PAI-2 CONJUGATES FOR THE TREATMENT AND IMAGING OF CANCER

Abstract: A method of detecting, visualising or treating cells which express a uPA/uPAR complex, in particular cancerous cells, with a PAI-2 conjugate molecule is described. The PAI-2 conjugate molecule comprises PAI-2 or a functional derivative, homologue, analogue, chemical equipment or mimetic thereof, which PAI-2 is bound, linked or otherwise associated with a toxin or label.

Description: PAI-2 CONJUGATES FOR THE TREATMENT AND IMAGING OF CANCER TECHNICAL FIELD The present invention relates to a method of treating a condition in a mammal, which condition is characterised by the undesirable, detrimental or otherwise unwanted growth of uPAR expressing cells and agents useful for same. More particularly, the present invention contemplates a method of treating said condition by specifically targeting the subject cells utilising plasminogen activator inhibitor or functional derivative, equivalent, homologue, analogue or mimetic thereof coupled to a toxin such as an alpha particle emitting radioisotope. The method of the present invention is useful, inter alia, in the targeted treatment of conditions such as neoplasms and, in particular, metastatic cancers. The invention also relates to toxin labelled plasminogen activator inhibitor molecules, pharmaceutical compositions comprising same and methods for their manufacture . The invention further relates to the detection or visualisation of tumors with labelled plasminogen activator inhibitor molecules. BACKGROUND ART Note: Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. The acquisition of the malignant phenotype involves a number of initiation and progression factors linked together in a multistep process (Meyer and Hart 1998: Haber and Fearon, 1998). Malignant tumours are potentially lethal because of the ability of cells within the tumour to invade and spread (metastasise) throughout the body (Meyer and Hart 1998). The process of metastasis can be summarised in the following steps: escape of tumour cells from a primary tumour mass and invasion into surrounding histologically normal tissue, intravasation and extravasation (entry into and exit from the vasculature or lymphatic system), and growth and survival (via angiogenesis) of tumour cells at a secondary site. One of the major failures in the treatment of cancer is poor detection and eradication of metastases before vital organ functions are compromised, resulting in minimal long-term survival benefit (Allen, 1999). Thus, effective cancer treatment entails primary tumour resection followed by removal of all metastases arising from the primary. The latter would require (1) identification of a marker specific to metastatic cells, and (2) development of a ligand that can be used to specifically target and kill the cells expressing the marker. Proteolytic enzymes such as urokinase plasminogen activator (herein referred to as "uPA") play a role in tumour angiogenesis and metastatic cell migration; both of which are processes that require tissue barriers to be breached (reviewed in Andreasen et al., 1997). Under normal physiological conditions, most cells express very little or no uPA (Pollanen et al , 1991). Urokinase plasminogen activator converts zymogen plasminogen into the highly active protease plasmin, which has broad specificity towards integral extracellular matrix (ECM) molecules (eg: type IV collagen, vitronectin, proteoglycan, fibronectin and laminin) (Pollanen et al, 1991). Plasmin also contributes to ECM remodelling by activating zymogen metalloproteases (MMPs) which more thoroughly degrade the collagen structural components (Pollanen et al. , 1991). While plasminogen can also be converted to plasmin by tissue plasminogen activator (tPA), tPA is primarily responsible for fibronolysis (Lijnen and Collen, 1982; Pollanen et al., 1991). In contrast uPA is primarily involved in pericellular proteolysis as it binds to its specific cell-surface receptor uPAR (Pollanen et al, 1991). The activities of uPA and plasmin are physiologically inhibited by the serpins plasminogen activator inhibitors type 1 and 2 (PAI-1 and PAI-2) and alpha 2-antiplasmin, respectively (Pollanen et al , 1991). The uPA system and MMPs, such as MMP-9, have been shown to act cooperatively in allowing tumour cells to breach the vascular wall (Kim et al. , 1999). Prior art "magic bullet" style treatments for neoplastic conditions have been extensively investigated but, to date, have met with little or no success. Specifically, most previous attempts at such targeted treatment have relied on the use of antibodies directed to various cell surface molecules expressed by cancer cells. However, such approaches have suffered from many drawbacks including: (i) the surface molecules to which the antibodies are directed have not been uniquely expressed by the cancer cells. Accordingly, such treatments have also been toxic to a significant number of normal cells. (ii) the antibodies which have been utilised in such treatments have been of mouse origin or have been "humanised" mouse antibodies. The use of such antibodies has led to immunological complications associated with the HAMA response. That is. repeated dosing of humans with murine antibodies, such as humanised antibodies, has led to the development of anti-murine antibodies thereby causing both rapid clearance of the murine antibodies and the production of immune complexes which cause the HAMA response. (iii) the antibody-cell surface molecule complexes are often internalised. Accordingly, the toxin which is coupled to the subject antibody is less effective. (iv) the strength of binding of most antibodies to a target cell surface molecule is low with a dissociation constant of approximately 10 "6 M being common. (v) in coupling a toxin to the antibody the point of linkage cannot usually be predicted. Depending on the structure of a given antibody, coupling may occur at the antigen binding site region of the antibody thereby rendering the antibody useless. Further, prior art radiocolloid therapy is not suitable for adjunctive therapy as it is not selective of cancer cells. To the extent that beta and gamma emitting radionuciides have been coupled to specific monoclonal antibodies, problems have been experienced with most of the dose leaving the cancer cell. Therefore, therapeutic doses cannot be achieved without inducing severe complications. Accordingly, there is a need to develop a more effective method of targeting neoplastic cells for treatment, which method provides both improved selectivity in terms of its targeting function and improved delivery of a toxic signal. In terms of the delivery of a toxic signal, there is a need to develop a method which provides both a maximal dose of the subject toxin to the target cell but with minimal impact upon proximally located non- tarεet cells. SUMMARY OF THE INVENTION In work leading up to the present invention the inventors have determined that plasminogen activator inhibitors (herein referred to as "PAI") and in particular PAI-2, can be used as a targeting molecule for specific delivery of a toxin since cancer cells express the uPA/uPAR complex while non-diseased cells express little or no uPA/uPAR complex. Further, the inventors have determined that the coupling of an alpha particle emitting radioisotope to PAI-2 does not inhibit binding of PAI-2 to uPA and still further, that the labelled PAI-2-uPA/uPAR complex is internalised by the targeted cell thereby providing maximal impact of the high energy radioactive emission on the target cell and minimal impact on proximal cells. This is due to the alpha-emitter being highly toxic over a short range only. Finally, unlike the observed dissociation constant of 10 "6 M with respect to antibody /antigen interactions, coupling of PAI-2 to uPA is extremely strong, exhibiting a dissociation constant in the order of 10 " M. thereby minimising dissociation of the radiolabelled PAI-2 and consequently decreasing the risk of a toxic impact on localised non-target cells. Accordingly, a first aspect of the present invention is directed to a method of treating a condition in a mammal, which condition is characterised by the undesirable, detrimental or otherwise unwanted growth of cells expressing a uPA/uPAR complex, said method comprising administering to said mammal an effective amount of PAI or functional derivative, equivalent, homologue, analogue or mimetic thereof, which PAI is bound, linked or otherwise associated with a toxin, for a time and under conditions sufficient to down-regulate the growth of said cells. In a particularly preferred embodiment the plasminogen activator inhibitor (PAI) is PAI-2. Preferably the toxin is a radioisotope. and more preferably is an alpha particle emitting radioisotope which includes, but is not limited to, Tb-149 or Bi-213. More preferably the alpha particle emitting radioisotope is Bi-213. In a preferred embodiment the subject cell growth is proliferation, and the subject down- regulation is killing off the proliferating cells. The condition being treated is preferably cancer, more preferably a metastatic cancer which includes, but is not limited to. breast cancer, prostatic cancer and/or colorectal cancer. According to a second aspect the present invention contemplates a method of down- regulating the growth of cells expressing a uPA/uPAR complex, said method comprising contacting said cells with an effective amount of PAI or functional derivative, equivalent, homologue. analogue or mimetic thereof, which PAI is bound, linked or otherwise associated with a toxin. In a particularly preferred embodiment said PAI is PAI-2, whilst said toxin is preferably an alpha particle emitting radioisotope. In a preferred embodiment the subject cell growth is proliferation, and the subject down- regulation is killing off the proliferating cells. According to a third aspect the present invention relates to the use of PAI or functional derivative, equivalent, homologue. analogue or mimetic thereof, which PAI is bound, linked or otherwise associated with a toxin, in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by the undesirable, detrimental or otherwise unwanted growth of cells expressing a uPA/uPAR complex. In a particularly preferred embodiment said PAI is PAI-2, whilst said toxin is an alpha- particle emitting radioisotope. Even more preferably said condition is a neoplasm, still more preferably a metastatic malignancy. According to a fourth aspect the present invention relates to a PAI-2 conjugate molecule comprising PAI-2 or functional derivative, homologue, analogue, chemical equivalent or mimetic thereof, which PAI-2 is bound, linked or otherwise associated with a toxin or label. Preferably the toxin or label is a radioisotope which can be detected with any means of radioactive detection, and more preferably is an alpha emitting radioisotope which includes, but is not limited to, Tb-149 or Bi-213. Alternatively, the radioisotope is a gamma ray emitting particle which incudes, but is not limited to, radioactive technetium (Te-99); or F-18 labelling, which is detected by Position Emission Tomography (PET). According to a fifth aspect the present invention relates to a pharmaceutical composition comprising a PAI-2 conjugate molecule comprising PAI-2 or functional derivative, homologue, analogue, chemical equivalent or mimetic thereof, which PAI-2 is bound, linked or otherwise associated with a toxin or label in association with one or more pharmaceutically acceptable carriers and/or diluents. The pharmaceutical composition is preferably formulated for intravenous or subcutaneous application. According to sixth aspect the present invention provides a method of detecting or visualising cancerous cells, said method comprising contacting cells with a PAI-2 conjugate molecule comprising PAI-2 or functional derivative, homologue, analogue, chemical equivalent or mimetic thereof, which PAI-2 is bound, linked or otherwise associated with a label capable of being detected, and determining the cells which express a uPA/uPAR complex by detection or visualisation. Preferably the label is a radioisotope which can be detected by any means of radioactive detection, which radioisotope is preferably an alpha particle emitting radioisotope, a gamma ray emitting particle or a radioisotope which is detectable by Position Emission Tomography (PET). The cells may be contacted with the PAI-2 conjugate molecule either in vitro or in vivo. In one embodiment the method is used to detect and visualise cancerous cells such as tumors and the location thereof in a mammalian body. The method may also be used to monitor changes in cancerous cells in the mammalian body to determine progression of cancer throughout the body, and to monitor the development of tumors, effectiveness of cancer treatment and therapy, and regression, remission or modulation of cancer in the body. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" , and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. DETAILED DESCRIPTION Reference to "PAI" should be understood as a reference to any PAI or functional derivative, equivalent, homologue, analogue or mimetic thereof. In this regard, the PAI may be of any suitable form, such as a mature molecule, a precursor form of said mature molecule, mutant, polymorphic variant or a derivative, homologue, equivalent, analogue or mimetic thereof which exhibits at least one of the functional activities of said PAI. In a particularly preferred embodiment, said PAI is PAI-2, a glycoprotein of the serine protease inhibitor type and which exists in both glycosylated and unglycosylated forms. (Andreasen, 1990). Without limiting the present invention in any way, PAI-2 exhibits several advantages over PAI-1. First, PAI-2 is very stable in vitro compared to PAI-1 which is oxidation sensitive and easily inactivated (Kruithof et al.. 1995). Secondly, PAI- 2 is approximately 10.000 fold less active towards t-PA than PAI-1 and would not lead to the side effect that fibrino lysis is inhibited. Thirdly, high blood levels of PAI-2 are thought less likely to cause any other adverse side effects since high levels of PAI-2 are found during late pregnancy (in the non-pregnant state blood levels of PAI-2 are not detectable) and are not associated with toxicity (Kruithof et al. , 1996). Reference to "toxin" should be understood as a reference to any suitable toxin which achieves the object of providing a signal which reduces, prevents or otherwise inhibits the proliferation, differentiation or maintenance of subject cell (herein referred to as "down- regulating the growth" of said cell). The subject toxin may act by a variety of means including providing its signal via direct contact with a subject cell or emitting a molecule or particle, such as radiation in the case of a radioactive isotope toxin, which provides the signal to the subject cell. Preferably the toxin is a radioisotope and even more preferably a radioisotope which is highly toxic over a short range and exhibits a short half life thereby minimizing the occurrence of inadvertent toxicity on proximally located non-target cells. Most particularly, said radioisotope is an alpha particle emitting radioisotope. Examples of alpha-emitting radioisotopes suitable for use in the method of the present invention include, but are not limited to, Tb-149 or Bi-213. It should be understood that the toxin which is utilised in the method of the present invention may be in a purified, partially purified or unpurified form. It may also form a component of a larger molecule. The toxin may be naturally occurring or it may be synthetically or recombinantly produced. Reference to "growth" of a cell should be understood as a reference to the proliferation, differentiation and/or maintenance of viability of the subject cell, while "down-regulating the growth" of a cell is a reference to reducing, preventing or inhibiting the proliferation, differentiation and/or maintenance of viability of the subject cell. In a preferred embodiment the subject growth is proliferation and the subject down-regulation is killing. In this regard, killing may be achieved either by delivering a fatal hit to the cell or by delivering to the cell a signal which induces the cell to apoptose. Reference to "treatment" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a mammal is treated until total recovery. Accordingly, "treatment" includes amelioration of the symptoms or severity of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. Reference to an "effective amount" means an amount necessary to at least partly attain the desired response. It should be understood that the cell which is treated according to the method of the present invention may be treated in vitro or in vivo. A cell which is treated in vitro may be one that has been removed from the body of a mammal. For example, cells or tissue comprising neoplastic cells may be removed from a mammal, treated according to the method of the present invention to down-regulate or induce apoptosis of the neoplastic cells and then returned to the mammal. Alternatively, the in vitro cell may be a cell line in respect of which it is sought to down-regulate or modulate its apopotic behaviour via the methods disclosed herein. In accordance with the preferred aspect of the present invention, the cell may be a neoplastic cell (such as a malignant cell) located in vivo and the induction of apoptosis would be achieved by applying the method of the present invention in vivo. It should be understood that where reference is made to a specific cell type which is located in vivo, such as a breast cancer cell, prostate cancer cell or colorectal cancer cell, this cell may be located at the expected site in the mammal or. if a primary cancer has metastasised. the subject cancer may be located in another region of the patient's body. For example, it may form part of a secondary tumor (metastasis) which is located, for example, in the liver, lymph node or bone. The present invention is directed to inhibiting the unwanted growth of cells expressing a uPA/uPAR complex. This should be understood as a reference to cells expressing uPAR (the uPA receptor) to which is coupled a uPA molecule. The uPA molecule which has bound to the uPAR may be derived from any source and has not necessarily been produced from the subject cell in an autocrine fashion - although it should be understood that this possibility is not excluded. Without limiting the present invention to any one theory or mode of action. PAI-2 (to which has been coupled a toxin) will interact with the uPA molecule which, in rn, has interacted with the uPAR expressed by the subject cell. The PAI-2 is thereby specifically targeted to cells which express a uPAR to which is bound uPA. Still without limiting the present invention in any way, uPAR is expressed by neoplastic cells and is not expressed at significant levels by non-neoplastic cells. Reference to "interact" should be understood as a reference to any form of interaction. Said interaction may occur via the formation of bonds such as covalent bonds, hydrogen bonds, van Der Waals forces or via any other mechanism of interaction. Reference to "neoplastic cell" should be understood, in the context of the present invention, as a reference to a cell exhibiting abnormal growth (as hereinbefore defined) and which cell expresses uPAR. The neoplastic cell may be a benign cell or a malignant cell. Preferably, the cell is malignant. Without limiting the present invention in any way, uPAR is overexpressed in metastatic cancers including, but not limited to, breast cancer, prostate cancer and colorectal cancer. "Functional derivatives and mimetics" include fragments, parts, portions, mutants, and mimetics from natural, synthetic or recombinant sources including fusion proteins exhibiting any one or more of the functional activities of the subject PAI or toxin. To the extent that the subject PAI or toxin is a protein, derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences including fusions with other peptides, polypeptides or proteins. Homologues of a PAI or toxin contemplated herein include, but are not limited to, molecules derived from different species. Chemical and functional equivalents of PAI or toxin should be understood as molecules exhibiting any one or more of the functional activities of PAI or toxin, respectively, and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening. The derivatives of PAI or toxin include fragments having particular epitopes of parts of the entire PAI protein or toxin fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, PAI or derivative thereof may be fused to a molecule to facilitate its delivery to a cell. "Analogues" of PAI or toxin contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH_ι; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 -phosphate followed by reduction with NaBH The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide. Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH. Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 1. TABLE 1 Non-conventional Code Non-conventional Code amino acid amino acid α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrate Mgabu D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap D-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanine Anap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec D-α-methylvaline Dmval N-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine Dnmcys N-(3 ,3-diphenylpropyl)glycine Nbhe D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr D-N-methvlhistidine Dnmhis N-(hydroxyethyl))glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-r-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine Mmet L-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithine Morn L-α-methylphenylalanine Mphe L-α-methylproline Mpro L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2, 2-diphenyl- Nmbc ethylamino)cyclopropane Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH2) n spacer groups with n = l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero- bifunctional reagents which usually contain an amino-reactive moiety such as N- hydroxysuccinimide and another group specific-reactive moiety. Without limiting the present invention to any one theory or mode of action, it is thought that PAI-2 exhibits high selectivity to neoplastic cells at their most malignant phase. In this regard, PAI-2 functions as a "homing molecule" for cancer cells which express uPA. bound to uPA receptors, on their surface. Upon binding of radiolabelled PAI-2 to the cell surface uPA/uPAR complex, the entire molecular complex is internalised. Once internalised, the high energy radioactive emission kills the subject cell with little or no effect on proximally located non-target cells. Due to the absence of such side effects, together with the short half-life and rapid decay of the selected alpha particle emitting radioisotope, there is little or no detrimental effect to the subject as a whole. The high turnover cell toxicity of radiolabelled PAI-2 is thought to derive from the tight, essentially irreversible binding of PA-2 to uPA. Still without limiting the present invention in any way, Tb-149 and Bi-213. in particular, chelate to PAI-2 to form very stable bonding. In this regard, any suitable method may be utilised to achieve chelation. Preferably, the chelators cDTPAa and CHX-A are utilised. Alpha radiation can then kill the subject cancer cells in 1-5 nuclear hits. The linear energy transfer (LET) for the α particle, being very much greater than that for the beta rays, causes a high relative biological effectiveness over a much shorter range. As a result, a much greater fraction of the total energy is deposited in cells with alphas and very few nuclear hits are required to kill a cell (Lloyd et al.. 1979. Kassis et al.. 1986). Further, the short half life of alpha-emitters is particularly suitable for the killing of cancer cells and pre-angiogenic lesions while simultaneously releasing only a low radiation dose to normal tissue. The alpha therapeutic ratio is thought to be two orders of magnimde greater than that for a high energy beta emitter and is therefore the preferred form of toxin for use in the method of the present invention. Administration of the toxin labelled PAI, in the form of a pharmaceutical composition, may be performed by any convenient means. The toxin labelled PAI of the pharmaceutical composition are contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends. for example, on the human or animal and the toxin chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 μg to about 10 mg of toxin labelled PAI may be administered per kilogram of body weight per day. For example from about 0.1 μg-5 mg, 10 μg-5 mg or 100 μg-1 mg. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The toxin labelled PAI may be administered in any convenient manner such as by the intravenous, intraperitoneal. intramuscular, subcutaneous or intradermal. Preferably, the toxin-labelled PAI is administered intravenously or subcutaneously. In accordance with these methods, the toxin labelled PAI defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order. The term "mammal" should be understood as a reference to a human, primate, livestock animal (eg. sheep, pig, cow. horse, donkey) laboratory test animal (eg. mouse, rat. rabbit, guinea pig) companion animal (eg. dog, cat) or captive wild animal (eg. fox, kangaroo, deer). Preferably, the mammal is a human. The pharmaceutical forms of the PAI-2 conjugate molecule of the present invention suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof. Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired. The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.1 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.1 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. The present invention is further described by the following non-limiting Examples and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS Each of the Figures in turn illustrate the following: Figure 1 Formation of SDS-stable complex of radio-conjugated PAI-2 with UPA, indicating activity of PAI-2; Figure 2 ITLC human serum stability results of PAI-2-cDTPAa and BSA-cDTPAa conjugates labelled with Bi-213, indicating purity of radio-conjugated PAI- 2 in ITLC; Figure 3 MDA-MB-231 cell survival with radio-conjugate PAI-2; Figure 4 MCF-7 cell survival with radio-conjugate PAI-2; Figure 5 Confocal microscopy of endocytosis of radio-conjugate PAI-2 by MDA- MB231 cells; Figure 6 Alpha-PAI-2 tolerance study in nude mice (3 mCi/kg). Figure 7 Alpha-PAI-2 tolerance study in nude mice (6 mCi/kg, i.v. injection). Figure 8 TAT Breast Cancer in vivo model. 25 μCi radio-conjugate PAI-2 (Bi-213- cDTPA-PAI2) injected 2 days after tumor cells; Figure 9 TAT Breast Cancer in vivo model. 50 μCi radio-conjugate PAI-2 (Bi-213- cDTPA-PAI2) injected 2 days after tumor cells: Figure 10 TAT Breast Cancer in vivo model. 25 μCi radio-conjugate PAI-2 (Bi-213- cDTPA-PAI2) injected 7 days after tumor cells; Figure 11 TAT Breast cancer in vivo model. 25 μCi radio-conjugate PAI-2 (Bi-213- cDTPA-PAI2) injected 14 days after tumor cells. Figure 12 Alpha-PAI-2 or non-specific alpha-protein administered 2 days post inoculation of human breast cancer MDA-MB-231 cells in nude mice. (a) Alpha-PAI2 treatment 1.5 mCi/kg -25 μCi/mouse (n = 5) (b) Alpha-PAI2 treatment 6.0 mCi/kg -100 μCi/mouse (n = 5) (c) Alpha-PAI2 treatment 3.0 mCi/kg -50 μCi/mouse (n = 5) (d) Control Treatment 6.0 mCi/kg -100 μCi/mouse (n = 5) Figure 13 Systemic TAT with PAI in PC-3 tumours after 2 days cell inoculation Figure 14 Systemic TAT post tumour appearance in first week Figure 15 Systemic TAT post tumour appearance m fourth week EXAMPLE 1 PREPARATION OF PAI-2 ALPHA-EMITTER CONJUGATE Production The rare earth nuchde i49 Tb is produced on a tandem, cyclotron or linear accelerator using high energy heavy ions such as boron or carbon or mtrogen ions to bombard targets of Praseodymium, eg Pr( 12 C,4n) or Neodymium Nd( 12 C,5n) at higher energies Metal targets are rolled and mounted on a frame A thm target has 1 mg cm 2 , a thick target has 30 mg cm 2 A catcher foil is used to collect Tb ions in the thm foil geometry Tb-149 is also produced via the spallation reaction of high energy charged particles, eg protons on a high atomic number target such as tantalum The ions are passed through a magnetic mass analyser to select the A = 149 component of the yield The Tb- lmmunoconjugates are made using the longer lived isotope Tb-152 as an exact analogue An alternative α radiolabel is Bι-213, which is produced by α decay from Ac-225 the Bι-213 is eluted from the Ac-225 with 250 μL of fresh 0 15 M HI followed by 250 μL water The activity of the Bι-213 is assayed against the Au-198 setting in the dose calibrator The first elution is not used as it contains cold Bi Purification In the production of Tb-149, the product nuclides are separated from the thick target by dissolution in 6 M nitric acid, the sample is irradiated to dryness and yield determined by gamma ray spectroscopy . The residue is dissolved in 0.16 M -hydro xyisobutyric acid and passed through a cation exchange column (particle size 13 μm). The pH of the eluant is adjusted to 5 by aqueous ammonia. Elution was under a pressure of 7 kg cm "2 at a flow rate of 0.5 mL min "! . Terbium fractions are dried gently and heated to 450 degrees to destroy the Tb-isobutyrate complex. The residue is dissolved in dilute nitric or hydrochloric acid for the radiolabeling procedure. Bi-213 is eluted with 0.15 M hydriodic acid and the pH of the eluant adjusted as above. Labelling The purified product is chelated to molecules which target specific cancer cells. A number of different chelation procedures are available in the literature which use cDTPAa. DTPA-CHX", DOTA (l,4,7,10-tetraazacyclododecane-N,N,N,N,tetraacetic acid), and TETA. The labelling procedure is a modification of the method used by Izard et al (1992). Briefly, both chelators ie. cDTPAa and CHX-A" were prepared in chloroform and were purified under a stream of Nitrogen. A chelator: protein molar ratio of 20: 1 and 4: 1 was maintained for cDTPAa and CHX-A" respectively. After a 45-minute on-ice incubation of the chelator and PAI2 (Biotech Australia), the conjugate was purified on a PD-10 column (Pharmacia Biotech) using 0.5 M sodium acetate at pH 5.5 as the eluting buffer. This was followed by the addition of Bi-213 and TB-149 or TB-152). After 20-minute incubation, the ratio-conjugate (RI) was again purified on another PD-10 column using PBS at pH 7.0 as the eluting buffer. The protein recovery is > 97% as determined by instant thin layer chromatography (ITLC) of the fractions obtained. A similar procedure was followed for labelling the non-specific protein, bovine serum albumin (BSA). The inhibitory activity of cDPTAa-conjugated PAI2 was confirmed by its ability to form complexes after 40 min incubation at 20 °C with active uPA. Complex formation was detected by a molecular mass shift by SDS-PAGE (12% non-reducing gel)(Figure 1). Stability of radioconjugated protein The radioconjugates were incubated with fresh human serum at 37°C for 45 min (equivalent to one half-life for Bi-213). Samples were then analysed by ITLC (Figure 2). Radioconjugates remain at the origin whereas any free label runs at the solvent front. Similar experiments have been performed with Tb radioconjugates over long time periods. EXAMPLE 2 IN VITRO ANALYSIS OF PAI-2 ALPHA EMITTER CONJUGATE Tb-149 and Bi-213 ARCs have been produced and their stability, labelling efficiency, targeting and in vitro cell survival (37% survival for 2 μCi) with MDA-MB-231 and MCF-7 breast cancer cell lines have been tested. The following ARCs have been prepared, by the inventors, for the first time: Tb-149.cDTPAa.PAI2 Tb-149.CHX.PAI2 Bi-213.cDTPAa.PAI2 Bi-213.CHX.PAI2 The method of the present invention is suitable for any early stage metastatic cancer which express high levels of uPA-uPAR. Cell survival Cell survival data have been obtained for two cell lines (MDA-MB-231 and MCF-7) in the presence of plasminogen with or without the specific uPA activity blocking agent {gly-gly-arg chloromethylketone (EGR-CMK), as this agent effectively inhibits PAI2 binding; Hang et al. 1998} . The 37% survival dose (D 0 ) values are shown in Table 2. TABLE 2. Percentage cell survival at 5 μCi activity of alpha-proteins or at excess concentrations of cDTPA-PAI-2 and PAI-2 compared to controls. 1 - and + indicate without and with EGR-CMK pre-treatment. ND = not determined. 2 Values shown are the means ± DK (n = 6, each experiment performed in triplicate) 3 Values shown are the means ± SK (n = 2, each experiment performed in triplicate). 4 Protein concentration of alpha-PAI-2 was approximately 2.5 μg/mL. Conclusions: a) EGR-CMK significantly improves survival (by a factor of 2.5. Table 2) as a result of : i_nh,- : i,b_i.t : ion of . t,h . e - P T ^A Λ T I-2. i : n_ . terac _ti • o _n „ w ^ ce u u | ar U PA, proving the specific cytotoxicity of α-PAI2. b) MDA-MB-231 and MCF-7 have similar survivals (i.e. about 2 μCi for 37%), as shown in Figures 3 and 4. This does not imply similar cellular uPA levels but rather the high toxicity of the α-PAI2. c) No cell killing was observed with the freshly isolated normal human leukocytes (Table 2) reflecting that non-targeted cells are immune from α-PAI2. Endocvtosis The α-PAI2 complex is endocytosed in breast cancer cells, as shown by confocal microscopy in Figure 5. Some cells shown in Figure 5 have been stably transfected with the green fluorescent protein (GFP). This uncloned population was then incubated with TRITC- (red fluorescene) labeled PAI2 (10 μg/ml) for 1 h at room temperature before being viewed under the confocal microscope. The red fluorescent spots are evidence of internalisation and accumulation into vesicles (probably endosomes and lysosomes). Internalisation improves the cytotoxicity for isolated cells as the probability for α tracks crossing the nucleus is increased. EXAMPLE 3 TOLERANCE OF ALPHA-PAI-2 IN MICE Alpha-PAI-2 was administered by intra-peritoneal injection in adult (10-12 weeks), male BALB/c nude mice at a dose of 3 mCi/kg (i.e. 3 times the accepted tolerance dose in humans for an alpha-labelled antibody; D. Scheinberg, Memorial Sloan Kettering Institute, personal communication to B. Allen). A 10% short-term weight loss was observed in 2 of 3 mice with recovery by day 11. The third mouse lost approximately 10% body weight at day 7 and was stable at this level. Cold chelated-PAI-2 (DTTA- PAI-2), cold PAI-2 and the control (saline) mouse all showed small variations in weight. These mice were monitored for 100 days and the mouse weights are shown in Figure 6. Overall, the mice were clinically unaffected for the time period observed. To investigate the tolerance of mice to intravenous administration of the drug, alpha- PAI-2 (6 mCi/kg) was administered by tail vein injection in 8-10 week old female Arc(s) nude mice (Figure 7). There were five mice (noted n = 5) per dose. Weight changes were minimal (less than % 10) within the first 7 days, in most mice no change was noted. Similar results were obtained using a non-specific monoclonal antibody. All mice gained weight to adult levels and remained stable over the study period shown. These experiments demonstrate that alpha-PAI-2 is well tolerated in these animals, to a dose of 6 mCi/kg. EXAMPLE 4 BREAST CANCER MODEL Two million human breast cancer cells [MDA-MB-231 (mycoplasma free)] in sterile phosphate buffered saline (PBS) were injected sub-cutaneously into the mammary fat pad of first pair breasts on both sides of 4-6 week old female nude mice. At 2, 7 and 14 days post cell innoculation, each mouse was injected s.c. at the site of tumour cell inoculation with either PBS (control) or varying doses of α-PAI2 in PBS (described as Bi-213.cDPTAa.PAI2 in figures titles; specific activity 3-4 μCi/10μL). The maximum volume injected at any one time was 100-120 μl/site. Note that all mice were handled similarly and kept under similar feeding and housing conditions. The results are shown in figures 8, 9, 10 and 11. The tumour volume was recorded for each mouse at various time points since innoculation of breast cancer cells (indicatd on x-axis of figures as days after cell injection). The skin in area of innoculation was monitored for any signs of inflammation or damage - none were recorded. Also weight and over-all well-being was monitored from time of cell innoculation. These were all normal except for a small decrease in weight ( " 10%) immediately after alpha-PAI2 which was quickly recovered (data not shown). A similar phenomenon was seen with alpha-PAI2 treatment of mice without tumours in the alpha-PAI2 tolerance study (Figure 6 from Example 3). The mice are monitored for another 2-3 weeks after which they are sacrificed for the purpose of performing histological analysis of tissues associated with the injection site, including auxiliary lymph node tissue for the presence of cancer cells. Figures 8, 9, 10 and 11 show that significant reduction in tumor volume occurred with injection of radio-conjugate PAI-2. This was apparent with two doses of PAI-2, 25μCi (Fig. 8) or 50μCi (Fig. 9) injected 2 days after the inoculation of cancer cells into the mammary fat pad of the mouse. It was also apparent when the radio-conjugate PAI-2 was injected 7 days (Fig.10) or 14 days (Fig. 11) after the tumor cells. EXAMPLE 5 INTRAVENOUS TARGETED ALPHA THERAPY (TAT) WITH ALPHA-PAI-2 IN BREAST CANCER XENOGRAFTED NUDE MICE A further study was carried out with intravenous administration of the drug. Two million human breast cancer cells [MDA-MB-231 (mycoplasma free)] in sterile phosphate buffered saline (PBS) were injected sub-cutaneously into the mammary fat pad of first pair breasts on both sides of 4-6 week old female nude mice. At 2 days post-cell inoculation, each mouse was administered either alpha-PAI-2 (1.5 mCi/kg, 3 mCi/kg or 6 mCi/kg) or non-specific alpha-MAb (6 mCi/kg) by tail vein injection. There were five mice (noted n = 5) per dose. The maximum volume injected at any one time was 100-120 μl/site. Note that all mice were handled similarly and kept under similar feeding and housing conditions. The mmour volume was recorded for each mouse at various time points since inoculation of breast cancer cells (indicated on x-axis of figures as days after administration). The skin in the area of inoculation was monitored for any signs of inflammation or damage - none was recorded. Also weight and overall well-being was monitored from time of cell inoculation. There was no change except for a small decrease in weight ( " 10%) imediately after alpha-PAI-2 treatment in some mice which was quickly recovered (data not shown). The results are presented in Figure 12, and clearly show that alpha-PAI-2 administered intravenously can inhibit mmour growth. There is also a dosage effect - 6 mCi/kg being more effective than 3 mCi/kg, which was more effective than 1.5 mCi/kg. The mice were sacrificed at the last time point for mmour size measurements for the purpose of performing histological analysis of tissues associated with the cell inoculation site, as well as axillary lymph node tissue for the presence of cancer cells. By macroscopic inspection axillary lymph nodes appeared unaffected compared to nonspecific protein control mice that had enlarged and hardened lymph nodes. EXAMPLE 6 ALPHA-PAI-2 INHIBITION OF PROSTATE CANCER For the investigation of prostate cancer, cells from the human prostate adenocarcinoma, PC-3. were harvested and washed in culture medium. Then 2 x 10 6 cells were resuspended in 100 μL of the RPMI-1640 serum-free medium and 100 gL of matrigel (Becton Dickinson, Bedford MA) and injected via 18-gauge needle into the s.c. space of the right flank region of male 6-8 week old athymic nude mice, BALB/c (nu/nu). Because PC-3 cells require additional factors or stromal cells, it is necessary to co-inject the cells with matrigel to induce mmour growth in nude mice. Alpha-PAI-2 (referred to as TAT in these instances) was inoculated intravenously (i.e. systemic administration) either 2 days (Figure 13), 7 days (Figure 14), or 4 weeks after PC-3 cell inoculation (Figure 15). Each of these experiments contained 4 groups of 5 animals each, inoculated with a non-specific alpha-labelled antibody at 6 mCi/kg., or alpha-PAI-2 at 1.5 mCi/kg, 3 mCi/kg, or 6 mCi/kg. Each animal received a single injection of the drug, in 100 μl of solution. At one week, the mmour volume was approximately 46 m 3 , and at 4 weeks, the mmour volume was approximately 74 mm 3 . Tumour progression was documented by measuring volumes once weekly using calipers during the experiments, and their volumes were calculated by the following formula: length x width x height x 0.52 in millimeters. Data points for both experiments were expressed as average mmour volume ± SE of mean based on 4-7 determinations. The incidence of lymph node metastases is also reported, by observation. No mice died in these experiments. The results are presented in Figures 13, 14, and 15, and clearly show a dose related inhibition of prostate mmour cell growth in these animals, from the administration of alpha-PAI-2. The times of admimstration of alpha-PAI-2 by tail vein injection (i.v.) are indicated in these figures by an arrow. Similar results have been obtained from local administration of alpha-PAI-2 directly into the s.c. (sub-cutaneous) mmour. EXAMPLE 7 DIAGNOSTIC IMAGING OF CANCER Those skilled in the art will recognise that the same invention described herein can be used to identify the sites of tumours within the body. Thus, PAI-2 can locate tumours which express uPA with high specificity, as described above. It follows that if the PAI-2 so used is labelled with any agent that can be detected, the mmour location can be visualised. Examples of such detectable agents which could be coupled to PAI-2 include, but are not limited to. the following: radioactive Bi-213, as described in this application, which can be detected with any means of radioactive detection, such as a gamma camera; radioactive technetium (Tc-99), similarly detected; or 18-fluoro labelling, which is detected by Positron Emission Tomography (PET). The detection of sites of mmour growth has utility in the diagnosis of cancer, and in the identification of metastasis, that is. the spread of cancer from the primary mmour. It also has utility in defining the sites accessible for therapy, using any of the existing therapeutic modalities for cancer, including the alpha-PAI-2 described herein. The progression of cancer, and the effectiveness of treatment, can also be monitored using this PAI-2 targetting method for imaging of cancerous cells . These data from the Examples above clearly demonstrate the imaging and anti-tumor effectiveness of the radio-conjugated PAI-2 molecules of the present invention. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, feamres, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or feamres. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour. BIBLIOGRAPHY: Allen, B.J. and Blagojevic. N. (1996) Nuclear Medicine Communications 17:40-47. Allen, B.J. (1999) Nuclear Medicine Communications 20.205-207. Andreasen, P. A. , Kjoller, L. , Christensen, L. and Duffy, M.J. (1997) Int. J. Cancer 72:1-22. Bloomer, W.D. et al. (1984) Int. J. Radiat. Oncol. Biol. Phys. 10:341. Duffy, M.J. , Maguire. T.M. , McDermott, E.W. , et al. (1999) (/. Surg. Oncol. 71:130- 135. Gandolfo, G.M. , Conti, L. and Vercillo, M. (1996) Anticancer Res. 76:2155-2159. Gansow, O.A., Wu, C. (1995) Advanced methods of radiolabeling monoclonal antibodies with therapeutic radionuciides, Ed: Goldenberg D M, CRC Press, pp 63-76. Foekens, J.A.. Buessecker, F. , Peters, H.A. , et al. (1995) Cancer Res. 55:1423-1427. 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Claims: CLAIMS: 1. A method of treating a condition in a mammal, which condition is characterised by the undesirable, detrimental or otherwise unwanted growth of cells expressing a uPA/uPAR complex, said method comprising administering to said mammal an effective amount of PAI-2 or functional derivative, equivalent, homologue, analogue or mimetic thereof, which PAI is bound, linked or otherwise associated with a toxin, for a time and under conditions sufficient to down-regulate the growth of said cells. 2. A method of claim 1. wherein the toxin is a radioisotope. 3. A method of claim 2. wherein the radioisotope is an alpha particle emitting radioisotope. 4. A method of claim 3. wherein the alpha particle emitting radioisotope is Tb-149 or Bi-213. 5. A method of any of claims 1 to 4, wherein the cell growth is proliferation, and the down-regulation is killing of the proliferating cells. 6. A method of any of claims 1 to 5. wherein the condition being treated is cancer. . A method of claim 6. wherein the cancer is a metastatic cancer. 8. A method of claim 7. wherein the metastatic cancer is breast cancer, prostatic cancer and/or colorectal cancer. 9. A method of down-regulating the growth of cells expressing a uPA/uPAR complex, said method comprising contacting said cells with an effective amount of PAI-2 or functional derivative, equivalent, homologue, analogue or mimetic thereof, which PAI-2 is bound, linked or otherwise associated with a toxin. A method of claim 9, wherein the toxin is an alpha particle emitting radioisotope. A method of claim 10. wherein the alpha particle emitting radioisotope is Tb-149 or Bi-213. Use of PAI-2 or functional derivative, equivalent, homologue, analogue or mimetic thereof, which PAI-2 is bound, linked or otherwise associated with a toxin, in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by the undesirable, detrimental or otherwise unwanted growth of cells expressing a uPA/uPAR complex. Use of claim 12, wherein the toxin is an alpha-particle emitting radioisotope. Use of claim 13, wherein the alpha particle emitting radioisotope is Tb-149 or Bi- 213. Use of any of claims 12 to 14, wherein the condition is a neoplasm Use of claim 15, wherein the neoplasm is a metastatic malignancy. Use of claim 16, wherein the metastatic malignancy is breast cancer, prostatic cancer and/or colorectal cancer. A PAI-2 conjugate molecule comprising PAI-2 or functional derivative, homologue, analogue, chemical equivalent or mimetic thereof, which PAI-2 is bound, linked or otherwise associated with a toxin or label. A PAI-2 conjugate of claim 18, wherein the toxin or label is a radioisotope which can be detected with any means of radioactive detection. A PAI-2 conjugate of claim 19, wherein the radioisotope is an alpha particle emitting radioisotope A PAI -2 conjugate of claim 20, wherein the alpha particle emitting radioisotope is Tb-149 or Bi-213. A PAI-2 conjugate of claim 21, wherein the PAI-2 conjugate molecule is selected from the group Tb-149.cDTPAa.PAI2 Tb-149.CHX.PAI2 Bi-213.cDTPAa.PAI2 Bi-213.CHX.PAI2 A PAI-2 conjugate of claim 19, wherein the radioisotope is a gamma ray emitting particle A PAI -2 conjugate of claim 23, wherein the gamma ray emitting particle is technetium (Te-99). A PAI-2 conjugate of claim 19, wherein the radioisotope is F-18, which is detectable by Position Emission Tomography (PET). A pharmaceutical composition comprising a PAI-2 conjugate molecule comprising PAI-2 or functional derivative, homologue, analogue, chemical equivalent or mimetic thereof, which PAI-2 is bound, linked or otherwise associated with a toxin or label in association with one or more pharmaceutically acceptable carriers and/or diluents. A pharmaceutical composition of claim 26, wherein the pharmaceutical composition is formulated for intravenous or subcutaneous application. A method of detecting or visualising cancerous cells, said method comprising contacting cells with a PAI-2 conjugate molecule comprising PAI-2 or functional derivative, homologue, analogue, chemical equivalent or mimetic thereof, which PAI-2 is bound, linked or otherwise associated with a label capable of being detected, and determining the cells which express a uPA/uPAR complex by detection or visualisation. A method of claim 28, wherein the label is a radioisotope which can be detected by any means of radioactive detection. A method of claim 29, wherein the radioisotope is an alpha particle emitting radioisotope, a gamma ray emitting particle or a radioisotope which is detectable by Position Emission Tomography (PET). A method of any of claims 28 to 30, wherein the cancerous cells are associated with breast cancer, prostatic cancer and/or colorectal cancer. A method of any of claims 28 to 31, wherein the cells are contacted with the PAI- 2 conjugate molecule in vitro. A method of any of claims 28 to 31, wherein the cells are contacted with the PAI- 2 conjugate molecule in vivo. A method of claim 33, wherein the method is used to detect and visualise cancerous cells in a mammalian body. A method of claim 34, wherein the method is used to visualise tumor location in the mammalian body. A method of claim 34 or claim 35, wherein the method is used to monitor changes in cancerous cells in the mammalian body to determine progression of cancer throughout the body, development of tumors, effectiveness of cancer treatment and therapy, or regression, remission or modulation of cancer in the body. A method for the manufacmre of a medicament comprising the step of bringing a PAI-2 conjugate molecule as claimed in any of claims 18 to 25 in association with one or more pharmaceutically acceptable carriers and/or diluents.

Inventors: ALLEN BARRY JOHN, BUNN CLIVE LEIGHTON, RANSON MARIE, ALLEN, BARRY, JOHN, BUNN, CLIVE, LEIGHTON, RANSON, MARIE
Applicants: ALLEN BARRY JOHN, BIOTECH AUSTRALIA PTY LTD, BUNN CLIVE LEIGHTON, MEDICAL SCITEC AUSTRALIA PTY L, RANSON MARIE, UNIV WOLLONGONG, ALLEN, BARRY, JOHN, BIOTECH AUSTRALIA PTY LIMITED, BUNN, CLIVE, LEIGHTON, MEDICAL SCITEC AUSTRALIA PTY LTD, RANSON, MARIE, UNIVERSITY OF WOLLONGONG
Application year: 2001
Main classification: A61K38
IPC codes: A61K38, A61K45, A61K51, A61P35, C07K14, C12Q1, G01N33