WO2000064480A1 - Treatment, imaging and diagnosis of disease using an agent which binds alfa5-integrin - Google Patents

Abstract

A compound comprising a moiety which selectively binds α5-integrin and a further moiety. The compounds are useful in treating, diagnosing and imaging diseases in which undesirable new blood vessel formation occurs, including cancer.

Description

TREATMENT, IMAGING AND DIAGNOSIS OF DISEASE USING AN AGENT WHICH BINDS ALFA5-INTEGRIN

The present invention relates to the treatment, imaging and diagnosis of disease in a patient; in particular it relates to the treatment, imaging and diagnosis of cancer.

Cancer is a major killer. Although in recent years the understanding of cancer has greatly improved, and new treatments have become available, new and improved methods of treatment are still desirable.

Most tumours, particularly solid tumours, rely on the production of new blood vessels via angiogenesis for a supply of nutrients and oxygen to sustain their growth. Other diseases such as psoriasis and retinopathies also involve undesirable angiogenesis. Vascular targeting is a known therapeutic approach to the treatment of several pathologies, notable amongst these are attempts at targeting the tumour vasculature (1 , 2; also, see Tumour Angiogenesis, Bicknell, R. , Lewis, C.E. and Ferrara, N. (1997), Oxford University Press, Oxford).

Proposed tumour vascular targets include endoglin, an endothelial cell proliferation marker that is purported to be upregulated in endothelial cells in miscellaneous human solid tumours (Thorpe & Burrows (1995) Breast Cancer Res. & Treatment 36, 237-251 ; Burrows et al (1995) Clin. Chem. Res. 1, 1623-1634); vascular endothelial growth factor (VEGF; Brekken et al (1998) Cancer Res. 58, 1952-1958; WO 96/06641); and vascular cell adhesion molecule- 1 (VCAM-1; Ran et al (1998) Cancer Res. 58, 4646- 4653). Complete tumour regressions were not observed using VCAM-1 as at target for "coaguligands" (antibodies fused to tissue factor), since VCAM-1 is only present on a minority of tumour vessels. RGD- and NGR-containing peptides isolated following phage display have also been used to target tumour vasculature (Arap et al (1998) Science 279, 377-380), and αvβ3 and other αv-containing integrins have been used as targets (WO 97/10507; WO 98/10795)

Microvascular endothelial cells have been the subject of extensive research given their involvement in a wide range of pathologies including (inter alia) tumour angiogenesis, inflammation, arthritis, psoriasis and atherosclerosis (proliferation of the vasa vasorum within the vascular smooth muscle wall). Normal endothelial cells are a remarkably quiescent cell type, undergoing division approximately once every 1000 days, however, cell division can occur every 1 - 2 days during periods of extensive angiogenesis.

Endothelial cells are of haematopoeitic lineage and are known to be very rich in the number of mRNAs expressed when compared to other cell types (3). Thus, human umbilical vein endothelial cells have been shown to express over 1000 distinct mRNA sequences.

We have shown that out of this very large number of mRNAs which is expressed, a surprisingly small number show differential expression, and one of those molecules that does show differential expression is cc5- integrin.

A first aspect of the invention provides a compound comprising a moiety which selectively binds α5-integrin and a further moiety. By "a moiety which selectively binds α5-integrin" we mean any suitable such moiety which binds α5-integrin but does not substantially bind other molecules which are expressed on the surface of cells of the vascular tissue. In particular, the moiety does not substantially bind other integrin subunits such as αv integrin or β3 integrin. The compound comprising the binding moiety is one which preferably, in use, is able to localise to areas of new vascular growth, such as those associated with disease loci including tumour sites, but not localise substantially to other areas of the vasculature where there is no new vascular growth.

It is believed that in mature endothelial cells, such as those lining old blood vessels, the distribution of α5-integrin is polarised such that it is not present on the lumenal surface. In contrast, in cells in new blood vessels α5-integrin expression is not polarised and α5 integrin is present on the lumenal surface and is accessible to a moiety which selectively binds α5- integrin.

Typically, the compound, following administration to the patient, accumulates at the target site (ie the site of new vascular growth) such that at around 10 to 30 minutes after administration the target site has many times (and typically at least 100-times or preferably at least 1000-times) the concentration of compound compared to a non-target site such as parts of the vasculature where no new vascular growth is occurring.

Preferably the binding moiety is able to bind to α5-integrin with high affinity. For example, the binding constant for the binding of the binding moiety to α5-integrin is preferably between 10^"7 and 10^"10 M. Typically the binding moiety is an anti-α5 -integrin antibody. Such antibodies are described in, for example, Mould et al (1997) J. Biol. Chem. 272, 17283-17292, and Akiyama et al (1989) J. Cell Biol. 109, 863-875, incorporated herein by reference. In any case, suitable anti-α5-integrin antibodies may be made using methods well known in the art since the α5- integrin amino acid sequence is known, and α5-integrin may be expressed using recombinant DNA methods. The sequence for human fibronectin receptor alpha subunit (ie α5-integrin) is deposited in GenBank with accession number X06256. It is also described in Argraves et al (1987) J. Cell Biol. 105, 1183-1190. Bauer et al (1993) J. Cell Biol. Ill, 209-221 describes expression vectors expressing α5 -integrin.

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 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 which bind to α5-integrin.

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).

That antigenic specificity is conferred by variable domains and is independent of the constant domains is 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 mean molecules wherein the V_H and V_L partner domains are linked via a flexible oligopeptide.

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 circumstance, 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.

As mentioned above, monoclonal antibodies which will bind to α5-integrin are already known but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies can readily be prepared to α5- integrin. Suitable monoclonal antibodies to α5-integrin may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques" , H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications^'" , J G R Hurrell (CRC Press, 1982).

Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).

Suitably prepared non-human antibodies can be "humanized" 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 anti-α5 integrin antibodies but they may be prepared using methods known in the art that do not require immunisation of humans. For example, transgenic mice are available which contain, in essence, human immunoglobulin genes (see Vaughan et al (1998) Nature Biotechnol. 16, 535-539.

The moiety which selectively binds α5-integrin may also be a peptide. Suitable α5-integrin-binding peptides (or at least peptides which bind to α5βl integrin) are described in WO 95/14714, incorporated herein by reference. In particular, peptides having the amino acid sequence RRETAWA and CRRETAWAC, including cyclic peptides, are purported to bind α5βl integrin. Peptides which selectively bind α5 integrin may be made using pep tide phage display technology, such as that described in Scott & Smith (1990) Science 249, 386-390.

The further moiety may be any further moiety which confers on the compound a useful property with respect to the treatment or imaging or diagnosis of diseases or other conditions or states which involve undesirable neovasculature formation. Such diseases or other conditions or states are described in more detail below. In particular, the further moiety is one which is useful in killing or imaging neovasculature associated with the growth of a tumour. Preferably, the further moiety is one which is able to kill the endothelial cells to which the compound is targeted.

In a preferred embodiment of the invention the further moiety is directly or indirectly cytotoxic. In particular the further moiety is preferably directly or indirectly toxic to cells in neovasculature or cells which are in close proximity to and associated with neovasculature.

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. In one embodiment the cytotoxic moiety is a cytotoxic chemotherapeutic agent. 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; d-unethyltriazenorniidazole-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 (mithramycin) 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 (α^'s-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,p -DDD) and aminoglutethimide; 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 compounds 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 R-N = C = N-R', where R and R' 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 tumor 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 functional 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 crosslinking. However, it is recognized 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 a further 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 compound 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 EDTA or another chelating agent may be attached to the binding moiety and used to attach ^mIn or "Υ. Tyrosine residues may be labelled with ¹²⁵I or ¹³¹I.

The cytotoxic moiety may be a suitable indirectly cytotoxic polypeptide. In a particularly preferred embodiment, the indirectly cytotoxic polpeptide is a polypeptide which has enzymatic activity and can convert a relatively non-toxic prodrug into a cytotoxic drug. When the targeting moiety is an antibody this type of system is often referred to as ADEPT (Antibody- Directed Enzyme Prodrug Therapy). The system requires that the targeting moiety locates the enzymatic portion to the desired site in the body of the patient (ie the site expressing α5-integrin, such as new vascular tissue associated with a tumour) 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-tumor 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.)

Clearly, any α5-integrin binding moiety may be used in place of an anti- α5-integrin antibody in this type of directed enzyme prodrug therapy system.

The enzyme and prodrug of the system using an α5-integrin 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 dihydrofolate 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 Roffler et al, 1991); a doxorubicin prodrug activated by a human β-glucuronidase (Bosslet et al, 1994); further doxorubicin prodrugs activated by coffee bean - 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 (eg methotrexate-alanine) activated by carboxypeptidase A (Kuefner et al, 1990, Vitols et al, 1992 and Vitols et al, 1995). These and others are included in the following table.

(This table 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 1, 223).

Suitable enzymes for forming part of the enzymatic portion 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 eg thrombolysin (for thrombin prodrugs); hydrolases, such as phosphatases (eg alkaline phosphatase) or sulphatases (eg 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 CB1954 and analogues), azoreductase (for azobenzene mustards) and DT-diaphorase (for CB1954); oxidases, such as glucose oxidase (for glucose), xanthine oxidase (for xanthine) and lactoperoxidase; DL-racemases, catalytic antibodies and cyclodextrins.

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 compound but it is necessary only for it to be active when (a) it is in combination with the rest of the compound and (b) the compound is attached to, adjacent to or internalised in target cells.

When each moiety of the compound 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 α5-integrin 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 compound 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 compound 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 compound. Conceivably, the two portions of the compound may overlap wholly or partly.

The DNA is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention.

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) Ada Oncol. 34, 571-587; Shenoy & Singh (1992) Clin. Invest. 10, 533-551; Mitchell et al (1989) Int. J. Radiat. 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 (see for example, 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 photofrin (see, for example, Dougherty et al (1998) /. 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.

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) Anticancer 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 moiety which selectively binds to α5 -integrin is typically present on the surface of the delivery vehicle. For example, the moiety which selectively binds to α5-integrin, 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 moiety which selectively binds to α5 -integrin 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 1, 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 preexisting 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 moiety which selectively binds to cc5-integrin 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 anti-α5- integrin 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-1 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. 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 further embodiment of the invention, the further moiety comprised in the compound of the invention is a readily detectable moiety.

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 compound 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 compounds 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 technetium-99m or iodine- 123 for scinitgraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as iodine-123 again, iodine-131 , indium- I l l , fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Clearly, the compound 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 compound of the invention in known ways. For example, if the binding moiety is a polypeptide it may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine- 19 in place of hydrogen. Labels such as ^99mTc, ¹²³I, ^I86Rh, ⁱ⁸⁸Rh and ^nιIn 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 er al (1978) Biochem. Biophys. Res. Comm. 80, 49-57) can be used to incorporate iodine-123. Reference ("Monoclonal Antibodies in Immunoscintigraphy" , J-F Chatal, CRC Press, 1989) describes other methods in detail.

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 bispecific antibody wherein one binding site comprises the moiety which selectively binds to α5-integrin 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. A compound, such as an antibody which selectively binds to α5-integrin, 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) Q J Nucl. Med. 40, 234-251.

In a preferred embodiment of the invention, the moiety which selectively binds to α5-integrin and the further moiety are polypeptides which are fused.

A second aspect of the invention comprises a nucleic acid molecule encoding a compound of the first aspect of the invention.

Suitable nucleic acid molecules may readily be synthesised or constructed by the person skilled in the art using routine methods such as those described in Sambrook et al (1989) Molecular cloning, a laboratory manual, Cold Spring Harbor Press, Cold Spring Harbor, New York. Typically the nucleic acid is DNA, but it may be RNA. In the following, where DNA is used, unless the context indicates to the contrary, this also includes RNA.

The DNA is then expressed in a suitable host to produce a polypeptide comprising the compound of this aspect of the invention. Thus, the DNA encoding the polypeptide constituting the compound of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such techniques include those disclosed in US Patent Nos. 4,440,859 issued 3 April 1984 to Rutter et al, 4,530,901 issued 23 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to Crowl, 4,677,063 issued 30 June 1987 to Mark et al, 4,678,751 issued 7 July 1987 to Goeddel, 4,704,362 issued 3 November 1987 to Itakura et al, 4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 July 1988 to Toole, Jr. et al, 4,766,075 issued 23 August 1988 to Goeddel et al and 4,810,648 issued 7 March 1989 to Stalker, all of which are incorporated herein by reference.

The DNA encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the namre of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.

Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus) , plant cells, animal cells and insect cells.

The vectors include a procaryotic replicon, such as the ColEl ori, for propagation in a procaryote, even if the vector is to be used for expression in other, non-procaryotic, cell types. The vectors can also include an appropriate promoter such as a procaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.

A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical procaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.

A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.

An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers his3, trpl, leu2 and ura3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).

A variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules. Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3 '-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyse the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.

A desirable way to modify the DNA encoding the polypeptide of this aspect of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.

In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.

The compounds of the invention are useful in treating, imaging or diagnosing disease, particularly diseases in which there may be undesirable neovasculature formation, as described in more detail below.

Thus, a further aspect of the invention provides a compound according to the first aspect of the invention for use in medicine. Typically, the compound is packaged and presented as a medicament or as an imaging agent or as a diagnostic agent for use in a patient.

A still further aspect of the invention provides a pharmaceutical composition comprising a compound according to the first aspect of the invention and a pharmaceutically acceptable carrier.

The carrier(s) must be "acceptable" in the sense of being compatible with the compound 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.

Typically the pharmaceutical compositions or formulations of the invention are for parenteral administration, more particularly for intravenous administration.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

As discussed above, a number of diseases and conditions involve undesirable neovasculature formation. Neovasculature formation is associated with cancer, psoriasis, atherosclerosis, menorrhagia, arthritis (both inflammatory and rheumatoid), macular degeneration, Paget's disease, retinopathy and its vascular complications (including proliferative and of prematurity, and diabetic), benign vascular proliferations and fibroses.

By cancer is included Kaposi's sarcoma, leukaemia, lymphoma, myeloma, solid carcinomas (both primary and secondary (metastasis), vascular tumours including haemangioma (both capillary and juvenile (infantile)), haemangiomatosis and haemagioblastoma. Thus, a further aspect of the invention comprises a method of treating a patient who has a disease in which angiogenesis contributes to pathology the method comprising the step of administering to the patient an effective amount of a compound of the first aspect of the invention wherein the further moiety of the compound is one which either directly or indirectly is of therapeutic benefit to the patient.

Typically, the disease is associated with undesirable neovasculature formation and the treatment reduces this to a useful extent.

A still further aspect of the invention comprises a method of treating cancer the method comprising administering to the patient an effective amount of a compound of the first aspect of the invention wherein the further moiety of the compound is one which either directly or indirectly is of therapeutic benefit to the patient.

The tumours that may be treated by the metiiods of the invention include any tumours which are associated with new blood vessel production.

The term "tumour" is to be understood as referring to all forms of neoplastic cell growth, including tumours of the lung, liver, blood cells, skin, pancreas, stomach, colon, prostate, uterus, breast, lymph glands and bladder. Solid tumours are especially suitable. However, blood cancers, including leukaemias and lymphomas are now also believed to involve new blood vessel formation and may be treated by the methods of the invention.

Typically in the above-mentioned methods of treatment, the further moiety is one which destroys or slows or reverses the growth of the neovasculature.

Neovasculature formation occurs during the menstrual cycle. Prevention of neovasculature formation is believed to be a method of contraception. The corpus luteum, which makes progesterone, is highly vascularized. The compounds of the invention may be useful in preventing progesterone production by the corpus luteum in which case no implant would be sustained. Thus, a yet still further aspect of the invention provides a method of contraception the method comprising administering to the female an effective amount of a compound of the first aspect of the invention wherein the further moiety of the compound is one which is either directly or indirectly cytotoxic and/or which destroys or slows or reverses the growth of the neovasculature.

The compounds of the invention which comprise a readily detectable moiety are useful in imaging regions of the body where new blood vessel formation is occurring. Thus, a further aspect of the invention provides a method of imaging a region of neovasculature in the body of a patient, the method comprising administering to the patient an effective amount of a compound of the invention which comprises a readily detectable moiety. The method is useful in locating disease areas where undesirable new blood vessel formation is occurring, including cancer.

A further aspect of the invention provides a method of imaging cancer in the body of a patient the method comprising administering to the patient an effective amount of a compound of the invention which comprises a readily detectable moiety.

Imaging cancer in a patient is useful because it can be used to determine the size of a tumour and whether it is responding to treatment. Since metastatic disease involves new blood vessel formation, the method is useful in assessing whether metastasis has occurred.

It will readily be appreciated that, depending on the particular compound used in treatment, imaging or diagnosis, the timing of administration may vary and the number of other components used in therapeutic systems disclosed herein may vary.

For example, in the case where the compound of the invention comprises a readily detectable moiety or a directly cytotoxic moiety, it may be that only the compound, in a suitable formulation, is administered to the patient. Of course, other agents such as immunosuppressive agents and the like may be administered.

In respect of compounds which are detectably labelled, imaging takes place once the compound has localised at the target site.

However, if the compound is one which requires a further component in order to be useful for treatment, imaging or diagnosis, the compound of the invention may be administered and allowed to localise at the target site, and then the further component administered at a suitable time thereafter.

For example, in respect of the ADEPT and ADEPT-like systems above, the binding moiety-enzyme moiety compound is administered and localises to the target site. Once this is done, the prodrug is administered.

Similarly, for example, in respect of the compounds wherein the further moiety comprised in the compound is one which binds a further component, the compound may be administered first and allowed to localise at the target site, and subsequently the further component is administered.

Thus, in one embodiment a biotin-labelled anti-α5-integrin antibody is administered to the patient and, after a suitable period of time, detectably labelled streptavidin is administered. Once the streptavidin has localised to the sites where the antibody has localised (ie the target sites) imaging takes place. It is believed that the compounds of the invention wherein the further moiety is a readily detectable moiety may be useful in determining the angiogenic status of tumours or other disease states in which angiogenesis contributes to pathology. This may be an important factor influencing the namre and outcome of future therapy.

Thus, a further aspect of the invention provides a method of diagnosis of a patient to determine if they have a disease in which angiogenesis contributes to pathology, the method comprising administering to the patient an effective amount of a compound of the invention wherein the compound comprises a readily detectable moiety.

The method may be particularly useful for the diagnosis of cancer, in particular the diagnosis of metastatic disease.

Administration of a compound of the invention wherein the compound comprises a readily detectable label may be useful in determining the degree of angiogenesis which may be useful for prognosis.

Still further aspects of the invention provide the use of the appropriate compounds of the invention in the manufacture of (1) medicaments for treating diseases in which angiogenesis contributes to pathology, including cancer, (2) a contraceptive agent, (3) an agent for imaging a region of neovasculature formation in the body of a patient, (4) an agent for imaging cancer in the body of a patient, (5) a diagnostic agent and (6) a prognostic agent.

A further aspect of the invention provides a kit of parts (or a therapeutic system) comprising (1) a compound of the invention wherein the further moiety is a cytotoxic moiety which is able to convert a relatively non-toxic prodrug into a cytotoxic drug and (2) a relatively non-toxic prodrug. The kit of parts may comprise any of the compounds of the invention and appropriate prodrugs as herein described.

A still further aspect of the invention provides a kit of parts (or a therapeutic system) comprising (1) a compound of the invention wherein the further moiety is able to bind selectively to a directly or indirectly cytotoxic moiety or to a readily detectable moiety and (2) any one of a directly or indirectly cytotoxic moiety or a readily detectable moiety to which the further moiety of the compound is able to bind.

For example, a kit of parts may contain an anti-α5-integrin antibody labelled with biotin and streptavidin labelled with a readily detectable label as defined above.

The invention will now be described in more detail by reference to the following Figures and Examples wherein:

Figure 1. Differential hybridisation of human cDNA arrays with HDMEC cDNA probes. A pair of identical Atlas™ human cDNA arrays (a) were hybridised separately with radiolabelled first-strand cDNA probes made from quiescent (b) and EGF-treated (c) HDMECs.

Figure 2. Semi-quantitative RT-PCR analysis of mRNA expression of selected genes in HDMECs. M - molecular weight markers. Culture conditions: Q - quiescent cells, E - EGF-treated cells, V - VEGF-treated cells and C - a confluent culture exposed to ECGS. Figure 3. Cell cycle distribution of the HDMEC populations. HDMECs in the EGF-treated proliferative (A) or quiescent (B) cultures were harvested by incubation in 2% EDTA and fixed in ethanol for 30 min. Flow cytometric analysis of the DNA content was carried out using FACSCan (Becton Dickinson).

Figure 4. FACS analysis confirming upregulation of α5 integrin protein in proliferating HDMEC.

Example 1: α5 integrin is expressed in human microvascular endothelial cells in the proliferative state but not in the quiescent state

Abbreviations

DBI, diazepam binding inhibitor; ECGS, endothelial cell growth supplement; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3- phosphate dehydrogenase; HDMEC, human dermal microvascular endothelial cells; MCP-1 monocyte chemotactic protein- 1; N-cadherin, neural cadherin; PCR, polymerase chain reaction; VE-cadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor.

Endothelial cells are known to be a rich source of transcriptional gene expression. Recent technological advances now permit the detailed profiling of mRNA expression using arrays of known cDNAs on blots. We have used such arrays to examine expression mRNA by primary isolates of human foreskin microvascular endothelial cells in the proliferative and quiescent state. Cells were stimulated by a combination of known growth factors for these cells including vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and 'endothelial cell growth supplement (ECGS)' either alone or in combination. Despite the expression of many mRNAs, surprisingly few showed a change with proliferative status. Thus, of 588 mRNAs examined only one, namely monocyte chemotactic protein- 1 (MCP-1), showed a decrease on treatment with EGF. A combination of image grabbing followed by subtractive densitometry showed that several mRNA increased but few to a significant extent. Confluence was found to deliver an overriding off signal to both EGF and VEGF induced transcription. In the light of the possibility of selective vascular targeting, of particular interest was the increase in expression of the mRNA for the cell surface proteins VE- and N-cadherin and α5, αv, βl and β3 integrins. The α5 integrin offers a previously unrecognized opportunity for vascular targeting.

Many genes that were identified as having a particular function have subsequently been found to have additional functions. An important step in defining the functions of these genes is to determine their expression profiles in a range of tissues and under in vitro conditions. Traditional methods such as Northern blot analysis focus on only one gene at a time, in contrast the recent development of multiple cDNAs arrayed onto one blot permits the simultaneous analysis of the transcriptional expression of many genes. While this technique does not enable the identification of novel genes, it is useful in the functional profiling of known genes which in turn may lead to a better understanding of their biological role. The technique has recently been applied to a comparison of genes expressed in glioblastoma multiforme versus normal brain tissue (4), the identification of inflammatory disease-related genes (5), the characterisation of changes in mRNA expression following the introduction of a tumour suppressor gene into a melanoma (6) and the induction of heat shock and phorbol ester regulated genes in human T cells (7). Human dermal microvascular endothelial cells (HDMECs) isolated from foreskin show a growth response to both epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). Employing gridded cDNA blots (Atlas™ human cDNA expression array I) we have characterised transcriptional expression in quiescent and EGF- and VEGF- stimulated HDMECs. The most surprising result was the small number of mRNAs that showed differential expression. Where changes in expression of mRNAs appeared of interest, this was confirmed by semi- quantitative RT-PCR. Concordance between the results from the arrays and the RT-PCR was seen in all cases.

Materials and Methods

Cell culture: Primary human dermal microvascular endothelial cells (HDMECs) isolated from neonatal foreskin and shown to be von Willebrand factor positive, to take up acetylated LDL and to be smooth muscle α-actin negative were obtained from Clonetics (San Diego, CA). Cultures were routinely set up by seeding cells ( ^" 5000 cells/cm² of growth area) in gelatin-coated plasticware (Falcon) and maintained as described (8) in a complete growth medium consisting of endothelial basal medium (modified MCDB131 , EBM) (Clonetics) supplemented with 5 % foetal calf serum, EGF (10 ng/ml), ECGS (30 μg/ml), heparin (90 μg/ml), hydrocortisone (1 μg/ml), gentamycin (50 μg/ml) and amphotericin B (50 ng/ml). For some experiments, VEGF₁₆₅ was used at 100 ng/ml instead of EGF. Early passage ( < 9) HDMECs were used throughout the study. Subconfluent ( < 80%) exponentially growing HDMECs were used as proliferating cells. To quiesce the cells they were treated with basal medium supplemented with 5 % foetal calf serum for 5 days prior to the isolation of RNA.

Preparation of RNA and synthesis of the cDNA probes: Steady-state, total cellular RNA was isolated from cells using a standard single-step guanidinium isothiocyanate extraction (9). Poly(A)⁺ RNA was purified by two cycles of affinity chromatography using the QuickPrep mRNA purification kit (Pharmacia Biotech). First-strand cDNA probes were synthesised using 1 μg of each poly(A)⁺ RNA population with CDS primer mix (Clontech), α-³²P dCTP (10 μCi/μl, Amersham) and Moloney murine leukaemia virus reverse transcriptase.

Differential hybridisation of cDNA expression arrays: The ³²P-labelled single-stranded cDNA probes prepared from proliferating and quiescent HDMECs were separately hybridised in ExpressionHyb™ hybridisation solution overnight to a pair of identical commercial filters (Atlas™ human cDNA expression array I, Clontech) dotted with 588 cDNAs of known sequence and 9 house keeping genes (Fig la). Following high-stringency washing, the hybridisation pattern was analysed by autoradiography and quantified by image grabbing and subtractive densitometry.

Gene specific RT-PCR analysis: 10 μg of total RNA was treated with DNase I (2 units/μl) at 37°C for 30 minutes and reverse transcribed to first-strand cDNA using the conditions described above but without radiolabel. Subsequent PCR amplifications were carried out with gene- specific primers in 50-μl reactions for 25 thermocycles (denaturation at 94 °C, annealing at 55 °C and extension at 72 °C each for 30 seconds). For comparison of expression levels, cDNAs were first normalised to GAPDH product (100 bp) by PCR amplification of serially diluted cDNAs with a fixed amount of the competitive template, ie genomic GAPDH fragment (192 bp) containing intron F. The normalised cDNA aliquots were then used for amplification of selected genes using the following primers: 5'-AGCATGAAAGTCTCTGCCGCCC (sense) and 5'- CAGATTCTTGGGTTGTGGAGT-3' (antisense) for MCP-1 ; 5'-

AAGTCGGCCAGGATGTCTCAGG-3 ' and 5'-

GGCACAGTAACCAAATCCAGTCTCTC-3 ' for DBI; 5'-

GCGCGTGAAGGTTTGCC AGT-3 ' and 5'-

GATACTGGGGCTCGGCGTGGAT-3 ' for N-cadherin; 5'- CCAAGCCCTACCAGCCCAAAGT-3' and 5'-

CCGAGTTGAGCACCGACACATC-3 ' for VE-cadherin; 5'-

AACGC AGTCCC ATCTCAAATCC-3 ' and 5'-

GCCCAACATCTTCTTCAGTCTC-3' for αv integrin; 5'-

GGACGGGCTGGATGACTTGCTG-3 ' and 5'- CTGAGCCTTGTCCTCTATCCGG-3' for α5 integrin; 5'-

TAGC AAAGGAAC AGC AGA-GAAG-3 ' and 5'-

TGGACAAGGTGAGCAATAGAAG-3' for βl integrin; 5'-

ATGAGGAGGTGAAGAAGCAGAG-3 ' and 5'-

GGTGGCATTGAAGGATAGAGAC-3 ' for β3 integrin and 5'- CCACTCTGGGAAACCTGACAAC-3' and 5'-

CCTGGATCGCTCGCTCTGAAAC-3' for β5 integrin. Competitive amplification of GAPDH was carried out using oligos 5'-

GACAACAGCCTCAAGATCATCA-3' and 5'-

GTCCTTCCACGATACCAAAGTT-3' to monitor the acmal amount of cDNA used in each reaction. Amplification and size of the generated fragments were evaluated by electrophoresis of 10 μl of PCR reactions in a 2% agarose gel. FACS analysis of a5 integrin expression

HDMECs were treated exactly as for the isolation of mRNA for the hybridisation studies. After incubation with 10 μg/ml of mouse anti- human α5 integrin (SNAKA 52), the cells were incubated with rabbit anti- mouse FITC before FACS analysis.

Results

Expression of mRNAs in quiescent and EGF-stimulated proliferating endothelium. HDMECs were allowed to quiesce or received prolonged treatment with EGF to induce the proliferative state. That the cells were indeed quiescent or proliferating was confirmed by flow cytometric analysis of the DNA content. A conservative estimate showed that the cells in the S and G2/M phases were 56.1 % and 30.5 % of the population in the proliferating and the quiescent HDMEC cultures, respectively (Figure 3). RNA was then reverse transcribed to first-strand cDNA and hybridised with the human cDNA arrays (Fig 1). Table 1 and 2 document the expression profiles of 9 house keeping genes and those showing a difference in signal intensity after being normalised to the GADPH and subtractive densitometry. Of the 9 house keeping genes both the 23 kDa highly basic protein and β-actin appeared to be elevated in the EGF- treated proliferating HDMECs. Despite the expression of many RNAs, surprisingly few showed a significant increase and only one, monocyte chemotactic protein- 1 (MCP-1), showed a decrease. Confluent EGF- treated cells failed to show induction of even the few RNAs that were induced on treatment of a sub-confluent culture (data not shown), suggesting that cell-cell contact overrides the effect of EGF. Examination of differentially expressed mRNAs of interest by semi- quantitative PCR. The expression of 9 mRNAs of interest was examined by semi-quantitative PCR. In each case the preliminary result from the expression array was validated by the PCR analysis (Fig 2 and Table 3). Differential mRNA expression in response to chronic exposure to different growth factors, namely, EGF and VEGF, was also examined (Fig 2 and Table 3). MCP-1 was the only mRNA to show a fall in expression in proliferating cells. This fall in expression was specific for prolonged exposure to EGF and not VEGF or ECGS (Fig 2). The most marked upregulation was seen with VE-cadherin and the α5 integrin (Fig 2). Upregulation of α5 protein in proliferating HDMEC was confirmed by FACS analysis (Figure 4).

Discussion

It has been known for several years that the protein profile of the endothelial surface varies with the proliferative status of the cells. Thus, as early as 1991 Clarke and West showed by surface iodination of total protein, followed by two dimensional electrophoresis that novel proteins appeared in lysates of proliferating cells not seen in those from quiescent counterparts (10, 11). At that point in time the prospect of taking a spot on an autoradiograph through to a sequence was daunting. With the advent of molecular biology, new techniques rapidly became available for the identification of differentially expressed proteins. First amongst these was subtractive hybridisation, a technique that proved challenging because of the requirement for substantial quantities of high purity poly(A)⁺ RNA. Anchored PCR differential display (12) offered much hope and has indeed proved of great utility, either in the identification of differentially expressed known mRNAs or the identification of novel mRNAs. Nevertheless, PCR differential display and the related techniques are severely limited in the number of messages that may be examined. We have used an alternative that enables the screening of transcriptional RNA expression using cDNA arrays in which substantial numbers of known cDNAs are gridded on the blot. We have been able to transcriptionally profile the microvascular endothelial cell under different conditions of culture.

In this study, mRNA expression in human dermal microvascular endothelial isolates (HDMECs) was characterised when the cells were in either a quiescent or a proliferative state. A chronic proliferative stimulus was applied to the cells to mimic the situation that the angiogenic endothelial cell would experience in vivo. This differs from studies in which the cell receives a single inductional dose of growth factor to look for acute mRNA induction. Nevertheless, different intracellular signals from, for example, EGF or VEGF binding to their receptors may lead to chronic expression of different mRNAs. To our knowledge this is not known and was an aspect that we particularly wanted to examine here.

Table 3 documents mRNAs that were differentially induced by either EGF or VEGF but not vice versa.

The pattern of mRNA expression in HDMECs was found to be highly reproducible for cells cultured under given conditions and to be markedly different when compared to that for other cells of haematopoeitic lineage such as leukocytes or to whole tissues such as placenta (13). The most striking finding was that despite expression of a comparatively large number of cDNAs by the endothelial cells, few were differentially expressed between the proliferating and quiescent cells, indeed, only one, MCP-1 , showed a decrease on proliferation. Consistent with this, it has previously been shown that inhibitors of endothelial cell proliferation (interleukin-1 and tumour necrosis factor-α) stimulated release of MCP-1 from endothelial cells (14). Thus, it appears that MCP-1 is upregulated in inflammatory but downregulated in purely proliferative endothelial sites. It is not known why endothelial cells should make MCP-1 although they are generally considered along with other stromal cells to be important effector cells during inflammation. A downregulation of MCP-1 expression in proliferating HDMECs was unexpected in that a previous smdy has shown its expression to be upregulated on treatment of large vessel endothelium with basic fibroblast growth factor (15).

These differing observations serve to emphasise the heterogeneity in endothelium from different vascular beds and the difficulty in extrapolating from the smdy of one endothelial cell type.

Among the upregulated mRNAs in the proliferating cells of interest are the endothelial specific Tie-1 (strongly upregulated) and Tie-2 (upregulated) tyrosine kinase receptors (Figure 2 and Table 2). The ligand for Tie-2 is angiopoietin-1 , that for Tie-1 is not known. It is thought that in angiogenesis a growth factor (eg VEGF) acts on the endothelial cells to induce proliferation and then angiopoietin-1 acts later to promote vessel differentiation [24, 25]. An upregulation of Tie-2 on the proliferating cells accords with this model.

Of particular interest in this smdy was differential expression of cell surface molecules that could potentially be of use for vascular targeting. Surface molecules that showed an upregulation in the differential hybridisation were then examined by semi-quantitative PCR analysis. The results from the PCR analysis were all in complete agreement with the results from the hybridisation studies. Thus, Figure 2 confirms that the N- and VE-cadherins and the αv, α5, βl and β3 integrins are all upregulated on proliferating cells. Amongst these, this is the first report of upregulation of the α5 integrin on proliferating endothelium. Collo and Pepper [26] have reported an upregulation of the mRNA and total α5 integrin protein in multiple wounded monolayers of bovine microvascular endothelial cells. Upregulation correlated with a reduction in cell density but not proliferation.

Several mRNAs are known to be differentially expressed by endothelium. Notable amongst these are follistatin which is expressed by migrating and proliferating but not quiescent endothelium (16). Osteopontin, PC4 and a novel mRNA with homology to calmodulin-dependent protein kinases that have been shown to be differentially expressed when endothelial cells form bes in 3D culmre (17). Previous work has implicated a role for the βl integrin in angiogenesis. Thus, teratomas formed by ectopic injection of embryonic stem (ES) cells normally undergo extensive vascular isation. This vascular isation was greatly attenuated in teratomas formed from βl null ES cells (18). Further, while VEGF induced endothelial proliferation and formation of extensive branching blood vessels in normal embryoid bodies, it had no effect in βl null embryoid bodies. Other studies have confirmed that angiogenesis promoted by VEGF is mediated via the αl βl and α2βl integrins (19). These studies showed that VEGF stimulated an association of the βl subunit with the α units but had no effect on βl mRNA expression (19). This contrasts with EGF that stimulates a direct upregulation of the βl message in the same endothelial cell type. Contact inhibition was found to deliver an overriding signal to both EGF and ECGS mRNA induction. This is of interest when considering what signal may initiate activation of the quiescent endothelial cell in vivo. One of the first known events that occurs on activation of the quiescent endothelial cell is secretion of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA). Neither of these were present in the cDNA array, however, the mRNA of the uPA receptor showed an increase and it is known that uPA must bind to its receptor before it has proteolytic activity.

Expression of E-selectin and the VEGF receptor KDR is known to be upregulated on tumour endothelium (20, 21). The former is probably an inflammatory event, although several studies have implicated that E- selectin expression correlates with proliferation in the endothelial cell, being specifically associated with the G2/M phases of cell cycle. Thus, angiostatin upregulates E-selectin on endothelium and this is thought to be because it traps the cells in the G2/M phase (22). What induces KDR remains controversial. Overexpression of these proteins permits vascular targeting strategies to be explored, whether these be at the protein or transcriptional level (23). The novel finding of strongly increased expression of α5 integrin on the proliferating endothelial cells identifies one such target. However, the most surprising outcome to emerge from this smdy is that there may not be as many molecular targets on the endothelial cell as may once have been supposed.

Table 1. Expression pattern of house keeping genes in quiescent versus proliferating (chronically treated with EGF) HDMECs. Nine house keeping genes were included in the human cDNA arrays for the normalisation of the mRNA abundance, along with plasmid and bacteriophage DNAs as negative controls to confirm specificity of hybridisation.

Table 2. Differentially expressed mRNAs in quiescent versus proliferating (chronically treated with EGF) HDMECs.

Table 3. Differentially expressed mRNAs in VEGF- and EGF-stimulated HDMECs compared to quiescent controls.

Table 1

Position GenBank Signal Signal Differential in cDN-4 Name of genes/proteins accession intensity in intensity in expression⁸ array I number quiescent proliferating HDMEC* HDMEC*

House keeping genes

G5 Ubiquitin M26880 + +

G6 Phospholipase A2 M86400 - - ₌

G7 Hypoxanthine-guanine phosp oribosyltransferase V00 30 - - ~

G 12 Glyceryaldehyde 3-phosphate dehydrogenase X01677 +++ +++ = (G3PDH) G 13 α-tubulin K00558

G 14 HLA Class I antigen, C-4 α chain Ml 1886 - - = 4 σ^,

G 19 β-actin X00351 + ++ T

G20 23 Da Highly basic protein X56932 +++ +++--- T

G21 Ribosomal protein S9 U14971 + + ~

Negative controls

Gl , G8, i 315 Blank spots G2, G9, ( 316 Ml3 mpl8 (+) strand DNA - - G3, G 10, G17 λ-DNA

G4, Gi l , G18 pUC18 - -

*Signal intensity: -, negative; +-++++, positive.

^Differential expression: = largely unchanged; T, up-regulated.

Table 2

Position Broad category GenBank Signal Signal Differential in cDNA Name of genes/proteins accession intensity in intensity in expression⁸ array I (alternative names in parentheses) number quiescent proliferating HDMEC* HDMEC*

Oncogenes/tumour suppressors

A ll Transcnption factor API J041 1 1 - + T (c-jun proto-oncogene)

A2a Tyrosine-protein kinase receptor TIE-2 L06139 +/- t

A2b Insulin-like growth factor binding protein 2 M35410 +/ T

(IGFBP-2) A3a Ski oncogene X15218 +/- t

A3ι Transforming protein rhoA L25080 -/+ + T (Proto-oncogene rhoA, multidrug resistance protein) Cell cycle control proteins A5c Tyrosine-protein kinase receptor UFO M76125 + T

A5h Prothymosm α M26708 + ++ T A5ι Cell surface glycoprotein MUC18 M28882 +/- + T

A51 40S nbosoomal protein S19 M81757 -/+ + t

A6g Cychn Dl (Gl/S-specιfιc) X59798 +/- + T

A6h Cyclin D2 (Gl/S-specific) D13639 -/+ +/- t

A61 Cyc n Bl (G2/mιtotιc-specιfιc) M25753 - +/- t

A7b Cyclin Gl (Gl/mitotic-specific cychn G) U47414 - +/- T

Modulators/effectors/intracellular transducers

B i g Tyrosine-protein kinase TIE-1 X60957 ++ +++ T

B2a Urokinase plasminogen activator surface receptor U08839 +/- (uPAR, GPI-anchored form) t B3d Transcnption factor p65 LI 9067 ; + t

B3h Protein kinase MLK-3 (mixed lineage kinase 1) L32976 - + t

B5b MacMARCKS X70326

Stre.ss response proteins

B7h Natural killer cell enhancing factor B (NKEFB) L19185 - -/+ T B7k Heat shock protein 86 X07270 ++ +++ T B71 Heat shock 27-k-Da protein 1 X54079 +++ A-A-A-A- T

Apoptosis-asociated proteins

Cld MDM2 protein (p53 associated) Z 12020 -/+ +/- T C2e Apopain (cysteine protease CPP32 isoform α) U 13737 + + T? C2f DAD-1 (Defender against cell death 1) D 15057 + + T? C2g Nucleoside diphosphate kinase B L16785 ++ +++ (c-myc transcnption factor [puf]) T

C2h BAX (β) L22474 - + T

C2ι GRB (growth factor receptor-bound protein 2) L2951 1 -/+ + T

C3a Glutathione S-transferase M4 X08020 -/+ +/- T -p..

C3b Glutathione S-transferase P X I 5480 + ++ OO

T

C3d Glutathione peroxidase M21304 - +/- T

C4g ICH-2 protease (ICErel-II) U28014 +/- + t

C5e HDLC1 (cytoplasmic dynein light chain 1) U32944 +/- + T

DNA synthesis/repair/recombination proteins

C7b Peroxidase dismutase (superoxide dismulase 1 K00065 - +/- [Cu Zn]) T C7d LTV excision repair protein RAD 23 D21090 - +/- (xeroderma pigmentosum group C repair T complementing protein p58/HHR23B)

C7f Growth arrest and DNA-da age-inducible protein M60974 - +/- GADD45 t

C7g Growth arrest and DNA-damage-inducible protein S40706 - +/- 153 T DNA binding/transcription factors

Dll 60S πbosomal protein L6 (DNA-binding protein X69391 ++ +++ TAX)

D3j RNA polymerase II elongation factor SIII (PI 5 L34587 +/- + subunit) D3k Guanine nucleotide-binding protein G-S (α M 14631 ++ +++ subunit)

D5a Y box binding protein- 1 M82234 +++ ++++

(nuclease-sensitive element DNA-binding protein) Cell surface antigens/adhesion

E5d Vitronectin receptor α (integrin αv, CD51) Ml 4648 +/- + T E6a NADH-ubiquinone oxidoreductase B 18 subunit M33374 - + (cell adhesion protein [SQM]) T

E6b Neural-cadherin (N-cadherin) M34064 - +/- T

E6j Platelet membrane glycoprotein Ilia J02703 - +/- t

E7e Fibronectin receptor (α subunit) (integrin α5) X06256 -/+ + t ^

E7f Fibronectin receptor (β subunit) (integrin βl) X07979 + ++ T

E7g integrin oc6 X53586 -/+ +/- t Extracellular signaling/communication proteins

F2c Glucose-6-phosphate isomerase (neuroleukin) K03515 -/+ + T F3a Monocyte chemotactic protein 1 (MCP-1) M24545 +++ ++ i F4d Thymosin β-10 M92381 +++ ++++ T F4e Connective tissue growth factor M92934 ++ +++ t F5b Macrophage inflammatory protein-2-α (MIP2α) X53799 -/+ + t F5f Interleukin-8 Y0O787 - + t

*Signal intensity: -, negative; -/+, very weak; +/-, weak; +, positive; ++-++++, strong signal. 'Differential expression: =, unchanged; T, up-regulated; i, down-regulated.

Table 3

—-----—--— --------------------------------------------------._^^

Treatment or confluence of cells mRNA :GF VEGF Confluent

MCP-1 i = ~

DBI = T s

N-cadhenn t = T

VE-cadherin* TT TT T αv-integπn t T T α5-ιntegπn TT TT TT βl-integπn TT T TT β3-ιntegπn T TTT T TTT T T β5-integnn = = =

*VE-cadherin was not present on the cDNA array but was examined by RT-PCR in the light of potential cross reactivity with N-cadhenn. ⁶β5 integπn did not show a change in expression in the differential hybndization but was included as a control for RT-PCR.

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Claims

1. A compound comprising a moiety which selectively binds α5- integrin and a further moiety.

2. A compound according to Claim 1 wherein the moiety which selectively binds α5-integrin is an antibody.

3. A compound according to Claim 1 wherein the moiety which selectively binds α5-integrin is a peptide.

4. A compound according to any one of Claims 1 to 3 wherein the further moiety is a directly or indirectly cytotoxic moiety.

5. A compound according to Claims 1 to 3 wherein the further moiety is a readily detectable moiety.

6. A compound according to Claim 4 wherein the cytotoxic moiety is a directly cytotoxic chemotherapeutic agent.

7. A compound according to Claim 4 wherein the cytotoxic moiety is a directly cytotoxic polypeptide.

8. A compound according to Claim 4 wherein the cytotoxic moiety is a moiety which is able to convert a relatively non-toxic prodrug into a cytotoxic drug.

9. A compound according to Claim 4 wherein the cytotoxic moiety is a radiosensitizer.

10. A compound according to Claim 4 wherein the cytotoxic moiety comprises a nucleic acid molecule.

11. A compound according to Claim 10 wherein the nucleic acid molecule encodes a directly or indirectly cytotoxic polypeptide.

12. A compound according to Claim 10 wherein the nucleic acid molecule is directly cytotoxic.

13. A compound according to Claim 4 wherein the cytotoxic moiety comprises a radioactive atom.

14. A compound according to Claim 13 wherein the radioactive atom is any one of phosphorus-32, iodine-125, iodine-131 , indium-I l l , rhenium- 186, rhenium- 188 or yttrium-90.

15. A compound according to Claim 5 wherein the readily detectable moiety comprises a radioactive atom.

16. A compound according to Claim 15 wherein the radioactive atom is selected from any one of technetium-99m or iodine- 123.

17. A compound according to Claim 5 wherein the readily detectable moiety comprises a suitable amount of any of iodine- 123, iodine- 131 , indium-I l l , fluorine- 19, carbon-13, nitrogen- 15, oxygen-17, gadolinium, manganese or iron.

18. A compound according to any one of Claims 1 to 3 wherein the further moiety is able to bind selectively to a directly or indirectly cytotoxic moiety or to a readily detectable moiety.

19. A compound according to Claim 1 wherein the moiety which selectively binds α5-integrin and the further moiety are polypep tides which are fused.

20. A nucleic acid molecule encoding a compound according to Claim 19.

21. A host cell comprising a nucleic acid molecule according to Claim 20.

22. A compound according to any one of Claims 1 to 19 for use in medicine.

23. A pharmaceutical composition comprising a compound according to any one of Claims 1 to 19 and a pharmaceutically acceptable carrier.

24. A method of treating a patient who has a disease in which angiogenesis contributes to pathology the method comprising the step of administering to the patient an effective amount of a compound according to any one of Claims 1 to 4 and 6 to 14.

25. A method according to Claim 24 wherein the disease is any one of cancer, psoriasis, diabetic retinopathy, atherosclerosis or menorrhagia.

26. A method of treating cancer the method comprising the step of administering to the patient an effective amount of a compound according to any one of Claims 1 to 4 and 6 to 14.

27. A method of contraception the method comprising administering to the female an effective amount of a compound according to any one of Claims 1 to 4 and 6 to 14.

28. A method of imaging a region of neovasculature in the body of a patient the method comprising administering to the patient an effective amount of a compound according to any one of Claims 1 to 3, 5 and 15 to 17.

29. A method of imaging a cancer in the body of a patient the method comprising administering to the patient an effective amount of a compound according to any one of Claims 1 to 3, 5 and 15 to 17.

30. A method of diagnosis of a patient to determine if they have a disease in which angiogenesis contributes to pathology, the method comprising administering to the patient an effective amount of a compound according to any one of Claims 1 to 3, 5 and 15 to 17.

31. A method of determining the degree of angiogenesis associated with a cancer or a metastases thereof the method comprising administering to the patient an effective amount of a compound according to any one of Claims 1 to 3, 5 and 15 to 17.

32. Use of a compound according to any one of Claims 1 to 4 and 6 to 14 in the manufacture of a medicament for treating a disease which involves the production of new vasculature.

33. Use according to Claim 32 wherein the disease is any one of cancer, psoriasis, diabetic retinopathy, atherosclerosis or menorrhagia.

34. Use of a compound according to any one of Claims 1 to 4 and 6 to 14 in the manufacture of a medicament for treating cancer.

35. Use of a compound according to any one of Claims 1 to 4 and 6 to

14 in the manufacture of a contraceptive agent.

36. Use of a compound according to any one of Claims 1 to 3, 5 and

15 to 17 in the manufacture of an agent for imaging a region of neovasculature in the body of a patient.

37. Use of a compound according to any one of Claims 1 to 3, 5 and 15 to 17 in the manufacture of an agent imaging a cancer in the body of a patient.

38. Use of a compound according to any one of Claims 1 to 3, 5 and 15 to 17 in the manufacture of a diagnostic or prognostic agent for a disease in which angiogenesis contributes to the pathology.

39. A kit of parts comprising a compound according to Claim 8 and a relatively non-toxic prodrug.

40. A kit of parts comprising a compound according to Claim 18 and any one of a directly or indirectly cytotoxic moiety or a readily detectable moiety to which the said compound is able to bind via its further moiety.

41. Any novel method of treating or imaging cancer as herein disclosed.