WO2009019493A1

WO2009019493A1 - Antibodies binding human prostate epithelial cells

Info

Publication number: WO2009019493A1
Application number: PCT/GB2008/002733
Authority: WO
Inventors: Tahereh Kamalati; Lakjaya Buluwela
Original assignee: Imperial Innovations Ltd
Current assignee: Ip2ipo Innovations Ltd
Priority date: 2007-08-09
Filing date: 2008-08-11
Publication date: 2009-02-12
Anticipated expiration: 2010-02-09
Also published as: GB0715542D0

Abstract

An isolated recombinant antibody that binds to a normal human prostate epithelial cell and/or cultured human prostate tumour cell to a greater extent than to a prostate fibroblast or muscle cell, the recombinant antibody comprising at least one, two, three, four or five, or six, CDR sequence selected from a) GFTFSSYA (CDRl sequence); ISYDGSNK (CDR2 sequence); ARVYFRL WGQGTLVTV (CDR3 sequence); SSNIGSNY C(CDRl sequence); RNN (CDR2 sequence); AAWDDSLPVFGGGTKLTVLGAAA (CDR3 sequence); with no or up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitutions in each CDR sequence; or b) GDSVSSNSAA (CDRl sequence); TYYRSKWYN (CDR2 sequence); ARLVDASFDYWGQGTLVTV (CDR3 sequence); QGISSW (CDRl sequence); AAS (CDR2 sequence); QQANSFRTFGQGTKVEIKRAAA (CDR3 sequence); with no or up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitutions in each CDR sequence. The antibody is considered to be useful in imaging, diagnosis and treatment of cancer, particularly prostate cancer.

Description

ANTIBODIES BINDING HUMAN PROSTATE EPITHELIAL CELLS

The present invention relates to methods and reagents useful in relation to diagnosis and treatment of cancer, for example prostate cancer.

Cancer is a serious disease and a major killer. Although there have been advances in the diagnosis and treatment of certain cancers in recent years, there is still a need for improvements in diagnosis and treatment.

Carcinoma of the prostate has become a most significant disease in many countries and it is the most commonly diagnosed malignancy in men in the western world, its occurrence increasing significantly with age. By the year 2018 it is expected to be the biggest killer with 50% of the male population suffering from it (80% by age 80 years). Recent evidence suggests that prostate cancer is also increasing amongst younger men as well (Br J Cancer (1999) 79, 13-17). These increases and the recent deaths of many public figures from prostatic cancer have served to highlight the need to do something about this cancer. It has been suggested that the wider availability of screening may limit mortality from prostate cancer.

Prostate cancer screening currently includes a rectal examination and measurement of prostate specific antigen (PSA) levels. These methods lack specificity as digital rectal examination has considerable inter-examiner variability (Smith & Catalona (1995) Urology 45, 70-74).

For cancers such as prostate cancer, present screening methods are unsatisfactory; there is no reliable method for diagnosing the cancer, or predicting or preventing its possible metastatic spread, which is the main cause of death for most patients.

Imaging of cancer cells is a potentially important tool in the diagnosis, management and understanding of cancer. However, there are a very limited number of imaging agents for prostate cancer and thus new probes are urgently needed.

It is an object of the invention to provide tools and associated methods useful in imaging, diagnoses and prognoses of prostate cancer, and for aiding the clinician in the management of cancer, particularly prostate cancer. In particular, an object of the invention is to provide tools and associated methods for detecting primary or metastatic prostate cancer.

Further objects of the invention include the provision of methods of treatment of cancer, in particular prostate cancer, and methods of identifying compounds which may be useful in treating, diagnosing or imaging cancer.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

We have employed Single Chain Antibody Fragment (scFv) Phage Display to isolate human recombinant antibodies specific to the cell surface of normal human prostate epithelial cells, with the aim of generating reagents for primary prostate cell purification. This has been achieved by screening a semi-synthetic human antibody phage display library (the "Griffin.1" library; [I]) directly on live, primary normal human prostate epithelial cells, after subtraction of the library on live, primary normal human prostate mesenchymal cells. Our screen has generated 15 unique scFv clones with specificity for the cell surface of prostate epithelial cells.

Characterisation of these clones has resulted in identification of two antibody clones, scFv 95.2 and scFv 98.1, which exhibit reactivity with normal prostate epithelial cells, with little detectable binding to prostate fibroblasts or muscle cells. Further, both scFv 95.2 and scFv 98.1 bind efficiently to cultured prostate tumour cells and are strongly reactive with tumour cells in sections of prostate cancer tissue. The binding increases with increasing tumourogenicity of prostate epithelial cells. As will be well known to those skilled in the art, tumourogenicity can be assessed by ability to form tumours, for example in mice. It can be expressed as the % of introduced cells generating tumours, for example in mice. Tumourogenicity can also be assessed by the behaviour of cells in soft agar, as will be well known to those skilled in the art. Increasing tumourogenicity is considered to reflect increasing metastatic potential.

We consider that these antibodies and related antibodies are useful for the in vivo targeting of human prostate cancer and as therapeutic, diagnostic and imaging agents.

Single-chain antibody variable region fragments, scFv's, are expressed as a single polypeptide and, hence, are ideal building blocks to make fusion proteins with therapeutic as well as tumour-localising properties (2). scFv antibodies directed against growth factors and their receptors (3, 4), chemokines and their receptors (5), oncogenes (6) and transcription factors (7) have been effective in inhibiting tumour growth. Further, to date, several bacteriophage-derived antibodies have been generated with clinical cancer applications (8). scFv antibodies offer great flexibility in the development of targeting agents. Compared to whole IgG molecules and other antibody fragments, they represent affinity reagents which, by virtue of their small size, can penetrate into tumour masses more rapidly and, clear more quickly from normal tissues. This is important, as effective targeted therapy relies on efficient antibody retention after clearance from normal tissue.

A first aspect of the invention provides an isolated recombinant antibody that binds to a normal human prostate epithelial cell and/or cultured human prostate tumour cell to a greater extent than to a human prostate fibroblast or muscle cell, the recombinant antibody comprising at least one, two, three, four or five, or more preferably six, CDR sequence selected from a) GFTFSSYA (CDRl of IGHV3-30 like portion of 98.1); ISYDGSNK (CDR2 of IGHV3-30 like portion of 98.1); ARVYFRL WGQGTLVTV (CDR3 of IGHV3-30 like portion of 98.1); SSNIGSNY (CDRl of IGLV1-47 like portion of 98.1); RNN (CDR2 of IGLV1-47 like portion of 98.1); AAWDDSLPVFGGGTKLTVLGAAA (CDR3 of IGLV1-47 like portion of 98.1); with no or up to 1, 2, 3 or less preferably 4, 5, 6, 7, 8, 9 or 10 conservative or non- conservative amino acid substitutions in each CDR sequence; or

b) GDSVSSNSAA (CDRl of IGHV6-1 like portion of 95.2); TYYRSKWYN (CDR2 of IGHV6-1 like portion of 95.2); ARLVD ASFD YWGQGTLVTV (CDR3 of IGHV6-1 like portion of 95.2); QGISSW (CDRl of IGKVl- 37 like portion of 95.2); AAS (CDR2 of IGKVl- 37 like portion of 95.2); QQANSFRTFGQGTKVEIKRAAA (CDR3 of IGKVl- 37 like portion of 95.2); with no or up to 1, 2, 3 or less preferably 4, 5, 6, 7, 8, 9 or 10 conservative or non- conservative amino acid substitutions in each CDR sequence.

The antibody is typically a scFv antibody. The antibody typically has human framework regions and human CDR sequences but may alternatively have non- human framework sequences, for example mouse or rat framework sequences. The framework and CDR sequences are typically human or may be modified in order to make them more antigenically similar to human sequences as well known to those skilled in the art. The CDR sequences may have been arrived at using "affinity maturation" techniques based on the CDR sequences given above, for example in order further to improve the affinity or selectivity of the antibody binding. Suitable techniques will be well known to those skilled in the art, for example including techniques described in Pavoni et al (2006) BMC Cancer 6, 41.

In relation to option a) above the antibody may comprise at least one, two, three, four or five, or more preferably six, framework sequence selected from RGAVESGGGVVQPGRSLRLSCAAT (FRl from IGHV3-30 like portion of 98.1); MHWVRQAPGKGLEWVAV (FR2 from IGHV3-30 like portion of 98.1); YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (FR3 from IGHV3-3O like portion of 98.1); QSVLTQPPS ASGTPGQRVTISCSGS (FRl from IGLV1-47 like portion of 98.1); VYWYQQLPGTAPKLLIY (FR2 from IGLV1-47 like portion of 98.1);

QRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC (FR3 from IGLV 1-47 like portion of 98.1), with no or up to 1, 2, 3, 4, 5, or less preferably 6, 7, 8, 9 or 10 conservative or non- conservative amino acid substitutions in each framework sequence.

In relation to option b) above the antibody may comprise at least one, two, three, four or five, or more preferably six, framework sequence selected from QVQLQQSGPGLVKPSQTLSLTCAIS (FRl from IGHV6-1 like portion of 95.2); WNWIRQSPSRGLEWLGR (FR2 from IGHV6-1 like portion of 95.2); DYAVS VKSRITINPDTSKNQFSLQLNSVTPEDTAVYYC (FR3 from IGHV6-l like portion of 95.2); DIQLTQSPSSVSASVGDRVTITCRAS (FRI from IGKVl- 37 like portion of 95.2); LAWYQQKPGKAPKLLIY (FR2 from IGKVl- 37 like portion of 95.2); SLQSGVPSRPSGSGSGTDFTLTISSLQPEDF ATYYC (FR3 from IGKVl- 37 like portion of 95.2), with no or up to 1, 2, 3, 4, 5, or less preferably 6, 7, 8, 9 or 10 conservative or non- conservative amino acid substitutions in each framework sequence.

The antibody may comprise as the linker between the heavy-chain and light-chain like portions the sequence SSGGGGSGGGGSGGSAL with no or up to 1, 2, 3, or less preferably 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitutions.

The antibody may also comprise one or more tags to aid purification or identification, as well known to those skilled in the art, for example a His tag, for example HHHHHHGAA; or a Myc tag, for example EQKLISEEDLN. The antibody may also comprise a leader sequence to aid secretion of the antibody, for example the sequence MKYLLPTAAAGLLLLAAQPAMA.

The antibody may have or comprise the sequence

MKYLLPTAAAGLLLLAAQPAMA RGAVESGGGVVQPGRSLRLSCAAT GFTFSSYA MHWVRQAPGKGLEWVAV ISYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYC ARVYFRLWGQGTLVTV SS GGGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGS SSNI GSNYVYWYQQLPGTAPKLLIY RNNQRPSGVPDRFSGSKSGTSAS LAISGLRSEDEADYYC AAWDDSLPVFGGGTKLTVLGAAAHHHHHH GAAEQKLISEEDLN (98.1 sequence) or

MKYLLPTAAAGLLLLAAQPAMAQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNS AAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLN SVTPEDTAVYYCARLVDASFDYWGQGTLVTVSSGGGGSGGGGSGGSALDIQLTQ SPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQANSFRTFGQGTKVEIKRAAAHHHHHH GAAEQKLISEEDLN (95.2 sequence) with no or up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitution; or a sequence having at least 80, 85, 90 or 95% identity with either sequence.

By "antibody" we include not only whole immunoglobulin molecules but also fragments thereof such as Fab, F(ab')2, Fv and other fragments thereof that retain the antigen-binding site. Similarly the term "antibody" includes genetically engineered derivatives of antibodies such as single chain Fv molecules (scFv) and single domain antibodies (dAbs) . The term also includes antibody-like molecules which may be produced using phage-display techniques or other random selection techniques for molecules. The term also includes all classes of antibodies, including: IgG, IgA, IgM, IgD and IgE.

The variable heavy (V_H) and variable light (V_L) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).

Antigenic specificity is conferred by variable domains and is independent of the constant domains, known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V_H and V_L partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293-299.

By "ScFv molecules" we include molecules wherein the V_H and V_L partner domains are linked via a flexible linker oligopeptide. As noted above the linker may have the sequence SSGGGGSGGGGSGGSAL.

The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration to the target site. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

Whole antibodies, and F(ab')₂ fragments are "bivalent". By "bivalent" we mean that the said antibodies and F(ab')₂ fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining site. Although the antibody may be a polyclonal antibody, it is preferred if it is a monoclonal antibody. In some circumstances, particularly if the antibody is going to be administered repeatedly to a human patient, it is preferred if the monoclonal antibody is a human monoclonal antibody or a humanised monoclonal antibody.

Suitably prepared non-human antibodies can be "humanised" in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.

The antibodies may be human antibodies in the sense that they have the amino acid sequence of human antibodies with specificity as defined herein but they may be prepared using methods known in the art that do not require immunisation of humans, for example as described herein.

A second aspect of the invention provides an isolated recombinant antibody that binds to a normal human prostate epithelial cell and/or cultured human prostate tumour cell to a greater extent than to a prostate fibroblast or muscle cell, and competes with the binding of an antibody of the first aspect of the invention, preferably an antibody having the sequence set out above, to a normal human prostate epithelial cell and/or cultured human prostate tumour cell.

Suitable antibodies can be identified by means of a screen. A suitable method or screen for identifying antibodies, peptides or other molecules which selectively bind a target cell, protein or polypeptide may comprise contacting the target cell, protein or polypeptide with a test antibody, peptide or other molecule under conditions where binding can occur, and then determining if the test antibody, molecule or peptide has bound the target cell, protein or peptide. The ability of the antibody of the first aspect of the invention to block such binding can be assessed. Methods of detecting binding between two moieties are well known in the art of biochemistry. Preferably, the known technique of phage display is used to identify antibodies, peptides or other ligand molecules suitable for use as binding moieties. Examples of how suitable screens can be performed are provided in the Examples.

A further aspect of the invention provides a recombinant polynucleotide encoding an antibody of the invention. A still further aspect of the invention provides a polynucleotide encoding at least one, two, three, four or five, or six CDR sequences selected from a) GFTFSSYA (CDRl of IGHV3-30 like portion of 98.1); ISYDGSNK (CDR2 of IGHV3-30 like portion of 98.1); ARVYFRLWGQGTLVTV (CDR3 of IGHV3-30 like portion of 98.1); SSNIGSNY (CDRl of IGLV1-47 like portion of 98.1); RNN (CDR2 of IGLVl- 47 like portion of 98.1); AAWDDSLPVFGGGTKLTVLGAAA (CDR3 of IGLVl -47 like portion of 98.1); with no or up to 1, 2, 3 or less preferably 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitutions in each CDR sequence; or

Such sequences encoding individual CDR sequences are considered to be useful in assembling a polynucleotide that encodes an antibody that has the desired binding characteristics (or a fragment thereof).

A further aspect of the invention provides an expression vector comprising a recombinant polynucleotide of the invention. The genetic constructs of the invention can be prepared and manipulated using methods well known in the art. The present invention also relates to a host cell transformed with a genetic (preferably DNA construct) construct of the present invention. The host cell can be either 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., Bethesda, MD, USA, and RRl 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 includeBHKl, 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-I cells available from the ATCC as CRL 1650.

A further aspect of the invention provides an agent comprising an antibody of the invention. The antibody may comprise or be bound covalently or non-covalently to a therapeutic or imaging or detection agent or may be provided in a kit alongside a therapeutic or imaging or detection agent. The recombinant antibody of the invention allows, for example, the therapeutic or imaging agent to be targeted to prostate cancer cells, or the detection agent to bind to prostate cancer cells in a patient sample. The Figures show that the difference in intensity of binding is sufficient to allow cancer cells to be distinguished from non-cancer cells.

The therapeutic or imaging or detection agent may be bound to the antibody (which may be tagged, as discussed further below) via one or more selective non- covalent interactions, for example selective antibody-antigen interaction or biotin- avidin/streptavidin interaction. In an example, the recombinant antibody may comprise an antigen, biotin or avidin/strepatvidin tag, for example a His-6 tag or a Myc tag, as well known to those skilled in the art and as described in the Examples. The imaging agent may be attached to a further entity, for example a further antibody. Such arrangements will be well known to those skilled in the art, are discussed further below and examples are also shown in the present Examples. For imaging or therapy it is preferred that the antibody comprises or is bound covalently to a therapeutic or imaging or detection agent. For imaging or therapy in a human patient it is preferred that the antibody has human CDR and framework regions, as discussed above.

Agents indicated to be suitable as imaging agents (as discussed further below) may also be useful as detection agents. Additional detection agents will be well known to those skilled in the art and include Alexa 488, a fluorescent label for in vitro work, available from Invitrogen Ltd, 3 Fountain Drive, Inchinnan Business Park, Paisley. UK PA4 9RF. Other suitable fluorescent labels will be well known to those skilled in the art and include others available from Invitrogen (for example under the trademark Molecular Probes™). Isotope labels may be particularly suitable for in vivo use, as discussed further below.

The therapeutic agent may be or comprise a cytotoxic moiety; more preferably, the cytotoxic moiety is directly and/or indirectly cytotoxic.

By "directly cytotoxic" we include the meaning that the moiety is one which on its own is cytotoxic. By "indirectly cytotoxic" we include the meaning that the moiety is one which, although is not itself cytotoxic, can induce cytotoxicity, for example by its action on a further molecule or by further action on it.

Preferably, the cytotoxic moiety is a directly cytotoxic chemo therapeutic agent. Optionally, the cytotoxic moiety is a directly cytotoxic polypeptide. Cytotoxic chemotherapeutic agents are well known in the art. Cytotoxic chemotherapeutic agents, such as anticancer agents, include: alkylating agents including nitrogen mustards such as mechlorethamine (HN₂), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); Antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2'-deoxycoformycin). Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (rnithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes. Miscellaneous agents including platinum coordination complexes such as cisplatin (cw-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o,/?'-DDD) and amino glutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.

Various of these agents have previously been attached to antibodies and other target site-delivery agents, and so agents of the invention comprising these agents may readily be made by the person skilled in the art. For example, carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159; incorporated herein by reference) may be used to conjugate a variety of agents, including doxorubicin, to antibodies or peptides. Carbodiimides comprise a group of compounds that have the general formula Ri- N=C=N-R₂, where Ri and R₂ can be aliphatic or aromatic, and are used for synthesis of peptide bonds. The preparative procedure is simple, relatively fast, and is carried out under mild conditions. Carbodiimide compounds attack carboxylic groups to change them into reactive sites for free amino groups.

The water soluble carbodiimide, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is particularly useful for conjugating a functional moiety to a binding moiety and may be used to conjugate doxorubicin to tumour homing peptides. The conjugation of doxorubicin and a binding moiety requires the presence of an amino group, which is provided by doxorubicin, and a carboxyl group, which is provided by the binding moiety such as an antibody or peptide.

In addition to using carbodiimides for the direct formation of peptide bonds, EDC also can be used to prepare active esters such as N-hydroxysuccinimide (NHS) ester. The NHS ester, which binds only to amino groups, then can be used to induce the formation of an amide bond with the single amino group of the doxorubicin. The use of EDC and NHS in combination is commonly used for conjugation in order to increase yield of conjugate formation (Bauminger & Wilchek, supra, 1980).

Other methods for conjugating a cytotoxic moiety to a binding moiety also can be used. For example, sodium periodate oxidation followed by reductive alkylation of appropriate reactants can be used, as can glutaraldehyde cross-linking. However, it is recognised that, regardless of which method of producing a conjugate of the invention is selected, a determination must be made that the binding moiety maintains its targeting ability and that the functional moiety maintains its relevant function. In one embodiment of the invention, the cytotoxic moiety is a cytotoxic peptide or polypeptide moiety by which we include any moiety which leads to cell death. Cytotoxic peptide and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor and the like. Methods for linking them to targeting moieties such as antibodies are also known in the art. The use of ricin as a cytotoxic agent is described in Burrows & Thorpe (1993) Proc. Natl. Acad. Sci. USA 90, 8996-9000, incorporated herein by reference, and the use of tissue factor, which leads to localised blood clotting and infarction of a tumour, has been described by Ran et al (1998) Cancer Res. 58, 4646-4653 and Huang et al (1997) Science 275, 547-550. Tsai et al (1995) Dis. Colon Rectum 38, 1067-1074 describes the abrin A chain conjugated to a monoclonal antibody and is incorporated herein by reference. Other ribosome inactivating proteins are described as cytotoxic agents in WO 96/06641. Pseudomonas exotoxin may also be used as the cytotoxic polypeptide moiety (see, for example, Aiello et al (1995) Proc. Natl. Acad. Sci. USA 92, 10457-10461; incorporated herein by reference).

Certain cytokines, such as TNFα and IL-2, may also be useful as cytotoxic agents.

Certain radioactive atoms may also be cytotoxic if delivered in sufficient doses. Thus, the cytotoxic moiety may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-I l l, rhenium- 186, rhenium- 188 or yttrium-90, or any other isotope which emits enough energy to destroy neighbouring cells, organelles or nucleic acid. Preferably, the isotopes and density of radioactive atoms in the agents of the invention are such that a dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000 cGy) is delivered to the target site and, preferably, to the cells at the target site and their organelles, particularly the nucleus. The radioactive atom may be attached to the binding moiety in known ways. For example, a chelating agent may be attached to the binding moiety and used to attach ¹¹¹In or ⁹⁰Y. Tyrosine residues may be directly labelled with ¹²⁵I or ¹³¹I.

The cytotoxic moiety may be a suitable indirectly-cytotoxic polypeptide. In a particularly preferred embodiment, the indirectly cytotoxic polypeptide is a polypeptide which has enzymatic activity and can convert a non-toxic and/or relatively non-toxic prodrug into a cytotoxic drug. When the binding moiety is an antibody this type of system is often referred to as ADEPT (Antibody-Directed Enzyme Prodrug Therapy). The system requires that the binding moiety locates the enzymatic portion to the desired site in the body of the patient and after allowing time for the enzyme to localise at the site, administering a prodrug which is a substrate for the enzyme, the end product of the catalysis being a cytotoxic compound. The object of the approach is to maximise the concentration of drug at the desired site and to minimise the concentration of drug in normal tissues (see Senter, P.D. et al (1988) "Anti-tumour effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate" Proc. Natl. Acad. Sci. USA 85, 4842-4846; Bagshawe (1987) Br. J. Cancer 56, 531-2; and Bagshawe, K.D. et al (1988) "A cytotoxic agent can be generated selectively at cancer sites" Br. J. Cancer. 58, 700-703.)

In a preferred embodiment, the invention provides an agent wherein the cytotoxic moiety is capable of converting a non-cytotoxic prodrug into a cytotoxic drug.

The enzyme and prodrug of the system using a targeted enzyme as described herein may be any of those previously proposed. The cytotoxic substance may be any existing anti-cancer drug such as an alkylating agent; an agent which intercalates in DNA; an agent which inhibits any key enzymes such as dihydro folate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinases or topoisomerase; or an agent which effects cell death by interacting with any other cellular constituent. Etoposide is an example of a topoisomerase inhibitor. Reported prodrug systems include: a phenol mustard prodrug activated by an E. coli β-glucuronidase (Wang et al, 1992 and Ro filer et al, 1991); a doxorubicin prodrug activated by a human β-glucuronidase (Bosslet et al, 1994); further doxorubicin prodrugs activated by coffee bean α-galactosidase (Azoulay et al, 1995); daunorubicin prodrugs, activated by coffee bean α-D-galactosidase (Gesson et al, 1994); a 5-fluorouridine prodrug activated by an E. coli β-D- galactosidase (Abraham et al, 1994); and methotrexate prodrugs {e.g. methotrexate-alanine) activated by carboxypeptidase A (Kuefher et al, 1990, Vitols et al, 1992 and Vitols et al, 1995). These and others are included in Table A, below.

Table A:

Table A is adapted from Bagshawe (1995) Drug Dev. Res. 34, 220-230, from which full references for these various systems may be obtained; the taxol derivative is described in Rodrigues, M.L. et al (1995) Chemistry & Biology 2, 223).

Suitable enzymes for forming part of the enzymatic portion a agent of the invention include: exopeptidases, such as carboxypeptidases G, Gl and G2 (for glutamylated mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs) and aminopeptidases (for 2-α-aminocyl MTC prodrugs); endopeptidases, such as e.g. thrombolysin (for thrombin prodrugs); hydrolases, such as phosphatases (e.g. alkaline phosphatase) or sulphatases (e.g. aryl sulphatases) (for phosphylated or sulphated prodrugs); amidases, such as penicillin amidases and arylacyl amidase; lactamases, such as β-lactamases; glycosidases, such as β-glucuronidase (for β-glucuronomide anthracyclines), α-galactosidase (for amygdalin) and β-galactosidase (for β-galactose anthracycline); deaminases, such as cytosine deaminase (for 5FC); kinases, such as urokinase and thymidine kinase (for gancyclovir); reductases, such as nitroreductase (for CB 1954 and analogues), azoreductase (for azobenzene mustards) and DT-diaphorase (for CB 1954); oxidases, such as glucose oxidase (for glucose), xanthine oxidase (for xanthine) and lactoperoxidase; DL-racemases, catalytic antibodies and cyclodextrins.

Preferably, the prodrug is relatively non-toxic compared to the cytotoxic drug. Typically, it has less than 10% of the toxicity, preferably less than 1% of the toxicity as measured in a suitable in vitro cytotoxicity test.

It is likely that the moiety which is able to convert a prodrug to a cytotoxic drug will be active in isolation from the rest of the agent of the invention but it is necessary only for it to be active when (a) it is in combination with the rest of the agent of the invention and (b) the agent of the invention is attached to, adjacent to or internalised in target cells.

When each moiety of the agent of the invention is a polypeptide, the two portions may be linked together by any of the conventional ways of cross-linking polypeptides, such as those generally described in O'Sullivan et al (1979) Anal.

Biochem. 100, 100-108. For example, the binding moiety may be enriched with thiol groups and the further moiety reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP).

Amide and thioether bonds, for example achieved with m-maleimidobenzoyl-N- hydroxysuccinimide ester, are generally more stable in vivo than disulphide bonds. Alternatively, the agent may be produced as a fusion compound by recombinant DNA techniques whereby a length of DNA comprises respective regions encoding the two moieties of the agent of the invention either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the agent. Conceivably, the two portions of the agent may overlap wholly or partly.

The cytotoxic moiety may be a radiosensitizer. Radiosensitizers include fluoropyrimidines, thymidine analogues, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3- aminobenzodiamide, etanixadole, pimonidazole and misonidazole (see, for example, McGinn et al (1996) J Natl. Cancer Inst. 88, 1193-11203; Shewach & Lawrence (1996) Invest. New Drugs 14, 257-263; Horsman (1995) Acta Oncol. 34, 571-587; Shenoy & Singh (1992) CHn. Invest. 10, 533-551 ; Mitchell et al (1989) Int. J. Radial Biol. 56, 827-836; Iliakis & Kurtzman (1989) Int. J. Radiat. Oncol. Biol. Phys. 16, 1235-1241; Brown (1989) Int. J. Radiat. Oncol. Biol. Phys. 16, 987-993; Brown (1985) Cancer 55, 2222-2228).

Also, delivery of genes into cells can radiosensitise them, for example delivery of the p53 gene or cyclin D (Lang et al (1998) J Neurosurg. 89, 125-132; Coco Martin et al (1999) Cancer Res. 59, 1134-1140).

The further moiety may be one which becomes cytotoxic, or releases a cytotoxic moiety, upon irradiation. For example, the boron- 10 isotope, when appropriately irradiated, releases α particles which are cytotoxic (for example, see US 4, 348, 376 to Goldenberg; Primus et al (1996) Bioconjug. Chem. 7, 532-535).

Similarly, the cytotoxic moiety may be one which is useful in photodynamic therapy such as photo frin (see, for example, Dougherty et al (1998) J Natl. Cancer Inst. 90, 889-905). The further moiety may comprise a nucleic acid molecule which is directly or indirectly cytotoxic. For example, the nucleic acid molecule may be an antisense oligonucleotide which, upon localisation at the target site is able to enter cells and lead to their death. The oligonucleotide, therefore, may be one which prevents expression of an essential gene, or one which leads to a change in gene expression which causes apoptosis. Alternatively, the cytotoxic moiety is a nucleic acid molecule encoding a directly and/or indirectly cytotoxic polypeptide.

Examples of suitable oligonucleotides include those directed at bcl-2 (Ziegler et al (1997) J Natl. Cancer Inst. 89, 1027-1036), and DNA polymerase α and topoisomerase Ilα (Lee et al ( 1996) A nticancer Res. 16, 1805-1811.

Peptide nucleic acids may be useful in place of conventional nucleic acids (see Knudsen & Nielsen (1997) Anticancer Drugs 8, 113-118).

In a further embodiment, the binding moiety may be comprised in a delivery vehicle for delivering nucleic acid to the target. The delivery vehicle may be any suitable delivery vehicle. It may, for example, be a liposome containing nucleic acid, or it may be a virus or virus-like particle which is able to deliver nucleic acid. In these cases, the binding moiety is typically present on the surface of the delivery vehicle. For example, the binding moiety, such as a suitable antibody fragment, may be present in the outer surface of a liposome and the nucleic acid to be delivered may be present in the interior of the liposome. As another example, a viral vector, such as a retroviral or adenoviral vector, is engineered so that the binding moiety is attached to or located in the surface of the viral particle thus enabling the viral particle to be targeted to the desired site. Targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle. Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein. Targeted retroviruses are also available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre- existing viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).

Immunoliposomes (antibody-directed liposomes) may be used in which the binding moiety is an antibody. For the preparation of immuno-liposomes MPB- PE (N-[4-(p-maleimidophenyl)-butyryl]-phosphatidylethanolamine) is synthesised according to the method of Martin & Papahadjopoulos (1982) J Biol. Chem. 257, 286-288. MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface. The liposome is conveniently loaded with the DNA or other genetic construct for delivery to the target cells, for example, by forming the said liposomes in a solution of the DNA or other genetic construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 μm and 0.2 μm pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min. Freshly prepared MPB-PE-liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight. The immunoliposomes are separated from unconjugated antibodies by ultracentrifugation at 80 000 x g for 45 min. Immunoliposomes may be injected intraperitoneally or directly into the tumour.

The nucleic acid delivered to the target site may be any suitable DNA which leads, directly or indirectly, to cytotoxicity. For example, the nucleic acid may encode a ribozyme which is cytotoxic to the cell, or it may encode an enzyme which is able to convert a substantially non-toxic prodrug into a cytotoxic drug (this latter system is sometime called GDEPT: Gene Directed Enzyme Prodrug Therapy).

Ribozymes which may be encoded in the nucleic acid to be delivered to the target are described in Cech and Herschlag "Site-specific cleavage of single stranded

DNA" US 5,180,818; Altman et al "Cleavage of targeted RNA by RNAse P" US

5,168,053, Cantin et al "Ribozyme cleavage of HIV-I RNA" US 5,149,796; Cech et al "RNA ribozyme restriction endoribonucleases and methods", US 5,116,742; Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endonucleases and methods", US 5,093,246; and Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods; cleaves single-stranded RNA at specific site by transesterification", US 4,987,071, all incorporated herein by reference. Suitable targets for ribozymes include transcription factors such as c-fos and c-myc, and bcl-2. Durai et al (1997) Anticancer Res. 17, 3307-3312 describes a hammerhead ribozyme against bcl-2.

EP 0 415 731 describes the GDEPT system. Similar considerations concerning the choice of enzyme and prodrug apply to the GDEPT system as to the ADEPT system described above.

The nucleic acid delivered to the target site may encode a directly cytotoxic polypeptide.

Alternatively, the further moiety may comprise a polypeptide or a polynucleotide encoding a polypeptide which is not either directly or indirectly cytotoxic but is of therapeutic benefit. Examples of such polypeptides include anti-proliferative or anti-inflammatory cytokines, and anti-proliferative, immunomodulatory or factors influencing blood clotting which may be of benefit in medicine, for example in the treatment of cancer.

The further moiety may usefully be an inhibitor of angiogenesis such as the peptides angiostatin or endostatin. The further moiety may also usefully be an enzyme which converts a precursor polypeptide to angiostatin or endostatin. Human matrix metallo -proteases such as macrophage elastase, gelatinase and stromolysin convert plasminogen to angiostatin (Cornelius et al (1998) J Immunol. 161, 6845-6852). Plasminogen is a precursor of angiostatin. In a preferred embodiment, the invention provides an agent further comprising a readily detectable moiety. Such an agent may be useful as an imaging or diagnostic agent.

By a "readily detectable moiety" we include the meaning that the moiety is one which, when located at the target site following administration of the agent of the invention into a patient, may be detected, typically non-invasively from outside the body and the site of the target located. Thus, the agents of this embodiment of the invention are useful in imaging and diagnosis.

Typically, the readily detectable moiety is or comprises a radioactive atom which is useful in imaging. Suitable radioactive atoms include ^99mTc and ¹²³I for scintigraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRJ) such as ¹²⁵I, ¹²³I again, ¹³¹I, ¹¹¹In, ¹⁹F, ¹³C, ¹⁵N, ¹⁷O, gadolinium, manganese or iron. Clearly, the agent of the invention must have sufficient of the appropriate atomic isotopes in order for the molecule to be readily detectable.

The radio- or other labels may be incorporated in the agent of the invention in known ways. For example, if the binding moiety is a polypeptide it may be biosynthesised or may be synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine- 19 in place of hydrogen. Labels such as ^99mTc, ¹²³I, ¹⁸⁶Rh, ¹⁸⁸Rh and ^{1 11}In can, for example, be attached via cysteine residues in the binding moiety. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Comm. 80, 49-57) can be used to incorporate ¹²³I. Reference ("Monoclonal Antibodies in Immunoscintigraphy", J-F Chatal, CRC Press, 1989) describes other methods in detail.

Preferably, the readily detectable moiety comprises a radioactive atom, such as, for example technetium-99m or iodine- 123, Iodine- 125 or Iodine-131. Alternatively, the readily detectable moiety may be selected from the group comprising: iodine- 125, iodine- 123; iodine-131 ; indium-I l l ; fluorine- 19; carbon- 13; nitrogen- 15; oxygen- 17; gadolinium; manganese; iron.

In a preferred embodiment, the invention provides an agent further comprising a moiety capable of selectively binding to a directly or indirectly cytotoxic moiety, or to a readily detectable moiety.

In a further preferred embodiment of the invention the further moiety is able to bind selectively to a directly or indirectly cytotoxic moiety or to a readily detectable moiety. Thus, in this embodiment, the further moiety may be any moiety which binds to a further compound or component which is cytotoxic or readily detectable.

The further moiety may, therefore be an antibody which selectively binds to the further compound or component, or it may be some other binding moiety such as streptavidin or biotin or the like. The following examples illustrate the types of molecules that are included in the invention; other such molecules are readily apparent from the teachings herein.

A bispecifϊc antibody wherein one binding site comprises the binding moiety (which selectively binds to a normal human prostate epithelial cell and/or cultured human prostate tumour cell to a greater extent than to a prostate fibroblast or muscle cell, as defined herein) and the second binding site comprises a moiety which binds to, for example, an enzyme which is able to convert a substantially non-toxic prodrug to a cytotoxic drug.

Alternatively, the agent may comprise an antibody of the invention, to which is bound biotin. Avidin or streptavidin which has been labelled with a readily detectable label may be used in conjunction with the biotin labelled antibody in a two-phase imaging system wherein the biotin labelled antibody is first localised to the target site in the patient, and then the labelled avidin or streptavidin is administered to the patient. Bispecific antibodies and biotin/streptavidin (avidin) systems are reviewed by Rosebrough (1996) QJNucl. Med. 40, 234-251.

In a preferred embodiment of the invention, the binding moiety and the further moiety are polypeptides which are fused.

A further aspect of the invention provides a pharmaceutical composition comprising an antibody, agent or kit of parts of the invention and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is included that the formulation is sterile and pyrogen free. Suitable pharmaceutical carriers are well known in the art of pharmacy. The carrier(s) must be "acceptable" in the sense of being compatible with the agent of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free; however, other acceptable carriers may be used. Methods of manufacturing a pharmaceutical composition or medicament using an active agent, such as the agent of the invention, for example an antibody, are well known to persons skilled in the art of medicine and pharmacy.

A further aspect of the invention provides an antibody, agent, kit of parts or pharmaceutical composition of the invention for use in medicine.

A further aspect of the invention provides an antibody, agent, kit of parts or pharmaceutical composition of the invention for therapy, diagnosis or imaging of cancer, for example prostate cancer.

The recombinant antibody may have a therapeutic effect even if not associated with a further therapeutic agent. We consider that the antibody can induce cell cycle arrest (block at Gl) - see Examples and Figure 2.

A further aspect of the invention provides a method of treating or imaging of cancer, for example prostate cancer, the method comprising the step of administering an effective amount of an appropriate antibody, kit of parts or pharmaceutical composition of the invention to a patient.

By "effective amount" we include an amount of the agent of the invention that is, as appropriate, sufficient to image or prevent and/or reduce a condition associated with the particular cancer to be treated, for example prostate cancer. For example, an effective amount may prevent and/or reduce the size and/or rate of growth and/or spread of a tumour. An effective amount may prevent and/or reduce the cellular differentiation of a cancer cell, thereby preventing and/or reducing metastasis. Appropriate tests for determining the therapeutic effect and most appropriate route of administration of an agent, composition or medicament of the invention will be known to those skilled in the relevant arts of medicine

A further aspect of the invention provides a method of diagnosing (or aiding in diagnosing) cancer, for example prostate cancer in a patient, the method comprising the step of detecting the level or presence or absence of binding of an antibody, agent or kit of parts of the invention to a sample from the patient.

It will be understood by those skilled in the relevant arts of molecular and cellular biology that the agent of the invention could be used to identify cells, such as cancer cells, potentially susceptible to treatment using an antibody, agent or kit of parts as defined herein and/or a medicament as defined herein and/or a pharmaceutical composition as defined herein. For example, the agent of the invention could be used (in accordance with the methods of the invention and those described in the accompanying Examples) as a diagnostic reagent to detect cancer cells, for example prostate cancer cells, in a test sample from a patient. It would be clear to a skilled person that the identification of cells binding to an agent of the invention may indicate that the patient would be suitable for delivering a cytotoxic moiety using the agent of the invention and therefore susceptible to treatment using the agent and/or medicament and/or pharmaceutical composition of the invention. It will be appreciated that determining whether the cell displays a binding response associated with cancer or if imaging indicates the presence of cancer, may in itself be diagnostic of cancer or it may be used by the clinician as an aid in reaching a diagnosis.

For example, in relation to prostate cancer, it is useful if the clinician undertakes a histopatho logical examination of biopsy tissue or measures plasma PSA level (in relation to prostate cancer) or carries out external digital examination or carries out imaging. The possibility of using the blood IGF-I level has also been suggested (Chan et al (1998) Science 279, 563-566).

The method may be useful in determining the susceptibility of a human patient to cancer, diagnosing cancer in a human patient or predicting the relative prospects of a particular outcome of a cancer in a human patient.

Thus, the method the invention may be useful in prognosis or aiding prognosis. The method may be used as an adjunct to known prognostic methods such as histopathological examination of biopsy tissue, or measurement of plasma PSA levels (in relation to prostate cancer), external digital examination or other imaging techniques.

It will be appreciated that the imaging or diagnostic techniques of the invention will be useful to the clinician in determining how to manage the cancer in the patient. For example, since binding to the antibody or agent of the invention, is considered to be associated with increasing tumourogenicity, the clinician may use the information concerning binding to the antibody or agent (either using imaging techniques or ex vivo binding tests), to facilitate decision making regarding treatment of the patient. Thus, if the response is indicative of a low metastatic potential of said prostate cancer, unnecessary radical surgery may be avoided. Similarly, if the response is indicative of a high metastatic potential of said cancer, for example prostate cancer, radical surgery (ie prostatectomy) may be the preferred treatment. Imaging may also indicate where surgery may be required or what drug treatment is appropriate.

Although it is believed that any sample containing cells derived from the patient may be useful in the methods of the invention, it is preferred if the cells are derived from a sample of the tissue in which cancer is suspected or in which cancer may be or has been found. For example, if the tissue in which cancer is suspected or in which cancer may be or has been found is prostate, it is preferred if the sample containing cells is derived from the prostate of the patient. Samples of prostate may be obtained by surgical excision, laproscopy and biopsy, endoscopy and biopsy, and image-guided biopsy. The image may be generated by ultrasound or technetium-99-labelled antibodies or antibody fragments which bind or locate selectively at the prostate, such as antibodies of the present invention.

The sample may be directly derived from the patient, for example, by biopsy of the tissue, or it may be derived from the patient from a site remote from the tissue, for example because cells from the tissue have migrated from the tissue to other parts of the body. Alternatively, the sample may be indirectly derived from the patient in the sense that, for example, the tissue or cells therefrom may be cultivated in vitro, or cultivated in a xenograft model. Thus, although the cells derived from the patient may have been physically within the patient, they may alternatively have been replicated from cells which were physically within the patient. The tumour tissue may be taken from the primary tumour or from metastases. The sample may be lymph nodes, lymph or blood and the spread of disease detected.

Although any sample containing cells derived from the patient is useful in the methods of the invention, it is preferred (in relation to prostate cancer) if the sample is selected from the group consisting of prostate tissue, blood, urine or semen. Prostate tissue can be obtained from a patient using standard surgical techniques. Cells derived from the prostate are found in small numbers in the urine and in the blood. Although it is preferred that the sample containing cells from the patient is, or is derived directly from, a cell of the patient, such as a prostate cell, a sample indirectly derived from a patient, such as a cell grown in culture, is also included within the invention. The tumour tissue may be taken from the primary tumour or from metastases, and particularly may be taken from the margins of the tumour.

It will be appreciated that the aforementioned methods may be used for presymptomatic screening of a patient who is in a risk group for cancer. For example, men older than about 60 years are at greater risk of prostate cancer than men below the age of 35. Similarly, the methods may be used for the pathological classification of tumours such as prostate tumours.

It is preferred (in relation to prostate cancer) that if blood, semen, lymphatic circulation or urine is the source of the said sample containing cells derived from the patient that the sample is enriched for prostate-derived tissue or cells. Enrichment for prostate cells may be achieved using, for example, cell sorting methods such as fluorescent activated cell sorting (FACS) using a prostate- selective antibody such as one directed to prostate-specific antigen (PSA) or prostate specific membrane antigen (PSMA). Alternatively, enrichment may be achieved using magnetic beads or other solid support, for example a column, coated with such a prostate-specific antibody, for example an anti-PSA antibody or an antibody of the present invention.

The source of the said sample also includes biopsy material and tumour samples, including fresh or frozen tissue. Tissue (preferably fresh tissue) may be dissociated into single cells by gentle enzymatic treatment.

It is preferred that if the sample containing cells derived from the patient is not a substantially pure sample of the tissue or cell type in question that the sample is enriched for the said tissue or cells. Cells may be analysed individually, for example following single-cell immobilisation techniques. Methods by which single cells may be analysed include methods in which the technique of Laser Capture Microdissection (LCM) is used. This technique may be used to collect single cells or homogeneous cell populations for analysis and is described in, for example, Jin et al (1999) Lab Invest 79(4), 511-512; Simone et al (1998) Trends Genet 14(7), 272-276; Luo et al (1999) Nature Med 5(1), 117-122; Arcuturs Updates, for example June 1999 and February 1999; US 5,859,699 (all incorporated herein by reference). The cells of interest are visualised, for example by immunohistochemical techniques, and transferred to a polymer film that is activated by laser pulses. The technique may also be used for isolation of cells which are negative for a particular component. Microscopes useful in performing LCM are manufactured by Arcturus Engineering, Inc., 1220 Terra Bella Avenue, Mountain View, CA 94042, USA.

LCM may be used with other isolation or enrichment methods. For example, LCM may be used following a method which enriches the sample for the target cell type.

Any published documents referred to herein are hereby incorporated by reference.

The invention will now be described by reference to the following, non-limiting Example and Figures.

Figure 1. Reactivity of scFv clones 95.2 and 98.1 with human prostate cells as assessed by flow cytometry. FACS histograms demonstrating the binding of soluble scFv to prostate cancer cell lines (1), normal prostate cells (2) and primary tumour derived cell lines (3). Antibody binding was carried out at 4 °C using unfixed cells. Bound scFv 95.2 ( *) was detected through its His tag by using anti-6X His antibody followed by Alexa 488 ( +).

Figure 2. Reactivity of scFv clones 95.2 and 98.1 with human prostate cells and tissue as assessed by Immunocytochemistry and immunohistochemistry. 1. Reactivity of scFv 95.2 and 98.1 with formaldehyde fixed but not permeablised prostate epithelial cells. Particulate deposits (arrow). 2. Reactivity of scFv antibodies with normal and pathogenic human prostate tissue sections, embedded in paraffin. Bound scFv antibody was detected through its His tag by using anti- 6X His antibody followed by Alexa 488 or HRP labeled antibody. 3. Biological effect of scFv 95.2 and 98.1 on the cell cycle distribution of LNCaP cells. Figure 3. Amino acid sequence of clone 95,2 Figure 4. Amino acid sequence of clone 98.1

Example 1

Using scFv phage display we have successfully generated a panel of 15 human framework recombinant scFv antibodies specific to the cell surface of normal human prostate epithelial cells. Characterisation of this panel has demonstrated that two of these antibodies, scFv 95.2 and scFv 98.1, additionally exhibit a strong reactivity with prostate tumour cells that increases with the grade of tumour. We aim to characterise further scFv 92.5 and scFv 98.1 by identifying their target antigen and defining the expression profile of their target antigen in normal and malignant human tissue. We aim to develop further scFv 92.5 and scFv 98.1 for the in vivo targeting of human prostate cancer.

We seek to characterise the pharmacokinetic properties of scFv 95.2 and scFv 98.1, identify their target antigens and further develop these antibodies for therapeutic and diagnostic applications in prostate cancer.

Plan of investigation and methodology

Initial characterisation of scFv 95.2 and scFv 98.1 We have used flow cytometric analysis and fluorescent confocal microscopy to examine the reactivity of scFv 95.2 and scFv 98.1 with normal and malignant cells derived from the human prostate. Flow cytometric histograms presented in Figurel show that scFv 95.2 and scFv 98.1 have reactivity with normal primary human prostate epithelial cells while little reactivity is detectable to primary prostate fibroblasts and primary prostate muscle cells. Additionally, scFv 95.2 and scFv 98.1 both exhibit strong reactivity with three human prostate cancer cell lines derived from metastatic tumours, namely LNCaP, PC3 and DU 145. Further, scFv 95.2 and scFv 98.1 also exhibit reactivity with the 957/hTERT (not tumourogenic in mice), RC-92a/hTERT (partially tumourogenic in mice) and RC-5T/hTERT (fully tumourogenic in mice) cell lines, each of which have been derived by telomerase immortalisation of cells from primary human prostate tumours (9). As can be seen, scFv 95.2 and scFv 98.1 ' s reactivity with this panel of cells increases with increasing tumourogenicity. These results indicate that expression of the target epitope for scFv 95.2 and scFv 98.1 may be up regulated during the course of tumour progression. Using fluorescent confocal microscopy to visualise scFv 95.2 and scFv 98.1 binding on formaldehyde fixed un- permeablised cells, we have found that the target epitope for both antibodies is expressed on the cell surface, with a distinct punctuate appearance (Figure 2). Moreover, the detection of particulate aggregates at the periphery of the cells suggests that the target molecule for scFv 95.2 may also be shed or secreted. Notably, the expression pattern of the target epitope for scFv 98.1 is distinct from that of scFv 95.2, not only in its appearance but also in its cellular distribution. Specifically, in normal human prostate epithelial cells, the target epitope for scFv 98.1 appears to be concentrated at the polar tips of the cells (Figure 2) while in prostate tumour cells it appears to be uniformly expressed on the entire surface of the cells (Figure 2). Additionally, the absence of detectable particulate aggregates indicates that the target molecule for scFv 98.1 may be cell surface bound but not secreted. However, the possibility exists that the target molecule for scFv 98.1 is secreted but not laid down in aggregates as seen with that for scFv 95.2. To date, several prostate-specific proteins that are cell surface bound and/or secreted have been reported. However, a comparison of the expression profiles of these compared to that of the epitope for scFv 95.2 and scFv 98.1 in several established prostate tumour cell lines (as presented in Figure 1), strongly suggests that the molecules recognised by scFv 95.2 and scFv 98.1 are distinct from any of the above markers of prostate epithelial cells (Table 1).

Table 1. Comparison of expression of the target epitope of scFv clones with a spectrum of prostate specific protein in established human prostate tumour cell lines.

In order to further examine the reactivity of scFv 92.5 and scFv 98.1 with human prostate tumours, we have used a human prostate cancer tissue microarray composed of 10 normal and 20 prostate cancer tissue samples (LF-PR-I, Cytomyx Ltd). scFv 95.2 exhibited reactivity with 7 out of 10 normal and 17 out of 20 tumour samples present on this array. Similarly, scFv 98.1 exhibited reactivity with prostate epithelia in all the normal and tumour samples present on the array. These results are promising and can be followed up with further studies using arrays of graded prostate tumour tissues. Representative fields for one normal sample and one adenocarcinoma are shown in Figure. 2. Towards characterisation of biological affects of scFv 95.2 and scFv98.1, we have found that the target epitope for scFv 95.2 is not trypsin sensitive while that of scFv98.1 does exhibit sensitivity to trypsin. Additionally, binding of scFv 95.2 and scFv98.1 to their target molecules does not elicit antibody induced cellular internalisation. This is a desirable feature in antibodies used for in vivo targeting. Further, addition of scFv 95.2 to growing cultures of LNCaP cells induces a significant Gl arrest of cell growth (Figure 2). Similarly, addition of scFv 98.1 to growing cultures of LNCaP also elicits a significant Gl arrest of cell growth in these cells (Figure 2).

Further Experimental work

Identification of the target antigen for scFv 95.2 and scFv 98.1

Homology searches of antibody sequence databases show that scFv 95.2 and scFv

98.1 are not previously described antibodies and, therefore, have unknown binding epitopes. As the target epitope for both antibodies appear to be expressed in abundance on the cell surface of PC3 and RC58-T/hTERT prostate tumour cell lines, we use these cell lines to generate membrane fractions for use in Western blot and immunoprecipitation studies for the target antigen. Primary prostate fibroblast and muscle cells are used as negative controls. We have already shown that both antibodies can recognise their target epitopes on formaldehyde fixed cells and tissues, suggesting that they are likely to be useful for Western blot analysis. In these experiments, the bound scFv will be detected via the c-myc or 6X Histidine tag encoded as part of the antibody framework and for which secondary antibodies are available.

These studies identify the size of the scFv 95.2 and scFv 98.1 bound molecules. Once blotting and probing conditions are established, we go on to develop the use of both antibodies in probing whole cell lysates, so that we can determine the relative abundance of their target protein in prostate cancer cell lines and tissue samples, including primary prostate epithelial cells, 957/hTERT, RC-92a/hTERT and RC-5T/hTERT, which represent the gradation from normal to highly tumourogenic cells. These cells are all derived in the same way from different primary tumours.

The identification of the molecules recognised by scFv 95.2 and scFV 98.1 is attempted by two approaches. In the first, we use 2-Dimensional protein electrophoresis (isoelectric focussing in the first dimension, SDS-PAGE in the second) of membrane fractions from PC3 and RC58-T/hTERT in combination with immunoblotting to identify the recognised protein. Once the position of the recognised proteins is defined, it can be re-located on parallel, counter stained gels and excised as spots for protein sequencing by mass spectroscopy (see below). In a second approach, immuprecipitation is used to purify the target molecule bound by scFv 95 and scFv 98.1 for protein sequencing. Initially, this is done by binding membrane fractions from PC3 and RC58-T/hTERT cells with each antibody and using anti-c-myc/anti-His bound to protein A/G-Sepharose to retrieve scFv bound molecules. Bound proteins are then released with O.lmM glycine-HCl (pH 2.7), precipitated with trichloroacetic acid and resolved by SDS-PAGE electrophoresis. Subsequent to Coomassie staining, proteins purified from the tumour cell lines are excised for protein sequencing. This is performed using matrix-assisted laser desorption ionisation (MALDI) time-of-flight mass spectroscopy and quadrupoles, time-of-flight (TOF) mass spectroscopy.

The successful identification of the antigen for scFv 95.2 and scFv 98.1 leads to further studies, where other approaches, such as gene expression analysis, are used to examine the expression of the target molecule in normal and tumour prostate cells and tissues.

Evaluation of specificity of binding of scFv 95.2 and scFv 98.1 to human tissue. In order to evaluate the efficacy of use of scFv 95.2 and scFv 98.1 for targeted therapy of prostate cancer it is important to define the tissue specificity of binding, primarily to avoid undesirable side effects. In this context, the ideal antigen for tumour targeting should be expressed at high levels on the cell surface of tumour cells or tumour supporting cells and be absent, or expressed at a very low level, on vital organs. However, to date few, if any, truly tumour specific antigens have been identified (18). Nonetheless, molecular targets with less restricted expression have proven useful for antibody therapy. Specifically, ErbB2 the antigen targeted by the approved humanised mAb Herceptin is over-expressed in 20-30% of human breast and ovarian cancers. However, ErbB2 is also expressed at low levels in epithelial cells from a variety of organs (19). Similarly, expression of PMSA the antigen targeted by 7E11-C5.3, a murine monoclonal antibody approved for imaging prostate cancer, increases with tumour progression (20). However, PSMA is also expressed on a variety of normal epithelial cells in the body. Additionally, A33, a human antigen that is a promising molecular target for antibody therapy of colon cancer, is expressed in both normal and malignant colon epithelia (21). Our initial data shows that scFv 95.2 and scFv 98.1 both bind to normal prostate epithelial cells as well as prostate tumour cells. We use formalin fixed as well as frozen format tissue micrroarrays representing whole body, normal tissue surveys to elucidate whether our antibodies bind to tissues other than prostate. Further, we have already established that both scFv 95.2 and scFv 98.1 can effectively stain low-grade human prostate tumours (Figure 2). Since, our results indicate that expression of the target epitope for scFv95.2 and scFv 98.1 may increase with disease progression (Figurel), we aim to further substantiate these findings by utilising AccuMax prostate cancer tissue microarrays, which represent stage II (where the cancer involves more tissue within the prostate but is organ confined), III (where the cancer has spread outside the prostate to nearby tissue) and IV (where the cancer has metastasised to lymph node or other tissues) prostate cancer tissue samples with full clinical details. Furthermore, we aim to use tumour microarrays representing whole body cancer surveys in order to establish whether scFv 95.2 and scFv 98.1 can bind to tumours arising from organs other than prostate. A tumour crossreactive antibody that could be used for the treatment of a range of cancers of similar tissue type would be a unique and highly valuable tool. The tissue miccroarrays intended for use in this proposal are purchased from Stretton Scientific, Cytomyx Ltd and TriStar Technology Group, LLC. The clinical application of scFv 95.2 and scFv 98.1 in prostate cancer We are particularly keen to develop targeted antibodies for imaging and therapeutic purposes in prostate cancer. In this context, we have already established that scFv 95.2 and scFv 98.1 antibodies can both induce cell cycle arrest in prostate tumour cells in vitro and hence elicit an anti-growth effect. In order to develop these antibodies for in vivo, targeting, the biophysical and pharmacokinetic properties of scFv 95.2 and scFv 98.1 are determined. Specifically, an optimised radio labelling procedure is established in order to ensure that the radio labelling procedure does not compromise optimal functionality of the antibodies. To do this, both antibodies are radio labelled with Iodine 125 using the IODO-GEN method (22) and their in vitro binding kinetics determined using saturation binding (Scatchard) assays. Finally, the biophysical and pharmacokinetic properties of scFv 95.2 and scFv 98.1 are determined with a view to the development of these antibodies for in vivo targeting i.e.; imaging or targeted therapy. Specifically, scFv radio labelled with γ-emitting technetium-99m, 99m TC, the preferred radionuclide for clinical imaging (23), is injected in to nude Balb/c mice bearing human prostate tumour xenografts and biodistribution studies conducted to evaluate the blood clearance and organ distribution for this antibody.

To do this, 8-10 wk old mice are inoculated, in the right/left flank, with 0.1 ml cell suspension containing 5X106 LNCaP cells (human prostate cancer cell line) suspended in Matrigel. After 14-18 days, tumours (100-300 mg) develop (approx. 5-8 mm). The tumour-bearing mice are used in biodistribution studies, radio labelled metabolites studies and imaging studies.

In the biodistribution studies, blood clearance of the antibody is examined together with its organ distribution. To do this, tumour-bearing mice are injected via the tail vein with 0.1 ml saline containing radio labelled antibody. At selected time points after the injection (5 min, 30 min, 60 min, 4 hr, 24 hrs and 48 hrs) mice are sacrificed by exsanguinations via cardiac puncture under general anaesthesia (isofluorane inhalation). Blood, plasma, urine, faeces and tissues are obtained by dissection (tumour, liver, kidney, thyroid, spleen, lungs, heart, stomach, small intestine, large intestine, brain, muscle, skin and bone). Radioactivity in tissue and body fluids is determined. For each time point, 3 animals are studied. Hence a total of 18 animals is required for the biodistribution studies.

In parallel, the rate of metabolism of the radiolabeled scFv is determined using HPLC. In order to examine the rate of metabolism of the radio labelled antibody, High-Performance Liquid Chromatography (HPLC) will be employed to determine radiolabeled antibody metabolites in tissue and plasma samples. To do this tumour bearing animals are injected with the radiolabeled antibody as above and at time points indicated above, further tissue and plasma samples obtained specifically for HPLC. In this experiment, 3 animals are utilised per time point. Hence a total of 18 mice is required.

Furthermore, scFv 95.2 and scFv 98.1 are radiolabeled with Iodine 124 (positron emitting radionuclide) as established above, and their ability to selectively target human prostate tumours xenografted in nude mice evaluated using Small Animal Positron Emission Tomography. The tail veins of tumour-bearing mice are cannulated after induction of anaesthesia (isofluorane/N₂O/O₂) and the mouse placed within a thermostatically controlled jig. After injection of the radiolabeled antibody in PBS via the tail cannula, dynamic emission scans are acquired on a small animal PET scanner or, images acquired using a γ camera depending on the radiolabel utilised. The mice are scanned at 0-60 min, 4 hrs and 24 hrs post antibody injection. This experiment requires a total of 5 mice.

The above uses may require modifications to improve antibody thermal stability or valency (generation of diabodies). This may constitute engineering of the antibody in order to obtain affinity maturation.

References 1. Griffiths AD et al, (1994) EMBO J. 13, 3245-3260.

2. Shamara D et al., (2006) Nature Reviews (Drug Discovery) 5, 147-159.

3. Schmidt M et al., (1999) Oncogene, 18, 1711-1721.

4. Vitaliti A et al., (2000) Cancer Res. 60, 4311-4314. 5. Schmidt M et al., (1999) Oncogene, 18, 1711-1721.

6. Vitaliti A et al., (2000) Cancer Res. 60, 4311-4314.

7. Vaday GG et al., (2004) Clin Cancer Res. 10, 5630-5639.

8. Cochet O et al., (1999) Cancer Detect Prev. 23, 506-510.

9. Gu Y et al (2006) Exp Cell Res. 312, 831-843. 10. Corey E et al. (1998) Prostate 35, 135-143.

11. Veeramani S et al. (2005) Endocrine -Related Cancer 12, 805-822.

12. Ross S et al., (2002) Cancer Res. 62, 2546-2553.

13. Gu z (2000) Oncogene 19, 1288-1296.

14. Israeli RS (1996) Cancer Res. 54, 1807-1811. 15. Su Z-Z (1996) Proc. Natl. Acad. ScL 93, 7252-7257.

16. Chen ME et al., (1998) J Biol Chem. 273, 17618-17625.

17. Hubert RS (1999) Proc. Natl. Acad. ScL 96, 14523-14528

18. Hellstrom KE and Helstrom I (2002) Tumour antigens in Bertino Jr (Ed.), Encyclopedia of cancer, 2nd Edition. Academic Press, san diego, CA, v IV, p459. 19. Disis ML and Cheeever MA (1997) Adv. Cancer Res. 71, 343-371.

20. Murphy GP et al., (1998) Cancer 83, 2259-2269.

21. Heath JK et al., (1997) Proc Natl Acad Sci USA. 94, 469-474.

22. Collingridge DA et al., (2002) Cancer Res. 62, 5912-5919.

23. George AJ et al., (1995) Proc. Natl. Acad. ScL USA. 92, 8358-8362.

Claims

1. An isolated recombinant antibody that binds to a normal human prostate epithelial cell and/or cultured human prostate tumour cell to a greater extent than to a prostate fibroblast or muscle cell, the recombinant antibody comprising at least one, two, three, four or five, or six, CDR sequence selected from a) GFTFSSYA (CDRl sequence); ISYDGSNK (CDR2 sequence); ARVYFRLWGQGTLVTV (CDR3 sequence); SSNIGSNY C(CDRl sequence); RNN (CDR2 sequence); AAWDDSLPVFGGGTKLTVLGAAA (CDR3 sequence); with no or up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative or non- conservative amino acid substitutions in each CDR sequence; or

b) GDSVSSNSAA (CDRl sequence); TYYRSKWYN (CDR2 sequence); ARLVDASFDYWGQGTLVTV (CDR3 sequence); QGISSW (CDRl sequence);

AAS (CDR2 sequence); QQANSFRTFGQGTKVEIKRAAA (CDR3 sequence); with no or up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitutions in each CDR sequence.

2. The antibody of claim 1 wherein the antibody is a scFv antibody.

3. The antibody of claim 1 or 2 having or comprising the sequence

MKYLLPTAAAGLLLLAAQPAMA RGAVESGGGVVQPGRSLRLSCAAT GFTFSSYA MHWVRQAPGKGLEWVAV ISYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYC ARVYFRLWGQGTLVTV SS GGGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGS SSNI GSNYVYWYQQLPGTAPKLLIY RNNQRPSGVPDRFSGSKSGTSAS LAISGLRSEDEADYYC AAWDDSLPVFGGGTKLTVLGAAAHHHHHH GAAEQKLISEEDLN or MKYLLPTAAAGLLLLAAQPAMAQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNS AAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLN SVTPEDTAVYYCARLVDASFDYWGQGTLVTVSSGGGGSGGGGSGGSALDIQLTQ SPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQANSFRTFGQGTKVEIKRAAAHHHHHH GAAEQKLISEEDLN.

4. An isolated recombinant antibody that binds to a normal human prostate epithelial cell and/or cultured human prostate tumour cell to a greater extent than to a prostate fibroblast or muscle cell, and competes with the binding of an antibody according to any one of claims 1 to 3, to a normal human prostate epithelial cell and/or cultured human prostate tumour cell.

5. A recombinant polynucleotide encoding an antibody according to any one of the preceding claims.

6. A recombinant polynucleotide encoding at least one, two, three, four or five, or six, CDR sequences selected from a) GFTFSSYA (CDRl sequence); ISYDGSNK (CDR2 sequence); ARVYFRL WGQGTLVTV (CDR3 sequence); SSNIGSNY C(CDRl sequence); RNN (CDR2 sequence); AAWDDSLPVFGGGTKLTVLGAAA (CDR3 sequence); with no or up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitutions in each CDR sequence; or

b) GDSVSSNSAA (CDRl sequence); TYYRSKWYN (CDR2 sequence);

ARLVDASFDYWGQGTLVTV (CDR3 sequence); QGISSW (CDRl sequence);

AAS (CDR2 sequence); QQANSFRTFGQGTKVEIKRAAA (CDR3 sequence); with no or up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitutions.

7. An expression vector comprising a recombinant polynucleotide according to claim 5 or 6.

8. A host cell transformed with a recombinant polynucleotide according to claim 5 or 6 or expression vector according to claim 7.

9. An agent or kit of parts comprising an antibody according to any one of claims 1 to 4 and a further moiety.

10. The agent or kit of parts of claim 9 comprising a therapeutic moiety.

11. The agent or kit of parts of claim 10 wherein the therapeutic moiety is a cytotoxic moiety

12. The agent or kit of parts of claim 9 comprising a readily detectable moiety.

13. The agent or kit of parts of claim 12 wherein the readily detectable moiety is or comprises a radioactive atom which is useful in imaging.

14. The agent or kit of parts according to Claim 13 wherein the radioactive atom is technetium-99m or iodine- 123.

15. The agent or kit of parts according to Claim 13 wherein the readily detectable moiety is selected from the group comprising: iodine-125; iodine-123; iodine-131; indium-I l l ; fluorine-19; carbon-13; nitrogen-15; oxygen-17; gadolinium; manganese; iron.

16. The agent or kit of parts according to any one of Claims 9 to 15 further comprising a moiety capable of selectively binding to a directly or indirectly cytotoxic moiety.

17. The agent or kit of parts according to any one of Claims 9 to 15 further comprising a moiety capable of selectively binding to a readily detectable moiety.

18. A pharmaceutical composition comprising an antibody, agent or kit of parts according to any one of the preceding claims and a pharmaceutically acceptable carrier.

19. An antibody, agent, kit of parts or pharmaceutical composition according to any one of the preceding claims for use in medicine.

20. An antibody, agent, kit of parts or pharmaceutical composition according to any one of the preceding claims for use in therapy, diagnosis or imaging of cancer, for example prostate cancer.

21. A method of treating or imaging of cancer, for example prostate cancer, the method comprising the step of administering an effective amount of an appropriate antibody, kit of parts or pharmaceutical composition according to any one of the preceding claims to a patient.

22. A method of diagnosing (or aiding in diagnosing) cancer, for example prostate cancer in a patient, the method comprising the step of detecting the level or presence or absence of binding of an antibody, agent or kit of parts according to any one of the preceding claims to a sample from the patient.