WO2007101676A2

WO2007101676A2 - Treatment, diagnosis and imaging of mantle cell lymphoma

Info

Publication number: WO2007101676A2
Application number: PCT/EP2007/001962
Authority: WO
Inventors: Carl Arne Krister Borrebaeck; Sara Ek
Original assignee: Immunovia Ab
Priority date: 2006-03-09
Filing date: 2007-03-07
Publication date: 2007-09-13
Also published as: GB2435882A; WO2007101676A3; GB0604779D0

Abstract

The present invention provides a use a compound comprising a binding moiety which selectively binds to a protein listed in Table 1 in the treatment, imaging, diagnosis or prognosis of mantle cell lymphomas (MCL). Optionally, the compound also comprises a further moiety. Preferably, the binding moiety is an antibody or antigen- binding fragment thereof. Advantageously, the further moiety is a directly or indirectly cytotoxic moiety or a readily detectable moiety. The invention also provides methods of imaging MCL cells, methods of diagnosing or prognosing MCL in an individual and methods of treating an individual with MCL A further aspect of the present invention provides a method of identifying cells associated with MCL, the method comprising analysing the pattern of gene expression in a sample of cells to be tested and comparing it to the pattern of gene expression in a sample of known MCL cells. Preferably, the cells to be tested are identified as MCL cells if the expression of one or more genes encoding a protein listed in Table 1 is upregulated compared to normal B -cells.

Description

TREATMENT, DIAGNOSIS AND IMAGING OF DISEASE

The present invention relates to peptides, polypeptides and proteins overexpressed in Mantle cell lymphomas (MCL) and use of these peptides, polypeptides and proteins or nucleotide sequences encoding them, or molecules which bind thereto, in imaging and/or diagnosis and/or treatment of MCL.

The term lymphoma refers to all the malignancies of the lymphocytes, with B and T cell malignancies being the most commonplace. The classification of lymphomas is still under debate and far from conclusive. The current classification being used is the Revised European American Classification of Lymphomas (Harris et al., 1994, Blood 84:1361-1392). However, this system needs to be further divided to allow as precise diagnosis as possible, thereby permitting the optimal treatment for the patients.

Several gene expression studies of different types of B-cell lymphomas have been published. For example, Golub et al. (1999) Science 286:531-537 have shown that acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) can be distinguished based on their gene expression analysis. In addition, Alizadeh et al. (2000) Nature 403:503-511 found two distinct types of diffuse large B-cell lymphoma (DLBCL) when analysing the gene expression of these tumours on the Lymphochip.

B-cells go through several differentiation stages during development. The immature B-cell migrates from bone-marrow to the lymph node where the naive B-cell is stimulated to migrate into the B-cell follicles, forming Germinal Centres, before finally differentiating into an antibody secreting plasma cell. For each of these different stages a malignant counterpart has been found that resembles the normal B- cell origin (Pascual et al., 1997, Baillieres CHn Haematol 10:525-538).

Mantle cell lymphomas (MCLs) are believed to derive from a naive B-cell, although the relationship with the CD5⁺ B-I cells also has been discussed (Pascual et al., supra; Capello et al., 2000, Haematologica 85:195-201). MCLs are indolent and show no somatic mutation of their immunoglobulin genes, thus resembling their naive B-cel! origin. The median age of disorder for MCL patients is 60 years and the median survival is 2 to 5 years with a poor response to conventional therapeutic regimens (Weisenburger et al., 2000, Am J Hematol 64: 190-196). Seventy per cent of the patients have bone marrow involvement and the male to female predominance is 3: 1 (Kurtin, 1998, Adv Anat Pathol 5:376-398). MCL cells express the pan-B-cell markers CD 19, CD20, CD22, CD79a and CD79b and are negative for CDlO and CD23 (Kurtin, 1998, Adv Anat Pathol 5:376-398). MCL tumours can grow in three different growth patterns, mantle zone, nodular and diffuse pattern. There are also several morphological variants with different median survival (monocytoid B-cell like, pleomorphic or anaplastic variant, large cell variant and blastoid variant) that fill the criteria for being designated as MCLs (CD20⁺ neoplasms that are CD43⁺, CD5⁺, CD23^", CDlO^" and cyclin Dl positive) (Kurtin, 1998, Adv Anat Pathol 5:376-398). Follicular dendritic cell meshwork positive for CD21, CD23 and CD35 can often be seen in the MCL tumour tissue (Kurtin, 1998, Adv Anat Pathol 5:376-398). The t(l l:14)(ql3:q32) translocation that leads to the translocation of bcl-1 and over expression of cyclin Dl is a characteristic feature of MCL and is seen rarely in other types of non-Hodgkin's lymphoma (Kurtin, 1998, Adv Anat Pathol 5:376-398; Bentz et al, 2000, Genes Chromosomes Cancer 27:285-294). Cyclin Dl promotes the Gl to S phase transition and is believed to be one of the main features contributing to the malignant behaviour of MCL.

MCL is recognised as one of the most severe forms of lymphoma, generally exhibiting a resistance to conventional chemotherapy and a rapid clinical progression in many cases to incurable malignancies with a median survival time of 3 years (Kurtin 1998 supra). Hence, there is a need for improved therapies for the treatment of MCL and improved diagnostic methods. A first aspect of the invention provides a compound comprising a binding moiety which selectively binds to one of the proteins or polypeptides listed in Table 1 for the treatment and/or imaging and/or diagnosis of MCL.

A second aspect of the invention provides a compound according to the first aspect of the invention for use as a medicament. Preferably, the medicament is for the imaging, diagnosis, prognosis or treatment of MCL.

A third aspect of the invention provides a use of a compound according to the first aspect of the invention in the manufacture of an agent for imaging MCL cells in a body of an individual. Thus, the invention provides the use of a compound according to the first aspect of the invention for imaging MCL cells, either in vivo or in vitro.

A fourth aspect of the invention provides a use of a compound according to the first aspect of the invention in the manufacture of a diagnostic or prognostic agent for MCL. Thus, the invention provides the use of a compound according to the first aspect of the invention for diagnosing or prognosing MCL.

A fifth aspect of the invention is the use of a compound according to the first aspect of the invention wherein in the manufacture of a medicament for treating MCL. Thus, the invention provides the use of a compound according to the first aspect of the invention for treating MCL. It will be appreciated by persons skilled in the art that the medicament may be used for prophylactic and therapeutic purposes.

By "selectively bind" we include binding moieties which bind at least 10-fold more strongly to one of the proteins listed in Table 1 than to another polypeptide; preferably at least 50-fold more strongly and more preferably at least 100-fold more strongly. Table 1

Ectonucleotide L35594 NP 006200.2 SEQ ID 21 SEQ ID 50 Antibody pyrophosphate 250041- 250035

/phosphodieste SEQ ID 51 rase 2

(autotoxin)

Intestinal cell AB023153 NP _055735.1 SEQ ID 22 SEQ ID 62 Antibody kinase 250075-252364a

SEQ ID 52 Antibody

250075-

252364b

SEQ ID 53 Antibody

250045-242363

Nuclear S77154 NP_. _.006177.1 Isoform a - SEQ ID 54 Antibody receptor SEQ ID 23 250057 subfamily 4,

NP_. _.775263.1 Isoform b - group A, SEQ ID 24 member 2

NP_. _.775264.1 Isoform c -

SEQ ID 25

Aryl- AB002305 NP_ _.055677.3 SEQ ID 26 SEQ ID 55 Antibody hydrocarbon 250038- 250098 receptor nuclear translocator 2

Serine (or D83174 NP_ _001226.2 SEQ ID 27 SEQ ID 56 Antibody cysteine) 250053 proteinase inhibitor, clade

H member 1 precursor

Four and a AF063002 NP_ .001440.2 SEQ ID 28 SEQ ID 57 Antibody half LIM 250116- domains 1 SEQ ID 58 250027- 250091

Preferably, the binding moiety selectively binds to a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID Nos 1 to 32 and natural variants thereof. Most pereferably the polypeptide is SEQ ID No 20.

SEQ ID 20 is the polypeptide sequence of a galectin, LGALS3BP/ Mac-2 BP/ 9OK (NP_OO5558.1 , table 1). The galectins are a family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. LGALS3BP/ Mac-2 BP/ 9OK (NP_005558.1, table 1), is a highly glycosylated, secreted protein extensively studied hi human cancer.

LGALS3BP has been found elevated in the serum of patients with cancer and hi those infected by the human immunodeficiency virus (HTV). It appears to be implicated in immune response associated with natural killer (NK) and lymphokine- activated killer (LAK) cell cytotoxicity. The native protein binds specifically to a human macrophage-associated lectin known as Mac-2 aiid also binds gaiectin 1. High expression levels of LGALS3BP are associated with a shorter survival, the occurrence of metastasis or a reduced response to chemotherapy in patients with different types of malignancies. It is hypothesized that different galectins and gaiectin ligands with overlapping or opposite functions, expressed in different tumors during the different steps of the metastatic cascade might play a crucial role in tumor progression Grassadonia et al. , (2004) Glycoconj J. 19(7-9): p. 551-6.

In the present invention we show that the mRNA and protein corresponding to LGALS3BP is overexpressed in mantle cell lymphoma. In example 2 it is shown that that specific knock-down of LGALS3BP induces decreased proliferation in an in vitro model of MCL, indicative of a important role in the malignant behavior of MCL and, thus, demonstrating LGALS3BP constitutes a suitable target for therapy of patients afflicted by MCL.

By "natural variants" we include, for example, allelic variants. Typically, these will vary from the given sequence by only one or two or three, and typically no more than 10 or 20 amino acid residues. Typically, the variants have conservative substitutions.

Variants of the above polypeptides include polypeptides comprising a sequence with at least 60% identity to the amino acid sequences of SEQ ID Nos 1 to 32, preferably at least 70% or 80% or 85% or 90% identity to said sequences, and more preferably at least 95%, 96%, 97%, 98% or 99% identity to said amino acid sequences.

Percent identity can be determined by, for example, the LALIGN program (Huang and Miller, Adv. Appl. Math. (1991) 12:337-357) at the Expasy facility site fhttp://www.ch.embnet.org/software/L ALIGN_form.html^") using as parameters the global alignment option, scoring matrix BLOSUM62, opening gap penalty -14, extending gap penalty —4. Alternatively, the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.

Conveniently, the binding moiety selectively binds to a polypeptide having a binding site with the amino acid sequence selected from the group consisting of SEQ ED Nos 33 to 61 and natural variants thereof. Most preferably the binding site sequence is SEQ ED Nos 48 and/or 49.

The binding sites were identified as protein epitope signature tags (PrESTs) Agaton et al. (2003) MoI Cell Proteomics 2 pp 405-414. A PrEST represents a unique region comprising 100-150 amino acid residues of a protein. PrEST design is based on the selection of a protein region with as low as possible similarity to protein regions from other genes.

Preferably, the polypeptide is a human polypeptide.

Optionally the compound of the first aspect of the invention also comprises a further moiety.

Advantageously, the binding moiety and further moiety are covalently attached.

In one embodiment of the first aspect of the invention, the binding moiety is an antibody.

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 which bind to one of the proteins identified in Table 1.

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. ScL 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. ScL 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 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.

Suitable monoclonal antibodies 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 Application ", SGR Hurrell (CRC Press, 1982). Polyclonal antibodies may be produced which are polyspecific or monospecific. It is preferred that they are monospecific.

Chimaeric antibodies are discussed by Neuberger et al (1998, 8^th International Biotechnology Symposium Part 2, 792-799).

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 for one of the proteins identified in Table 1 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.

In an alternative embodiment, the binding moiety is a polypeptide. Polypeptide binding moieties can be identified by means of a screen. A suitable method or screen for identifying peptides or other molecules which selectively bind a target protein or polypeptide may comprise contacting the target protein or polypeptide with a test peptide or other molecule under conditions where binding can occur, and then determining if the test molecule or peptide has bound the target protein or peptide. 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 peptides or other ligand molecules suitable for use as binding moieties. An alternative method includes the yeast two hybrid system.

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 MCL. In particular, the further moiety is one which is useful in killing or imaging cells associated with MCL. Preferably, the further moiety is one which is able to kill the 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 MCL cells.

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 methyhnelamines 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); Antimetabohtes 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-tnioguanine; 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, phcamycin (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 (czs-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MTH); 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, carbodiirrήde conjugation (Bauminger & Wiichek (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 R₁- N=C=N-R₂, where R] 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 & Wiichek, 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 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 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. ScL 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. ScL 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 ^l ' ¹In 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 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 (i.e. the MCL cells) 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. ScL 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 binding moiety with specificity for one of the proteins identified in Table 1 may be used in place of an antibody in this type of directed enzyme prodrug therapy system.

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 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 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 the Table 2 below.

Table 2

(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 2, 223). Suitable enzymes for forming part of the enzymatic portion a compound ol 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 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-Unking 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 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 invention also provides a kit of parts (or a therapeutic system) comprising (1) a compound of the invention wherein the further 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 disclosed.

The invention also 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 or a readily detectable moiety to which the further moiety of the compound is able to bind.

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) Clin. Invest. 10, 533-551; Mitchell et al (1989) Int. J. Radial Biol. 56, 827- 836; Iliakis & Kurtzman (1989) Int. J. Radial Oncol. Biol. Phys. 16, 1235-1241; Brown (1989) Int. J. Radial 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 photofrin (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.

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 & Nieisen (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 adeno virus-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 μjTi 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-lipo somes 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 (i.e. MCL cell) 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 antiinflammatory cytokines, and anti-proliferative, immunomodulatory or factors influencing blood clotting which may be of benefit in treating MCL.

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 ^SSmTc and ^liJI for scintigraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as ¹²³I again, ¹³¹I, ¹¹¹In, ¹⁹F, ¹³C, ¹⁵N, ¹⁷O, 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 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 ¹¹¹In 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.

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 binding moiety (which selectively binds to a protein listed in Tabie 1) 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 compound may comprise an antibody which selectively binds to a protein listed in Table 1 , 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 binding moiety and the further moiety are polypeptides which are fused.

The compounds of the first of the invention are useful in treating, imaging or diagnosing MCL, as described in more detail below.

A sixth aspect of the invention provides a pharmaceutical composition comprising a compound according to 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 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. Other formulations and administration routes are discussed in the Examples.

A seventh aspect of the invention provides a method of imaging MCL cells in the body of an individual, the method comprising administering to the individual an effective amount of a compound according to the first aspect of the invention wherein the further moiety is a readily detectable moiety.

hi a preferred embodiment of this aspect of the invention, the method comprises the further step of detecting the location of the compound in the individual.

Detecting the compound or antibody can be achieved using methods well known in the art of clinical imaging and diagnostics. The specific method required will depend on the type of detectable label attached to the compound or antibody. For example, radioactive atoms may be detected using autoradiography or in some cases by magnetic resonance imaging (MRI) as described above.

An eighth aspect of the invention provides a method of diagnosing or prognosing MCL in an individual, the method comprising administering to the individual an effective amount of a compound according to the first aspect of the invention wherein the further moiety is a readily detectable moiety. The method may be one which is an aid to diagnosis.

In a preferred embodiment of this aspect of the invention, the method of diagnosing, or aiding diagnosis of, MCL in an individual comprises the further step of detecting the location of the compound in the individual.

A ninth aspect of the invention provides a method of treating an individual in need of treatment, the method comprising administering to the individual an effective amount of a compound according to the first aspect of the invention wherein the further moiety is a cytotoxic or therapeutic moiety. Preferably, the patient in need of treatment has MCL.

It will be appreciated that the compounds of the invention may act directly on MCL cells by binding selectively to a protein or polypeptide listed in Table 1.

Alternatively, the compound may act indirectly by interfering with (e.g. inhibiting or preventing) the interaction between a protein or polypeptide listed in Table 1 and a second moiety, wherein the interaction is essential for growth of MCL cells. For example, the compound may bind to a site on the protein or polypeptide listed in Table 1 which interacts with the further moiety, or vice versa, thereby interfering with the interaction between the protein or polypeptide and the further moiety. Alternatively, the compound may act via an allosteric mechanism to interfere with the interaction between the protein or polypeptide and the further moiety.

Preferably, the compound interferes with the interaction between cellular growth factors or cell surface receptor thus affecting proliferation, differentiation or maturation of the cancer cell, e.g. as has been shown for the IL-13/IL-13 receptor interactions. The therapeutic efficacy is thus achieved by interfering with normal cellular functions that are specifically over-expressed in the malignant cell, giving that particular cell a specific signal. It will be further appreciated that, depending on the particular compound used in imaging, diagnosis or treatment, 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.

hi 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 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 {i.e. the target sites) imaging takes place.

A tenth aspect of the invention provides a method of introducing genetic material selectively into MCL cells the method comprising contacting the cells with a compound according to the first aspect of the invention as described above wherein the further moiety is a nucleic acid, or a compound comprising (i) a binding moiety which selectively binds to a protein listed in Table 1 and (ii) a nucleic acid.

Preferably, the binding moiety is an antibody. Typically, the binding moiety is comprised in a delivery vehicle and preferably, the delivery vehicle is a liposome, as described in further detail above. In this embodiment, the further moiety is nucleic acid and is comprised within the liposome, also as described above. Typically, the method is used in gene therapy, and the genetic material is therapeutically useful. Therapeutically useful genetic material includes that which encodes a therapeutic protein.

An eleventh aspect of the invention provides a nucleic acid molecule encoding a compound according to the first aspect of the invention or a binding moiety thereof. By "nucleic acid molecule" we include DNA, cDNA and mRNA molecules, which may be single- or double-stranded.

A twelfth aspect of the invention provides an expression vector comprising a nucleic acid molecule according to the tenth aspect of the invention. By "expression vector" we mean one which is capable, in an appropriate host, of expressing a polypeptide encoded by the nucleic acid molecule.

Such vectors may be useful in expressing the encoded compound or binding moiety thereof in a host cell for production of useful quantities of the compounds of the invention. A variety of methods have been developed to operably link nucleic acid molecules, especially DNA, to vectors, for example, via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted into 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, e.g. generated by endonuclease restriction digestion, 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 polymerising activities.

The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a larger 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 site 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 the invention is to use PCR. This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art.

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

The DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention or binding moiety thereof. Thus, the DNA encoding the polypeptide 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 compound of the invention or binding moiety thereof. 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 (or in the case or retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention or binding moiety thereof may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.

The companion DNA will depend upon the nature 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 expression vector 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 typically include a prokaryotic replicon, such as the CoIEl on, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of 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 prokaryotic 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-I 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 URAS. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).

Other vectors and expression systems are well known in the art for use with a variety of host cells.

A thirteenth aspect of the invention provides a recombinant host cell comprising a nucleic acid molecule or vector of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and rypicaiiy 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, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic and kidney cell lines. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CRL 1658 and 293 cells which are human embryonic kidney cells. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.

Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sd. USA 69, 2110 and Sambrook et al

(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,

Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY.

The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from

Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.

Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cells, bacterial cells, insect cells and vertebrate cells. For example, many bacterial species may be transformed by the methods described in Luchansky et al (198S) MoI. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5 PEB using 6250V per cm at 25 μFD.

Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol 194, 182.

Successfully transformed cells, i.e. cells that contain a DNA construct of the present invention, can be identified by well-known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. MoI. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.

In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity.

Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.

The host cell may be a host cell within an non-human animal body. Thus, transgenic non-human animals which express a compound according to the first aspect of the invention (or a binding moiety thereof) by virtue of the presence of the transgene are included. Preferably, the transgenic non-human animal is a rodent such as a mouse. Transgenic non-human animals can be made using methods well known in the art.

A fourteenth aspect of the invention provides a method of producing a compound of the invention or binding moiety thereof, the method comprising expressing a nucleic acid molecule according to the tenth aspect of the invention or culturing a host cell according to the twelfth aspect of the invention.

Methods of cultivating host cells and isolating recombinant proteins are well known in the art. It will be appreciated that, depending on the host cell, the compounds of the invention (or binding moieties thereof) produced may differ. For example, certain host cells, such as yeast or bacterial cells, either do not have, or have different, post-translational modification systems which may result in the production of forms of compounds of the invention (or binding moieties thereof) which may be post- translationally modified in a different way.

It is preferred that compounds of the invention (or binding moieties thereof) are produced in a eukaryotic system, such as a mammalian cell.

According to a less preferred embodiment, the compounds of the invention (or binding moieties thereof) can be produced in vitro using a commercially available in vitro translation system, such as rabbit reticulocyte lysate or wheatgerm lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system may be coupled to a transcription system, such as the TNT transcription-translation system (Promega). This system has the advantage of producing suitable rnRNA transcript from an encoding DNA polynucleotide in the same reaction as the translation.

Preferably, the production method of this aspect of the invention comprises a further step of isolating the compounds of the invention (or binding moieties thereof) produced from the host cell or from the in vitro translation mix. Preferably, the isolation employs an antibody which selectively binds the expressed polypeptide of the invention.

As discussed above, in a preferred embodiment of the compounds of the first aspect of the invention the binding moiety comprises an antibody or antigen-binding fragment thereof

Antibodies can be raised in an animal by immunising with an appropriate peptide. Appropriate peptides include the proteins listed in Table 1 and fragments thereof. Alternatively, with today's technology, it is possible to make antibodies as defined herein without the need to use animals. Such techniques include, for example, antibody phage display technology as is well known in the art. Appropriate peptides, as described herein, may be used to select antibodies produced in this way.

It will be appreciated that, with the advancements in antibody technology, it may not be necessary to immunise an animal in order to produce an antibody. Synthetic systems, such as phage display libraries, may be used. The use of such systems is included in the methods of the invention and the products of such systems are "antibodies" for the purposes of the invention.

It will be appreciated that such antibodies which recognise one of the proteins listed in Table 1 and variants or fragments thereof are useful research reagents and therapeutic agents, particularly when prepared as a compound of the invention as described above. Suitably, the antibodies of the invention are detectably labelled, for example they may be labelled in such a way that they may be directly or indirectly detected. Conveniently, the antibodies are labelled with a radioactive moiety or a coloured moiety or a fluorescent moiety, or they may be linked to an enzyme. Typically, the enzyme is one which can convert a non-coloured (or non-fluorescent) substrate to a coloured (or fluorescent) product. The antibody may be labelled by biotin (or streptavidin) and then detected indirectly using streptavidin (or biotin) which has been labelled with a radioactive moiety or a coloured moiety or a fluorescent moiety, or the like or they may be linked to any enzyme of the type described above.

A fifteenth aspect of the invention provides a method of identifying cells associated with MCL, the method comprising analysing the pattern of gene expression in a sample of cells to be tested and comparing it to the pattern of gene expression in a sample of known MCL cells.

Preferably, the known MCL cells are characterised by the upregulation of expression of one or more genes encoding a protein listed in Table 1 compared to normal B-cells.

In a preferred embodiment of the fourteenth aspect of the invention, the method further comprises comparing the pattern of gene expression in a sample of cells to be tested with the pattern of gene expression in a control sample.

Conveniently, the control sample comprises normal B-cells.

Advantageously, the cells to be tested are identified as MCL cells if the expression of one or more genes encoding a protein listed in Table 1 is upregulated compared to normal B-cells.

Preferably, the cells to be tested are identified as MCL cells if the expression of genes encoding all of the proteins listed in Table 1 is upregulated compared to normal B- cells.

By "upregulated" we mean that the expression of the gene is increased by at least 10% compared to expression of the same gene in normal B-cells. Preferably, expression of the gene is increased by at least 20%, 30%, 40% or 50%. Most preferably expression of the gene is increased by at least 50%.

A further aspect of the invention provides a method of distinguishing between different types or stages of MCL, the method comprising analysing the pattern of gene expression in a sample of cells to be tested and comparing it to the pattern of gene expression in a sample of MCL cells of a known type or stage.

A further aspect of the invention is a method of screening for a molecule with efficacy in the treatment of MCL, the method comprising the steps of:

(i) contacting a molecule to be tested with a protein listed in Table 1 (or a fragment or binding site thereof); and

(ii) detecting the presence of a complex containing the protein (or fragment thereof) and the molecule to be tested.

In a preferred embodiment, the test molecule is a polypeptide.

Suitable peptide ligands that will bind to a protein listed in Table 1 (or a fragment thereof) may be identified using methods known in the art.

One method, disclosed by Scott and Smith (1990) Science 249, 386-390 and Cwirla et al (1990) Proc. Natl. Acad. ScL USA 87, 6378-6382, involves the screening of a vast library of filamentous bacteriophages, such as Ml 3 or fd, each member of the library having a different peptide fused to a protein on the surface of the bacteriophage. Those members of the library that bind to a protein listed in Table 1 (or a fragment thereof) are selected using an iterative binding protocol, and once the phages that bind most tightly have been purified, the sequence of the peptide ligands may be determined simply by sequencing the DNA encoding the surface protein fusion. Another method that can be used is the NovaTope (TM) system commercially available from Novagen, Inc., 597 Science Drive, Madison, WI 53711. The method is based on the creation of a library of bacterial clones, each of which stably expresses a small peptide derived from a candidate protein in which the ligand is believed to reside. The library is screened by standard lift methods using the antibody or other binding agent as a probe. Positive clones can be analysed directly by DNA sequencing to determine the precise amino acid sequence of the ligand.

Further methods using libraries of beads conjugated to individual species of peptides as disclosed by Lam et al (1991) Nature 354, 82-84 or synthetic peptide combinatorial libraries as disclosed by Houghten et al (1991) Nature 354, 84-86 or matrices of individual synthetic peptide sequences on a solid support as disclosed by Pirrung et al m US 5143854 may also be used to identify peptide ligands.

It will be appreciated that screening assays which are capable of high throughput operation will be particularly preferred. Examples may include cell based assays and protein-protein binding assays. An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used. For example, an assay for identifying a compound capable of modulating the activity of a protein kinase may be performed as follows. Beads comprising scintillant and a polypeptide that may be phosphorylated may be prepared. The beads may be mixed with a sample comprising the protein kinase and ³²P-ATP or ³³P-ATP and with the test compound. Conveniently this is done in a 96-well format. The plate is then counted using a suitable scintillation counter, using known parameters for P or P SPA assays. Only ³²P or ³³P that is in proximity to the scintillant, i.e. only that bound to the polypeptide, is detected. Variants of such an assay, for example in which the polypeptide is immobilised on the scintillant beads via binding to an antibody, may also be used.

Other methods of detecting polypeptide/polypeptide interactions include ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Fluorescence Energy Resonance Transfer (FRET) methods, for example, well known to those skilled in the art, may be used, in which binding of two fluorescent labelled entities may be measured by measuring the interaction of the fluorescent labels when in close proximity to each other.

Alternative methods of detecting binding of a polypeptide to macromolecules, for example DNA, RNA, proteins and phospholipids, include a surface plasmon resonance assay, for example as described in Plant et al (1995) Analyt Biochem 226(2), 342-348. Methods may make use of a polypeptide that is labelled, for example with a radioactive or fluorescent label.

A further method of identifying a compound that is capable of binding to a protein listed in Table 1 (or a fragment thereof) is one where the polypeptide is exposed to the compound and any binding of the compound to the said polypeptide is detected and/or measured. The binding constant for the binding of the compound to the polypeptide may be determined. Suitable methods for detecting and/or measuring (quantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be performed, for example, using a method capable of high throughput operation, for example a chip-based method. New technology, called VLSIPS™, has enabled the production of extremely small chips that contain hundreds of thousands or more of different molecular probes. These biological chips or arrays have probes arranged in arrays, each probe assigned a specific location. Biological chips have been produced in which each location has a scale of, for example, ten microns. The chips can be used to determine whether target molecules interact with any of the probes on the chip. After exposing the array to target molecules under selected test conditions, scanning devices can examine each location in the array and determine whether a target molecule has interacted with the probe at that location.

Biological chips or arrays are useful in a variety of screening techniques for obtaining information about either the probes or the target molecules. For example, a library of peptides can be used as probes to screen for drugs. The peptides can be exposed to a receptor, and those probes that bind to the receptor can be identified. See US Patent No. 5,874,219 issued 23 February 1999 to Rava et al.

It will be understood that it will be desirable to identify compounds that may modulate the activity of the a protein listed in Table 1 in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the interactions between the said and the interacting polypeptide are substantially the same as between a said naturally occurring polypeptide and a naturally occurring interacting polypeptide in vivo.

It will be appreciated that in the method described herein, the ligand may be a drug- like compound or lead compound for the development of a drug-like compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential. The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, nonselective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

Alternatively, the methods may be used as "library screening" methods, a term well known to those skilled in the art. Thus, for example, the method of the invention may be used to detect (and optionally identify) a polynucleotide capable of expressing a polypeptide activator of a protein listed in Table 1. Aliquots of an expression library in a suitable vector may be tested for the ability to give the required result.

Preferably, the compound decreases the activity of the protein listed in Table 1. For example, the compound may bind substantially reversibly or substantially irreversibly to the active site of said protein. In a further example, the compound may bind to a portion of said protein that is not the active site so as to interfere with the binding of the said protein to its ligand. In a still further example, the compound may bind to a portion of said protein so as to decrease said protein's activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said protein's activity, for example in the activation of the said protein by an "upstream activator".

In a yet further aspect of the invention, the polypeptides or proteins of table 1 or natural variants thereof can be used in a pharmaceutical formulation. Furthermore, immunological formulations (i.e. vaccines) may be prepared by combining a protein or polypeptide listed in table 1 or natural variant thereof with an adjuvant. Active immunisation of the patient is preferred. In this approach, one or more polypeptides or polynucleotides or expression vectors are prepared in an immunogenic formulation containing suitable adjuvants and carriers and administered to the patient in known ways. Suitable adjuvants include Freund's complete or incomplete adjuvant, muramyl dipeptide, the "Iscoms" of EP 109 942, EP 180 564 and EP 231 039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141). "Pluronic" is a Registered Trade Mark. The patient to be immunised is a patient requiring to be protected from mantle cell lymphoma.

The invention also includes a method of vaccinating an individual against mantle cell lymphoma, the method comprising administering to the individual a protein or polypeptide listed in table 1 or natural variant thereof. The dose and frequency is determined by the physician

The invention also includes the use of a protein or polypeptide listed in table 1 or natural variant thereof in the manufacture of a vaccine for vaccinating an individual against mantle cell lymphoma.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

Figurel. - The functional effect of the knock-down is assessed using a proliferation assay and Granta 519, as a MCL in vitro model. Specific knock-down of the target gene and suitable control is performed using sequence specific probes (siRNA) for the (i) target gene, LGALS3BP1, (ii) a positive control, Eg5, (iii) a negative control (scrambled sequence) and (iv) un-knocked cells as reference (electroporation control). The error bars show the variation over the triplicates. EXAMPLES

Example 1

Materials and methods

Selection of candidate genes

The MCL-associated genes were derived, using gene expression profiling (U95v2, Afrymetric Inc., Santa Clara, CA) of tumor samples from 19 patients diagnosed with MCL and 11 samples of normal pediatric tonsil B cells (Malmoe University Hospital), as previously described Ek et al. (2004) Haematologica 89, pp686-95 and Ek et al. (2002) Cancer Res. 62 pp4398-4405. The different B cell populations used were: naϊve B cells (CD19⁺, IgD⁺, CD380, pre-activated B cells (CD19⁺, CD23⁺, CD38^"), centroblasts (CD19⁺, CD38⁺, CDIl⁺), centrocytes (CD19⁺, CD38⁺, COlT) and memory B cells (CD19⁺, CD38^", IgD^'). When filtering the data for differentially regulated genes, all samples were scaled to a fixed median value, as recommended by Affymetric Inc. in the U95v2 product literature.

After filtering of the data, three main categories of genes were selected:

1. Genes that were quantitatively over expressed (>2 fold) in the majority (>80%) of MCL patient samples, compared to normal B cell populations.

2. Genes that were qualitatively (i.e. present vs. absent call) over expressed in the majority (>80%) of MCL patient samples, compared to normal B cell populations. These genes were not expressed in any of the normal B cell populations analyzed.

3. Genes that were qualitatively over expressed in a sub-group (>30%) of MCL patients compared to normal B cell populations. These genes were not expressed in any of the normal B cell populations analyzed. Generation and Analysis of the Affinity Reagents

Suitable Protein Epitope Signature Tags (PrESTs), representing unique regions for each target protein, were designed using bioinformatic tools Lindskog et al. (2005) Biotechniques 38 pp723-727 and with the human genome sequence as template Hubbard et al. (2005) Nucleic Acid Res. 33 D447-53. In the design of the PrESTs, transmembrane regions and signal peptides were avoided and an amino acid sequence, with a size between 100 and 150 amino acids, with low homology to other human proteins was selected to decrease the risk of cross-reactivity of antibodies to other human proteins. The cloning and protein expression were performed, as previously described by Agaton et al. (2003) MoI Cell Proteomics 2 pp405-414. The mono-specific antibodies were subsequently obtained by affinity purification of rabbit polyclonal antisera, as described by Nilsson et al. (2005) Proteomics 5 4327- 37. The antibodies were quality controlled using a protein array and Western blots procedure, as described by Uhlen et al. (2005) MoI Cell Proteomics 4 1920-32.

Case Selection

A total of 25 cases of Non-Hodgkin's lymphoma were collected from the files of the Department of Pathology, The University of Schleswig-Holstein, Campus Lύbeck, between 2004 and 2005. AU cases were diagnosed and classified using morphologic and immunophenotypic criteria specified in Jaffe et al. (2001) World Health Organization Classification of lymphoid neoplasms (IARC Press) p 121-235. The group of B-cell NHL cases assessed included: 8 mantle cell lymphoma (6 small cell or classical and 2 blastoid), 4 follicular lymphoma (all grade 1 or 2), 1 nodal marginal zone B-cell lymphoma, 2 lymphoplasmacytic lymphoma and 3 small lymphocytic lymphoma/chronic lymphocytic leukemia. Non-neoplastic lymphoid tissues were included, represented by tonsils with follicular hyperplasia and tonsils with EBV infection (M. Pfeiffer). All cases were analyzed using either tissue microarrays (n = 25) or full tissue sections (n = 2). The tissue microarrays included duplicate cores from all cases and were constructed, using a manual tissue arrayer (AlphaMetrix Biotech GmbH, 63322 Rόdermark, Germany). All neoplasms assessed were fixed in 10% buffered formalin, routinely processed and embedded in paraffin.

Immunohistochemical Methods

The immunohistochemical (EHC) methods used in this study have been described previously Hsu et al. (1981) J Histochem Cytochem 29 p577-80. Briefly, heat- induced antigen retrieval was performed prior to IHC staining. Sections were incubated with a polyclonal antibody (0.1 - lμg/ slide) at room temperature for 30 minutes. Detection of signal was achieved, using the ChemMate™ EnVision™ Detection Kit Peroxidase/DAB, Rabbit/Mouse LSAB+ kit (DAKO, Hamburg, Germany) that contains secondary biotinylated goat anti rabbit/mouse antibody and streptavidin/horseradish peroxidase complex, according to the manufacturer's recommendations. 3,3'-diaminobenzidine/H₂O₂ (DAKO) was used as chromogen and slides were counterstained with hematoxylin.

Scoring of Positive Cells in IHC

Defined staining patterns were noticed for the various antibodies and a cut-off of 2% of stained cells was used to categorized an antibody as positive. Cytoplasmic or nuclear staining was also considered and expression was graded visually as: negative, weak or moderate/strong. A lymphoma case was considered weakly positive if cytoplasmic staining was similar to or less than that of normal cells, and moderate to strongly positive if cytoplasmic staining was similar to or greater than that of normal cells. Results

Filtering for MCL-associated genes and subsequent PrEST selection

After comprehensive analysis of differentially expressed genes, where all 19 MCL samples and 5 normal B cell populations, derived from 11 different tonsils, were compared we chose 21 genes. These were used to generate the subsequent PrEST (prtoein epitope signature tags) identification. Since we focused on gene products potentially useful for prognosis and therapy, all the selected genes were quantitatively or qualitatively overexpressed in MCL, as compared to normal B cell populations (Table 1). Furthermore, the selection of genes were focused mainly on poorly or presently non-characterized gene products, since we considered these to be most interesting when searching for novel tumor target candidates.

Generation of PrEST-specific antibodies

One or two unique PrESTs were identified for each of the 21 genes, using the designed algorithm. All antibodies had after purification by affinity chromatography concentrations in the interval of 0.02 - 0.4 mg/ml and did pass our quality control, including PrEST microarray analysis (Uhlen et al (2005) supra). The antibodies were subsequently analyzed by IHC on several different lymphomas.

Immunohistochemistry analysis

The quality-controlled, monospecific antibodies were then evaluated, using EHC on paraffin-embedded tissue sections from MCL, follicular lymphoma (FL), chronic lymphocytic lymphoma (CLL), marginal zone (MGZ)-lymphoma and tonsils. A 2% cut-off of positive cells was set to determine the antibody reactivity, although in most positive cases the percentage of positive cells was over 50% of tumor cells or defined cell populations, respectively. The tested antibodies showed reactivity to one or more of the tissues, in particular the reactive antibodies showed staining of MCL tumor ceiis. The reactivity pattern of the MCL-associated antibodies we found that eight antibodies ( Antibodies 15, 33, 34, 38, 41, 53, 60, 93) were able to separate MCL from the other lymphoma types that were tested, such as FL and CLL.

Example 2 - Knock down function ofLGALS3BP (NP_005558.1) identified by example 1

Cell culture The cell line, Granta 519 (ACC 12) was obtained from Deutsche Sammlung von Mikroorganismen un Zellkulturen (DSMZ) GmH (Braunschweig, Germany). The cell line was cultured in a humidified atmosphere at 37°C, using a 5% CO₂ atmosphere. The cells were maintained at approximately 1x10⁶ cells/ml.

siRNA design and transfection

Granta 519 was chosen as an in vitro model system for functional analysis of MCL using siRNA. A unique small interfering RNAs (siRNAs) towards the LGALS3BP1 (NP_005558.1) gene was designed, using an on-line tool at www.ambion.com. The siRNA was produced and purified by Ambion (Austin, TX). Freeze-dried siRNAs were suspended in nuclease-free water (Ambion) at a final concentration of 20 μM and transfection was carried using an Amaxa nucleofection Device (Amaxa Biosystems, Cologne, Germany). The 0-17 program and Cell line Nucleofector Solution T was used. For every transfection, 2.5xlO⁶ cells were electroporated with 100 pmol of siRNA. The sequence for the target gene is shown below:

LGALSBP Sequence (5 '->3') Sense: CCUCACCGAGGAUACCUACtt

Antisense: GUAGGUAUCCUCGGUGAGGtt

Proliferation assay To evaluate the functional effect of gene silencing, the proliferation of transfected Granta 519 cells was assessed by a [Η] -thymidine incorporation assay. 3 hours after nucleofection, cells were transferred to a 96-well plate (approximately 100 000 cells to each well, in triplicates). After 40 hours, cells were pulsed by the addition of 0.5 μCi/well of [³H]-thymidine (Amersham Biosciences) and incubated for 8h. After 48 hours, cells were harvested and the incorporation of [³H] -thymidine was assessed using a 1450 Micro Beta Liquid Scintillation Counter (Pharmacia, Uppsala, Sweden).

Results and discussion

Using siRNA specific for LGALS3BP1 (NP_005558.1), a decrease in proliferation was detected in the MCL in vitro model, Granta 519. The magnitude of the decrease was comparable to the positive control Eg5, a motor protein known to be essential for mitosis and which upon knock-down causes cell cycle arrest. As the degree of decrease was comparable, the results indicate that LGALS3BP1 is highly involved in the survival and proliferation of MCL tumor cells.

Example 3 - Pharmaceutical formulations

A further aspect of the invention provides a pharmaceutical formulation comprising a compound according to the first aspect of the invention in admixture with a pharmaceutically or veterinarily acceptable adjuvant, diluent or carrier.

Preferably, the formulation is a unit dosage containing a daily dose or unit, daily sub- dose or an appropriate fraction thereof, of the active ingredient.

The compounds of the invention may be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

In human therapy, the compounds of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The compounds of invention may also be administered via intracavernosal injection.

The polypeptides, molecules and pharmaceutical formulations of the present invention may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.

The polypeptides, molecules and pharmaceutical formulations of the present invention can be administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.

Electroporation therapy (EPT) systems can also be employed for the administration of proteins and peptides. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.

Proteins and peptides can also be delivered by electroincorporation (EI). EI occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In EI, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.

An alternative method of protein and peptide delivery is the ReGeI injectable system that is thermo-sensitive. Below body temperature, ReGeI is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.

Protein and peptide pharmaceuticals can also be delivered orally. The process employs -a natural process for oral -uptake of -vitamin B₁₂ in the body to-co-deliver- proteins and peptides. By riding the vitamin B₁₂ uptake system, the protein or peptide can move through the intestinal wall. Complexes are synthesised between vitamin B₁₂ analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B₁₂ portion of the complex and significant bioactivity of the drug portion of the complex.

Proteins and polypeptides can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient. See Derossi et al. (1998), Trends Cell Biol 8, 84-87. Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy- propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The compounds of the invention can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

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. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyopbilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

For oral and parenteral administration to human patients, the daily dosage level of the compounds of the invention will usually be from lmg/kg to 30 mg/kg. Thus, for example, the tablets or capsules of the compound of the invention may contain a dose of active compound for administration singly or two or more at a time, as appropriate.

The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case.

There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.

The compounds of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydro fluoroalkane such as 1,1,1,2-tetrafluoroethane

(HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff delivers an appropriate dose of a compound of the invention for delivery to the patient. It will be appreciated that he overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the compounds of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The compounds of the invention may also be transdermally administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.

For ophthalmic use, the compounds of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the compounds of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Generally, in humans, oral or topical administration of the compounds of the invention is the preferred route, being the most convenient. In circumstances where the recipient suffers from a swallowing disorder or from impairment of drug absorption after oral administration, the drug may be administered parenterally, e.g. sublingually or buccally.

For veterinary use, a compound of the invention is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

Claims

1. Use of a compound comprising a binding moiety which selectively binds to a protein or polypeptide listed in Table 1 in the preparation of a medicament for treating mantle cell lymphoma (MCL).

2. Use of a compound comprising a binding moiety which selectively binds to a protein listed in Table 1 in the preparation of a diagnostic or prognostic agent for MCL.

3. Use of a compound comprising a binding moiety which selectively binds to a protein listed in Table 1 in the preparation of an agent for detecting MCL cells.

4. A use according to any one of Claims 1 to 3 wherein the binding moiety selectively binds to a polypeptide having an amino acid sequence selected from the group consisting of SEQ E) Nos 1 to 33 and natural variants thereof.

5. A use according to claim 4 wherein the binding moiety selectively binds to a polypeptide having an amino acid sequence of SEQ ID Nos 20 and natural variants thereof.

6. A use according to any one of Claims 1 to 3 wherein the binding moiety selectively binds to a polypeptide having a binding site with the amino acid sequence selected from the group consisting of SEQ E) Nos 34 to 61, and natural variants thereof.

7. A use according to any claim 6 wherein the binding moiety selectively binds to a polypeptide having a binding site with the amino acid sequence of SEQ E) Nos 48 and/or 49, and natural variants thereof.

8. A use according to any one of the preceding claims wherein the binding moiety is an antibody.

9. A use according to any one of the preceding claims wherein the binding moiety is a peptide.

10. A use according to any one of the preceding claims wherein the compound also comprises a further moiety.

11. A use according to claim 10 wherein the further moiety is a directly or indirectly cytotoxic moiety.

12. A use according to Claim 11 wherein the cytotoxic moiety is a directly cytotoxic chemotherapeutic agent.

13. A use according to Claim 11 or 12 wherein the cytotoxic moiety is a directly cytotoxic polypeptide.

14. A use according to Claim 11 wherein the cytotoxic moiety is a moiety which is able to convert a relatively non-toxic prodrug into a cytotoxic drug.

15. A use according to Claim 11 wherein the cytotoxic moiety is a radiosensitizer.

16. A use according to any of claims 10 to 15 wherein the further moiety comprises a nucleic acid molecule.

17. A use according to Claim 16 wherein the nucleic acid molecule is a cytotoxic nucleic acid.

18. A use according to Claim 16 wherein the nucleic acid moiecule encodes a directly or indirectly cytotoxic polypeptide.

19. A use according to Claim 16 wherein the nucleic acid molecule is directly cytotoxic.

20. A use according to Claim 16 wherein the nucleic acid encodes a therapeutic polypeptide.

21. A use according to Claim 11 wherein the cytotoxic moiety comprises a radioactive atom.

22. A use according to Claim 21 wherein the radioactive atom is phosphorus-32, iodine-125. iodine-131, indium-Ill, rhenium- 186, rhenium- 188 or yttrium-90.

23. A use according to Claim 10 wherein the further moiety is a readily detectable moiety.

24. A use according to Claim 23 wherein the readily detectable moiety comprises a radioactive atom.

25. A use according to Claim 24 wherein the radioactive atom is technetium-99m or iodine-123.

26. A use according to Claim 23 wherein the readily detectably moiety comprises a suitable amount of any one of iodine-123, iodine-131, indium- 111, fluorine- 19, carbon- 13. nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.

27. A use according to Claim 10 wherein the further moiety is able to bind selectively to a directly or indirectly cytotoxic moiety.

28. A use according to Claim 10 wherein the further moiety is able to bind selectively to a readily detectable moiety.

29. A use according to Claim 10 wherein the selective binding moiety and the further moiety are polypeptides which are fused.

30. A compound comprising a binding moiety which selectively binds to a protein or polypeptide listed in Table 1.

31. A compound according to Claim 30 wherein the binding moiety selectively binds to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID Nos 1 to 33, and natural variants thereof.

32. A compound according to Claim 31 wherein the binding moiety selectively binds to a polypeptide having an amino acid sequence of SEQ ID No 20, and natural variants thereof.

33. A compound according to Claim 30 wherein the binding moiety selectively binds to a polypeptide having a binding site with the amino acid sequence selected from the group consisting of SEQ ID Nos 34 to 61, and natural variants thereof.

34. A compound according to Claim 33 wherein the binding moiety selectively binds to a polypeptide having a binding site with the amino acid sequence of

SEQ ID Nos 48 and/or 49, and natural variants thereof.

35. A compound according to any one of Claims 30 to 34 wherein the binding moiety is an antibody.

36. Λ compound according to any one of Claims 30 to 34 wherein the binding moiety is a peptide.

37. A compound as claimed in any of claims 30 to 36 also comprising a further moiety.

38. A compound according to claim 37 wherein the further moiety is a directly or indirectly cytotoxic moiety.

39. A compound according to Claim 38 wherein the cytotoxic moiety is a directly cytotoxic chemotherapeutic agent.

40. A compound according to Claim 38 wherein the cytotoxic moiety is a directly cytotoxic polypeptide.

41. A compound according to Claim 38 wherein the cytotoxic moiety is a moiety which is able to convert a relatively non-toxic prodrug into a cytotoxic drug.

42. A compound according to Claim 38 wherein the cytotoxic moiety is a radiosensitizer.

43. A compound according to claim 37 wherein the further moiety comprises a nucleic acid molecule.

44. A compound according to Claim 43 wherein the nucleic acid molecule is a cytotoxic nucleic acid.

45. A compound according to Claim 44 wherein the nucleic acid molecule encodes a directly or indirectly cytotoxic polypeptide.

46. A compound according to Claim 44 wherein the nucleic acid molecule is directly cytotoxic.

47. A compound according to Claim 44 wherein the nucleic acid encodes a therapeutic polypeptide.

48. A compound according to Claim 38 wherein the cytotoxic moiety comprises a radioactive atom.

49. A compound according to Claim 48 wherein the radioactive atom is phosphorus-32, iodine-125, iodine-131, indium-I l l, rhenium-186, rhenium- 188 or yttrium-90.

50. A compound according to Claim 37 wherein the further moiety is a readily detectable moiety.

51. A compound according to Claim 50 wherein the readily detectable moiety comprises a radioactive atom.

52. A compound according to Claim 51 wherein the radioactive atom is technetium-99m or iodine- 123.

53. A compound according to Claim 50 wherein the readily detectably moiety comprises a suitable amount of any one of iodine-123, iodine-131, indium- 111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

54. A compound according to Claim 37 wherein the further moiety is able to bind selectively to a directly or indirectly cytotoxic moiety.

55. A compound according to Claim 37 wherein the further moiety is able to bind selectively to a readily detectable moiety.

56. A compound according to Claim 37 wherein the selective binding moiety and the further moiety are polypeptides which are fused.

57. A pharmaceutical composition comprising a compound according to any one of Claims 30 to 56 and a pharmaceutically acceptable carrier.

58. A compound according to any one of Claims 30 to 56 for use in medicine.

59. A compound according to any one of Claims 30 to 56 for use in the treatment, imaging, diagnosis or prognosis of MCL.

60. A nucleic acid molecule encoding a compound according to any one of Claims 30 to 56 or a binding moiety thereof.

61. An expression vector comprising a nucleic acid molecule according to Claim 60.

62. A recombinant host cell comprising a nucleic acid molecule according to Claim 60.

63. A recombinant host cell according to Claim 62 wherein the host cell is a bacteria] cell.

64. A recombinant host cell according to Claim 62 wherein the host cell is a mammalian cell.

65. A method of producing a compound according to any one of Claims 30 to 56 or a binding moiety thereof comprising expressing a nucleic acid molecule according to Claim 60 or an expression vector according to Claim 61 or culturing a host cell according to any one of Claims 62 to 64.

66. A kit of parts comprising a compound according to Claim 41 and a relatively non-toxic prodrug.

67. A kit of parts comprising a compound according to Claim 54 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.

68. A method of imaging MCL cells in the body of an individual, the method comprising administering to the individual an effective amount of a compound according to Claim 54.

69. A method of diagnosing or prognosing MCL in an individual, the method comprising administering to the individual an effective amount of a compound according to Claim 54.

70. A method according to Claim 68 or 69 further comprising the step of detecting the location of the compound in the individual.

71. A method of treating an individual with MCL, the method comprising administering to the individual an effective amount of a compound according to any one of Claims 30 to 56.

72. A method according to Claim 71, wherein the compound interferes with the interaction between a protein or polypeptide listed in Table 1 and a second moiety which is essential for the growth of MCL cells.

73. A method of introducing genetic material selectively into MCL cells, the method comprising contacting the cells with a compound according to any one of Claims 30 to 56.

74. A method of identifying cells associated with MCL. the method comprising analysing the pattern of gene expression in a sample of cells to be tested and comparing it to the pattern of gene expression in a sample of known MCL cells.

75. A method of distinguishing between different types or stages of MCL, the method comprising analysing the pattern of gene expression in a sample of cells to be tested and comparing it to the pattern of gene expression in a sample of MCL cells of a known type or stage.

76. A method according to Claim 74 or 75 wherein the known MCL cells are characterised by the upregulation of expression of one or more genes encoding a protein listed in Table 1 compared to normal B-cells.

77. A method according to Claim 74 or 75 further comprising comparing the pattern of gene expression in a sample of cells to be tested with the pattern of gene expression in a control sample.

78. A method according to Claim 77 wherein the control sample comprises normal B-cells.

79. A method according to any one of Claims 74 to 78 wherein the cells to be tested are identified or distinguished as MCL cells if the expression of one or more genes encoding a protein listed in Table 1 is upregulated compared to normal B-cells.

80. A method according to any one of Claim 79 wherein the cells to be tested are identified or distinguished as MCL cells if the expression of genes encoding all of the proteins listed in Table 1 is upregulated compared to normal B-cells.

81. A method of screening for a molecule with efficacy in the treatment of MCL, the method comprising the steps of:

(i) contacting a molecule to be tested with a protein listed in Table 1 (or a fragment or binding sequence thereof); and

82. A pharmaceutical composition comprising protein or polypeptide listed in table 1 or natural variant thereof and a pharmaceutically acceptable carrier.

83. An immunological formulation comprising a protein or polypeptide listed in table 1 or natural variant thereof and an adjuvant.

84. A method of vaccinating an individual against mantle cell lymphoma, the method comprising administering to the individual a protein or polypeptide listed in table 1 or natural variant thereof.

85. Use of a protein or polypeptide listed in table 1 or natural variant thereof in the manufacture of a vaccine for vaccinating an individual against mantle cell lymphoma.

86. A compound substantially as described herein.

87. A method of imaging MCL cells substantially as described herein.

88. A method of diagnosing or prognosing MCL in an individual substantially as described herein.

89. A method of treating an individual with MCL substantially as described herein.

90. A method of vaccinating an individual against MCL substantially as described herein.

91. A kit of parts substantially as described herein.

92. A pharmaceutical composition or immunological formulation substantially as described herein.

93. A method of identifying cells associated with MCL substantially as described herein.