the absence of factors (a and d) or after overnight incubation widi 20 ng/ml of wt-HGF/SF (b and e) or HPl (c and ). In control cultores (a and d) cells exhibited a typical epithelial morphology. The effects of wt-HGF/SF and HPl on cell dissociation and morphology were undistinguishable. Fig. 6 shows the clearance, tissue distribution and activity in vivo of wt- HGF/SF and HPl. a) Clearance curves of I25 I -labelled wt-HGF/SF and HPl from rat serum. Six adult rats were injected eidier with 125 I -labeled wt-HGF/SF or HPl in me jugular vein and blood samples taken at die times shown. The concentration of wt-HGF/SF (solid squares) and HPl (open squares) remaining in serum (TCA insoluble radioactivity) is shown as a fraction of die total radioactivity injected. For each time point me level of mutant HPl in blood was at least 5 times higher than for wt-HGF/SF. b) Distribution of I-labelled wt-HGF/SF and HPl in rat tissues. Two hours after injection, two animals were sacrificed and, after perfusion, several organs (liver, kidney, lung, heart, testis spleen) were homogenised and die amount of TCA-insoluble radioactivity measured. The level of tissue radioactivity was 2-3 fold higher for HPl than for wt- HGF/SF, although die pattern of organ distribution was die same with die two proteins. c) Stimulation of DNA synthesis in mouse liver by wt-HGF/SF and HPl. wt-HGF/SF and HPl were administered intravenously over a period of 3 days (total dose 277.5 /-ig/animal distributed in 5 injections). At die time in which die animals received the last dose of factor, BrdU was injected intraperitoneally (75 μg/g body weight) and 12 hours later die animals were sacrificed. BrdU-labelled cells were counted on frozen liver sections (at least 8 different sections and a total of 120 fields per animal). Data are the mean +_ sd for 4 (wt- HGF/SF) or 3 (HPl) animals. Figure 7 shows die amino acid sequence of human hepatocyte growth factor from the Swiss Prot database accession no. P 14210. Nucleotide sequences encoding HGF or information relating to such sequences are given in Seki et al (1991) Gene 102, 213-219; Miyazawaka et al (1989) Biochem. Biophys. Res. Comm. 163, 967-973; Seki et al (1990) Biochem. Biophys. Res. Comm. 172, 321-327; Nakamura et al (1989) Nature 342, 440-443; Weidner et al (1991) Proc. Natl. Acad. Sci. USA 88, 7001-7005; Yushiyama et al (1991) Biochem. Biophys. Res. Comm. 175, 660-667; and Lokker et al (1992) EMBO J. 11, 2503-2510 all of which are incorporated herein by reference. Abbreviations: EGF: epidermal growth factor; FGF: fibroblast growth factor; HB-EGF: heparin-binding EGF; HGF/SF: hepatocyte growdi factor/scatter factor; HSPGs: heparan sulphate proteoglycans; MES: 2-[N- morpholino] ethane sulfonic acid; NK2: a truncated variant of HGF/SF corresponding to die N terminal domain, kringle 1 and kringle 2; SPR; surface plasmon resonance; VEGF: vascular endodielial growdi factor. Example 1: Engineered mutants of HGF/SF with reduced binding to heparan sulphate proteoglycans and improved activity in vivo Summary HGF/SF is a high molecular weight, heparin-binding, polypeptide growdi factor diat plays important roles in mammalian development and tissue regeneration. In order to identify the amino acids involved in receptor or heparin binding, we have engineered a number of HGF/SF mutants in which several clusters of solvent-accessible residues in die hairpin structure of die N-terminal domain or in kringle 2 have been replaced. Two of the mutants (HPl : R73E, R76E, R93E and HP2: R73E, R76E, K78E) were of particular interest as diey exhibited greatly decreased affinity for heparin but full mitogenic and motogenic activities on target cells in cultore. The affinity of HPl for heparin was more than 50 fold lower compared to wild type HGF/SF, as established from the binding kinetics of the NK2 fragments of me two proteins. The change in affinity in HPl was the result of a decreased on-rate ( ~ 5 x 10 4 M "1 s ~l vs 1.75 x 10 6 M 1 s 1 ) and was paralleled by decreased binding to cell-surface and matrix heparan sulphate proteoglycans. Important differences were observed in the behaviour of wild type HGF/SF and HPl in vivo: the mutant protein exhibited a delayed clearance from blood, higher tissue levels and a 2.7 fold higher activity in inducing DNA syndiesis in normal, adult murine liver. Thus we have engineered mutant forms of HGF/SF which, by virtue of decreased affinity for heparan sulphate, exhibit greater activity in vivo. These results have implications for understanding die role of heparan sulphate proteoglycans in HGF/SF activity and for dierapeutic applications of die growth factor. In order to dissect die receptor and heparin binding sites in HGF/SF, we generated diree-dimensional models of die individual domains (29) to help design a number of site-specific HGF/SF mutants. The available X-ray structures of antidirombin (30) and FGF-heparin complexes (31) indicate diat the heparin binding sites are composed primarily of clusters of positively charged residues making electrostatic contact widi negatively charged groups in me glycosaminoglycan. We dierefore searched die surface of the hairpin structure and kringle 2 of HGF/SF for clusters of positively charged amino acids, identified diree such clusters (two in the hairpin structore and one in kringle 2) and introduced specific mutations at diese sites. Materials and Methods Cell lines Cell lines were eimer purchased from ATCC or previously described in (7) and (32). Cells were routinely grown in DMEM supplemented widi 10% fetal calf serum at 37°C and 10% C0 2 . In vitro mutagenesis of the hairpin structure and kringle 2 A double stranded oligonucleotide (5'-CA CAG TCA GGA CAT CAT CAT CAT CAT CAT TAA GGA TCC TCT AGA GGT AC -3') coding for six histidine residues and a stop codon was cloned into me Kpn I restriction site of the 2.2 kb Bam HI/Kpn I fragment of the HGF/SF cDNA. In initial experiments, a cDNA encoding wt- HGF/SF fused to a C-terminal heamagglutinin tag was used (26). For die generation of point mutants of die hairpin structore or kringle 2, codon substitutions were introduced into the cDNA by annealing mismatched primers in PCR reactions. The PCR fragments were cloned into the cDNA and mutations confirmed by sequencing. The point mutants generated in this way are listed in me legend to Fig. 1. For deletion of die hairpin structore, an additional Pst I restriction site was created at nt 300 and, by partial digestion, a fragment coding for amino acids 68 to 100 of HGF/SF was deleted. Transient expression of wt and mutant HGF/SF in Neuro 2a cells cDNAs encoding mutant HGF/SFs (10 μg each) were transiently transfected into Neuro 2a cells as described (26). For immunoprecipitation, one day after transfection die Neuro 2a cells were labelled overnight widi 20 μCi/ml of 35 S-cysteine (Amersham) and HGF/SF was immunoprecipitated from the supernatant (1.5 ml) using 2 μg of anti-HGF/SF antibody (clone 34) and 100 ml of protein A-Sepharose 4B beads. The precipitate was washed three times with 50 mM Tris-Cl pH 8.5, 100 mM NaCl, 1 mM EDTA, 1 mg/ml ovalbumin and 5 g/1 Triton X-100 and used for SDS gel electrophoresis and phosphoimager analysis. Expression of full length wt-HGF/SF and HPl in insect cells For large scale production of wt -HGF/SF and HPl die corresponding cDNAs were fused to a C-terminal histidine tag and cloned into die Baculovirus expression vector pVL1393. Recombinant virus was generated by cotransfection of 2 μg of expression plasmid and 0.5 μg Baculogold- DNA (Pharmingen, La Jolla, USA), plaque purified, propagated and used to infect 5 litre batches of SF9 cells. wt-HGF/SF and HPl were purified from the supernatant after extensive dialysis against PBS and affinity chromatography on 5 ml Ni 2+ -chelate columns (Qiagen, Germany) using a linear gradient of imidazole (0-0.4 M in 0.1 M NaCl, 50 mM MES pH 6.0) followed by cation exchange chromatography (8). The purity of the preparations was typically 80-90% from Coomassie stained SDS-gels. Expression of wt-NK2 and HP1-NK2 in P. pastoris cDNAs encoding wt-NK2 or HP1-NK2 were generated by PCR amplification of a human HGF/SF cDNA and cloned into the P. pastoris expression vector pPIC 9K (Invitrogen Inc.) creating an in frame fusion with the α-mating signal peptide of S. cerevisiae. Linearised, recombinant pPIC 9K plasmids encoding wt-NK2 and HP1-NK2 plasmids were used for transformation of die GS115, his ' strain of P. pastoris by spheroplasting. Transformed clones were initially selected on his ' plates and subsequently on G418 plates for multicopy integration (33). Selected clones were used for large-scale expression of die recombinant proteins in shake flasks. The cultore supernatant was harvested 72 hours after induction and the supernatant was clarified by centrifugation followed by filtration. wt-NK2 was initially purified on heparin-Sepharose4B (Pharmacia) followed by ion-exchange chromatography on S-Sepharose (Pharmacia) using a gradient of NaCl (0.25-1.00 M) in 0.05 M MES, pH 6.0. HP1-NK2 was purified directly by ion-exchange chromatography on S-Sepharose. Monomer and dimer species of wt-NK2 and HPl NK2 were separated by size-exclusion chromatography on a Superdex75 column (Pharmacia) in 0.05 MES, 0.25 M NaCl, buffer, pH 6.0. The yield of biologically active protein was between 5-10 mg/litre. Heparin and MET ELISAs For heparin ELISA, 96 well plates (Nunc, Multisorp) were incubated widi 200 μl/well of high molecular weight heparin (200 μg/ml, Sigma H3393). After blocking, purified HGF/SF and supernatants of Neuro 2a cells expressing wt- and mutant HGF/SF proteins were incubated for 2 hours and detected widi eitiier the rabbit anti tag antibody HA 11 (Hiss diagnostics) or widi an anti-HGF/SF mouse monoclonal antibody (clone 34) followed by incubation widi HRP-conjugated second antibody. For MET ELISA, a soluble fusion protein consisting of die extracellular domain(s) of MET and die hinge, CH2 and CH3 domain of immunoglobulin γl was produced (GH, unpublished results) and purified by protein A-Sepharose affinity chromatography to ~ 80% homogeneity. Wells were coated witii full lenguJ wt-HGF/SF or HPl overnight at 4°C, blocked, and die MET-Ig fusion protein was added (200 ng/ml) for 2 hours followed by incubation with goat anti-human IgG antibody and HRP-conjugated rabbit anti-goat IgG antibody. For colour development 0.66 mg/ml ortho-phenyl diamine in 50 mM phosphate buffer pH 6.0 was used as HRP substrate (100 μl/well). Heparin binding kinetics of full length wt-HGF/SF and HPl and their NK2 fragments Kinetic analysis of die binding of wild-type and mutant proteins was carried out by surface plasmon resonance (SPR) using a BIAcore 2000 instrument (BIAcore Ltd., St. Albans, Herts., UK). 10 mg/ml high molecular weight heparin (Sigma H3393) was biotinylated widi a diree fold excess of NHS-LC-biotin (Pierce 21336) in 1 ml of 100 mM phosphate buffer pH 8.0 for 1 hour at RT. Biotinylated heparin was separated from free biotin derivatives by sequential passage through two 2ml Excellulose columns (Pierce) and used for capture on die surface of streptavidin-coated SA sensor chips. All experiments were carried out at an operating temperature of 25 °C. The running buffer (EB) in all runs was PBS containing 0.2mM EDTA, 0.5 g/1 NaN 3 0.05 g/1 p20 detergent (BIAcore Ltd). In order to prevent possible leakage of heparin from the test surfaces (flowcells 2-4) to die control one (flowcell 1), surface 1 was blocked with two 20 μl injections of 250 ng/ml biotin in EB at a flow-rate of 5 μl/min. Surface 2 was then coated at 5 μl/min with 10 ml of 2 nM heparin-biotin in EB. Approximately 25 RU were bound in this example; since a small increase in signal may occur due to crosslinking of the dextran by die long heparin molecules, an exact estimate of the heparin bound at low levels is not possible. The surface was tiien blocked widi biotin as for surface 1. To prevent absorption or denatoration of die protein samples for SPR analysis, these were routinely diluted in 0.2 mg/ml BSA in 0.2 mM EDTA which had been pre-absorbed onto Heparin-Sepharose4B (Pharmacia Biotech, St. Albans) for 30 min at room temperature. For analysis of me various proteins, samples were pumped at 15 μl/min. Regeneration of me surface was by sequential "QUICKINJECT/EXTRACLEAN" of 10 μl of 0.1 g/1 SDS in water followed by 2 x 10 μl of 1M NaCl. Finally 15 ml of 0.2 mg/ml BSA in EB were injected to block any non-specific binding sites. Data files from the automated analysis runs were analysed by non-linear curve fitting (34) using die BIAcore BIAevaluation software (BIAcore Ltd). Protein concentrations were measured by die BCA reagent (Pierce) and the biological activity of each batch of protein confirmed in die MDCK assay (7) to ensure consistent specific activity. Cell biological assays Colony scatter assays of MDCK and mink lung cells were carried out as previously described (7). For measurement of DNA synmesis, mink lung cells were plated at a density of 5,000-10,000 cells/well in 24 well plate and serum-starved by incubation in DMEM for 48 hours. Different concentrations of HGF/SF and HPl were dien added and incubation continued for 24 hours in DMEM containing 100 μg/ml BSA before addition of 3 H-thymidine (1 μCi/well) and a final incubation for 16 hours. Radioactivity insoluble in 5 % TC A was measured by scintillation counting after solubilization in 0.2 M NaOH. Total binding of wt-HGF/SF and HPl to mink lung cells Mink lung cells were grown to confluence in 24 well plates. After two washes with medium, cells were incubated at 4°C for 30 min. Serial dilutions of HGF/SF and HPl in PBS were men added to me cells and incubated for anotiier 15 min at 4°C. Unbound factor was removed by diree washes with PBS and cells fixed widi 4% paraformaldehyde in PBS. After two further washes the cells were incubated with a mouse anti HGF/SF monoclonal antibody (clone 34) which reacted in an identical manner widi wt-HGF/SF and HPl followed by incubation wim FITC conjugated anti-mouse IgG. Clearance and tissue distribution of wt-HGF/SF and HPl in rats 10 μg of pure HGF/SF and HPl were iodinated by a solid phase method using Iodogen (Pierce) by Biotez, Berlin, Germany. After me reaction, 1 mg/ml BSA was added and free Iodine was removed by multiple filtration on a Centrisat filtration apparatus (Sartorius) widi a 30 kDa cut off. The specific activities were calculated to be 1.85 MBq/mg for HGF/SF and 1.95 MBq/mg for HPl . The biological activities of the proteins before and after iodination was compared in a motility assay using MDCK cells and determined to be 80% after iodination. Adult rats (3 per groups) were anaesthetised, a cadieter implanted into the jugular vein and a single iv bolus injection of 0.2 ml of 125 I-HGF/SF was given (0.45 MBq for wt- HGF/SF and 0.38 MBq for HPl per animal). After 1, 3, 5, 7, 10 and 60 mins, 2 hours and 20 hours, 200 μl samples of blood were taken via the catheter. Blood was allowed to clot and centrifuged at 10,000 g for 5 min at 4°C. For each sample 100 μl were diluted widi an equal volume of PBS and 2 ml of ice-cold 10% TCA were added. After 30 min on ice, samples were centrifuged and die precipitate washed widi 5% TCA before counting. For measurement of tissue radioactivity, animals were sacrificed 2 hours after injection of 125 I-labelled wt-HGF/SF or HPl and perfused widi PBS. Organs were taken out, weighted, homogenised and the amount of TCA precipitatable radioactivity determined. Effect of wt-HGF/SF and HPl on DNA synthesis in adult liver The ability of wild-type HGF/SF and HPl to induce DNA syntiiesis in liver cells was measured following intravenous administration of recombinant proteins and cell labelling with BrdU. Briefly, female animals (6-8 weeks old; 19.5 to 26.9 g of body weight) were injected twice daily widi either PBS, wild-type HGF/SF or HPl in toe tail vein (55.5 μg/dose) for diree days. At die time of the last injection of factor, each animal received BrdU (75 μg/g body weight) was injected intraperitoneally and 12 hours later die animals were sacrificed, die liver was dissected and frozen sections were prepared. Sections were fixed in 70% edianol (5 mins), treated widi 2.4 M HC1 for 10 mins at 37 °C and nuclei diat had incorporated BrdU were stained widi anti-BrdU antibody (Sigma, 1 :400) followed by peroxidase-conjugated anti-mouse IgG (Jackson Laboratories, 1:500). Results Hairpin structure and kringle 2 mutants of HGF/SF The N-terminal domain of HGF/SF contains four cysteine residues mat are conserved in die HGF/SF homologues HGFl/MSP and plasminogen. Studies on plasminogen (35) indicated diat ti-iis domain forms an hairpin structore stabilised by two disulphide bonds. A diree dimensional model of the hairpin structore of HGF/SF suggested die presence of several positively charged residues which were absent in die corresponding models of HGFl/MSP and plasminogen (29). Six different point mutants of die hairpin structore of HGF/SF were constructed (HPl to HP6) in which a total of 10 amino acid residues were substituted. The strategy involved mutation of positive to negative, negative to positive and hydrophobic to polar (see legend to Fig. 1 for the sequence of die mutants). The activity of these point mutants was compared to diose of wild type (wt-) HGF/SF and HPΔ, a mutant in which die whole hairpin structore (amino acids 68 to 100) was deleted. In addition, two multiple mutants of kringle 2 were constructed: in mutant Kr2-1 a cluster of four positive residues on die surface of the kringle (H241, R242, K244 and R249) was mutated; in mutant Kr2-2 three residues were mutated (R281, W282 and Y284) that had been predicted to form a hydrophobic pocket on the surface of kringle 2 similar to the one present in plasminogen kringle 4 (29). For initial studies, mutants were expressed by transient transfection in die Neuro2A cell line (Fig. la) and characterized for heparin binding (Fig. lb) and biological activity (Fig. lc). As expected HPD, die mutant lacking me hairpin structore, exhibited neidier heparin binding nor biological activity. The point mutants fell in diree different groups: group 1 (HPl, HP2 and HP4) exhibited normal signalling activity but much decreased (HPl and HP2) or decreased (HP4) heparin-binding; group 2 (HP3) showed a severe loss in bodi signalling and heparin-binding and group 3 (HP5 and HP6) behaved essentially like wild-type HGF/SF (Fig. lb and c). The first kringle 2 mutant, Kr2-1 showed severely reduced heparin- binding and biological activity whereas the second one, Kr2-2, was indistinguishable from wt-HGF/SF (Fig. lb and c). From mese results we can conclude that a first cluster of positively charged residues in the hairpin structore (R73, R76) is involved in heparin but not in MET binding and diat residues R281, W282 and Y284 are involved in neimer. It also appears that two extra clusters of positively charged residues, one in the hairpin structore (K91 and K94) and one in kringle 2 (H241 , R242, K24 and R 249) may participate bom in MET and heparin binding. Binding kinetics of wt-HGF/SF, HPl and their NK2 fragments to heparin Full-length wt-HGF/SF and HPl (one of die two mutants showing greatly reduced heparin-binding but normal biological activity) were expressed in insect cells in order to analyse dieir kinetics of binding to heparin by surface plasmon resonance (SPR). These experiments revealed important differences in the binding kinetics of HPl . The binding to heparin of wt-HGF/SF (4 to 30 nM) revealed a single first- order dissociation rate constant (k off ) of ~ 1 x 10 ~3 s 1 . The bulk of die binding during association could be fitted to a single-site model widi an association rate constant (k on ) of — 1 x 10 6 M "1 s 1 , giving a molar dissociation constant (K j ) of ~ 1 x 10 "9 M. HPl (4 to 80 nM) showed much reduced heparin binding (data not shown). However we considered tiiat die difference observed between wt-HGF/SF and HPl might have been affected by die presence of dimeric or oligomeric forms of the two proteins, a point that could not be established due to low recovery from size-exclusion columns. We therefore expressed die NK2 fragments of wt- HGF/SF and HPl (ie all the domains shown to be involved in heparin binding, Mizuno et al., 1994) in the methylotropic yeast P. pastoris and used diese for furdier kinetic analysis. wt-NK2 and HP1-NK2 purified from the supernatant of P. pastoris cultures consisted of a mixture of NK2 monomers and dimers diat could be readily separated on Superdex-75 columns, allowing die binding kinetics to heparin to be established widi bonafide monomeric species. Sensorgrams overlays of the binding to heparin of wt-NK2 (Fig. 2a) and HP1-NK2 (Fig. 2b) confirmed major differences in heparin binding. For wt-NK2, the dissociation curves were slower at die higher concentrations and apparent k off values varied between 1 and 4 x 10 "3 s ' (Fig. 2c, solid squares). In contrast, die dissociation of HP1-NK2 (Fig. 2b) rose with concentration to an apparent k^ value of 6-7 x 10 "3 s 1 (Fig. 2c, open squares). Analysis of the association phase of wt-NK2 (Fig. 2a) showed complex kinetics. Assuming a single class of binding sites and taking data from the earliest (fastest) phase, plots of the observed association rate constant ("K s ") vs C for concentrations up to 300 nM gave a straight line (r 2 = 0.997) from which we calculated a k ^ of 1.75 x 10 6 M 1 s 1 (Fig. 2d, full squares). In the later stages of binding a second phase witii much slower association was discernible but attempts to fit models based on two classes of binding sites were inadequate. Taking die fastest value for k^ of 4 x 10 "3 s 1 and a value for k on of 1.75 x 10 6 M "1 s 1 , a Kj value of 2.3 nM was calculated for wt-NK2. For HP1-NK2, analysis of die early phase of binding (assuming me presence of a single class of binding sites) gave a - ^ value of ~ 5 x 10 4 M "1 s 1 from the K- s v-s C plot (Fig. 2d, open squares). This figure was ~ 35 fold lower than for wt-NK2. Taking a value of 6 x 10 ~3 s 1 for k^ (Fig. 2c, open squares), a K j of 120 nM was calculated for HP1-NK2. The binding affinity of HP1-NK2 is tiierefore over 50 fold lower than for wt- NK2. Binding of wt-HGF/SF and HPl to HSPGs and MET and activity of the two proteins on target cells in vitro The binding of wt-HGF/SF and HPl to cell surface and matrix heparan sulphates was investigated in several cell types, including die carcinoma lines HepG2 and Hep3B and mink lung cells. The latter have recently been shown to bind large amounts of HGF/SF and its truncated forms NK1 and NK2, via HSPGs (36). By immunofluorescence, the total binding of HPl to HepG2, Hep3B and mink lung cells was much lower man for wt-HGF/SF (data not shown). These results were confirmed and extended by a quantitative solid phase assay and a representative experiment, illustrating d e binding of full lengdi wt-HGF/SF and HPl to mink lung cells, is shown in Fig. 3. The total binding of wt-HGF/SF to mink lung cells increased steadily from 10 to 675 ng/ml whereas binding of HPl was hardly measurable over die same range of concentrations. Exogenous heparin (0.1 mg/ml) competed effectively for the binding of wt-HGF/SF to die cells and reduced it to levels only slightly higher dian diose of HPl. This confirms diat die bulk of cell-bound wt-HGF/SF was associated with HSPGs and diat HPl failed to bind to such molecules. In marked contrast widi die reduced binding of HPl to heparin and cell- associated HSPGs, me binding of HPl to a soluble form of the MET receptor and its ability to induce stimulation of DNA syntiiesis in mink lung cells were only marginally lower man for wt-HGF/SF (Fig. 4a and 4b respectively). Similar results were obtained widi the mouse keratinocyte line MK (data not shown). Equally, die ability of HPl to induce dissociation and scattering of mink lung cells and MDCK cells were indistinguishable from wt-HGF/SF (compare panels b vs c and e vs f in Fig. 5). Thus, in spite of a considerable decrease in binding affinity for heparin and HSPGs, die ability of the HPl mutant to induce DNA synthesis and cell movement on target cells was indistinguishable from wt-HGF/SF. In vivo activity of wt-HGF/SF and HPl We investigated die effect of the HPl mutation on the behaviour of the factor in vivo. For this, wt-HGF/SF and HPl were labelled widi 125 -I and injected in die jugular vein of adult rats. The concentration of title two proteins in serum and in several organs was men measured in die form of TCA-insoluble radioactivity. Fig. 6a shows die clearance curve for wt- HGF/SF and HPl from serum. In agreement with previous studies (23, 37, 38), we observed a rapid clearance of wt-HGF/SF from the circulation ( > 98% over the first 15 minutes). The clearance of HPl was much slower and, at every time point tested, the concentration of HPl in plasma was 5-10 fold higher tiian wt-HGF/SF up to 20 hours after injection (Fig. 6a). The slower clearance of HPl from serum was associated widi an increased amount of die mutant factor in several organs including liver, kidney, lung, heart, testis and spleen (Fig. 6b). The relative distribution of die HPl mutant and wt-HGF/SF among different organs was similar but the concentration of HPl was 2-3 fold higher dian wt-HGF/SF (Fig. 6b). The ability of wt-HGF/SF to induce DNA syndiesis in adult liver was investigated next after, intravenous injection of me two proteins. As expected, in die absence of factors DNA syndiesis was very low but it increased considerably after administration of either HGF/SF or HPl (Fig. 6c). However, stimulation by HPl was 2.7 fold higher dian for wt- HGF/SF (Fig. 6c) in line widi die increased level of die factor in serum and liver (Fig. 6a and 6b). Discussion The heparin and receptor binding sites of HGF/SF Previous studies had shown that the hairpin structore of the N terminal domain of HGF/SF is important for both heparin (24) and receptor binding (25-28). In this study we show diat die heparin and receptor binding sites of HGF/SF can be separated by protein engineering and diat mutants forms of HGF/SF with much reduced heparin binding have superior biological activity in vivo. Given tiiat both die HPl and HP2 mutants had much decreased affinity for heparin but biological activities comparable to wt-HGF/SF on target cells in cultore, we suggest diat the mutations in common (R73E and R76E) are important for heparin binding, a conclusion supported by preliminary characterization of the R73E, R76E double mutant. In contrast, substitution of a second cluster of positively charged residues in die hairpin structore (K91, R93 and K94) or of a separate cluster of positively charged residues in kringle 2 (H241, R242, K244 and R 249), abolished both heparin- binding and HGF/SF signalling in target cells. We note diat alanine scanning mutagenesis of die N-terminal domain of HGF/SF (39) failed to identify residues in die hairpin structore essential for MET binding and signalling. This may be due to die fact diat multiple substitutions of neighbour residues in die hairpin structore (such as die ones diat we have introduced) are required to produce measurable changes. The degree of heparin-binding activity was not reported by Lokker et al (39). Binding kinetics of wt-HGF/SF and its NK2 fragment to heparin Our data indicate that full lengdi wt-HGF/SF binds heparin widi high affinity (K j = ~ 1 nM). This figure compares widi a value of 0.6 nM from a separate study in which binding of full lengώ wt-HGF/SF to 35 S -heparin was measured following separation of bound and free 35 S-heparin by ultrafiltration (23). In order to ensure that the measured kinetics resulted from the interaction of monomeric protein species widi heparin (and dius establish accurate kinetics for die HPl mutant) we relied on monomeric NK2 fragments expressed in P. pastoris. The dissociation kinetic of wt-NK2 revealed a significant effect on apparent k^ values at die highest concentrations of protein suggesting cooperative binding of NK2 to heparin (data not shown). Analysis of die stoichiometry of binding at or near saturation indicates approximately 4 wt-NK2 molecules per 16 kDa heparin. Less certain estimates for wt-HGF/SF on low-heparin surfaces provided a value of 3.5 molecules of HGF/SF per heparin molecule. These values suggest iat die heparin-binding site of NK2 and HGF/SF encompasses — 14 monosaccharide units, which is consistent widi die findings of Lyon et al. (40) who reported diat heparan sulphate fragments of 12-14 units were required for high-affinity binding to HGF/SF. The molar dissociation constant of full lengώ HGF/SF for heparin (I = InM) compares with a value of 50 nM obtained for die interaction of acidic FGF and heparin under similar experimental conditions (41). The on rates of HGF/SF and FGF are similar (1.75 vs 1.0 x 10 6 M "1 s 1 respectively) and die difference in the affinity is attributable to die off-rate (1 x 10 "3 s "1 vs 6 x 10 "2 s 1 for HGF/SF and FGF). The decreased affinity for heparin of HPl has been achieved by reducing me on-rate (Fig. 2d), a result in line widi die emerging role of electrostatic interactions on association rates (42). Different roles for heparan sulphates in the HGF/SF and FGF systems The HPl mutant has provided a number of insights on die role of heparin- binding in HGF/SF activity. HGF/SF is one of a number of growdi factors that bind HSPGs but toe role of heparan sulphate in growdi factor activity is generally not clear except in die case of FGF. Widi FGF ώere is evidence diat diree components (ligand, receptor and heparan sulphate) are required for signalling. This conclusion is supported by transfection experiments (2,3) and biochemical studies widi purified components (43). HSPGs probably act by dimerising FGF dius promoting receptor dimerization and signalling (44), an interpretation consistent wim the recent structures of FGF-heparin complexes (31). Our results indicate a different role for HSPGs in die HGF/SF system. Although anomer stody suggests diat heparin enhances die activity of HGF/SF and the NK1 and NK2 fragments on cells in cultore (36), we find diat die receptor binding and biological activity of the HPl mutant are essentially indistinguishable from wt-HGF/SF in spite of a > 50 fold loss in heparin binding. Our results do, however, raise die question of die physiological function of HGF/SF binding to HSPGs. The in vivo data presented here suggest a role for HSPGs in promoting the internalisation and degradation of HGF/SF in tissues (Fig. 6a and 6b). Thus HSPGs may represent a means of clearing HGF/SF in vivo. Indeed there is a striking parallel between me increased DNA syndiesis activity of HPl in liver (Fig. 6c) and die increased level of factor in die tissue (Fig. 6b). It is possible to speculate on other possible roles for HSPGs. For example, HSPGs may help localize HGF/SF to selected cell or tissue comparto ents, as required for die establishment of a 'morphogen gradient' during embryogenesis (45,46). An example of this process may be the developing limb, where migration of myogenic precursor cells in the bud is known to depend critically on HGF/SF and MET (47). Applications of heparin-deficient HGF/SF mutants in vivo There has been considerable interest recently in potential dierapeutic applications of HGF/SF and several studies have addressed die question of die pharmacokinetics of the growdi factor in vivo. The results of these studies indicated HGF/SF is cleared very rapidly from the circulation (23, 37, 38) and diat die half life of the growth factor in plasma could be extended by complexing it widi eiώer heparin (23) or other polyanions, such as dextran sulphates (48). HGF/SF infused as a complex with dextran sulphate also had significantly higher activity in inducing hepatocyte proliferation in adult animals (48). Our work demonstrates mat equal or superior results can be achieved widi engineered forms of HGF/SF, such as the HPl mutant. The mutant has a longer residence time in die circulation (Fig. 6a), greater tissue levels (Fig. 6b) and is more active than wt-HGF/SF in inducing DNA syndiesis in adult liver (Fig. 6c). It ώerefore overcomes the need to complex HGF/SF with heparin-like compounds in order to improve pharmacokinetics and activity. Although it is well established that polypeptide growdi factors exert their effects on target cells via specific membrane receptors (1), it has become clear in recent years ώat a number of these proteins also bind cell surface and matrix proteoglycans, especially of toe heparan sulphate type (HSPGs). The biological significance of mis dual interaction is well understood in the case of fibroblast growth factor (FGF). Cells expressing the FGF receptor but lacking membrane-bound heparan sulphate fail to respond to basic-FGF (2,3) implying heparan sulphate as an essential co- receptor for FGF signalling. Odier growdi factors, such as toe epidermal growdi factor (EGF) - related polypeptides amphiregulin (4) and heparin binding - EGF (HB-EGF) (5) as well as several splice variants of vascular endodielial growth factor (VEGF) (6) also require cell-bound heparan sulphate for activity although toe role of HSPGs in toese systems is less defined. Hepatocyte growth factor/scatter factor (HGF/SF) (7-12) is a high- molecular weight polypeptide growdi factor distinct from the otoers and widi a domain structore related to diat of die blood proteinase precursor plasminogen. Like plasminogen, HGF/SF is produced as an inactive, single chain polypeptide which is converted proteolytically into a two- chain species (13-15). Two-chain HGF/SF induces growdi, movement and differentiation in target cells via a specific membrane tyrosine kinase receptor encoded by die c-MET protooncogene (16,17). HGF/SF binds tightly to d e MET receptor widi a Kj in the sub nanomolar range (18,19). HGF/SF also binds heparin (20,21). This does not appear to affect the binding affinity for a soluble form of the MET receptor although it does increase receptor phosphorylation and mitogenicity on target cells (22). HGF/SF-heparin complexes also exhibit sustained plasma levels and reduced hepatic clearance in vivo (23). The modular structore of HGF/SF has facilitated die identification of die domains responsible for receptor and heparin-binding. Deletion of toe hairpin structore of the N-terminal domain (a domain homologous to plasminogen' s activation peptide) or kringle 2 results in mutants with reduced apparent affinities for heparin (24). By contrast, deletions of eimer kringle 1, 3, 4 or the proteinase domain, have little or no effect (24). Thus the heparin binding site(s) of HGF/SF appear to be contain in the hairpin structore of the N terminal domain and kringle 2. The same domains, however, are critical for MET binding and signalling (25-28). Example 2: Treatment with variant HGF Patients with acute liver disease eitiher due to viral infection or chemical/drug injury are treated wito die variant HGF by daily iv injections in saline at toe dose of 1-100 μg/kg toroughout the acute phase of the disease. The development of toe disease is monitored by measuring the plasma levels fetoprotein, γ-globulin, SGOT, SGPT and γ-GT. References 1. Ullrich, A. and Schlessinger, J. (1990) Cell 61: 203-212. 2. 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Claims: CLAIMS 1. A variant hepatocyte growth factor (HGF) which is substantially incapable of binding a heparan sulphate proteoglycan but which is capable of binding to die HGF receptor for use in medicine. 2. A variant hepatocyte growth factor (HGF) according to Claim 1 wherein a positively-charged amino acid residue in the hairpin loop structore of wild-type HGF has been replaced widi an amino acid residue with no charge or a negative charge for use in medicine. 3. A variant human hepatocyte growth factor (HGF) according to Claim 1 or 2 wherein at least amino acid residue R73 has been replaced by an amino acid residue wim no charge or widi a negative charge for use in medicine. 4. A variant human hepatocyte growth factor (HGF) according to Claim 1 or 2 wherein at least amino acid residue R76 has been replaced by an amino acid residue widi no charge or with a negative charge for use in medicine. 5. A variant human hepatocyte growth factor (HGF) according to any one of the preceding claims wherein both amino acid residues R73 and R76 have been replaced independently wiώ an amino acid residue with no charge or witii a negative charge for use in medicine. 6. A variant human hepatocyte growth factor (HGF) comprising amino acid residue replacements R73E and R76E for use in medicine. 7. A variant human hepatocyte growth factor (HGF) comprising amino acid residue replacements R73E, R76E and R93E for use in medicine. 8. A variant human hepatocyte growth factor (HGF) comprising amino acid residue replacements R73E, R76E and K78E for use in medicine. 9. A variant human hepatocyte growth factor (HGF) consisting of human HGF with amino acid replacements R73E and R76E for use in medicine. 10. A variant human hepatocyte growth factor (HGF) consisting of human HGF with amino acid replacements R73E, R76E and R93E for use in medicine. 11. A variant human hepatocyte growth factor (HGF) consisting of human HGF with amino acid replacements R73E, R76E and K78E for use in medicine. 12. A variant hepatocyte growth factor (HGF) according to Claim 1 which antagonises toe action of wild-type HGF for use in medicine. 13. A variant hepatocyte growth factor (HGF) according to Claim 12 wherein toe variant HGF further comprises a mutation which confers resistance in the variant HGF to proteolytic cleavage by enzymes capable of in vivo conversion of HGF into its two-chain form for use in medicine. 14. A variant human hepatocyte growth factor (HGF) according to Claim 13 which have an amino acid alteration at or adjacent to any of amino acids 493, 494, 495 and 496 of toe wild-type human HGF. 15. A pharmaceutical composition comprising a variant hepatocyte growth factor (HGF) as defined in any of the preceding claims and a pharmaceutically acceptable carrier. 16. A method of treating a patient in need of treatment with a hepatocyte growth factor or an antagonist thereof the meώod comprising administering to the patient an effective amount of a variant HGF as defined in any one of Claims 1 to 14. 17. A method according to Claim 16 wherein the patient has cancer. 18. Use of a variant hepatocyte growdi factor (HGF) as defined in any one of Claims 1 to 10 in toe manufacture of a medicament for treating a patient in need of treatment with a HGF or an antagonist thereof. 19. Use as defined in Claim 18 wherein the patient has cancer. 20. A variant hepatocyte growth factor (HGF) which is substantially incapable of binding heparan sulphate proteoglycan but which is capable of binding to toe HGF receptor provided that toe variant HGF is not a variant of human HGF in which the replacements (a) R73E, R76E and R93E or (b) R73E and R76E or (c) K91E, R93E and K94E have been made. 21. A variant hepatocyte growth factor (HGF) according to Claim 20 wherein a positively-charged amino acid residue in the hairpin loop structore of wild-type HGF has been replaced wiώ an amino acid residue with no charge or with a negative charge. 22. A variant human hepatocyte growth factor (HGF) according to Claim 20 or 21 wherein at least amino acid residue R73 has been replaced by an amino acid residue with no charge or wito a negative charge. 23. A variant human hepatocyte growdi factor (HGF) according to Claim 20 or 21 wherein at least amino acid residue R76 has been replaced by an amino acid residue with no charge or with a negative charge. 24. A variant human hepatocyte growdi factor (HGF) according to any one of Claims 20 to 23 wherein bom amino acid residues R73 and R76 have been replaced independently with an amino acid residue with no charge or with a negative charge. 25. A variant human hepatocyte growth factor (HGF) comprising amino acid residue replacements R73E and R76E. 26. A variant human hepatocyte growth factor (HGF) comprising amino acid residue replacements R73E, R76E and R93E. 27. A variant human hepatocyte growth factor (HGF) comprising amino acid residue replacements R73E, R76E and K78E. 28. A variant human hepatocyte growth factor (HGF) consisting of human HGF with amino acid replacements R73E and R76E. 29. A variant human hepatocyte growto factor (HGF) consisting of human HGF wito amino acid replacements R73E, R76E and R93E. 30. A variant hepatocyte growto factor (HGF) according to Claim 20 which antagonises die action of wild-type HGF. 31. A variant hepatocyte growto factor (HGF) according to Claim 30 wherein the variant HGF further comprises a mutation which confers resistance in toe variant HGF to proteolytic cleavage by enzymes capable of in vivo conversion of HGF into its two chain form. 32. A variant human hepatocyte growth factor (HGF) according to Claim 31 which have an amino acid alteration at or adjacent to any of amino acids 493, 494, 495 and 496 of toe wild-type human HGF. 33. A polynucleotide encoding a variant hepatocyte growto factor according to any one of Claims 20 to 30. 34. A vector comprising a polynucleotide according to Claim 33. 35. A host cell comprising a polynucleotide or vector according to Claim 33 or 34. 36. A method of producing a variant hepatocyte growth factor (HGF) the method comprising cultoring a cell as defined in Claim 35 and isolating the variant HGF therefrom. 37. Any novel variant hepatocyte growto factor (HGF) as herein disclosed.

Inventors: BIRCHMEIER WALTER, GHERARDI ERMANNO, HARTMANN GUIDO, BIRCHMEIER, WALTER, GHERARDI, ERMANNO, HARTMANN, GUIDO
Applicants: BIRCHMEIER WALTER, GHERARDI ERMANNO, HARTMANN GUIDO, IMP CANCER RES TECH, BIRCHMEIER, WALTER, GHERARDI, ERMANNO, HARTMANN, GUIDO, IMPERIAL CANCER RESEARCH TECHNOLOGY LIMITED
Application year: 1998
Main classification: C12N15
IPC codes: A61K38, A61K45, A61K48, A61P1, A61P35, C07K14, C12N1, C12N5, C12N15, C12P21