WO2003034069A2

WO2003034069A2 - Tcmp 03 protein for use in cancer therapy

Info

Publication number: WO2003034069A2
Application number: PCT/GB2002/004740
Authority: WO
Inventors: Jonathan Alexander Terrett
Original assignee: Oxford Glycosciences (Uk) Ltd
Priority date: 2001-10-18
Filing date: 2002-10-18
Publication date: 2003-04-24
Also published as: WO2003034069A3; AU2002336185A1; GB0125072D0

Abstract

The present invention relates to a protein, designated TCMP 03, and to compositions comprising the protein, including vaccines, antibodies that are immunospecific for the protein, and to agents that modulate the expression or activity of the protein. The use of these compositions in the diagnosis, screening and treatment of cancer is also provided.

Description

PROTEIN FOR USE IN CANCER THERAPY

Treatment of most cancer types is usually via surgery, chemotherapy, radiotherapy or biological therapy. However, some tumors become refractory to such treatments, as the cancer cells develop resistance to chemotherapy drugs or lose their hormone sensitivity, leading to recurrent or metastatic disease, which is often incurable. More recently, attention has focused on the development of immunological therapies (Green, M.C., et al., (2000) Cancer Treat. Rev. 26, 269- 286; Davis, I.D. (2000) Immunol. Cell Biol. 78, 179-195; Knuth, A. et al, (2000) Cancer Chemother Pharmacol. 46, S46-51; Shiku, H., et al., (2000) Cancer Chemother. Pharmacol. 46, S77-82; Saffran, D.C., et al., (1999) Cancer Metastasis Rev. 18, 437-449), such as cancer vaccines and monoclonal antibodies (mAbs), as a means of initiating and targeting a host immune response against tumour cells. In 1998 the FDA approved the use of Herceptin (Stebbing, J. et al, (2000) Cancer Treat. Rev. 26, 287-290; Dillman, R.O. (1999) Cancer Biother. Radiopharm. 14, 5-10; Miller, K.D. & Sledge, G.W. (1999) Invest. New Drugs 17, 417-427), a mAb that recognises the erbB2/HER2-neu receptor protein, as a treatment for metastatic breast cancer. In combination with chemotherapy, Herceptin has been shown to prolong the time to disease progression, when compared to patients receiving chemotherapy alone (Baselga, J., et al., (1998) Cancer Res. 58, 2825-2831). Herceptin, however, is only effective in treating the 10-20% of patients whose breast tumours over-express the erbB2 protein. Thus, the identification of other suitable targets or antigens for immunotherapy of other cancers in addition to breast cancer has become increasingly important. An ideal protein target for cancer immunotherapy should have a restricted expression profile in normal tissues and be over-expressed in tumours, such that the immune response will be targeted to tumour cells and not against other organs. In addition, the protein target should be exposed on the cell surface, where it will be accessible to therapeutic agents. Tumour antigens have been identified for a number of cancer types, by using techniques such as differential screening of cDNA (Hubert, R.S., et al, (1999) Proc. Natl. Acad, Sci. USA 96, 14523-14528; Lucas, S., et al, (2000) Int. J. Cancer 87, 55-60) and the purification of cell-surface antigens that are recognised by tumour-specific antibodies (Catimel, B., et al, (1996) J. Biol. Chem. 271, 25664-25670).

We have identified a protein, designated TCMP 03. Its' sequence is provided in Figure 1 (SEQ ID No.1 and SEQ ID No.2) and is also available under the accession number AAF24042 in the GenBank database (held by the National Institute of Health (NIH), available at http://www.ncbi.nlm.nih.gov/) where it is provided only as a conceptual translation. Furthermore, we have detected and quantified expression levels of this protein and shown that TCMP 03 expression is elevated in breast tumour cell lines and clinical samples, as well as in some leukaemia cell lines and clinical samples. This suggests that it may be a suitable target for screening for, therapy or diagnosis of cancer.

The term 'cancer' (or 'carcinoma') is a malignant new growth that arises from epithelium, found in skin or, more commonly, the lining of body organs, for example, breast, prostate, lung, kidney, pancreas, stomach or bowel. A carcinoma tends to infiltrate into adjacent tissue and spread (metastasise) to distant organs, for example to bone, liver, lung or the brain. As used herein the term cancer includes both metastatic tumour cell types, such as but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such as but not limited to, breast cancer, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer and ovarian cancer.

Accordingly the present invention provides a method of screening for and/or diagnosis of cancer in a subject, and/or monitoring the effectiveness of cancer therapy, which comprises the step of detecting and/or quantifying in a biological sample obtained from said subject a TCMP03 polypeptide which: a) comprises or consists of the amino acid sequence shown in Figure 1 (SEQ ID Nol); b) is a derivative having one or more amino acid substitutions, modifications, deletions or insertions relative to the amino acid sequence shown in Figure 1 (SEQ ID No 1) and which retains the activity of TCMP 03; or c) is a fragment of a polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID No 1), which is at least ten amino acids long and has at least 70% homology over the length of the fragment.

Levels of said polypeptides can be compared to a previously determined reference range or control.

In the context of the present invention, the biological sample can be obtained from any source, such as a fluid sample, e.g. blood, serum or lymph or a tissue sample, e.g. breast tissue. When looking for evidence of metastasis, one would look at major sites of metastasis such as, lymph nodes, liver, lung and/or bone. The polypeptides described in a) to c) above are hereinafter referred to as TCMP 03 polypeptides. The term 'polypeptides' includes peptides, polypeptides and proteins. These are used interchangeably unless otherwise specified.

TCMP 03 polypeptides may be in isolated or recombinant form, and may be fused to other moieties. In particular, fusions of the polypeptides of the present invention with localisation- reporter proteins such as the Green Fluorescent Protein (U.S. 5,625,048, 5,777,079, 6,054,321 and 5,804,387) or the DsRed fluorescent protein (Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L. & Lukyanov S. A. (1999). Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotech. 17:969-973.) are specifically contemplated by the present invention. They are provided in substantially pure form, that is to say, they are free, to a substantial extent, from other proteins. Thus, a polypeptide of the present invention may be provided in a composition in which it is the predominant component present (i.e. it is present at a level of at least 50%; preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 98%, or at least 99% when determined on a weight/weight basis excluding solvents or carriers).

In order to more fully appreciate the present invention, polypeptides within the scope of a)-c) above will now be discussed in greater detail. Polypeptides within the scope of a)

A polypeptide within the scope of a), may consist of the particular amino acid sequence given in Figure 1 (SEQ ID No 1) or may have an additional N-terminal and/or an additional C-terminal amino acid sequence relative to the sequence given in Figure 1 (SEQ ID No 1). Additional N-terminal or C-terminal sequences may be provided for various reasons.

Techniques for providing such additional sequences are well known in the art. Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems. For example, an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone Somatostatin by fusing it at its N-terminus to part of the β galactosidase enzyme (Itakwa et al, Science 198: 105-63 (1977)).

Additional sequences can also be useful in altering the properties of a polypeptide to aid in identification or purification. For example, a fusion protein may be provided in which a polypeptide is linked to a moiety capable of being isolated by affinity chromatography. The moiety may be an antigen or an epitope and the affinity column may comprise immobilised antibodies or immobilised antibody fragments which bind to said antigen or epitope (desirably with a high degree of specificity). In such circumstances the fusion protein can usually be eluted from the column by addition of an appropriate buffer.

Additional N-terminal or C-terminal sequences may, however, be present simply as a result of a particular technique used to obtain a polypeptide of the present invention and need not provide any particular advantageous characteristic to the polypeptide of the present invention. Such polypeptide are also within the scope of the present invention.

Whatever additional N-terminal or C-terminal sequence is present, it is preferred that the resultant polypeptide should exhibit the immunological activity of the polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID No 1). Polypeptides within the scope of b)

It will be appreciated by the person skilled in the art that polypeptides defined in b) above are variants of the polypeptide given in a) above, provided that such variants exhibit the activity of the polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID No 1). Alterations in the amino acid sequence of a protein can occur which do not affect the activity of a protein. These include amino acid deletions, insertions and substitutions and can result from alternative splicing and/or the presence of multiple translation start sites and stop sites. Polymorphisms may arise as a result of the infidelity of the translation process. Thus changes in amino acid sequence may be tolerated which do not affect the protein's function. The skilled person will appreciate that various changes can often be made to the amino acid sequence of a polypeptide which has a particular activity to produce variants (sometimes known as "muteins") having at least a proportion of said activity, and preferably having a substantial proportion of said activity. Such variants of the polypeptides described in a) above are within the scope of the present invention and are discussed in greater detail below. They include allelic and non-allelic variants. An example of a variant is a polypeptide as defined in a) above, apart from the substitution of one or more amino acids with one or more other amino acids. The skilled person is aware that various amino acids have similar properties. Thus, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions, it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: - phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains);

- lysine, arginine and histidine (amino acids having basic side chains);

- aspartate and glutamate (amino acids having acidic side chains);

- asparagine and glutamine (amino acids having amide side chains); and

- cysteine and methionine (amino acids having sulphur-containing side chains). Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions.

Amino acid changes relative to the sequence given in a) above can be made using any suitable technique e.g. by using site-directed mutagenesis (Hutchinson et al, (1978) J. Biol. Chem.

253:6551). Amino acid deletions or insertions may also be made relative to the amino acid sequence given in a) above. Thus, for example, amino acids which do not have a substantial effect on the activity of the polypeptide, or at least which do not eliminate such activity, may be deleted. Such deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced - for example, dosage levels can be reduced.

Amino acid insertions can also be made, for example, to alter the properties of a polypeptide

(e.g. to assist in identification, purification or expression, as explained above in relation to fusion proteins).

It should be appreciated that amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids.

Whether or not natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

Whatever amino acid changes are made (whether by means of substitution, insertion or deletion), preferred polypeptides of the present invention have at least 50% sequence identity with a polypeptide as defined in a) above, more preferably the degree of sequence identity is at least 75%, or at least 80%, or at least 85%. Sequence identities of at least 90%, or at least 95%, or at least

98%, or at least 99% are most preferred.

The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g. gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e. % identity = # of identical positions/total # of positions x 100). The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.

USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers & Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis & Robotti (1994) Comput. Appl Bioscl, 10 :3-5; and FASTA described in Pearson & Lipman (1988) Proc. Natl. Acad. Sci. 55:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

The preferred mathematical algorithm is that of the BLAST™ software available from NCBI (Altschul, S.F. et al, 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, D.J. 1993, Nature Genet. 3:266-272. Madden, T.L. et al, 1996, Meth. Enzymol. 266: 131-141; Altschul, S.F. et al, 1997, Nucleic Acids Res. 25:3389-3402); Zhang, J. & Madden, T.L. 1997, Genome Res. 7:649- 656).

Where high degrees of sequence identity are present there will be relatively few differences in amino acid sequence. Thus for example they may be less than 20, less than 10, or even less than 5 differences over the length of the polypeptide. Polypeptides within the scope of c)

As discussed supra, it is often advantageous to reduce the length of a polypeptide. Preferably, the resultant reduced length polypeptide still has a desired activity or can give rise to useful antibodies. Feature c) of the present invention therefore covers fragments of polypeptides a) or b) above.

The skilled person can determine whether or not a particular fragment has activity using techniques well known to those skilled in the art. Preferred fragments are at least 10 amino acids long. They may be at least 20, at least 50 or at least 100 amino acids long.

The polypeptides of the present invention find use in a therapeutic approach to cancer. The skilled person will appreciate that for the preparation of one or more polypeptides of the invention, the preferred approach will be based on recombinant DNA techniques.

The invention also provides a method of screening for and/or diagnosis of cancer in a subject, and/or monitoring the effectiveness of cancer therapy, which comprises the step of detecting and/or quantifying in a biological sample obtained from said subject a TCMP03 nucleic acid molecule which: d) comprises or consists of the DNA sequence shown in Figure 1 (SEQ ID No 2) or its RNA equivalent; or is e) a sequence which is complementary to the sequences of d); f) a sequence which codes for the same polypeptide, as the sequences of d) or e); or g) a sequence which codes for a polypeptide as defined by the TCMP 03 polypeptides of b) or c) above.

Levels of said nucleic acids can be compared to a previously determined reference range or control.

It is preferred if sequences of g) have e.g. at least 50%, at least 75%, at least 80%, or at least 85%, or at least 90% or 95% , or at least 98%, or at least 99% sequence identity with that of SEQ ID No.2.

The polypeptides of the present invention can be coded for by a large variety of TCMP03 nucleic acid molecules, taking into account the well known degeneracy of the genetic code. They can be inserted into vectors and cloned to provide large amounts of DNA or RNA. Suitable vectors may be introduced into host cells to enable the expression of polypeptides of the present invention using techniques known to the person skilled in the art.

The term 'RNA equivalent' when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule, allowing for the fact that in RNA 'U' replaces 'T' in the genetic code. The nucleic acid molecule may be in isolated, recombinant or chemically synthetic form.

Techniques for cloning, expressing and purifying proteins and polypeptides are well known to the skilled person. DNA constructs can readily be generated using methods well known in the art. These techniques are disclosed, for example in J. Sambrook et al, Molecular Cloning 2^nd Edition, Cold Spring Harbour Laboratory Press (1989); in Old & Primrose Principles of Gene Manipulation 5th Edition, Blackwell Scientific Publications (1994); and in Stryer [Biochemistry 4th Edition, W H Freeman and Company (1995)]. Modifications of DNA constructs and the proteins expressed such as the addition of promoters, enhancers, signal sequences, leader sequences, translation start and stop signals and DNA stability controlling regions, or the addition of fusion partners may then be facilitated.

Normally the DNA construct will be inserted into a vector, which may be of phage or plasmid origin. Expression of the protein is achieved by the transformation or transfection of the vector into a host cell which may be of eukaryotic or prokaryotic origin. Such vectors and suitable host cells form additional aspects of the present invention.

Knowledge of the nucleic acid structure can be used to raise antibodies and for gene therapy. Techniques for this are well-known by those skilled in the art.

By using appropriate expression systems, polypeptides of the present invention may be expressed in glycosylated or non-glycosylated form. Non-glycosylated forms can be produced by expression in prokaryotic hosts, such as E. coli.

Polypeptides comprising N-terminal methionine may be produced using certain expression systems, whilst in others the mature polypeptide will lack this residue.

Preferred techniques for cloning, expressing and purifying a substance of the present invention are summarised below: Polypeptides may be prepared natively or under denaturing conditions and then subsequently refolded. Baculoviral expression vectors include secretory plasmids (such as pACGP67 from Pharmingen), which may have an epitope tag sequence cloned in frame (e.g. myc, V5 or His) to aid detection and allow for subsequent purification of the protein. Mammalian expression vectors may include pCDNA3 and pSecTag (both Invitrogen), and pREP9 and pCEP4 (invitrogen). E. coli systems include the pBad series (His tagged - Invitrogen) or pGex series (Pharmacia).

In addition to nucleic acid molecules coding for polypeptides, referred to herein as "coding" nucleic acid molecules, nucleic acid molecules that are complementary to coding nucleic acid molecules are described and referred to as "hybridising" nucleic acid molecules . These include mRNA molecules and complementary DNA Molecules (e.g. cDNA molecules). Hybridising nucleic acid molecules can be useful as probes or primers.

Desirably such hybridising molecules are at least 10 nucleotides in length and preferably are at least 25 or at least 50 nucleotides in length. The hybridising nucleic acid molecules preferably hybridise to nucleic acids within the scope of (d), (e), (f) or (g) above specifically.

Desirably the hybridising molecules will hybridise to such molecules under stringent hybridisation conditions. One example of stringent hybridisation conditions is where attempted hybridisation is carried out at a temperature of from about 35°C to about 65°C using a salt solution, which is about 0.9 molar. However, the skilled person will be able to vary such conditions as appropriate in order to take into account variables such as probe length, base composition, type of ions present, etc.

Manipulation of the DNA encoding the protein is a technique for both modifying proteins and for generating large quantities of protein for purification purposes. This may involve the use of PCR techniques to amplify a desired nucleic acid sequence. Thus the sequence data provided herein can be used to design primers for use in PCR so that a desired sequence can be targetted and then amplified to a high degree.

Typically primers will be at least five nucleotides long and will generally be at least ten nucleotides long (e.g. fifteen to twenty-five nucleotides long). In some cases, primers of at least thirty or at least thirty-five nucleotides in length may be used.

As a further alternative chemical synthesis may be used. This may be automated. Relatively short sequences may be chemically synthesised and ligated together to provide a longer sequence.

In addition to being used as primers and/or probes, hybridising nucleic acid molecules of the present invention can be used as anti-sense molecules to alter the expression of substances of the present invention by binding to complementary nucleic acid molecules. This technique can be used in anti -sense therapy.

A hybridising nucleic acid molecule may have a high degree of sequence identity along its length with a nucleic acid molecule within the scope of (d)-(g) above (e.g. at least 50%, at least 75% or at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity). As will be appreciated by the skilled person, the higher the sequence identity a given single stranded nucleic acid molecule has with another nucleic acid molecule, the greater the likelihood that it will hybridise to a nucleic acid molecule which is complementary to that other nucleic acid molecule under appropriate conditions.

Thus, the nucleic acid molecules may have one or more of the following characteristics: 1 ) they may be DNA or RNA; 2) they may be single or double stranded;

3) they may be provided in recombinant form i.e. covalently linked to a 5' and/or a 3' flanking sequence to provide a molecule which does not occur in nature;

4) they may be provided without 5' and/or 3' flanking sequences which normally occur in nature; 5) they may be provided in substantially pure form. Thus they may be provided in a form which is substantially free from contaminating proteins and/or from other nucleic acids; and

6) they may be provided with introns or without introns (e.g. as cDNA).

The invention also provides diagnostic kits, comprising a capture reagent (e.g. an antibody) against a TCMP 03 polypeptide as defined above. In addition, such a kit may optionally comprise one or more of the following:

(1) instructions for using the capture reagent for diagnosis, prognosis, therapeutic monitoring or any combination of these applications; (2) a labelled binding partner to the capture reagent;

(3) a solid phase (such as a reagent strip) upon which the capture reagent is immobilised; and

(4) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use or any combination thereof. If no labelled binding partner to the capture reagent is provided, the anti-polypeptide capture reagent itself can be labelled with a detectable marker, e.g. a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.

The methods for diagnosis according to the present invention may be performed using a number of methods known to those skilled in the art, including without limitation, immunoprecipitation followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, 2 dimensional gel electrophoresis, immunocytochemistry, immunohistochemistry, immunoassays, e.g. western blots, radioimmunoassays, ELISA, (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.

A convenient means for such detection or quantification involves the use of antibodies. Thus, the polypeptides of the invention will also find use in raising antibodies. Hence, another aspect of the present invention provides antibodies, which bind to a TCMP 03 polypeptide of the invention. Preferred antibodies bind specifically to TCMP 03 polypeptides so that they can be used to identify, purify and/or inhibit the activity of such polypeptides. The antibodies may be monoclonal, polyclonal, chimeric, humanised or bispecific or conjugated to a therapeutic moiety, detectable label, second antibody or a fragment thereof, a cytotoxic agent or cytokine according to the circumstances of their use.

Thus, the TCMP 03 polypeptide of the invention may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Antibodies of the invention include, but are not limited to polyclonal, monoclonal, bispecific, humanized or chimeric antibodies, single chain antibodies, Fab fragments and F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobulin molecules of the invention can be of any class (e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule. In yet another aspect the invention provides the use of an antibody that specifically binds to at least one TCMP 03 polypeptide for screening and / or diagnosis of cancer, preferably breast cancer or leukaemia.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies, which recognize a specific domain of a polypeptide of the invention, one may assay generated hybridomas for a product which binds to a polypeptide fragment containing such domain. For selection of an antibody that specifically binds a first polypeptide homolog but which does not specifically bind to (or binds less avidly to) a second polypeptide homolog, one can select on the basis of positive binding to the first polypeptide homolog and a lack of binding to (or reduced binding to) the second polypeptide homolog.

For preparation of monoclonal antibodies (mAbs) directed toward a polypeptide of the invention any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAbs of the invention may be cultivated in vitro or in vivo. Monoclonal antibodies can be produced in germ-free animals utilizing known technology (PCT US90/02545).

The monoclonal antibodies include but are not limited to human monoclonal antibodies and chimeric monoclonal antibodies (e.g. human-mouse chimeras). A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a human immunoglobulin constant region and a variable region derived from a murine mAb (See, e.g. US 4,816,567; and US 4,816397). Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g. US 5,585,089). Chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in WO 87/02671; EP 184,187; EP 171,496; EP 173,494; WO 86/01533; US 4,816,567; EP 125,023; Better et al., 1988, Science 240: 1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80: 1553-1559; Morrison, 1985, Science 229: 1202-1207; Oi et al., 1986, Bio Techniques 4:214; US 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060.

Completely human antibodies for therapeutic treatment of human patients can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g. all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., US 5,625,126; US 5,633,425; US 5,569,825; US 5,661,016; and US 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g. a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Bio/technology 12:899-903). The antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g. human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g. using labelled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT Application No. PCT/GB91/01134; PCT Publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g. as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240: 1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in US 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240: 1038- 1040 (1988). The invention further provides for the use of bispecific antibodies, which can be made by methods known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al., 1991, EMBO J. 10:3655-3659. According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details for generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 1986, 121:210.

The invention provides functionally active fragments, derivatives or analogs of the anti-polypeptide immunoglobulin molecules. Functionally active means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies (i.e. tertiary antibodies) that recognize the same antigen that is recognized by the antibody from which the fragment, derivative or analog is derived. Specifically, in a preferred embodiment the antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art. The present invention provides antibody fragments such as, but not limited to, F(ab')2 fragments and Fab fragments. Antibody fragments which recognize specific epitopes may be generated by known techniques. F(ab')2 fragments consist of the variable region, the light chain constant region and the CHI domain of the heavy chain and are generated by pepsin digestion of the antibody molecule. Fab fragments are generated by reducing the disulfide bridges of the F(ab')2 fragments. The invention also provides heavy chain and light chain dimmers of the antibodies of the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g. as described in US 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any other molecule with the same specificity as the antibody of the invention. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may be used (Skerra et al., 1988, Science 242: 1038-1041). In other embodiments, the invention provides fusion proteins of the immunoglobulins of the invention (or functionally active fragments thereof), for example in which the immunoglobulin is fused via a covalent bond (e.g. a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not the immunoglobulin. Preferably the immunoglobulin, or fragment thereof, is covalently linked to the other protein at the N-terminus of the constant domain. As stated above, such fusion proteins may facilitate purification, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system.

The immunoglobulins of the invention include analogs and derivatives that are either modified, i.e. by the covalent attachment of any type of molecule as long as such covalent attachment that does not impair immunospecific binding. For example, but not by way of limitation, the derivatives and analogs of the immunoglobulins include those that have been further modified, e.g. by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc. Additionally, the analog or derivative may contain one or more non-classical amino acids.

The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the polypeptides of the invention, e.g. for imaging or radio-imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc. and for radiotherapy.

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression technique.

Recombinant expression of antibodies, or fragments, derivatives or analogs thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g. as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, the nucleic acid encoding the antibody may be obtained by cloning the antibody. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence. If an antibody molecule that specifically recognizes a particular antigen is not available (or a source for a cDNA library for cloning a nucleic acid encoding such an antibody), antibodies specific for a particular antigen may be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies. Alternatively, a clone encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries (e.g. as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid encoding at least the variable domain of the antibody molecule is obtained, it may be introduced into a vector containing the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g. WO 86/05807; WO 89/01036; and US No. 5,122,464). Vectors containing the complete light or heavy chain for co-expression with the nucleic acid to allow the expression of a complete antibody molecule are also available. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitution(s) or deletion(s) necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydryl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis, in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551) based methods, etc.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human antibody constant region, e.g. humanized antibodies.

Once a nucleic acid encoding an antibody molecule of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the antibodies by expressing nucleic acid containing the antibody molecule sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing an antibody molecule coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY). The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.

The host cells used to express a recombinant antibody of the invention may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecules. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 198, Gene 45: 101; Cockett et al., 1990, Bio/Technology 8:2). A variety of host-expression vector systems may be utilized to express an antibody molecule of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g. E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g. Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g. baculovirus) containing the antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of antibody is to be produced, for the generation of pharmaceutical compositions comprising an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). In mammalian host cells, a number of viral-based expression systems (e.g., an adenovirus expression system) may be utilized.

As discussed above, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g. glycosylation) and processing (e.g. cleavage) of protein products may be important for the function of the protein.

For long-term, high-yield production of recombinant antibodies, stable expression is preferred. For example, cells lines that stably express an antibody of interest can be produced by transfecting the cells with an expression vector comprising the nucleotide sequence of the antibody and the nucleotide sequence of a selectable (e.g. neomycin or hygromycin), and selecting for expression of the selectable marker. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule. The expression levels of the antibody molecule can be increased by vector amplification

(for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA. Once the antibody molecule of the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of an antibody molecule, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Alternatively, any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns and histidine- tagged proteins are selectively eluted with imidazole-containing buffers. In a preferred embodiment, antibodies of the invention or fragments thereof are conjugated to a diagnostic or therapeutic moiety. The antibodies can be used for diagnosis or to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See US 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive

125 131 i l l 99 nuclides include I, I, In and Tc. Antibodies of the invention or fragments thereof can be conjugated to a therapeutic agent or drug moiety to modify a given biological response. The therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, a biological response modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g. Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982), EP 0624 377.

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described in US 4,676,980.

An antibody with or without a therapeutic moiety conjugated to it can be used as a therapeutic that is administered alone or in combination with cytotoxic factor(s) and/or cytokine(s). In yet another aspect, the present invention provides methods for screening for anti-cancer agents, preferably anti-breast cancer agents or anti-leukaemia agents, that modulate the expression or activity of a TCMP 03 polypeptide or the expression of a TCMP 03 nucleic acid molecule. These agents may be useful in the treatment of cancer, preferably breast cancer or leukaemia.

In a further aspect, the present invention provides methods for screening for anti-cancer agents, preferably anti-breast cancer agents or anti-leukaemia agents that interact with a TCMP 03 polypeptide or a TCMP 03 nucleic acid molecule.

Agents identified through the screening methods of the invention are potential therapeutics for use in the treatment of cancer, preferably breast cancer or leukaemia.

Agents can be selected from a wide variety of candidate agents. Examples of candidate agents include but are not limited to, nucleic acids (e.g. DNA and RNA), antibodies, carbohydrates, lipids, proteins, polypeptides, peptides, peptidomimetics, small molecules and other drugs. Agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is suited to e.g. peptide libraries, while the other approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145; US 5,738,996; and US 5,807,683).

Examples of suitable methods based on the present description for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90:6909; Erb et al, 1994, Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al, 1994, J. Med. Chem. 37:2678; Cho et al, 1993, Science 261:1303; Carrell et al, 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al, 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al, 1994, J. Med. Chem. 37: 1233.

Libraries of compounds may be presented, for example, presented in solution (e.g. Houghten, 1992, Bio/Techniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (US 5,223,409), spores (US 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al, 1992, Proc. Natl. Acad. Sci. USA 89: 1865-1869) or phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al,. 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310). In one embodiment, agents that modulate the expression of a polypeptide are identified in a cell-based assay system. Accordingly, cells expressing a TCMP 03 polypeptide are contacted with a candidate agent or a control agent and the ability of the candidate agent to alter expression of the TCMP 03 polypeptide is determined. In a further embodiment, the expression of the TCMP 03 polypeptide may be compared to a reference range or control. If desired, this assay may be used to screen a plurality (e.g. a library) of candidate agents. The cell, for example, can be of prokaryotic origin (e.g. E. coli) or eukaryotic origin (e.g. yeast or mammalian). Further, the cells can express a TCMP 03 polypeptide endogenously or be genetically engineered to express a TCMP 03 polypeptide. The ability of the candidate agents to alter the expression of a TCMP 03 polypeptide can be determined by methods known to those of skill in the art, for example and without limitation, by flow cytometry, radiolabelling,a scintillation assay, immunoprecipitation or western blot analysis.

In another embodiment, a cell-based assay system is used to identify agents capable of modulating the activity of a TCMP 03 polypeptide. In such an assay, the activity of a TCMP 03 polypeptide is measured in a population of cells that naturally or recombinantly express a TCMP 03 polypeptide, in the presence of a candidate agent. In such an assay, the activity of a TCMP 03 is measured in a population of cells that naturally or recombinantly express a TCMP 03, in the presence of agent and in the absence of a candidate agent (e.g. in the presence of a control agent) and the activity of the TCMP 03 polypeptide is compared. The candidate agent can then be identified as a stimulator or inhibitor of the activity of a TCMP 03 polypeptide based on this comparison. In a further embodiment, the activity of a TCMP 03 polypeptide can be measured in the presence or absence of a candidate agent. Preferably, the activity of a TCMP 03 polypeptide is compared to a reference range or control. In another embodiment, agents, such as, an enzyme, or a biologically active portion thereof, which is responsible for the production or degradation of a TCMP 03 polypeptide, or is responsible for the post-translational modification of a TCMP 03 polypeptide can be identified. In a primary screen, substantially pure, native or recombinantly expressed TCMP 03 polypeptides or cellular extract or other sample comprising native or recombinantly expressed TCMP 03 polypeptides are contacted with a plurality of candidate agents, for example but without limitation, a plurality of agents presented as a library, that may be responsible for the processing of a TCMP 03 polypeptide, in order to identify such agents. The ability of the candidate agent to modulate the production, degradation or post-translational modification of a TCMP 03 polypeptide can be determined by methods known to those of skill in the art, including without limitation, flow cytometry, radiolabelling, a kinase assay, a phosphatase assay, immunoprecipitation and western blot analysis.

In yet another embodiment, cells expressing a TCMP 03 polypeptide are contacted with a plurality of candidate agents. The ability of such an agent to modulate the production, degradation or post-translational modification of a TCMP 03 polypeptide can be determined by methods known to those of skill in the art, including without limitation, flow cytometry, radiolabelling, kinase assay, phosphatase assay, immunoprecipitation and Western blot analysis.

In one embodiment, agents that modulate the expression of a polypeptide are identified by contacting cells (e.g. cells of prokaryotic origin or eukaryotic origin) expressing a TCMP 03 polypeptide with a candidate agent or a control agent (e.g. phosphate buffered saline; PBS) and determining the expression of a TCMP 03 polypeptide or RNA encoding a TCMP 03 polypeptide. The level of expression of a TCMP 03 polypeptide or mRNA encoding said polypeptide in the presence of the candidate agent is compared to the level of expression of a TCMP 03 polypeptide or mRNA encoding said polypeptide in the absence of the candidate agent (e.g. in the presence of a control agent). The candidate agent can then be identified as a modulator of the expression of a TCMP 03 polypeptide based on this comparison. For example, when expression of a TCMP 03 polypeptide (or its mRNA) is significantly greater in the presence of the candidate agent than in its absence, the candidate agent is identified as a stimulator of expression of a TCMP 03 polypeptide. Alternatively, when expression of a TCMP 03 polypeptide (or its mRNA) is significantly less in the presence of the candidate agent than in its absence, the candidate agent is identified as an inhibitor of the expression of the polypeptide. The level of expression of a TCMP 03 polypeptide or its' encoding mRNA can be determined by methods known to those of skill in the art. For example, mRNA expression can be assessed by Northern blot analysis or RT-PCR, and protein levels can be assessed, without limitation, by western blot analysis. In another embodiment, agents that modulate the expression of a TCMP 03 polypeptide are identified in an animal model. Examples of suitable animals include, but are not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats. Preferably, the animal used represents a model of TCMP 03. In accordance with this embodiment, the candidate agent or a control agent is administered (e.g. orally, rectally or parenterally such as intraperitoneally or intravenously) to a suitable animal and the effect on the expression of a TCMP 03 polypeptide (or its mRNA) is determined. Changes in the expression of a polypeptide can be assessed by the methods outlined above.

In yet another embodiment, a TCMP 03 polypeptide is used as a "bait protein" in a two- hybrid assay or three hybrid assay to identify other proteins that bind to or interact with the polypeptide (see e.g. US 5,283,317; Zervos et al, 1993, Cell 72:223-232; Madura et al. 1993, J. Biol. Chem. 268: 12046-12054; Bartel et al, 1993, Bio/Techniques 14:920-924; Iwabuchi et al, 1993, Oncogene 8: 1693-1696; and WO 94/10300). As those skilled in the art will appreciate, such binding proteins are also likely to be involved in the propagation of signals by a TCMP 03 polypeptide, for example, they may be upstream or downstream elements of a signalling pathway involving a TCMP 03 polypeptide.

Alternatively, polypeptides that interact with a TCMP 03 polypeptide can be identified by isolating a protein complex comprising a TCMP 03 polypeptide and identifying the associated polypeptides using methods known in the art such as mass spectrometry (e.g. Blackstock, W. & Weir, M. 1999, Trends in Biotechnology, 17: 121-127; Rigaut, G. 1999, Nature Biotechnology, 17: 1030-1032; Husi, H. 2000, Nature Neurosci. 3:661-669; Ho, Y. et al, 2002, Nature, 415: 180-183; Gavin, A. et al, 2002, Nature, 415: 141-147).

One skilled in the art will also appreciate that a TCMP03 polypeptide may also be used in a method for the structure-based design of an agent, in particular a small molecule which acts to modulate (e.g. stimulate or inhibit) the activity of said polypeptide, said method comprising:

1) determining the three-dimensional structure of said polypeptide;

2) deducing the three-dimensional structure of the likely reactive or binding site(s) of the agent;

3) synthesising candidate agents that are predicted to react or bind to the deduced reactive or binding site; and

4) testing whether the candidate agent is able to modulate the activity of said polypeptide.

It will be appreciated that the method described above is likely to be an iterative process.

This invention further provides novel agents identified by the above-described screening methods and uses thereof for treatments as described herein.

When a reference is made herein to a method of treating or preventing a disease or condition using a particular agent or combination of agents, it is to be understood that such a reference is intended to include the use of that agent or combination of agents in the preparation of a medicament for the treatment or prevention of the disease or condition. The term 'treatment' includes either therapeutic or prophylactic therapy.

As used herein "active agent" refers to the TCMP 03 polypeptides, TCMP03 nucleic acid anti-TCMP03 antibodies and also to agents that interact with, or modulate the expression or activity of TCMP03 polypeptides, e.g. small molecules. As discussed herein, active agents of the invention find use in the treatment of cancer, preferably for use in the treatment of breast cancer or leukaemia.

Thus, in a further aspect, the present invention provides a pharmaceutical formulation comprising at least one active agent, optionally together with one or more pharmaceutically acceptable excipients, carriers or diluents. Preferably, the pharmaceutical formulation is for use as a vaccine and so any additional components will be acceptable for vaccine use. In addition, the skilled person will appreciate that one or more suitable adjuvants may be added to such vaccine preparations.

The medicament will usually be supplied as part of a sterile, pharmaceutical composition, which will normally include a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient).

It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions).

Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof.

Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.

For the preparation of solutions and syrups, excipients which may be used include for example water, polyols and sugars. For the preparation of suspensions, oils (e.g. vegetable oils) may be used to provide oil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986). Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For infections of the eye or other external tissues, for example mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffmic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which may be administered, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulisers or insufflators.

Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations. Pharmaceutical compositions adapted for parenteral administration include aqueous and non- aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like). The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the active agents of the invention.

Dosages of the active agents of the invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice.

To administer an active agent of the invention by certain routes of administration, it may be necessary to coat the active agent with, or co-administer the active agent with, a material to prevent its inactivation. For example, the active agent may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. The compositions may contain from 0.1% by weight, preferably from 10-60% by weight, of the active agent, depending on the method of administration.

Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active agent per dose. Such a unit may contain for example lOOmg/kg to O.Olmg kg depending on the condition being treated, the route of administration and the age, weight and condition of the patient. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.

It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of an active agent will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular mammal being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e. the number of doses of an active agent given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

Dosage regimens are adjusted to provide the optimum desired response (e.g. a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active agent for the treatment of sensitivity in individuals.

Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e. combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one anti-tumour agent or other conventional therapy.

In certain embodiments, antibodies of the invention can be formulated to ensure proper distribution in vivo, for example, in liposomes. For methods of manufacturing liposomes, see e.g. US 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see e.g. Ranade, V. 1989, J. Clin. Pharmacol. 29: 685). Exemplary targeting moieties include folate or biotin (see, e.g. US 5,416,016); mannosides (Umezawa et al, 1988, Biochem. Biophys. Res. Comm. 153: 1038); antibodies (Bloeman, P. et al., 1995, FEBS Lett. 357: 140; Owais, M. et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; psi 20 (Schreier et al, 1994, J. Biol. Chem. 269: 9090); see also Keinanen, K. & Laukkanen, M., 1994, FEBS Lett. 346: 123; Killion, J. & Fidler, I., 1994, Immunomethods 4: 273. In one embodiment of the invention, the active agents of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the active agents in the liposomes are delivered by bolus injection to a site proximal to the therapeutic target, e.g. the tumour.

Where a composition is fluid, the term 'fluid' includes compositions to the extent that it is deliverable by syringe or orally. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Proper fluidity can be maintained, for example, by use of a coating such as lecithin, by maintenance of required particle size in the case of dispersion and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols, such as manitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

When the active agent is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier. Additional aspects of the present invention include:

(i) the use of at least one active agent in the preparation of a medicament for the treatment of a subject with cancer. In particular, the preparation of vaccines and/or compositions comprising or consisting of antibodies is a preferred embodiment of this aspect of the invention;

(ii) a method for the treatment of cancer in a subject, which comprises administering to said subject a therapeutically effective amount of at least one active agent of the invention; and

(iii) an active agent for use in the treatment of cancer.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. The invention will now be described with reference to the following examples, which should not in any way be construed as limiting the scope of the present invention. The examples refer to the figures in which:

Figure 1: shows the nucleotide sequence (SEQ ID No. 2) and predicted amino acid sequence (SEQ ID No 1) of TCMP 03; Figure 2: shows the distribution of TCMP 03 mRNA levels in normal tissues. Levels of mRNA were quantified by real time RT-PCR and are expressed as the number of copies ng^"1 cDNA;

Figure 3: shows the expression of TCMP 03 in normal and tumor breast tissues. Samples

1-40 are breast tumor samples, of which samples 25-40 were isolated from patients with lymph node metastasis. The final 2 samples are from normal breast tissue (reduction mammoplasties). TCMP 03 mRNA levels are expressed as the number of copies ng^"1 cDNA; and

Figure 4: shows the expression of TCMP 03 in chronic lymphoid leukaemia clinical samples and in leukaemia/lymphoma cell lines. TCMP 03 mRNA levels are expressed as the number of copies ng^'1 cDNA.

Example 1: Expression of TCMP 03 mRNA in human tissues

We used real time quantitative RT-PCR (Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M., Real time quantitative PCR, Genome Res 6, 986-994 (1996); Morrison, T.B., Weis, J.J. & Wittwer, C.T., Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification, Biotechniques 24, 954-958 (1998)) to analyse the distribution of TCMP 03 mRNA in normal human tissues (Fig. 2) and in metastatic or non-metastatic breast cancer tissues (Fig 3). Quantification of TCMP 03 mRNA bv RT-PCR

Real time RT-PCR was used to quantitatively measure TCMP 03 expression in three samples sets: normal tissue mRNAs (Clontech), breast cancer tissues (removed during surgery) and normal breast tissue (removed during breast reduction mammoplasty). Ethical approval for the normal and tumor breast samples was obtained at surgery (University of Oxford, UK). The primers used for PCR were as follows:

Sense: 5' gggtctcatcaggtcagcatgg 3' (SEQ ID No 3) Antisense: 5' ctgcacgcccagtttagcacac 3' (SEQ ID No 4)

Reactions containing lOng cDNA, SYBR green sequence detection reagents (PE Biosystems) and sense and antisense primers were assayed on an AB 17700 sequence detection system (PE Biosystems). The PCR conditions were 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 40 cycles of 95°C for 15s, 65°C for lmin. The accumulation of PCR product was measured in real time as the increase in SYBR green fluorescence, and the data were analysed using the Sequence Detector program vl.6.3 (PE Biosystems). Standard curves relating initial template copy number to fluorescence and amplification cycle were generated using the amplified PCR product as a template, and were used to calculate TCMP 03 copy number in each sample.

The distribution of TCMP 03 mRNA expression in normal samples was restricted to a few tissues, with the highest expression levels in kidney and brain tissues, moderate expression in testis and thymus tissues and low expression in all the other tissues examined. To examine the expression of this gene in breast cancer tissues, TCMP 03 mRNA levels were quantified in 40 tumor samples, 16 of which were isolated from patients with lymph node metastasis (samples BC25-BC40 in Figure 3) and in 2 samples of control (non-cancerous) breast tissue. The expression of TCMP 03 was significantly elevated in 19 (-50%) of the tumor samples with respect to the control tissues. However, there was no association between TCMP 03 expression in breast cancer tissue and lymph node metastasis.

Figure 4 shows the expression of TCMP 03 in 10 clinical samples taken from subjects with chronic lymphoid leukaemia (CLL 1 to CLL 10). One sample shows a massive upregulation of TCMP 03, suggesting that some leukaemias have elevated levels of TCMP 03. In addition, Figure 4 shows the level of TCMP 03 expression in normal spleen and thymus samples, as well as in a number of leukaemia/lymphoma cell lines: Raji/Daudi/Ca46/Namalwa (these are human Burkitt's lymphoma-derived cell lines); HL60 (promyelocytic human leukaemia cell line); K562 (chronic myelogenous human leukaemia cell line). Likewise, a subset of 3 cell lines (Daudi, HL60 and K562) show greatly increased levels of TCMP 03 expression (expression levels of 10,000 or higher mRNA copy number per ng cDNA), whereas the other four cell lines have expression levels below 2,000 mRNA copy number per ng cDNA.

Thus TCMP 03 shows a restricted pattern of expression in normal human tissues and is elevated in breast tumours and some leukaemias, suggesting that this protein has potential as a therapeutic target.

The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Functionally equivalent methods and apparatus within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications and variations are intended to fall within the scope of the appended claims. The contents of each reference, patent and patent application cited in this application is hereby incorporated by reference in its entirety.

Claims

1. A method of screening for and/or diagnosis of cancer in a subject, and/or monitoring the effectiveness of cancer therapy, which comprises the step of detecting and/or quantifying in a biological sample obtained from said subject:

(i) a TCMP 03 polypeptide which: a) comprises or consists of the amino acid sequence shown in Figure 1 (SEQ ID Nol); b) is a derivative having one or more amino acid substitutions, modifications, deletions or insertions relative to the amino acid sequence shown in Figure 1 (SEQ ID No 1) and which retains the activity of TCMP 03; or c) is a fragment of a polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID No 1), which is at least ten amino acids long and has at least 70% homology over the length of the fragment; or

(ii) a nucleic acid molecule which: d) comprises or consists of the DNA sequence shown in Figure 1 (SEQ ED No 2) or its RNA equivalent; e) a sequence which is complementary to the sequence of d); f) a sequence which codes for the same polypeptide, as the sequences of d) or e); or g) a sequence which codes for a polypeptide as defined in b) or c).

2. The method of claim 1, wherein the level of said polypeptide or said nucleic acid is compared to a previously determined reference range or control.

3. An antibody, functionally-active fragment, derivative or analogue thereof, that specifically binds to one or more TCMP 03 polypeptides as defined in claim l(i).

4. An antibody according to claim 3 wherein the antibody is monoclonal, polyclonal, chimeric, humanised or bispecific, or is conjugated to a therapeutic moiety, detectable label, second antibody or a fragment thereof, a cytotoxic agent or cytokine.

5. The method according to claim 1, wherein the step of detecting comprises:

(a) contacting the sample with a capture reagent that is specific for a polypeptide as defined in claim l(i); and

(b) detecting whether binding has occurred between the capture reagent and said polypeptide in the sample.

6. The method according to claim 5, wherein step (b) comprises detecting the captured polypeptide using a directly or indirectly labelled detection reagent.

7. The method according to claim 5 or 6, wherein the capture reagent is immobilised on a solid phase.

8. The method according to any one of claims 5 to 7, wherein the polypeptide is detected and/ or quantified using an antibody as defined in claim 3 or 4.

9. A diagnostic kit comprising a capture reagent specific for a TCMP 03 polypeptide as defined in claim l(i), reagents and instructions for use.

10. A method of screening for anti-cancer agents that interact with a polypeptide as defined in claim l(i), said method comprising:

(a) contacting said polypeptide with a candidate agent; and

(b) determining whether or not the candidate agent interacts with said polypeptide.

11. The method according to claim 10, wherein the determination of interaction between the candidate agent and a TCMP 03 polypeptide comprises quantitatively detecting binding of the candidate agent and said polypeptide.

12. A method of screening for anti-cancer agents that modulate

(a) the expression or activity of a TCMP 03 polypeptide as defined in claim l(i), or

(b) the expression of a nucleic acid molecule as defined in claim l(ii), comprising:

(i) comparing the expression or activity of said polypeptide, or the expression of said nucleic acid molecule, in the presence of a candidate agent with the expression or activity of said polypeptide, or the expression of said nucleic acid molecule, in the absence of the candidate agent or in the presence of a control agent; and

(ii) determining whether the candidate agent causes the expression or activity of said polypeptide, or the expression of said nucleic acid molecule, to change.

13. The method of claim 12, wherein the expression or activity level of said polypeptide, or the expression level of said nucleic acid molecule is compared with a predetermined reference range.

14. The method of claim 12 or 13, which additionally comprises selecting an agent which modulates the expression or activity of said polypeptide, or the expression of said nucleic acid molecule for further testing, or therapeutic or prophylactic use as an anti-cancer agent.

15. An agent identified by the method of any of claims 10-14 which interacts with, or causes the expression or activity of said polypeptide, or the expression of said nucleic acid molecule, to change.

16.

(i) a TCMP 03 polypeptide as defined in claim l(i);

(ii) a nucleic acid molecule as defined in claim l(ii);

(iii) an antibody as defined in claim 3 or 4; or

(iv) an agent as defined in claim 15; for use in medicine.

17. A pharmaceutical formulation comprising:

(i) a TCMP 03 polypeptide as defined in claim l(i);

(ii) a nucleic acid molecule as defined in claim l(ii); (iii) an antibody as defined in claims 3 or 4; or (iv) an agent as defined in claim 15; together with a pharmaceutically acceptable carrier or excipient.

18. The use of:

(i) a TCMP 03 polypeptide as defined in claim l(i);

(ii) a nucleic acid molecule as defined in claim l(ii); (iii) an antibody as defined in claims 3 or 4; or (iv) an agent as defined in claim 15; in the manufacture of a medicament for the treatment of a subject with cancer.

19. A method for the treatment of cancer in a subject, which comprises administering to said subject a therapeutically effective amount of

(i) at least one TCMP 03 polypeptide as defined in claim l(i);

(ii) a nucleic acid molecule as defined in claim l(ii);

(iii) an antibody as defined in claims 3 or 4; or

(iv) an agent as defined in claim 15.

20. The method of any one of claims 1-2, 5-8, 10-14 or 19 or the use of claim 18 wherein the cancer is selected from breast cancer or leukaemia.