WO2001057068A1 - Mucin-1 derived antigens and their use in immunotherapy - Google Patents

Abstract

Peptides and polypeptides capable of eliciting the immune response are disclosed which comprise an amino acid sequence substantially corresponding to that of an epitope of the non-VNTR, non-leader region of mucin. The peptides or polypeptides are useful themselves, or in the form of fusion proteins or conjugate compounds with carbohydrate polymers, in the prevention or treatment of disease states, particularly carcinomas such as adenocarcinomas. DNA vaccines and uses of the peptides and polypeptides for pulsing dentritic cells for in vivo transfer are also disclosed.

Description

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 modern 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 lOOKd) which are produced by many epithelial cells and tumours (1). 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 (2). There are twelve forms of known human mucin designated MUCl to MUC12 (see, for example, 3, 4, 26, 27). MUCl 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 MUCl 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 with severe side-effects. There is, accordingly, an urgent need for therapeutic compounds and methods for the prevention/treatment of tumours.

Prior art 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 MUCl to elicit cytotoxic T cells in vitro

(71). 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 MUCl and such lack of dominance may result in cytotoxic T lymphocytes (CTLs) to other epitopes, not LLLLTVLTV (SEQ ID NO:l).

In work leading to the present invention, the inventors surprisingly found that when a mannan-conjugate of HMFG (whole MUCl) was used for immunisation, non-VNTR, non-leader regions of MUCl could be selectively antigenic. This is the first time that cytotoxic T cells to non-VNTR regions of

MUCl have been demonstrated in mice immunised with whole MUCl. 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:

Accordingly, in the first aspect, 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 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

MUCl). Examples of specific tumour antigens are carcinoembryonic antigen (CEA) from colon and other cancers or, indeed, antigens extracted from any tumour expressing MUCl;

Preferably, the immune response elicited by the peptide or polypeptide is a cell mediated immune response, particularly one involving the activation of cytotoxic T cells against cells expressing aberrantly glycosylated mucin (e.g. 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 naturally- occurring 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, Nα- alkylamino 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. (7). 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. (7). Fusion proteins may involve fusion of the peptide or polypeptide to a carrier protein selected from glutathione-S-transferase, β-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.g. 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 (e.g. keyhole limpet hemocyanin) using any of the well established procedures in the art (e.g. 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 MUCl (e.g. amino acids 22 to 131, or amino acids 402 to 473 of human MUCl according to NCBI database Accession No. M61170 (see also figure 1); although an amino acid sequence from the transmembrane region of the non-leader, non-VNTR region of human MUCl 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)

SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQG QDVTLAPATEPASGS AATW (SEQ ID NO : 4)

S APDNRPAL (SEQ ID NO : 6) NSSLEDPSTDYYQELQRDISE (SEQ ID NO: 7)

TQFNQYKTEAASRVNL (SEQ ID NO : 8)

AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO : 9)

YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10)

In a second aspect, the present invention provides a compound comprising a conjugate of the peptide or polypeptide of the first aspect 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-β-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, α-D-galactopyranose 6-sulphate, fructose and α 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 first aspect 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 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 ε 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 l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide 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- N-maleimido-1, 8-octane. Derivatizing agents such as a methyl-3-[(p-azido- phenyl)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.

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 a third 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 first aspect of the invention, or a fusion protein comprising the peptide or polypeptide of the first aspect of the invention, and, optionally, an adjuvant and/or a pharmaceutically acceptable carrier.

In a fourth 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 second aspect of the invention and, optionally, an adjuvant and/or a pharmaceutically acceptable carrier.

Suitable adjuvants for use in the vaccine of the third or fourth aspect 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 third or fourth aspect 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 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 third or fourth aspect 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 lμg to 10,000μg 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 50μg to 5,000μg, still more preferably lOOμg to l,000μg, and even more preferably lOOμg to 500μg. 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 (e.g. one or more of GM-CSF, G-CSF, M-CSF, TNFα or β, interferon α or γ, any of ILl 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.

In a fifth 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 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, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

In a sixth 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 second aspect, 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 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 non- toxic 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. (7).

In yet another aspect, the present invention provides a compound comprising a conjugate between MUCl 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

MUCl is human MUCl (e.g. HMFG) and the carbohydrate polymer is a polymer of mannose, particularly oxidised mannose, or is oxidised mannan. The carbohydrate polymer may be conjugated to the MUCl at an amount within the range, for example, of about 1-10 mg per mg of MUCl, preferably about 5-8 mg per mg of MUCl, more preferably about 7 mg per mg of MUCl. 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 MUCl protein (NCBI database Accession No. M61170). Figure 2: Assay for HMFG and mannan. (a) Inhibition of binding of anti-MUCl antibody to HMFG by competitor preparations of HMFG (m) and mannan-HMFG (1). (b) Binding of mannan-HMFG (1) and HMFG (m) to anti- MUCl antibody and Con A detected by a radioimmunoassay.

Figure 3: A2K^DMUCl double transgenic mice were immunised with mannan-HMFG and splenocytes were used in CTL assays. Cytotoxic activity of the effector cells were measured on ⁵¹Cr-labelled MCF7 with (n) or without cold K562 (1); BT20 (p) or ME272 (m).

Figure 4: C5 BL/6 and BALB/c mice were immunised with mannan- HMFG and splenocytes were used in CTL assays. Lysis of P815 (a) or RMA (c) cells pulsed with various 9-mer peptides from the intracellular peptide 471- 493; Lysis of P815 (b) or RMA (d) cells pulsed with various 9-mer peptides from the extracellular peptides 33-103 and 51-70 and (e) Lysis of P815 cells pulsed with YYQELQRDI (SEQ ID NO: 35) and RMA-MUCl cells pulsed with SAPDNRPAL (SEQ ID NO: 36). As controls for peptide pulsing and antigen- specific 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 ⁵¹Cr-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 ⁵¹Cr-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 MUCl-expressing carcinomas of the breast and other tissues [35]. MUCl 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 MUCl 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 MUCl when whole tumour cells or mucin extracts (HMFG) are used to immunise mice for the production of antibodies [40].

Because of this focus and the finding that non-HLA restricted CTL also are directed to the VNTR, almost all interest in MUCl 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 MUCl 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 (H-2^d), C57BL/6 (H-2^b), human MUCl transgenic mice (obtained from B. Acres (Transgene, Strasbourg, France)), transgenic HLA- A*0201/K^b mice (H-2^b) (obtained from The Scripps Clinic and Research Foundation, La Jolla, CA.) and double transgenic mice (A2K^DMUC1) were bred at The Austin Research Institute (ARI). The human MUCl in the MUCl transgenic mice (back crossed to DBA/2) is under the control of the human MUCl promoter; MUCl is expressed in the lung bronchioles, β-islets of the pancreas, kidney tubules and stomach [43]. The HLA-A*0201/K^D mice express a transgene composed of the αl and α2 domains of HLA-A*0201 and the α3 contains the transmembrane and cytoplasmic domains of H-2K^D [44]. The double transgenic mice were screened for expression of the HLA- A*0201/K'^:, and human MUCl transgenes by flow cytometry with antibodies to HLA-A*0201 and MUCl. RMA-MUCl is a MUCl transfected (C57BL/6 (H- 2 )) lymphoma cell line [45]. Tm211 is a MUCl transfected P815 mastocytoma (DBA/2 origin; H-2^d) obtained from B. Acres (Transgene,

Strasbourg, France) [46]. All mouse cell lines were maintained in Dulbecco's modified Eagles medium (DMEM) with lOOIU/ml penicillin, lOOμg/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 CO2 incubator at 37°C. BALB/c, C57BL/6 and double transgenic A2K^DMUCl mice were immunised intraperitoneally with 3 injections of 5μg mannan-HMFG or HMFG on days 0, 10, 17 while HLA- A*0201/K^b mice were injected once.

2.2 Synthetic Peptides

Peptides (Table 1) were synthesised at the ARI; the purity of the peptides (>95%) was determined by mass spectroscopy.

2.3 Conjugation of HMFG to Mannan

HMFG was isolated from human milk [49] and coupled to mannan. Mannan (1ml, 14mg/ml) in phosphate buffer (0.1M, pH6.0) was treated with sodium periodate (lOOμl, 0.1M) and incubated at 4°C for 30 min [48]. Ethanediol (lOμl) was added for 30 mins at 4° 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 epi topes. 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 (K^b, SIINFEKL; SEQ ID NO: 11) and papillomavirus-16 E7 protein (D^b, RAHYNWTF; SEQ ID NO: 12) gives scores of 17 and 6 respectively. 2.5 Cytotoxic T Cell and Cftotoxic T Cell Precursor (CTLp) Frequency Assays CTL assays were performed as described [37, 39, 48]. Briefly, 7 to 10 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 ⁵¹Cr release assay was performed with lxlO⁴ 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 incubation with 9-mer peptides (25μg/ml) [37] . For CTL assays with A2K^bMUCl double transgenic effectors, MCF7 (MUCl+HLA-A*0201+) and BT20 (MUCl+HLA-A*020r) breast cancer cell lines or the ME272 (MUCl" 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 (lxlO³ - 1.28xl0⁵). Cells were cultured in U-bottomed microtitre trays, with 5xl0⁵ mitomycin C treated BALB/c (H-2^d), C57BL/6 (H- 2^b) or HLA-A*0201/K^b spleen cells, in DMEM supplemented with 10% foetal calf serum, 5μM of various MUCl peptides (Table 1) or HMFG and 10 U/ml rhIL-2. Seven days later, each microculture was assayed for cytotoxicity by replacing lOOμl of culture medium with 100 μl target cell suspension containing 10^{4 51}Cr-labelled Tm211 (H-2^d), RMA-MUCl (H-2^b) Tumour or EBV transformed human B cells (HLA-A*0201) or MCF7 as targets. As a specificity control non-MUCl expressing P815(H-2^d) or RMA(H-2^b) cells were used. Cytotoxic activity was considered to be present if in each well ⁵¹Cr release was found 3 standard deviations above the mean isotope release from 10⁴ effectors cultured with stimulators only or from stimulator cells with peptide only or rhIL2 only. A linear relationship (0.987< r^ < 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 μl of 10 μg/ml HMFG in bicarbonate buffer (0.2M, pH9.0) overnight at 4°C or 1 hr at 37°C and non-specific binding was blocked with 2% bovine serum albumin (BSA). Various concentrations of HMFG or mannan-HMFG were incubated with anti-MUCl antibody (VA2 [57], 1/200 supernatant) for 3 hr and lOOμl was added to PVC microtitre well plates coated with HMFG. After washing with phosphate buffered saline (PBS) containing 0.05% Tween 20, 50μl of sheep anti-mouse immunoglobulin 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(3- ethylbenzthiazoline) 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-MUCl antibody (BC2 [58]) in bicarbonate buffer overnight and non-specific binding blocked as described above. HMFG or Mannan-HMFG was then added to the wells and incubated for 1 hr at RT followed by washing extensively with PBS containing 0.05% Tween 20. Fifty μl 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 (120μl) was added to the wells, and plates counted in a β-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 μg/ml while for the mannan-HMFG was 20μg/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- MUCl antibody bound to the plate and ¹²⁵I-labelled Con-A for the read out

(Fig lb). Non-conjugated HMFG did not bind ¹²⁵I-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 MUCl (Table 2). It was apparent that immunisation with mannan-HMFG leads to CTL reacting with epitopes from the whole of MUCl, i.e., from both the VNTR and non-VNTR region. The responses were :- a) HMFG. When whole MUCl (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. b 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, i.e., by immunising with HMFG, anti-VNTR CTL were produced, results similar to those found previously by immunising with mannan-conjugated VNTR peptides [47] . 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. d) 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 MUCl sequences, and not due to non-specific killing by NK cells or other cells, P815 target cells were used with a non-MUCl 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 MUCl VNTR (47) and, thus, conjugation of HMFG to mannan is necessary for generating a strong CTLp frequency in mice. 3.3 CTL Responses to mannan-HMFG in C57BL/6 mice

C57BL/6 were immunised with mannan-HMFG and in vitro stimulated with the same antigens used for the BALB/c mice (Table 2). There was a CTLp frequency of 1/13,500 for whole HMFG and 1/12,500 for the VNTR region peptide pl-30 (Table 2). 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-MUCl peptide, T4N1, for stimulation and also using the non-MUCl transfected parent RMA cell line as the target. Thus, C57BL/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 HLA-

Transgenic HLA-A*0201/K^b 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) i.e., whole HMFG is as immunogenic as VNTR (Table 2). Further, when an extracellular peptide (31-55) was used, the CTLp frequency was 1/40,000, i.e., 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 MUCl peptides.

3.5 Cellular Immune Responses to Mannan-HMFG in A2I<pMUCl Double Transgenic Mice To ascertain the ability of MUCl CTL to lyse MUCl positive breast cancer cells A2K^bMUCl double transgenic mice injected with mannan HMFG 3 times were stimulated in vitro with either HMFG, the VNTR peptide (pl- 30), extracellular peptides (31-55, 344-364) or intracellular peptides (408-423, 471-493, 507-526) (Table 2). 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 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-MUCl CTL. As seen in Figure 2, MUCl CTL lysed 55% of MUC1+ MCF7 (HLA-A*0201) breast carcinoma cells at an E:T ratio of 12:1 and was reduced to 17% when incubated in the presence of cold ^~ K562 targets. The MUCl CTL were HLA restricted as no lysis was detected when the MUC1+ BT20 (HLA-Al) breast cancer cell line was used. The MUCl CTL did not lyse the MUCl -ve melanoma cell line ME272. Thus, immunisation of A2K^bMUCl mice with mannan-HMFG resulted in specific Class I restricted CTL that can lyse tumour cells expressing native MUCl and, moreover, anti-MUCl CTL can be generated in mice in the presence of endogeneously expressed human MUCl. 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-2^d-restricted peptides (intracellular region MUCl)

Several peptides (NYGQLDIFP(K^d) SEQ ID NO: 13; YGQLDIFPA(D^d) SEQ ID NO: 14; KNYGQLDIF(L^d), SEQ ID NO: 15) were contained in 471-493 (CTLp frequency= 1/12,500) and had predicted scores 6, 6 and 10 respectively (Table 3). 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(D ) (SEQ ID NO: 14), KNYGQLDIF(L^d) (SEQ ID NO: 15). The pulsed cells were not lysed by Mannan-HMFG derived CTL from BALB/c mice (Figure 3a), i.e., the CTL epitopes were not predicted accurately by the algorithm. The MUCl VNTR peptides SAPDTRPAP(D^d) (SEQ ID NO: 16) and APDTRPAPG (L^d) (SEQ ID NO: 17) identified previously as CTL epitopes in the VNTR region [38], were used as positive controls and 62% and 50% lysis at an E:T ratio of 50:1 was obtained. The listeriolysin K^d peptide (GYKDGNEYI; SEQ ID NO: 18) and HTV D^d peptide (RKSIRIQRGPGRAFVTIGKGKGKGY; SEQ ID NO: 19), used as negative controls, did not give rise to lysis (Fig. 3a).

Predicted H-2 -restricted peptides fextracellular region MUCH

A number of 9-mer peptides in the extracellular region are predicted to be CTL epitopes [(AVSMTSSVL(K^d), SEQ ID NO: 20; TTQGQDVTL(K^d), SEQ ID NO: 21; NAVSMTSSV(K^d), SEQ ID NO: 22; TSATQRSSV(K^d), SEQ ID NO: 23; SSTTQGQDV(K^d), SEQ ID NO: 24; SVPSSTEKN(D^d), SEQ ID NO: 25; EPASGSAAT(L^d), SEQ ID NO: 26; SPGSGSSTT(L^d), SEQ ID NO: 27; VPSSTEKNA(L ), SEQ ID NO: 28; TPGGEKETS(L^d), SEQ ID NO: 29; TSATQRSSV(L^d), SEQ ID NO: 30; SSTTQGQDV(L^d), 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 3). Of these, four were made (AVSMTSSVL(K^d), SEQ ID NO; 20, NAVSMTSSV(K^d), SEQ ID NO: 22; VPSSTEKNA(L^d), SEQ ID

NO: 28; SVPSSTEKN (D^d), SEQ ID NO: 25) and tested. Three of the four peptides were indeed presented and one was not. The synthetic peptides AVSMTSSVL(K^d), SEQ ID NO: 20; NAVSMTSSV(K^d), SEQ ID NO: 22 and VPSSTEKNA(L^d), SEQ ID NO: 28 sensitised P815 target cells with 77%, 80% 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-2^b restricted peptides. Even though there were fewer identified peptide epitopes for C57BL/6 mice, there are a large number of potential CTL epitopes present in the peptides, albeit with low scores (Table 3). The 9-mer CRRKNYGQL (D^b, K^b), 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 MUCl VNTR peptides APGSTAPPA (D ), SEQ ID NO: 33 and SAPDTRPAP (K^b), SEQ ID NO: 16 were t sed as positive specificity controls, where lysis of 70% and 80% were obtained while no lysis was detected for the ovalbumin K^b 9-mer SIINFEKL, SEQ ID NO: 11 and Adenovirus D^b 9-mer (used as negative specificity controls). The 9-mer peptides STEKNAVSM(D ),

SEQ ID NO: 34; AVSMTSSVL(D ), SEQ ID NO: 20 and AVSMTSSVL(K^b), 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 ( -20% 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 MUCl molecule from the intracellular region (YYQELQRDI(K^d), SEQ ID NO: 35 score 2880) and extracellular region N-terminal to the VNTR (SAPDNRPAL(D^b), 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 MUCl 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 MUCl fusion protein containing 5 repeats of the VNTR linked to mannan (MFP) and this generated strong cellular responses to MUCl characterised by the production of IFN-γ, 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 MUCl antigens as in a Phase I clinical trial using MFP, 4 of 15 patients generated proliferative responses, 13 of 25 showed high levels of MUCl specific serum antibody and 2 of 10 generated CTL to MUCl [59]. 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, 60], and studies thus far have concentrated on the MUCl 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 MUCl 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 MUCl: none resulted and none were found in an international study. Scanning the whole MUCl 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 MUCl epitopes but dependent on natural antigen processing for their presentation, and showed that cellular immune responses to the non-VNTR regions of the MUCl can be generated which are as effective as those generated to the VNTR and further both HLA-A*0201 and A2K^bMUCl 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

MUCl, the VNTR and also to intracellular peptides in mannan-HMFG immunised BALB/c, C57BL/6, HLA-A*0201/K and double transgenic A2K^bMUCl 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 3). 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: 20; NAVSMTSSV, SEQ ID NO: 22 and VPSSTEKNA, SEQ ID NO: 28) were identified as functional CTL epitopes in the lysis assays (Figure 3b).

In C57BL/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 MUCl sequence using the T cell epitope algorithm for mouse K^d, D^d, L^d, K^b, D , K^k, and human HLA-Al, 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 (D^b), (SEQ ID NO: 36) and YYQELRDI (K^d) (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 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 4). For example, SAPDTRPAP (SEQ ID NO: 16) peptide has been confirmed to be a K^b-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 ([38]). Similarly, the Yx, L and D^d was not predicted accurately [38]. 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 MUCl 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 MUCl protein has been delivered in a vaccinia construct [46, 63], as a construct in DNA immunisation [64], 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 MUCl (as HMFG) should be stressed. Other studies, in mice and humans have used non- glycosylated peptides which have led to antibody production in both MUCl 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 A2K^bMUCl transgenic mice. Mannan-HMFG gave a higher CTLp frequency in A2K^bMUCl 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 non- glycosylated form of MUCl VNTR [47], i.e., both glycosylated and non- glycosylated forms of the VNTR were equally immunogenic provided they are presented with oxidised mannan. Clearly, the carbohydrate coating did not obscure the underlying peptide. Thus, mannan-HMFG is able to break tolerance in A2K^bMUCl transgenic mice by producing CTLs to peptides in the VNTR, the extracellular region and the intracellular region in MUCl. These res ύts reinforce the concept that MUCl should be a useful target in therapy.

The use of mannan-HMFG in humans warrants some discussion in that MUCl 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 MUCl VNTR conjugated to mannan no autoimmunity was detected, however, careful dose escalation studies and monitoring is necessary [59]. 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 [70]. Such a deviation may occur when using whole MUCl.

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 5 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 5) compared to the positive controls (VNTR peptides conjugated to mannan). 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 [72]). 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.

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28. Mandelboimo, 0., Berke 0., Fridkin, M., Feldman, M., Eisenstein, M., and Eisenbach, L., CTh Induction by a Tumour-associated Antigen Octapeptide Derived from a Murine Lung Carcinoma, Nature, 369,67-71, 1994.

29. Goodman GE, Hellstrom I, Yelton DE, Murray JL, OHara 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 with non-small-cell lung, colon, and breast cancer. Cancer Immunol. Immunother. 1993;36:267-273.

30. Apostolopoulos V, McKenzie IFC. Gellular mucins: targets for immunotherapy. Crit. Rev. Immunol. 1994;14:293-309. 31. Finn OJ, Jerome KR, Henderson RA, Pecher G, Domenech N, Magarian- Blander J, Barratt-Boyes SM. MUC-1 epithelial tumor mucin-based immunity and cancer vaccines. Immunol. Rev. 1995;145:61-89.

32. Schrier DM, Stemmer SM, Johnson T, Kasliwal R, Lear J, Matthes S, Taffs S, Dufton C, Glenn SD, Butchko G et al. High-dose 90YMx diethylenetriaminepentaacetic acid (DTPA)-BrE-3 and autologous hematopoietic stem cell suppoή (AHSCS) for the treatment of advanced breast cancer: a phase I trial. Cancer Res. 1995;55(23 Suppl):592lS-5924S.

33. Richman CM, DeNardo SJ, O'Grady LF, DeNardo GL. Radioimmunotherapy for breast cancer using escalating fractionated doses of ¹³¹I-labeled chimeric L6 antibody with peripheral blood progenitor cell transfusions. Cancer Immunol. Immunother. 1993;36: 267-273.

34. MacLean GD, Miles DW, Rubens RD, Reddish MA, Longenecker BM. Enhancing the effect of THERATOPE STn-KLH cancer vaccine in patients with metastatic breast cancer by pretreatment with low-dose intravenous cyclophosphamide. Immunother. Emphasis Tumor Immunol. 1996;19:309- 316.

35. Apostolopoulos V, McKenzie IFC, Pietersz GA. Breast cancer immunotherapy -Current status and Future prospects. Immunol. 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 T∑ 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 1 are MHC-restricted. . Immunol.

1995;1155:5089-5094.

38. Apostolopoulos V, Haurum J, McKenzie IFC. MUCl peptide epitopes associated with 5 different H2 class I molecules. Eur. T. Immunol. 1997;27:2579-2587. 39. Apostolopoulos V, Karanikas V, Haurum JS, McKenzie IFC. Induction of

HLA-A2-restricted CTLs to the Mucin 1 human breast cancer antigen.

T. Immunol. 1997;159:5211-5218.

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

Cancer Cells 1990;2:75-78. 41. Jerome KR, Barnd DL, Boyer CM, Papadimitriou JT, McKenzie IFC, Bast

RC, Finn OJ. Adenocarcinoma reactive cytotoxic T lymphocytes recognise an epitope present on the protein core of epithelial mucin molecules. Cellular Immunity and Immunotherapy of Cancer 1990;321-328.

42. Barnd DL, Lan MS, Metzgar RS, Finn OJ. Specific major histocompatibility complex - unrestricted recognition of Tumor-associated mucins by human cytotoxic T cells. Proc Nat. Acad. Sci. USA 1989;86:7159- 7163.

43. Acres B., Apostolopoulos V., Balloul JM., Wreschner D., Xing PX., Hadji DA., Spetz JF., Bisouarne N., Kieny MP and McKenzie IFC. MUCl specific cjrtotoxic T cell precursor analysis in human MUCl transgenic mice immunised with human MUCl vaccines. Cancer Immunol. Immunother. (In Press)

44. Vitiello A, Marchesini D, Furze J, Sherman LA, Chesnut RW. Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-murine class I major histocompatibility complex. T. Exp. Med. 1991;173:1007.

45. Graham RA, Stewart LS, Peat NP, Beverley P, Taylor-Papadimitriou J. MUCl-based immunogens for tumor therapy: development ofmurine model systems. Tumor Targeting 1995;1:211.

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

47. Jarasch E, Bruder G, Keenan TW, Franke WW. Redox constituents in milk fat globule membranes and rough endoplasmic reticulum from lactating gland. T. Cell Biol. 1977;73:223-241. 48. Apostolopoulos V, Pietersz GA, McKenzie IFC. Cell-mediated immune responses to MUCl fusion protein coupled to mannan. Vaccine 1996;74:930- 938.

49. Parker KC, Bednarek MA, Hull LK, Utz U, Cunningham B, Zweerink HJ, Biddison WE, Coligan JE. Sequence motifs impoήant for peptide binding to the human MHC class I molecule, HLA-A2. T. Immunol. 1992;149:3580-3587.

50. Parker KC, Bednarek MA, Coligan JE. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. T. Immunol. 1994;152:163-175.

51. Screpanti I, Felli MP, Toniato E, Meco D, Martinotti S, Frati L, Santoni A, Gulino A. Enhancement of natural-killer-cell susceptibility of human breast- cancer cells by estradiol and v-Ha-ras oncogene. Int. T. Cancer 1991;47:445- 449.

52. Chen Q, Jackson H, Gibbs P., Davis ID, Trapani J, Cebon J. Spontaneous T cell responses to melanoma differentiation antigens from melanoma patients and healthy subjects. Cancer Immunol. Immunother. 1998;47:191-197.

53. Taswell, C. Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. I. Immunol. 1981;126:1614-1619.

54. Fazekas de St. Groth S. The evaluation of limiting dilution assays. T. Immunol. Methods 1982;49:R11.

55. Lefkovits I, Waldmann H. Limiting dilution analysis of the cells of the immune system. I. The cloned basis of the immune response. T. Immunol. Today 1984;5:265.

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

57. Apostolopoulos V, Xing P-X, Trapani JA, McKenzie IFC. Production of anti-breast cancer monoclonal antibodies using glutathione-S-transferase- MUCl bacterial fusion protein. Brit. T. Cancer 1993;67:713-720. 58. Xing P-X, Tjandra TJ, Stacker SA, Teh JG, Thompson CH, McLaughlin PJ, McKenzie IFC. Monoclonal antibodies reactive with mucin expressed in breast cancer. Immunol. Cell Biol. 1989;67:183-195.

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 mannan- MUCl fusion protein. I Clin. Invest. 1997;100:2783-2792.

60. Domenech N, Henderson RA, Finn OJ. Identification of an HLA-All- restricted epitope from the tandem repeat domain of the epithelial tumor antigen mucin. T. Immunol. 1995;155:4766-4774. 61. Lipford GB, Hoffman M, Wagner H, Heeg K. Primary in viyo responses to ovalbumin. Probing the predictive value of the I<P motif T. Immunol. 1993;150:1212-1222.

62. Pamer EG, Harty JT, Be van MJ. Precise prediction of a dominant class I MHC-restricted epitope oflisteria monocytogenes. Nature 1991;353:852-855. 63. Akagi J, Hodge JW, McLaughlin JP, Gritz L, Mazzara G, Kufe D, Schlom J, Kantor JA. Therapeutic antitumor response after immunization with an admixture of recombinant vaccinia viruses expressing a modified MUCl gene and the murine T-cell costimulatory molecule B7. T. Immunother. 1997;20:38- 47.

64. Graham RA, Burchell JM, Beverley P, Taylor-Papadimitriou J. Intramuscular immunization with MUCl cDNA can protect G57 mice challenged with MUCl -expressing syngeneic mouse Tumor cells. Int. T. Cancer 1996;65:664-670.

65. Gong J, Chen L, Chen D, Kashiwaba M, Manome Y,Tanaka T, Kufe D. Induction of antigen-specific antitumor immunity with adenovirus-transduced dendritic cells. Gene Ther. 1997;4:1023-1028.

66. Pecher G, Finn OJ. Induction of cellular immunity in chimpanzees to human tumor-associated antigen mucin by vaccination with MUC-1 cDNA- transfected 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 MUCl in a human MUCl transgenic murine model. Cancer Res. 1998;58:315-321.

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

69. Reddish M, MacLean GD, Koganty RR, Kan-Mitchell J, Jones V, Mitchell MS, Longenecker BM. Anti-MUCl class I restricted CTLs in metastatic breast cancer patients immunized with a synthetic MUCl peptide. Int. T- Cancer 1998;76:817-823. 70. Apostolopoulos V, Lofthouse SA, Popovski V, Chelvanayagam G,

Sandrin MS, McKenzie IFC. Peptide mimics of a tumor antigen induce functional cytotoxic T cells. Nature Biotech. 1998;16:276-280.

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

72. Lai, W.C. and Bennett, M. DNA Vaccines. Crit. Rev. Immunol. 1998; 18: 449-484. 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 MUCl and Sequences of the Synthetic Peptides

N-Terminal VNTR Tranpmeiηbrane Cytoplasmic tail

MUC1

Extracellular region

Cp 13-32 (SEQ 3D NO 39)

(C)PAHGVTSAPDTRPAPGSTAP. pl -30 PDTRPAPGSTAPPAHGVTSAPDTRPAPGST (SEQ ID NO 40)

31-55 TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO 2)

51-70 RSSVPSSTEKNAVSMTSSVL (SEQ ID NO 3)

33-103 Glutathione -S-transferase fusion protein

SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLS

SHSPGSGSSTTQGQDV TLAPATEPASGSAATW (SEQ ID NO: 4)

229-237 SAPDNRPA (SEQ ID NO: 6) 344-364 NSSLEDPSTDYYQELQRDISE (SEQ ID NO: 7) Intracellular region 408-423 TQFNQYKTEAASRVNL (SEQ ID NO 8) 471-493 AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO 9) 507-526 (C)YVPPSSTDRSPYEKVSAGNG (SEQ ID NO 41) Mouse CD4 T4N1 KTLVLGKEQESAELPCECY (SEQ ID NO: 42)

Table 2 CTLp Frequencies in Spleens of Mice Immunised With mannan-HMFG

π

Table 3 Mice --mmunised with mannan-HMFG: CTLp Frequencies to Various non-NNTR Peptides and their Predicted CTL Epitopes

ω

CO

Table 4 Experimentally Determined and Predicted Mouse and Human CTL Epitopes in the MUCl VNTR

O OS

Sequence Listing:

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 1 5

<210> 2

<211> 25

<212> PRT

<213> Homo sapiens

<400> 2

Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr 1 5 10 15

Ser Ala Thr Gin Arg Ser Ser Val Pro 20 25

<210> 3

<211> 20

<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 15

Ser Ser Val Leu 20

<210> 4

<211> 71

<212> PRT

<213> Artificial Sequence <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 15

Thr Gin Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Val Ser 20 25 30

Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly Ser Gly Ser Ser 35 40 45

Thr Thr Gin Gly Gin Asp Val Thr Leu Ala Pro Ala Thr Glu Pro Ala 50 55 60

Ser Gly Ser Ala Ala Thr Trp ^* 65 70

<210> 5

<211> 473

<212> PRT

<213> Homo sapiens •

<400> 5

Met Thr Pro Gly Thr Gin Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15

Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30

Gly Glu Lys Glu Thr Ser Ala Thr Gin Arg Ser Ser Val Pro Ser Ser 35 40 45

Thr Glu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His 50 55 60

Ser Pro Gly Ser Gly Ser Ser Thr Thr Gin Gly Gin Asp Val Thr Leu 65 70 75 80

Ala Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gin 85 90 95

Asp Val 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 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 lie Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala 210 215 220

Ser His Ser 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 lie Ser Asn Leu Gin Phe 260 265 270

Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gin Glu Leu Gin 275 280 285

Arg Asp lie Ser Glu Met Phe Leu Gin lie Tyr Lys Gin Gly Gly Phe 290 ^" 295 300

Leu Gly Leu Ser Asn lie Lys Phe Arg Pro Gly Ser Val Val Val Gin 305 310 315 320

Leu Thr Leu Ala Phe Arg Glu Gly Thr lie Asn Val His Asp Val Glu 325 330 335

Thr Gin Phe Asn Gin Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu 340 345 350

Thr lie Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala 355 360 365

Gin Ser Gly Ala Gly Val Pro Gly Trp Gly lie Ala Leu Leu Val Leu 370 375 380

Val Cys Val Leu Val Ala Leu Ala He Val Tyr Leu He Ala Leu Ala 385 390 395 400

Val Cys Gin Cys Arg Arg Lys Asn Tyr Gly Gin Leu Asp He Phe Pro 405 410 415

Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr 420 425 430

His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu 435 440 445

Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro 450 455 460 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 5

<210> 7

<211> 21

<212> PRT

<213> Homo sapiens

<400> 7

Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gin Glu Leu Gin 1 5 10 15

Arg Asp He Ser Glu 20

<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 15

<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 He Phe 1 5 10 15

Pro Ala Arg Asp Thr Tyr His 20

<210> 10 <211> 20

<212> PRT

<213> Homo sapiens

<400> 10

Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 1 5 10 15

Ala Gly Asn Gly 20

<210> 11

<211> 8

<212> PRT

<213> Homo sapiens

<400> 11

Ser He He Asn Phe Glu Lys Leu 1 5

<210> 12

<211> 9

<212> PRT

<213> papillomavirus-16

<400> 12

Arg Ala His Tyr Asn He Val Thr Phe 1 5

<210> 13

<211> 9

<212> PRT

<213> Homo sapiens

<400> 13

Asn Tyr Gly Gin Leu Asp He Phe Pro 1 5

<210> 14

<211> 9

<212> PRT

<213> Homo sapiens

<400> 14

Tyr Gly Gin Leu Asp He Phe Pro Ala 1 5 <210> 15

<211> 9

<212> PRT

<213> Homo sapiens

<400> 15

Lys Asn Tyr Gly Gin Leu Asp He Phe 1 5

<210> 16

<211> 9

<212> PRT

<213> Homo sapiens

<400> 16

Ser Ala Pro Asp Thr Arg Pro Ala Pro 1 5

<210> 17

<211> 9

<212> PRT

<213> Homo sapiens

<400> 17

Ala Pro Asp Thr Arg Pro Ala Pro Gly 1 5

<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 Glu Tyr He ^■ 1 5

<210> 19

<211> 25

<212> PRT

<213> Human immunodeficiency virus

<400> 19

Arg Lys Ser He Arg He Gin Arg Gly Pro Gly Arg Ala Phe Val Thr 1 5 10 15 He Gly Lys Gly Lys Gly Lys Gly Tyr 20 25

<210> 20

<211> 9

<212> PRT

<213> Homo sapiens

<400> 20

Ala Val Ser Met Thr Ser Ser Val Leu 1 5

<210> 21

<211> 9

<212> PRT

<213> Homo sapiens

<400> 21

Thr Thr Gin Gly Gin Asp Val Thr Leu 1 5

<210> 22

<211> 9

<212> PRT

<213> Homo sapiens

<400> 22

Asn Ala Val Ser Met Thr Ser Ser Val 1 5

<210> 23

<211> 9

<212> PRT

<213> Homo sapiens

<400> 23

Thr Ser Ala Thr Gin Arg Ser Ser Val 1 5

<210> 24

<211> 9

<212> PRT

<213> Homo sapiens

<400> 24

Ser Ser Thr Thr Gin Gly Gin Asp Val 1 5 <210> 25

<211> 9

<212> PRT

<213> Homo sapiens

<400> 25

Ser Val Pro Ser Ser Thr Glu Lys Asn ' 1 5

<210> 26

<211> 9

<212> PRT

<213> Homo sapiens

<400> 26

Glu Pro Ala Ser Gly Ser Ala Ala Thr 1 5

<210> 27

<211> 9

<212> PRT

<213> Homo sapiens

<400> 27

Ser Pro Gly Ser Gly Ser Ser Thr Thr 1 5

<210> 28

<211> 9

<212> PRT

<213> Homo sapiens

<400> 28

Val Pro Ser Ser Thr Glu Lys Asn Ala 1 5

<210> 29

<211> 9

<212> PRT

<213> Homo sapiens

<400> 29

Thr Pro Gly Gly Glu Lys Glu Thr Ser 1 5 <210> 30

<211> 9

<212> PRT

<213> Homo sapiens

<400> 30

Thr Ser Ala Thr Gin Arg Ser Ser Val 1 5

<210> 31

<211> 9

<212> PRT

<213> Homo sapiens

<400> 31

Thr Ala Pro Pro Ala His Gly Val Thr 1 5

<210> 32

<211> 9

<212> PRT

<213> Homo sapiens

<400> 32

Cys Arg Arg Lys Asn Tyr Gly Gin Leu 1 5

<210> 33

<211> 9

<212> PRT

<213> Homo sapiens

<400> 33

Ala Pro Gly Ser Thr Ala Pro Pro Ala 1 5

<210> 34

<211> 9

<212> PRT

<213> Homo sapiens

<400> 34

Ser Thr Glu Lys Asn Ala Val Ser Met 1 5

<210> 35 <211> 9

<212> PRT

<213> Homo sapiens

<400> 35

Tyr Tyr Gin Glu Leu Gin Arg Asp He 1 5

<210> 36

<211> 9

<212> PRT

<213> Homo sapiens

<400> 36

Ser Ala Pro Asp Asn Arg Pro Ala Leu 1 5

<210> 37

<211> 9

<212> PRT

<213> Homo sapiens

<400> 37

Ser Thr Ala Pro Pro Ala His Gly Val 1 5

<210> 38

<211> 32

<212> PRT

<213> Homo sapiens

<400> 38

Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly 1 5 10 15

Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro 20 25 30

<210> 39

<211> 21

<212> PRT

<213> Homo sapiens

<400> 39

Cys Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 1 5 10 15 Gly Ser Thr Ala Pro 20

<210> 40

<211> 30

<212> PRT

<213> Homo sapiens

<400> 40

Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly 1 5 10 15

Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr 20 25 30

<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 15

Ser Ala Gly Asn Gly 20

<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 15

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 He Phe 1 5 10 15 Pro Ala Arg Asp Thr Tyr His 20

<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 15

<210> 45

<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 15

Thr Gin Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Val Ser 20 25 30

Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly Ser Gly Ser Ser 35 40 45

Thr Thr Gin Gly Gin Asp Val Thr Leu Ala Pro Ala Thr Glu Pro Ala 50 55 60

Ser Gly Ser Ala Ala Thr Trp 65 70

<210> 46

<211> 9

<212> PRT

<213> Homo sapiens

<400> 46

Cys Tyr Val Pro Pro Ser Ser Thr Asp 1 5

<210> 47 <211> 9 ^"<212>. PRT <213> Homo sapiens

<400> 47

Gin Phe Asn Gin Tyr Lys Thr Glu Ala <210> 48

<211> 9

<212> PRT

<213> Homo sapiens

<400> 48

Gin Tyr Lys Thr Glu Ala Ala Ser Arg 1 5

<210> 49

<211> 9

<212> PRT

<213> Homo sapiens

<400> 49

Thr Glu Ala Ala Ser Arg Val Asn Leu 1 5

<210> 50

<211> 9

<212> PRT

<213> Homo sapiens

<400> 50

Ser Leu Glu Asp Pro Ser Thr Asp Val 1 5

<210> 51

<211> 8

<212> PRT

<213> Homo sapiens

<400> 51

Asn Ala Val Ser Met Thr Ser Val 1 5

<210> 52

<211> 8

<212> PRT

<213> Homo sapiens

<400> 52

Ser Thr Glu Lys Asn Val Ser Met 1 5 <210> 53

<211> 9

<212> PRT

<213> Homo sapiens

<400> 53

Val Pro Pro Ser Ser Thr Asp Arg Ser 1 5

<210> 54

<211> 9

<212> PRT

<213> Homo sapiens

<400> 54

Phe Asn Gin Tyr Lys Thr Glu Ala Ala 1 5

<210> 55

<211> 9

<212> PRT

<213> Homo sapiens

<400> 55

Gly Gly Glu Lys Glu Thr Ser Ala Thr 1 5

<210> 56

<211> 9

<212> PRT

<213> Homo sapiens

<400> 56

Thr Gly Ser Gly His Ala Ser Ser Thr 1 5

<210> 57

<211> 9

<212> PRT

<213> Homo sapiens

<400> 57

Thr Glu Ala Ala Ser Arg Val Asn Leu 1 5 <210> 58

<211> 9

<212> PRT

<213> Homo sapiens

<400> 58

Asn Ser Ser Leu Glu Asp Pro Ser Thr 1 5

<210> 59

<211> 9

<212> PRT

<213> Homo sapiens

<400> 59

Asp Pro Ser Thr Asp Tyr Tyr Gin Glu 1 5

<210> 60

<211> 9

<212> PRT

<213> Homo sapiens

<400> 60

Pro Ser Thr Asp Tyr Tyr Gin Glu Leu 1 5

<210> 61

<211> 9

<212> PRT

<213> Homo sapiens

<400> 61

Ser Ser Thr Glu Lys Asn Ala Val Ser 1 5

<210> 62

<211> 9

<212> PRT

<213> Homo sapiens

<400> 62

Arg Ser Pro Tyr Glu Lys Val Ser Ala 1 5

<210> 63 <211> 9 <212> PRT

<213> Homo sapiens

<400> 63

Ser Ser Leu Glu Asp Pro Ser Thr Asp 1 5

<210> 64

<211> 9

<212> PRT

<213> Homo sapiens

<400> 64

Lys Glu Thr Ser Ala Thr Gin Arg Ser 1 5

<210> 65

<211> 9

<212> PRT

<213> Homo sapiens

<400> 65

Pro Asp Thr Arg Pro Ala Pro Gly Ser 1 5

<210> 66

<211> 9

<212> PRT

<213> Homo sapiens

<400> 66

Ala Pro Pro Ala His Gly Val Thr Ser 1 5

Claims

Claims:

1. 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.

2. A peptide or polypeptide according to claim 1, wherein said peptide or polypeptide consists of an amino acid sequence derived from the non-VNTR, non-leader region of a mucin.

3. A peptide or polypeptide according to claim 1, wherein said epitope is from an extracellular region of the non-VNTR, non-leader region of a mucin.

4. A peptide or polypeptide according to claim 1, wherein said epitope is from an intracellular region of the non-VNTR, non-leader region of a mucin.

5. A peptide or polypeptide according to claim 1, wherein said epitope is from a transmembrane region of the non-VNTR, non-leader region of a mucin.

6. A peptide or polypeptide according to any one of the preceding claims, wherein said mucin is mucin 1 (MUCl).

7. A peptide or polypeptide according to claim 6, wherein said mucin 1 is human mucin 1.

8. A peptide or polypeptide according to claim 7, wherein said human mucin 1 is human milk fat globule membrane antigen (HMFG).

9. A peptide or polypeptide according to claim 3, wherem 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).

10. A peptide or polypeptide according to claim 4, wherein said epitope has the amino acid sequence: YYQELQRDI (SEQ ID NO: 35).

11. A peptide or polypeptide according to claim 1 or 2, 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: TGSGHASS TPGGEKETS ATQRSSVP (SEQ ID NO: 2), RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3),

SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQG QDVTLAPATEPASGSAATW (SEQ ID NO: 4), SAPDNRPAL (SEQ ID NO: 6), NSSLEDPSTDYYQELQRDISE (SEQ ID NO: 7), TQFNQYKTEAASRVNL (SEQ ID NO: 8), AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: 9) and YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10).

12. A fusion protein comprising a peptide or polypeptide according to any one of the preceding claims with a suitable carrier protein.

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

14. A fusion protein according to claim 13, 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- β-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, α-D-galactopyranose 6-sulphate, fructose, α 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.

15. A fusion protein according to claim 14, wherein said carbohydrate polymer comprises at least 20 monomer units.

16. A fusion protein according to claim 15, wherein said carbohydrate polymer comprises more than 1000 monomer units.

17. A fusion protein according to claim 16, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

18. A fusion protein according to any one of claims 13-17, wherein the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer comprising mannose.

19. A fusion protein according to any one of claims 13-17, wherein the carbohydrate polymer is a polymer of oxidised mannose or is oxidised mannan.

20. A peptide or polypeptide according to any one of claims 1-11 coupled to a suitable carrier protein.

21. A peptide or polypeptide according to claim 20, wherein said peptide or polypeptide and/or said carrier protein is conjugated to a carbohydrate polymer.

22. A peptide or polypeptide according to claim 21, 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-β-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, α-D-galactopyranose 6-sulphate, fructose, α 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.

23. A peptide or polypeptide according to claim 22, wherein said carbohydrate polymer comprises at least, 20 monomer units.

24. A peptide or polypeptide according to claim 23, wherein said carbohydrate polymer comprises more than 1000 monomer units.

25. A peptide or polypeptide according to claim 24, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

26. A peptide or polypeptide according to any one of claims 21-25, wherein the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer comprising mannose.

27. A peptide or polypeptide according to any one of claims 21-25, wherein the carbohydrate polymer is a polymer of oxidised mannose or is oxidised mannan.

28. A compound comprising a conjugate of a peptide or polypeptide according to any one of claims 1-11 and a carbohydrate polymer.

29. A compound comprising a conjugate of mucin 1 and a carbohydrate polymer, such that the conjugate is capable of eliciting a cell mediated immune response in a human or other animal.

30. A compound according to claim 29, wherein said mucin 1 is human mucin 1.

31. A compound according to claim 30, wherein said human mucin 1 is human milk fat globule membrane antigen (HMFG).

32. A compound according to any one of claims 28-31, 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-β-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, α-D-galactopyranose 6-sulphate, fructose, α 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.

33. A compound according to claim 32, wherein said carbohydrate polymer comprises at least 20 monomer units.

34. A compound according to claim 33, wherein said carbohydrate polymer comprises more than 1000 monomer units.

35. A compound according to claim 34, wherein said carbohydrate polymer comprises more than 10,000 monomer units.

36. A compound according to any one of claims 28-35, wherein the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer comprising mannose.

37. A compound according to any one of claims 28-35, wherein the carbohydrate polymer is a polymer of oxidised mannose or is oxidised mannan.

38. A vaccine or therapeutic agent comprising a peptide or polypeptide according to any one of claims 1-11 or 20-27 or a fusion protein according to any one of claims 12-19 and, optionally, an adjuvant and/or a pharmaceutically acceptable carrier.

39. A vaccine or therapeutic agent comprising a conjugate compound according to any one of claims 28-37 and, optionally, an adjuvant and/or a pharmaceutically acceptable carrier.

40. 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 according to any one of claims 1-11 or 20-27 or a fusion protein according to any one of claims 12-19, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

41. 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 any one of claims 28-37, optionally in combination with an adjuvant and/or a pharmaceutically acceptable carrier.

42. A method of preventing or treating a carcinoma in a subject, said method comprising administering to said subject a vaccine or therapeutic agent according to claim 38 or 39.

43. A method according to claim 42, wherein said carcinoma is an adenocarcinoma.

44. A method according to claim 43, wherein said adenocarcinoma is breast cancer.

45. The use of a peptide or polypeptide according to any one of claims 1-11 or 20-27, a fusion protein according to any one of claims 12-19, or a conjugate compound according to any one of claims 28-37, to pulse dendritic cells for in vivo transfer and use as a vaccine.

46. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the peptide or polypeptide of any one of claims 1-11 or a fusion protein according to any one of claims 12-19.

47. A DNA vaccine comprising a nucleic acid molecule according to claim 46.