AU780395B2 - Mucin-1 derived antigens and their use in immunotherapy - Google Patents

Description

WO 01/57068 PCT/AU01/00090 1 Mucin-1 derived antigens and their use in immunotherapy Field of the Invention: This invention relates to the immunotherapy of disease states, such as the immunotherapy of carcinomas.

Background of the Invention: Cancer is a major cause of death and severe trauma in modem society.

Cancer is not limited to one group; the young, old, males, females and peoples of all races may contract cancer, although cancer in children is relatively rare, perhaps with the exception of childhood leukemia. In western society, cancer of the colon and lung cancer are major diseases. In women, breast cancer is the most common form of cancer.

Many cancers are accompanied by overproduction of human mucin.

Mucins are heavily glycosylated proteins (greater than about 100Kd) which are produced by many epithelial cells and tumours Mucins found on cancer cells are different in some respects to those on normal epithelial cells, in that some mucins have a deficiency in their carbohydrate coat which leaves the protein core exposed There are twelve forms of known human mucin designated MUC1 to MUC12 (see, for example, 3, 4, 26, 27). MUC1 is the most ubiquitous. The various mucins all have very similar properties, that is, they are transmembrane glycoproteins, all having a variable number of repeated amino acid sequences, which have a high content of serine, threonine and proline. Overproduction of aberrantly glycosylated mucins (either non-glycosylated or a deficiency in glycosylation) is characteristic of tumours of the breast, ovary, pancreas, colon, lungs, prostate and other tumours of secretory tissue. The cDNA sequences of the respective protein cores of the human mucins MUC1 to MUC7 have been cloned and characterised and have been found to contain highly repetitive central portions of varying numbers of repeats of particular amino acid motifs (known as VNTR's).

The surgery associated with tumour removal is traumatic to the patient, often disfiguring, and costly. Established chemotherapeutic and radiation procedures for tumour treatment which may be carried out in place of or in conjunction with surgical procedures are often debilitating and associated 141328216 Received 8 May 2002 2 with severe side-effects. There is, accordingly, an urgent need for therapeutic compounds and methods for the prevention/treatment of tumours.

Prior ait relating to mucins mainly concerns the use of VNTR as a possible treatment or prophylactic for cancer. In one case, there has been a report of using the leader sequence of MUC1 to elicit cytotoxic T cells in vitro However, studies involving this peptide, LLLLTVLTV (SEQ ID NO: 1) were conducted in vitro which is not necessarily indicative of how the peptide would behave in vivo. In addition, the epitope may not necessarily be a dominant T cell epitope of MUC1 and such lack of dominance may result in cytotoxic T lymphocytes (CTLs) to other epitopes, not LLLLTVLTV (SEQ ID NO:1).

In work leading to the present invention, the inventors surprisingly found that when a mannan-conjugate of HMFG (whole MUC1) was used for immunisation, non- VNTR, non-leader regions of MUC1 could be selectively antigenic. This is the first time that cytotoxic T cells to non-VNTR regions of MUC1 have been demonstrated in mice immunised with whole MUC1. This means that the non-VNTR peptides could have high affinity for the major histocompatability complex (MHC) class 1. This is surprising in view of the fact that VNTR peptides display low affinity for MHC class 1. The inventors' studies were conducted in vivo.

Disclosure of the Invention: The present invention provides a method for inducing a cell mediated immune response against mucin which comprises administering to a subject an effective amount of: a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin, said sequence comprising an epitope of said mucin non-VNTR, non-leader region, (ii) a fusion protein comprising a peptide or polypeptide according to and a suitable carrier protein, or (iii) a conjugate compound comprising a peptide or polypeptide according to and a carbohydrate polymer, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

AMENDED SHEET

IPENAU

PCT/AUO1/00090 14 1 328216 Received 8 May 2002 2a A further aspect of the invention is a method of preventing or treating a carcinoma in a subject, said method comprising administering to said subject a vaccine or therapeutic agent comprising an effective amount of: a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin, said sequence comprising an epitope of said mucin non-VNTR, non-leader region, (ii) a fusion protein comprising a peptide or polypeptide according to and a suitable carrier protein, or (iii) a conjugate compound comprising a peptide or polypeptide according to and a carbohydrate polymer, and an adjuvant and/or a pharmaceutically acceptable carrier.

The invention also provides a method for inducing a cell mediated immune response against mucin which comprises administering to a subject an effective amount of: a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin, said sequence comprising an epitope of said mucin non-VNTR, non-leader region, wherein said epitope has an amino acid sequence selected from: AVSMTSSVL (SEQ ID NO: 20), NAVSMTSSV (SEQ ID NO: 22), VPSSTEKNA (SEQ ID NO: 28), SAPDNRPAL (SEQ ID NO: 36) and YYQELQRDI (SEQ ID (ii) a fusion protein comprising a peptide or polypeptide according to and a suitable carrier protein, or (iii) a conjugate compound comprising a peptide or polypeptide according to and a carbohydrate polymer, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

Another aspect of the invention is a method for inducing a cell mediated immune response against mucin which comprises administering to a subject an effective amount of: a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin, said sequence comprising an epitope of AMENDED SHEET

WFEA/AU

PCT/AU01/00090 141328216 Received 8 May 2002 2b said mucin non-VNTR, non-leader region, wherein said peptide or polypeptide comprises an amino acid sequence substantially corresponding to one of the following amino acid sequences or an immunogenic fragment thereof: TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO: RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3),

SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLA

PATEPASGSAATW (SEQ ID NO: SAPDNRPAL (SEQ ID NO: 6), NSSLEDPSTDYYQELQRDISE (SEQ ID NO: TQFNQYKTEAASRVNL (SEQ ID NO: AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10) and CYVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 41), (ii) a fusion protein comprising a peptide or polypeptide according to and a suitable carrier protein, or (iii) a conjugate compound comprising a peptide or polypeptide according to and a carbohydrate polymer, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

A further aspect of the invention is an isolated peptide or polypeptide as hereinbefore defined, said peptide or polypeptide being capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin, said sequence comprising an epitope of said mucin non-VNTR, non-leader region, wherein said epitope has an amino acid sequence selected from: AVSMTSSVL (SEQ ID NO: NAVSMTSSV (SEQ ID NO: 22), VPSSTEKNA (SEQ ID NO: 28), YYQELQRDI (SEQ ID NO: 35) and SAPDNRPAL (SEQ ID NO: 36).

Another aspect of the invention is an isolated peptide or polypeptide as hereinbefore defined, said peptide or polypeptide being capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence substantially corresponding to one of the following amino acid sequences or an immunogenic fragment thereof:

SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLA

PATEPASGSAATW (SEQ ID NO: SAPDNRPAL (SEQ ID NO: 6), AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10) and CYVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 41).

AMENDED SHEET I EAi.AU 141328216 PCT/AU01/00090 Received 8 May 2002 2c Accordingly, the present invention provides a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide comprises an amino acid sequence substantially corresponding to that of an epitope of the non- VNTR, non-leader region of a mucin.

It is to be understood that the term "polypeptide" as used in the preceding paragraph and hereinafter does not encompass full-length mucin protein.

Preferably, the peptide or polypeptide consists entirely of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin (and which includes an epitope). However, the peptide or polypeptide may include an additional amino acid sequence(s) derived from other regions of a mucin (including the VNTR and/or leader region). As such, the peptide or polypeptide may also include an epitope(s) from the VNTR and/or leader AMENDED SHEET

IPFA/AU

WO 01/57068 PCT/AU01/00090 3 region. Furthermore, the peptide or polypeptide may include an additional amino acid sequence(s) derived from other natural or artificial sources (e.g.

the peptide or polypeptide may include a heterologous leader and/or signal sequence, or an amino acid sequence substantially corresponding to that of an epitope from an antigen from any tumour type or other source expressing MUC1). Examples of specific tumour antigens are carcinoembryonic antigen (CEA) from colon and other cancers or, indeed, antigens extracted from any tumour expressing MUC.I Preferably, the immune response elicited by the peptide or polypeptide is a cell mediated immune response, particularly one involving the activation of cytotoxic T dells against cells expressing aberrantly glycosylated mucin such as those characteristic of breast, overy, pancreas, colon, lung and prostate tumourigenic cells).

The term "substantially corresponding" as used herein in relation to amino acid sequences is intended to encompass minor variation(s) in the particular amino acid sequence which do/does not substantially alter the biological activity of the particular amino acid sequence. For example, in relation to the amino acid sequence of an epitope of a non-VNTR, non-leader region of a mucin, the term "substantially corresponding" encompasses variation(s) of that sequence (which variation(s) may be found in naturallyoccurring variant sequences or otherwise) where the epitopic activity is substantially unaltered, i.e. the epitope variant is still capable of eliciting a substantially equivalent immune response. Such variations may include conservative amino acid substitutions. Conservative amino acid substitutions envisaged are: G, A, V, I, L, M; D, E; N, Q: S, T; K, R, H; F, Y, W, H; and P, Naalkylamino acids.

The peptide or polypeptide according to the invention may be derived from natural sources, synthesised according to standard techniques or produced recombinantly. Peptide synthesis may be employed for polypeptides containing up to about a hundred amino acids. Generally, for polypeptides containing about twenty or more amino acids, the preferred means of production is recombinant expression in a host cell. Procedures for expression of recombinant proteins in prokaryotic and eukaryotic host cells are well established, see, for example, Sambrook, et al. WO 01/57068 PCT/AU01/00090 4 The peptide or polypeptide may be part of a fusion protein. Procedures for expression of fusion proteins in prokaryotic and eukaryotic host cells are well established, see, for example, Sambrook, et al. Fusion proteins may involve fusion of the peptide or polypeptide to a carrier protein selected from glutathione-S-transferase, P-galactosidase, or any other protein or part thereof, particularly those which enable affinity purification utilising the binding or other affinity characteristics of the protein to purify the resultant fusion protein. The fusion protein may involve fusion of the peptide or polypeptide according to the invention to the C-terminal or N-terminal of the carrier protein. The exact nature of the fusion protein will depend upon the vector system in which the fusion protein is produced. An example of a bacterial expression vector is pGEX which can be used to produce a fusion protein consisting of glutathione-S-transferase with a peptide, polypeptide or protein of interest. The carrier protein may or may not be cleaved from the peptide or polypeptide of the invention following expression. The fusion protein may be treated with mild periodate oxidation.

As mentioned above, expression of the peptide or polypeptide, or a fusion protein comprising the peptide or polypeptide, may be achieved using a host cell, e.g. a prokaryotic E.coli or B. subtilis) or eukaryotic (baculovirus, CHO cells, COS cells or yeast) host cell expression system. In some of these systems, for example, baculovirus or yeast, glycosylation of the peptide, polypeptide or fusion protein can be achieved by introducing well known glycosylation motifs.

Similarly, the peptide or polypeptide may be simply coupled to a suitable carrier protein keyhole limpet hemocyanin) using any of the well established procedures in the art treatment with glutaraldehyde).

Preferably, the peptide or polypeptide according to the present invention comprises an amino acid sequence derived from human mucin 1.

More preferably, the peptide or polypeptide comprises an amino acid sequence derived from human milk fat globule membrane antigen (HMFG).

Even more preferably, the peptide or polypeptide comprises an amino acid sequence derived from the extracellular region or intracellular region of the non-leader, non-VNTR region of human MUC1 amino acids 22 to 131, or amino acids 402 to 473 of human MUC1 according to NCBI database Accession No. M61170 (see also figure although an amino acid sequence from the transmembrane region of the non-leader, non-VNTR region of 141328216 PCT/AU01/00090 Received 8 May 2002 human MUC1 may also be suitable. Still more preferably, the peptide or polypeptide comprises an amino acid sequence substantially corresponding to one of the following amino acid sequences or an immunogenic fragment thereof: TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO: 2) RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3)

SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLA

PATEPASGSAATW (SEQ ID NO: 4) SAPDNRPAL (SEQ ID NO: 6) NSSLEDPSTDYYQELQRDISE (SEQ ID NO: 7) TQFNQYKTEAASRVNL (SEQ ID NO: 8) AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: 9) YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: In one aspect, the present invention provides a compound comprising a conjugate of the peptide or polypeptide as hereinbefore described and a carbohydrate polymer.

Preferably, the carbohydrate polymer is a polymer of a carbohydrate selected from the group consisting of glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-0-methyll-D-galactose, 2-0-acetyl-1-Dxylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, a-D-galactopyranose 6-sulphate, fructose and a abequose, conformation and configuration isomers thereof, or a carbohydrate formed of two or more different monomer units. The number of repeated monomer units in the polymer is not important but generally carbohydrate polymers would comprise at least twenty monomer units, preferably in excess of one hundred monomer units, more preferably in excess of one thousand monomer units, and still more preferably in excess of ten thousand monomer units or more. Carbohydrate polymers may be a mixture of polysaccharide chains of varying molecular weights. More preferably, the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer containing mannose units. Most preferably, the carbohydrate polymer is a polymer of oxidised mannose.

The peptide or polypeptide according to the present invention may be conjugated to a carbohydrate polymer according to standard techniques well known in the art of carbohydrate chemistry for the derivatization and reaction of polysaccharides and monosaccharides. Carbohydrates may be oxidised with AMENDED

SHEET

IPEA/AU

141328216 PCT/AU01/00090 Received 8 May 2002 6 conventional oxidising reagents such as sodium periodate to give a polyaldehyde which can then be directly reacted with the peptide or polypeptide where amino functional groups on the peptide chain (such as the M amino group of lysine) react with the aldehyde groups which may be further reduced to form a Schiff base.

Polysaccharide chains may be first activated with cyanogen bromide and the activated polysaccharide then reacted with a diamine, followed by conjugation to the peptide or polypeptide to form a conjugate which may, optionally, then be oxidized.

The carbohydrate and polypeptide may be derivatised with bifunctional agents in order to cross-link the carbohydrate and polypeptide. Commonly used cross-linking agents include 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, Nhydroxysuccinimide esters, for example, esters with 4-azidosalicyclic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3,3'dithiobis(succinimidyl-propionate), and bifunctional maleimides such as bis-Nmaleimido-1, 8-octane. Derivatizing agents such as a methyl-3-[(p-azidophenyl)dithio] propioimidate yield photactivitable intermediates which are capable of forming cross-links in the presence of light. Oxidized carbohydrates may be reacted with hydrazine derivatives of antigens to give a conjugate. Alternatively, carbohydrates may be reacted with reagents such as carbonyl diimidazole followed by reaction with antigen, which after oxidation gives the desired conjugate.

The coupling of the peptide or polypeptide to a carbohydrate involves converting any or all of the functional groups on the carbohydrate to reactive groups and thereafter reacting the reactive groups on the carbohydrate with reactive groups on the polypeptide. Carbohydrate polymers are replete with hydroxide groups, and in some instances, carboxyl groups (such as in idruionate), ester groups (such as methylgalacturonate) and the like. These groups may be activated according to standard chemical procedures. For example, hydroxyl groups may be reacted with hydrogen halides, such as hydrogen iodide, hydrogen bromide and hydrogen chloride to give the reactive halogenated polysaccharide. Hydroxy groups may be activated with phosphorous trihalides, active metals (such as sodium ethoxide, aluminium isopropoxide and potassium tert-butoxide), or esterified (with groups such as tosyl chloride or acetic acid) to form reactive groups which can be then reacted with reactive groups on the polypeptide to form one or more bonds. Other functional groups on carbohydrates apart from hydroxyl groups may be activated to give reactive groups according to standard techniques.

AMENDED

SHEET

IPEA/AU

PCT/AU01/00090 141328216 141328216 Received 8 May 2002 7 The carbohydrate polymer may be purified from a natural source or otherwise synthesised in accordance with standard techniques. Carbohydrates are available commercially from many suppliers.

The carbohydrate polymer is preferably conjugated to the peptide or polypeptide at any amount which permits the peptide or polypeptide to elicit a cell mediated immune response in a human or other animal. Such an amount may be within the range, for example, of about 0.1-10 mg per mg of the peptide or polypeptide.

Fusion proteins as described above and peptides or polypeptides otherwise coupled to a suitable carrier protein as described above, may also be coupled to a carbohydrate polymer (especially oxidised mannose). Similarly, the carbohydrate polymer is preferably conjugated to the fusion protein at any amount which permits the fusion protein to elicit a cell mediated immune response in a human or other animal. In this case however, the amount may be within the range, for example, of about 1-10 mg per mg of the fusion protein, more preferably about 5-8 mg per mg of the fusion protein.

In another aspect, the present invention provides a vaccine against disease states, particularly human disease characterised by tumour cells expressing mucin or a subunit thereof, wherein said vaccine comprises the peptide or polypeptide of the present invention, or a fusion protein comprising the peptide or polypeptide of the present invention, and, optionally, an adjuvant and/or a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides a vaccine against disease states, particularly human disease characterised by tumour cells expressing mucin or a subunit thereof, wherein said vaccine comprises the conjugate compound of the present invention and, optionally, an adjuvant and/or a pharmaceutically acceptable carrier.

Suitable adjuvants for use in the vaccine of the present invention include any of those well known in the art such as Quil A. QS-21 Iscoms, liposomes, alum, salts, oil, emulsions, etc.

The vaccine of the present invention may be administered to human patients to protect against various disease states including cancer cell growth, and in particular, the growth of tumours of secretory tissues, such as tumours of the breast, colon, lung, pancreas, prostate, and the like. Subjects may be immunised with the vaccine to AMENDED SHEET

IPEA/AU

141328216 PCT/AU01/00090 Received 8 May 2002 8 protect against tumour formation of secretory tissues. Alternatively, subjects suffering from tumours may be immunised with the vaccine as part of a therapeutic regimen for tumour treatment. By way of example, to protect women from breast cancer, women may be immunised with the vaccine pre- or post-puberty and may receive one or more injections, preferably an initial immunisation followed by one or more booster injections separated by several months to several years. The route of immunisation is no different from conventional human vaccine administration.

Accordingly, the vaccine of the present invention may be administered subcutaneously, intramuscularly, orally, intravenously, and the like.

The amount of a peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention which is delivered to a subject is not critical or limiting. However, an effective amount of a peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention, is one which will stimulate an immune response. In this regard, the effective amount may vary according to the immune status of the subject (i.e.

depending on whether the subject is immunosuppressed or immunostimulated), the judgement of the attending physician or veterinarian, whether the vaccine is to be used to prevent or treat a disease state or to prevent tumour formation, or whether the vaccine is to be used in the treatment of an existing tumour. By way of example, subjects may receive from 1g to 10,000tg of the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention, more preferably 50tg to 5,000pg, still more preferably 100pg to 1,000tg, and even more preferably 100pg to 500gg. Adjuvants are not generally required. However, adjuvants may be used for immunisation.

The peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention may be administered to subjects in concert with a cytokine or other immune regulator one or more of GM-CSF, G-CSF, M-CSF, TNFca or P, interferon a or y, any of IL1 through IL13, or any other cytokine). The immune regulator may be administered at the same or different time as the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention, optionally as part of a multi-component administration form.

AMENDED

SHEET

IPEA/AU

141328216 PCT/AUO 1/00090 141328216 Received 8 May 2002 9 In another aspect, the present invention provides a method for inducing a cell mediated immune response against mucin which comprises administering to a subject an effective amount of the peptide or polypeptide as hereinbefore described (which may be coupled to a suitable carrier protein), or a fusion protein comprising the peptide or polypeptide of the present invention, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides a method for inducing a cell mediated immune response against mucin which comprises administering to a subject an effective amount of a conjugate compound according to the present invetion, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

The administration to human and animal subjects of the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention may provoke a potentiated cellular response of activated T-lymphocytes which are cytotoxic to cells expressing mucins. A potential benefit of this invention arises from the fact that humans and animals may be protected against cancer prior to tumour growth, as the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention of the invention may provoke a cellular immune response to cytotoxic T-cells which kill tumour cells expressing mucin. This invention is applicable to the immunisation against tumours of secretory tissue, such as adenocarcinomas, more particularly, tumours of the breast, ovary, pancreas, colon, lung, prostate and the like.

The peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention may also be used as, or as a component of, therapeutic agents for the treatment of patients suffering from cancer, as a part of the overall treatment for eradication or reduction of the cancer. Thus, the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention may be administered to subjects suffering from cancer either before or after surgery to AMENDED

SHE-T

IPEA/AU

WO 01/57068 PCT/AU01/00090 remove a tumour. Preferably, the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention is administered as part of a chemotherapeutic regime following tumour excision. In such circumstances, the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention is administered in an amount consonant with standard chemotherapeutic regimes for the administration of cytotoxic compounds for use in tumour treatment It is believed that the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein and conjugate compound according to the present invention possess the advantage of being substantially nontoxic on administration to humans or animals, and as a consequence, are well tolerated.

In a further aspect, the present invention relates to the use of the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention in the treatment of adenocarcinoma, particularly breast cancer.

In a still further aspect, the present invention relates to the use of the peptide or polypeptide (which may be coupled to a suitable carrier protein), fusion protein or conjugate compound according to the present invention to pulse dendritic cells for in vivo transfer and use as a vaccine.

In yet a still further aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the peptide or polypeptide of the first aspect (which may be coupled to a suitable carrier protein), or a fusion protein comprising the peptide or polypeptide of the first aspect The nucleic acid molecule may be incorporated into a transfer or expression vector, or used in a DNA vaccine. Such nucleic acid molecules may be produced according to standard techniques either by cloning or synthesis as described in, for example, Sambrook et al. In yet another aspect, the present invention provides a compound comprising a conjugate between MUC1 and a carbohydrate polymer such as those discussed above, such that the conjugate is capable of eliciting a cell mediated immune response in a human or other animal. Preferably, the MUC1 is human MUC1 HMFG) and the carbohydrate polymer is a WO 01/57068 PCT/AU01/00090 11 polymer of mannose, particularly oxidised mannose, or is oxidised mannan.

The carbohydrate polymer may be conjugated to the MUC1 at an amount within the range, for example, of about 1-10 mg per mg of MUC1, preferably about 5-8 mg per mg of MUC1, more preferably about 7 mg per mg of MUC1.

The conjugate compound may be used in a vaccine or as a therapeutic agent in a manner akin to that discussed above.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

The invention will now be described with reference to the following non-limiting Examples and accompanying figures.

Brief description of the accompanying figures: Figure 1: Provides the amino acid sequence of a human MUC1 protein (NCBI database Accession No. M61170).

Figure 2: Assay for HMFG and mannan. Inhibition of binding of anti-MUC1 antibody to HMFG by competitor preparations of HMFG and mannan-HMFG Binding of mannan-HMFG and HMFG to anti- MUC1 antibody and Con A detected by a radioimmunoassay.

Figure 3: A2KbMUC1 double transgenic mice were immunised with mannan-HMFG and splenocytes were used in CTL assays. Cytotoxic activity of the effector cells were measured on 5 1 Cr-labelled MCF7 with or without cold K562 BT20 or ME272 Figure 4: C57BL/6 and BALB/c mice were immunised with mannan- HMFG and splenocytes were used in CTL assays. Lysis of P815 or RMA (c) cells pulsed with various 9-mer.peptides from the intracellular peptide 471- 493; Lysis of P815 or RMA cells pulsed with various 9-mer peptides WO 01/57068 PCT/AU01/00090 12 from the extracellular peptides 33-103 and 51-70 and Lysis of P815 cells pulsed with YYQELQRDI (SEQ ID NO: 35) and RMA-MUC1 cells pulsed with SAPDNRPAL (SEQ ID NO: 36). As controls for peptide pulsing and antigenspecific cell lysis, known peptide antigens were used and are shown in each panel and described in the text.

Figure 5: Balb/c mice were immunised with mannan-507-KLH and splenocytes were used for CTL assays. The lysis of "Cr-labelled P815 target cells unpulsed or pulsed with Cpl3-32 or 507 peptide at various effector:target ratios were measured.

Figure 6: Balb/c mice were immunised with mannan-471-KLH and splenocytes were used for CTL assays. The lysis of "Cr-labelled P815 target cells unpulsed or pulsed with Cpl3-32 or 471 peptide at various effector:target ratios were measured.

Abbreviations: The following abbreviations are used in the Examples: ELISA: enzyme linked immunosorbent assay DTH: delayed type hypersensitivity FP: fusion protein GST: glutathione-S-transferase HMFG: human milk fat globule Kd: kilodalton KLH: keyhole-limpet haemocyanin PAGE: polyacrylamide gel electrophoresis PBS: phosphate buffered saline SDS: sodium dodecyl sulphate Tc: cytotoxic T-lymphocytes EXAMPLE 1 1. INTRODUCTION Immunotherapeutic approaches for the treatment of breast cancer have included the use of monoclonal antibodies and the generation of cytotoxic T lymphocytes (CTL) [29-34]. The identification of target antigens, the availability of recombinant proteins and cytokines have given impetus to immunotherapy. Thus, there are new means by which to generate an effective cytotoxic T cell response to MUC1-expressing carcinomas of the breast and WO 01/57068 PCT/AU01/00090 13 other tissues MUC1 is a particularly attractive target for the generation of CTL: it is immunogenic in mice for the production of antibodies and, more recently, the present inventors have described CD8+ CTL, and the MHC Class I H-2 and HLA-A*0201 binding peptides have been mapped in the VNTR [36-39]. Furthermore, in cancer cells, there is up to a 100 fold increase in the amount of mucin [40] and there should be a significant amount of MUCI peptide available to be bound by Class I molecules. The reason for the focus on the VNTR peptides is clear: it is the most immunogenic region in MUC1 when whole tumour cells or mucin extracts (HMFG) are used to immunise mice for the production of antibodies Because of this focus and the finding that non-HLA restricted CTL also are directed to the VNTR, almost all interest in MUC1 for CTL induction has concentrated on VNTR peptides [37, 41, 42]. In contrast, the present example relates to the induction of CTL to non-VNTR epitopes, in the extracellular and intracellular parts of MUC1 identified by immunising mice with native mucin (HMFG) obtained from human breast milk, or by immunising with peptides as described herein.

2. MATERIALS AND METHODS Mice and Tumour Cells BALB/c C57BL/6 human MUC1 transgenic mice (obtained from B. Acres (Transgene, Strasbourg, France)), transgenic HLA- A*0201/Kb mice (H-2b)(obtained from The Scripps Clinic and Research Foundation, La Jolla, CA.) and double transgenic mice (A2KbMUC1) were bred at The Austin Research Institute (ARI). The human MUC1 in the MUC1 transgenic mice (back crossed to DBA/2) is under the control of the human MUC1 promoter; MUC1 is expressed in the lung bronchioles, P-islets of the pancreas, kidney tubules and stomach The HLA-A*0201/Kb mice express a transgene composed of the al and ac2 domains of HLA-A*0201 and the t3 contains the transmembrane and cytoplasmic domains of H-2Kb [44].

The double transgenic mice were screened for expression of the HLA- A*0201/Kb and human MUC1 transgenes by flow cytometry with antibodies to HLA-A*0201 and MUC1. RMA-MUC1 is a MUC1 transfected (C57BL/6 (H- 2 lymphoma cell line Tm211 is a MUC1 transfected P815 mastocytoma (DBA/2 origin; H-2d) obtained from B. Acres (Transgene, Strasbourg, France) All mouse cell lines were maintained in Dulbecco's WO 01/57068 PCT/AU01/00090 14 modified Eagles medium (DMEM) with 100IU/ml penicillin, l00g/ml Streptomycin and 10% foetal calf serum (all from Commonwealth Serum Laboratories (CSL), Melbourne) and human cell lines in RPMI with the same additives in a 7% humidified C02 incubator at 37°C. BALB/c, C57BL/6 and double transgenic A2KbMUC1 mice were immunised intraperitoneally with 3 injections of 5pg mannan-HMFG or HMFG on days 0, 10, 17 while HLA- A*0201/Kb mice were injected once.

2.2 Synthetic Peptides Peptides (Table 1) were synthesised at the ARI; the purity of the peptides was determined by mass spectroscopy.

2.3 Conjugation of HMFG to Mannan HMFG was isolated from human milk [49] and coupled to mannan.

Mannan (lml, 14mg/ml) in phosphate buffer (0.1M, pH6.0) was treated with sodium periodate (100tl, 0.1M) and incubated at 4 0 C for 30 min [48].

Ethanediol (10gl) was added for 30 mins at 40 to stop the reaction, and the mixture was passed through a PD10 column (Pharmacia Biotech, Sweden), equilibrated in bicarbonate buffer (0.2 M, pH 9.0) and the oxidised mannan fraction was mixed with 1 mg of HMFG overnight at room temperature to give mannan-HMFG.

2.4 T Cell Epitope Prediction There are several CTL epitope prediction algorithms available and in this study we used the program developed by Dr Kenneth Parker available on the internet (bimas.dcrt.nih.gov/molbio/hla_bind/) to identify potential T cell epitopes. This program is based on scores given to the amino acids at each of the positions from 1-9 from input sequences by comparison with the reported databases [49, 50]. Higher numerical values for the 9-mer predict increased likelihood of being a T cell epitope. For example, the T cell epitope for ovalbumin (Kb, SIINFEKL; SEQ ID NO: 11) and papillomavirus-16 E7 protein

(D

b RAHYNIVTF; SEQ ID NO: 12) gives scores of 17 and 6 respectively.

2.5 Cytotoxic T Cell and Cytotoxic T Cell Precursor (CTLp) Frequency Assays CTL assays were performed as described [37, 39, 48]. Briefly, 7 to days after the final immunisation, splenocytes were harvested, washed and resuspended in growth medium and serially diluted in 96-well microtitre plates. A standard 3 hr 5 1 Cr release assay was performed with 1x10 4 peptide pulsed or untreated P815 or RMA cells as targets at various effector:target ratios. Peptide pulsed P815 or RMA target cells were prepared by overnight WO 01/57068 PCT/AU01/00090 incubation with 9-mer peptides (25pg/ml) [37] For CTL assays with A2KbMUC1 double transgenic effectors, MCF7 (MUC1+HLA-A*0201+) and (MUC1+HLA-A*0201-) breast cancer cell lines or the ME272 (MUC1- HLA-A*0201+) melanoma cell line was used as targets. All of these human tumour cell lines are susceptible to cell mediated lysis [39, 51, 52]. CTLp frequencies were determined from a minimum of 32 replicates, for at least 6 effector cell numbers (1x10 3 1.28x10 5 Cells were cultured in U-bottomed microtitre trays, with 5x10 5 mitomycin C treated BALB/c C57BL/6 (H- 2 b or HLA-A*0201/Kb spleen cells, in DMEM supplemented with 10% foetal calf serum, 5tM of various MUC1 peptides (Table 1) or HMFG and 10 U/ml rhIL-2. Seven days later, each microculture was assayed for cytotoxicity by replacing 100l of culture medium with 100 1 l target cell suspension containing 10 4 5 1 Cr-labelled Tm211 RMA-MUC1 (H-2b) Tumour or EBV transformed human B cells (HLA-A*0201) or MCF7 as targets. As a specificity control non-MUC1 expressing P815(H-2d) or RMA(H-2b) cells were used. Cytotoxic activity was considered to be present if in each well 1 Cr release was found 3 standard deviations above the mean isotope release from 10 4 effectors cultured with stimulators only or from stimulator cells with peptide only or rhIL2 only. A linear relationship (0.987< r 2 1) existed between the number of responder cells, represented on a linear scale, and the frequency of negative wells on a logarithmic scale. CTLp frequencies were determined as the inverse of responder cell dose required to generate 37% negative wells [53-55]. CTLp frequency assays were performed three times and the individual frequencies did not differ by more than 20% from the mean value. However, it should be noted that the CTLp frequency in immunised mice are directly correlated with tumour protection (28).

2.6 Inhibition ELISA An antibody inhibition ELISA was performed to compare the activity of HMFG before and after conjugation to mannan. Polyvinyl chloride plates were coated with 70 p1 of 10 g/ml HMFG in bicarbonate buffer (0.2M, overnight at 4 0 C or 1 hr at 37 0 C and non-specific binding was blocked with 2% bovine serum albumin (BSA). Various concentrations of HMFG or mannan-HMFG were incubated with anti-MUC1 antibody (VA2 1/200 supernatant) for 3 hr and loO1l was added to PVC microtitre well plates coated with HMFG. After washing with phosphate buffered saline (PBS) containing 0.05% Tween 20, 50l of sheep anti-mouse immunoglobulin WO 01/57068 PCT/AU01/00090 16 conjugated to horseradish peroxidase (Amersham, UK) was added and incubated for a further 1 hr at RT. After washing with PBS/Tween20, the plate was developed with the chromogenic substrate 2,2"-azino-di(3ethylbenzthiazoline) sulphonate (ABTS) (Amersham, UK) and the absorption at 405 nm recorded.

2.7 Radioimmunoassay A sandwich radioimmunoassay was performed to ascertain that the mannan was covalently linked to HMFG. A microtitre plate was coated with serial dilutions of anti-MUC1 antibody (BC2 in bicarbonate buffer overnight and non-specific binding blocked as described above. HMFG or Mannan-HMFG was then added to the walls and incubated for 1 hr at RT followed by washing extensively with PBS containing 0.05% Tween 20. Fifty 0l of radiolabelled concanavalin A, which binds specifically to mannan but not HMFG, was then added and the plate incubated for a further 1 hr followed by washing with PBS/Tween 20. Microscint-O (120pl) was added to the wells, and plates counted in a p-scintillation counter.

3. RESULTS 3.1 Preparation and Characterisation of mannan-HMFG The activity of the HMFG after conjugation to mannan was determined by inhibition ELISA; the 50% inhibitory concentration for HMFG was 22 gg/ml while for the mannan-HMFG was 20pg/ml (Fig. la), i.e. HMFG retained full reactivity.after conjugation to mannan. The integrity of the mannan- HMFG complex was shown by a sandwich radioimmunoassay using anti- MUC1 antibody bound to the plate and 1 2 5 I-labelled Con-A for the read out (Fig Ib). Non-conjugated HMFG did not bind 12 5 I-Con-A while mannan- HMFG bound demonstrating mannan to be linked to HMFG.

3.2 CTL Responses to mannan-HMFG in BALB/c Mice Spleen cells, from BALB/c mice immunised with mannan-HMFG, were stimulated in vitro with different peptides (from both VNTR and non-VNTR regions, Table 1) and CTLp were determined by testing on target cells expressing native MUC1 (Table It was apparent that immunisation with mannan-HMFG leads to CTL reacting with epitopes from the whole of MUC1, from both the VNTR and non-VNTR region.

The responses were WO 01/57068 PCT/AU01/00090 17 a) HMFG. When whole MUC1 (HMFG) protein was used as the source of stimulating peptides, a CTLp frequency of 1/9,700 was obtained. Clearly HMFG is immunogenic for CTL production in BALB/c mice and can be processed to yield peptides presented by Class I molecules.

bl VNTR. When VNTR peptides Cpl3-32 and pl-30 were used to stimulate, CTLp frequencies of 1/7000 (Cpl3-32) and 1/13,200 (pl-30) resulted, by immunising with HMFG, anti-VNTR CTL were produced, results similar to those found previously by immunising with mannan-conjugated VNTR peptides This is the first description of such CTL obtained by immunising with native mucin which is glycosylated.

c) Extracellular regions. When in vitro stimulation was with peptides containing amino acids 31-55, 51-70, 33-103, 344-364, CTL could be detected with a frequency of 1/19,500 (31-55); 1/10,000 (51-70); 1/20,150 (33-103) and 1/36,800 (344-364). Thus, CTL can be produced to non-VNTR regions from the extracellular region; this is the first description of such CTL.

dl Intracellular regions. Three different, non-overlapping intracellular peptides containing amino acids 408-423, 471-493, 507-526, were examined using the approach described above. CTLp frequencies of 1/30,000 (408-423), 1/12,500 (471-493) and 1/22,500 (507-526) were obtained, amino acids 471- 493 being the most effective to restimulate cytolytic cells.

To demonstrate that the CTL were specific for MUC1 sequences, and not due to non-specific killing by NK cells or other cells, P815 target cells were used with a non-MUC1 peptide, T4N1, as the pulsed antigen, CTLp either were not detected or the frequencies were 1/200,000 and were considered to be negative (not shown). Of the different regions, 3 were of equivalent immunogenicity (using CTLp frequency as a measure) extracellular (51-70) VNTR (Cpl3-32) intracellular (471-493), all of which gave a high frequency of 1/10,000.

In contrast, immunising BALB/c mice with non-conjugated HMFG, and stimulating with the VNTR peptide Cpl3-32, the CTLp frequency was 1/80,500. This frequency is similar to the CTLp frequency of 1/95,000 obtained with mannan conjugated to a recombinant bacterial fusion protein containing 5 repeats of the MUC1 VNTR (47) and, thus, conjugation of HMFG to mannan is necessary for generating a strong CTLp frequency in mice.

WO 01/57068 PCT/AU01/00090 18 3.3 CTL Responses to mannan-HMFG in C57BI6 mice C57Bl/6 were immunised with mannan-HMFG and in vitro stimulated with the same antigens used for the BALB/c mice (Table There was a CTLp frequency of 1/13,500 for whole HMFG and 1/12,500 for the VNTR region peptide pl-30 (Table Of the non-VNTR extracellular peptides, CTL were detected only to one extracellular peptide (344-364) with a frequency of 1/24,500. CTL were not detected to any of the intracellular peptides. Again, the specificity of the CTL were confirmed by using a non-MUC1 peptide, T4N1, for stimulation and also using the non-MUC1 transfected parent RMA cell line as the target. Thus, C5 7BL/6 mice can respond to both VNTR and non-VNTR peptides, but there were no responses to certain peptides to which BALB/c mice responded.

3.4 Cellular Immune Responses to mannan-HMFG in Transgenic HIA- A*0201/Kb Mice Transgenic HLA-A*0201/Kb mice were immunised once with mannan- HMFG (not x3 as used above), stimulated in vitro with either HMFG, the VNTR peptide (pl-30) or one of the extracellular peptides (31-55). The CTLp were measured on human EBV HLA-A*0201+ cells (see below) and frequencies were 1/39,000 (HMFG), and 1/33,000 (VNTR pl-30), which compare favourably with immunisation with mannan-VNTR peptide (1/48,000) whole HMFG is as immunogenic as VNTR (Table Further, when an extracellular peptide (31-55) was used, the CTLp frequency was 1/40,000, the same as that found for VNTR. Thus, HLA-A*0201 can present extracellular and VNTR peptides. It should be noted that, the target cell being EBV transformed B cells, which expresses HLA-A*0201 but not H- 2 b class I molecules (expressed by the immunised mice), the CTLs detected were restricted to HLA-A*0201 presenting MUC1 peptides.

Cellular Immune Responses to Mannan-HMFG in A2KbMUC1 Double Transgenic Mice To ascertain the ability of MUC1 CTL to lyse MUC1 positive breast cancer cells A2KbMUC1 double transgenic mice injected with mannan HMFG 3 times were stimulated in vitro with either HMFG, the VNTR peptide (plextracellular peptides (31-55, 344-364) or intracellular peptides (408-423, 471-493, 507-526) (Table There was a CTLp frequency of 1/2,000 for the whole HMFG and 1/8,000 for the VNTR region peptide pl-30. CTL were detected to the extracellular peptides 31-55 and 344-364 with a frequency of WO 01/57068 PCT/AU01/00090 19 1/2,000 and 1/11,000 respectively. Of the intracellular peptides, CTL were detected for only peptide 408-423 with a frequency of 1/20,000.

Spleens of the immunised mice were used in a direct CTL assay to ascertain specificity of the anti-MUC1 CTL. As seen in Figure 2, MUC1 CTL lysed 55% of MUC1+ MCF7 (HLA-A*0201) breast carcinoma cells at an E:T ratio of 12:1 and was reduced to 1796 when incubated in the presence of cold K562 targets. The MUC1 CTL were HLA restricted as no lysis was detected when the MUC1+ BT20 (HLA-A1) breast cancer cell line was used. The MUC1 CTL did not lyse the MUC1 -ve melanoma cell line ME272.

Thus, immunisation of A2KbMUC1 mice with mannan-HMFG resulted in specific Class I restricted CTL that can lyse tumour cells expressing native MUC1 and, moreover, anti-MUC1 CTL can be generated in mice in the presence of endogeneously expressed human MUC1.

3.6 T Cell Epitope Prediction and Mapping To precisely map the T cell epitopes involved in CTLp generation, a large number of overlapping 9-mer peptides would have to be synthesised and used in CTL assays. Instead, a CTL epitope prediction program was used to select putative immunogenic peptides and these were synthesised to test their antigenicity.

Predicted H-2d-restricted peptides (intracellular region MUC1) Several peptides (NYGQLDIFP(Kd) SEQ ID NO: 13; YGQLDIFPA(Dd) SEQ ID NO: 14; KNYGQLDIF(Ld), SEQ ID NO: 15) were contained in 471-493 (CTLp frequency= 1/12,500) and had predicted scores 6, 6 and 10 respectively (Table To ascertain if the predicted 9-mers are presented by the Class I molecules, cytotoxic T cell assays were performed using spleen cells from mannan-HMFG immunised mice as effectors and P815 target cells were pulsed with the synthetic peptides. These were NYGQLDIFP(K d (SEQ ID NO: 13), YGQLDIFPA(Dd) (SEQ ID NO: 14), KNYGQLDIF(Ld) (SEQ ID NO: The pulsed cells were not lysed by Mannan-HMFG derived CTL from BALB/c mice (Figure 3a), the CTL epitopes were not predicted accurately by the algorithm. The MUC1 VNTR peptides SAPDTRPAP(Dd) (SEQ ID NO: 16) and APDTRPAPG (Ld) (SEQ ID NO: 17) identified previously as CTL epitopes in the VNTR region were used as positive controls and 62% and lysis at an E:T ratio of 50:1 was obtained. The listeriolysin Kd peptide (GYKDGNEYI; SEQ ID NO: 18) and HIV Dd peptide WO 01/57068 PCT/AU01/00090 (RKSIRIQRGPGRAFVTIGKGKGKGY; SEQ ID NO: 19), used as negative controls, did not give rise to lysis (Fig. 3a).

Predicted H-2d-restricted peptides (extracellular region MUC1) A number of 9-mar peptides in the extracellular region are predicted to be CTL epitopes [(AVSMTSSVL(Kd), SEQ ID NO: 20; TTQGQDVTL(Kd), SEQ ID NO: 21; NAVSMTSSV(Kd), SEQ ID NO: 22; TSATQRSSV(Kd), SEQ ID NO: 23; SSTTQGQDV(Kd), SEQ ID NO: 24; SVPSSTEKN(Dd), SEQ ID NO: EPASGSAAT(Ld), SEQ ID NO: 26; SPGSGSSTT(Ld), SEQ ID NO: 27; VPSSTEKNA(Ld), SEQ ID NO: 28; TPGGEKETS(Ld), SEQ ID NO: 29; TSATQRSSV(Ld), SEQ ID NO: 30; SSTTQGQDV(Ld), SEQ ID NO: 24] and were contained in peptide 33-103 (CTLp frequency= 1/20,150) with scores of 58, 40, 29, 10, 10, 2.9, 39, 39, 36, 30, 10 and 10) respectively. A subset of these peptides were also contained in the 51-70 peptide(CTLp frequency=1/10,000) (Table Of these, four were made (AVSMTSSVL(Kd), SEQ ID NO; 20, NAVSMTSSV(Kd), SEQ ID NO: 22; VPSSTEKNA(Ld), SEQ ID NO: 28; SVPSSTEKN SEQ ID NO: 25) and tested. Three of the four peptides were indeed presented and one was not The synthetic peptides AVSMTSSVL(Kd), SEQ ID NO: 20; NAVSMTSSV(Kd), SEQ ID NO: 22 and VPSSTEKNA(Ld), SEQ ID NO: 28 sensitised P815 target cells with 77%/6, and 78% lysis at E:T of 50:1 respectively, while SVPSSTEKN (with the lowest predictive value) was inactive (Figure 3b). Therefore, AVSMTSSVL, SEQ ID NO: 20; VPSSTEKNA, SEQ ID NO: 28 and NAVSMTSSV, SEQ ID NO: 22 are CTL epitopes in peptides 33-103 and 51-70.

Predicted H-2b restricted peptides.

Even though there were fewer identified peptide epitopes for C5 7BU16 mice, there are a large number of potential CTL epitopes present in the peptides, albeit with low scores (Table The 9-mer CRRKNYGQL (Db, Kb), SEQ ID NO: 32 was contained in 471-493 (CTLp not detected) and had scores of 10 and 1.4. It weakly sensitised RMA targets to lysis by mannan-HMFG CTL with 20 lysis at a E:T of 50:1 and 42% lysis at E:T of 100:1 (Figure 3c).

The MUC1 VNTR peptides APGSTAPPA SEQ ID NO: 33 and SAPDTRPAP SEQ ID NO: 16 were used as positive specificity controls, where lysis of 70% and 80% were obtained while no lysis was detected for the ovalbumin Kb 9-mer SIINFEKL, SEQ ID NO: 11 and Adenovirus Db 9-mer (used as negative specificity controls). The 9-mer peptides STEKNAVSM(Db), SEQ ID NO: 34; AVSMTSSVL(Db), SEQ ID NO: 20 and AVSMTSSVL(Kb), WO 01/57068 PCT/AU01/00090 21 SEQ ID NO: 20 were contained in the peptides 33-103 and 51-70 with scores of 15, 10 and 1.2. All of these three peptides weakly sensitised RMA targets to lysis at 50:1 and -40% lysis at E:T of 100:1) (Figure 3d). There were no CTL reactive to peptides 31-55 and 51-70 in C57BL/6 mice.

Two high scoring CTL epitopes predicted from the whole MUC1 molecule from the intracellular region (YYQELQRDI(Kd), SEQ ID NO: score 2880) and extracellular region N-terminal to the VNTR (SAPDNRPAL(Db), SEQ ID NO: 36 score 4723) with scores of 2880 and 4723 sensitized RMA and P815 target cells to 50% lysis at an E:T of 50:1 (Figure 3e). Therefore, several T cell epitopes are present in the non-VNTR regions of the MUC1 molecule and 9-mer peptides can be presented by target cells to CTL generated by mannan-HMFG immunisation.

4. DISCUSSION Previous immunisation studies by the present inventors used a MUC1 fusion protein containing 5 repeats of the VNTR linked to mannan (MFP) and this generated strong cellular responses to MUC1 characterised by the production of IFN-y, IL-12, very little IgG2a antibody and protection from tumour growth [36, 48]. Immune responses in humans have also shown promise for the therapeutic use of MUC1 antigens as in a Phase I clinical trial using MFP, 4 of 15 patients generated proliferative responses, 13 of showed high levels of MUC1 specific serum antibody and 2 of 10 generated CTL to MUC1 However, in vitro peptide binding studies and in vivo studies using transgenic HLA-A*0201 mice demonstrated that the VNTR sequences can only be presented by HLA-A*0201 and HLA-A*1101 [39, and studies thus far have concentrated on the MUC1 VNTR because of its preferential immunogenicity in mice, at least for antibodies, and because of evidence from humans implicating the VNTR in immune responses. Other protein sequences of MUC1 have not been examined for their cellular immunity. In the past, the present inventors have sought monoclonal antibodies to non-VNTR regions in mice immunised with MUC1: none resulted and none were found in an international study. Scanning the whole MUC1 sequence for potential T cell epitopes predicted many previously untested peptides. The inventors have therefore immunised mice with mannan conjugated HMFG, to provide all possible MUC1 epitopes but dependent on natural antigen processing for their presentation, and showed WO 01/57068 PCT/AU01/00090 22 that cellular immune responses to the non-VNTR regions of the MUC1 can be generated which are as effective as those generated to the VNTR and further both HLA-A*0201 and A2KbMUC1 transgenic mice could be immunised, indicating that humans should also be able to be immunised.

Cellular responses could be detected to the extracellular region of MUC1, the VNTR and also to intracellular peptides in mannan-HMFG immunised BALB/c, C57BIJ6, HLA-A*0201/Kb and double transgenic A2KbMUC1 mice. Immunised BALB/c mice developed CTL that could respond to more non-VNTR CTL epitopes than C57BL/6 mice, in which only the 344-364 peptide and SAPDNRPAL (SEQ ID NO: 36) was recognised by CTL (Table 2, Figure 3e).

Of the various peptides used for restimulation, several possible candidate 9-mer epitopes could be predicted using the peptide motif search program (Table In BALB/c mice, the precursor frequency for the 471-493 peptide was 1/12,500, however the predicted epitope peptides NYGQLDIFP (SEQ ID NO: 13), YGQLDIFPA (SEQ ID NO: 14) and KNYGQLDIF (SEQ ID NO: 15) were not able to sensitise P815 targets for lysis by mannan-HMFG CTL (Figure 3a). Therefore, either the stimulating CTL epitope was not correctly identified by the algorithm or these synthetic peptides were not appropriately processed and presented by the target cells. In contrast, several 9-mers present in the 33-103 and 51-70 sequences (AVSMTSSVL SEQ ID NO: NAVSMTSSV, SEQ ID NO: 22 and VPSSTEKNA, SEQ ID NO: 28) were identified as functional CTL epitopes in the lysis assays (Figure 3b).

In C57BI/6 mice, the CRRKNYGQL, (SEQ ID NO: 32), STEKNAVSM (SEQ ID NO: 34) and AVSMTSSVL (SEQ ID NO: 20) peptides from the 51-70 and 471-493 sequences sensitised RMA cells for lysis however no CTLp were identified by restimulation with the larger peptides. This observation could result from the three 9-mers not being processed and presented by the MUC1+ cells.

Further analysis of the entire MUC1 sequence using the T cell epitope algorithm for mouse Kd, Dd, Ld, Kb, Db, Kk, and human HLA-A1, HLA- A*0201, HLA-A3 and HLA-A24 epitopes show several candidate 9-mers for presentation by mouse or human cells. Of these 9-mer peptides, SAPDNRPAL (SEQ ID NO: 36) and YYQELRDI (Kd) (SEQ ID NO: 35) were synthesised and both were very efficient in sensitising P815 or RMA cells for lysis by mannan-HMFG CTL (Figure 3e). It is apparent in this study and others that WO 01/57068 PCT/AU01/00090 23 the prediction of CTL epitopes is not always accurate. A comparison of the predicted and experimentally determined T cell epitopes for the VNTR region illustrates that the lower scores do not necessarily predict a lack of presentation or antigenicity (Table For example, SAPDTRPAP (SEQ ID NO: 16) peptide has been confirmed to be a Kb-restricted epitope by class I stabilisation when incubated with the TAP defective RMA-S cells as well as by lysis of peptide pulsed RMA cells (Figure 3c), however the predicted score is only 0.004 Similarly, the Kk, Ld and Dd was not predicted accurately The HLA-A*0201 T cell epitope, STAPPAHGV (SEQ ID NO: 37) identified independently by epitope mapping [39] was predicted albeit with a low score. The prediction algorithms act as a guide, to the probability of antigen presentation, but the in vivo response will be defined by antigen processing, immunodominance, T-cell repertoire, glycosylation and other unknown factors [61, 62].

The whole MUC1 protein in purified form has not previously been used to immunise mice to generate cellular immunity, although several other immunisation methods have been used. The whole MUC1 protein has been delivered in a vaccinia construct [46, 63], as a construct in DNA immunisation in transfected dendritic cells [65] and in transfected EBV- B cells [66] In none of these studies was the specificity of the CTL ascertained. However, the importance of using glycosylated MUC1 (as HMFG) should be stressed. Other studies, in mice and humans have used nonglycosylated peptides which have led to antibody production in both MUC1 transgenic mice [67] and in humans [59, 68, 69]; in these studies it was considered that B cell and at times T cell tolerance had been overcome but, with respect to antibodies, the non-glycosylated peptides represent novel antigens and the response is not surprising. However, in the studies described herein, native glycosylated mucin (HMFG) linked to mannan successfully primed CTL in several strains of mice including A2KbMUC1 transgenic mice.

Mannan-HMFG gave a higher CTLp frequency in A2KbMUC1 mice (1/2000) compared to BALB/c or C57B1/6 mice and could be due to either the different strain of mice or to the presence of a higher affinity HLA-A*0201 CTL epitope. In BALB/c mice, HMFG gave a CTLp frequency of 1/80,500. This was comparable to the CTLp frequency in mice immunised with a nonglycosylated form of MUCi VNTR both glycosylated and nonglycosylated forms of the VNTR were equally immunogenic provided they are WO 01/57068 PCT/AU01/00090 24 presented with oxidised mannan. Clearly, the carbohydrate coating did not obscure the underlying peptide. Thus, mannan-HMFG is able to break tolerance in A2KbMUCl transgenic mice by producing CTLs to peptides in the VNTR, the extracellular region and the intracellular region in MUC1.

These results reinforce the concept that MUC1 should be a useful target in therapy.

The use of mannan-HMFG in humans warrants some discussion in that MUC1 is present on some normal cells such as pancreas, kidney. Hence, it is possible that immune responses may be generated to these tissues and give rise to autoimmunity. Thus far in our clinical trials using MUC1 VNTR conjugated to mannan no autoimmunity was detected, however, careful dose escalation studies and monitoring is necessary The HMFG obtained directly from donors is likely to be less preferred for use and recombinant material may be more appropriate. However, using recombinant material, the high level of glycosylation of the HMFG should be kept in mind. Presumably, a eukaryotic vector will be necessary. Thirdly, we have recently shown that the VNTR peptides can deviate the immune response towards antibodies, because of a cross reaction with existing, natural human antibodies Such a deviation may occur when using whole MUC1.

EXAMPLE 2 The non-VNTR peptides were coupled to keyhole limpet hemocyanin (KLH) using gluteraldehyde and then reacted with oxidised mannan as follows: Two mg of the peptide 471 or 507 was dissolved in 1.75 ml phosphate buffer and mixed with 0.25 ml KLH (2mg/ml), treated with 1ml of 0.25% glutaraldehyde and allowed to mix in the dark overnight at room temperature. The mixture was dialysed into phosphate buffer overnight The dialysed mixture was mixed with 1 ml oxidised mannan prepared as described in European Patent Application No. 94303817.4 and allowed to stand overnight.

BALB/c mice (6-8 weeks) were immunised intraperitoneally with micrograms Mannan-peptide KLH on days 0, 10 and 17 and CTL activity in splenocytes determined as described. Non-VNTR peptide conjugated to mannan showed positive responses in the CTL assay (Figures 4 and compared to the positive controls (VNTR peptides conjugated to mannan).

WO 01/57068 PCT/AU01/00090 EXAMPLE 3 The non-leader, non-VNTR peptides and polypeptides may also be used for the preparation of DNA vaccines. This can be performed by using established procedures in DNA cloning and nucleic acid vaccination. For example, the nucleic acid sequence encoding one or more of the non-leader, non-VNTR peptides and polypeptides, with necessary restriction enzyme sites at the 3 and 5' ends can be synthesised in a automated DNA synthesiser and cloned into a suitable vector such as pcDNA3 or pSV3 The clones can be screened for incorporation of the nucleic acid sequences by restriction enzyme digests or protein expression. The DNA can then be injected into various sites in humans and other animals for immunisation.

WO 01/57068 WO 0157068PCT/AU01/00090 26 References: 1. Gendler, Papadimitriou, J. Duhig, Rotlibard, J. Burchell, J., A highy immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats., T. Blot. Chem., 263:12820-12823, 1988.

2. Harisch, F. G. Uhlenbruck, Structures of Neutral 0-Linked polylactosaminoglycans on human skim milk mucins, 1. Biol. Chem., 264: 8 72- 883, 1989.

3. MarJolijn, J. Ligtenberg, M. 1. Vos, H. Anneiniek M. C., Gennissen, A. M. C. Hilkens, J. Episialin, A carcinoma-associated mucin is generated by a polymorphic gene encoding splice variants with alternating amino termini, T. Biol. Chiem., 265:5573-5578, 1990.

4. Crocker, G. Price, M. R, Gen etic polymorphism of high molecular weight glycoproteins: A comparative study in normal individuals and breast cancer pati ents, Br. T. Cancer, 55:651-652, 1987.

Barnd, D. Lan, M. Metzger, R. Finn, 0. Specific, major histocompatibiliiy complex-unrestricted recognition of tumour-associated mucins by human cytotoxic T-cells, Proc. Natl. Acad. Sci. USA., 86:7159-7163, 1989.

6. Jerome, K. Barnd, D. Boyer, C. Taylor-Papadimitriou, J., McKenzie, I. F. Bast, R, and Finn, 0. Adenocarcinoma reactive cytotoxic T-lymphocytes recognize an epitope present on the protein core of epithelial mucin molecules. Cellular immunity and immunotherapy of cancer, 321-328, 1990.

7. Sambrook, et al., Molecular Cl oning: A Lczboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, 1989.

8. Xing, P. Tjandra, J. Stacker, S. A, 'rel, J. Thompson, C. H., McLaughlin, P. and McKenzie, 1. F. Mon oclonal antibodies reactive with mucin expressed in breast cancer, Immunol. Cell Biol., 67:183-185, 1989.

9. Apostolopoulos, Xing, P. Trapani, J. A. and McKenzie, I F. C., Production of anti-breast cancer monoclonal antibodies using a glutathione-Stransferase-MUCJ bacterial fusion protein, Br. 1. Cancer, 67:713-720, 1993.

Siddiqul, Abe, Hayes, Shani, Yunis, E. Kufe, D., Isolation and sequencing of a cDNA coding for the human DF3 breast WO 01/57068 WO 0157068PCT/AU01/00090 27 carcinoma-associated antigen, Proc. Nail. Acad. Sci. USA., 85:2320-2323, 1988.

11. Smith, D. B. Johnson, K. Single-step purficati on of polyp eptides expressed in Escherichia coli as fusions with glutathion e S-transferase, Gee 67:31-40, 1988.

12. Tomonari, K. A rat antibody against a structure functi on ally related to the mouse Tcell eceptoe/T3 complex, Immunogenetics. 28:455458, 1988.

13. Pierres, Naquet, Van Agthoven, Bekklioucha, Denizot, F., Mishal, Schmitt-Verhulst, A-M. and Pierres, A rat anti-mouse T4 monoclonal antibody (Ff129. 19) inhibits the proliferation of Ia-reactive T-cell.

clones and delineates two phenotypically distinct (7'4 Lyt-2, and T4-, Lytsubsets among anti-Ia cytolytic T-cell clones, T. Immunol., 132:2775- 2782, 1984 14. Ledbetter, J. A. and Herzenberg, L. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens, Immunol.. Rev. 47:63- 1979.

Harlow, D. and Lane, A Laboratoiy Manual E. Harlow and D. Lane eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 271, 1988.

16. Apostolopoulos, Xing, P. Trapani, J. A. and McKenzie, 1. F. C., Production of anti-breast cancer monoclonal antibodies using a glutathione-Straizsferase-MUCI bacterial fusion protein, Br. T. Cancer, 67(4):713-720, 1993.

17. Xing, P. Tjandra, J. Stacker, S. Teh, J. Thompson, C. H., McLaughlin, P. and McKenzie, I. F. Monoclonal antibodies reactive with m ucin expressed in breast cancer, hnmunol.Cell Biol.. 6 7:18 3-185, 1989.

18. Devine, P. Clark, B. Birrell, 0. Layton, 0. Ward, B. 0, Alewood, P. F. and McKenzie, L F. The breast tumor-associated epitope defined by monoclonal antibody 3E1. 2 is an 0-linked mucin carbohydrate containing N-glycolylneurainic acid, Cancer Res., 51:5826-5836, 1991.

19. Hareuvem., MX, Gautier, Kieny, M. Wreschner, Cham-bon, P.

and Lathe, Vaccination against tumor cells expressing breast cancer epithelial tumor antigen, Proc. Nail. Acad. Sci. USA, 87:9498-9502, 1990.

Pierres, Naquet, VanAgthoven, Bekkhoucha, Denizot, F., Mishal, Schmitt-Verhulst, A-M. and Pierres, A rat anti-mouse T4 monoclonal antibody (129. 19) inhibits the proliferation of Ia-reactive T-cell clones and delineates two phenoiypicaLly distinct (T4, Lyt-2,3-, and T4-, Lyt- WO 01/57068 WO 0157068PCT/AU01/00090 28 2,3k) subsets among anti-Ia cytolytic T-cell clones, 1. lImmunol.. 132:2 775- 2782, 1984.

21. Miller, R. A. and Stutman, Monoclonal antibody to Lyt 2 antigen blocks H-5 21- and H-2K- specific mouse cytotoxic T-cells, Nature, 296:76-78, 1982.

22. Tomonari, A rat antibody against a structure fimnctionally related to the mouse T-cell receptor/T3 complex, Immunogenetics, 28:455-458, 1988.

23. Auchincloss, Moses, Conti, Sundt, Smith, Sachis, D. H.

and Winn, H.L Rejection of transgenic skin expressing a xeno-classl antigen is CD4-dependent and CD8-independent, Transpl. Proc.. 22(3): 1059-1060, 1990.

24. Fierres, Naquet, Van Agthoven, Bekkhoucha, Denizot, F., Mishal, Schmitt-Verhulst, A-M. and Pierres, A rat anti-mouse T4 monoclonal antibody (H129. 19) inhibits the proliferation of Ia-reactive T-cell clones and delineates two phenotypicaLly distinct Lyt-2, and T4-, Lytsubsets among anti-Ia cytolytic T-cell1 clones, T. Inimunol., 132:2775- 2782, 1984.

Ledbetter, J. A. and Herzenberg, L. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens, Irnmunol. Rev.. 47:63- 1979.

26. Apostolopoulos, Xing, P. X, and McKenzie 1. F. New Trends in the Development of a Breast Cancer Vaccine, Cancer Forum, 17:11-116, 1993.

27. Bobek, Tsai, BesbrockA. Levine, M. Molecular Cloning Sequence and Specificity of Expression of the Gene Encoding the Low Molecular. Weight Human Salivary Mucin (MUC7), T. Biol. Chem., 268:20563- 20569, 1993.

28. Mandelboimo, Berke Fridkin, Feldman, Eisenstein, M., and Eisenbach, CTh Induction by a Tumour-associated Antigen Octapeptide Derived from. a Murine Lung Carcinoma, Nature, 369,67-71, 1994.

29. Goodman GE, Helistrom I, Yelton DE, Murray JL, O'7ara S, Meaker E,.

Zeigler L, Palazollo P, Nicaise C, Usakewicz; J et al. Phase I trial of chimeric (human-mouse) monoclonal antibody L6 in patients width non-small-cell lung, colon, and breast cancer. Cancer Immunol. Immunother. 1993;36:267-2 73.

Apostolopoulos V, McKenzie IFC. Cell ular mucins: targets for immunotherapy. Crit. Rev. Immunol. 1994;14: 293-309.

WO 01/57068 WO 0157068PCT/AU01 /00090 29 31. Finn OJ, Jerome KR, Henderson RA, Pecher G, Domenech N, Magarian- Blander J, B arratt-Boyes SM. MUC-1 epithelial tumor xxucin -based imnmunity and cancer vaccines. ]Inmunol. Rev. 1995;145:61-89.

32. Schrier DM, Stemmer SM, Johnson T, Kasliwal R, Lear J, Mattlies S, Taffs S, Dufton C, Glenn SD, Butchko G et al. High-dose 9OYA~z diethylenetriaminepentaacetic acid (DTPA-BrE-3 and autologous hem atopoi etic stem cell support (AHS CS) for the treatment of advanced breast cancer: a phase I trial. Cancer Res. 1995;55(23 Suppl):5921S-5924S.

33. Richmnan CM, DeNardo SJ, O'Grady LF, DeNardo GL.

Radioimmunotlierapy for breast cancer using escalating fraction ated doses of 13 1 1-labeled chimeric L6 antibody with peripheral blood progenitor cell transfusions. Cancer Immunol. hnmunother. 1993;36: 267-2 73.

34. MacLean GD, Miles DW, Rubens RD, Reddish MA, Longenecker BM.

Enhancing the effect of THERATOPE S~h-KLM cancer vaccine in patients with metastatic breast cancer bypretreatmnent with low-dose intravenous cyclophosphaidde. Iminuuiother. Emphasis Tumor Immunol. 1996;19:309- 316.

Apostolopoulos V, McKenzie IFC, Pietersz GA. Breast cancer immnunotherapy -Current status and Future prospects. Tmmunol. Cell. Biol.

1996;74:457-464.

36. Apostolopoulos V, Pietersz GA, Loveland BE, Sandrin MS, McKenzie IFC. Oxidative/reductive conjugation of mannan to antigen selects for Ti or T2 immune responses. Proc. Nat. Acad. Sci. USA 1995;92:10128-10132.

37. Apostolopoulos V, Loveland BE, Pietersz GA, McKenzie IFC. CTL in mice immunized with human Mucin I are MHC-restricted. T. Immunol.

1995;1155 :5089-5094.

38. Apostolopoulos V, Haurum J, McKenzie IFC. MIUCi peptide epitopes associated with 5 different H2 class I molecules. Eur. 1. Immunol.

1997;27:2579-2587.

39. Apostolopoulos V, Karanicas V, HauruinJS, McKenzie EFC. Induction of IA-A2-restricted CTLs to the Mucin 1 human breast cancer antigen.

McKenzie WFC, Xing P-X. Mucins in Breast Cancer-Recent Advances.

Cancer Cells 1990;2:75-78.

41. Jerome KR, Barnd DL, Boyer CM, Papadiniitriou JT, McKenzie lEC, Bast RC, Finn OJ. Adenocarcinoma reactive cytotoxic Tlymphocytes recognise an WO 01/57068 ~VO 0157068PCT/AUOI/00090 epitope present on the protein core of epithelial mucin molecules. Cellular Immunitv and Imimunotherapy of Cancer 1990;321-3 28.

42. Barnd DL, Lan MS, Metzgar RS, Finn OJ. Specific major histocompatibility complex unrestricted recognition of Tumor-associated mucins by humnan cytotoic T cells. Proc. Nat. Acad. Sci. USA 1989;8 6:7159- 7163.

43. Acres Apostolopoulos Balloul jMk, Wreschner Xing PX., Hadji DA., Spetz JFR, Bisouarne Kieny MP and McKenzie IFC, MWC1 specific cytotoxic T cell precursor analysis in h uman MUCI trcznsgenic mice immunised with human MUCI vaccines. Cancer Immunol. Inununother. (In Press) 44. Vitiello A. Marchesini D, Furze J, Sherman LA, Cliesnut RW. Analysis of the IHZA-restricted influenza-specific cytotoxic Tlymphocyte response in transgenic mice carrying a chimeric human-murine class I major histocompatibility complex. T.Ep.M 1991;173:1007.

Graham RA, Stewart LS, Peat NP, Beverley P, Taylor-Papadlinitriou J.

MUCl-based immunogens for tumor therapy: development of murine model systems. Tumor Targtling 1995;1:211.

46. Acres RB, Hareuveni M, Balloul JM, Kieny M4P. Vaccinia virus MUCI immunization of mice: immune response and protection against the growth of murine tumors bearing the MUCI antigen. T. lInmunother. 1993;14:136-143.

47. Jarasch E, Bruder G, Keenan TW, Franke WVW. Redox constituents in ifk fat globule membranes and rough endoplasmic reticulum from lactating gland. T. Cell Biol. 1077;73:223-241.

48. Apostolopoulos V, Pietersz GA, McKenzie IFC. Cell-mediated immune responses to MUCi fusion protein coupled to mann an. Vaccine 1996;74:930- 938.

49. Parker KC, Bednarek MA, Hull LK, Utz U, Cunningham B, Zweeiink HJ, Biddison WE, Coligan JE. Sequence motifs important for peptide binding to the human MHC class I molecule, IHA-A2. 1. Immunol. 1992;149:35 80-3 58 7.

Parker KC, Bednarek MAN, Coligan JE. Scheme for raning potential I-IA-A2 binding peptides based on independent binding of indivdualpeptide side-chains. 1. Immunol. 1994;152:163-175.

51. Screpanti 1, Felli AV, Toniato, E, Meco D, Mfartinotti S, Frati L, Santoni A, Gulino A. Enhancement of natural-killer-cell susceptibility of human breast- WO 01/57068 WO 0157068PCT/AU01/00090 31 cancer cdls by estradiol and v-Ha-ras on cogene. Int. 1. Cancer 1991;47:445- 449.

52. Chen Q Jackson H, Gibbs Davis ED, Trapani J, Cebon J. Spontaneous T cell responses to melanoma diffrentiation antigens from melanoma patients and healthy subjects. Cancer Inimunol. lImmunother. 1998;47:191-19 7.

53. Taswell, C. Limiting dilution assays for the determination of im:un ocompetent cell frequen cies. I. Data analysis. 1. Immunol.

1981;126:1614-1619.

54. Fazekas de St. Groth S. The evaluation of limiting dilution assays.

T. Immunol. Methods 1982;49:R11.

Lefkovits 1, Waidmaun H. Limiting dilution analysis of the cells of the immune system. L. The clonal basis of the immune response. 1. Immunol.

Today 1984;5:265.

56. Pietersz GA, Wenjun~ L, Popovski V, Apostolopoulos V, and McKenzie IFG. Parameters for using mannan-MUCI fusion protein to induce cellular immunity. Cancer Immunol. Inununother. 1998,45:321.

7. Apostolopoulas V, Xing P-X, Trapani JA, McKenzie IFG. Production of anti-breast cancer monocilon al antibodies using glutathione-S-transferase- MUC1 bacteria) fusion protein. Brit. 1. Cancer 1993;67:713-720.

58. Xing P-X, Tjandra JJ, Stacker SA, Teh JG, Thompson CH, McLaughlin PJ, McKenzie IFC. Mon oclonal antibodies reactive wvith mucin expressed in breast cancer. Immunol. Cell B~iol. 1989;07:183-195.

59. Karanikas VA, Hwang L, Pearson J, Ong CS, Apostolopoulos V, Vaughan HA, Xing P-X, Pietersz GA, Tait BD, Thynne G, McKenzie IFC.

Immune responses of patients with adenocarcinoma vaccinated with maruian- MUCi fuzsion protein. J. Clin. Invest 199 7;100:2 783-2792.

Domenech N, Henderson RA, F inn OJ. Identification of an HIA-Al 1restricted epitope from the tandem repeat domain of the epithelial tumor antigen mucin. 1. Immunol. 1995;155:4766-4774.

61. Lipford GB, Hoffman M, Wagner HL Heeg K. Primaiy in vivo responses to ovolburnin. Probing the predi ctive value of the Kb motif. T. mmno1.

1993;150:1212-1222.

62. Panier EG, Harty JT, Bevan MW. Precise prediction of a dominant class I MUG-restricted epitope of listeria monocytogenes. Nature 1991;35 3:852-855.

63. Akagi J, Hodge JW, McLaughlin JP, Gritz L, Mazzara G, Kufe D, Schiom J, Kantor JA. Therapeutic antitumor response after immunization with an WO 01/57068 WO 0157068PCTIAUOI/00090 32 admixture of recombinant vaccinia viruses expressing a modified MUCi gene and the murine T-cell costimulatoiy molecule B7. 1. Immunother. 1997;20:38- 47.

64. Graham RA, Bm'chell JM, Beverley P, Taylor-Papadim-itriou J.

Intramuscular immunization with MUCi cDNA can protect C57nmice challenged with MUCI -expressing syngeneic mouse Tumor cells. Int. 1. Cancer 1996;65 :664-670.

Gong J, Chen L, Chen D, Kashiwaba M, Manome Y,Tanaka T, Kufe D.

Induction of antigen-specific antitumor immunity writh adenovirus-transduced den dritic, cells. Gene Ther. 1997;4:1023-1028.

66. Pecher G, Finn OJ. Induction of cell ular immunity in chimpanzees to human tumor-associated antigen mucin by vaccination with MUC-1 cDNAfransfected Epstein -Barr virus-immortalized autologous B cells. Proc. Nat.

Acad. Sci. USA 1996;93:1699-1704.

67. Rowse GJ, Tempero RM, VenLith ML, Hollingsworth MA, Gendler SJ.

Tolerance and immunity to M4UCi in a human MUCI transgenic murine model. Cancer Res. 1998;58:315-321.

68. Goydos JS, Elder E, Whiteside TL, Finn OJ, Lotze Mr. A phase I trial of a synthetic mucin peptide vaccine. Induction of specific immune reactivity in patients idth adenocarcinoma. 1. Surg. Res. 1996; 63:298- 304.

69. Reddish M, MacLean GD, Koganty RR, Kan-Mitchell J, Jones V, Mitchell MS, Longenecker BM. Anti -MUCI class I restricted CTLs in metastatic breast cancer patients immunized with a synthetic MUCi peptide. mt. I Cancer 1998;76:817-823.

70. Apostolopoulos V, Lofthouse SA, Popovski V, Chelvanayagam G, Sandrin MfS, McKenzie JFG. Peptide mimics of a tumor antigen inducefunctional cytotoxic Tcells. Nature Biotech. 1998;16:276-280.

71. Brossart, R Heinrich KS, Stuhier G, Behnke L, Reicherdt VC, Stevanovic S, A, Rammensee HG, Kanz L, Brugger W. Identification of HLA-A2 restricted T cell epitopes derived from the MUCI tumor antigen for broadly applicable vaccine therapies. Blood, 1999; 93(12). 4309-4317.

72. Lai, W.C. and Bennett, M. DNA Vaccines. Grit Rev. Immunol. 1998; 18: 449-484.

WO 01/57068 PCT/AU01/00090 33 It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Table 1 Diagramatic Structure of MUCI and Sequences of-the Synthetic Peptides* N-Terminal VNTR' TmeI'rn Cytoplasmic tail 1 1 I HI 33' 423 1 1452 .544

PDTRPAPGSTAPPAHGVTSAPDTIRPAPGSTAP

1 13 .32 .(SEQ ID NO: 38)

MUCI

E~race~ular regiod CpI3-32 P1-30 31.55 51-70 33-103 229-237 344-364 Intraoelular region 408-423 471-493 507-526 Mouse CD4 T4NI (C)PAHGVTrSAPDTRPAPGSTAP. (SEQ ED NO: 39) PDTRPAPGSTAPPAHGVTSAPDTROAPGST (SEQ IID NO: TGSOHASSTPGGEKETSATORSSVP (SEQ DD NO: 2) RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3) Glutathione -S-Vansferase fusion protein

SGHASSTPGGEKETSATORSSVPSSTEKNAVSMTSSVLS

SHSPGSGSSTTQODVTLAPATEPASGSAATW (SEQ ID NO: 4) SAPIDNRPAL (SEQ ID NO: 6) NSSLEDPSTDYYQELORDISE (SEQ IID NO: 7) TQFNQYKTEAASAVNL .(SEQ ED NO: 8) AVGOCRRKNYGQLDIFPARDTYH (SEQ ID NO: 9) (C)YVPPSSTDRSPYEKVSAGNO (SEQ ID NO: 41) KTLVLGKEOESAELPCEGY (SEQ ID NO: 42) Table 2 CTI-p Frequencies In Spleens of Mice Immunised With mannan-HMFG Restimulating Peptide Details CTLp Antigen Frequency Inimunized Strain C57BLB(KbDb) B3ALB/C HLAO0201/Kb A2KbMUCj *(Kd[)dLd) Target Cell MUCI-RMA Target MUfC1- EBV +pep MCF7 P815 Whole MUCI -HMFa 1/13,500 1/9.700 1139,000 1/2,000 Extracellular region Cpl3n32.. ND 1/7,000 ND ND P'1-so 1/12,600 1/13,200 1133,000 1/1,000 31-556 Not Detected 1/1 9,500 1/40,000 1/2,000 51-70 Not Detected 1/10,000 ND ND 33-103 ND 1/20,150 ND ND 3441364* 1/24,500 1/38.800 ND 1/11,000 lntaceflkulr ralan :408-423 Not Detected 1/30,000 ND 1/20,000 471-493 Not Detected 1/12,1500 ND Not detected 0756 Not Detected 1/22,500 ND Not detected INon-MUC1 TN Not Detected Not Detected Not detected Not Detected Table 3 Mfice Immunised with mannan-HFG: CTLp Frequencies to Various non-VNTR Peptides and their Predicted CTL Epitopes Peptide Used for CThp Frequency Predicted 9-mers and Score for the Various 1H2 Haplotypes Stimulation ALB/c C57BI/6 KdCTL CTL L' CTL D' aii CTL 471-493 1/12,500 not NYGQLDLFP 00/ YOQLDIFPA 0% KNYGQLDIF 0% CRRICNYQL 20% CRRKNY()QL AVCQCRRKNYGQLDIFP detected 6 6 10 10 1.4 ARDTYH (SEQ ID NO: 13) (SEQ MDNO- 14) (SEQ ID NO: 13) (SEQID NO: 32) (SEQrD NO: 32) (SEQ ID NO: 507-526 112,500 not CYVPI'SSID VPPWSTRS VPPSSTRS RSPYEKVSA VPPSSTDRS CYVI'PSSTDRSPYEKVSA detected 12 2.4 45 0.99 0.6 ONO (SEQ MD NO: 41) (SEQ ED NO: 46) ID NO- 53) MD NO: 53) __(SEQ ID NO: 62) (SEQ ID NO: 53) 40&-423 1/30,000 not QFNQYKTEA FNQYKTEAA TEAASRVNL TEAASRVNL FNQYICTEAA TGFNQYKTEAASRYNL detected 29 2.2 6.0 0.1 0.72 (SEQ MD NO: 44) (SEQ ED NO* 47) (SEQ ID NO- 54) (SEQ ID NO: 57) (SEQ MD NO: 57) (SEQ MD NO: 54)

QYK-EAASP.

(SEQ MDNO- 48)

TEAASRVNL

*5.8 ED NO:.49) 344-364 1536,800 1M24,500 YYQELQR-DL YYQELQRDI NSSLEDPST SSLEDPSTD PSTYYQEL NSSLEDPSTDYYQELQRD 2880 *1.9 5 4.4 2.9 LSE (SEQ ED NO: 7) (SEQ ID NO- 35) (SEQ IDNO: 35) (qEQ MD NO- 59) (SEQ ID NO: 63) (SEQ MD No- DPSTDYYQE

YYQELQRDI

3.6 1.1 (SEQ MD NO: 59) (SEQ ID NO-

PSTDYYQEL

(SEQED NO: 60)

SLEDPIV

14 ID NO, 50) 33-103 U120,150 not done AV$ISSVL 77% S.VPSSTEKN 0% EPASGSAAT STEKNAVSM 19% AVSMISSV 18% SQHASSTPOOEKETSATQ 58 2S 39 15 1.2 RSSVPSSThIDNAVSMTSS (SEQ MD NO: 20) (SEQ ID NO: 25) (SEQ ED NO: 26) (SEQ ID NO: 34) (SEQ ED NO:

VLSSHSPOSGSSTFQGQD

VTLAPATEPASOSAATW

(SEQ ID NO- ITQGQDVrL SPGSGSSTT AVSMTSMV 18% 39 10.1 ID NO: 21) (SEQ ID NO: 27) fR_(SQ MD NO-. 20) NAVSMTSV SO0A VPSSTEKNA 78%/ 29 36 ID NO: 5 1) (SEQ MD NO:28) TSATQRSSV TPGGEKETS ID NO: 23) (SEQ ID NO: 29) SSTrQGQDV TSATORSSV ID NO: 3 1) (SEQ ID NO: 23)

SSTI'QGQDV

~(SEQ ID NO: 3 1) 1-70 1110,000 not AVSMTSSVL SVPSSIN VPSSTEKNA STEKNAVSM AVSMTSSVL RSSVPSSTEKNAVSMTSS detected 53 2.9 36 15 1.2 VL (SEQ ID NO: 3) (SEQ ID NO: 20) (SEQ IDNO: 25)- (SEQ ID NO: 28) MD NO: 34) __(SEQ ID NO: NAVSMTSV AVS6mTsvL AVSMTSSVL 29 5.0 (SEQ ID NO: 51) (SEQ ID NO: 20) ID29 STEKNVSM SSTEKq4AVS (SEQrD NO: 52) (SEQ M NO-

NAVSMTSV

(SEQ ID NO: 31-55 1119,500 not TSATQRSSV GGEI(ETSAT TPGOEXETS TSATQRSSV KETSATQRS TOSOHASTOEETA detected 10 2.0 30 2.6 0.24 TQRSSVP (SEQ ID NO: 23) (SEQ ID NO: 55) (SEQ ID NO: 29) (SEQ ID NO: 23) (SEQ MD NO: 64) (EMNO2)TOSGHASST

TSATQRSSV

10 I_ (SEQU)NO:56) (SEQ M NO, Table 4 Experimentally Determined and Predicted Mouse and Human C1L Epitopes in the MUC1 VNTR Haplotype Experimentally Predicted Score for T Predicted T cell epitopes Predicted Score from determined T cell cell epitope from algorithm epitope algorithm Kb SAPDTRPAP 0.004 APPAHGVTS 0.330 (SEQID NO:6) (SEQID NO: 66) TAFPAHGVT 0.300 (SEQ ID NO: 31) ND STAPPAHGV 12.000 _SE ID NO:37) kk PDTRPAPGS 0.200 STAPPAHGV SE ID NO:6 (SEQID NO: 37) APDTRPAPG 0.900 APPAHGVTS 45.00 (SEQ ID NO: 17) (SEQ ID NO: 66) APGSTAPPA 30.00 SEQ ID NO:33) D d SAPDTRPAP 0.086 STAPPAHGV 12.00 (SEQ D) NO: 6) (SEQ ID NO: 37) HLA-A*0201 SrAPPAHGV 0.966 STAPPAHGV 0.986 (SEQIDNO:37) (SEQ ID NO:3 0_ WO 01/57068 WO 0157068PCT/AU01/00090 39 Seguence Listig: SEQUENCE LISTING <110> The Austin Research Institute <120> Antigens and their use in immunotherapy <160> 66 <170> Patentln Ver. 2.1 <210> 1 <211> 9 <212> PRT <213> Homo sapiens <400> 1 Leu Leu Leu Leu Thr Val Leu Thr Val <210> 2 <211> <212> PRT <213> Homo sapiens <400> 2 Thr Gly Ser Gly His 1 5 Ser Ala Thr Gin Arg Ala Ser Ser Ser Ser Val Thr Pro Gly Gly Glu Lys Glu Thr 10 <210> 3 <211> <212> PRT <213> Homo sapiens <400> 3 Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Val Ser Met Thr 1 5 10 Ser Ser Val Leu <210> 4 <211> 71 <212> PRT <213> Artificial Sequence WO 01157068 PCT/AUO1/00090 <220> <223> Description of Artificial Sequence: Glutathione-S-transferase fusion protein <400> 4 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 1 5 10 Thr Gin Arg Ser Ser Val Pro Ser Ser Thr Giu Lys Asn Ala Val Ser 25 Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly Ser Gly Ser Ser 40 Thr Thr Gin Gly Gin Asp Val Thr Leu Ala Pro Ala Thr Giu Pro Ala 55 Ser Gly Ser Ala Ala Thr Trp, <210> <211> 473 <212> PRT <213> Homo sapiens- <400> Met Thr Pro Gly Thr Gin Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Giy 25 Gly Glu Lys Glu Thr Ser Ala Thr Gin Arg Ser Ser Val Pro Ser Ser 40 Thr Giu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His 55 Ser Pro Gly Ser Gly Ser Ser Thr Thr Gin Gly Gin Asp Val Thr Leu 70 75 Ala Pro Ala Thr Giu. Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gin 90 Asp Vai Thr Ser Val Thr Arg Pro Ala Leu Gly Ser Thr Thr Pro Pro 100 105 110 Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro Gly Ser 115 120 125 Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro 130 135 140 Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro 145 150 155 160 WO 01/57068 WO 0157068PCT/AUOI/00090 41 Asp Asn Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val 165 170 175 Thr Ser Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His 180 185 190 Asn Gly Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr 195 200 205 Pro Phe Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala 210 215 220 Ser His Set Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Thr Val 225 230 235 240 Pro Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gin Leu Ser Thr 245 250 255 Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gin Phe 260 265 270 Asn Ser Set Leu Glu Asp Pro Sex Thr Asp Tyr Tyr Gin Giu Leu Gin 275 280 285 Arg Asp Ile Ser Giu Met Phe Leu Gin Ile Tyr Lys Gin Gly Gly Phe 290 .295 300 Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Giy Ser Val Val Val Gin 305 310 315 320 Leu Thr Leu Ala Phe Arg Giu Gly Thr Ile Asn Val His Asp Val Giu 325 330 335 Thr Gin Phe Asn Gin Tyr Lys Thr Giu Ala Ala Ser Arg Tyr Asn Leu 340 345 350.

Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala 355 360 365 Gin Ser Gly Ala Giy Vai Pro Gly Trp Giy Ile Ala Leu Leu Vai Leu 370 375 -380 Val Cys Val Leu Vai Ala Leu Ala Ile Vai Tyr Leu Ile Ala Leu Ala 385 390 395 400 Val Cys Gin Cys Arg Arg Lys Asn Tyr Gly Gin Leu Asp Ile Phe Pro 405 410 415 Ala Arg Asp Thr Tyr His Pro Met Ser Giu Tyr Pro Thr Tyr His Thr 420 425 430 His Gly Arg Tyr Vai Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Giu 435 440 445 Lys Vai Ser Ala Gly Ann Gly Gly Ser Ser Leu Ser Tyr Thr Ann Pro 450 455 460 WO 01/57068 WO 0157068PCT/AU01/00090 42 Ala Val Ala Ala Thr Ser Ala Asn Leu 465 470 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Ser Ala Pro Asp Asn Arg Pro Ala Leu 1 <210> 7 <211> 21 <212> PRT <213> Homo sapiens <400> 7 Asn Ser Ser Leu Giu Amp Pro Ser Thr Asp Tyr Tyr Gin Glu Leu Gln Arg Asp Ile Ser Glu <210> 8 <211> 16 <212> PRT <213> Homo sapiens <400> 8 Thr Gin Phe Asn Gin Tyr Lys Thr Glu Ala Ala Ser Arg Val Asn Leu 1 5 10 <210> 9 <211> 23 <212> PRT <213> Homno sapiens <400> 9 Ala Val. Cys Gin Cys 1 5 Arg Arg Lys Asn Tyr Gly Gin Leu Amp Ile Phe 10 Pro Ala Arg Asp Thr Tyr His <210> WO 01/57068 WO 0157068PCT/AU01/00090 43 <211> <212> PRT <213> Homo sapiens <400> Tyr Val Pro Pro Ser Ser Thr Asp AMg Ser Pro Tyr Glu Lys Val Ser 1 5 10 Ala Gly Asn <210> 11 <211> 8 <212> PRT <213> Homo sapiens <400> 11 Set Ile Ile 1 <210> 12 <211> 9 <212> PRT <213> papil <400> 12 Arg Ala His 1 Asn Phe Giu Lys Leu L ama virus -16 Tyr Asn Ile Val Thr Phe <210> 13 <211> 9 <212> PRT <213> Homo sapiens <400> 13 Asn Tyr Gly 1 Gin Leu Asp Ile Phe Pro <210> 14 <211> 9 <212> PRT <213> Homo sapiens <400> 14 Tyr Gly Gin Leu Asp Ile Phe Pro Ala WO 01/57068 WO 0157068PCT/AU01/00090 44 <210> <211> 9 <212> PRT <213> Homo sapiens <400> Lys Asn Tyr Gly Gin Leu Asp Ile Phe 1 <210> 16 <211> 9 <212> PRT <213> Homo sapiens <400> 16 Ser Ala Pro Asp Thr Arg Pro Ala Pro 1 <210> 17 <211> 9 <212> PRT <213> Homo sapiens <400> 17 Ala Pro Asp Thr Arg Pro Ala Pro Gly 1 <210> 18 <211> 9 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: listeriolysin Kd peptide <400> 18 Gly Tyr Lys Asp Gly Asn Giu Tyr Ile 1 <210> 19 <2'1l> <212> PRT <213> Human immunodeficiency virus <400> 19 Arg Lys Ser Ile Arg Ile Gin Arg Gly Pro Gly Arg Ala Phe Val Thr 1. 5 10 WO 01/57068 WO 0157068PCT/AUOI/00090 Ile Gly Lys Gly Lys Gly Lys Gly Tyr <.210> <211> 9 <212> PRT <213> Homo sapiens <400> Ala Val Ser 1 Met Thr Ser Ser Val Leu <210> 21 <211> 9 <212> PRT <213> Homo sapiens <400> 21 Thr Thr Gin 1 Gly Gin Asp Val Thr Leu <210> 22 <211> 9 <212> PRT <213> Homo sapiens <400> 22 Asn Ala Val 1 Ser Met Thr Ser Ser Val.

<210> 23 <211> 9 <212> PRT <213> Homo sapiens <400> 23 Thr Sex Ala 1 Thr Gin Arg Set Ser Val <210> 24 <211> 9 <212> PRT <213> Homo sapiens <400> 24 Ser Set Thr Thr Gin Gly Gin Asp Val 1 WO 01/57068 WO 0157068PCTIAU01/00090 <210> <211> 9 <212> PRT <213> Homo sapiens <400> Ser Val Pro 11 Ser Ser Thr Glu Lys An <210> 26 <211> 9 <212> PRT <213> Homo sapiens <400> 26 Glu Pro Ala 1 Ser Gly Ser Ala Ala Thr <210> 27 <211> 9 <212> PRT <213> Homo sapiens <400> 27 Ser Pro Gly 1 Ser Gly Set Ser Thr Thr <210> 28 <211> 9 <212> PRT <213> Homno sapiens <400> 28 Val Pro Ser 1 Ser Thr Glu Lys Asn Ala <210> 29 <211> 9 <212> PRT <213> Homo sapiens <400> 29 Thr Pro Gly Gly Glu Lys Glu Thr Ser WO 01/57068 WO 0157068PCT/AUOI/00090 <210> <211> 9 <212> PRT <213> Homo sapiens <400> Thr Ser Ala 1 Thr Gin Arg Ser Ser Val <210> 31 <211> 9 <212> PRT <213> Homo sapiens <400> 31 Thr Ala Pro 1 Pro Ala His Gly Val Thr <210> 32 <211> 9 <212> PRT <213> Homo sapiens <400> 32 Cys Arg Arg 1 Lys Asn Tyr Gly Gin Leu <210> 33 <211> 9 <212> PRT <213> Homo sapiens <400> 33 Ala Pro Gly 1 Ser Thr Ala Pro Pro Ala <210> 34 <211> 9 <212> PRT <213> Homo sapiens <400> 34 Ser Thr Glu Lys Asn Ala Val Ser Met <210> WO 01/57068 WO 0157068PCTAU1OOO90 <211> 9 <212> PRT <213> Homao sapiens <400> Tyr Tyr Gin 1 Giu Leu Gin Arg Asp Ile <210> 36 <211> 9 <212> PRT <213> Homo sapiens <400> 36 Ser Ala Pro 1 Asp Asn Arg Pro Ala Leu <210> 37 <211> 9 <212> PRT <213> Homo sapiens <400> 37 Ser Thr Ala Pro Pro Ala His Gly Val <210> 38 <211> 32 <212> PRT <213> Homo sapiens <400> 38 Pro Asp Thr Arg Pro 1 5 Ala Pro Gly Ser Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Axg Pro 25 Ala Pro Gly Ser Thr Ala Pro <210> 39 <211> 21 <212> PRT <213> Homo sapiens <400> 39 Cys Pro Ala His Gly Val Thr Ser Ala Pro Asp Tbr Arg Pro Ala Pro 1 5 10 WO 01/57068 WO 0157068PCT/AU01/00090 49 Gly Ser Thr Ala Pro <210> <211> <212> PRT <213> Homo sapiens <400> Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly 1 5 10 Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr 25 <210> 41 <211> 21 <212> PRT <213> Homo sapiens <400> 41 Cys Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val 1 5 10 Ser Ala Gly Asn Gly <210> 42 <211> 19 <212> PRT <213> Mus musculus <400> 42 Lys Thr Leu Val Leu Gly Lys Glu Gin Glu Ser Ala Glu Leu Pro Cys 1 5 10 Glu Cys Tyr <210> 43 <211> 23 <212> PRT <213> Homo sapiens <400> 43 Ala Val Cys Gin Cys Arg Arg Lys Asn Tyr Gly Gin Leu Asp Ile Phe 1 5 10 WO 01/57068 WO 0157068PCT/AUOI/00090 Pro Ala Arg Asp Thr Tyr His <210> 44 <211> 16 <212> PRT <213> Homo sapiens <400> 44 Thr Gly Phe Asn Gin Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu 1 5 10 <210> <211> 71 <212> PRT <213> Homo sapiens <400> Ser Gly His Ala Ser Ser Thr Pro Gly Gly Giu Lys Glu Thr Ser Ala 1 5 10 Thr Gin Arg Ser Ser Val Pro Ser Ser Thr Giu Lys Asn Ala Val Sex 25 Met Thr Ser Ser Val Leu Sex Ser His Ser Pro Gly Ser Gly Ser Ser 40 Thr Thr Gin Gly Gin Asp Val Thr Leu Ala Pro Ala Thr Giu Pro Ala 55 Ser Gly Ser Ala Ala Thr Trp <210> 46 <211> 9 <212> PRT <213> Homo sapiens <400> 46 Cys Tyr Val Pro Pro Ser Ser Thr Asp 1 <210> 47 <211> 9 <212> PRT <213> Homo sapiens <400> 47 Gin Phe Asn Gin Tyr Lys Thr Giu Ala WO 01/57068 WO 0157068PCT/AUOI/00090 <210> 48 <211> 9 <212> PRT <213> Homo sapiens <400> 48 Gin Tyr Lys Thr Glu Ala Ala Ser Arg 1 <210> 49 <211> 9 <212> PRT <213> Homo sapiens <400> 49 Thr Glu Ala 1 Ala Ser Arg Val Asn Leu <210> <211> 9 <212> PRT <213> Homo sapiens <400> Ser Leu Glu Asp Pro Ser Thr Asp Val 1 <210> 51 <211> 8 <212> PRT <213> Homo sapiens <400> 51 Asn Ala Val 1 Ser Met Thr Sex Val <210> 52 <211> 8 <212> PRT <213> Homo sapiens <400> 52 Ser Thr Glu Lys Asn Val Ser Met 1 WO 01/57068 WO 0157068PCT/AU01/00090 <210> 53 <211> 9 <212> PRT <213> Homo sapiens <400> 53 Val Pro Pro 1 Ser Ser Thr Asp Arg Ser <210> 54 <211> 9 <212> PRT <213> Homo sapiens <400> 54 Phe Asn Gin 1 Tyr Lys Thr Glu Ala Ala <210> <211> 9 <212> PRT <213> Homo sapiens <400> Gly Gly Glu

I

Lys Glu Thr Ser Ala Thr <210> 56 <211> 9 <212> PRT <213> Homo sapiens <400> 56 Thr Gly Ser 1 Gly His Ala Ser Ser Thr <210> 57 <211> 9 <212> PRT <213> Homo sapiens <400> 57 Thr Glu Ala Ala Sex Arg Val Asn Leu 1 WO 01/57068 WO 0157068PCT/AUOI/00090 <210> 58 <211> 9 <212> PRT <213> Homo sapiens <400> 58 Aasn Ser Ser 1 Leu G2lu Asp Pro Ser Thr <210> 59 <211> 9 <212> PRT <213> Homno sapiens <400> 59 Asp Pro Ser 1 Thr Asp Tyr Tyr Gin Glu <210> <211> 9 <212> PRT <213> Homo sapiens <400> Pro Ser Thr 1 Asp Tyr Tyr Gin Glu Leu <210> 61 <211> 9 <212> PRT <213> Homo sapiens <400> 61 Ser Ser Thr 1 Glu Lys Asn Ala Val Ser <210> 62 <211> 9 <212> PRT <213> Homo sapiens <400> 62 Arg Ser Pro Tyr Glu Lys Val Ser Ala <210> 63 <211> 9 WO 01/57068 WO 0157068PCT/AU01/00090 <212> PRT <213> Homo sapiens <400> 63 Ser Ser Leu 1 Glu Asp Pro Ser Thr Asp <210> 64 <211> 9 <212> PRT <213> Homo sapiens <400> 64 Lys Glu Thr 1 Ser Ala Thr Gin Arg Ser <210> <211> 9 <212> PRT <213> Homo sapiens <400> Pro Asp Thr 1 Arg Pro Ala Pro Gly Ser <210> 66 <211> 9 <212> PRT <213> Homo sapiens <400> 66 Ala Pro Pro Ala His Gly Val Thr Ser EDITORIAL NOTE APPLICATION NUMBER 29877/01 The following Sequence Listing pages 1/15 to 15/15 are part of the description. The claims pages follow on pages 55 to 66.

WO 01/57068 1/15 SEQUENCE LISTING <110> The Austin Research Institute <120> Antigens and their use in immunotherapy <160> 66 <170> Patentln Ver. 2.1.

PCT/AU01/00090 <210> 1 <211> 9 <212> PRT <213> Homo sapiens <400> 1 Leu Leu Leu Leu Thr 1 <210> 2 <211> <212> PRT <213> H-omo sapiens <400> 2 Thr Gly Ser Gly His 1 5 Ser Ala Thr Gin Arg Val Leu Thr Val Ala Ser Ser Ser Ser Val Thr Pro Gly Gly Glu Lys GJlu Thr 10 <210> 3 <211> <212> PRT <213> Homo sapiens <400> 3 Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Val Ser Met Thr 1 5 10 Ser Ser Val Leu <210> 4 <211> 71 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Glutathione-S-trans ferase <400> 4 Ser Gly His Ala Ser Ser Thr Pro 1 5 Sequence: fusion protein Gly Gly Glu Lys Glu Thr Ser Ala 10 WO 01/57068 Thr Gin Met Thr Thr Thr Set Gly Arg Ser Set Ser Ser Val Gin Gly Gin Ser Ala Ala PCT/AU01/00090 2/15 Val Pro Ser Ser *Thr Glu Lys Asn Ala Val Set 25 Leu Ser Ser His Sex Pro Gly Set Gly Ser Ser 40 Asp Val Thr Leu Ala Pro Ala Thr Glu Pro Ala 55 Thr Trp <210> <211> 473 <212> PRT <213> Homuo sapiens <400> Met Thr Pro Gly Thr 1 5 Val Leu Thr Val Val Gly Glu Lys Glu Thr Thr Glu Lys Asn Ala Ser Pro Gly Ser Gly Ala Pro Ala Thr Glu Asp Val Thr Ser Val :100 Ala His Asp Val Thr 115 Thr Ala Pro Pro Ala 130 Ala Pro Gly Set Thr 145 Asp Asn Arg Pro Ala 165 Thr Ser Ala Set Gly 180 Phe Phe Gly His Gin Arg Thr Set Thr Gin Gly Ser Ala Leu 105 Asp Asn Thr Set Ala His Thr Ala 170 Gly Set Asn Gly Thr Set Ala Axg AlaThTrThPoAlSe 95200 205 Lys Set Thr WO 01/57068 PCT/AUO1/00090 3/15 Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala 215 Thr Asp Ala 230 Ser Asn His Phe Leu Ser Asp Pro Set 280 Met Phe Leu 295 Ile Lys Phe 310 Arg Glu Gly Tyr Lys Thr Ser Val Ser 360 Vai Pro Gly 375 Ala Leu Ala 390 Arg Lys Asn His Pro Met Pro Pro Ser 440 Asn Gly Gly 455 Ser Ala Asn 470 220 Thr His 235 Ser Pro Ile Set Tyr Tyr Tyr Lys 300 Gly Set 315 Asn Val Ala Set Pro Phe Ile Ala 380 Tyr Leu 395 Gin Leu Ty~r Pro Asp Arg Leu Ser 460 His Gin Asn Gin 285 Gin Val His Arg Pro 365 Leu Ile Asp Thr Ser 445 Tyr Thr Set 255 Gin Leu Giy Val Val 335 Asn Set Val Leu Phe 415 His Tyr Asn <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Set Ala Pro Asp Asn )krg Pro Ala Leu 1 WO 01/57068 WO 0157068PCT/AUOI/00090 4115 <210> 7 <211> 21 <212> PRT <213> Homo sapiens <400> 7 Asn Ser Ser Leu Giu Asp Pro Ser Thr Asp Tyr Tyr Gin Glu Leu Gin 1 5 10 Arg Asp Ile Ser Glu <210> 8 <211> 16 <212> PRT <213> Homo sapiens <400> 8 Thr Gin Phe Asn Gin Tyr Lys Thr Glu Ala Ala Ser Arg Val Asn Leu 1 5 10 <210> 9 <211> 23 <212> PRT <213> Homo sapiens <400> 9 Ala Val Cys Gin Cys Arg Arg Lys Asn Tyr Gly Gin Leu Asp Ile Phe 1 5 10 Pro Ala Arg Asp Thr Tyr His <210> <211> <212> PRT <213> Homo sapiens <400> Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 1 5 *10 Ala Gly Asn Gly <210> 11 <211> 8 <212> PRT <213> Homo sapiens WO 01/57068 WO 0157068PCT/AU1/00090 5/15 <400> 11.

Ser Ile Ile 1 <210> 12 <211> 9 <212> PRT <213> papil] <400> 12 Arg Ala His 1 Asn Phe Giu Lys Leu Lomavirus-16 Tyr Asn Ilie Val Thr Phe <210> 13 <211> 9 <212> PRT <213> Homo sapiens <400> 13 Asn Tyr Gly 1 Gin Leu Asp Ile Phe Pro <210> 14 <211> 9 <212> PRT <213> Homo sapiens <400> 14 Tyr Gly Gin 1 Leu Asp Ile Phe Pro Ala <210> <211> 9 <212> PRT <213> Homo sapiens <400> Lys Asn Tyr 1 Gly Gin Leu Asp Ile Phe <210> 16 <211> 9 <212> PRT <213> Homo sapiens <400> 16 Ser Ala Pro Asp Thr Arg Pro Ala Pro 1 WO 01/57068 WO 0157068PCT/AU01/00090 6/15 <210> 17 <211> 9 <212> PRT <213> Homo sapiens <400> 17 Ala Pro Asp Thr Arg Pro Ala Pro Gly 1 <210> 18 <211> 9 <212> PRT <213> Unknown organism <220> <223> Description of Unknown organism: listeriolysin Kd peptide <400> 18 Gly Tyr Lys 1 <210> :19 <211> <212> PRT <213> Human immunodefi ciency virus <400> 19 Arg Lys Ser Ile Arg 1 5 Ile Gin Arg Gly Lys Gly Gly Pro Gly Arg Ala Phe Val Thr 10 Ile Gly Lys Gly Lys <210> <211> 9 <212> PRT <213> Homo sapiens <400> Ala Val Ser 1 Met Thr Ser Ser Val Leu <210> 21 <211> 9 <212> PRT <213> Homo sapiens <400> 21 Thr Thr Gin Giy Gin Asp Val Thr Leu 1 PCTI/AU01I00O 9 0 WO 0170687/15 <210> 22 <211> 9 <212> PP- spin <213> H01m saien <400> 22 VlSt.mtrr SSet 'Val Asfl Ala 5 e le i e <210> 23 <211> 9 <213> flolU sapiens <400> 23r~ <4ho Ser la ThEr Gin A-g Set Set Val <22.0> 24 <211> 9 <212> PwR <213> ,OoO sapiens <400> 24 rGi Sl GnAPVa set set Thr ThE G5 l i a <210' <211> 9 <212> PPRT ain <213> Hlo sain <400> 25 rSe h l LyAs Se Val Po Se 5h 1~ y

S

<210> 26 <211>-9 <212> Pp.T sain <213> IiOrUOsain <400> 26 e Sl SeAl AaTh Glu Pro Ala Se 5l e l l h <21o> 27 <211> 9 <212>

PRT

<213> 1{oWmo sapiens 1pCT!AU0I0( 9 0 WO 011s706 8 8/15 <400> 2'1 Spro Gly Ser GlY Ser Ser Thr Thr 1 <210> 29 <211> 9 <212> PRT sain <213> Hli~OO pin <400> 28 SrSrT. l y -nIl Val- Pro 5 e h 3ULsAf l 1 <210> 29 <211> 9 <212> PRT sain <213> H-oOO pin <400> 29 Thr Pro Gly Gly G1u Lys G1u Thr Ser <210> <211> 9 <212>

PRT

<213> Hlomo sapi-ens <400> 30 ThE Ser Ala ThE, G-l P~r SeE SeE Val- 1 <210> 31 <211> 9 <212> PRT sain <213> HofOO pin <400> 31 PrAlHi lVaTh Thr Ala Pro 5 l i l a h <210> 32 <211> 9 <2'12> PRT ain <213> HOMO sa5 n <400> 32 Ly sTy l nLe Cys Axg Arg 5 y G~ i e pcTIAu()l/00 90 WO 01157068 <210> 33 <211> 9 <212> PPT sain <213> HlifOMO pif <-400> 33 erTh Ala PrO pro Ala Ala Pr Gly SrT 1 <210> 34 <211> 9 <212> PP~T sain <213> HlttOO pin 9/15 <,400> 34 Ser hr Gu Ly P-5 SeE TE Gi i~y A~5 Val Ser 14et <210> <211> 9 <212> PRT sain <213> HoOO p~n <400> 35 Tyr Tyr Gin 1 G1, 1.eu Gin PArg Asp Ile <210> 36 <211> 9 <212>

PFIT

<213> Homfo sapiens <400> 36 Ser Ala Pro

I

.Asp A.f A~ ro Ala LSu <,210> 31 <211> 9 <212> PP- spin 4213> HoOO pin <400> 31 Ser Thr Ala PrO pro Ala His GlY Val 5 21>38 21>32 4212> P PR s pi n <213> HOoO ain <400> 38 PCTIAUO1/000 9

O

WO 01/571068 Pro Asp Thr zArg 1 Val Thr Ser Ala 1011 proj~aHis Ala Y pro Ala Pro GlY Ser Thr Aa Pro 15 laHs

I

Jr AS Thr Arg Pr Ala Pro GlY Ser Thr Ala Pro <210> 39 <211> 21 <212> PRT apen <213> H011'0apen <400> 39 S l CSPro .a sGl a 3 'rser Ala Pro Asp Thr Xr Pr Ala pro 15 GySer Thr Ala Pro Gly 20 <210> <211> <212> 'PT sain <213> HoOO p3e~ <400> 40 TrAgpro Ala Pro ASP

X

Pro Gly Ser ThE Axg 25o rhr Ala Pro pr 10 AaHis GlY 15 Ala Pro GlY Ser Thr Val Thr Ser Ala Pro ASP <210> 41 <211> 21 <212> PRT spin <2'13> HOMDO sapiensuLysVa <400> 41 poPro S~e erThr AS 10 e PO~ G1 y cys Tyr Val Pral Ser A-la Gly Asn GIlY <210> 42 <211> 19 <212> pHkT msu <213> lAusAl l e PrCy <400> 2 G11 G3.f Gl-U Ser Al15ULe r y <400> 4210 L ThE LeU Val Leu Gly Lys

G

1 WO 01/57068 C/01(09 11/15 Giu Cys Tyr <210> 43 <211> 23 <212> PRT <213> Homo sapiens <400> 43 sTy lGn eAsIePh Ala Val Cys Gin Cys Arg Arg LysAnTy GyGiLeAs liPe 1 5 10 is pro Ala Arg Asp Thr Tyr His <210> 44 <211> 16 <212> PRT <213> Homo sapiens <400> 44 Thr Gly Phe Asn Gin Tyr Lys Thr Glu Ala Ala Scr Arg Tyr Asn Lets 1 5 .10 <210> <211> 71 <212> PRT <213> Homo sapiens <400> 45 Ser Gly His Ala Ser Ser Thr Pro Gly Gly GlU Lys GlU Thr Ser Ala 1 5 10 Thr Gin Arg Ser Ser Val Pro Ser Ser Thr Giu Lys Asn Ala Val Scr 25 Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly Ser Giy Ser 40 Thr Thr Gin Gly Gin Asp Val Thr Leu Ala Pro Ala Thr Giu Pro Ala 55 Ser Gly Scr Ala Ala Thr Trp <210> 46 <211> 9 <212> PRT <213> Homo sapiens <400> 46 Cys Tyr Val Pro Pro 5cr Ser Thr Asp vCTIJAU01lI00 9 0 WO 01157068 1.1 <210> 47 <211> 9 <212> IoITl sapiens <4003> Hom 4 As n Gln Tyr LaYS ThE Glu A~la 1 <210> 48 <211> 9 <212> PPRT sain <213> HliOO pin <400> 48 G1U Ty Lays Thr G1U- Ala Ala Ser Ax 1 5 <210> 49 <211> 9 <212> pRT Jn <213> H~OMO s apin <400> 49 e kgVa s e ThEr G3AI Ala Ala1 5~ r a.Af <210> <211> 9 <212> PRT sain <213> HoOO pin <-400> 50 As r e h ApVa Ser Leu GlU A 5 e:TEApV] <210> 51 <211' 8 <212> PRT sain <213> HioOO pif <400> 51 1 eMe h eVa Asfl Ala Va 1 5.:$tTr e:Va 1 <210> 52 <211>'B8 pCTIA'UO0I0 90 WO 01/57068 13115 <212> PRT sain 4213> H~oOO pIe 4400> 52 LsAS a e e Set Thr G3lU 5y ~lv3 e e 1 <210> 53 <211> 9 <212> PRT sain <213> HoOO pin <400> 53 VtPS r e Val Pro Pr Set S~ 5h .P E e <210> 54 <211> 9 <212> pRT sain <-213> 11010sapen <400> 54 rLy h l apa Pile Asfl Gln Ty~ 5y h 3~ l <210> <211> 9 <212> PIT sain <213> HOMO spin <400> 5 ys G3.U Tilt Set Ala Tilt Gly Gly G-u 1 .<210> 56 -<2311> 9 <212> PRT sain .<213> HOMO spin <400> 56 lHiAl e eTh Tilt Gly Se G 5 i l StsrTl <210> 5'1 <211> 9 <212> PRT sain <213> HOMO spin <400> 57 rAgVa s e Til rG.u Akla Ala eAtVa SLe 15 PCT/AUOI/000 9 0 WO 01/57068 <210> 58 <211> 9 <212>

PRT

<213> HOMuO sapiens <400> 58pr eTh Asn Ser ser Leu Glu ASpPr SeTh 14/15 <210> 59 <211> 9 <212> PRT <213> HOMfO sapiens <400> 59 Asp pro Set

I

Thr Asp Tyr Tyr Gin G1u 5 <210> <211> 9 <212>

PRT

<213> Homo sapiens <400> 60 y y l l e pro Ser Thr Asp Tr 5y i 1 e <210> 61 <211> 9 <212>

PRT

<213> Homo sapiens <400> 61 Ser Ser Thr G1U Lys A~fl Ala Val ser 1 <210> 62 <211> 9 <212> PgT <213> Homo sapiens <400> 62 Axq Ser Pro 1 <210> 63 <211> 9 <212>

PRT

Tyr Giu Lys Val Ser Ala W O 011 068 C 'TIA U OlI J0O 9

O

4213> 110mb sapienls <400> 63 be A SP pr Ser Thr

MSP

Ser Ser 1 <210> 64 <211> 9 <212> PpT sapiens <213> II0Mb <400> 64 5er la T1h Gfl PAE ser 1 <210> <211> 9 <212> PW] en <213> 11,.o~ sapin <400> 65 Pro A3la pr GlY Ser Pro ALsp TrAr 1 <210> 66 <211> 9 <212> PP-T sain <-213> 1-Ombsano n <400> 66 pLia flis G]-Y Val Thr Ser Ala Pro

Claims (71)

1. A method for inducing a cell mediated immune response against mucin which comprises administering to a subject an effective amount of: a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin, said sequence comprising an epitope of said mucin non-VNTR, non-leader region, (ii) a fusion protein comprising a peptide or polypeptide according to and a suitable carrier protein, or (iii) a conjugate compound comprising a peptide or polypeptide according to and a carbohydrate polymer, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

2. A method according to claim 1, wherein said epitope is from an extracellular region of the non-VNTR, non-leader region of a mucin.

3. A method according to claim 1, wherein said epitope is from an intracellular region of the non-VNTR, non-leader region of a mucin.

4. A method according to claim 1, wherein said epitope is from a transmembrane region of the non-VNTR, non-leader region of a mucin.

5. A method according to any one of the preceding claims, wherein said mucin is mucin 1 (MUC1).

6. A method according to claim 5, wherein said mucin 1 is human mucin 1.

7. A method according to claim 6, wherein said human mucin 1 is human milk fat globule membrane antigen (HMFG). AMENDED SH- IPEAIAU 141328216 Received 8 May 2002 56

8. A method according to claim 2, wherein said epitope has an amino acid sequence selected from: AVSMTSSVL (SEQ ID NO: 20), NAVSMTSSV (SEQ ID NO: 22), VPSSTEKNA (SEQ ID NO: 28) and SAPDNRPAL (SEQ ID NO: 36).

9. A method according to claim 4, wherein said epitope has the amino acid sequence: YYQELQRDI (SEQ ID NO: A method according to claim 1, wherein said peptide or polypeptide comprises an amino acid sequence substantially corresponding to one of the following amino acid sequences or an immunogenic fragment thereof: TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO: RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3), SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLA PATEPASGSAATW (SEQ ID NO: SAPDNRPAL (SEQ ID NO: 6), NSSLEDPSTDYYQELQRDISE (SEQ ID NO: TQFNQYKTEAASRVNL (SEQ ID NO: AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10) and CYVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 41).

11. A method according to claim 1, wherein said peptide or polypeptide consists of an amino acid sequence substantially corresponding to one of the following amino acid sequences or an immunogenic fragment thereof: TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO: RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3), SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLA PATEPASGSAATW (SEQ ID NO: SAPDNRPAL (SEQ ID NO: 6), NSSLEDPSTDYYQELQRDISE (SEQ ID NO: TQFNQYKTEAASRVNL (SEQ ID NO: AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10) and CYVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 41).

12. A method according to claim 1, wherein said fusion protein is conjugated to a carbohydrate polymer.

13. A method according to claim 12, wherein the carbohydrate polymer is a polymer of a carbohydrate monomer unit selected from the group consisting of AMENDED rH-f IPEA/AU PCT/AU01/00090 141328216 Received 8 May 2002 57 glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-0-methyll-D-galactose, 2-0-acetyl-o-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, a-D-galactopyranose 6- sulphate, fructose, a abequose and conformation and configuration isomers thereof, or is a polymer of a carbohydrate formed of two or more different types of said carbohydrate monomer units.

14. A method according to claim 13, wherein said carbohydrate polymer comprises at least 20 monomer units. A method according to claim 14, wherein said carbohydrate polymer comprises more than 1000 monomer units.

16. A method according to claim 15, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

17. A method according to any one of claims 12-16, wherein the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer comprising mannose.

18. A method according to any one of claims 12-16, wherein the carbohydrate polymer is a polymer of oxidised mannose or is oxidised mannan.

19. A method according to claim 1, wherein said carbohydrate polymer of said conjugate compound is a polymer of a carbohydrate monomer unit selected from the group consisting of glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-0-methyll-D-galactose, xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, a-D-galactopyranose 6-sulphate, fructose, a abequose and conformation and configuration isomers thereof, or is a polymer of a carbohydrate formed of two or more different types of said carbohydrate monomer units. A method according to claim 19, wherein said carbohydrate polymer comprises at least 20 monomer units. AMENDED SH2CET IPEA/AU PCT/AU01/00090 141328216 Received 8 May 2002 58

21. A method according to claim 20, wherein said carbohydrate polymer comprises more than 1000 monomer units.

22. A method according to claim 21, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

23. A method according to any one of claims 19-22, wherein the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer comprising mannose.

24. A method according to any one of claims 19-22, wherein the carbohydrate polymer is a polymer of oxidised mannose or is oxidised mannan. A method of preventing or treating a carcinoma in a subject, said method comprising administering to said subject a vaccine or therapeutic agent comprising an effective amount of; a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin, said sequence comprising an epitope of said mucin non-VNTR, non-leader region, (ii) a fusion protein comprising a peptide or polypeptide according to and a suitable carrier protein, or (iii) a conjugate compound comprising a peptide or,polypeptide according to and a carbohydrate polymer, and an adjuvant and/or a pharmaceutically acceptable carrier.

26. A method according to claim 25, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin.

27. A method according to claim 25, wherein said epitope is from an extracellular region of the non-VNTR, non-leader region of a mucin.

28. A method according to claim 25, wherein said epitope is from an intracellular region of the non-VNTR, non-leader region of a mucin. AMENDED SHEr- IPEA/AU PCT/AU01/00090 141328216 Received 8 May 2002 59

29. A method according to claim 25, wherein said epitope is from a transmembrane region of the non-VNTR, non-leader region of a mucin. A method according to any one of claims 25-29, wherein said mucin is mucin 1 (MUC1).

31. A method according to claim 30, wherein said mucin 1 is human mucin 1.

32. A method according to claim 31, wherein said human mucin 1 is human milk fat globule membrane antigen (HMFG).

33. A method according to claim 27, wherein said epitope has an amino acid sequence selected from: AVSMTSSVL (SEQ ID NO: 20), NAVSMTSSV (SEQ ID NO: 22), VPSSTEKNA (SEQ ID NO: 28) and SAPDNRPAL (SEQ ID NO: 36).

34. A method according to claim 28, wherein said epitope has the amino acid sequence: YYQELQRDI (SEQ ID NO: A method according to claim 25, wherein said peptide or polypeptide comprises an amino acid sequence substantially corresponding to one of the following amino acid sequences or an immunogenic fragment thereof: TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO: RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3), SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLA PATEPASGSAATW (SEQ ID NO: SAPDNRPAL (SEQ ID NO: 6), NSSLEDPSTDYYQELQRDISE (SEQ ID NO: TQFNQYKTEAASRVNL (SEQ ID NO: AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10) and CYVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 41).

36. A method according to claim 26, wherein said peptide or polypeptide consists of an amino acid sequence substantially corresponding to one of the following amino acid sequences or an immunogenic fragment thereof: TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO: RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3), AMENDED SHEET (PEA/AU PCT/AU01/00090 141328216 Received 8 May 2002 SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTrQGQDVTLA PATEPASGSAATW (SEQ ID NO: SAPDNRPAL (SEQ ID NO: 6), NSSLEDPSTDYYQELQRDISE (SEQ ID NO: TQFNQYKTEAASRVNL (SEQ ID NO: AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10) and CYVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 41).

37. A method according to claim 25, wherein said fusion protein is conjugated to a carbohydrate polymer.

38. A method according to claim 37, wherein the carbohydrate polymer is a polymer of a carbohydrate monomer unit selected from the group consisting of glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-0-methyll-D-galactose, 2-0-acetyl-j-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, a-D-galactopyranose 6- sulphate, fructose, a abequose and conformation and configuration isomers thereof, or is a polymer of a carbohydrate formed of two or more different types of said carbohydrate monomer units.

39. A method according to claim 38, wherein said carbohydrate polymer comprises at least 20 monomer units. A method according to claim 39, wherein said carbohydrate polymer comprises more than 1000 monomer units.

41. A method according to claim 40, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

42. A method according to any one of claims 38-41, wherein the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer comprising mannose.

43. A method according to any one of claims 38-41, wherein the carbohydrate polymer is a polymer of oxidised mannose or is oxidised mannan. AMENDED SHEET (PEA/AU PCT/AU01/00090 141328216 141328216 Received 8 May 2002 61

44. A method according to claim 25, wherein said carbohydrate polymer of said conjugate compound is a polymer of a carbohydrate monomer unit selected from the group consisting of glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-0-methyll-D-galactose, xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, a-D-galactopyranose 6-sulphate, fructose, a abequose and conformation and configuration isomers thereof, or is a polymer of a carbohydrate formed of two or more different types of said carbohydrate monomer units.

45. A method according to claim 44, wherein said carbohydrate polymer comprises at least 20 monomer units.

46. A method according to claim 45, wherein said carbohydrate polymer comprises more than 1000 monomer units.

47. A method according to claim 46, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

48. A method according to any one of claims 44-47, wherein the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer comprising mannose.

49. A method according to any one of claims 44-47, wherein the carbohydrate polymer is a polymer of oxidised mannose or is oxidised mannan.

50. A method for inducing a cell mediated immune response against mucin which comprises administering to a subject an effective amount of; a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin, said sequence comprising an epitope of said mucin non-VNTR, non-leader region, wherein said epitope has an amino acid sequence selected from: AVSMTSSVL (SEQ ID NO: 20), NAVSMTSSV (SEQ ID NO: 22), VPSSTEKNA (SEQ ID NO: 28), SAPDNRPAL (SEQ ID NO: 36) and YYQELQRDI (SEQ ID AMENDeD H 'PE4/AU 141328216 PCT/AU01/00090 Received 8 May 2002 62 (ii) a fusion protein comprising a peptide or polypeptide according to and a suitable carrier protein, or (iii) a conjugate compound comprising a peptide or polypeptide according to and a carbohydrate polymer, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

51. A method for inducing a cell mediated immune response against mucin which comprises administering to a subject an effective amount of; a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin, said sequence comprising an epitope of said mucin non-VNTR, non-leader region, wherein said peptide or polypeptide comprises an amino acid sequence substantially corresponding to one of the following amino acid sequences or an immunogenic fragment thereof: TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO: RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3), SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLA PATEPASGSAATW (SEQ ID NO: SAPDNRPAL (SEQ ID NO: 6), NSSLEDPSTDYYQELQRDISE (SEQ ID NO: TQFNQYKTEAASRVNL (SEQ ID NO: AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10) and CYVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 41), (ii) a fusion protein comprising a peptide or polypeptide according to and a suitable carrier protein, or (iii) a conjugate compound comprising a peptide or polypeptide according to and a carbohydrate polymer, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

52. An isolated peptide or polypeptide as hereinbefore defined, said peptide or polypeptide being capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non- leader region of a mucin, said sequence comprising an epitope of said mucin non- VNTR, non-leader region, wherein said epitope has an amino acid sequence selected AMENDED SHEET IPEAIAU 63 from: AVSMTSSVL (SEQ ID NO: 20), NAVSMTSSV (SEQ ID NO: 22), VPSSTEKNA (SEQ ID NO: 28), YYQELQRDI (SEQ ID NO: 35) and SAPDNRPAL (SEQ ID NO: 36).

53. An isolated peptide or polypeptide as hereinbefore defined, said peptide or polypeptide being capable of eliciting an immune response, wherein said peptide or polypeptide consists of an amino acid sequence substantially corresponding to one of the following amino acid sequences or an immunogenic fragment thereof: SAPDNRPAL (SEQ ID NO: AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: 9), YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10) and CYVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 41).

54. An isolated peptide or polypeptide as hereinbefore defined, said peptide or polypeptide being capable of eliciting an immune response, wherein said peptide or polypeptide consists of the following amino acid sequence: SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASG SAATW (SEQ ID NO: 4). A fusion protein comprising a peptide or polypeptide according to any one of claims 52 to 54 with a suitable carrier protein.

56. A fusion protein according to claim 55, wherein said fusion protein is conjugated to a S carbohydrate polymer.

57. A fusion protein according to claim 56, wherein the carbohydrate polymer is a polymer of .25 a carbohydrate monomer unit selected from the group consisting of glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-0-methyll-D-galactose, acetyl-3-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, a-D-galactopyranose 6-sulphate, fructose, a abequose and conformation and configuration isomers thereof, or is a polymer of a carbohydrate formed of two or more different types of said carbohydrate monomer units.

58. A fusion protein according to claim 57, wherein said carbohydrate polymer comprises at least 20 monomer units.

59. A fusion protein according to claim 57, wherein said carbohydrate polymer comprises more than 1000 monomer units. 141726212 64 A fusion protein according to claim 57, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

61. A fusion protein according to any one of claims 56 to 60, wherein the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer comprising mannose.

62. A fusion protein according to any one of claims 56 to 60, wherein the carbohydrate polymer is a polymer of oxidised mannose or is oxidised mannan.

63. A peptide or polypeptide according to any one of claims 52 to 54 coupled to a suitable carrier protein.

64. A peptide or polypeptide according to claim 63, wherein said peptide or polypeptide and/or said carrier protein is conjugated to a carbohydrate polymer. A peptide or polypeptide according to claim 64, wherein the carbohydrate polymer is a polymer of a carbohydrate monomer unit selected from the group consisting of glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, galactose, 2-0-acetyl-p-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, 20 methyl galacturonate, a-D-galactopyranose 6-sulphate, fructose, a abequose and conformation *o and configuration isomers thereof, or is a polymer of a carbohydrate formed of two or more different types of said carbohydrate monomer units.

66. A peptide or polypeptide according to claim 65, wherein said carbohydrate polymer comprises at least 20 monomer units.

67. A peptide or polypeptide according to claim 65, wherein said carbohydrate polymer comprises more than 1000 monomer units.

68. A peptide or polypeptide according to claim 65, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

69. A peptide or polypeptide according to any one of claims 64 to 68, wherein the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer comprising mannose. 141726212 A peptide or polypeptide according to any one of claims 64 to 68, wherein the carbohydrate polymer is a polymer of oxidised mannose or is oxidised mannan.

71. A compound comprising a conjugate of a peptide or polypeptide according to any one of claims 52 to 54 and a carbohydrate polymer.

72. A compound according to claim 71, wherein the carbohydrate polymer is a polymer of a carbohydrate monomer unit selected from the group consisting of glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-0-methyll-D-galactose, acetyl-p-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, a-D-galactopyranose 6-sulphate, fructose, a abequose and conformation and configuration isomers thereof, or is a polymer of a carbohydrate formed of two or more different types of said carbohydrate monomer units.

73. A compound according to claim 72, wherein said carbohydrate polymer comprises at least monomer units.

74. A compound according to claim 72, wherein said carbohydrate polymer comprises more than 1000 monomer units.

75. A compound according to claim 72, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

76. A compound according to any one of claims 71 to 75, wherein the carbohydrate polymer is *5 a polymer of mannose or is a carbohydrate polymer comprising mannose.

77. A compound according to any one of claims 71 to 75, wherein the carbohydrate polymer is o a polymer of oxidised mannose or is oxidised mannan. 0) 78. A vaccine or therapeutic agent comprising a peptide or polypeptide according to any one of claims 52 to 54 and 63 to 70 or a fusion protein according to any one of claims 55 to 62 and, optionally, an adjuvant and/or a pharmaceutically acceptable carrier.

79. A vaccine or therapeutic agent comprising a conjugate compound according to any one of claims 71 to 77 and, optionally, an adjuvant and/or a pharmaceutically acceptable carrier. 141726212 A method according to any one of claims 25 to 49, wherein said carcinoma is an adenocarcinoma.

81. A method according to claim 80, wherein said adenocarcinoma is breast cancer.

82. The use of a peptide or polypeptide according to any one of claims 52 to 54 and 63-70, a fusion protein according to any one of claims 55 to 62, or a conjugate compound according to any one of claims 71 to 77, to pulse dendritic cells for in vivo transfer and use as a vaccine.

83. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the peptide or polypeptide of any one of claims 52 to 54 or a fusion protein according to any one of claims to 62.

84. A DNA vaccine comprising a nucleic acid molecule according to claim 83. Dated: 6 January 2005 Patent Attorneys for the Applicant SBLAKE DAWSON WALDRON PATENT SERVICES o• o* oo 141726212