WO2011009173A1

WO2011009173A1 - Cancer immunotherapy

Info

Publication number: WO2011009173A1
Application number: PCT/AU2010/000934
Authority: WO
Inventors: Kristen Radford
Original assignee: Mater Medical Research Institute
Priority date: 2009-07-23
Filing date: 2010-07-23
Publication date: 2011-01-27

Abstract

This invention relates generally to use of a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] in immunotherapy, especially immunotherapy for cancers, including hormone-related cancers such as prostate, breast, or ovarian cancer.

Description

"CANCER IMMUNOTHERAPY"

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from Australian provisional application 2009903449, the entire contents of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates generally to use of a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence

FLGYLILGV [SEQ ID NO:1] in immunotherapy, especially immunotherapy for cancers, including hormone-related cancers such as prostate, breast, or ovarian, cancer.

BACKGROUND OF THE INVENTION

[0003] Prostate cancer is the most common type of cancer found in American men, other than skin cancer, and the second leading cause of cancer death in men. Breast cancer is the most common cancer among women in the United States, other than skin cancer, and is the second leading cause of cancer death in women, after lung cancer. Ovarian cancer is the ninth most common cancer in women (not counting skin cancer) and ranks fifth as the cause of cancer death in women (American Cancer Society, access via http://www.cancer.org).

[0004] Primary therapy of these cancers using surgical removal and radiotherapy cures a significant number of patients but many will develop metastatic disease. There are currently no effective treatments for metastatic prostate, breast or ovarian cancer.

Conventional treatment with surgery, chemotherapy or radiotherapy has had only a limited impact on survival in these cancers and their toxicity may be significant. Thus, there is an urgent need for the development of efficacious, non-toxic treatments for these malignancies.

[0005] The concept of immunotherapy is the use of the immune system to attack cancer cells. Immunotherapy is where the patient's immune system is stimulated to attack the malignant cells that are responsible for the disease, and is attractive because of the potential efficacy, lack of side effects and ability to be combined with other therapies, including chemotherapy and radiotherapy.

[0006] The identification of tumour-associated antigens (TAAs) that can serve as cytotoxic T lymphocyte (CTL) targets is essential to analysing and generating clinical immune therapies. A number of TAAs have been identified for prostate cancer, including prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), and prostate stem cell antigen (PCSA). However, the use of multiple TAAs and several epitopes to maximise response and minimise immune escape by antigen loss tumour variants is now favoured by clinicians (Forsberg, O. et al. , 2009 Prostate 69: 70- 81). Thus, the identification of additional TAAs remains an important priority for clinical immunotherapy.

[0007] Human kallikrein (KLK) 4 is a serine protease belonging to the PSA-related kallikrein family. KLK4 mRNA and its protein product, kallikrein 4 (k4 or hK4), have limited expression in normal tissue but they are expressed in normal prostate cells and also in seminal fluid albeit at considerably lower levels than PSA. In contrast, KLK4IWΛ is significantly overexpressed in the majority of prostate cancers, where it appears to play a role in prostate cancer development and progression. KLK4 is also expressed in breast cancer and ovarian cancer.

[0008] The specificity of expression of hK4 warranted its investigation as a therapeutic cancer vaccine candidate. In work leading up to the present invention, the inventors identified a peptide, referred to herein as hK4A or peptide A, which is a dominant TAA epitope that is endogenously processed by host cells including antigen-presenting cells (e.g., dendritic cancer cells) and which is capable of inducing strong immune responses against cancer cells (e.g. , prostate cancer cells). This discovery has been reduced to practice in novel compositions and methods described below.

SUMMARY OF THE INVENTION

[0009] Thus, the present invention is predicated in part on the surprising discovery of a peptide from human kallikrein 4, which is a cancer tumour-associated antigen (TAA) epitope. The peptide is referred to herein as peptide A, and has the sequence FLGYLILGV [SEQ ID NO: I]. The discovery has many applications, especially in cancer immunotherapy.

[0010] Accordingly, in one aspect the present invention provides an immunogenic composition, the composition comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: 1] or a nucleic acid molecule from which the peptide is expressible.

[0011] In another aspect, the present invention provides a composition for stimulating an immune response in a subject, the composition comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a nucleic acid molecule from which the peptide is expressible. In certain embodiments, the immune response is a T-cell mediated response (e.g., a cytotoxic lymphocyte (CTL) mediated immune response). In another aspect, the present invention provides a composition for preventing or treating a cancer (e.g. , a hormone- related cancer such as prostate cancer, breast cancer, or ovarian cancer) in a subject, the composition comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a nucleic acid molecule from which the peptide is expressible.

[0012] In some embodiments, the composition comprises an immunostimulatory antigen-presenting cell or antigen-presenting cell precursor that presents a peptide or a processed form thereof on its surface, wherein the peptide comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NOrI].

[0013] In some embodiments, the composition comprises a peptide comprising, consisting or consisting essentially of an amino acid corresponding to the corresponding to the sequence FLGYLILGV [SEQ ID NO:1], wherein the peptide is in particulate form (e.g., attached to, contained within, or otherwise associated with a particle).

[0014] In some embodiments, the composition comprises a peptide comprising, consisting or consisting essentially of an amino acid corresponding to the corresponding to the sequence FLGYLILGV [SEQ ID NO:1] and an antigen-binding molecule (e.g., an antibody). In certain embodiments, the peptide is conjugated to or otherwise associated with the antigen-binding molecule.

[0015] In some embodiments, the composition comprises a nucleic acid molecule from which a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is expressible.

Suitably, the nucleic acid molecule comprises a nucleotide sequence that encodes the peptide and that is operably connected to a regulatory polynucleotide.

[0016] In another aspect, the present invention provides an immunogenic composition, the composition comprising a molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence

corresponding to the sequence FLGYLILGV [SEQ ID NO: I]. [0017] In yet another aspect, the present invention provides a composition for stimulating an immune response in a subject, the composition comprising a molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I]. In certain embodiments, the immune response is a T-cell mediated response {e.g., a cytotoxic lymphocyte (CTL) mediated immune response). In another aspect, the present invention provides a composition for preventing or treating a cancer (e.g., a hormone-related cancer such as prostate cancer, breast cancer, or ovarian cancer) in a subject, the composition comprising a molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I].

[0018] In some embodiments, the composition comprises T-lymphocytes that are primed to respond to a peptide comprising, consisting or consisting essentially of an amino acid corresponding to the sequence FLGYLILGV [SEQ ID NO: I]. In certain embodiments, the T lymphocytes are cytotoxic (CTL) T lymphocytes.

[0019] In some embodiments, the composition comprises an antigen-binding molecule (e.g., an antibody) that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence

FLGYLILGV [SEQ ID NO:1].

[0020] In another aspect, the present invention provides a method for producing a composition of the present invention.

[0021] In some embodiments, the method comprises a method for producing an immunostimulatory antigen-presenting cell or antigen-presenting cell precursor, the method comprising contacting an antigen-presenting cell or antigen-presenting cell precursor with a peptide that comprises, consists or consists essentially of an amino acid sequence

corresponding to the sequence FLGYLILGV [SEQ ID NO:1], or a nucleic acid molecule from which the peptide is expressible, for a time and under conditions sufficient for the peptide or a processed form thereof to be presented by the antigen-presenting cell or antigen-presenting cell precursor.

[0022] In some embodiments, the method comprises producing T-lymphocytes that are primed to respond to a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], the method comprising contacting antigen-presenting cells that present the peptide or a processed form thereof on their surface with a population of T lymphocytes for a time and under conditions sufficient to produce T lymphocytes that are primed to respond to the peptide ("primed T lymphocytes").

[0023] Another aspect of the present invention provides methods for stimulating an immune response in a subject. In certain embodiments, the immune response is a T-cell mediated response (e.g., a cytotoxic lymphocyte (CTL) mediated immune response). A further aspect of the present invention provides methods for preventing or treating a cancer (e.g. , a hormone-related cancer such as prostate cancer, breast cancer, or ovarian cancer) in a subject. These methods generally comprise administering to the subject an effective amount of a composition as broadly described above.

[0024] Yet another aspect of the present invention provides the use of a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], a nucleic acid molecule from which the peptide is expressible, or a molecule that is immuno-interactive with the peptide, in the manufacture of a medicament for stimulating an immune response or for preventing or treating a cancer in a subject. In certain embodiments, the immune response is a T-cell mediated response (e.g., a cytotoxic lymphocyte (CTL) mediated immune response). In some embodiments, the cancer is a hormone-related cancer (e.g., prostate cancer, ovarian cancer, or breast cancer). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows graphs of induction of human K4 (hK4)-specific CTL responses in four healthy donors (HDl, HD2, HD3, and HD4) (individual results of Example 2). Dendritic cells (DCs) were loaded with hK4 peptides A-E and used to stimulate autologous PBMC from each healthy donor in vitro. Cultures were restimulated on a weekly basis with hK4 A-E peptide - pulsed irradiated PBMC. Induction of specific responses were assessed prior to each stimulation by ELISPOT using T2 cells pulsed with individual hK4 peptides A-E as stimulators. A response to a given peptide was deemed positive only if the number of IFN-γ producing cells following stimulation with T2 cells pulsed with specific "test" peptide was significantly higher than those stimulated with control T2 cells by student's paired t-test of triplicate wells. Data represent the number of specific IFN-γ producing cells ("test" minus "control") of positive responses to each peptide. [0026] Figure 2 shows graphs of induction of hK4-specific CTL responses in four prostate cancer patients (PCaI, PCa2, PCa3, and PCa4) (individual results of Example 2). DCs were loaded with hK4 peptides A-E and used to stimulate autologous PBMC. Cultures were restimulated on a weekly basis with hK4 A-E peptide - pulsed irradiated PBMC.

Induction of specific responses were assessed prior to each stimulation by ELISPOT using T2 cells pulsed with individual hK4 peptides A-E as stimulators. A response to a given peptide was deemed positive only if the number of IFN-γ producing cells following stimulation with T2 cells pulsed with specific "test" peptide was significantly higher than those stimulated with control T2 cells by student's paired t-test of triplicate wells. Data represent the number of specific IFN-γ producing cells ("test" minus "control") of positive responses to each peptide.

[0027] Figure 3 shows consolidated results from Example 2 investigating induction of hK4-specific CTL responses where (a) shows hK4 A-E-specific responses induced in a representative donor (PCa3) by ELISPOT after three stimulations. Data represent the mean ± SD of the number of specific IFN-γ producing cells per 10⁴ CD8⁺ T cells from triplicate wells. A response to a given peptide was deemed positive only if the number of IFN-γ producing cells was significantly higher than those stimulated with control T2 cells by student's unpaired-test. Data was analysed by 1 way ANOVA were p<0.05 is significant; (b) shows maximum responses induced to hK4 peptides A-E by ELISPOT in healthy donors (open symbols) and prostate cancer patients (closed symbols). Data represent the mean number of specific IFN-γ producing cells (T2 cells pulsed with specific peptide-control T2 cells) per 10⁴ CD8⁺ T cells. Data was analysed by 1 way ANOVA where p<0.05 is significant. Donors were as follows: HDl = 0 [open diamond]; HD2 = α [open square]; HD3 = V [open triangle];

HD4 = o [open circle]; PCaI = * [closed star]; PCa2 =• [closed circle]; PCa3 = * [closed asterisk]; PCa4 = T [closed triangle]; PCa5 =♦ [closed diamond]; PCa6 =■ [closed square]; (c) shows expansion of hK4A-specific CD8⁺ T cells in a representative donor (PCa5) using hK4A/HLA-A*0201 pentamer.

[0028] Figure 4 shows graphs of the generation of CD8⁺ CTL clones specific for hK4 peptides A and C as described in Example 3. CTL clones specific for hK4A peptide (a and c) or hK4C peptide (b and d) were isolated from polyclonal CTL by an IFN-γ secretion assay followed by flow cytometry sorting and cloning by limit dilution. The specificity of each clone was assessed by examining the number of IFN-γ producing cells by ELISPOT following stimulation with T2 cells pulsed with specific or irrelevant control (a and b), and by their capacity to lyse T2 cells pulsed with specific (c) or irrelevant (d) pulsed peptide by ⁵¹Cr release assay; (e) Clones do not cross-react with other hK4 peptide epitopes. Clones specific for hK4A (A5) or hK4C (C39) were screened for reactivity against hK4 peptides A-E by ELISPOT; (f) A5 (anti-hK4A) and Cl 7 (anti-hK4C) clones recognise target MHC/peptide complexes with high avidity down to 5pg/ml.

[0029] Figure 5 shows expression of KLK4-254 (Exon 1 -4) and KLK4-205 (Exon 2-5) transcripts in the prostate cancer cell lines LNCaP (HLA- A2⁺), PC-3 (HLA- A2^') and HLA-A2⁺ ovarian cancer cell lines CaOV3 and OVCAR-3 by PCR.

[0030] Figure 6 shows some results from Example 4, being graphs which show that hK4A but not hK4C is endogenously processed and presented by tumour cells and DC, where (a) shows CTL clone specific for hK4A lyses hK4 A-pulsed but not control T2 by ⁵¹Cr release assay. The same clone lyses HLA-A*0201⁺hK4⁺ prostate and ovarian cancer cell lines LNCaP and CaO V3 but not control HLA-A*0201^" prostate cell line PC3 (c), and MoDC transfected with KLK4 RNA (e). A CTL clone specific for hK4C lyses hK4 C-pulsed but not control T2 by ⁵¹Cr release assay (b) but does not lyse HLA-A*0201⁺hK4⁺ expressing tumour cell lines (d).

[0031] Figure 7 shows generation of hK4A-specific responses using DC from prostate cancer patients electroporated with KLK4-254 mRNA as described in Example 4. The percentage of hK4A/HLA-A*0201 pentamer positive CD8+ T cells after 4 stimulations from 1 of 2 representative donors (PCa5) is shown.

[0032] Figure 8 shows graphs showing that peptide/MHC pentamer complexes can monitor induction of hK4A-specific immune responses. hK4A-specific pentamers were synthesised by Proimrnune and used to monitor the induction of hK4-specific responses in a healthy donor (a and b) and prostate cancer patient (c and d). Data represent the percentage of hK4A pentamer specific CD8⁺ T cells in precursor PBMC (a and c) or after 3 rounds of in vitro stimulation (b and d). (e) shows hK4A pentamer staining of hK4A-specific CTL clone A5.

[0033] Figure 9 shows the results of Example 6, showing induction of CD8⁺ T cell responses specific for hK4A, PSA_154-I63, PAP_299-307 and PSMA_7H-719 in HLA-A*0201⁺ prostate cancer patients (closed symbols) or healthy donors (open symbols) using specific peptide/ HLA-A* 0201 pentamers. Data represent the maximum percentage of pentamer positive CD8⁺ T cells for each peptide for up to 4 stimulations. Data was analysed by 1 way ANOVA where p<0.05 is significant.

[0034] Figure 10 shows the CD8⁺ T cell response to hK4A in a HLA-Al I⁺ prostate cancer patient. DC from a HLA-A*02017HLA-Al l⁺prostate cancer patient were loaded with hK4A peptide and used to stimulate autologous CD8⁺ T cells. Cultures were restimulated weekly with autologous hK4A-ρulsed PBMC for 4 stimulations and the percentage of peptide-specific CTL was analysed using specific hK4A/MHC pentamer. Data represent the maximum percentage of hK4A-specific pentamer positive CD8⁺ T cells during the culture period. DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0036] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0037] The term "about" is used herein to refer to conditions (e.g., amounts, concentrations, time etc) that vary by as much as 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% to a specified condition.

[0038] By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins

(antibodies), immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.

[0039] By "autologous" is meant something (e.g. , cells, tissues etc) derived from the same organism.

[0040] The term "allogeneic" as used herein refers to cells, tissues, organisms etc that are of different genetic constitution. [0041] By "antigen" is meant all, or part of, a protein, peptide, or other molecule or tnacromolecule capable of stimulating an immune response in a vertebrate animal, especially a mammal. Such antigens are also reactive with antibodies from animals immunised with that protein, peptide, or other molecule or macromolecule.

[0042] By "biologically active fragment" is meant a fragment of a full-length parent peptide or polypeptide which fragment retains an activity of the parent peptide or polypeptide. As used herein, the term "biologically active fragment" includes deletion mutants and small peptides, for example of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous amino acids, which comprise an activity of the parent peptide polypeptide. Peptides of this type may be obtained through the application of standard recombinant nucleic acid techniques as, for example, described in Sambrook et al. MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), in particular Sections 16 and 17; Ausubel et al CURRENT

PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al. CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.

Alternatively, peptides of this type may be synthesised using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS : A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989), see particularly Chapter 9, or by Roberge et al. (1995 Science 269: 202). Alternatively, peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid

chromatographic (HPLC) techniques.

[0043] As used herein, the term "cis-acting sequence" or "cis-regulatory region" or similar term shall be taken to mean any sequence of nucleotides which is derived from an expressible genetic sequence wherein the expression of the genetic sequence is regulated, at least in part, by the sequence of nucleotides. Those skilled in the art will be aware that a cis- regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of any structural gene sequence. [0044] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0045] The terms "construct" and "synthetic construct" are used interchangeably herein to refer to heterologous nucleic acid sequences that are operably linked to each other and may include sequences providing the expression of a polynucleotide in a host cell and optionally sequences that provide for the maintenance of the construct.

[0046] As used herein, "culturing," "culture" and the like refer to the set of procedures used in vitro where a population of cells (or a single cell) is incubated under conditions which have been shown to support the growth or maintenance of the cells in vitro. The art recognises a wide number of formats, media, temperature ranges, gas concentrations etc. which need to be defined in a culture system. The parameters will vary based on the format selected and the specific needs of the individual who practices the methods herein disclosed. However, it is recognised that the determination of culture parameters is routine in nature.

[0047] By "derivative" is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. The term "derivative" also includes within its scope alterations that have been made to a parent sequence including additions, or deletions that provide for functionally equivalent molecules.

[0048] By "effective amount", in the context of stimulating an immune response or preventing or treating a cancer, is meant the administration of that amount of composition to an individual in need thereof, either in a single dose or as part of a series, that is effective for that stimulation, prevention or treatment. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. [0049] By "expression vector" is meant any autonomous genetic element capable of directing the synthesis of a protein encoded by the vector. Such expression vectors are known by practitioners in the art.

[0050] The term "gene" is used in its broadest context to include both a genomic DNA region corresponding to the gene as well as a cDNA sequence corresponding to exons or a recombinant molecule engineered to encode a functional form of a product.

[0051] Reference herein to "immuno-inter active" includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.

[0052] "Inactivation" of a cell is used herein to indicate that the cell has been rendered incapable of cell division to form progeny. The cell may nonetheless be capable of response to stimulus, or biosynthesis and/or secretion of cell products such as cytokines. Methods of inactivation are known in the art. Preferred methods of inactivation are treatment with toxins such as mitomycin C, or irradiation. Cells that have been fixed or permeabilised and are incapable of division are also examples of inactivated cells.

[0053] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state.

[0054] Reference herein to "immunodeficient" includes reference to any condition in which there is a deficiency in the production of humoral and/or cell-mediated immunity.

[0055] A composition is "immunogenic" if it is capable of either: a) generating an immune response against an antigen (e.g., a tumour antigen) in a naϊve individual; or b) reconstituting, boosting, or maintaining an immune response in an individual beyond what would occur if the compound or composition was not administered. A composition is immunogenic if it is capable of attaining either of these criteria when administered in single or multiple doses.

[0056] By "modulating" is meant increasing or decreasing, either directly or indirectly, the level and/or functional activity of a target molecule. For example, an agent may indirectly modulate the said level/activity by interacting with a molecule other than the target molecule. In this regard, indirect modulation of a gene encoding a target polypeptide includes within its scope modulation of the expression of a first nucleic acid molecule, wherein an expression product of the first nucleic acid molecule modulates the expression of a nucleic acid molecule encoding the target polypeptide. In certain embodiments, "modulation" or "modulating'^'1 means that a desired/selected response is more efficient (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), more rapid (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), greater in magnitude (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), and/or more easily induced (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more) than if the antigen had been used alone.

[0057] By "obtained from" is meant that a sample such as, for example, a cell sample, a nucleic acid extract or polypeptide extract is isolated from, or derived from, a particular source of the host. For example, the extract may be obtained from a tissue or a biological fluid isolated directly from the host.

[0058] The term "5 ' non-coding region" is used herein in its broadest context to include all nucleotide sequences which are derived from the upstream region of an expressible gene, other than those sequences which encode amino acid residues which comprise the polypeptide product of said gene, wherein 5' non-coding region confers or activates or otherwise facilitates, at least in part, expression of the gene.

[0059] The term "oligonucleotide" as used herein refers to a polymer composed of a multiplicity of nucleotide units (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotides and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs),

phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule may vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotides, but the term can refer to molecules of any length, although the term

"polynucleotide" or "nucleic acid" is typically used for large oligonucleotides.

[0060] The term "operably connected" or "operably linked" as used herein means placing a structural gene under the regulatory control of a promoter, which then controls the transcription and optionally translation of the gene. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the genetic sequence or promoter at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting; i.e., the gene from which the genetic sequence or promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the genes from which it is derived.

[0061] The terms "patient," "subject," "hosf or "individual" used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, most particularly a human, for whom prophylaxis or therapy is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, humans, any member of the subphylum Chordata including primates, rodents (e.g. , mice rats, guinea pigs), lagomorphs (e.g. , rabbits, hares), bovines (e.g. , cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards etc), and fish. The subject may be in need of prophylaxis or treatment for a cancer, however, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0062] The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotides in length.

[0063] "Polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same.

[0064] Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5', of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. Preferred promoters according to the invention may contain additional copies of one or more specific regulatory elements to further enhance expression in a cell, and/or to alter the timing of expression of a structural gene to which it is operably connected. [0065] The term "recombinant polynucleotide" as used herein refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature. For example, the recombinant polynucleotide may be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.

[0066] By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant polynucleotide.

[0067] The term sequence "identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base {e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, GIy, VaI, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu, Asn, GIn, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention,

"sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[0068] The term sequence "similarity" refer to the percentage number of amino acids that are identical or constitute conservative amino acid substitutions as defined in Table 1 below. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al, 1984 Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0069] As used herein "stimulating" an immune or immunological response refers to initiating, boosting, or maintaining the capacity for the host's immune system to react to a target substance or antigen, such as a tumour cell, at a level higher than would otherwise occur. The immune response that is stimulated may be a primary or a secondary immune response. Stimulating a "primary" immune response refers herein to eliciting specific immune reactivity in a subject in which previous reactivity was not detected; for example, due to lack of exposure to the target antigen, refractoriness to the target, or immune suppression. Stimulating a "secondary" response refers to the reinitiation, boosting, or maintenance of reactivity in a subject in which previous reactivity was detected; for example, due to natural immunity, spontaneous immunisation, or treatment using one or several compositions or procedures.

[0070] By "vector" is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art. 2. Compositions

[0071] The present invention is based at least in part on the discovery of a peptide from the human kallikrein 4 molecule (hK4 or KLK4) that is a cancer tumour-associated antigen (TAA) epitope. The peptide is referred to herein as peptide A or hK4A. Peptide A is located in the signal sequence region of exon 1 of hK4 in the hK4-254 isoform, at residues 11-19, and has the sequence FLGYLILGV [SEQ ID NO:1]. The inventors have demonstrated that peptide A is endogenously processed by host cells including antigen-presenting cells (e.g., dendritic cancer cells), and which is capable of inducing strong immune responses against cancer cells (e.g., prostate cancer cells). Thus the present inventors propose that peptides that comprise, consist or consist essentially of an amino acid sequence corresponding to peptide A are useful in immunotherapy, especially cancer immunotherapy.

[0072] Accordingly, in one aspect the present invention provides an immunogenic composition, the composition comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a nucleic acid molecule from which the peptide is expressible.

[0073] In another aspect, the present invention provides a composition for stimulating an immune response in a subject, the composition comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a nucleic acid molecule from which the peptide is expressible. In certain embodiments, the immune response is a T-cell mediated response (e.g., a cytotoxic lymphocyte (CTL) mediated immune response). In another aspect, the present invention provides a composition for preventing or treating a cancer (e.g., a hormone- related cancer such as prostate cancer, breast cancer, or ovarian cancer) in a subject, the composition comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a nucleic acid molecule from which the peptide is expressible.

[0074] In another aspect, the present invention provides an immunogenic composition, the composition comprising a molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence

corresponding to the sequence FLGYLILGV [SEQ ID NO: I].

[0075] In yet another aspect, the present invention provides a composition for stimulating an immune response in a subject, the composition comprising a molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I]. In certain embodiments, the immune response is a T-cell mediated response (e.g., a cytotoxic lymphocyte (CTL) mediated immune response). In another aspect, the present invention provides a composition for preventing or treating a cancer (e.g., a hormone-related cancer such as prostate cancer, breast cancer, or ovarian cancer) in a subject, the composition comprising a molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I]. 2.1 Peptides

[0076] The compositions of the present invention may comprise a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I].

[0077] As used in the above statement and in similar statements elsewhere in this specification, the term "comprises" (and the like) means the peptide includes the amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], and may also include any one or more other elements. Thus, the amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is a mandatory element, and any other elements are optional and may or may not be present. Other elements may include, for example, additional amino acid residues at either end of the amino acid sequence, and/or other molecules.

[0078] As used in the above statement and in similar statements elsewhere in this specification, the term "consists essentially of (and the like) means that the peptide includes the amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], and may also include one or more other elements, provided those elements do not interfere with or contribute to the activity or action of the peptide. Thus, the amino acid sequence

corresponding to the sequence FLGYLILGV [SEQ ID NO: 1] is a mandatory element, and other elements are optional and may or may not be present, depending upon whether or not they affect the activity or action of the peptide. For example, where a composition "consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], the amino acid sequence may comprise additional amino acid residues {e.g., by as much as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more additional residues) at either end of the amino acid sequence, and/or may be conjugated or otherwise associated with other molecules {e.g., a protecting moiety such as an N-terminal blocking residue {e.g. , pyroglutamate)), provided those additional residues or molecules do not substantially modulate the immunogenic properties of the amino acid sequence.

[0079] As used in the above statement and in similar statements elsewhere in this specification, the term "consists of (and the like) means that the peptide includes, and is limited to, the amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I]. Thus, the phrase "consists of indicates that the amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: 1] is a mandatory element, and that no other elements (such as amino acid residues at either end of the amino acid sequence or other molecules) may be present. [0080] As used in the statement "amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1]" and similar statements in this specification, the term "corresponding to" or "corresponds to" (and the like) means an amino acid sequence that displays substantial similarity and/or identity to the sequence FLGYLILGV [SEQ ID NO:1], and that can stimulate the desired immune response. In general, the peptide displays at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 % similarity and/or identity to the sequence FLGYLILGV [SEQ ID NO: I].

[0081] In certain embodiments, the sequence of the amino acid may differ from the sequence FLGYLILGV [SEQ ID NO:1] by at least one amino acid substitution, addition, and/or deletion.

[0082] Substituted amino acids may include conservative amino acid substitutions. Non-conservative substitutions may be tolerated, depending on the location of the substituted residue in the peptide, and other factors known to those skilled in the art. Exemplary conservative substitutions are shown in table 1 :

[0083] TABLE 1: Exemplary conservative amino acid substitutions

[0084] Alternatively or in addition, substituted amino acids or added amino acids can be any non-naturally occurring amino acids or derivatives thereof. Non-naturally occurring amino acids include chemical analogues of a corresponding naturally occurring amino acid. Examples of unnatural amino acids and derivatives include, but are not limited to, 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, nor leucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids.

[0085] In some embodiments, the peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is or is a derivative of a homolog or isoform of the sequence FLGYLILGV [SEQ ID NO:1], or displays at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 % similarity and/or identity to a homolog or isoform of the sequence FLGYLILGV [SEQ ID NOrI]. A "homolog" is a molecule from a different species and which is related by descent from a common ancestral DNA sequence. The term "homolog" may apply to the relationship between genes separated by the event of speciation or to the relationship between genes separated by the event of genetic duplication. Human, rat, mouse, dog and chimpanzee K4 homologs are known. For example, k4 homologs having the following accession numbers are listed on the NCBI database: AF228497 for human DNA, NM_004917 for human mRNA, NM_019928 for mouse mRNA, NMJ)Ol 004101 for rat mRNA, BN000381 for rat precursor mRNA, XM_524486 for chimpanzee mRNA (predicted from genomic sequence), and XM__001116184 for Rhesus monkey mRNA (predicted from genomic sequence). An "isoform" is a peptide that has the same function as another peptide but which is encoded by a different polynucleotide and may have small differences in its sequence.

[0086] The peptide may be of any suitable size. A number of factors can influence the choice of peptide size. The size of a peptide can be chosen such that it includes, or corresponds to the size of T cell epitopes and their processing requirements. Practitioners in the art will recognise that class I-restricted T cell epitopes usually range between 8-10 amino acid residues in length. The epitopes may or may not require natural flanking residues. If placed next to unnatural flanking residues, such epitopes can generally require 2 to 3 natural flanking amino acid residues to ensure they are efficiently processed and presented. From the foregoing, it is advantageous, but not essential, that the size of the peptide is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acid residues. Suitably, the size of the peptide is no more than about 60, 50, 40, 30 amino acid residues. In certain advantageous

embodiments, the size of the peptide is sufficient for presentation by an MHC class I molecule of an antigen presenting cell.

[0087] The peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] may be a biologically active fragment of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I]. Reference herein to a "fragment" means a molecule which contains at least about five contiguous amino acids. The biologically active fragment may have the activity associated with the full-length amino acid sequence and/or may have an altered activity. An "altered activity" includes an enhanced activity or loss of a detrimental activity. The biologically active fragment may have the ability to activate an antigen- presenting cell (i.e. the antigen-presenting cell or precursor thereof processes and presents the fragment), as described herein.

[0088] In certain embodiments, the peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] may comprise, consist or consist essentially of a sequence XiX₂X₃X₄XsX₆X₇XsXg, wherein the amino acid residues that are likely to be anchor residues (/. e. , those residues that engage complementary pockets located in the MHC molecule) are amino acid residues that are identical or related to amino acid residues in the sequence FLGYLILGV [SEQ ID NO:1], where "related" means that they have similar chemical features or properties (e.g., aromatic, aliphatic, small). The anchor residue can be readily identified by consulting the available algorithms, including SYFPEITHI and BIMAS algorithms described herein.

[0089] As used herein, an "aromatic amino acid residue" is an amino acid residue which includes an aromatic ring, including those selected from the group consisting of phenylalanine, histidine, tryptophan, and tyrosine. As used herein, an "aliphatic amino acid residue" is an amino acid residue with an aliphatic side chain, including those selected from the group consisting of isoleucine, valine, leucine, alanine, proline, and methionine. As used herein, a "small amino acid residue" is an amino acid residue with a side chain that is not sufficiently large, even if polar groups are lacking, to confer hydrophobicity, including those selected from the group consisting of proline, glycine, serine, alanine and threonine.

[0090] Methods well known in the art can be used to determine whether an amino acid sequence X₁X₂X₃X₄X₅XeXyXsXp, is a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: 1] as herein defined. The predictive methods include consulting the available algorithms, including SYFPEITHI and BIMAS algorithms described herein, and those described in US 2010-0168398 which discusses both "statistical" and "structure-related" methods that can be used to predict whether a particular peptide will bind to an MHC class I molecule. The "statistical" methods are typically based on experimentally obtained affinity data, whilst the "structure-related" methods are typically based on available 3D structural information of MHC molecules. See also, Madden, D. R. et al, 1992 Cell 70: 1035-1048; FaIk, K. et al, 1991 Nature 351 : 290-296; Matsumura, M. et al, 1992 Science 257: 927-934; Saper, M. A. et al, 1991 J. MoI Biol. 219: 277-312; and Latron, F. et al, 1992 Science 257: 964-967. As well as predictive methods, peptides can be synthesised and tested for activity, including in assays such as those known in the art (see for example Fruci, D. et al. 1993 Human Immunology 38(3): 187-192) and described herein (see, for example, the Examples section).

[0091] The peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] may comprise a peptide that has been suitably modified, for example, by lipid modification to modify its physico-chemical properties.

[0092] The peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] may be prepared in recombinant form using standard protocols as, for example, described in Sambrook et al. MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), in particular Sections 16 and 17; Ausubel et al CURRENT PROTOCOLS IN

MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6. Typically, the peptide may be prepared by a procedure including the steps of (a) providing an expression vector from which the peptide is expressible; (b) introducing the vector into a suitable host cell; (c) culturing the host cell to express recombinant peptide from the vector; and (d) isolating the recombinant peptide.

[0093] Alternatively, the peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] can be synthesised using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL

APPROACH (IRL Press at Oxford University, Oxford, England, 1989) or by Roberge et al. (1995 Science 269: 202). Syntheses may employ, for example, either t-butyloxycarbonyl (t- Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) chemistries (see Chapter 9.1 of Coligan et al. supra; Stewart and Young, 1984, SOLID PHASE PEPTIDE SYNTHESIS, 2^nd ed. Pierce Chemical Co., Rockford, 111, 1994; and Atherton and Shephard, supra).

2.2 Nucleic acid molecules from which the peptides are expressible

[0094] The compositions of the present invention may comprise a nucleic acid molecule from which a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is expressible.

[0095] In some embodiments, the nucleic acid molecule may comprise a naturally- occurring polynucleotide encoding a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I]. In certain embodiments, the nucleic acid molecule comprises the sequence TTC CTG GGG TAC CTC ATC CTT GGT GTC [SEQ ID NO: 2].

[0096] In some embodiments, the nucleic acid molecule may comprise a variant of a naturally-occurring polynucleotide encoding a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: 1], including but not limited to a nucleic acid molecule engineered using recombinant techniques. In one example of a variant, the codon composition of the polynucleotide is modified to permit enhanced expression of the peptide by replacing existing or naturally- occurring codons in the peptide-encoding polynucleotide with synonymous codons that have a higher translational efficiency than the existing or naturally occurring codons. In some embodiments, the codon composition of the peptide-encoding polynucleotide is modified using methods as set forth in detail in WO 99/02694 and WO 00/42215. Briefly, these methods are based on the observation that translational efficiencies of different codons vary between different cells or tissues and that these differences can be exploited, together with codon composition of a gene, to regulate expression of a peptide in a particular cell or tissue type. Thus, for the construction of codon-optimised polynucleotides, at least one existing codon of a parent polynucleotide is replaced with a synonymous codon that has a higher translational efficiency in a target cell or tissue than the existing codon it replaces. Although it is preferable to replace all the existing codons of a parent nucleic acid molecule with synonymous codons which have that higher translational efficiency, this is not necessary because increased expression can be accomplished even with partial replacement. Suitably, the replacement step affects 5, 10, 15, 20, 25, 30%, more preferably 35, 40, 50, 60, 70% or more of the existing codons of a parent polynucleotide.

[0097] In some embodiments, the nucleic acid molecule hybridises under at least low, medium, high or very high stringency conditions to a nucleic acid molecule that comprises a nucleotide sequence encoding a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO : 1 ] , or a complement thereof.

[0098] As used herein, the term "hybridises" or "hybridisation" is used to refer to the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or DNA- RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA, U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" are used herein to refer to the hybridisation potential of paired nucleotides in complementary nucleic acid strands.

Matched nucleotides hybridise efficiently, such as the classical A-T (or A-U) and C-G base pairs mentioned above. Mismatches are other combinations of nucleotides that do not hybridise efficiently.

[0099] The phrase "hybridising specifically to" and the like refer to the binding, duplexing, or hybridising of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture {e.g., total cellular DNA or RNA).

[0100] As used herein, the term "hybridises under low stringency, medium stringency, high stringency or very high stringency" describes conditions for hybridisation and washing. Guidance for performing hybridisation reactions can be found in Ausubel et al, (1998, supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used. Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridisation at 42⁰C, and at least about 1 M to at least about 2 M salt for washing at 42⁰C. Low stringency conditions may also include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridisation at 65⁰C, and (i) 2 x sodium chloride/sodium citrate (SSC), 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA₅ 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at room

temperature. One embodiment of low stringency conditions includes hybridisation at 6 x SSC at about 45⁰C, followed by two washes in 0.2 x SSC, 0.1% SDS at least at 50⁰C (the temperature of the washes can be increased to 55°C for low stringency conditions). Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for

hybridisation at 42⁰C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55⁰C. Medium stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M

NaHPO₄ (pH 7.2), 7% SDS for hybridisation at 65°C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at 60-65⁰C. One embodiment of medium stringency conditions includes hybridising in 6 x SSC at about 45°C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 60 C. High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridisation at 42°C, and about 0.01 M to about 0.02 M salt for washing at 55°C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridisation at 65°C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65⁰C. One embodiment of high stringency conditions includes hybridising in 6 x SSC at about 45⁰C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65°C.

[0101] Other stringency conditions are well known in the art and a skilled addressee will recognise that various factors can be manipulated to optimise the specificity of the hybridisation. Optimisation of the stringency of the final washes can serve to ensure a high degree of hybridisation. For detailed examples, see Ausubel et al. {supra) at Chapters 2.10.1 to 2.10.16 and Sambrook et al. {supra) at Sections 1.101 to 1.104. 2.3 Antigen-presenting cells and antigen-presenting cell precursors

[0102] In some embodiments, the composition comprises an immunostimulatory antigen-presenting cell or antigen-presenting cell precursor that presents a peptide or a processed form thereof on its surface, wherein the peptide comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1].

[0103] Such antigen-presenting cells and antigen-presenting cell precursors include professional or facultative antigen-presenting cells. Professional antigen-presenting cells function physiologically to present antigens in a form that is recognised by specific T cell receptors so as to stimulate or anergise a T lymphocyte or B lymphocyte mediated immune response. Professional antigen-presenting cells not only process and present antigens in the context of the major histocompatibility complex (MHC), but also possess the additional immunoregulatory molecules required to complete T cell activation or induce a tolerogenic response. Professional antigen-presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone Kupffer cells, microglia, T cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells. Non-professional or facultative antigen-presenting cells typically lack one or more of the immunoregulatory molecules required to complete T lymphocyte activation or anergy. Examples of non- professional or facultative antigen-presenting cells include, but are not limited to, activated T lymphocytes, eosinophils, keratinocytes, astrocytes, follicular cells, microglial cells, thymic cortical cells, endothelial cells, Schwann cells, retinal pigment epithelial cells, myoblasts, vascular smooth muscle cells, chondrocytes, enterocytes, thymocytes, kidney tubule cells and fibroblasts. In some embodiments, the antigen-presenting cell is selected from monocytes, macrophages, B lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells. In certain advantageous embodiments, the antigen-presenting cell or precursor thereof expresses a molecule that can be used to isolate the cell and/or can be used to target the cell, including but not limited to CMRF56, CDl Ic, CD141, Clec9A, CLEC9A, DEC-205, MCL, DCL-I, macrophage mannose R, DC-SIGN or other DC or myeloid specific (lectin) receptors, and includes a dendritic cell or a Langerhans cell. Suitably the molecule is expressed on the antigen-presenting cell or antigen-presenting cell precursor at higher levels than on other cells. [0104] In another aspect, the composition of the present invention comprises (a) a peptide that comprises, consists or consists essentially of an amino acid sequence

corresponding to the sequence FLGYLILGV [SEQ ID NO: I]₅ a nucleic acid molecule from which the peptide is expressible, a delivery vehicle comprising the peptide as described in the following section, a modified form of the peptide as described in the following section, and (b) an antigen-presenting cell or antigen-presenting cell precursor.

2.3.1 Preparing antigen-presenting cells

[0105] Yet a further aspect of the present invention provides a method for producing an immunostimulatory antigen-presenting cell or antigen-presenting cell precursor, the method comprising contacting an antigen-presenting cell or antigen-presenting cell precursor with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], or a nucleic acid molecule from which the peptide is expressible, for a time and under conditions sufficient for the peptide or a processed form thereof to be presented by the antigen-presenting cell or antigen-presenting cell precursor.

[0106] Immunostimulatory antigen-presenting cells (APCs) or antigen-presenting cell precursors may be prepared according to any suitable method known to the skilled practitioner. Illustrative preparation methods are described by Albert et al. (WO 99/42564), Takamizawa et al. 1997 J Immunol, 158(5): 2134-2142, Thomas and Lipsky 1994 J Immunol 153(9): 4016-4028, 0'Doherty et al 1994 Immunology 82(3): 487-93, Fearnley et al. 1997 Blood, 89(10): 3708-3716, Weissman et al. 1995 Proc Natl Acad Sd USA, 92(3): 826-830, Freudenthal and Steinman 1990 Proc Natl Acad Sci USA 87(19): 7698-7702, Romani et al. 1996 J Immunol Methods 196(2): 137-151, Reddy et al. 1997 Blood 90(9): 3640-3646, Thurnher et al. 1997 Exp Hematol 25(3): 232-237, Caux et al. 1996 J Exp Med 184(2): 695- 706; Caux et al.1996 Blood 87(6): 2376-85, Luft et al. 1998 Exp Hematol 26(6): 489-500; UxSi et al 1998 J Immunol 161(4): 1947-1953, Cdla et al. 1999 J Exp Med 189(5): 821-829; Cella et al 1997 Nature 388(644): 782-787; Cella et al. 1996 J Exp Med 184(2): 747-572, Ahonen et al. 1999 Cell Immunol 197(1): 62-72 and Piemonti et. al 1999 J Immunol 162(11): 6473-6481).

[0107] In some embodiments, the antigen-presenting cells or antigen-presenting cell precursors are purified or isolated from a host, prepared and then re-introduced or reinfused into the host. Conveniently, antigen-presenting cells or precursors thereof can be obtained from the host to be treated either by surgical resection, biopsy, blood sampling (including from whole blood, fresh blood or a fraction thereof), or other suitable technique. Purification and isolation techniques may be utilised, and are well known to those skilled in the art. Such cells are referred to herein as "autologous" cells. In other embodiments, the antigen-presenting cells, antigen-presenting cell precursors, or cell lines are purified, isolated and/or cultured from a different source than the host. Such cells are referred to herein as "allogeneic" cells. Purification and isolation techniques may be utilised, and are well known to those skilled in the art. In certain embodiments, the antigen-presenting cells, antigen- presenting cell precursors, or cell lines are highly susceptible to treatment with at least one IFN as described in WO 01/88097 (i.e., implied high level expression of class I HLA).

[0108] In some embodiments, autologous or allogeneic antigen-presenting cells or antigen-presenting cell precursors may be purified or isolated from a sample using an antigen- binding molecule (e.g., an antibody) that is immuno-interactive with a molecule expressed on the surface of the antigen-presenting cell or antigen-presenting cell precursor, including but not limited to CMRF56, CDl Ic, CD141, Clec9A, CLEC9A, DEC-205, MCL, DCL-I, macrophage mannose R, DC-SIGN or other DC or myeloid specific (lectin) receptors.

Suitably the molecule is expressed on the antigen-presenting cell or antigen-presenting cell precursor at higher levels than on other cells.

[0109] In some embodiments, antigen-presenting cells or antigen-presenting cell precursors suitable for use in the compositions, methods and uses of the present invention are made by a process including contacting, activating, or 'pulsing' the antigen-presenting cells or precursor thereof with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] for a time and under conditions sufficient to permit the peptide to be internalised by the antigen-presenting cells or precursor thereof; and culturing the antigen-presenting cells or precursor thereof so contacted for a time and under conditions sufficient for the peptide to be processed for presentation by the antigen-presenting cells or antigen-presenting cell precursors. The cells presenting the peptide or processed form thereof can then be used to stimulate autologous or allogeneic T-cells in vitro or in vivo.

[0110] The antigen-presenting cell or precursor thereof may be contacted with the peptide in vitro. The amount of peptide to be placed in contact with antigen-presenting cells, or antigen-presenting cell precursors can be determined empirically by persons of skill in the art. Typically antigen-presenting cells or antigen-presenting cell precursors are incubated with peptide in vitro for about 1 to 6 hours at 37° C. Usually, for purified peptides, 0.1-10 μg/mL is suitable for producing antigen-presenting cells or antigen-presenting cell precursors presenting the peptide or processed form thereof. The peptide should be exposed to the antigen-presenting cells or antigen-presenting cell precursors for a period of time sufficient for those cells to internalise the peptide. The time and dose of peptide necessary for the cells to internalise and present the peptide or processed form thereof may be determined using pulse-chase protocols in which exposure to peptide is followed by a washout period and exposure to a read-out system e.g., peptide-reactive T cells. Once the optimal time and dose necessary for cells to express the peptide or processed form thereof on their surface is determined, a protocol may be used to prepare suitable antigen-presenting cells or antigen- presenting cell precursors. Those of skill in the art will recognise in this regard that the length of time necessary for an antigen-presenting cell or antigen-presenting cell precursor to process and present the peptide or processed form thereof may vary depending on the peptide or form of peptide employed, its dose, and the antigen-presenting cell or antigen-presenting cell precursor employed, as well as the conditions under which peptide loading is undertaken. These parameters can be determined by the skilled artisan using routine procedures.

[0111] In specific embodiments, the peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is solubilised in DMSO (e.g., 100% pure DMSO) at high concentration (e.g., 1 mg peptide/10-30 μL DMSO) so that large pools of peptide do not contain excessive amounts of DMSO when pulsed onto cells.

[0112] The antigen-presenting cell or precursor thereof may also be contacted with the peptide in vivo. As for in vitro methods, the peptide should be exposed to the antigen- presenting cells or antigen-presenting cell precursors for a period of time sufficient for those cells to internalise and present the peptide or processed form thereof. The requisite conditions may be readily determined by those skilled in the art using routine procedures.

2.3.1.1 Enhanced delivery of peptides

[0113] The delivery of the exogenous peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: 1 ] to an antigen-presenting cell or antigen-presenting cell precursor can be enhanced by methods known to practitioners in the art. For example, several different strategies have been developed for delivery of exogenous peptide to the endogenous processing pathway of antigen-presenting cells or their precursors, especially dendritic cells. These methods include the use of delivery vehicles, including targeted delivery. Alternatively or in addition, the methods include modification of the peptide to enhance delivery to antigen-present cells or their precursors.

[0114] The compositions of the present invention include compositions comprising a peptide that comprises, consists or consists essentially of an amino acid sequence

corresponding to the sequence FLGYLILGV [SEQ ID NO:1] and a delivery vehicle as described below, or a modified form of a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO : 1 ] as described below.

[0115] Suitable delivery vehicles include, but are not limited to microparticles including microspheres and nanospheres, polymeres, bacterial ghosts, bacterial

polysaccharides, gel matrices, cationic lipids, polylysine, bacteria, virus-like particles, viruses, gold particles, immune-stimulating complexes, liposomes, virosomes (e.g.,

immunopotentiating reconstituted influenza virosomes or IRIVs), GM-CSF, or antigen- binding molecules (e.g., antibodies). In these embodiments, the peptide may be attached to or encapsulated in the delivery vehicle. "Attached to" refers to attachment of the peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] to the delivery vehicle. For example, attachment may be achieved by means of a covalent linkage or an intermolecular interaction (e.g.

hydrogen-bonds or van der Waals forces). In illustrative examples, the peptide can be anchored on the surface of a lipid containing delivery vehicle, such as a liposome or virosome. "Encapsulated in" refers to the peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: 1] that is inside the delivery vehicle, such as microparticles, bacterial ghosts, attenuated bacteria, virus like particles, attenuated viruses, ISCOMs, liposomes and virosomes.

[0116] The contemplated methods include conjugation of the peptide to an antigen- binding molecule (e.g., an antibody) that is immuno-interactive with an antigen-presenting cell or antigen-presenting cell precursor, insertion of the peptide into pH-sensitive liposomes (Zhou and Huang, 1994 Immunomethods 4: 229-235), osmotic lysis of pinosomes after pinocytic uptake of soluble peptide (Moore et al. 1988 Cell 54: 777-785), coupling of the peptide to potent adjuvants (Aichele et aL 1990 J. Exp. Med. 171 : 1815-1820; Gao et al. 1991 J Immunol. 147: 3268-3273; Schulz et al. 1991 Proc. Natl. Acad. Sci. USA 88: 991-993; Kuzu et al. 1993 Euro. J. Immunol. 23: 1397-1400; and Jondal et al 1996 Immunity 5: 295- 302) and apoptotic cell delivery of the peptide (Albert et al 1998 Nature 392: 86-89; Albert et al 1998 Nature Med. 4: 1321-1324; WO 99/42564; and WO 01/85207). Recombinant bacteria {e.g., E. colϊ) or transfected host mammalian cells may be pulsed onto dendritic cells (as particulate peptide, or apoptotic bodies respectively) for peptide delivery. Recombinant chimeric virus-like particles (VLPs) have also been used as vehicles for delivery of exogenous heterologous peptide to the MHC class I processing pathway of a dendritic cell line

(Bachmann et al. 1996 Eur. J. Immunol. 26(11): 2595-2600).

[0117] In certain embodiments, the peptide is conjugated to an antigen-binding molecule, including an antibody, that is immuno-interactive with a molecule expressed on the surface of an antigen-presenting cell or an antigen-presenting cell precursor, including but not limited to CMRF56, CDl Ic₅ CD141, Clec9A, CLEC9A, DEC-205, MCL, DCL-I,

macrophage mannose R, DC-SIGN or other DC or myeloid specific (lectin) receptors.

[0118] Alternatively, or in addition, a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] may be linked to, or otherwise associated with, a cytolysin to enhance the transfer of the peptide into the cytosol of an antigen-presenting cell or precursor thereof for delivery to the MHC class I pathway. Exemplary cytolysins include saponin compounds such as saponin-containing Immune Stimulating Complexes (ISCOMs) (see e.g., Cox and Coulter, 1997 Vaccine 15(3): 248-256 and US 6,352,697), phospholipases (see, e.g., Camilli et al. 1991 J Exp. Med. 173: 751-754), pore-forming toxins (e.g., an α-toxin), natural cytolysins of gram-positive bacteria, such as listeriolysin O (LLO, e.g., Mengaud et al 1988 Infect. Immun. 56: 766-772 and Portnoy et al 1992 Infect. Immun. 60: 2710-2717), streptolysin O (SLO, e.g., Palmer et al 1998 Biochemistry 37(8): 2378-2383) and perfringolysin O (PFO, e.g. Rossjohn et al. Cell 89(5): 685-692). Where the antigen-presenting cell or precursor thereof is phagosomal, acid activated cytolysins may be advantageously used. For example, listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH conditions within the phagosome), thereby facilitating delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al 1992 Infect. Immun. 60: 2710-2717).

[0119] The cytolysin may be provided together with a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] in the form of a single composition or may be provided as a separate composition, for contacting the antigen-presenting cells or antigen-presenting cell precursors. In one embodiment, the cytolysin is fused or otherwise linked to the peptide, wherein the fusion or linkage permits the delivery of the peptide to the cytosol of the target cell. In another embodiment, the cytolysin and peptide are provided in the form of a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast. Suitably, when the delivery vehicle is a microbial delivery vehicle, the delivery vehicle is non- virulent. In a preferred embodiment of this type, the delivery vehicle is a non-virulent bacterium, as for example described in US 6,287,556, comprising a first polynucleotide encoding a non-secreted functional cytolysin operably linked to a regulatory polynucleotide which expresses the cytolysin in the bacterium, and a second polynucleotide encoding the peptide. Non-secreted cytolysins may be provided by various mechanisms, e.g., absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g., a functional signal sequence mutation), or poisoned microbes, etc.. A wide variety of nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well characterised strains, particularly laboratory strains of E. coli, such as MC4100, MC1061, DH5α, etc.. Other bacteria that can be engineered for the invention include well-characterised, nonvirulent, non-pathogenic strains of Listeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis, etc. In particular embodiments, the bacteria are attenuated to be non-replicative, non-integrative into the host cell genome, and/or non-motile inter- or intra-cellularly.

[0120] In some other embodiments, in order to enhance the class I presentation of the antigen, the peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is modified to comprise an intracellular degradation signal or degron. The degron is suitably a ubiquitin- mediated degradation signal selected from a destabilising amino acid at the amino-terminus of the peptide, a ubiquitin acceptor, a ubiquitin, a biologically active fragment of ubiquitin, or combination thereof.

[0121] Thus, in one embodiment, the peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is modified to include a destabilising amino acid at its amino-terminus so that the peptide so modified is subject to the N-end rule pathway as disclosed, for example, in US 5,093,242 and US 5,122,463. In a preferred embodiment of this type, the destabilising amino acid is selected from isoleucine and glutamic acid, more preferably from histidine tyrosine and glutamine, and even more preferably from aspartic acid, asparagine, phenylalanine, leucine, tryptophan and lysine. In an especially preferred embodiment, the destabilising amino acid is arginine.

[0122] Modification or design of the amino-terminus of the peptide can also be accomplished at the genetic level. Conventional techniques of site-directed mutagenesis for addition or substitution of appropriate codons to the 5' end of an isolated or synthesised peptide-encoding polynucleotide can be employed to provide a desired amino-terminal structure for the encoded peptide. For example, so that the peptide expressed has the desired amino acid at its amino-terminus, the appropriate codon for a destabilising amino acid can be inserted or built into the amino-terminus of the peptide-encoding sequence. Where necessary, a nucleic acid sequence encoding the amino-terminal region of a peptide can be modified to introduce one or more lysine residues in an appropriate context, which act as a ubiquitin acceptor as described in more detail below. This can be achieved most conveniently by employing DNA constructs encoding "universal destabilising segments". A universal destabilising segment comprises a nucleic acid construct which encodes a polypeptide structure, preferably segmentally mobile, containing one or more lysine residues, the codons for lysine residues being positioned within the construct such that when the construct is inserted into the coding sequence of the peptide-encoding polynucleotide, the lysine residues are sufficiently spatially proximate to the amino-terminus of the encoded peptide to serve as the second determinant of the complete amino-terminal degradation signal. The insertion of such constructs into the 5' portion of an peptide-encoding polynucleotide would provide the encoded peptide with a lysine residue (or residues) in an appropriate context for

destabilisation.

[0123] The codon for the amino-terminal amino acid of the peptide can be made to encode the desired amino acid by, for example, site-directed mutagenesis techniques currently standard in the field. Suitable mutagenesis methods are well known in the art. Alternatively, suitable methods for altering DNA are set forth, for example, in US 4,184,917, US 4,321,365 and US 4,351,901. Instead of in vitro mutagenesis, the synthetic polynucleotide can be synthesised de novo using readily available machinery. Sequential synthesis of DNA is described, for example, in US 4,293,652. However, it should be noted that the present invention is not dependent on, and not directed to, any one particular technique for constructing a polynucleotide encoding a modified peptide as described herein. [0124] If the peptide-encoding polynucleotide is a synthetic or recombinant polynucleotide, the appropriate 5' codon can be built-in during the synthetic process.

Alternatively, nucleotides for a specific codon can be added to the 5' end of an isolated or synthesised polynucleotide by ligation of an appropriate nucleic acid sequence to the 5' (amino-terminus-encoding) end of the polynucleotide. Nucleic acid inserts encoding appropriately located lysine residues (such as the "universal destabilising segments" described above) can suitably be inserted into the 5' region to provide for the second determinant of the complete amino-terminal degradation.

[0125] In certain embodiments, the modified peptide, which comprises a destabilising amino acid at its amino terminus, is fused or otherwise conjugated to a masking entity, which masks said amino terminus so that when unmasked the peptide will exhibit the desired rate of intracellular proteolytic degradation. Suitably, the masking entity is a masking peptide sequence. The fusion peptide is designed so that the masking peptide sequence fused to the amino-terminus of the peptide of interest is susceptible to specific cleavage at the junction between the two. Removal of the peptide sequence thus unmasks the amino-terminus of the peptide of interest and the half-life of the released peptide is thus governed by the predesigned amino-terminus. The fusion peptide can be designed for specific cleavage in vivo, for example, by a host cell endoprotease or for specific cleavage in an in vitro system where it can be cleaved after isolation from a producer cell (which lacks the capability to cleave the fusion peptide). Thus, in a preferred embodiment, the masking peptide sequence is cleavable by an endoprotease, which is preferably an endogenous endoprotease of a mammalian cell. Suitable endoproteases include, but are not restricted to, serine endoproteases (e.g., subtilisins and furins) as described, for example, by Creemers et al. (1998 Semin. CellDev. Biol. 9(1): 3- 10), proteasomal endopeptidases as described, for example, by Zwickl et al. (2000 Curr. Opin. Struct. Biol. 10(2): 242-250), proteases relating to the MHC class I processing pathway as described, for example, by Stolze et al. (2000 Nat. Immunol. 1 : 413-418) and signal peptidases as described, for example, by Dalbey et al (1997 Protein ScI 6 (6): 1129-1138). In a preferred embodiment of this type, the masking peptide sequence comprises a signal peptide sequence. Suitable signal peptides sequences are described, for example, by Nothwehr et al. (1990 Bioessays 12(10): 479-484), Izard et al. (1994 MoI Microbiol 13(5): 765-773), Menne, et al. (2000 Bioinformatics 16(8): 741-742) and Ladunga (2000 Curr. Opin. Biotechnol.

11(1): 13-18). Suitably, an endoprotease cleavage site is interposed between the masking peptide sequence and the peptide. [0126] A modified peptide with an attached masking sequence may be conveniently prepared by fusing a nucleic acid sequence encoding a masking peptide sequence upstream of another nucleic acid sequence encoding a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] which includes a destabilising amino acid at its amino- terminus. The codon for the amino-terminal amino acid of the peptide of interest is suitably located immediately adjacent to the 3' end of the masking peptide-encoding nucleic acid sequence.

[0127] In another embodiment, the peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is modified to include, or is otherwise associated with, an ubiquitin acceptor which is a molecule that preferably contains at least one residue appropriately positioned from the N- terminal of the antigen as to be able to be bound by ubiquitin molecules. Such residues preferentially have an epsilon amino group such as lysine. Physical analysis demonstrates that . multiple lysine residues function as ubiquitin acceptor sites (King et al. 1996 MoI. Biol. Cell 7: 1343-1357; King et al. 1996 Science TlA: 1652-1659). Examples of other ubiquitin acceptors include lad or Sindis virus RNA polymerase. Ubiquitination at the N-terminal of the protein specifically targets the protein for degradation via the ubiquitin-proteosome pathway.

[0128] Other protein processing signals that destabilise a peptide of interest and allow for enhanced intracellular degradation are contemplated in the present invention. These other methods may not necessarily be mediated by the ubiquitin pathway, but may otherwise permit degradation of proteins in the cytoplasm via proteosomes. For example, the present invention contemplates the use of other intracellular processing signals which govern the rate(s) of intracellular peptide degradation including, but not limited to, those described by Bohley et al. (1996 Biol. Chem. Hoppe. Seyler 377: 425-435). Such processing signals include those that allow for phosphorylation of the target protein (Yaglom et al. 1996 MoI. Cell Biol. 16: 3679-3684; Yaglom et al. 1995 MoI. Cell Biol. 15: 731-741). Also

contemplated by the present invention are modification of a parent peptide that allows for post-translational arginylation (Ferber et al. 1987 Nature 326: 808-811; Bohley et al. 1991 Biomed. Biochim. Acta 50: 343-346) of the peptide which can enhance its rate of intracellular degradation. Also contemplated is the use of certain structural features of proteins that can influence higher rates of intracellular protein turn-over, including protein surface hydrophobicity, clusters of hydrophobic residues within the protein (Sadis et al. 1995 MoI. Cell Biol. 15: 4086-4094), certain hydrophobic pentapeptide motifs at the protein's carboxy- terminus (C-terminus) {e.g., ARINV) as found on the C-terminus of ornithine decarboxylase (Ghoda et α/. 1992 MoI Cell Biol. 12: 2178-2185; Li e/ al. 1994 MoI Cell Biol. 14: 87-92), or AANDENYALAA as found in C-terminal tags of aberrant polypeptides (Keiler et al. 1996 Science 271 : 990-993) or PEST regions (regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T)), which are optionally flanked by amino acids comprising

electropositive side chains (Rogers et al. 1986 Science 234(4774): 364-368; Rogers et al. 1988, J Biol. Chem. 263: 19833-19842). Moreover, certain motifs have been identified in proteins that appear necessary and possibly sufficient for achieving rapid intracellular degradation. Such motifs include RXALGXIXN region (where X = any amino acid) in cyclins (Glotzer et al. 1991 Nature 349: 132-138) and the KTKRNYSARD motif in isocitrate lyase (Ordiz et al. 1996 FEBS Lett. 385: 43-46).

[0129] The present invention also contemplates enhanced cellular degradation of a parent peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] which may occur by the incorporation into that peptide of known protease cleavage sites. For example amyloid beta- protein can be cleaved by beta- and gamma-secretase (Iizuka et al. 1996 Biochem. Biophys. Res. Commun. 218: 238-242) and the two-chain vitamin K-dependent coagulation factor X can be cleaved by calcium-dependent endoprotease(s) in liver (Wallin et al. 1994 Thromb. Res. 73: 395-403).

[0130] In yet another embodiment, a parent peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is conjugated to a ubiquitin or a biologically active fragment thereof, to produce a modified peptide whose rate of intracellular proteolytic degradation is increased, enhanced or otherwise elevated relative to the parent peptide. In a preferred embodiment of this type, the ubiquitin or biologically active fragment is fused, or otherwise conjugated, to the parent peptide. Suitably, the ubiquitin is of mammalian origin, more preferably of human or other primate origin.

[0131] In one embodiment, the ubiquitin-peptide fusion protein is suitably produced by covalently attaching a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] to a ubiquitin or a biologically active fragment thereof. Covalent attachment may be effected by any suitable means known to persons of skill in the art. For example, peptide conjugates may be prepared by linking proteins together using bifunctional reagents. The bifunctional reagents can be homobifunctional or heterobifunctional.

[0132] Homobifunctional reagents are molecules with at least two identical functional groups. The functional groups of the reagent generally react with one of the functional groups on a peptide, typically an amino group. Examples of homobifunctional reagents include glutaraldehyde and diimidates. An example of the use of glutaraldehyde as a cross-linking agent is described by Poznansky et al. (1984 Science 223: 1304-1306). The use of diimidates as a cross-linking agent is described for example by Wang, et al. (1977

Biochemistry 16: 2937-2941). Although it is possible to use homobifunctional reagents for the purpose of forming a modified peptide according to the invention, skilled practitioners in the art will appreciate that it is difficult to attach different proteins in an ordered fashion with these reagents. In this regard, in attempting to link a first protein with a second protein by means of a homobifunctional reagent, one cannot prevent the linking of the first protein to each other and of the second to each other. Heterobifunctional crosslmking reagents are, therefore, preferred because one can control the sequence of reactions, and combine proteins at will. Heterobifunctional reagents thus provide a more sophisticated method for linking two proteins. These reagents require one of the molecules to be joined, hereafter called Partner B, to possess a reactive group not found on the other, hereafter called Partner A, or else require that one of the two functional groups be blocked or otherwise greatly reduced in reactivity while the other group is reacted with Partner A. In a typical two-step process for forming heteroconjugates, Partner A is reacted with the heterobifunctional reagent to form a derivatised Partner A molecule. If the unreacted functional group of the crosslinker is blocked, it is then deprotected. After deprotecting, Partner B is coupled to derivatised Partner A to form the conjugate. Primary amino groups on Partner A are reacted with an activated carboxylate or imidate group on the crosslinker in the derivatisation step. A reactive thiol or a blocked and activated thiol at the other end of the crosslinker is reacted with an electrophilic group or with a reactive thiol, respectively, on Partner B. When the crosslinker possesses a reactive thiol, the electrophile on Partner B preferably will be a blocked and activated thiol, a maleimide, or a halomethylene carbonyl (e.g., bromoacetyl or iodoacetyl) group. Because biological macromolecules do not naturally contain such electrophiles, they must be added to Partner B by a separate derivatisation reaction. When the crosslinker possesses a blocked and activated thiol, the thiol on Partner B with which it reacts may be native to Partner B. [0133] An example of a heterobifunctional reagent is N-succinimidyl 3-(2- pyridyldithio)propionate (SPDP) (see for example Carlsson et al, 1978, Biochem. J, 173: 723-737). Other heterobifunctional reagents for linking proteins include for example succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) (Yoshitake et al, 1979, EMr. J Biochem, 101 : 395-399), 2-iminothiolane (IT) (Jue et al, 1978, Biochemistry, 17: 5399-5406), and S-acetyl mercaptosuccinic anhydride (SAMSA) (Klotz and Heiney, 1962, Arch. Biochem. Biophys., 96: 605-612). All three react preferentially with primary amines (e.g., lysine side chains) to form an amide or amidine group which links a thiol to the derivatised molecule (e.g., a heterologous antigen) via a connecting short spacer arm, one to three carbon atoms long. Examples of heterobifunctional reagents comprising reactive groups having a double bond that reacts with a thiol group include SMCC mentioned above, succinimidyl m-maleimidobenzoate, succinimidyl 3-(maleimido)propionate,

sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl 4-(N- maleimidomethylcyclohexane- 1 -carboxylate and maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). In a preferred embodiment, MBS is used to produce the conjugate. Other heterobifunctional reagents for forming conjugates of two proteins are described for example in US 4,671,958 and US 5,241,078.

[0134] In an alternate embodiment, a ubiquitin-peptide fusion protein is suitably expressed by a synthetic chimeric polynucleotide comprising a first nucleic acid sequence, which encodes a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], and which is linked, upstream of, downstream of, and in reading frame with, a second nucleic acid sequence encoding a ubiquitin or biologically active fragment thereof.

[0135] The delivery vehicles described above can be used to deliver a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] to virtually any antigen-presenting cell or antigen-presenting cell precursor capable of endocytosis of the subject vehicle, including phagocytic and non-phagocytic antigen-presenting cells and their precursors. In embodiments when the delivery vehicle is a microbe, the subject methods generally require microbial uptake by the target cell and subsequent lysis within the antigen-presenting cell vacuole or antigen-presenting cell precursor vacuole (including phagosomes and endosomes).

[0136] In other embodiments, the peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is produced inside the antigen-presenting cell or antigen-presenting cell precursor by introduction of a suitable expression vector. The portion of the expression vector encoding the peptide may comprise a naturally-occurring polynucleotide sequence or a variant thereof, which has been engineered using recombinant techniques. In one example of a variant, the codon composition of a polynucleotide encoding a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is modified to permit enhanced expression of the peptide by replacing existing or naturally-occurring codons in the peptide-encoding nucleotide sequence with synonymous codons that have a higher translational efficiency than the existing or naturally occurring codons. In some embodiments, the codon composition of the peptide-encoding nucleotide sequence is modified using methods as set forth in detail in WO 99/02694 and WO 00/42215. Briefly, these methods are based on the observation that translational efficiencies of different codons vary between different cells or tissues and that these differences can be exploited, together with codon composition of a gene, to regulate expression of a peptide in a particular cell or tissue type. Thus, for the construction of codon-optimised polynucleotides, at least one existing codon of a parent polynucleotide is replaced with a synonymous codon that has a higher translational efficiency in a target cell or tissue than the existing codon it replaces. Although it is preferable to replace all the existing codons of a parent nucleic acid molecule with synonymous codons which have that higher translational efficiency, this is not necessary because increased expression can be accomplished even with partial replacement. Suitably, the replacement step affects 5, 10, 15, 20, 25, 30%, more preferably 35, 40, 50, 60, 70% or more of the existing codons of a parent polynucleotide.

[0137] The expression vector for introduction into the antigen-presenting cell or antigen-presenting cell precursor will be compatible therewith such that the peptide-encoding polynucleotide is expressible by the cell. For example, expression vectors of this type can be derived from viral DNA sequences including, but not limited to, adenovirus, adeno-associated viruses, herpes-simplex viruses and retroviruses such as B, C, and D retroviruses as well as spumaviruses and modified lentiviruses. Suitable expression vectors for transfection of animal cells are described, for example, by Wu and Ataai (2000 Curr. Opin. Biotechnol. 11(2): 205- 208), Vigna andNaldini (2000 J. Gene Med. 2(5) -.308-316), Kay et al. (2001 Nat. Med. 7(1): 33-40), Athanasopoulos et al (2000 Int. J. MoI. Med. 6(4): 363-375) and Walther and Stein (2000 Drugs 60(2): 249-271). Targeted delivery of the expression vector to the antigen- presenting cells or precursors thereof is contemplated. The expression vector is introduced into the antigen-presenting cell or precursor thereof by any suitable means which will be dependent on the particular choice of expression vector and antigen-presenting cell or antigen-presenting cell precursor employed. Such means of introduction are well-known to those skilled in the art. For example, introduction can be effected by use of contacting (e.g., in the case of viral vectors), electroporation, transformation, transduction, conjugation or triparental mating, transfection, infection membrane fusion with cationic lipids, high- velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitate, direct microinjection into single cells, and the like. Other methods also are available and are known to those skilled in the art. Alternatively, the vectors are introduced by means of cationic lipids, e.g., liposomes. Such liposomes are commercially available (e.g., Lipofectin®, Lipofectamine™, and the like, supplied by Life Technologies, Gibco BRL,

Gaithersburg, Md.). It will be understood by persons of skill in the art that the techniques for assembling and expressing peptide-encoding nucleic acid molecules, immunoregulatory molecules and/or cytokines as described herein e.g., synthesis of oligonucleotides, nucleic acid amplification techniques, transforming cells, constructing vectors, expressions system and the like and transducing or otherwise introducing nucleic acid molecules into cells are well established in the art, and most practitioners are familiar with the standard resource materials for specific conditions and procedures.

[0138] In some embodiments, the antigen-presenting cells or antigen-presenting cell precursors that present on their surface a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a processed form of the peptide are obtained by isolating antigen-presenting cells or their precursors from a cell population or tissue to which modification of an immune response is desired. Typically, some of the isolated antigen-presenting cells or precursors will constitutively present the peptide or a processed form thereof or have taken up such peptide in vivo. In this instance, the delivery of exogenous peptide is not essential. Alternatively, cells may be derived from biopsies of healthy or diseased tissues, lysed or rendered apoptotic and then pulsed onto antigen-presenting cells (e.g., dendritic cells) or antigen-presenting cell precursors. In certain embodiments of this type, the antigen-presenting cells or antigen- presenting cell precursors represent cancer or tumor cells to which an immune response is required. Illustrative examples of cancers or tumor cells include prostate cancer, breast cancer, ovarian cancer, and cells obtained or derived from these cancers.

[0139] In some of the above embodiments, the cancer or tumor cells will constitute facultative or non-professional antigen-presenting cells or antigen-presenting cell precursors, and may in some instances require further modification to enhance their antigen-presenting functions. In these instances, the antigen-presenting cells or antigen-presenting cell precursors are further modified to express one or more immunoregulatory molecules, which include any molecules occurring naturally in animals that may regulate or directly influence immune responses including: proteins involved in antigen processing and presentation such as

TAP1/TAP2 transporter proteins, proteosome molecules such as LMP2 and LMP7, heat shock proteins such as gp96, HSP70 and HSP90, and major histocompatibility complex (MHC) or human leukocyte antigen (HLA) molecules; factors that provide co-stimulation signals for T cell activation such as B7 and CD40; factors that provide co-inhibitory signals for direct killing of T cells or induction of T lymphocyte or B lymphocyte anergy or stimulation of T regulatory cell (Treg) generation such as OX-2, programmed death- 1 ligand (PD-IL); accessory molecules such as CD83; chemokines; lymphokines and cytokines such as IFN s α, β and γ, interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-22, etc.), factors stimulating cell growth (e.g., GM-SCF) and other factors (e.g., tumor necrosis factors (TNFs), DC-SIGN, MIPIa, MlPlβ and transforming growth factor-β (TGF-β). In certain

advantageous embodiments, the immunoregulatory molecules are selected from a B7 molecule (e.g., B7-1, B7-2 or B7-3) and an ICAM molecule (e.g., ICAM-I and ICAM-2).

[0140] Instead of recombinantly expressing immunoregulatory molecules, antigen- presenting cells (e.g., cancer cells) or antigen-presenting cell precursors expressing the desired immunostimulatory molecule(s) may be isolated or selected from a heterogeneous population of cells. Any method of isolation/selection is contemplated by the present invention, examples of which are known to those of skill in the art. For instance, one can take advantage of one or more particular characteristics of a cell to specifically isolate that cell from a heterogeneous population. Such characteristics include, but are not limited to, anatomical location of a cell, cell density, cell size, cell morphology, cellular metabolic activity, cell uptake of ions such as Ca²⁺, K⁺, and H⁺ ions, cell uptake of compounds such as stains, markers expressed on the cell surface, protein fluorescence, and membrane potential. Suitable methods that can be used in this regard include surgical removal of tissue, flow cytometry techniques such as fluorescence-activated cell sorting (FACS), immunoaffinity separation (e.g., magnetic bead separation such as Dynabead™ separation), density separation (e.g., metrizamide, Percoll™, or Ficoll™ gradient centrifugation), and cell-type specific density separation. Desirably, the cells are isolated by flow cytometry or by immunoaffinity separation using an antigen-binding molecule that is immuno-interactive with the immunoregulatory molecule.

[0141] Alternatively, the immunoregulatory molecule can be provided to the antigen-presenting cells (e.g., cancer cells) or antigen-presenting cell precursors in soluble form. In some embodiments of this type, the immunoregulatory molecule is a B 7 molecule that lacks a functional transmembrane domain (e.g., that comprises a B7 extracellular domain), non-limiting examples of which are described by McHugh et al. (1998, Clin.

Immunol. Immunopathol. 87(l):50-59), Faas et α/. (2000, J Immunol. 164(12):6340-6348) and Jeannin et al. (2000, Immunity 13(3):303-312). In other embodiments of this type, the immunostimulatory protein is a B7 derivative including, but not limited to, a chimeric or fusion protein comprising a B 7 molecule, or biologically active fragment thereof, or variant or derivative of these, linked together with an antigen-binding molecule such as an

immunoglobulin molecule or biologically active fragment thereof. For example, a

polynucleotide encoding the amino acid sequence corresponding to the extracellular domain of the B7-1 molecule, containing amino acids from about position 1 to about position 215, is joined to a polynucleotide encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of human Ig Cγl, using PCR, to form a construct that is expressed as a B7Ig fusion protein. DNA encoding the amino acid sequence corresponding to a B7Ig fusion protein has been deposited with the American Type culture Collection (ATCC) in Rockville, Md., under the Budapest Treaty on May 31, 1991 and accorded accession number 68627. Techniques for making and assembling such B7 derivatives are disclosed for example in US 5,580,756. Reference also may be made to Sturmhoefel et al. (1999, Cancer Res. 59: 4964- 4972) who disclose fusion proteins comprising the extracellular region of B7-1 or B7-2 fused in frame to the Fc portion of IgG2a.

[0142] The half-life of a soluble immunoregulatory molecule may be prolonged by any suitable procedure if desired. Preferably, such molecules are chemically modified with polyethylene glycol (PEG), including monomethoxy-polyethylene glycol, as for example disclosed by Chapman et al. (1999, Nature Biotechnology 17: 780-783).

[0143] Alternatively, or in addition, the antigen-presenting cells (e.g., cancer cells) or antigen-presenting cell precursors are cultured in the presence of at least one IFN for a time and under conditions sufficient to enhance the antigen presenting function of the cells and washing the cells to remove the IFN(s). In certain advantageous embodiments of this type, the step of culturing may comprise contacting the cells with at least one type I IFN and/or a type II IFN. The at least one type I IFN is suitably selected from the group consisting of an IFN-α, an IFN-β, a biologically active fragment of an IFN-α, a biologically active fragment of an IFN-β, a variant of an IFN-α, a variant of an IFN-β, a variant of a said biologically active fragment, a derivative of an IFN-α, a derivative of an IFN-β, a derivative of a said

biologically active fragment, a derivative of a said variant, an analogue of IFN-α and an analogue of IFN-β. Typically, the type II IFN is selected from the group consisting of an IFN- γ, a biologically active fragment of an IFN-γ, a variant of an IFN-γ, a variant of said biologically active fragment, a derivative of an IFN-γ, a derivative of said biologically active fragment, a derivative of said variant and an analogue of an IFN-γ. Exemplary methods and conditions for enhancing the peptide-presenting functions of antigen-presenting cells or antigen-presenting cell precursors using IFN treatment are described in WO 2001/88097.

[0144] In some embodiments, the antigen-presenting cells (e.g., cancer cells), antigen-presenting cell precursors, or cell lines are suitably rendered inactive to prevent further proliferation once administered to the subject. Any physical, chemical, or biological means of inactivation may be used, including but not limited to irradiation (generally with at least about 5,000 cGy, usually at least about 10,000 cGy, typically at least about 20,000 cGy); or treatment with mitomycin-C (usually at least 10 μg/mL; more usually at least about 50 μg /mL).

[0145] The antigen-presenting cells or antigen-presenting cell precursors may be prepared to present the peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a processed form thereof by any number of means, such that the peptide or processed form thereof may be presented by those cells for potential modulation of other immune cells, including T lymphocytes and B lymphocytes, particularly for producing T lymphocytes and B

lymphocytes that are primed to respond to a specified antigen or group of antigens.

2.4 Particulate compositions

[0146] In some embodiments, the composition comprises a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], wherein the peptide is in particulate form (e.g., attached to, contained within, or otherwise associated with a particle).

[0147] Desirably, the particle is capable of being taken up (e.g., endocytosis or phagocytosis) by an immune cell such as, but not limited to, an antigen-presenting cell (e.g., a dendritic cell, macrophage or Langerhans cell). In some embodiments, the particle comprises a matrix, carrier or substrate. Representative particles are suitably dimensioned and include nanoparticles and microparticles. In some embodiments, the particle comprises a lipid matrix or carrier such as a cationic lipid, an anionic lipid, non-ionic and/or a zwitterionic lipid, e.g., polyglyceryl alkyl ethers, sphingolipids or a phospholipid (such as phosphatidylcholine). In specific examples of this type, the particle is liposomal. In other embodiments, the particle comprises a carrier particle, such as a metal particle (e.g., a tungsten, gold, platinum or iridium particle). In still other embodiments, the particle comprises a polymeric matrix or carrier, illustrative examples of which include biocompatible polymeric particles (e.g., particles fabricated with poly(lactide-co-glycolide)). In still other embodiments, the particle comprises a ceramic or inorganic matrix or carrier.

[0148] The compositions of the present invention may be encapsulated, adsorbed to, or associated with, particulate carriers. Such carriers can be used to selectively introduce the compositions to cells of the immune system. The particles can be taken up by professional antigen-presenting cells such as macrophages and dendritic cells, and/or can enhance antigen presentation through other mechanisms such as stimulation of cytokine release. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al. 1993 Pharm. Res. 10: 362-368; McGee J. P. et al. 1997 J

Microencapsul, 14(2): 197-210; O'Hagan D. T. etal 1993 Vaccine 11(2): 149-54.

2.4.1 Particles

[0149] In accordance with some embodiments of the present invention, the peptide may be attached to, contained within or otherwise associated with a particle. A variety of particles may be used in the invention, including but not limited to, liposomes, micelles, lipidic particles, ceramic/inorganic particles and polymeric particles, and are typically selected from nanoparticles and microparticles. The particles are suitably sized for phagocytosis or endocytosis by antigen-presenting cells. Antigen-presenting cells include both professional and facultative types of antigen-presenting cells. Professional antigen- presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone

Kupffer cells, microglia, T cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells. Examples of facultative antigen-presenting cells include but are not limited to activated T cells, astrocytes, follicular cells, endothelium and fibroblasts. In some embodiments, the antigen-presenting cell is selected from monocytes, macrophages, B-lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells. In specific embodiments, the antigen-presenting cell expresses CMRF56, CDl Ic, CD141, Clec9A, CLEC9A, DEC-205, MCL, DCL-I, macrophage mannose R, DC-SIGN or other DC or myeloid specific (lectin) receptors, and includes a dendritic cell. In illustrative examples, the particles have a dimension of less than about 100 μm, more suitably in the range of less than or equal to about 1 μm, although the particles may be as large as about 30 μm, and as small as a few nm. Liposomes consist basically of a phospholipid bilayer forming a shell around an aqueous core. Advantages include the lipophilicity of the outer layers which "mimic" the outer membrane layers of cells and that they are taken up relatively easily by a variety of cells. Polymeric vehicles typically consist of micro/nanospheres and

micro/nanocapsules formed of biocompatible polymers, which are either biodegradable (for example, polylactic acid) or non-biodegradable (for example, ethylenevinyl acetate). Some of the advantages of the polymeric devices are ease of manufacture and high loading capacity, range of size from nanometer to micron diameter, as well as controlled release and

degradation profile.

[0150] In some embodiments, the particles comprise an antigen-binding molecule on their surface, which is immuno-interactive with a marker that is expressed at higher levels on antigen-presenting cells {e.g., dendritic cells) than on non-antigen-presenting cells.

Illustrative markers of this type include CMRF56, CDl Ic₅ CD141 , Clec9A, CLEC9A, DEC- 205, MCL, DCL-I, macrophage mannose R, DC-SIGN or other DC or myeloid specific (lectin) receptors, as for example disclosed by Hawiger et al (2001 J Exp. Med. 194: 769), Kato et al (2003 J. Biol. Chem. 278: 34035), Benito et al (2004 J Am. Chem. Soc 126: 10355), Schjetne, et al (2002 Int. Immunol. 14: 1423) and van Vliet et al (2006 Nat.

Immunol. 211: 577-585).

[0151] The particles can be prepared from a combination of the bioactive agent(s), and a carrier matrix (e.g., surfactant, excipient or polymeric material). In some embodiments, the matrices are biodegradable and biocompatible, and optionally are capable of biodegrading at a controlled rate for delivery of a therapeutic or diagnostic agent. The particles can be made of a variety of materials. Both inorganic and organic materials can be used as well as polymeric and non-polymeric materials. Illustrative materials of this type include polar lipids, organic polymers and monomers, poly- and mono-saccharides, ceramic/inorganic materials, polypeptides and proteins. Other suitable materials include, but are not limited to, gelatin, polyethylene glycol, trehalose, dextran and chitosan. Particles with degradation and release times ranging from seconds to months can be designed and fabricated, based on factors such as the particle material.

2.4.2 Polymeric Particles

[0152] Polymeric particles may be formed from any biocompatible and desirably biodegradable polymer, copolymer, or blend. The polymers may be tailored to optimise different characteristics of the particle including: i) interactions between the bioactive agents to be delivered and the polymer to provide stabilisation of the bioactive agents and retention of activity upon delivery; ii) rate of polymer degradation and, thereby, rate of agent release profiles; iii) surface characteristics and targeting capabilities via chemical modification; and iv) particle porosity.

[0153] Surface eroding polymers such as polyanhydrides may be used to form the particles. For example, polyanhydrides such as poly[(p-carboxyphenoxy)-hexane anhydride] (PCPH) may be used. Biodegradable polyanhydrides are described in US 4,857,311.

[0154] In other embodiments, bulk eroding polymers such as those based on polyesters including poly(hydroxy acids) or poly(esters) can be used. For example, polyglycolic acid (PGA), polylactic acid (PLA), or copolymers thereof may be used to form the particles. The polyester may also have a charged or functionalisable group, such as an amino acid. In illustrative examples, particles with controlled release properties can be formed of poly(D,L-lactic acid) and/or ρoly(D,L-lactic-co-glycolic acid) ("PLGA").

[0155] Other polymers include poly(alkylcyanoacrylates), polyamides,

polycarbonates, polyalkylenes such as polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), polyvinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers of acrylic and methacrylic acids, celluloses and other polysaccharides, and peptides or proteins, or copolymers or blends thereof. Polymers may be selected with or modified to have the appropriate stability and degradation rates in vivo for different controlled drug delivery applications.

[0156] In some embodiments, particles are formed from functionalised polymers such as polyester graft copolymers, as described in Hrkach et al. (1995 Macromolecules 28: 4736-4739); and "Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class of

Functional Biodegradable Biomaterials" in Hydrogels and Biodegradable Polymers for Bioapplications, ACS Symposium Series No. 627, Raphael M. Ottenbrite et al, Eds., American Chemical Society, Chapter 8, pp. 93-101, 1996.

[0157] Materials other than biodegradable polymers may be used to form the particles. Suitable materials include various non-biodegradable polymers and various excipients. The particles also may be formed of the bioactive agent(s) and surfactant alone.

[0158] Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerisation, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art, provided that the conditions are optimised for forming particles with the desired diameter.

[0159] Methods developed for making microspheres for delivery of encapsulated agents are described in the literature, for example, as described in Doubrow, M., Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton,¹ 1992. Methods also are described in Mathiowitz and Langer (1987 J. Controlled Release 5, 13-22); Mathiowitz et al. (1987 Reactive Polymers 6: 275-283); and Mathiowitz et al. (1988 J. Appl. Polymer Sci. 35: 755-774) as well as in US 5,213,812, US 5,417,986, US 5,360,610, and US 5,384,133. The selection of the method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by

Mathiowitz et al. (1990 Scanning Microscopy 4: 329-340; 1992 J Appl. Polymer Sci. 45: 125-134); and Benita et al. (1984 J. Pharm. Sci 73: 1721-1724).

[0160] In solvent evaporation, described for example, in Mathiowitz et al., {\ 990), Benita; and US 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent, such as methylene chloride. Several different polymer concentrations can be used, for example, between 0.005 and 2.0 g/mL. The bioactive agent(s), either in soluble form or dispersed as fine particles, is (are) added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface-active agent such as poly( vinyl alcohol). The aqueous phase may be, for example, a concentration of 1% poly(vinyl alcohol) w/v in distilled water. The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid microspheres, which may be washed with water and dried overnight in a lyophiliser.

Microspheres with different sizes (between 0.1 and 1000 μm) and morphologies can be obtained by this method. [0161] Solvent removal was primarily designed for use with less stable polymers, such as the polyanhydrides. In this method, the agent is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride. The mixture is then suspended in oil, such as silicon oil, by stirring, to form an emulsion. Within 24 hours, the solvent diffuses into the oil phase and the emulsion droplets harden into solid polymer microspheres. Unlike the hot-melt microencapsulation method described for example in Mathiowitz et al. (1987 Reactive Polymers 6:275), this method can be used to make microspheres from polymers with high melting points and a wide range of molecular weights. Microspheres having a diameter for example between one and 300 microns can be obtained with this procedure.

[0162] With some polymeric systems, polymeric particles prepared using a single or double emulsion technique, vary in size depending on the size of the droplets. If droplets in water-in-oil emulsions are not of a suitably small size to form particles with the desired size range, smaller droplets can be prepared, for example, by sonication or homogenation of the emulsion, or by the addition of surfactants.

[0163] If the particles prepared by any of the above methods have a size range outside of the desired range, particles can be sized, for example, using a sieve, and optionally further separated according to density using techniques known to those of skill in the art.

[0164] The polymeric particles can be prepared by spray drying. Methods of spray drying, such as that disclosed in WO 96/09814 by Sutton and Johnson, disclose the preparation of smooth, spherical microparticles of a water-soluble material with at least 90% of the particles possessing a mean size between 1 and 10 μm.

2.4.3 Ceramic Particles

[0165] Ceramic particles may also be used to deliver the bioactive agents of the invention. These particles are typically prepared using processes similar to the well known sol-gel process and usually require simple and room temperature conditions as described for example in Brinker et al ("Sol-Gel Science: The Physics and Chemistry of Sol-Gel

Processing;" Academic Press: San Diego, 1990, p-60), and Avnir et al. (1994 Chem. Mater. 6: 1605). Ceramic particles can be prepared with desired size, shape and porosity, and are extremely stable. These particles also effectively protect doped molecules (polypeptides, drugs etc.) against denaturation induced by extreme pH and temperature (Jain et al. 1998 J. Am. Chem. Soc. 120: 11092-11095). In addition, their surfaces can be easily functionalised With different groups (LaI et al 2000 Chem. Mater. 12: 2632-2639; Badley et al 1990 Langmuir 6: 792-801), and therefore they can be attached to a variety of monoclonal antibodies and other ligands in order to target them to desired sites in vivo.

[0166] Various ceramic particles have been described for delivery in vivo of active agent-containing payloads. For example, GB 1 590 574 discloses incorporation of

biologically active components in a sol-gel matrix. WO 97/45367 discloses controllably dissolvable silica xerogels prepared via a sol-gel process, into which a biologically active agent is incorporated by impregnation into pre-sintered particles (1 to 500 μm) or disks.

WO 00/50349 discloses controllably biodegradable silica fibres prepared via a sol-gel process, into which a biologically active agent is incorporated during synthesis of the fibre. US 2004-0180096 describes ceramic nanoparticles in which a bioactive substance is entrapped. The ceramic nanoparticles are made by formation of a micellar composition of the dye. The ceramic material is added to the micellar composition and the ceramic nanoparticles are precipitated by alkaline hydrolysis. US 2005-0123611 discloses controlled release ceramic particles comprising an active material substantially homogeneously dispersed throughout the particles. These particles are prepared by mixing a surfactant with an apolar solvent to prepare a reverse micelle solution; (b) dissolving a gel precursor, a catalyst, a condensing agent and a soluble active material in a polar solvent to prepare a precursor solution; (c) combining the reverse micelle solution and the precursor solution to provide an emulsion and (d) condensing the precursor in the emulsion. US 2006-0210634 discloses adsorbing bioactive substances onto ceramic particles comprising a metal oxide (e.g., titanium oxide, zirconium oxide, scandium oxide, cerium oxide and yttrium oxide) by evaporation. Kortesuo et al. (2000 Int. J. Pharm. May 10; 200(2): 223-229) disclose a spray drying method to produce spherical silica gel particles with a narrow particle size range for controlled delivery of drugs such as toremifene citrate and dexmedetomidine HCl. Wang et al. (2006, Int. J. Pharm. 308(1-2): 160-167) describe the combination of adsorption by porous CaCO₃ microparticles and encapsulation by polyelectrolyte multilayer films for delivery of bioactive substances.

2.4.4 Liposomes

[0167] Liposomes can be produced by standard methods such as those reported by Kim et al (1983 Biochim. Biophys. Acta 728, 339-348); Liu et al (1992 Biochim. Biophys. Acta 1104: 95-101); Lee et al (1992 Biochim. Biophys. Acta. 1103: 185-197), Brey et al. (US 2002-0041861), Hass et al. (US 2005-0232984), Kisak et al (US 2005-0260260) and Smyth-Templeton et al (US 2006-0204566). Additionally, reference may be made to Copeland et al. (2005 Immunol. Cell Biol. 83: 95-105) who review lipid based particulate formulations for the delivery of antigen, and to Bramwell et al. (2005 Crit. Rev. Ther. Drug Carrier Syst. 22(2): 151-214; 2006, J Pharm. Pharmacol. 58(6): 717-728) who review particulate delivery systems for vaccines, including methods for the preparation of protein- loaded liposomes. Many liposome formulations using a variety of different lipid components have been used in various in vitro cell culture and animal experiments. Parameters have been identified that determine liposomal properties and are reported in the literature, for example, by Lee et al. (1992 Biochim. Biophys. Acta. 1103: 185-197); Liu et al. (1992 Biochim.

Biophys. Acta. 1104: 95-101); and Wang et al. (1989 Biochem. 28: 9508-951).

[0168] In some embodiments, preparative methods based on hydration of dried- lipid film are used, in which the lipids of choice (and any organic-soluble bioactive), dissolved in an organic solvent, are mixed and dried onto the bottom of a glass container under vacuum. The lipid film is rehydrated using an aqueous buffered solution containing any water-soluble bioactives to be encapsulated by gentle swirling. The hydrated lipid vesicles can then be further processed by extrusion, submitted to a series of freeze-thawing cycles or dehydrated and then rehydrated to promote encapsulation of bioactives. Liposomes can then be washed by centrifugation or loaded onto a size-exclusion column to remove unentrapped bioactive from the liposome formulation and stored at 4° C. The basic method for liposome preparation is described in more detail in Thierry et al. (1992 Nuc. Acids Res. 20: 5691-5698).

[0169] A particle carrying a payload of bioactive agent(s) can be made using the procedure as described in: Pautot et al. (2003 Proc. Natl. Acad. Sci. USA. 100(19): 10718-21). Using the Pautot et al. technique, streptavidin-coated lipids (DPPC, DSPC, and similar lipids) can be used to manufacture liposomes. The drug encapsulation technique described by Needham et al. (2001 Advanced Drug Delivery Reviews 53(3): 285-305) can be used to load these vesicles with one or more active agents.

[0170] The liposomes can be prepared by exposing chloroformic solution of various lipid mixtures to high vacuum and subsequently hydrating the resulting lipid films (DSPC/CHOL) with pH 4 buffers, and extruding them through polycarbonated filters, after a freezing and thawing procedure. It is possible to use DPPC supplemented with DSPC or cholesterol to increase encapsulation efficiency or increase stability, etc. A transmembrane pH gradient is created by adjusting the pH of the extravesicular medium to 7.5 by addition of an alkalinisation agent. A bioactive agent {e.g., a small molecule inhibitor of the NF-κB pathway, which is, for example, a weak base) can be subsequently entrapped by addition of a solution of the bioactive agent in small aliquots to the vesicle solution, at an elevated temperature, to allow accumulation of the bioactive agent inside the liposomes.

[0171] Other lipid-based particles suitable for the delivery of the bioactive agents of the present invention such as niosomes are described by Copeland et al. (2005 Immunol. Cell Biol. 83: 95-105).

2.4.5 Ballistic particles

[0172] The bioactive agents of the present invention may be attached to {e.g. , by coating or conjugation) or otherwise associated with particles suitable for use in needleless or "ballistic" (biolistic) delivery. Illustrative particles for ballistic delivery are described, for example, in: WO 02/101412; WO 02/100380; WO 02/43774; WO 02/19989; WO 01/93829; WO 01/83528; WO 00/63385; WO 00/26385; WO 00/19982; WO 99/01168; WO 98/10750; and WO 97/48485. It shall be understood, however, that such particles are not limited to their use with a ballistic delivery device and can otherwise be administered by any alternative technique (e.g., injection or microneedle delivery) through which particles are deliverable to immune cells.

[0173] The active agents can be coated or chemically coupled to carrier particles (e.g., core carriers) using a variety of techniques known in the art. Carrier particles are selected from materials which have a suitable density in the range of particle sizes typically used for intracellular delivery. The optimum carrier particle size will, of course, depend on the diameter of the target cells. Illustrative particles have a size ranging from about 0.01 to about 250 μm, from about 0.05 to about 50 μm, and from about 1 to about 10 μm; and a particle density ranging from about 0.1 to about 25 g/cm³. Non-limiting particles of this type include metal particles such as, tungsten, gold, platinum and indium carrier particles. Tungsten particles are readily available in average sizes of 0.5 to 2.0 μm in diameter. Gold particles or microcrystalline gold (e.g. , gold powder Al 570, available from Engelhard Corp., East Newark, NJ.) may also be used. Gold particles provide uniformity in size (available from Alpha Chemicals in particle sizes of 1-3 μm, or available from Degussa, South Plainfield, NJ. in a range of particle sizes including 0.95 μm) and low toxicity. Microcrystalline gold provides a diverse particle size distribution, typically in the range of 0.1 -5 μm. The irregular surface area of microcrystalline gold provides for highly efficient coating with the active agents of the present invention. [0174] Many methods are known and have been described for adsorbing, coupling or otherwise attaching bioactive molecules {e.g., hydrophilic molecules such as proteins and nucleic acids) onto particles such as gold or tungsten particles. In illustrative examples, such methods combine a predetermined amount of gold or tungsten with the bioactive molecules, CaCl₂ and spermidine. In other examples, ethanol is used to precipitate the bioactive molecules onto gold or tungsten particles (see, for example, Jumar et al. 2004 Phys. Med. Biol. 49: 3603-3612). The resulting solution is suitably vortexed continually during the coating procedure to ensure uniformity of the reaction mixture. After attachment of the bioactive molecules, the particles can be transferred for example to suitable membranes and allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular particle-mediated delivery instruments.

[0175] The formulated compositions may suitably be prepared as particles using standard techniques, such as by simple evaporation (air drying), vacuum drying, spray drying, freeze drying (lyophilisation), spray-freeze drying, spray coating, precipitation, supercritical fluid particle formation, and the like. If desired, the resultant particles can be dandified using the techniques described in WO 97/48485.

2.4.6 Surfactants

[0176] Surfactants which can be incorporated into, or used to fabricate, particles include phosphoglycerides. Exemplary phosphoglycerides include phosphatidylcholines, such as the naturally occurring surfactant, L-α-phosphatidylcholine dipalmitoyl ("DPPC"). The surfactants advantageously improve surface properties by, for example, reducing particle- particle interactions, and can render the surface of the particles less adhesive. The use of surfactants endogenous to the lung may avoid the need for the use of non-physiologic surfactants.

[0177] Providing a surfactant on the surfaces of the particles can reduce the tendency of the particles to agglomerate due to interactions such as electrostatic interactions, Van der Waals forces, and capillary action. The presence of the surfactant on the particle surface can provide increased surface rugosity (roughness), thereby improving aerosolisation by reducing the surface area available for intimate particle-particle interaction.

[0178] Surfactants known in the art can be used including any naturally occurring surfactant. Other exemplary surfactants include phospholipids such as diphosphatidyl glycerol (DPPG) or phosphatidylethanolamine; fatty alcohols or fatty acids such as palmitic acid or oleic acid polyoxyethylene-9-lauryl ether; ; sorbitan esters such as sorbitan trioleate (Span 85); bile salts; and amphiphilic polymers such as poloxamers or proteins.

2.5 Antigen-binding molecules compositions

[0179] In some embodiments, the composition comprises a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] and an antigen-binding molecule (e.g., an antibody).

[0180] In some embodiments, the antigen-binding molecule (e.g., an antibody) is immuno-interactive with a cell surface antigen. In certain embodiments, the antigen-binding molecule (e.g., antibody) specifically binds with the cell surface antigen. In some

embodiments, the cell surface antigen is present on an antigen-presenting cell (e.g. , dendritic cell) or an antigen-presenting cell precursor. Illustrative markers of this type include

CMRF56, CDl Ic, CDHl₅ Clec9A, CLEC9A, DEC-205, MCL, DCL-I, macrophage mannose R, DC-SIGN or other DC or myeloid specific (lectin) receptors, as for example disclosed by Hawiger et al (2001, J Exp Med 194, 769), Kato et al 2003, J Biol Chem 278, 34035), Benito et al. (2004, J Am Chem Soc 126, 10355), Schjetne, et al. (2002, Int Immunol 14, 1423) and van Vliet et al, 2006, Nat Immunol Sep 24; [Epub ahead of print])(van Vliet et al. , Immunobiology 2006, 211 :577-585). Suitably the antigen-presenting cell is immuno- interactive with a molecule that is expressed on the antigen-presenting cell or antigen- presenting cell precursor at higher levels than on other cells, such as non-antigen-presenting cells or precursors thereof.

[0181] Such compositions may permit effective targeting of the desired immune cells. Thus in certain embodiments, when the composition comprising the peptide and the antigen-binding molecule is administered to a subject, or is brought into contact with a sample comprising antigen-presenting cells or precursors thereof, the antigen-binding molecule targets the antigen-presenting cells or precursors thereof such that the peptide is brought close to, or in contact with, the antigen-presenting cells or precursors thereof enabling

internalisation by the antigen-presenting cells or precursors thereof and subsequent presentation. In this way, an immunostimulatory population of antigen-presenting cells or their precursors may be prepared either in vitro for subsequent administration to a subject, or in vivo in the subject.

[0182] The antigen-binding molecule may be an antibody, including a monoclonal antibody, a polyclonal antibody, or a chimeric antibody. [0183] In some embodiments, the peptide is conjugated to or otherwise associated with the antigen-binding molecule. For example, the antigen-binding molecule (e.g., an antibody) may be immuno-interactive with the peptide. Methods of producing such antibodies are well known in the art, and include those described in US 2007-0036812, US 2009-0274714, US 2010-0029571, and US 2010-0158931. In specific embodiments, the antigen-binding molecule comprises an antibody that specifically binds to the peptide.

Methods of conjugation are also well known in the art.

2.6 Nucleic acid compositions

[0184] In some embodiments, the composition comprises a nucleic acid molecule from which a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is expressible.

[0185] In some embodiments, the composition is formulated to administer or deliver the polynucleotide as "naked" DNA, for example as described in Ulmer et a 1993 Science 259:1745-49 and reviewed by Cohen, 1993 Science 259:1691-92. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

[0186] Suitably, the nucleic acid molecule comprises a nucleotide sequence that encodes the peptide and that is operably connected to a regulatory polynucleotide.

[0187] Exemplary compositions include vaccines, constructs or vectors, including but not limited to recombinant vaccines and expression vectors.

[0188] The regulatory polynucleotide suitably comprises transcriptional and/or translational control sequences, which will be compatible for expression in the cell or tissue type of interest. Typically, the transcriptional and translational regulatory control sequences include, but are not limited to, a promoter sequence, a 5' non-coding region, a cw-regulatory region such as a functional binding site for transcriptional regulatory protein or translational regulatory protein, an upstream open reading frame, ribosomal-binding sequences, transcriptional start site, translational start site, and/or nucleotide sequence which encodes a leader sequence, termination codon, translational stop site and a 3' non-translated region. Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. Promoter sequences contemplated by the present invention may be native to the organism of interest or may be derived from an alternative source, where the region is functional in the chosen organism. The choice of promoter will differ depending on the intended host. For example, promoters which could be used for expression in mammalian cells generally include the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, the β-actin promoter as well as viral promoters such as the SV40 large T antigen promoter, human cytomegalovirus (CMV) immediate early (IE) promoter, rous sarcoma virus LTR promoter, adenovirus promoter, or a HPV promoter, particularly the HPV upstream regulatory region (URR) may also be used. All these promoters are well described and readily available in the art. Alternatively, the promoter may be lineage specific and, in this regard, epithelial-specific promoters are particularly desirable such as, but not limited to, promoters of the following genes transglutaminase type 1, involucrin, loricrin, SPR genes and filagrin as well as those of keratin genes (e.g., KlO, K14. K5. K1).

[0189] The composition may also comprise a 3' non-translated sequence. A 3' non- translated sequence refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is characterised by effecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. Polyadenylation signals are commonly recognised by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. The 3' non-translated regulatory DNA sequence preferably includes from about 50 to 1,000 nucleotide base pairs and may contain transcriptional and translational termination sequences in addition to a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.

[0190] In some embodiments, the composition further contains a screenable marker gene to permit identification of cells containing the composition. Screenable genes (e.g., lacZ, gfp, etc) are well known in the art and will be compatible for expression in a particular cell or tissue type.

[0191] It will be understood, however, that expression of protein-encoding polynucleotides in heterologous systems is now well known, and the present invention is not directed to or dependent on any particular construct, vector, transcriptional control sequence or technique for its production. Rather, synthetic polynucleotides prepared according to the methods as set forth herein may be introduced into selected cells or tissues or into a precursors or progenitors thereof in any suitable manner in conjunction with any suitable synthetic construct or vector, and the synthetic polynucleotides may be expressed with known promoters in any conventional manner.

[0192] The compositions can be introduced into suitable host cells for expression using any of a number of non- viral or viral gene delivery vectors. For example, retroviruses (in particular, lenti viral vectors) provide a convenient platform for gene delivery systems. A coding sequence of interest (for example, a sequence useful for gene therapy applications) can be inserted into a gene delivery vector and packaged in retroviral particles using techniques known in the art. Recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.

[0193] In one illustrative embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. A selected nucleotide sequence that encodes a peptide that comprises, consists or consists essentially of an amino acid sequence

corresponding to the sequence FLGYLILGV [SEQ ID NO:1] can be inserted into a construct or vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject. Several illustrative retroviral systems have been described examples of which include: US 5,219,740; Miller and Rosman 1989 Bio Techniques 7: 980-990; Miller A. D. 1990 Human Gene Therapy 1 : 5-14; Scarpa et al. 1991 Virology 180: 849-852; Burns et al. 1993 Proc. Natl. Acad. ScL USA 90: 8033-8037; and Boris-Lawrie and Temin 1993 Cur. Opin. Genet. Develop. 3: 102-109).

[0194] In addition, several illustrative adenovirus-based systems have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimising the risks associated with insertional mutagenesis (see, e.g., Haj-Ahmad and Graham, 1986, J Virol. 57: 267-274; Bert et al., 1993, J Virol. 61: 5911-5921; Mittereder et al, 1994, Human Gene Therapy 5: 717-729; Seth et al, 1994, J Virol. 68: 933-940; Barr et al, 1994, Gene Therapy 1 : 51-58; Berkner, K. L., 1988, Bio Techniques 6: 616-629; and Rich et al, 1993, Human Gene Therapy 4: 461-476).

[0195] Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., US 5,173,414; US 5,139,941; WO 92/01070; WO 93/03769; Lebkowski et al 1988 Molec. Cell Biol 8: 3988-3996; Vincent et al 1990

Vaccines 90, Cold Spring Harbor Laboratory Press; Carter, B. J., 1992, Current Opinion in Biotechnology 3: 533-539; Muzyczka, N., 1992, Current Topics in Microbiol, and Immunol. 158: 97-129; Kotin, R. M., 1994, Human Gene Therapy 5: 793-801; Shelling and Smith, 1994, Gene Therapy 1: 165-169; and Zhou et al. , 1994, J Exp. Med. 179: 1867-1875.

[0196] Additional viral vectors useful for delivering the peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO : 1 ] by gene transfer include those derived from the pox family of viruses, such as vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] can be constructed as follows. The polynucleotide is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the expression products of interest into the viral genome. The resulting TK^ recombinant can be selected by culturing the cells in the presence of 5-BrdU and picking viral plaques resistant thereto.

[0197] Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the coding sequences of interest. The use of an Avipox vector is particularly desirable in human and other mammalian species since members of the Avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant Avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

[0198] Any of a number of alphavirus vectors can also be used for delivery of polynucleotide compositions of the present invention, such as those vectors described in US 5,843,723; US 6,015,686; US 6,008,035 and US 6,015,694. Certain vectors based on

Venezuelan Equine Encephalitis (VEE) can also be used, illustrative examples of which can be found in US 5,505,947 and US 5,643,576.

[0199] Moreover, molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al, J. Biol. Chem. 268:6866-69, 1993; and Wagner et al, Pr oc. Natl. Acad. ScI USA 89: 6099-6103, 1992, can also be used for gene delivery under the invention. [0200] In other illustrative embodiments, lentiviral vectors are employed to deliver an antigen-encoding polynucleotide into selected cells or tissues. Typically, these vectors comprise a 5' lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to one or more genes of interest, an origin of second strand DNA synthesis and a 3' lentiviral LTR, wherein the lentiviral vector contains a nuclear transport element. The nuclear transport element may be located either upstream (5') or downstream (3') of a coding sequence of interest (for example, a synthetic Gag or Env expression cassette of the present invention). A wide variety of lentiviruses may be utilised within the context of the present invention, including for example, lentiviruses selected from the group consisting of HIV, HIV-I , HIV-2, FIV, BIV, EIAV, MVV, CAEV, and SIV. Illustrative examples of lentiviral vectors are described in WO 00/66759, WO 00/00600, WO 99/24465, WO 98/51810, WO 99/51754, WO 99/31251, WO 99/30742, and WO 99/15641. Desirably, a third generation SIN lentivirus is used. Commercial suppliers of third generation SIN (self-inactivating) lentiviruses include Invitrogen (ViraPower Lentiviral Expression System). Detailed methods for construction, transfection, harvesting, and use of lentiviral vectors are given, for example, in the Invitrogen technical manual "ViraPower Lentiviral Expression System version B 050102 25-0501", available at http://www.invitrogen.com/Content/Tech- Online/molecular_biology/manualsjp-ps/virapower_lentiviral_system_man.pdf. Lentiviral vectors have emerged as an efficient method for gene transfer. Improvements in biosafety characteristics have made these vectors suitable for use at biosafety level 2 (B L2). A number of safety features are incorporated into third generation SIN (self-inactivating) vectors.

Deletion of the viral 3' LTR U3 region results in a provirus that is unable to transcribe a full length viral RNA. In addition, a number of essential genes are provided in trans, yielding a viral stock that is capable of but a single round of infection and integration. Lentiviral vectors have several advantages, including: 1) pseudotyping of the vector using amphotropic envelope proteins allows them to infect virtually any cell type; 2) gene delivery to quiescent, post mitotic, differentiated cells, including neurones, has been demonstrated; 3) their low cellular toxicity is unique among transgene delivery systems; 4) viral integration into the genome permits long term transgene expression; 5) their packaging capacity (6-14 kb) is much larger than other retroviral, or adeno-associated viral vectors. In a recent demonstration of the capabilities of this system, lentiviral vectors expressing GFP were used to infect murine stem cells resulting in live progeny, germline transmission, and promoter-, and tissue-specific expression of the reporter (Ailles, L. E. and Naldini, L., HIV-1-Derived Lentiviral Vectors. In: Trono, D. (Ed.), Lentiviral Vectors, Springer-Verlag, Berlin, Heidelberg, New York, 2002, pp. 31 -52). An example of the current generation vectors is outlined in FIG. 2 of a review by Lois et al. (Lois, C, Hong, E. J., Pease, S., Brown, E. J., and Baltimore, D., Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors, Science, 295 (2002) 868-872).

[0201] In certain embodiments, a polynucleotide may be integrated into the genome of a target cell. This integration may be in the specific location and orientation via

homologous recombination (gene replacement) or it may be integrated in a random, nonspecific location (gene augmentation). In yet further embodiments, the polynucleotide may be stably maintained in the cell as a separate, episomal segment of DNA. Such polynucleotide segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronisation with the host cell cycle. The manner in which the expression construct is delivered to a cell and where in the cell the polynucleotide remains is dependent on the type of expression construct employed.

[0202] Furthermore, other particulate systems and polymers can be used for the in vivo delivery of the compositions of the present invention. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest. Similarly, DEAE dextran- mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods. See, e.g., Feigner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for a review of delivery systems useful for gene transfer. Peptoids (US 5,831,005) may also be used for delivery of a construct of the present invention.

[0203] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering agents that are in nucleic acid form (e.g., constructs of the present invention). The particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun." For a description of such techniques, and apparatuses useful therefor, see, e.g., US 4,945,050; US 5,036,006; US 5,100,792; US 5,179,022; US 5,371,015; and US 5,478,744. In illustrative examples, gas- driven particle acceleration can be achieved with devices such as those manufactured by PowderMed Pharmaceuticals PLC (Oxford, UK) and PowderMed Vaccines Inc. (Madison, Wis.), some examples of which are described in US 5,846,796; US 6,010,478; US 5,865,796; US 5,584,807; and EP 0500 799. This approach offers a needle-free delivery approach wherein a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high speed within a helium gas jet generated by a hand held device, propelling the particles into a target tissue of interest. Other devices and methods that may be useful for gas-driven needle-less injection of compositions of the present invention include those provided by Bioject, Inc. (Portland, Oreg.), some examples of which are described in US 4,790,824; US 5,064,413; US 5,312,335; US 5,383,851; US 5,399,163; US 5,520,639 and US 5,993,412.

[0204] Alternatively, micro-cannula- and microneedle-based devices (such as those being developed by Becton Dickinson and others) can be used to administer nucleic acid constructs of the invention. Illustrative devices of this type are described in EP 1 092 444. Standard steel cannula can also be used for intra-dermal delivery. These methods and devices include the delivery of substances through narrow gauge (about 30 G) "micro-cannula" with limited depth of penetration, as defined by the total length of the cannula or the total length of the cannula that is exposed beyond a depth-limiting feature. It is within the scope of the present invention that targeted delivery of substances including nucleic acid constructs can be achieved either through a single microcannula or an array of microcannula (or

"microneedles"), for example 3-6 microneedles mounted on an injection device that may include or be attached to a reservoir in which the substance to be administered is contained. 2.7 T lymphocyte compositions

[0205] In some embodiments, the composition comprises T-lymphocytes that are primed to respond to a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], including when the peptide is presented by a cancer cell. In certain embodiments, the T lymphocytes are cytotoxic (CTL) T lymphocytes.

[0206] As used herein, the term "primed" means that the level and/or functional activity of the T-lymphocytes is more efficient (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), more rapid (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), greater in magnitude (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), and/or more easily induced (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more) than if the T-lymphocytdes had not been primed.

[0207] The T lymphocytes may be autologous or allogeneic. [0208] The T lymphocytes may be clones.

[0209] These compositions comprising primed T-lymphocytes can be prepared by any suitable method known in the art. In certain embodiments, antigen-presenting cells that express the peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a processed form thereof, including those described herein, may be presented to a population of T lymphocytes for a time and under conditions sufficient to produce T lymphocytes that are stimulated or primed to respond to the peptide, including when the peptide is presented by a cancer cell.

[0210] These compositions are especially useful in methods comprising adoptive transfer to immunodeficient individuals who are unable to mount normal immune responses.

For example, antigen-specific CD8⁺ CTL have been shown to be adoptively transferred for therapeutic purposes in individuals afflicted with HIV infection (Koup et al. 1991 J Exp.

Med. 174: 1593-1600; Carmichael et al. 1993 J Exp. Med. Ill: 249-256; and Johnson et al.

1992 J. Exp. Med. 175: 961-971), malaria (Hill et al. 1992 Nature 360: 434-439) and malignant tumours such as melanoma (Van der Brogen et al. 1991 Science 254: 1643-1647; and Young and Steinman 1990 J. Exp. Med. 171 : 1315-1332). See also Forsberg O. et al,

(2009 Prostate 69: 70-81).

2.8 Antigen-binding molecules immuno-interactive with the peptide

[0211] In some embodiments, the composition comprises an antigen-binding molecule (e.g. , an antibody) that is immuno-interactive with the peptide a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I].

[0212] In some embodiments, the antigen-binding molecule comprises an antibody that specifically binds to the peptide. Methods of producing such antibodies are well known in the art, and include those described in US 2007-0036812, US 2009-0274714, US 2010- 0029571, and US 2010-0158931.

[0213] In some embodiments, the antigen-binding molecule is conjugated to an toxin, including ricin.

[0214] Advantageously, the antigen-binding molecule (with or without conjugation to a toxin) binds to a cell expressing the peptide a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID

NO:1], and inactivates or kills the cell. 2.9 Ancillary components

[0215] In some embodiments, the composition further comprises an additional tumour antigen associated peptide or a nucleic acid molecule from which the additional peptide is expressible. Suitable additional peptides, include, but are not limited to peptides expressed by cancer cells, including but not limited to peptides derived from

prostase/kallikrein 4 (KLK4) including human prostase/KLK4 (hKLK4), cancer antigen 125 (CSA-125), cancer antigen-549 (CA-549), cancer antigen-15-3 (CA-15-3), cancer antigen-19- 9 (CA-19-9), cancer antigen-27.29 (CA-27.29), alpha-fetoprotein, early prostate cancer antigen-2, prostate specific antigen (PSA), prostatic acid phosphatase (PAP), prostate specific membrane antigen (PSMA), prostate-specific transglutaminase (TGM4), prostate stem cell antigen (PSCA), T-cell receptor γ-chain alternate reading frame protein (TARP), six transmembrane epithelial antigen prostate (STEAP), prostate-specific gene with homology to a G protein-coupled receptor (PSGR), transient receptor potential p8 (Trp-p8), prostate and testis expression (PATE), prostate, ovary, testis, and placenta expression (POTE), six transmembrane protein of prostate 1 (STAMP 1), androgen-induced bZIP (AIbZIP), novel prostate-specific antigen (NPSA), Prostein, PAGE-4, parathyroid hormone-related protein (PTH-rp), telomerase reverse transcriptase (TERT) including human TERT (hTERT), survivin, NY-ESO, NY-BR-I, HER-2/neu, ρ53, PRAME, MUCl, prostate cancer antigen- 1 (PCA-I),

[0216] In certain embodiments, the additional peptide(s) may be selected from the group consisting of hKLK4₁₅5.₁₆₄, hKLK4_117-i₂₆, PSA_16-24, PSA_53-6I, PSA_68-7?, PSA_141-I₅₀, PSAi_46-154, PSAi_52-I₆₀, PSA_154-163, PSA(VlY)i_S4-i63, PSA_162-170, PSA_165-174, PSA₂₄₈-2S7, PAP₁₃. ₂₂, PAPi_3-21, PAPn_2-I₂O₁ PAPi_35-I₄₃, PAPi_55-I₆₃, PAP₂I_3-221, PAP_248-257, PAP_299-30?, PSMA_4-12, PSMA_27-35, PSMA_178-186, PSMA_207-2I5, PSMA_227-235, PSMA_431-440, PSMA_441-450, PSMA_624-632, PSMA₇₁₁-₇₁9, TGM4₆i_2-62o, PSCA_7-I5, PSCAi_4-22, PSCA₂I_-30, PSCA_76-845PSCAi_05-113,

TARP(V28L)_27-35, TARP(P5L)_4-13, STEAP_165-173, STEAP_262-270, PSGR_202-2IO, Trp-p8_187-i₉₅, Trp-p8_596-605, Trp-p8_770-778, PATE_5-]3, POTE₃₂₃-S₃I, POTE_290-298, STAMP l_402-4]0, STAMPl_373- ₃₈₂/STEAP_232-24i, STEAP_86-94, STEAP_262-270, STEAP_292-300, STEAP(I2L)_292-300, AIbZIP_70-78, NPSA_20-28, Prostein₃i_-39, Prostein_292-300, PrOStCm_464-472, PrOStCIn_464-473, PTH-rp_36-44, PTH-rp_42- si, PTH-rp_59-67, PTH-rp₅9-68, PTH-rp_]02-m, PTH-rpi₆₅-i73, hTERT_30-38, hTERT(L9V)₃₀-38, hTERTi_67-175, hTERT_277-285, hTERT_324-332, hTERT_325-333, hTERT_342-350, hTERT₃₅i_-360, hTERT_46M69, hTERT_540-548, hTERT_572-580, hTERT(Rl Y)_572-580, hTERT_845-853, hTERT₈₆₅-873, hTERT_973-98i, hTERTi_O88-io₉₆, NY-ESO-, _157-i₆₅, NY-ESO-(1ORF2)_{M 1}, NY-BR-I_904-912, HER- 2/neu_369-3₇₇, HER-2/neu_654-662, HER-2/neu_5-i₃, HER-2/neu435-443, HER-2/neu_689-697, HER- 2/neu_665-673, HER-2/neu_952-961, HER-2/neu₄₈._56! HER-2/neu_1O23-1o₃₂, HER-2/neu_391-399, HER- 2/neu4_O2-41o, HER-2/neu4_66-474, HER-2/neu_650-658, p53_264-272, ρ53_65-73, PRAME_10O-108,

PRAME_142-151, PRAME₃O_0-309, PRAME_425-433, MUCl₉₅o-9S8, MUCl_12-20, survivin_5-14, survivin_18-28, survivin(Fl OK)_18-27, survivin(C9 Y)_38-46, survivin(Q 1 OY)_47-56, survivin_53-62, survivin_92-i₀₁, survivin(E2T)_93-101, survivin_95-io₄, and survivin(T2M)_96-i₀₄. Many suitable additional peptides are known in the art (Forsberg O. etal, 2009 Prostate 69: 70-81 and Kiessling, A. et al. 2008 European Urology 694-708).

[0217] In some embodiments, the compositions further comprise an adjuvant that enhances the effectiveness of the composition. Examples of adjuvants which may be effective include but are not limited to: CpG, polyI:C, LPS, surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin,

dimethyldioctadecylammonium bromide, N, N-dicoctadecyl-N', N'bis(2-hydroxyethyl- propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; mineral gels such as aluminum phosphate, aluminum hydroxide or alum; peptides such as muramyl dipeptide and derivatives such as N-acetyl- muramyl-L-threonyl-D-isoglutamine (thur-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- ethylamine (CGP 1983 A, referred to as MTP-PE), and RIBI, which contains three

components extracted from bacteria, monophosphoryl lipid A, dimethylglycine, tuftsin; oil emulsions; trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion; lymphokines; QuilA and immune stimulating complexes (ISCOMS). Suitably, the adjuvant delivers the antigen to the class I major histocompatibility (MHC) pathway. For example, such adjuvants include, but are not limited to, saponin- containing compounds (e.g., ISCOMs) and cytolysins, which mediates delivery of antigenic peptides to the cytosol of a target cell. The cytolysin may be linked to, or otherwise associated with, the peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or the nucleic acid molecule from which the peptide is expressible. In some embodiments, the cytolysin mediates transfer of the peptide from the vacuole (e.g., phagosome or endosome) to the cytosol of an antigen- presenting cell and in illustrative examples of this type, the cytolysin is a listeriolysin. The effectiveness of an adjuvant may be determined by those methods known in the art. [0218] In some embodiments, the composition further comprises one or more cytokines, which are suitably selected from IL-4, IL-13, flt3, SCF, IL-3, IL-6, GM-CSF, G- CSF, TNF-α, TNF-β, LT-β, IL-2, IL-7, IL-9, IL-15, IL-5, IL-lα, IL-lβ, IFN-γ, IL-17, IL-16, IL-18, HGF, IL-I l, MSP, FasL, TRAIL, TRANCE, LIGHT, TWEAK, CD27L, CD30L, CD40L, APRIL, TALL-1 , 4-1BBL, OX40L, GITRL, IGF-I, IGF-II, HGF, MSP, FGF-a, FGF- b, FGF-3-19, NGF, BDNF, NTs, Tpo, Epo, Angl-4, PDGF-AA, PDGF-BB, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, TGF-α, AR, BTC, HRGs, HB-EGF, SMDF, OB, CT-I, CNTF, OSM, SCF, Flt-3L, M-CSF, MK and PTN or their functional, recombinant or chemical equivalents or homologues thereof.

[0219] In some embodiments, the composition further comprises a

pharmaceutically acceptable excipient or carrier, including, but not limited to a solid or liquid filler, a diluent or encapsulating substance. In embodiments where the composition comprises cells {e.g., antigen-presenting cells or T lymphocytes), the composition may comprise a pharmaceutically acceptable excipient or carrier which is non-toxic to the cells. Such excipient or carrier may be, for example, water, the growth medium in which the cells were grown, or any suitable buffering medium such as saline, including saline with or without a physiological compatible buffer like phosphate or Hepes and nutrients such as dextrose, isotonic saline, physiologically compatible ions, or amino acids, and various culture media suitable for use with cell populations, particularly those devoid of other immunogenic components, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents. Carrying reagents, such as albumin and blood plasma fractions and non-active thickening agents, may also be used. Non-active biological components, to the extent that they are present in the composition, are preferably derived from a syngeneic animal or human as that to be treated, and are even more preferably obtained previously from the subject.

[0220] In some embodiments, the composition of the present invention is formulated for use with other known therapies, for example, in conjunction with surgical therapy, chemotherapy, radiotherapy, hormone therapy, antiangiogenic therapy, and/or another immunotherapy. 3. Preparation of immunostimulating compositions

[0221] The preparation of the compositions of the present invention uses routine methods known to persons skilled in the art, including those described in "Remington 's Pharmaceutical Sciences " Mack Publishing Co., Easton, Pa., latest edition.

[0222] The composition may be formulated for administration by any suitable route, including but not limited to oral, rectal, transmucosal, intestinal, parenteral,

intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular.

[0223] In certain embodiments, the composition comprises a formulation or vaccine, prepared as an injectable, either as liquid solution or suspension. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.

[0224] If soluble actives are employed, the soluble active ingredients can be formulated into the composition as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic basis such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic basis as

isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. In some embodiments, the pharmaceutically acceptable salt may be selected from a group including hydrochlorides, hydrobromides, hydroiodides, sulphates, bisulphates, nitrates, citrates, tartrates, bitartrates, phosphates, malates, maleates, napsylates, fumarates, succinates, acetates, terephthalates, pamoates and pectinates.

[0225] If desired, compositions or devices comprising the compositions suitable for sustained or intermittent release may be prepared.

4. Methods for stimulating immune responses

[0226] The compositions of the invention may be used for stimulating an immune response. Thus, the present invention also extends to methods for stimulating an immune response in a subject. Advantageously, the immune response is a cell-mediated immune response (e.g., a T-cell mediated response, which desirably includes CDS⁺ IFN-γ-producing T cells). A further aspect of the present invention provides methods for preventing or treating a cancer in a subject. As used herein, the term "cancer" refers to the uncontrolled growth of a cell of a subject and optionally the ability of this cell to migrate from the original site of the subject's body to a distant site. The cancer may be any cancer cell which contains, produces or expresses a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I]. In some

embodiments, the cancer cell is a hormone-related cancer cell. In some embodiments, the cancer is prostate cancer, breast cancer, or ovarian cancer.

[0227] By "preventing or treating", "prevent", "preventing", "prophylaxis ", "treatment, " "treat," "treated" and the like is meant to include both prophylactic and therapeutic treatment, including but not limited to preventing, relieving, altering, reversing, affecting, inhibiting the development or progression of, ameliorating, or curing (1) a disease or condition, such as a cancer, or (2) a symptom of the disease or condition, or (3) a predisposition toward the disease or condition, including conferring protective immunity to a subject. In some embodiments, these terms include reducing the size of or inhibiting the increase in the size of a tumour, reducing the number of cancer cells, reducing the size of a cancer cell, inhibiting the growth of a cancer cell, or killing a cancer cell.

[0228] "Protective immunity" (also referred to as "immunoenhancement" and the like) is well-known in the art, and means to increase the subject's capacity to respond to a disease-specific antigen (e.g., cancer antigen) i.e., cells primed to attack the antigen are increased in number, activity, and ability to detect and destroy the the antigen. Strength of immune response is measured by standard tests including: direct measurement of peripheral blood lymphocytes by means known to the art; natural killer cell cytotoxicity assays (see, e.g., Provinciali M. et al. 1992 J Immunol. Meth. 155: 19-24), cell proliferation assays (see, e.g., Vollenweider, I. And Groseurth, P. J. 1992 J. Immunol. Meth. 149: 133-135), immunoassays of immune cells and subsets (see, e.g., Loeffler, D. A., et al. 1992 Cytom. 13: 169-174);

Rivoltini, L., et al. 1992 Can. Immunol. Immunother. 34: 241-251); or skin tests for cell- mediated immunity (see, e.g., Chang, A. E. et al. 1993 Cancer Res. 53: 1043-1050).

[0229] Thus suitable subjects include, but are not limited to, individuals who have cancer, and those who do not have the cancer but are predisposed to or at risk of developing a cancer. Methods of diagnosing cancer in an individual are well known in the art (see for example US 2007/0036812 which describes a method of cancer diagnosis using an antibody that binds to the TAA peptide. It is envisaged that such a method could be used in the present invention with an antibody that binds a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] as herein described). Additionally, many methods to determine if a subject is predisposed to or at risk of developing a cancer are well known in the art.

[0230] The methods of the present invention generally comprise administering to the subject an effective amount of a composition of the present invention as herein defined.

[0231] In certain embodiments, these methods comprise increasing in the subject the number of antigen-presenting cells or antigen-presenting cell precursors that present a peptide that comprises, consists or consists essentially of an amino acid sequence

corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a processed form thereof on their surface, or increasing in the subject the number of T-lymphocytes that are primed to respond to a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I].

[0232] In some embodiments, the subject has an HLA-A haplotype. Illustrative types of this haplotype include HLA-A2 and HLA-Al 1. Accordingly, in some embodiments, the methods comprise identifying or selecting a subject whose haplotype is an HLA-A haplotype (e.g. , HL A- A2 or HLA-Al 1 ) and then administering to the subj ect an effective amount of the composition of the present invention as herein defined.

4.1 Route of administration and dosage

[0233] The methods of the present invention can employ any route of

administration which produces the desired immune response, and thus the composition being administered will be suitably formulated. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular. In certain embodiments, the method comprises injecting the subject with a composition of the present invention.

[0234] In still other embodiments, a composition of the present invention can be delivered via a particle bombardment approach, many of which have been described. In one illustrative example, gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, Wis.), some examples of which are described in US 5,846,796;

US 6,010,478; US 5,865,796; US 5,584,807; and EP 0500 799. This approach offers a needle- free delivery approach wherein a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high speed within a helium gas jet generated by a hand held device, propelling the particles into a target tissue of interest.

[0235] In a related embodiment, other devices and methods that may be useful for gas-driven needle-less injection of compositions of the present invention include those provided by Bioject, Inc. (Portland, Oreg.), some examples of which are described in

US 4,790,824; US 5,064,413; US 5,312,335; US 5,383,851; US 5,399,163; US 5,520,639 and US 5,993,412.

[0236] If desired, compositions or devices comprising the compositions suitable for sustained or intermittent release may be implanted in the body or topically applied thereto for the relatively slow release of the composition (or components thereof) into the body.

[0237] In some embodiments, where the composition comprises a nucleic acid molecule from which a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] is expressible, administration to a mammal, especially a human, may include delivery via direct oral intake, systemic injection, or delivery to selected tissue(s) or cells. Delivery of the constructs to cells or tissues of the mammal may be facilitated by microprojectile bombardment, liposome mediated transfection (e.g., lipofectin or lipofectamine), electroporation, calcium phosphate or DEAE-dextran-mediated transfection, for example. A discussion of suitable delivery methods may be found in Chapter 9 of Ausubel et al, (1994-1998, supra).

[0238] The step of introducing the expression vector into the selected target cell or tissue will differ depending on the intended use and species, and can involve one or more of non-viral and viral vectors, cationic liposomes, retroviruses, and adenoviruses such as, for example, described in Mulligan, R.C., (1993). Such methods can include, for example:

[0239] A. Local application of the expression vector by inj ection (Wolff et al. 1990 Science 247 (4949 Pt 1): 1465-1468), surgical implantation, instillation or any other means. This method can also be used in combination with local application by injection, surgical implantation, instillation or any other means, of cells responsive to the protein encoded by the expression vector so as to increase the effectiveness of that treatment. This method can also be used in combination with local application by injection, surgical implantation, instillation or any other means, of another factor or factors required for the activity of the protein. [0240] B . General systemic delivery by inj ection of DNA, (Calabretta et al, 1993), or RNA₅ alone or in combination with liposomes (Zhu et al, 1993), viral capsids or nanoparticles (Bertling et al, 1991) or any other mediator of delivery. Improved targeting might be achieved by linking the polynucleotide/expression vector to a targeting molecule (the so-called "magic bullet" approach employing, for example, an antigen-binding molecule), or by local application by injection, surgical implantation or any other means, of another factor or factors required for the activity of the protein encoded by the expression vector, or of cells responsive to the protein.

[0241] C. Injection or implantation or delivery by any means, of cells that have been modified ex vivo by transfection (for example, in the presence of calcium phosphate:

Chen et al, 1987; or of cationic lipids and polyamines: Rose et al, 1991), infection, injection, electroporation (Shigekawa et al, 1988) or any other way so as to increase the expression of the polynucleotide in those cells. The modification can be mediated by plasmid,

bacteriophage, cosmid, viral (such as adenoviral or retroviral; Mulligan, 1993; Miller, 1992; Salmons et al. , 1993) or other vectors, or other agents of modification such as liposomes (Zhu et al, 1993), viral capsids or nanoparticles (Bertling et al, 1991), or any other mediator of modification. The use of cells as a delivery vehicle for genes or gene products has been described by Barr et al, 1991 and by Dhawan et al, 1991. Treated cells can be delivered in combination with any nutrient, growth factor, matrix or other agent that will promote their survival in the treated subject.

[0242] In determining the dosage of the composition to be administered in the methods of the present invention, the physician or veterinarian may consider factors including the subject to be treated inclusive of the age, sex, weight and health condition thereof including the progression of the cancer (if any) in the subject over time, the composition being administered, and the route of administration. Precise amounts of the composition can also depend on the judgment of the practitioner. In any event, those of skill in the art may readily determine suitable dosages of the compositions of the invention without undue experimentation. Cell-containing compositions (e.g. antigen-presenting cells or T

lymphocytes) are suitably administered to a patient in the range of between about 10⁴ and 10¹⁰, and more preferably between about 10⁶ and 10⁸ treated cells/administration.

[0243] Single or multiple administrations of the composition can be carried out with dosage of each administration and pattern of administration being selected by the treating physician or veterinarian. [0244] The effectiveness of the compositions and/or methods of the present invention may be assessed using methods known in the art. For example, CTL lysis assays may be employed using stimulated splenocytes or peripheral blood mononuclear cells (PBMC) on peptide coated or recombinant virus infected cells using ⁵¹Cr or Alamar Blue™ labeled target cells. Such assays can be performed using for example primate, mouse or human cells (Allen et al, 2000, J Immunol. 164(9): 4968-4978 also Woodbeπy et al, infra). Alternatively, the efficacy of the immunisation may be monitored using one or more techniques including, but not limited to, HLA class I tetramer staining - of both fresh and stimulated PBMCs (see for example Allen et al. , supra), proliferation assays (Allen et al. , supra), ELISPOT assays and intracellular IFN-γ staining (Allen et al, supra), ELISA assays - for linear B cell responses; and Western blots of cell sample expressing the synthetic polynucleotides.

, [0245] In some embodiments, the methods of the present invention are combined with other known therapies, for example, with surgical therapy, chemotherapy, radiotherapy, hormone therapy, antiangiogenic therapy, and/or another immunotherapy.

5. Methods for monitoring immune responses

[0246] The present invention also relates to methods of monitoring an immune response in a subject. As used herein, "monitoring' includes detecting the presence of, measuring the magnitude of, or detecting a change in an immune response in a subject.

[0247] The subject may be an individual who is being treated for cancer, including by surgical therapy, chemotherapy, radiotherapy, hormone therapy, antiangiogenic therapy and/or immunotherapy. In some embodiments, the subject is an individual who has been administered a composition designed to stimulate an immune response, especially to a cancer. The composition may be a composition of the present invention, or another composition. In certain embodiments, the subject has been treated using a composition or method of the present invention. In these embodiments, the purpose may be to assess the effectiveness of a composition or method of the present invention in the subject, for example, by determining whether the therapy is treating a cancer in the subject. In certain other embodiments, the subject has not been treated using a composition or method of the present invention, but has been treated using a different therapy. In these embodiments, the purpose may be to assess the effectiveness of the immune response by the different therapy, for example, by

determining whether the therapy is treating a cancer in the subject. [0248] The methods may comprise any suitable method in the art, incorporating the discovery of the present invention that peptide A is a dominant TAA epitope that is endogenously processed.

[0249] Thus, suitable methods include the use of fluorescently labelled tetramers or pentamers as known in the art (Allen et al. supra). Briefly, a sample comprising T cells (such as a blood sample) is taken from the subject. The sample is then contacted with a

fluorescently labelled tetramer or pentamer of a complex, the complex comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] conjugated to an HLA molecule. Binding of the tetramer or pentamer to the sample is then measured, for example using flow cytometry methods, in order to monitor the immune response.

[0250] The methods may also comprise the use of an antibody that binds a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] as herein described. Other suitable methods include limiting dilution assays, enzyme-linked immunospot analysis (ELISPOT), proliferation and cytotoxicity assays, and delayed-type hypersensitivity testing (in vivo).

[0251] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples. EXAMPLES

Materials and Methods

[0252] The following Materials and Methods were used in the Examples described herein.

[0253] Blood samples

[0254] Peripheral blood mononuclear cells (PBMCs) were obtained by

venepuncture or apheresis from HLA-A*201 positive healthy donors and prostate cancer patients following informed consent obtained under ethical approval from the Mater Health Services Human Research Ethics Committee. Human AB serum was obtained from the Australian Red Cross Blood Service.

[0255] Healthy donors (HD) 1 (0) and 2 (α) were 45 and 46 year old males, HD3

(V) and 4 (o) were 40 and 25 year old females, respectively. [0256] Prostate cancer (PCa) patient 1 (^T) was aged 74 with stage T2a disease. PCa2 (•), aged 76, had undergone radical prostatectomy followed by biochemical recurrence and hormone sensitive disease. PCa3 (*), aged 70, had hormone refractory disease with bony metastases, and PCa4 (T), aged 73, had hormone refractory disease with nodal metastases. PCa5 (♦), male aged 68, had hormone sensitive disease. PCa6 (■), male aged 53, had hormone naive disease.

[0257] Antibodies, reagents and cell lines

[0258] The biotinylated CMRF-56 mouse monoclonal antibody (Ab) was prepared as previously described (Hock, B. D. et al, 1999 Tissue Antigens 53: 320-324 and Lopez, J. A. et al. , 2003 J Immunol Methods 274: 47-61). Fluorescein isothiocyanate (FITC-), and phycoerythrin (PE)-conjugated anti-CD3, CD 14, CD 19, CD20, CD56, and IgGl, peridinin chlorophyll protein (PerCP)-conjugated anti-CD4 and allophycocyanin (APC)-conjugated anti-HLA-DR, CD3, and IgG2a were purchased from BD Biosciences (San Jose, CA). FITC- conjugated anti-CD80, CD83, and PE-Cyanan5 -conjugated CD40 and CD86 were purchased from BD Pharmingen (San Diego, CA). IL-2 was provided by Roche (Basel, Switzerland) and IL-7 and β-2 -microglobulin was purchased from Sigma. All cell lines, including the prostate cancer cell lines LNCaP 1740 (HLA- A*201⁺ and KLK4⁺) and PC3 (HLA-A*201^" KLKf), ovarian carcinoma cell lines OVCAR-3 and CaOV3 (both HLA-A*201⁺ KLK4⁺), the myelogenous leukaemia cell line K562 and the TAP deficient T2 cell line were obtained from the ATCC (Manassas, Va) and maintained in the recommended media.

[0259] Peptides

[0260] Two separate computer algorithms were used to analyse the hK4 amino acid sequence for potential HLA-A*201 binding nonamer peptide sequences. The algorithm from the Biolnformatics and Molecular Analysis Section of the NIH (BIMAS, Parker, K. C, et al, 1994 J. Immunol. 152(1): 163-175, access via http ://www-bimas.cit.nih. gov) scores potential HLA-A*201 binding peptides based on the estimated half-time of dissociation of complexes containing peptide at 37⁰C and pH 6.5, whilst the SYFPEITHI algorithm (Rammensee, H. et al, 1999 Immunogeneetics 50: 213-219; access via http://www.syrpeithi.com) scores peptides according to amino acids in anchor and auxiliary anchor positions based on published motifs. Peptides with the highest scores obtained from both programs were synthesised using standard solid-phase synthesis (Auspep, Parkville, Australia) and stocks were reconstituted at lO mg/mL in DMSO. [0261] Peptides currently used in prostate cancer clinical trials (PAP-5₂₉9-30₇ ALDVYNGLL; PSA-3i_54-i₆₃ VISNDVCAQV; PSMA-2₇i_1-7i₉ ALFDIESKV (Bachem AG)) were also synthesised. In addition, the well-characterised HLA-A*201 -restricted epitope from influenza A matrix protein (FIuMP) was synthesised and used as a positive control for comparative purposes.

[0262] T2 binding assays

[0263] Peptide binding to HLA- A*201 was assessed by stabilisation of HLA- A*201 molecules on the surface of T2 cells by flow cytometry. T2 cells were washed twice in RPMI 1640 and resuspended in X- vivo 15 serum-free medium (Cambrex) in the presence of 1.25 μg/mL β-2-microglobulin. Serial dilutions of hK4A, hK4B, hK4C, hK4D, hK4E, PAP-5_299-307, PSA-31_54-163, and PSMA-2_711-7i₉, FIuMP peptides (50-0.75 μg/mL in dH₂O or 25% DMSO) were added and cells incubated at room temperature for 2 hours with occasional gentle re-suspension. Cells were then washed and stained with an anti-HLA-A2 Ab (HB 82 supernatant, ATCC) followed by FITC-labelled sheep anti-mouse antibody and analysed by flow cytometry. The HLA- A2 stabilisation ratio was calculated as the mean mean

fluorescence intensity (mean MFI) of HLA- A2 staining of T2 cells pulsed with test peptide divided by the MFI of control T2 cells without peptide at saturating (12 μg/mL)

concentrations of peptide.

[0264] CMRF-56⁺ DC preparation

[0265] CMRF-56^"1" DC preparations were isolated as previously described (Lopez,

J. A. et al, 2003 J Immunol Methods 274: 47-61). Briefly, PBMCs were isolated by Ficoll- density gradient separation and cultured for 16 hours in RPMI 1640 containing 2 mM glutamine, 10 mM HEPES, 1 mM pyruvate, 0.1 mM nonessential amino acids, 50 μM 2- mercaptoethanol, and 10% human pooled AB serum (complete medium [CM], Takayama, T. K., et al, 2001 Biochemistry 40(50): 15341-15348) at a density of 1 to 1.5 x 10⁷ cells/mL in non-tissue culture grade Petri dishes. Cells were collected by washing three times with cold PBS and resuspended at 6.6 x 10⁷/mL in cold separation buffer (PBS, 2 mM EDTA, 1% human pooled AB serum). Biotinylated CMRF-56 mAb was added for 15 minutes at 4⁰C. Cells were washed once in cold PBS and resuspended in separation buffer at 10⁸ cells/mL. 100 μL/10⁸ PBMC anti-biotin microbeads (Miltenyi Biotech, Sydney, Australia) were added for 15 minutes at 4⁰C. After washing once, cells were resuspended at 10⁸AnL and passed over a MACS LS magnetic column (Miltenyi) according to the manufacturer's instructions.

Alternatively, CMRF-56⁺ cells were isolated on the CliniMACS machine using the enrichment 1.1 program and a TS Tubing set (Miltenyi). The positively selected cell fraction was referred to as the CMRF-56⁺ preparation.

[0266] Generation of KLK4 mRNA

[0267] Full length KLK4 cDNA was cloned into the pGEM4Z cDNA vector. The vector was linearised with Spe I restriction enzyme, then purified using the Qiaquick PCR purification kit (Qiagen). Linearised cDNA was converted to mRNA using the mMessage mMachine kit T7 (Ambion) and purified using the MEGAclear kit (Ambion). The concentration of mRNA was determined using a nanodrop (Thermo Scientific).

[0268] Transfection of DC

[0269] DC isolated from donor PBMC by CMRF-56⁺ positive selection as described above were transfected with mRNA using a BioRad GenePulsar II. Briefly, DC were resuspended at 10⁷ cells/mL in RPMI. 200 μL of the cell suspension was mixed with 4 μg mRNA (CMRF-56⁺ DC) or 10 μg mRNA (MoDC) in a 0.2 cm gap cuvette (BioRad). Cells were electroporated at 350 V, 150 μF, then incubated on ice for 1 minute, prior to resuspension in complete AB media.

[0270] Induction of CTL cultures

[0271] The CMRF-56^4" preparation was re-suspended in X-vivo 15 medium and pulsed with either hK4 peptides, PSMA_7H-719, PSAi₅₄.₎₆₃, or PAP299-3₀7 peptides (5 μg/mL of each) for 2 hours at room temperature. Cells were washed once in CM and incubated with autologous PBMC responders at a 1 :5 ratio in CM, containing 10 ng/mL IL-7. IL-2 (25 U/mL) was added on day 3 and every 3 days thereafter. Cultures were restimulated with autologous irradiated (300OcGy) peptide-pulsed PBMC on day 12 and then at weekly intervals.

[0272] IFN-γ ELISPOT

[0273] The induction of peptide-specific T lymphocyte responses in the cultures was measured prior to every stimulation using an IFN-γ ELISPOT assay kit (Mabtech). In brief, nitrocellulose filtration plates (Millipore) were coated overnight at 4⁰C with 50 μg/mL IFN-γ capture antibody. After washing and blocking with CM, titrations of harvested CTL cultures were incubated with individual test (in triplicate), control, or no peptide pulsed T2 cells in the presence of 1.25 μg/mL β-2-microglobulin. After 18 hours culture (37⁰C, 5% CO₂), the plates were washed with PBS/0.01% Tween followed by PBS, and incubated with biotinylated IFN-γ detection antibody for 2 hours at 37⁰C before washing again. Spots were visualised by the addition of streptavidin-alkaline phosphatase for 1 hour at 37⁰C, followed by Sigma FAST™ substrate. Colour development was terminated with water, plates were dried and spots counted on an automated ELISPOT reader (Autoimmun Diagnostika). All dilutions were performed in triplicate against individual peptides and controls. The frequency of antigen-specific CTL was calculated from dilutions which fell within the linear sensitivity range of the assay and responses were classified as antigen-specific only where the number of spots in the test wells were significantly greater (p < 0.05 by Student's paired t-test) than those in the control wells at the same CTL dilution. The percentage of CD8⁺ T lymphocytes within the cultures was monitored by flow cytometry. Positive responses were then expressed as number of IFN-γ producing cells per 10⁴ or 10⁶ CD8⁺ T lymphocytes from the means of triplicate test minus control wells.

[0274] Clonal expansion of in-vitro generated CTL

[0275] When the CTL cultures generated greater than 1% specific CTLs detected by ELISPOT, attempts were made to isolate and expand clonal antigen-specific CTL using an IFN-γ secretion assay (Miltenyi Biotec). Briefly, cultures were incubated with specific peptide- or control-pulsed T2 cells overnight, then stained with IFN-γ catch reagent followed by incubation in CM for 45 minutes at 37⁰C to trap IFN-γ on the surface of secreting cells. Cells were stained with a PE-conjugated IFN-γ detection antibody along with anti-CD3-FITC, CD4-APC, CD8-PerCPC5.5 and CD56-PECy7. Single IFN-γ⁺ CD3⁺CD8⁺CD56- events were sorted by flow cytometry into round-bottom plates and expanded with irradiated allogeneic PBMC in the presence of 4 μg/mL phytohemagglutinin (PHA) and 100 LVmL IL-2.

Specificity of the expanded clones was tested by ⁵¹Cr release assay using peptide-pulsed T2 cells as targets. Clones were maintained in CM containing 100 LVmL IL-2 with intermittent restimulation with irradiated allogeneic PBMC and PHA as required.

[0276] Cytotoxicity assay

[0277] A standard 4 hour chromium (⁵¹Cr) release assay was used to examine the CTL cytotoxic capacity against peptide-pulsed T2 cells, RNA electroporated autologous DC and tumour cell lines. Washed targets were labelled with 100 μCi ⁵¹Cr and incubated with titrated doses of CTL for 4 hours in the presence of excess K562 cells to saturate any nonspecific NK-mediated lysis. Culture supernatants were harvested and counted on a Microbeta Trilux β-scintillation counter (Wallac). Percent specific lysis was calculated at 100 x (test count-spontaneous release control count) / (total release control count-spontaneous release control count).

[0278] Pentamer analysis

[0279] Induction of peptide specific MHC-class I restricted CTL was determined using pentamer technology prior to each stimulation. Briefly, CTL cultures were stained with the relevant Peptide/MHC APC-conjugated pentamers (Proimmune) in PBS/0.2% human serum albumin for 30 minutes in the dark at room temperature followed by staining with anti- human CD4-PE, CD3-FITC, CD14-PerPC, CD19-PerCP, CD8-FITC (all BD) and 7- aminoactinomycin D (Sigma). Cells were washed and analysed on a BD FACS Calibur flow cytometer. Pentamer positive^' cells were determined as those that were CD 14/19/7 AAD negative, CD8/Pentamer positive. A no pentamer stained control was used to set the gating strategy. The percentage of CD 8 positive pentamer cells was expressed as a percentage of the total CD8⁺ T lymphocytes.

[0280] Reverse transcription-PCR (RT-PCT)

[0281] Total RNA was isolated from cancer cells using TRIzol reagent (Invitrogen,

Mount Waverley, Victoria, Australia), treated with RNasefree DNase I (Invitrogen) and reverse-transcribed with random hexamers pd(N)6 (Roche Diagnostics, Brisbane,

Queensland, Australia) and Superscript II (Invitrogen), PCR with KLK4-specific primers K4ExlATGS (5'-ATGGCCACAGCAGGAAATCCC-S ') and K4Ex4AS (5'- CACGCACTGCAGCACGGT AG-3 '), (detects full length KLK4-254 transcript that includes the signal peptide codon) or K4Ex2S (5'-GCGGCACTGGTCATGGAAAACG-S') and K4Ex5AS (5'-CAAGGCCCTGCAAGTACCCG-S '), (detects exon 1 deleted KLK4-205 transcript that does not include the signal peptide codon) was performed with 1 μl cDNA, 0.5 units platinum Taq DNA polymerase (Invitrogen) and 35 cycles with annealing temperatures of 62⁰C. PCR for β2-microglobulin (β2M), which gave a 249 bp amplicon used as an internal control for RNA integrity, was performed using forward (5'-

TGAATTGCT ATGTGTCTGGGT-3') and reverse (5'-CCTCCATGATGCTGCTTACAT-S') primers for 32 cycles with similar PCR conditions. The PCR products were electrophoresed on a 1% (w/v) agarose gel and visualised by ethidium bromide staining. EXAMPLE 1

[0282] Identification, synthesis and characterisation of HLA-A*201 binding epitopes from hK4 [0283] The two computer algorithms (BIMAS and SYFPEITHI) were used to identify nonamer peptides sequences from hK4 that were predicted to bind efficiently to HLA-A*201. Five peptides with the highest binding scores from both algorithms (designated hK4A-E) were chosen for further analysis (Table 2). The hK4A peptide sequence is located in the signal sequence region of exon 1 that is only present in the hK4-254 isoform, whilst the peptide sequences B-E are located downstream in regions common to both hK4-254 and hK4- 205 isoforms.

[0284] PSA, PAP and PSMA peptides that have been previously used in prostate cancer clinical trials were included for comparative purposes. In addition, the well- characterised HLA- A*201 -restricted epitope from influenza A matrix protein (FIuMP) was used as a positive control for comparative purposes.

[0285] The five hK4 peptides (hK4A-E), and the PSA, PAP, PSMA and FIuMP peptides were synthesised. The binding affinity to HLA-A*201 of each peptide was then assessed using a T2 binding assay. The results are shown in Table 2.

[0286] Each of the five hK4 peptides increased expression of HLA-A*0201 on the surface of the T2 cells over the range of 0.75-50 μg/mL, with most peptides showing a stabilisation plateau at around 6-12 μg/mL.

[0287] At peptide saturation (12 μg/mL), the five hK4 peptides all bound HLA- A*201 with similar or higher affinity compared to the control strong HLA-A*201 binding epitope from FIuMP, as indicated by stabilisation ratios of greater than 1.4 in the T2 binding assay. Of the five hK4 peptides, the hK4A exon 1 signal sequence peptide demonstrated the highest affinity for HLA-A*201, consistent with the predictions from both algorithms.

[0288] TABLE 2: Peptide sequences, predicted binding and stabilisation of HLA-A2 on T2 cells

EXAMPLE 2

[0289] Induction of hK4 peptide-specific CD8⁺ T lymphocyte responses in healthy donors and prostate cancer patients

[0290] The ability of the hK4 peptides identified in Table 2 to induce CD8⁺ CTL responses (i.e. immunogenic responses) was examined in healthy donors and prostate cancer patients at various stages of their disease.

[0291] Individual results

[0292] PBMC from HL A- A*201 ⁺ healthy donors (HD 1 , HD2, HD3 , and HD4) were stimulated with autologous CMRF-56⁺DC pulsed with a pool of all five peptides. After 2 stimulations the cultures were monitored on a weekly basis by IFN-γ ELISPOT using individual peptides presented on T2 cells as stimulators. Robust CD8⁺ T lymphocyte responses ranging from 0.10-3.44% of CD8⁺ T lymphocytes could be generated against the hK4A peptide in all 4 healthy donors tested that peaked between 2-4 stimulations (results shown in Figure 1 and Table 3). Responses ranging from 0.04- 0.80% of CD8⁺ T lymphocytes were also detected against the hK4C peptide in all 4 healthy donors. Weaker responses were occasionally observed in some donors against the hK4B, D and E peptides (results shown in Figure 1 and Table 3).

[0293] As cancer patients are often immuno-compromised, it was decided to examine whether hK4 specific immune responses could be generated in patients with prostate cancer. The same method as above was used with PBMC from patients PCaI , PCa2, PCa3, and PCa4. Consistent with healthy donors, strong responses against the hK4A peptide, ranging from 0.10-2.08% of CD8⁺ T lymphocytes, could be detected in all 4 prostate cancer patients, regardless of their disease status (Figure 2 and Table 3). Responses ranging from 0.21-0.47% of CD8⁺ T lymphocytes could also be detected against the hK4C peptide in 3/4 prostate cancer patients. Some responses against hK4B, D and E were also observed in the majority of prostate cancer patients tested (Figure 2 and Table 3).

[0294] TABLE 3: Percentage of specific responses obtained to each hK4 peptide in healthy donors and PC patients

[0295] Although responses could be generated against all of the peptides in various donors, peptides A and C gave the highest overall responses in the majority of donors tested (8/8 donors for A and 7/8 donors for C).

[0296] Consolidated results

[0297] Additional prostate cancer patients PCa5 and PCa6 were recruited, and the same method as above for PCaI -4 was performed, to determine the ability of hK4 peptides to induce an immunogenic response.

[0298] The results shown separately above for HD 1-4 were consolidated, as were the results shown separate above for PCaI -4 along with the results for PCa5 and PCa6. These consolidated results are shown in Figure 3b.

[0299] Strong IFN-γ production specific for the hK4 A peptide was observed in healthy donors (Figure 3b, open symbols) and prostate cancer patients (Figure 3b, closed symbols). Both male (□ [open square] and 0 [open diamond], Figure 3b) and female (V [open triangle] and o [open circle], Figure 3b) healthy donors induced hK4A- specific responses. The maximum percentage of specific IFN-γ-producing cells was observed between 2-4 stimulations. Specific responses to the hK4B, C, D and E peptides were induced in some but not all of the healthy donors and prostate cancer patients tested (Figure 3a and 3b). However these were significantly lower in magnitude compared to the hK4A peptide.

[0300] To further investigate and confirm the ability of the hK4A peptide to induce CD8+ T cell responses, hK4A/HLA-A*0201 pentamers were synthesised and used to monitor the induction of K4A-specific CD8+ T cell responses (1) in 1 healthy donor and 1 prostate cancer patient in vitro (HDl and PCaI) and then subsequently (2) in 2 healthy donors and 10 prostate cancer patients.

[0301] The results for (1) are shown in Figure 8. Detectable levels of hK4A- specific CTL precursors were observed in unstimulated PBMC (Figure 8a and 8c) that could be expanded after several rounds of stimulation to levels similar to those detected using the IFN-γ ELISPOT assay (Figure 8b and 8d, Table 2). Furthermore, the majority of the cells in the CTL clonal cultures were stained positive for the pentamer, with irradiated allogeneic feeder cells accounting for the CD8^negpentamer^neg population evident in the cultures (Figure 8e).

[0302] Similarly, the results for (2) showed that, consistent with the

ELISPOT data, stimulation with hK4A peptide induced expansion of hK4A/HLA- A* 0201 -specific CD8+ T cells in all healthy donors and 8/11 prostate cancer patients (one representative - Pca5 - shown in Figure 3 c). The magnitude of the hK4A/HLA- A*0201 -specific response ranged from 1.29% to 80% of CD8⁺ T cells.

[0303] Collectively these data demonstrate that HLA-A* 0201 -restricted responses specific for hK4 can be generated in both male and female healthy donors as well as prostate cancer patients with the strongest response being to the hK4A epitope. The results also indicate that hK4A pentamers will be a useful tool for monitoring patient responses during the course of clinical trials. (Note from Example 7 below that hK4A is no longer believed to be HLA-A*0201 -restricted).

[0304] Based on the foregoing results, peptides A and C were chosen for further analysis.

EXAMPLE 3

[0305] Isolation and expansion of CD8⁺ T lymphocyte clones specific for hK4A and hK4C

[0306] CD8⁺ T lymphocytes specific for hK4A and hK4C peptides were isolated from the polyclonal cultures using an IFN-γ secretion assay and cloned by limiting dilution.

[0307] Two CTL clones specific for hK4A and four clones specific for hK4C were expanded and analysed to establish their specificity and functionality. [0308] All CTL clones secreted IFN-γ in response to T2 cells pulsed with their respective peptides, but not in response to control peptide pulsed T2 cells in ELISPOT assays (Figure 4a and 4b). Furthermore, each clone also lysed T2 cells pulsed with the respective hK4A or C peptides but not control T2 cells in a ⁵¹Cr release assay (Figure 4c and 4d).

[0309] Next, it was examined whether these clones demonstrated any cross- reactivity with other hK4 HLA-A*0201 restricted peptides. Clones specific for hK4A peptide did not cross-react with other HLA-A*0201 -restricted hK4 peptides B-E in IFN-γ ELISPOT assays (Figure 4e). Likewise, anti-hK4C CTL clones recognised the hK4C peptide exclusively and did not cross-react with hK4 peptides A, B, D, or E (Figure 4e).

[0310] Next the avidity of the clones was investigated by examining their ability to recognise T2 cells pulsed with titrating doses of specific peptide (Figure 4f). Clones specific for hK4C showed reactivity down to 5-50 pg/mL of specific peptide while hK4A clones showed an even higher avidity, reacting to as little as 0.5 pg/mL of specific peptide.

[0311] These data demonstrate that high avidity, functional CTL clones with specificity for the HLA-A*0201 -restricted peptides hK4A and hK4C can be expanded after in vitro stimulation. (Note from Example 7 below that hK4A is no longer believed to be HLA-A*0201 -restricted).

EXAMPLE 4

[0312] CTL specific for hK4A but not hK4C peptides recognise and lyse hK4-expressing cells

[0313] To determine whether the hK4A and hK4C peptides are endogenously processed and presented by tumour cells, the hK4A and hK4C specific CTL clones' ability to lyse peptide pulsed T2 cells, and to lyse ϋ-Zi^-expressing tumour cell lines was tested.

[0314] First, mRNA expression of both KLK4-254 and KLK4-205 transcripts in the target prostate cancer cell lines LNCaP (HLA- A*0201⁺) and PC-3 (HLA- A*0201^") and also the target ovarian carcinoma cell lines CaOV3 (HLA-A*0201⁺) and OVCAR-3 (HLA-A*0201⁺) were confirmed (Figure 5).

[0315] The hK4A clone [0316] The CTL clone specific for the hK4A peptide demonstrated strong lysis of peptide pulsed T2 cells (Figure 6a).

[0317] The clone also lysed the LNCaP and CaOV3 cell lines but not the control HLA-A*0201^" cell line PC3 (Figure 6c). Although the OVCAR-3 is HLA- A*0201⁺, it was resistant to CTL lysis, consistent with its low HLA-A2 expression and other data (Bondurant, K. L. et al, 2005 Cancer Res 11(9): 3446-3454).

[0318] Next, DC were electroporated with mRNA encoding the KLK4-254 transcript and used to induce CTL responses in a prostate cancer patient (PCa5). Using this approach, hK4A/HLA-A*0201 pentamer-specific CD8⁺ T cells were detected after 4 stimulations that represented 2% of the total CD8⁺ T cells (Figure 7).

[0319] These data make the crucial point that the hK4A epitope is

endogenously processed and presented by KLK4-254-expressmg tumour cell lines.

[0320] The hK4C clone

[0321] In contrast, the hK4C clones failed to lyse any of the cell lines tested (Figure 6d) despite their capacity to lyse hK4C peptide pulsed T2 cells very efficiently at low effectortarget ratios in the same assay (Figure 6b). Thus, the hK4C epitope is not endogenously processed and presented by HLA- A2⁺ tumour cell lines.

EXAMPLE 5

[0322] Transfection of MoDC

[0323] To further validate the hK4A peptide as a vaccine candidate, it was decided to examine whether this peptide could be processed and presented by dendritic cells (DCs). MoDC were transfected with KLK4 RNA or mock transfected, and used as targets against autologous hK4A specific CTL clones (Figure 6e). KLK4 RNA transfected but not the mock transfected DC were killed by the CTL demonstrating that DC, as well as tumour cell lines can process and present the hK4A peptide.

EXAMPLE 6

[0324] hK4A induces superior responses compared to PSA_154-163, PAP₂₉9. ₃₀₇ and PSMA₇₁₁..₇₁₉ peptides

[0325] The induction of strong CTL responses to the hK4A peptide in prostate cancer patients and the capacity of these CTL to lyse hK4-tumour cell targetrs makes the hK4 epitope an attractive candidate for immunotherapy. [0326] To validate this concept, pentamers were used to compare the magnitude of expansion of the K4A-specific CD8⁺ T cell responses with those specific for the known HLA-A*0201 -restricted epitopes from the prostate cancer TAA:

PSMA_711-719, PSA_154-163 and PAP_299-307. These have been used for DC vaccination in clinical trials (Lubaroff, D. M. et al, 2009 Clin. Cancer Res. 15(23): 7375-7380;

McNeel, D. G. et al, 2009 J CUn. Oncol. 27(25): 4047-4054; and Olson, W. C. et al, 2007 Recent CHn. Trials 2(3): 182-190).

[0327] Consistent with the data in Figure 3 , hK4A-sρecific CD8⁺ T lymphocytes were expanded in all 5 prostate cancer patients (Figure 9 closed symbols) and one healthy donor (Figure 9 open symbols). In contrast, CD8⁺ T cells specific for the PSA_154-163 and PAP2₉₉-₃o₇ peptides did not significantly expand in the same donors (Figure 9). PSMA₇n-₇i₉-specific CD8⁺ T cells were induced in most donors, however these were lower in magnitude compared to the responses induced by hK4A in the healthy donor and in 4/5 prostate cancer patients (Figure 9).

[0328] These data suggest that hK4A is an immunodominant prostate cancer tumour-associated antigen (TAA) epitope, which is at least as good as the PSMA₇₁₁-₇i9 epitope and superior to the PSA_154-163 and PAP_299-307 epitopes in current clinical use.

EXAMPLE 7

[0329] hK4A is not restricted to HLA-A2

[0330] Using the computer algorithms BIMAS and SYFPEITHI, a range of HLA haplotypes was screened to determine if hK4A epitope was predicted to bind to haplotypes other than HLA-A*0201.

[0331] The results are shown in Table 4 below. While the strongest overall binding prediction for hK4A was to HLA-A*0201 by both programs, it was also found that hK4A was predicted to bind to the haplotypes listed in Table 4. The binding score for each haplotype by either algorithm, and frequency of the allele in the Australian population is provided.

[0332] Table 4: Predicted binding scores for hK4A across various haployptes, and the frequency of each haplotype in the Australian population

[0333] The ability of a HLA-A*02017HLA-A11 ⁺ prostate cancer patient to develop an immune response against hK4A was then tested. Following 4 rounds of stimulation with hK4A peptide, 16.8% of CD8⁺ T cells bound to hK4A specific pentamer (Figure 10), representing a significant immune response to this epitope in a HLA-A*0201 negative donor.

DISCUSSION

[0334] Discussion of results

[0335] The identification of new immunogenic prostate cancer TAA is essential to develop effective immunotherapy for this disease. The above examples identified an immunogenic CTL epitope within hK4, that was endogenously processed by normal antigen presenting cells. This epitope, which was termed hK4A, enabled us to expand functional hK4A specific CTL in vitro from most healthy donors and prostate cancer patients. Most importantly, the hK4A epitope was endogenously processed and presented by tumour cells allowing the hK4A-specific CTL to lyse HLA-A*0201⁺ prostate and ovarian cancer cell lines expressing hK4. Furthermore, we found hK4A to be an immunodominant epitope that generated CTL in the majority of donors tested at least as effectively as PSMA and more effectively than the known PSA and PAP epitopes being used in current clinical trials. Thus hK4A appears to be suitable for use in cancer immunotherapy. Furthermore, whilst originally believed to be a HLA- A*0201 -restricted epitope, it now appears that hK4A is not restricted to HLA-A*0201, and includes at least HLA-Al I⁺.

[0336] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0337] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An immunogenic composition, the composition comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: 1] or a nucleic acid molecule from which the peptide is expressible.

2. A composition for stimulating an immune response in a subject, the composition comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a nucleic acid molecule from which the peptide is expressible.

3. The composition of claim 2, wherein the immune response is a T-cell mediated response.

4. The composition of claim 2 or claim 3, wherein the immune response is a cytotoxic lymphocyte (CTL) mediated immune response.

5. A composition for preventing or treating a cancer in a subject, the composition comprising a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a nucleic acid molecule from which the peptide is expressible.

6. The composition of claim 5, wherein the cancer is a hormone-related cancer.

7. The composition of claim 5 or claim 6, wherein the cancer is prostate cancer, breast cancer, or ovarian cancer.

8. The composition of any one of claims 1 to 7, wherein the composition comprises an immunostimulatory antigen-presenting cell or antigen-presenting cell precursor that presents a peptide or a processed form thereof on its surface, wherein the peptide comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I].

9. The composition of any one of claims 1 to 7, wherein the composition comprises a peptide comprising, consisting or consisting essentially of an amino acid corresponding to the corresponding to the sequence FLGYLILGV [SEQ ID NO:1], wherein the peptide is in particulate form.

10. The composition of claim 9, wherein the peptide is attached to, contained within, or otherwise associated with a particle.

11. The composition of any one of claims 1 to 7, wherein the composition comprises a peptide comprising, consisting or consisting essentially of an amino acid corresponding to the corresponding to the sequence FLGYLILGV [SEQ ID NO:1] and an antigen-binding molecule.

12. The composition of claim 11, wherein the peptide is conjugated to or otherwise associated with the antigen-binding molecule.

13. The composition of claim 11 or claim 12, wherein the antigen-binding molecule is an antibody.

14. The composition of any one of claims 1 to 7, wherein the composition comprises a nucleic acid molecule from which a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: 1 ] is expressible.

15. The composition of claim 14, wherein the nucleic acid molecule comprises a nucleotide sequence that encodes the peptide and that is operably linked to a regulatory polynucleotide.

16. An immunogenic composition, the composition comprising a molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I].

17. A composition for stimulating an immune response in a subject, the composition comprising a molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I].

18. The composition of claim 17, wherein the immune response is a T-cell mediated response.

19. The composition of claim 17 or claim 18, wherein the immune response is a cytotoxic lymphocyte (CTL) mediated immune response.

20. A composition for preventing or treating a cancer in a subject, the composition comprising a molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1].

21. The composition of claim 20, wherein the cancer is a hormone-related cancer.

22. The composition of claim 20 or claim 21, wherein the cancer is prostate cancer, breast cancer, or ovarian cancer.

23. The composition of any one of claims 16 to 22, wherein the composition comprises T-lymphocytes that are primed to respond to a peptide comprising, consisting or consisting essentially of an amino acid corresponding to the sequence FLGYLILGV [SEQ JD NOil].

24. The composition of claim 23, wherein the T lymphocytes are cytotoxic (CTL) T lymphocytes.

25. The composition of any one of claims 16 to 22, wherein the composition comprises an antigen-binding molecule that is immuno-interactive with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1].

26. The composition of claim 25, wherein the antigen-binding molecule is an antibody.

27. A method for producing an immunostimulatory antigen-presenting cell or antigen-presenting cell precursor, the method comprising contacting an antigen- presenting cell or antigen-presenting cell precursor with a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], or a nucleic acid molecule from which the peptide is expressible, for a time and under conditions sufficient for the peptide or a processed form thereof to be presented by the antigen-presenting cell or antigen-presenting cell precursor.

28. A method for producing T-lymphocytes that are primed to respond to a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], the method comprising contacting antigen-presenting cells that present the peptide or a processed form thereof on their surface with a population of T lymphocytes for a time and under conditions sufficient to produce T lymphocytes that are primed to respond to the peptide.

29. A method for stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of a composition of any one of claims 1 to 26.

30. The method of claim 29, wherein the immune response is a T-cell mediated response.

31. The method of claim 29 or claim 30, wherein the immune response is a cytotoxic lymphocyte (CTL) mediated immune response.

32. A method for preventing or treating a cancer in a subject, the method comprising administering to the subject an effective amount of a composition of any one of claims 1 to 26.

33. The method of claim 32, wherein the cancer is a hormone-related cancer.

34. The method of claim 32 or claim 33, wherein the cancer is prostate cancer, breast cancer, or ovarian cancer.

35. The method of any one of claims 29 to 34, wherein the method comprises increasing in the subject the number of antigen-presenting cells or antigen-presenting cell precursors that present a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1] or a processed form thereof on their surface.

36. The method of any one of claims 29 to 34, wherein the method comprises increasing in the subject the number of T-lymphocytes that are primed to respond to a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO: I].

37. The use of a peptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence FLGYLILGV [SEQ ID NO:1], a nucleic acid molecule from which the peptide is expressible, or a molecule that is immuno-interactive with the peptide, in the manufacture of a medicament for stimulating an immune response or for preventing or treating a cancer in a subject.

38. The use of claim 37, wherein the immune response is a T-cell mediated response.

39. The use of claim 37 or claim 38, wherein the immune response is a cytotoxic lymphocyte (CTL) mediated immune response.

40. The use of claim 37, wherein the cancer is a hormone-related cancer.

41. The use of claim 37 or claim 40, wherein the cancer is prostate cancer, ovarian cancer, or breast cancer.