WO2008113970A2

WO2008113970A2 - Peptides

Info

Publication number: WO2008113970A2
Application number: PCT/GB2008/000732
Authority: WO
Inventors: Shahriar Behboudi
Original assignee: Ucl Business Plc
Priority date: 2007-03-16
Filing date: 2008-03-05
Publication date: 2008-09-25
Also published as: WO2008113970A3

Abstract

The present invention relates to peptides comprising a fragment of the human alpha- fetoprotein molecule of SEQ ID NO: 1, wherein said peptides comprise: (a) amino acids 46-55 of SEQ ID NO: 1; (b) a variant of (a) in which one, two or three amino acids are deleted; or (c) a variant of (a) comprising one, two, three or four amino acid substitutions from the sequence of (a); wherein said peptide is capable of activating T cells that produce TGF-β and/or GM-CSF, and wherein said peptide does not comprise a B cell epitope, a CTL epitope or a ThI epitope.

Description

PEPTIDES

Field of the Invention

The present invention relates to peptides derived from human alpha-fetoprotein. In particular, the invention relates to peptide fragments of alpha-fetoprotein that include an epitope capable of stimulating activation of immune cells such as regulatory T cells. The invention also relates to cells stimulated by such a peptide and to the use of such peptides and cells.

Background to the Invention

Alpha-fetoprotein (AFP) is one of the earliest proteins to be synthesized by the embryonic liver and is found in fetal and maternal tissue fluids but its synthesis decreases after birth and only trace amounts are expressed in the normal adult liver. The AFP gene becomes reactivated in hepatocellular carcinoma (HCC) and also during hepatocyte regeneration (e.g. as in acute hepatitis). AFP is a diagnostic and prognostic serum marker and it also serves as a tumour rejection antigen and therefore is an attractive target for immunotherapy.

Little is known about the biological activities of AFP during fetal and prenatal development; however it has been suggested that this oncofetal protein has intrinsic immunoregulatory properties. The inventors have previously identified three immunodominant AFP-derived ThI epitopes and have shown that the presence of a potent anti- AFP CD4+ T cell responses is associated with better clinical outcome in HCC ''².

Summary of the Invention The inventors have found that an alpha-fetoprotein-derived epitope (AFP^.ss) is recognized by immune cells such as CTLA-4+ CD4+ Th3 cells and NK cells. These immune cells produce TGF-β, GM-CSF and IL-2, but not ThI, Th2, TrI or ThI 7 type cytokines, in a peptide specific manner. The expanded AFP_46-55Treg cells have immunoregulatory property as shown by their ability to inhibit T cell proliferation in vitro. Cell-to-cell contact is required for AFP_46-55 Treg cells to exert suppressive activity. The frequencies of circulating AFP_46-55-specific regulatory CD4+ T cells are significantly higher in hepatocellular carcinoma (HCC) patients over-expressing AFP than that in healthy individuals (p<0.01), suggesting that these cells may expand in vivo in response to encounter with the tumour antigen. The reduction of tumour burden by tumour necrosis-inducing treatment reduced the levels of circulating AFP_46-5sCD4+ Treg cells and shifted the balance in favor of ThI responses. These data suggest that self antigen- specific Treg cells are expanded in cancer patients and these cells may play an important role in the modulation of anti-tumour immunity.

Accordingly, the present invention provides a peptide comprising a fragment of the human alpha- fetoprotein molecule of SEQ ID NO: 1, wherein said peptide comprises:

(a) amino acids 46-55 of SEQ ID NO: 1 (b) a variant of (a) in which one, two or three amino acids are deleted; or

(c) a variant of (a) comprising one, two, three or four amino acid substitutions from the sequence of (a); wherein said peptide is capable of activating T cells that produce TGF-β and/or GM- CSF, and wherein said peptide does not comprise a B cell epitope, a CTL epitope or a ThI epitope.

The invention also provides: a vector capable of expressing a peptide of the invention; an isolated antigen presenting cell which presents a peptide of the invention; a method of producing regulatory T cells capable of inhibiting T cell proliferation, the method comprising

(a) contacting a sample from an individual with a peptide of the invention, wherein said sample comprises antigen presenting cells and T cells; or

(b) contacting a sample from an individual with an antigen presenting cell of the invention, wherein said sample comprises T cells. - an isolated regulatory T cell obtainable by such a method; a pharmaceutical formulation comprising a peptide, vector, antigen presenting cell or T cell of the invention and a pharmaceutically acceptable carrier. a method of inhibiting T cell proliferation comprising contacting a population of T cells with an antigen presenting cell or regulatory T cell of the invention. - a method of inhibiting T cell proliferation comprising:

(b) contacting a sample from an individual with an antigen presenting cell of the invention, wherein said sample comprises T cells; and contacting a population of T cells with said sample of (a) or (b) a method of treating or preventing a hypersensitivity reaction or of preventing or decreasing an immune response comprising administering to a patient in need thereof a peptide, vector, antigen presenting cell or regulatory T cell of the invention. - a peptide, vector, antigen presenting cell or regulatory T cell of the invention for use in treating or preventing a hypersensitivity reaction or of preventing or decreasing an immune response.

Brief Description of the Drawings Figure 1 : Identification of AFP-derived peptide epitope recognized by

CD3+CD4+ T cells. Short term T cell lines were generated in the presence of 62 different AFP-derived peptides. Cell lines were re-stimulated with relevant and irrelevant peptides or cultured in medium without peptide and peptide recognition was analyzed by using intracellular cytokine assay for GM-CSF (Figure IA). The percentages of CD4+ GM-CSF producing T cell are shown (Figure IB). The results represent two different experiments on different days. Similar pattern of response to AFP-derived peptides was detected in T cell lines generated from PBMC of 3 different HCC patients.

AFP46.5₅ CD4+ T cells were incubated with different concentration of AFP45.55 or AFP36₄.373 peptides and the percentage of GM-CSF producing CD4+ T cells was analyzed using intracellular cytokine assay (Figure 1C). To determine optimal peptide length required for T cell recognition, AFP_46-55CD4+ T cells were re-stimulated with AFP_46-55, AFP_47-55, AFP_44-57, AFP_42-55 or AFP_364-373 peptide and the induction of intracellular GM-CSF was analyzed using flow cytometry (Figure ID). The results represents two different experiments on different days.

AFP_46-55 CD4+ T cells were re- stimulated with the relevant or an irrelevant peptide or cultured in medium only and GM-CSF secretion was measured in the culture supernatant using ELISA assay (Figure IE). Each ELISA experiment was performed in triplicate and the results are representative of two different experiments on different days.

Figure 2: AFP_46-55 CD4+ T cells produce TGF-β in a dose dependent manner. AFP_46-55 CD4+ T cells were re-stimulated for 48 h in serum free medium with different concentrations of the relevant or an irrelevant peptide and the levels of total TGF-β was measured in the culture supernatant using an ELISA assay (Figure 2A). AFP_46-55 CD4+ T cell lines were stimulated with purified AFP (5 μg/ml), human serum albumin (5 μg/ml), AFP₄₆.₅₅ peptide or AFP_364-373 peptide for 48 h and the levels of total TGF-β was measured in culture supernatant (Figure 2B). Columns, each experiment were performed in duplicate and the results are representative of two different experiments on different days. CD4+, CD2+ or CD8+ cells were depleted from PBMCs isolated from an HCC patient and cultured in the presence of AFP_46-55 peptide and rIL-2. Cells were washed and cultured (3 x 10⁵ cells/ well) in serum free medium in the presence of AFP_46-55 peptide for 48 h. the levels of total TGF-β was measured in culture supernatant using ELISA (Figure 2C). Columns, each experiment were performed in duplicate and the results are representative of two different experiments on different days. AFP_46-55 CD4+ T cell lines were re-stimulated with AFP_46-55 peptide or AFP_364-

₃₇₃ (irrelevant peptide) and the percentage of active TGF-β produced by CD4+ T cells was detected using intracellular cytokine assay (Figure 2d). Representative data from three different T cell lines from different HCC patients are shown.

Figure 3A: Analysis of cytokine production by AFP_46-55 CD4+ T cells. Cell lines were re-stimulated with AFP_46-55 peptide or an irrelevant peptide and the production of IFN-γ, TNF-α, IL-5, IL-13, IL-17, IL-2 or IL-IO by CD4+ T cells was analyzed using intracellular cytokine assay. The percentages of cytokine producing CD4+ T cells are shown. Similar pattern of cytokine production was detected in 5 different T cell lines.

Figure 3B to D: AFP_46-55 peptide stimulated TGF-β, GM-CSF and IL-2 production by CD4+ T cells and NK cells but did not induce ThI , Th2, ThI 7 or TrI type cytokines. Cells were re-stimulated with AFP_46-55 peptide or an irrelevant peptide (AFP_364-373) and the production of IFN-γ, TNF-α, IL-5, IL-13, IL-17, IL-2, GM-CSF,

TGFβ and IL-10 by CD4+ T cells was analyzed using intracellular cytokine assay and surface staining for latent TGF-β. (B) A summary of cytokines produced by CD4 T cells from 6 different individuals is shown. Each symbol represents % cytokine producing CD4+ T cells from an individual. (C) The percentages of peptide-specific cytokine producing CD4+ T cells are shown. Two independent experiments were performed. (D) NK cells stimulated with AFP_46-55 peptide produce similar cytokine pattern as CD4+ T cells. Figure 4: Phenotypic characterization of peptide-specific and non-specific CD4+ T cells. AFP₄₆.₅₅ CD4+ T cell line was re-stimulated with AFP_46-55 peptide and the cells were stained with mAb to detect surface CD4, CD69L, CD45RO and GITR molecules, intracellular CTLA-4 and GM-CSF, intra-nucleus FOXP3. Isotype control Abs served as negative control staining. CD4+ GM-CSF producing T cells or CD4+ GM-CSF negative cells were gated and the expression levels of surface and intracellular molecules are shown. Representative data from T cell lines generated from PBMCs of 4 different HCC patients are shown. The results are representative of two different experiments on different days. Similar pattern of phenotypic characterization was observed in T cell lines generated from two different HCC patients.

Figure 5: AFP₄₆.₅₅ CD4+ T cells are developed from CD4+CD25+ and

CD4+CD25- T cells. CD4+ or CD8+ T cells were depleted from PBMCs and the remaining cells or PBMCs (no depletion) were cultured in medium in the presence of AFP_46-55 peptide and rIL-2 for 10 days. AFP_46-55 specific GM-CSF producing cells were analyzed using intracellular cytokine assay and the percentage of cytokine producing cells is shown (Figure 5a). Columns, each experiment are performed in duplicate and the results are representative of two different experiments on different days.

CD4+CD25+ were depleted from PBMCs and the remaining cells, PBMCs (no depletion) or PBMCs plus CD4+CD25+ T cells were cultured in the presence OfAFP₄₆. ₅₅ peptide and rIL-2 for 10 days. The frequency of peptide specific CD4+ T cells was determined using intracellular cytokine assay for GM-CSF (Figure 5b). CD4+CD25+ or CD4+CD25- T cells were stimulate with AFP_46-55 peptide pulsed autologous DC and cultured in medium containing rIL-2 for 10 days. Cells were re-stimulated with DC pulsed with AFP_46-55 or an irrelevant peptide and the frequencies of GM-CSF producing cells were determined using intracellular cytokine assay for GM-CSF. The percentage of GM-CSF producing cells is shown (Figure 5c). The results are representative of two different experiments on different days.

Figure 6: AFP_46-55 CD4+ T cells suppress T cell proliferation to anti-CD3 Ab stimulation in a cell-to-cell contact manner. CD4+CD25- T cells were cultured with AFP_46-55 CD4+ T cells in different ratio and anti-CD3 Ab induced T cells proliferation was assayed by adding 3H[thymidine] during the last 18h of culture. The levels of T cell proliferation is shown (Figure 6A). In transwell system, responding CD4+ T cells were cultured in outer wells with APCs. AFP_46-55 or AFP364-373 CD4+ T cells were cultured in inner wells with APCs. The cells in outer wells and inner wells were stimulated with anti-CD3 Ab. There was no detectable suppressive activity when responding cells were cultured in outer wells, regardless of the presence of AFP₄₆.₅₅ or AFP35₄.373 CD4+ T cells or none in the inner wells (Figure 6B). Each experiment was performed in triplicate and the results are representative of two different experiments on different days.

Figure 7: AFP_46-55 CD4+ T cells are expanded in HCC patients. The frequency of AFP_46-55 was detected in T cell lines generated from PBMCs of HCC patients (n=15) and healthy donors (n=10) using intracellular cytokine assay for GM-CSF (Figure 7A). Tumour embolisation reduces circulating^ AFP_46-55 CD4+ T cells. The frequency of

AFP_46-55 CD4+ T cells was determined before and three months after TACE/ TAE treatment in 5 HCC patients using intracellular cytokine assay for GM-CSF (Figure 7B). The results are representative of two different experiments on different days. Figure 8: Detection of AFP_46-55 specific-TGFβ releasing cells ex vivo. The frequency OfAFP_46-55 specific-TGFβ releasing T cells was analyzed in PBMCs of four healthy donors (HD-I, HD-2, HD-3 and HD-4) ex vivo using an ELISPOT assay for TGFβ. The results are presented as spot forming units (s.f.u) per 3 x 10$ cells and average spots with standard deviation (S. D.) is shown. The experiments were performed in triplicate and the results are representative of two experiments performed on different days.

Brief Description of the Sequence Listing

SEQ ID NO: 1 is the amino acid sequence of human alpha- fetoprotein (AFP). SEQ ID NO: 2 is the amino acid sequence of a fragment from amino acid 46 to amino acid 55 of SEQ ID NO: 1.

SEQ ID NO: 3 is the amino acid sequence of the mouse peptide corresponding to the human sequence of SEQ ID NO: 2.

SEQ ID Nos: 4 to 65 are the human AFP peptide sequences shown in Table 1 below. Detailed Description of the Invention

Fragments of AFP

The present invention derives from the identification and characterisation of the first AFP-derived epitope recognized by Th3 type regulatory T cells. The invention provides peptides comprising such an epitope.

It has been suggested that the administration of full length recombinant AFP in animal models and patients suffering from autoimmune disease may suppress anti-self immune responses³. However, since human AFP is a largely growth-promoting substance, the therapeutic injection of full-length (70 kDa), naive AFP into normal and/or diseased adult could potentially be hazardous and would require extensive preclinical toxicity testing. Moreover, the entire full-length, naϊve AFP molecule is bristling with innumerable potential biologically active sites. On stimulation, the biological response of these individual sites cannot be predicted or controlled and could produce undesired or dangerous side effects. Therefore, site-specific AFP-derived peptides as described herein offer a safer, more conservative approach for possible modulation of these multiple biological responses.

The present invention thus relates to particular fragments of the human AFP protein. The invention relates to these fragments, to peptides that comprise or consists essentially of such fragments, and to their uses. The amino acid sequence of full length human AFP is given in SEQ ID NO: 1.

Of particular interest are fragments of the AFP protein that comprise amino acids 46-55, i.e. to fragments comprising the amino acid sequence LATIFF AQFV (SEQ ID NO: 2). As reported in the Examples below, this peptide has been shown to be particularly effective in stimulating immune cells such as regulatory T cells. A peptide of the invention may therefore comprise, or consist essentially of, the amino acid sequence of SEQ ID NO: 2.

Other peptides of the invention may comprise variants of the sequence of SEQ ID NO: 2. Suitable variants may, for example, be different fragments of the full length human AFP sequence, derived from the same region as SEQ ID NO: 2.

Peptide fragments according to the invention may be derived by truncation, e.g. by removal of one or more amino acids from the N and/or C-terminal ends of a polypeptide. Fragments may also be generated by one or more internal deletions. For example, a variant of SEQ ID NO: 2 may comprise a fragment of SEQ ID NO: 2, i.e. a shorter sequence within the region amino acids 46-55 of SEQ ID NO: 1. This may include a deletion of one, two three, four or more amino acids from the N- terminal end of SEQ ID NO: 2 or from the C-terminal end of SEQ ID NO: 2. Such deletions may be made from both ends of SEQ ID NO: 2. For example, a shorter variant may comprise amino acids 47-55, 48-55, 46-54, 46-53, 47-54, 47-53, 48-54 or 48-53 of SEQ ID NO: 1.

A variant of SEQ ID NO: 2 may include additional amino acids from the human AFP protein sequence extending beyond the end(s) of SEQ ID NO: 2. A variant may be, for example, 11, 12, 13, 15, up to 20, up to 25, up to 30, up to 40, up to 50 or more amino acids in length. A variant may be a peptide that is less than 12, less than 15, less than 20, less than 25, less than 50 or less than 100 amino acids in length. Thus, a peptide of the invention may comprise a fragment of the human AFP sequence that comprises SEQ ID NO: 2. For example, a variant sequence may comprise amino acids 42-55 or 44-57 of SEQ ID NO: 1. The use of a shorter peptide has advantages in reducing the cost of production. The use of a shorter peptide also reduces the likelihood that sequences from the full length AFP protein that might be capable of having different effects will be present.

A variant may include a combination of the deletions and additions discussed above. For example, amino acids may be deleted from one end of SEQ ID NO: 2, but additional amino acids from the full length AFP protein sequence may be added at the other end of SEQ ID NO: 2.

A variant peptide may also include one or more amino acid substitutions from the amino acid sequence of human AFP or a fragment thereof. For example, a suitable variant may have an amino acid sequence which has more than 60%, or more than 70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90, 95 or 98% amino acid identity to the full length human AFP protein of SEQ ID NO: 1 or a fragment thereof, such as the fragments described herein. This level of amino acid identity may be seen across the full length of the sequence or over a part of the sequence, such as 8, 10, 12, 20, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full length polypeptide.

In connection with amino acid sequences, "sequence identity" refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters: Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters -Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized.

A variant peptide may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 50 or more amino acid substitutions from the full length human AFP amino acid sequence or a fragment thereof. Substitution variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid. Some properties of the 20 main amino acids which can be used to select suitable substituents are as follows:

A variant peptide may comprise an equivalent sequence derived from a different organism. For example, a variant peptide may comprise a peptide equivalent to amino acids 46-55 of the human AFP sequence, but derived from the AFP gene of a different organism. Such a species variant may derive from any organism that expresses an AFP protein. For example, the species variant may derive from a mammal such as a primate, rodent or a domestic or farm animal. A variant peptide may also comprise a variant of such a species variant sequence such as a deletion, addition or substitution variant as described herein.

The equivalent peptide in a different AFP gene may easily be determined by one of skill in the art. The equivalent sequence can be determined by sequence alignment. For example, in the human AFP protein, the peptide at amino acids 46-55 has the sequence LATIFF AQFV. In the mouse sequence, the equivalent peptide sequence varies from the human sequence by three amino acid substitutions: IATITFTQFV. The skilled person can easily confirm that a species variant has the same function as a human peptide of the invention by using one of the methods described herein. Such a method may be used to confirm that a species variant, or a variant thereof, is capable of activating suitable immune cells such as T cells in a human sample. Such a method may be used to confirm that a species variant, or a variant thereof, is capable activating suitable immune cells such as T cells, where the sample is derived from a non-human animal, such as the same animal from which the species variant sequence derives.

Further variants include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be modified, e.g. labelled, providing the function of the peptide is not significantly adversely affected.

Where the peptide has a sequence that varies from the sequence of SEQ ID NO: 1 or a fragment thereof, the variations may occur across the full length of the sequence, within the sequence of SEQ ID NO: 2, or outside the sequence of SEQ ID NO: 2. For example, the variations described herein, such as additions, deletions, substitutions and modifications, may occur within the sequence of SEQ ID NO: 2. A variant peptide may comprise or consist essentially of the amino acid sequence of SEQ ID NO: 2 in which one, two, three, four or more amino acid substitutions have been made. A variant peptide may comprise a fragment of human AFP that is larger than SEQ ID NO: 2. In this embodiment, the variations described herein, such as substitutions and modifications, may occur within the sequence of SEQ ID NO: 2, outside the sequence of SEQ ID NO: 2 but within a fragment of SEQ ID NO: 1 , or both.

Variants as described above may be prepared during synthesis of the peptide or by post- production modification, or when the peptide is in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.

As explained herein, the peptide of SEQ ID NO: 2 has the ability to activate immune cells, hi particular, the peptide of SEQ ID NO: 2 has the ability to activate immune cells capable of expressing TGF-β and GM-CSF. The peptide of SEQ ID NO: 2 has the ability to activate regulatory T cells. In particular, the peptide of SEQ ID NO: 2 is recognised by CTLA-4+ CD4+ Th3 cells. These T cells produce TGF-β and GM-CSF and IL-2, in a peptide specific manner, but not ThI, Th2, TrI or ThI 7 type cytokines. These T cells also have immunomodulatory ability as shown by their ability to inhibit T cell proliferation in vitro. In accordance with the invention, a variant peptide is preferably a functional variant of SEQ ID NO: 2. That is, a peptide of the invention is preferably capable of activating T cells having one, more or all of the characteristics set out above. For example, a peptide of the invention may activate regulatory T cells. The T cells activated by such a peptide may be Th3 cells, and may express FOXP3 and/or CTLA-4. The T cells may be FOXP3+ CTLA-4+ CD4+ Th3 cells or FOXP3- CTLA-4+ CD4+ Th3 cells. The T cells activated by such a peptide may produce TGF-β and/or GM-CSF in a peptide specific manner. The T cells may also produce IL-2. The T cells preferably do not produce cytokines that are characteristic of ThI, Th2, TrI or ThI 7 cells. For example, the T cells preferably do not produce one, more, or all of IFNγ, IL- 10, TNF-α, IL-5, IL-13 and IL-17. The T cells activated by such a peptide preferably have an immunomodulatory activity, such as the ability to inhibit the proliferation of other T cells, such as ThI cells.

The peptide of SEQ ID NO: 2 also has the ability to activate other immune cells. The inventors have found that the peptide of SEQ ID NO: 2 is capable of activating natural killer cells.

In particular, the peptide of SEQ ID NO: 2 is found to be capable of activating CD3 negative CD56 high or low NK cells. These NK cells produce IL-2, GM-CSF and TGF-β, but not ThI, Th2, TrI or ThI 7 type cytokines. A peptide of the invention may be capable of activating other immune cells having one, more or all of the characteristics set out above. For example, a peptide of the invention may activate NK cells. A peptide of the invention may activate CD3 negative CD56 high or low cells such as CD3 negative CD56 high or low NK cells. The cells activated by such a peptide may produce TGF-β and/or GM-CSF. The TGF-β and/or GM-CSF may be produced in a peptide specific manner. The cells may also produce IL-2. The cells preferably do not produce cytokines that are characteristic of ThI, Th2, TrI or Th 17 cells. For example, the cells preferably do not produce one, more or all of IFNγ, IL-IO, TNF-α, IL-5, IL-13 and IL-17. Thus, a peptide of the invention may activate one or more classes of immune cell. A peptide of the invention may activate regulatory T cells as described herein. A peptide of the invention may additionally or alternatively activate NK cells as described herein. A peptide of the invention may additionally or alternatively activate other cells. The cells activated by such a peptide may produce TGF-β and/or GM-CSF. The TGF-β and/or GM-CSF may be produced in a peptide specific manner. The cells may also produce IL-2. The cells preferably do not produce cytokines that are characteristic of ThI, Th2, TrI or Th 17 cells. For example, the cells preferably do not produce one, more or all of IFNγ, IL-10, TNF-α, IL-5, IL-13 and IL-17. Thus, the cells are preferably not B cells, CTL cells, ThI cells, Th2 cells, TrI cells or ThI 7 cells. The cells activated by a peptide of the invention may have an immunomodulatory activity, such as an immunosuppressive activity.

The ability of a peptide to activate suitable cells can be easily tested by a person skilled in this field. For example, the ability of a peptide to activate cells can be measured in vitro. A suitable method is described in the Examples in relation to T cells. Briefly, a sample of cells such as PBMCs (peripheral blood mononuclear cells), mononuclear cells isolated from lymph nodes or tumour infiltrating lymphocytes, is provided. These may be from a patient that it is desired to treat. These may be from an individual having hepatocellular carcinoma. These may be from a healthy individual. These cells are contacted with the peptide of interest or with antigen presenting cells that have been peptide-pulsed with the peptide of interest. IL-2 may also be added to stimulate T cell proliferation. TGFβ may be added to enhance T cell proliferation. This should lead to the expansion of a population of T cells that are activated by the peptide of the invention. Equivalent methods for testing the ability of a peptide to activate other cell types, such as other cells described herein, e.g. NK cells, will be apparent to one of skill in the art.

The phenotype and cytokine profile of the expanded cells can be determined using routine methods. For example antibodies can be used to stain for molecules or cytokines that are specific to the cell type of interest such as particular T cell specific or NK cell specific molecules or cytokines. Secreted cytokines can also be measured directly, for example by ELISA. Further suitable methods are well known in the art. Similarly, the activity of such cells may be measured using routine methods. For example, as described further below, the interaction between such cells and ThI cells may be assessed to determine whether cells such as T cells activated by a peptide of the invention, are capable of immunomodulatory effects, such as regulation of ThI cells. The peptide of SEQ ID NO: 2 is believed to form an epitope for Th3 cells. A peptide of the invention will preferably retain this epitope or an epitope capable of retaining the same function. As used herein, the term "epitope" generally refers to the site on a target antigen which is recognised by an immune receptor such as a T-cell receptor and/or an antibody. Preferably it is a short peptide derived from or as part of a protein. However the term is also intended to include peptides with glycopeptides and carbohydrate epitopes. A single antigenic molecule may comprise several different epitopes. The term "epitope" also includes modified sequences of amino acids or carbohydrates which stimulate responses which recognise the whole organism.

The peptide of the invention may be used as described herein to suppress the proliferation of lymphocytes such as ThI, Th2, TcI or B cells. It may be used to counter conditions or circumstances in which an unwanted or excessive immune response induced by T cells or B cells is present. In particular it may be used in conditions or circumstances where it is desired to reduce a ThI and/or TcI immune response in an individual. In view of this intended function, it is preferable if the peptide does not include sequences that may lead to stimulation of immune function, such as stimulation of a ThI and/or TcI cell response. The peptide of the invention preferably does not include any epitopes other than the Th3 cell epitope exemplified by SEQ ID NO: 2. For example, the peptide may include no ThI and/or TcI cell epitopes. The peptide may lack any epitopes that could lead to a ThI cell, Th2 cell, TcI cell, CTL or B cell immune response. Thus, the peptide may lack any ThI cell epitopes and/or Th2 cell epitopes and/or CTL epitopes and/or TcI cell epitopes and/or B cell epitopes.

Epitopes can be identified from knowledge of the amino acid and corresponding DNA sequences of the peptide or polypeptide, as well as from the nature of particular amino acids (e.g., size, charge, etc.) and the codon dictionary, without undue experimentation. See, e.g., Ivan Roitt, Essential Immunology, 1988; Janis Kuby, Immunology, 1992 e.g., pp. 79-81. Some guidelines in determining whether a protein or an epitope of interest will stimulate a response, include: peptide length - the peptide should be at least 8 or 9 amino acids long to fit into the MHC class I complex and at least 8-25, such at least as 13-25 amino acids long to fit into a class II MHC complex. These lengths are the minimum for the peptide to bind to the respective MHC complex. It is preferred for the peptides to be longer than these lengths because cells may cut peptides. The peptide should contain an appropriate anchor motif which will enable it to bind to the various class I or class II molecules with high enough specificity to generate an immune response. This can be determined, without undue experimentation, by comparing the sequence of the protein of interest with published structures of peptides associated with the MHC molecules.

Thus, the skilled artisan can ascertain an epitope of interest by comparing the protein sequence with sequences listed in the protein database. Such epitope scanning can be used to determine the likely locations of different epitopes types. For example, such epitope scanning can be used to determine whether the sequence of a given peptide is likely to contain any ThI, Th2, TcI, CTL or B cell epitopes. Such epitope scanning can be used to determine whether a variant of SEQ ID NO: 2 as described above is likely to maintain function as a Th3 epitope. An alternative method of identifying epitopes involves providing a library of short peptides which are fragments of the polypeptide sequence of interest. Each of these peptides may be assessed separately for their ability to identify an immune response against the full length polypeptide. Members of the library may be screened in groups or pools or individual members of the library, such as individual members of a single pool, may be assessed separately.

In fact, a number of epitopes are known in the human AFP sequence. For example, the Inventors have previously identified CD4+ ThI epitopes at amino acids 249-258, 364-373, 137-145, 570-578, 545-554, 489-497, 86-95 and 343-351 of the human AFP protein. A number of groups have identified CD8 T cell epitopes in the human AFP protein. For example, Liu et al (J. Immunol (2006) 177: 712-721) reported CD8 T cell epitopes at amino acids 1-9, 137-145, 158-166, 178-186, 218-226, 235-243, 306-315, 325-334, 485-493, 492-500, 507-516, 542-550, 547-556, 555-563 and 549- 557. Mizukoshi et al (Int J Cancer (2006) 118: 1194-1204) reported CD8 T cell epitopes at amino acids 403-411 , 424-432, 434-442, 357-365 and 414-424. WO 98/35981 reports CD8 T cell epitopes at amino acids 158-166, 12-20, 404-412, 441- 450, 178-186, 547-556, 555-563, 287-295, 1-9, 492-500, 235-243 and 542-550. Of these, amino acids 547-556, 492-500, 235-243 and 542-550 were reported to show high levels of specific cell killing. Preferably, a peptide of the invention will not include any of these sequences. A peptide of the invention may include part of one or more of these sequences, but will not include sufficient amino acids from any of these sequences to be capable of acting as an epitope for a ThI, CTL or B cell. Thus, a peptide of the invention will preferably not contain a functional ThI epitope such as one of the epitopes listed above. A peptide of the invention will preferably not contain a functional CTL epitope such as one of the CD8+ epitopes listed above or a functional B cell epitope.

For example, as indicated above, a CD8+ epitope is believed to exist at amino acids 1-9 of human AFP and a CD4+ ThI epitope is believed to exist at amino acids 86- 95 of human AFP. A peptide of the invention will preferably not comprise amino acids 1-9 or 86-95 of human AFP. A peptide of the invention may comprise a fragment of SEQ ID NO: 1 that lies between these two epitopes. For example, a peptide of the invention may comprise or consists essentially of a fragment of human AFP that lies between amino acids 9 and 86 of SEQ ID NO: 1 and that comprises SEQ ID NO: 2 or a variant thereof as described herein. A peptide of the invention may comprise part of one or both of these epitope sequences, insofar as the peptide does not comprise a functional CD4+ ThI or CD8+ epitope. For example, a peptide of the invention may consist essentially of or lie within amino acids 2-94 of the human AFP protein. Such a peptide may comprise a fragment of AFP that lies within amino acids 2 and 94 of the human AFP protein, such as a fragment of amino acids 2-94 that comprises SEQ ID NO: 2 or a variant thereof as described herein. Nucleic acids and Vectors

The peptides of the invention may be administered directly, or may be administered indirectly by expression from an encoding sequence. For example, a polynucleotide may be provided that encodes a peptide of the invention, such as any of the peptides described above.

The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide of the invention may be provided in isolated or purified form.

A nucleic acid sequence which "encodes" a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For the purposes of the invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

A peptide of the invention may thus be produced from or delivered in the form of a polynucleotide which encodes, and is capable of expressing, it. Any reference herein to the use, delivery or administration of a peptide of the invention is intended to include the indirect use, delivery or administration of such a peptide via expression from a polynucleotide that encodes it.

Polynucleotides of the invention can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The polynucleotide molecules of the present invention may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the peptide of the invention in vivo in a targeted subject. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors) which are suitable for use as reagents for nucleic acid immunization. Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide of the invention may be administered to a host subject. Preferably the polynucleotide is prepared and/or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a peptide of the invention.

The present invention thus includes expression vectors that comprise such polynucleotide sequences. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for expression of a peptide of the invention. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. Thus, a polypeptide of the invention may be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given regulatory sequence, such as a promoter, operably linked to a nucleic acid sequence is capable of effecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the nucleic acid sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

A number of expression systems have been described in the art, each of which typically consists of a vector containing a gene or nucleotide sequence of interest operably linked to expression control sequences. These control sequences include transcriptional promoter sequences and transcriptional start and termination sequences. The vectors of the invention may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. A "plasmid" is a vector in the form of an extrachromosomal genetic element. The vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example to allow in vivo expression of the polypeptide.

A "promoter" is a nucleotide sequence which initiates and regulates transcription of a polypeptide-encoding polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. It is intended that the term "promoter" or "control element" includes full- length promoter regions and functional (e.g., controls transcription or translation) segments of these regions.

A polynucleotide, expression cassette or vector according to the present invention may additionally comprise a signal peptide sequence. The signal peptide sequence is generally inserted in operable linkage with the promoter such that the signal peptide is expressed and facilitates secretion of a polypeptide encoded by coding sequence also in operable linkage with the promoter.

Typically a signal peptide sequence encodes a peptide of 10 to 30 amino acids for example 15 to 20 amino acids. Often the amino acids are predominantly hydrophobic. In a typical situation, a signal peptide targets a growing polypeptide chain bearing the signal peptide to the endoplasmic reticulum of the expressing cell. The signal peptide is cleaved off in the endoplasmic reticulum, allowing for secretion of the polypeptide via the Golgi apparatus. Thus, a peptide of the invention may be provided to an individual by expression from cells within the individual, and secretion from those cells. Alternatively, polynucleotides of the invention may be expressed in a suitable manner to allow presentation of a peptide of the invention by an antigen presenting cell (APC). For example, polynucleotides of the invention may be expressed in a suitable manner to allow presentation of a peptide of the invention by an MHC class II molecule or MHC-like molecule at the surface of an antigen presenting cell. For example, a polynucleotide, expression cassette or vector of the invention may be targeted to antigen presenting cells, or the expression of encoded peptide may be preferentially stimulated or induced in such cells.

In some embodiments, the polynucleotide, expression cassette or vector will encode an adjuvant, or an adjuvant will otherwise be provided. As used herein, the term "adjuvant" refers to any material or composition capable of specifically or non- specifically altering, enhancing, directing, redirecting, potentiating or initiating an antigen-specific immune response.

Polynucleotides of interest may be used in vitro, ex vivo or in vivo in the production of a peptide of the invention. Such polynucleotides may be administered or used in the manufacture of a medicament for the treatment of a disease, condition or symptom that is associated with a T cell response, such as any of the diseases, conditions or disorders described herein.

Methods for gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859 and 5,589,466. The nucleic acid molecule can be introduced directly into the recipient subject, such as by standard intramuscular or intradermal injection; transdermal particle delivery; inhalation; topically, or by oral, intranasal or mucosal modes of administration. The molecule alternatively can be introduced ex vivo into cells that have been removed from a subject. For example, a polyncleotide, expression cassette or vector of the invention may be introduced into APCs of an individual ex vivo. Cells containing the nucleic acid molecule of interest are reintroduced into the subject such that an immune response can be mounted against the peptide encoded by the nucleic acid molecule. The nucleic acid molecules used in such immunization are generally referred to herein as "nucleic acid vaccines."

APCs and Regulatory T cells

There are two subsets of CD4⁺ Treg cells - natural and adaptive - that differ in terms of their development, specificity, mechanism of action and dependence on T-cell receptor and co-stimulatory signalling. It has been reported that antigen-induced or adaptive Treg cells consist of several types such as Tr-I and Th3 cells, which produce IL-10, and TGF-β, respectively.

Antigen-induced CD4+ effector and Treg recognize peptides presented by MHC class II molecules but play distinctively different roles in regulating host immune responses against cancer or other diseases. CD4+ effector T cells (ThI) play an important role in maintaining an effective anti-tumour immunity, whereas regulatory T cells can suppress host immune response and induce self tolerance. The identification of MHC-class II restricted tumour antigens capable of stimulating ThI cells is pivotal in developing effective cancer vaccines. However, the majority of tumour associated antigens are self antigens with the ability to stimulate Treg cells that can inhibit the development of an effective anti-tumour immunity. To avoid unwanted expansion of regulatory T cells by vaccines targeting tumour antigens, it is crucial to identify CD4+ Treg epitopes within tumour-associated antigen sequences. On the other hand, the expansion of Treg in auto-immune diseases could suppress anti-self immune responses. Therefore, MHC class Il-restricted T cell epitopes with the ability to induce the expansion of Treg in vivo could be used in the treatment of auto-immune diseases. Moreover, it is clear that desirable peptides for therapeutic vaccines should be promiscuous T cell epitopes, which could be recognized by CD4+ T cells with different alleles, allowing broad population coverage. As explained above, the peptides of the invention can be used to activate T cells.

In particular the peptides of the invention can activate and thus expand a population of regulatory Th3 cells. The invention thus relates to a method of producing a population of regulatory T cells.

This may be achieved by contacting a peptide of the invention with a population of antigen presenting cells (APCs). This will allow for presentation of the peptide of the invention, or an epitope contained therein, to T cells. Such antigen presenting cells may thus present the peptide of the invention or part of that peptide on an MHC class II molecule at the surface of the antigen presenting cells. The antigen presenting cells that present the peptide of the invention may then be contacted with a population of T cells in order to allow activation and/or expansion of suitable regulatory T cells within that population. These two steps may be carried out separately, by first contacting with APCs and then with T cells, or simultaneously, by contacting the peptide of the invention with a sample that contains both APCs and T cells. For example, the method may comprise contacting a sample of PBMCs from an individual with a peptide of the invention.

Such a method may result in the specific activation and expansion of T cells that recognise the peptide of the invention. Additional components may be included in the cell composition to assist this activation and expansion. For example, IL-2 and/or TGFβ may be added to stimulate T cell proliferation. TGFβ has been shown to act as a growth factor for maintenance or expansion of FOXP3 expressing regulatory T cells.

Such a method may produce an expanded population of regulatory T cells. The invention also encompasses APCs that present a peptide of the invention, T cells that recognise a peptide of the invention and T cells that have been activated by a peptide of the invention. Such cells may have been produced, or may be obtainable by, a method as described herein. Such cells may be provided in isolated form. Such cells may be produced in vitro, in vivo or ex vivo.

The T cells produced by such a method may be Th3 cells, and may express FOXP3 and/or CTLA4 as described herein. Preferably the T cells are FOXP3+ CTLA- 4+ CD4+ Th3 cells or FOXP3- CTLA-4+ CD4+ Th3 cells. The T cells produced by such a method may express TGF-β and/or GM-CSF in a peptide specific manner. The T cells may also express low levels of IL-2. The T cells preferably do not produce cytokines that are characteristic of ThI, Th2, TrI or ThI 7 cells. For example, the T cells preferably do not produce one, more, or all of IFNγ, IL-IO, TNF-α, IL-5, IL- 13 and IL- 17. The T cells produced by such a method preferably have an immunomodulatory activity, such as the ability to inhibit the proliferation of other T cells, such as ThI cells.

As explained above, the peptides of the invention may also activate other immune cells. For example the peptides of the invention may also activate immune cells that produce TGF-β and/or GM-CSF. Such cells may also produce IL-2. For example, the peptides of the invention may activate NK cells.

Natural killer (NK) cells mediate innate responses against pathogens and modulate the function of other immune cells through production of cytokines. Similar to ThI and Th2 cells, human NK cells can differentiate into cell populations with distinct patterns of cytokine secretion. For example, it has been shown that IFN-gamma producing NK cells mediate inflammatory responses and IL- 13 producing NK cells contribute to IgE production by B cells. Regulatory NK cells with the ability to suppress antigen specific T cell responses have been identified. Moreover, it has been shown that NK cells are able to produce regulatory cytokines such as IL-10 and TGF-β. The peptides of the invention may be used to activate and thus expand a population of other immune cells. The peptides of the invention may thus be used to produce a population of immune cells. The immune cells produced by such a method may be NK cells. The cells may be CD3 negative CD56 high or low cells such as CD3 negative CD56 high or low NK cells. The cells produced by such a method may produce TGF-β and/or GM-CSF. The TGF-β and/or GM-CSF may be produced in a peptide specific manner. The cells may also produce IL-2. The cells preferably do not produce cytokines that are characteristic of ThI, Th2, TrI or Th 17 cells. For example, the cells preferably do not produce one, more or all of IFNγ, IL-IO, TNF-α, IL-5, IL- 13 and IL-17. The cells produced by such a method may have an immunomodulatory activity, such as an immunosuppressive activity.

Such cells may be produced by methods known in the art. For example, a peptide of the invention may be expressed by an antigen presenting cell as described above. Such an antigen presenting cell may be contacted with a population of cells in order to allow activation and/or expansion of suitable cells within that population. Suitable methods are as described above in relation to T cells. Such an antigen presenting cell may express a peptide of the invention and present all or part of that peptide on an MHC class II molecule or another MHC-like molecule at its surface.

Such methods may result in the specific activation and/or expansion of cells that recognise the peptide of the invention. Cells that recognise a peptide of the invention and cells that have been activated by a peptide of the invention may be provided. Such cells may have been produced, or may be obtainable by, a method as described herein. Such cells may be provided in isolated form. Such cells may be produced in vitro, in vivo or ex vivo. Such methods of producing activated cells may be carried out in vitro, ex vivo, or in vivo. For example, such a method may be used in vitro to produce a population of APCs or an expanded population of cells (such as T cells) that may be subsequently used in therapy. Such a method may be carried out ex vivo on a sample of cells that have been obtained from a patient. The APCs or cells (such as T cells) produced in this way therefore form a pharmaceutical agent that can be used in the treatment of that patient. The cells should be accepted by the immune system of the patient because they derive from that patient. Delivery of cells that have been produced in this way to the individual from whom they were originally obtained, thus forms a therapeutic embodiment of the invention.

Formulations and compositions

The molecules and cells of the invention may be provided in an isolated, substantially isolated, purified or substantially purified form. For example, a peptide of the invention may be provided substantially free from other peptides. A cell of the invention may be substantially free of other cell types.

The molecules and cells of the invention may be provided in a composition. Formulation of a composition comprising a molecule, or cell of the invention can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan.

For example, compositions containing one or more molecules or cells of the invention can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or vehicle. These excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen- free water) prior to parenteral administration of the reconstituted composition. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.

Other parentally-administrable compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Alternatively, the peptides or polynucleotides of the present invention may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules. The formulated compositions will include an amount of the peptide, polynucleotide or cell of interest which is sufficient to mount an immunological response. An appropriate effective amount can be readily determined by one of skill in the art. Such an amount will fall in a relatively broad range that can be determined through routine trials. The compositions may contain from about 0.1% to about 99.9% of the peptide, polynucleotide or cells and can be administered directly to the subject or, alternatively, delivered ex vivo, to a sample derived from the subject, using methods known to those skilled in the art.

Therapeutic methods The present invention relates to molecules such as peptides, polynucleotides and vectors that are capable of inhibiting the proliferation of T cells and that can be used to achieve such inhibition. Such molecules may be capable of achieving an immunomodulatory effect, such as an immunosuppressive effect. The molecules, cells or compositions of the invention can thus be used in the treatment or prevention of any disease, condition or symptom which is associated with an unwanted or increased immune response, in particular a T cell response, that is any disease, condition or symptom which is a direct or indirect result of a T cell mediated immune response, or which results from a disease or condition to which the presence of a T cell response contributes. The molecules, cells or compositions of the invention can be used to generate an immunosuppressive effect in a patient in need thereof. It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment.

According to the present invention, a molecule or cell of the invention may be employed alone as part of a composition, such as but not limited to a pharmaceutical composition or a vaccine composition or an immunotherapeutic composition to prevent and/or treat a condition associated with an unwanted immune response such as an unwanted T cell response, such as an unwanted ThI response. The in vivo or ex vivo administration of the composition may be for either "prophylactic" or "therapeutic" purpose.

Prophylaxis or therapy includes but is not limited to the production in, or administration to, an individual of an activated population of cells such as T cells that are capable of specifically recognising a peptide of the invention and that are capable of inhibiting or reducing an immune response such as a T cell response, such as a ThI response. As used herein, the term "therapeutic" or "treatment" includes: the prevention, reduction or complete elimination of a T cell response; and the prevention, reduction or elimination of symptoms, a disease or condition associated with a T cell response. Prophylaxis or therapy also encompasses alleviating, reducing, curing or at least partially arresting symptoms and/or complications resulting from or associated with an unwanted T cell response.

When provided prophylactically, the molecule, cell or composition of the present invention is typically provided in advance of any symptom. The prophylactic administration of the molecule, cell or composition of the present invention is to prevent or ameliorate a subsequent T cell response. When provided therapeutically, the molecule, cell or composition of the present invention is typically provided at or shortly after the onset of a symptom of the T cell response, or a symptom of the disease or condition characterised by a T cell response. For example, the molecule, cell or composition of the present invention may be provided either prior to or during a transplant or after the transplant, such as after the onset of transplant rejection. Immune responses are known to be linked to numerous specific medical conditions, such as autoimmune disease and transplant rejection. The molecules, cells or compositions of the invention may therefore be used in the prevention or treatment of any of these specific conditions. Accordingly, the present invention relates to a molecule, cell or composition of the invention for use in a method of therapy, in particular in a method or treating or preventing a disease, disorder or symptoms associated with or caused by a T cell response. Such a disease, disorder or symptom may be directly associated with a T cell response, such as a ThI or Th2 cell response that is modulated by an immune cell of the invention such as a T cell or NK cell of the invention. Such a disease, disorder or symptom may be indirectly associated with such a response. For example, the suppression of a CD4+ T cell response may inhibit T cell dependent B-cell maturation or function. The disease, disorder or symptom may thus result from downstream effect of the altered T cell function, such as altered B cell function. The molecules, cells or compositions of the invention may thus also be used in the manufacture of a medicament for treating or preventing such a disease, disorder or condition. In particular, the molecules, cells and compositions of the invention are proposed for the treatment or prevention of a hypersensitivity response such as an autoimmune disease or allergy, or transplant rejection. A number of hypersensitivity reactions can be triggered by unwanted T cell response to host or foreign antigens. These include autoimmune disease, allergy, delayed type hypersensitivity and transplant rejection.

Many autoimmune diseases are known. Of particular relevance here are autoimmune diseases that are mediated, at least in part, by T cell responses. Examples of such autoimmune diseases include rheumatoid arthritis, multiple sclerosis, insulin- dependent diabetes mellitus (type-I diabetes) and psoriasis. Other autoimmune reactions such as Rhesus factor reactions and lupus (SLE) have been linked to the production of a Th2 response. The molecules, cells or compositions of the invention may be used in the treatment of any of these diseases. They may also be used in the treatment of other conditions that are characterised by an unwanted T cell response. One example of such a condition is infection or inflammation in a normally immune- privileged site in the body. For example, uveitis is an inflammatory disease of the eye that can be caused by infection of the eye or as part of an automimmune disease. Allergic reactions such as asthma, allergic rhinitis, eczema, urticaria and anaphylaxis are all related to Th2 responses. Delayed type hypersensitivity is caused by over-stimulation of immune cells such as ThI cells and can result in chronic inflammation and cytokine release. The molecules, cells or compositions of the invention may be used in the treatment of such symptoms and reactions.

The transplant or grafting of cells, tissues or organs from one individual to another often leads to the generation of an immune response in the host individual directed against the foreign cells. The suppression or reduction of T cell activation and T cell mediated immune responses therefore has utility in the prevention or treatment of such transplant rejection. The molecules, cells or compositions of the invention may therefore be used in the therapeutic or prophylactic treatment of transplant rejection.

The invention also provides a method of treating or preventing any of these diseases, disorders or symptoms comprising administering to a subject in need thereof a peptide, cell or composition of the invention. In one aspect, the invention relates to methods that are carried out ex vivo. For example, as explained above, a sample from a patient may be treated outside the body with a peptide of the invention in order to activate immune cells from the patient such as T cells and/or NK cells from the patient in that sample. For example, the sample may comprise PBMCs. The sample may be treated to purify or increase the concentration of PBMCs before treatment with the peptide. The sample may be treated to remove components that might affect the activation of T cells by a peptide of the invention. For example, the sample may be treated or purify to remove components such as cytokines or potential antigens.

Such a method may involve contacting a peptide of the invention with a sample from an individual, wherein the sample comprises antigen presenting cells (APCs). This may be carried out under suitable conditions to allow the APCs to present the peptide, or a fragment thereof, such as on MHC class II molecules or MHC-like molecules, at the cell surface. Those APCs may then be used in a therapeutic or prophylactic method as described herein. For example, APCs that present a peptide of the invention may be administered to an individual in order to inhibit or reduce a T cell response in that individual. Preferably, the APCs are returned to the individual from which they were originally obtained.

Such a method may involve the activation of immune cells such as T cells and/or NK cells ex vivo. For example, a peptide of the invention or an APC that presents such a peptide may be contacted with a sample from an individual such that cells such as T cells in the sample may be activated. Where such a method uses a peptide of the invention, the sample from the individual will preferably comprise both APCs and T cells. Where such a method uses an APC of the invention, the sample from the individual will preferably comprise T cells. The contacting step is preferably carried out under suitable conditions to allow the peptide of the invention to activate T cells and optionally other immune cells, such as NK cells, in the sample. Additional components may be used to facilitate T cell activation and expansion. For example, IL- 2 may be used to facilitate the expansion and proliferation of activated T cells in the sample.

The immune cells such as T cells that have been activated in this way may be used in a therapeutic or prophylactic method as described herein. For example, such activated cells may be administered to an individual in order to inhibit or reduce a T cell response in that individual. Preferably, the activated cells are returned to the individual from which they were originally obtained. This therefore provides to the patient an activated and preferably expanded population of cells such as T cells that specifically recognise the peptide of the invention. As described herein, such cells may act to inhibit T cell proliferation in the patient and can therefore have the therapeutic effects in the patient described herein. Optionally, after contacting with the peptide, the sample may be tested to determine whether suitable APCs, T cells or other cells have been activated. This testing can be carried out using any of the methods described herein. For example, the presence of suitable activated NK cells or T cells can be tested by assessing or quantifying the presence of cytokines such as TGF-β or GM-CSF in the sample, or by screening a proportion of said cells for characteristic markers as described above. Alternatively, the sample can be tested for the ability to inhibit T cell proliferation. Suitable methods for carrying out such tests are described herein and further methods would be known to a person skilled in this technical field. Such testing can be used to ensure that the material returned to the patient does include suitable activated T cells as described herein.

In a further aspect, the invention relates to methods that are carried out in vivo. For example, a peptide, cell or composition of the invention may be administered to a patient. For example, a peptide, cell or composition of the invention may be administered to a patient for any of the therapeutic or prophylactic purposes described herein. An APC of the invention may be an APC as described above that presents a peptide of the invention such as an APC that presents the peptide at its surface using a MHC class II molecule. A T cell of the invention may be a T cell as described above that specifically recognises a peptide of the invention. An immune cell may be a T cell, NK cell or other cell that has been activated by a peptide of the invention. The APC or T cell, NK cell or other cell may be derived from the patient. For example, the cell may have been produced by an ex vivo method as described above. The cell may be not derived from the patient.

Subject to be treated

The present invention relates in particular to the treatment or prevention of diseases or other conditions which are associated with an unwanted immune response such as an unwanted T cell dependent response. These treatments may be used on any animal which is susceptible to such a response. The subject to be treated may be any member of the subphylum cordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The terms do not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The methods described herein are intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly. If a mammal, the subject will preferably be a human, but may also be a domestic livestock, laboratory subject or pet animal. The molecules, cells or compositions of the present invention may thus be used in the treatment of any such species. As indicated above, a suitable peptide may be selected based upon the species to be treated. For example a peptide may derive from the native AFP polypeptide sequence of that species. A peptide may be tested using a sample from that species to confirm that it is capable of activating a suitable response.

Combined therapy

In one aspect, a molecule, cell or composition of the invention may be used in combination with another therapeutic agent. The therapeutic agent may be, for example, an agent which has an immunosuppressive effect, or an agent used in the treatment of a condition which is associated with a T cell response. The molecule, cell or composition of the invention is preferably administered in an amount which is sufficient to augment the effects of the other therapeutic agent or vice versa. Numerous other agents may be used to reduce T cell responses or in the treatment of conditions which are associated with T cell responses. For example, many autoimmune conditions are currently treated with non-steroidal anti-inflammatory drugs (NSAIDS), biological response modifiers (BRMs), immunosuppressive drugs, corticosteroids and physical and/or occupational therapy. The other therapeutic agent may be an agent that potentiates the effects of the molecule of the invention. For example, the other agent may be an immunomodulatory molecule or an adjuvant which enhances the response to the peptide or cell of the invention.

In one embodiment, therefore, a peptide, cell or composition of the invention is used for therapy in combination with one or more other therapeutic agents. The therapy may be any of the treatments or therapeutic methods described herein.

The two agents may be administered separately, simultaneously or sequentially. The two may be administered in the same or different compositions. Accordingly, in a method of the invention, the subject may also be treated with a further therapeutic agent.

A composition may therefore be formulated which comprises a molecule and/or cell of the invention and also one or more other therapeutic molecules. A composition of the invention may alternatively be used simultaneously, sequentially or separately with one or more other therapeutic compositions as part of a combined treatment. Thus the invention also provides the use of a molecule or cell of the invention, and a further therapeutic agent in the manufacture of one or more medicament(s) for the prevention or reduction of a T cell response or for the prevention or treatment of a disease, condition or symptom associated with a T cell response as described herein.

Delivery methods

Once formulated the compositions can be delivered to a subject in vivo using a variety of known routes and techniques. For example, a composition can be provided as an injectable solution, suspension or emulsion and administered via parenteral, subcutaneous, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, intravenous injection using a conventional needle and syringe, or using a liquid jet injection system. Compositions can also be administered topically to skin or mucosal tissue, such as nasally, intratracheally, intestinal, rectally or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, and active or passive transdermal delivery techniques.

A suitable route of administration may be determined by the skilled practitioner depending upon the particular symptom, disease or condition to be treated. Administration may be local to the site or tissue of interest, or may be systemic. Where a peptide of the invention is to be administered, it is preferred to administer the peptide to a site in the body where it will have the ability to contact suitable antigen presenting cells, and where it, or they, will have the opportunity to contact immune cells such as T cells of the individual. Where an APC is to be administered, it is preferred to administer the APC to a site in the body where it will have the ability to contact, and activate, suitable immune cells such as T cells of the individual. T cells of the invention are believed to act via direct cell-cell contact on CD4+ T cells to inhibit a T cell response. Where a T cell of the invention is to be administered, it is preferred to administer the T cell to a site in the body where it will have the ability to contact other T cells that may be responsible for, or associated with, the unwanted T cell response. An immune cell such as a T cell of the invention may therefore be administered directly to the location of an unwanted T cell response. For example, in the case of rheumatoid arthritis, a molecule (e.g. a peptide), cell or composition of the invention may be administered directly to the affected joints, or may be administered systemically in order to create a more general effect. In the case of uveitis, a molecule, cell or composition of the invention may be administered directly to the site of inflammation in the eye.

Delivery regimes

The molecules (e.g. peptides), cells or compositions are administered to a subject in an amount that is compatible with the dosage formulation and that will be prophylactically and/or therapeutically effective. An appropriate effective amount will fall in a relatively broad range but can be readily determined by one of skill in the art by routine trials. The "Physicians Desk Reference" and "Goodman and Gilman's The Pharmacological Basis of Therapeutics" are useful for the purpose of determining the amount needed.

As used herein, the term "prophylactically or therapeutically effective dose" of a peptide of the invention means a dose in an amount sufficient to activate immune cells such as T cells having the characteristics described herein. A "prophylactically or therapeutically effective dose" of a molecule (e.g. peptide) or cell of the invention means a dose in an amount sufficient to suppressor reduce a T cell response, particularly a ThI response in the individual, and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from a disease, such as an autoimmune disease, which is associated with such a T cell response.

Prophylaxis or therapy can be accomplished by a single direct administration at a single time point or by multiple administrations, optionally at multiple time points. Administration can also be delivered to a single or to multiple sites. Those skilled in the art can adjust the dosage and concentration to suit the particular route of delivery. In one embodiment, a single dose is administered on a single occasion. In an alternative embodiment, a number of doses are administered to a subject on the same occasion but, for example, at different sites. In a further embodiment, multiple doses are administered on multiple occasions. Such multiple doses may be administered in batches, i.e. with multiple administrations at different sites on the same occasion, or may be administered individually, with one administration on each of multiple occasions (optionally at multiple sites). Any combination of such administration regimes may be used.

Dosages for administration will depend upon a number of factors including the nature of the composition, the route of administration and the schedule and timing of the administration regime. Suitable doses of a molecule of the invention may be in the order of up to 15μg, up to 20μg, up to 25μg, up to 30μg, up to 50μg, up to lOOμg, up to 500 μg or more per administration. For some molecules of the invention, the dose used may be higher, for example, up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg or higher. Such doses may be provided in a liquid formulation, at a concentration suitable to allow an appropriate volume for administration by the selected route.

Kits

The invention also relates to a combination of components described herein suitable for use in a treatment of the invention which are packaged in the form of a kit in a container. Such kits may comprise a series of components to allow for a treatment of the invention. For example, a kit may comprise two or more different peptides and/or cells of the invention, or one or more peptides or cells of the invention and one or more additional therapeutic agents suitable for simultaneous administration, or for sequential or separate administration. The kit may optionally contain other suitable reagent(s), control(s) or instructions and the like.

References

1. Alisa, A. et al. Analysis of CD4+ T-CeIl Responses to a Novel {alpha} - Fetoprotein-Derived Epitope in Hepatocellular Carcinoma Patients. Clin Cancer

Res 11, 6686-94 (2005).

2. Ayaru, L. et al. Unmasking of {alpha} -Fetoprotein-Specifϊc CD4+ T Cell Responses in Hepatocellular Carcinoma Patients Undergoing Embolization. J Immunol 178, 1914-22 (2007). 3. Murgita, R. A. Recombinant human alpha- fetoprotein as an immunosuppressive agent. United States Patent 6,774,108(2004).

4. Bruix, J., SaIa, M. & Llovet, J. M. Chemoembolization for hepatocellular carcinoma. Gastroenterology 127, S 179-88 (2004).

5. Casares, N. et al. CD4+/CD25+ regulatory cells inhibit activation of tumor- primed CD4+ T cells with IFN-gamma-dependent antiangiogenic activity, as well as long-lasting tumor immunity elicited by peptide vaccination. J Immunol 171, 5931-9 (2003).

6. Wang, H.Y. et al. Tumor-specific human CD4+ regulatory T cells and their ligands: implications for immunotherapy. Immunity 20, 107-18 (2004). 7. Wang, H. Y., Peng, G., Guo, Z., Shevach, E.M. & Wang, R.F. Recognition of a new ARTCl peptide ligand uniquely expressed in tumor cells by antigen- specific CD4+ regulatory T cells. J Immunol 174, 2661-70 (2005). 8. Shevach, E.M. From vanilla to 28 flavors: multiple varieties of T regulatory cells. Immunity 25, 195-201 (2006). 9. Larche, M. & Wraith, D. C. Peptide-based therapeutic vaccines for allergic and autoimmune diseases. Nat Med 11, S69-76 (2005). 10. Butterfield, L.H. et al. T-cell responses to HLA-A*0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer. Clin Cancer Res 9, 5902-8 (2003). 11. Bei, R. et al. Cryptic epitopes on alpha-fetoprotein induce spontaneous immune responses in hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis patients. Cancer Res 59, 5471-4 (1999).

EXAMPLES

Materials and Methods

Table 1 : Alpha-fetoprotein derived peptides

Synthetic peptides and cell lines

In total 62 peptides spanning the AFP sequence were synthesized by Mimotopes Pty Ltd. (Clayton Victoria, Australia) (Table 1).

Patient recruitment

The study was approved by Cromwell, the London Clinic, joint UCL/UCLH hospital's ethical committees and all patients gave written informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of treatment-naive patients with HCC or healthy donors. In five cases, circulating AFP46-55-specifc CD4+ T cell responses were analyzed in HCC patients before and 3 months after trans-arterial chemo/embolisation (TACE/ TAE). Trans-arterial TACE or TAE involves the injection of coils or particles into the hepatic artery with or without a chemotherapeutic agent respectively, leading to obstruction of the hepatic artery branch supplying the tumour. This produces necrosis of the well-vascularised tumour and a reduction in tumour burden⁴.

Generation of T cell lines and FACS analysis

T cell lines were generated as described previously¹. In brief, PBMCs were re- suspended in AIM-V medium (Gibco) and cultured in duplicate with individual peptides (1 μM). Recombinant IL-2 (25 IU/ ml) was added on day 2-3 of culture and cells were analysed after a total of 10-12 days of culture. AFP-specifϊc T cell lines were incubated for 5 hours at 37°C with AFP-derived peptides (1 μM) or peptide-pulsed or protein pulsed antigen presenting cells (EBV-B cell lines, HepG2 cells or autologous monocytes) and Brefeldin A. Cells were surface stained with antibodies to CD3, CD4, CD8, CD25, TCRαβ, HLA-DR and GITR. The cells were then permeabilised and fixed using Cytofix/Cytoperm™ (BD PharMingen, Cowley, UK). Afterwards, the cells were stained for intracellular molecules (PE or FITC conjugated GM-CSF, TGF-β, IL-2, IFN-γ, IL-10, TNF-α, IL-5, IL-13, IL-17, CTLA-4) or isotype controls, washed twice and the frequency of peptide-specific T cell responses quantified by flow cytometry. An immunological response/responder was defined as a two fold increase in frequency of cytokine-producing cells above control peptides or proteins. Intracellular staining for Foxp3 protein was performed by using FITC-conjugated FOXP3 antibody, fixation and permeabilization buffers provided by the Foxp3 kit (eBioscience) according to the manufacturer's instructions. AFP46-55-spcific T cell lines or control-T cell lines were washed and cultured in serum free medium in the presence of relevant or irrelevant peptides for 48h and the levels of TGF-β and GM-CSF were measured in culture supernatants by ELISA kits (R&D, Abingdon, UK).

Purification ofCD4⁺CD25^~ and CD4⁺ CD25⁺ cells

CD4⁺ CD25⁺ cells were isolated from PBMC with the CD4⁺CD25⁺ regulatory T- cell isolation kit (Miltenyi Biotec), with a Midi Macs separator unit, according to the manufacturer's instructions. The efficiency of CD4⁺CD25⁺ T-cell depletion was >90%. Untreated PBMC or PBMC depleted of CD4⁺CD25⁺ cells were then used for producing short-term T-cell lines. To purify CD4⁺CD25^~ T cells, CD25 were depleted from PBMCs and then CD4+ T cells were isolated using antibody coated beads (Dynal) from the remaining cells. The purity of CD4⁺CD25^" T cells was > 99%.

Proliferation Assays

CD4+CD25- T cells (2 x 10⁵) isolated from PBMCs by antibody coated beads were cultured for 5 days in 96-well plates containing 5 x 10^ CD3-depleted APCs, 0.5 μg/ ml anti-CD3 mAb, and different numbers of regulatory (AFP4g.₅₅) or effector (AFP_364-373 peptide) CD4+ T cells in medium containing 10% human serum. The proliferation of responder T cells by the incorporation of [³H]thymidine for the last 18 hr of culture. Cells were harvested and radioactivity counted in scintillation counter. All experiments were performed in triplicates.

Transwell experiments were performed in 24-well plates with pore size 0.4 μm

(Coring Costar). Purified naϊve CD4+ T cells (I x 10^) were cultured in the outer wells in medium containing 0.5μg/ml anti-CD3 antibody and 2 x 10^ APCs. Equal numbers of AFP46-55-CD4+ T cells or AFP_364-373-CD4+ T cells were added into inner wells in the same medium containing anti-CD3 and 2 x 10^ APCs. The cells in inner and outer wells were harvested separately and transferred into 96 well plates after 3 days culture. [³H]thymidine was added, and the cells were cultured for another 18 hr before being harvested for counting the radioactivity with a liquid scintillation counter. ELISA assay

GM-CSF and total TGF-β secretions in culture supernatants were determined by ELISA after 48 hours of incubation of the T cell lines with peptides or proteins (AFP46-55 CD4+ T cells and AFP364-373 CD4+ T cells) (R&D Systems). AFP364- 373 CD4+ T cells were used as control for AFP46-55 T cell lines.

ELISPOT assay

TGFβ-releasing cells were detected upon specific peptide stimulation using an ELISPOT assay ex vivo. Nitrocellulose-backed plates (96-well, MAHA S45; Millipore) were coated with mouse anti-human latent TGFβ capture antibody overnight at 4⁰C. The wells were washed five times with PBS and blocked using blocking buffer (1 % BSA, 5 % sucrose PBS) for 2 hours. PBMCs and the peptides were then added into the wells and incubated for 18 hours at 37°C in 5% CO₂. The wells were washed with wash buffer (0.05% Tween 20 in PBS), then lμg/ml of secondary biotin-conjugated anti- human latent TGFβ antibody (R & D systems) was added and incubated at 4°C overnight. The color development was done using ELISPOT blue color module (R & D systems). After 30 min, the wells were washed with tap water, dried and the spots counted.

Statistical analysis

The Mann- Whitney test was used to compare the frequencies of AFP46-55- specific GM-CSF producing CD4+ T cells in healthy and cancer patients. The statistical significance was defined at p<0.05 for all analysis done.

Results

Identification of a CD4+ T cell epitope from AFP and its cytokine profiles

It has been suggested that T cells can recognize self antigens and develop into regulatory T cells. However, there is very little information about antigen specificity of regulatory T cells. To identify and characterize CD4+ T regulatory cells recognizing AFP, peripheral mononuclear cells (PBMCs) were isolated from hepatocellular carcinoma (HCC) patients and cultured in the presence of the 62 different AFP-derived peptides (Table 1). Ten days later, the presence of AFP-derived peptide-specific CD3⁺CD4⁺ T cells was analyzed using intracellular cytokine staining for GM-CSF, a cytokine produced by ThI, Th2 and CD4+ regulatory T cells. AFP₄₆.₅₅ CD4+ T cell lines recognized AFP_46-55 peptide (LATIFFAQFV) but not an irrelevant peptide [AFP_364-373; AFP-derived ThI epitope ⁵] and produced GM-CSF. No response was detected in T cell lines expanded in the presence of other AFP-derived peptides (Table 1) (Figure IA, B). The response to 62 different AFP-derived peptides was analyzed in 3 different HCC patients. AFP₄₆_₅₅-specific CD4+ T cell response was detected in all three patients (data not shown), suggesting that AFP_46-55 peptide has a unique ability to stimulate a subset of CD4+ T cells in a dose dependent manner (Figure 1C). To identify the optimal length of peptide sequence, AFP₄₆.₅₅ CD4⁺ T cells were stimulated with AFP_47-55 (9 amino acid long), AFP44-57 (14 amino acid long), AFP42-55 (14 amino acid long) and the frequency of peptide-specifϊc GM-CSF producing CD4+ T cells were analyzed. AFP_46-55 CD4+ T cells recognized AFP_47-55, AFP_44-57, AFP₄2_-55 but not an irrelevant peptide (AFP_364-373) and produced peptide-specifϊc GM-CSF. However, the frequency of GM-CSF-producing cells among CD4⁺ T cells was higher in cells stimulated with AFP_46-55 (Figure 1 D). These results were confirmed by testing T cell lines generated from 3 different HCC patients (data not shown).

The release of GM-CSF by peptide-specific CD4+ T cells was analyzed using ELISA assay. AFP_46-55 or AFP_364-373 T cell lines were washed and stimulate with peptides (triplicate, 2 x 10⁵ cells/well) for 48 hrs. The levels of GM-CSF were measured in the supernatants of the T cell lines using ELISA for GM-CSF. T cell lines generated in the presence OfAFP_46-55 but not AFP_364-373 peptide produced high levels of

GM-CSF (Figure IE). Stimulation OfAFP_364-373 T cell lines (ThI cell line) or T cells cultured in the presence of medium only with AFP_46-55 peptide did not induce GM-CSF production above the background (data not shown). To study the role of IL-2, IL-7 and IL- 15 on the generation and expansion of

AFP-specific CD4+ T cells, PBMCs were cultured in the presence or absence of these cytokines in different combinations. AFP_46-55 CD4+ T cells were not expanded in the absence of IL-2 (no recombinant cytokine, IL-7 alone, IL- 15 alone or IL-7 + IL-15) suggesting that IL-2 is essential for the expansion of these cells. However, the highest levels OfAFP_46-55 CD4+ T cells were detected in cultures expanded in the presence of

IL-2 (25 IU/ml), IL-7 (20 ng/ml) and IL- 15 (20 ng/ml) (data not shown). AFP ^* ₄6-5₅ CD4+ T cells produce TGF-β in a dose dependent manner

TGF-β is an immuno-regulatory cytokine produced by some Treg. To test the ability of T cell lines to produce antigen specific TGF-β, AFP₄G_-55 ^or AFP_364-373 T cell lines generated from PBMC of a patients with HCC were washed and stimulated (2 x 10⁵ cells/well) with various concentrations of AFP₄G_-55 peptide or AFP_364-373 peptide in serum free medium for 48 hrs. The levels of total TGF-β were measured in the culture supernatant using an ELISA for TGF-β. AFP_46-55 T cell lines stimulated with AFP₄G_-55 produced TGF-β in a dose dependent manner (Figure 2A). AFP_364-373 T cell lines re- stimulated with AFP_364-373 or AFP_46-55 peptides did not produce TGF-β. The depletion of CD4+ cells or CD2+ cells prior to peptide re-stimulation but not CD8+ T cells significantly reduced peptide specific TGF-β production by AFP_46-55 T cell lines

(Figure 2B), suggesting that CD4+ T cells are the source of TGF-β. To test the recognition of purified AFP by AFP₄₆.₅₅ CD4+ T cells, AFP₄₆.₅₅ CD4+T cell lines or a control T cell line (AFP_364-373 T cell line) were cultured with purified AFP (5 μg/ml) or a control protein (human serum albumin) for 48h. The levels of total TGF-β were measured in the culture supernatant using an ELISA for TGF-β. AFP₄G_-55 CD4+ T cell lines stimulated with purified AFP but not with control protein produced high levels of TGF-β (Figure 2C). To further characterize TGF-β producing cells, AFP_46-55 T cell line were re-stimulated with AFP₄G_-55 ^{or an} irrelevant peptide and the production of peptide- specific TGF-β was analyzed using flow cytometry to detect intracellular TGF-β and/or membrane bound latent TGF-β. AFP_46-55 CD4+ T cell recognized AFP_46-55 peptide but not an irrelevant peptide and produced TGF-β (Figure 2D). Peptide specific intracellular TGF-β production was detected in all patients tested (data not shown).

AFP₄₆_₅₅ CD4+ T cells do not produce ThI, Th2, ThI 7 type cytokines

Antigen-specific CD4+ T cells are classified as ThI or Th2 based on their ability to produce different cytokine profiles. To classify AFP_46-55 CD4+ T cells, we analyzed their ability to produce ThI or Th2 type cytokines upon peptide recognition. AFP_46-55 - specific CD4+ T cells were generated from an HCC patients and their ability to produce ThI or Th2 type cytokines was evaluated using intracellular cytokine assays. AFP_46-55

CD4+ T cells did not produce ThI type (IFN-γ, TNF-α), Th2 (IL-13, IL-5), TrI (IL-10) or ThI 7 (IL-17) type cytokines. However, AFP_46-55 -specific CD4+ T cells recognized the relevant peptide and produced GM-CSF and low levels of IL-2 (Figure 3 A and C). AFP_46-55 T cell lines generated from 3 different HCC patients and 2 healthy controls produced similar pattern of cytokine production (data not shown). NK cells were identified as CD3 negative CD56 high or low cells. The ability of these cells to respond to the peptide and produce cytokines was analysed using intracellular cytokine assay and surface staining for latent TGF-β. NK cells produced IL-2, GM-CSF and TGF-β but did not produce ThI, Th2, TrI or ThI 7 type cytokines (Figure 3C).

AFP₄₆_₅₅ CD4+ T cells express CTLA-4

Then, we analyze the phenotypic characterization OfAFP_46-55 CD4+ T cells.

The expression levels of surface and intracellular molecules (CD4, CD45RO, CD62L, CTLA-4 and GITR) were analyzed using flow cytometry. AFP_46-55 CD4+ GM-CSF producing T cells expressed surface CD45RO, GITR and intracellular CTLA-4. Nonspecific CD4+ T cells (cells not producing GM-CSF) did not express GITR or CTLA-4 (Figure 4). AFP_46-55 CD4+ T cells expressed CD3, TCRαβ, CD25 but not CD8, HLA-

DR, CD 14 or CD 16 (data not shown). Similar pattern of surface molecule expression was detected in AFP_46-55 CD4+ T cells generated from four different HCC patients and 2 healthy donors (data not shown).

AFP_46.55 T cells can be generated from both CD4+CD25+ and CD4⁺CD25- cells

The depletion of CD4+ T cells but not CD8+ T cells from PBMCs reduced the generation of AFP-specific GM-CSF producing T cells, suggesting that these cells are derived from CD4+ T cells (Figure 5a). It has been shown that the depletion of

CD4⁺CD25⁺ T cells improves T cell proliferation and development of antigen-specific anti-tumour ThI and TcI responses ²². To examine the role of regulatory T cells in the generation Of AFP_46-55 CD4+ T cells, CD4+CD25+ T cells were depleted from PBMCs of an HCC patient. The depletion of CD4⁺CD25⁺ reduced the frequencies OfAFP_46-55 CD4+ T cells (Figure 5b). To confirm these findings, CD4+CD25+ and CD4+CD25- T cells were isolated from PBMCs of an HCC patient and cultured in medium containing rIL-2 and stimulate with autologous DC-pulsed AFP_46-55 peptide for 10 days. Similar level of peptide-specific GM-CSF producing CD4+ T cells was detected in both cultures, suggesting that AFP₄₆.₅₅-CD4+ T cells can be generated from both

CD4+CD25+ and CD4+CD25" T cell populations (Figure 5c).

AFP46-55 -specific CD4+ T cells suppress T cell proliferation in vitro in a contact dependent manner

Inhibition of T proliferation is a unique functional characteristic of regulatory T cells. T cell clones were generated and peptide-specific T cell clones were selected based on their ability to produce GM-CSF using ELISA assay. To examine whether AFP₄₆.₅₅ CD4+ T cells inhibit T cell proliferation, CD4+CD25" T cells were purified from human PBMCs as responding T cells and co-cultured with AFP_46-55 CD4+ T cell clone or AFP_364-373-CD4+ T cell clone in growth medium containing purified APCs and anti-CD3 antibody for 5 days. T cell proliferation was analyzed using a standard proliferation assay. AFP_46-55 CD4+ T cells but not AFP_364-373-specific CD4+ T cells (ThI type cytokine producing cells) inhibited anti CD3 -induced T cell proliferation in a dose dependent manner (Figure 6A). AFP_46-55 CD4+ T cells generated from two other patients with HCC gave similar results, suggesting that these cells can suppress T cell proliferation (data not shown). These results suggest that AFP_46-55 CD4+ T cells can be classified as CD4+ Treg cells. Transwell experiments were performed to test whether cell-cell contact is required for AFP_46-55 CD4+ T cells to exert their suppressive activity. Co-culture of CD4+ naive T cells with AFP_46-55 CD4+ T cells in the presence of anti-CD3 and CD3- depleted APCs inhibited T cell proliferation of naive CD4+ T cells in vitro (1 :1 ratio). However, in transwell experiments, AFP_46-55 CD4+ T cells when cultured in the inner well containing medium with anti-CD3 and the purified APCs, did not inhibit the proliferative activity of CD4+ naive T cells cultured in the outer well containing medium, anti-CD3 and APCs (Figure 6B). Taken together, these results indicate that AFP_46-55 CD4+ T cells inhibit T cell proliferation in a contact dependent manner.

Analysis of AFP '4^.55 CD4+ T cells in HCC patients and healthy donors

AFP is expressed by the majority of HCC and we have shown that anti-AFP ThI can be induced in HCC patients¹'². Moreover, here we have shown that CD4+T cells isolated from HCC patients can recognize an AFP-derived epitope and develop into GM-CSF producing regulatory T cells. However, it was not clear whether CD4+ T cells isolated from healthy donors who had been exposed to high levels of AFP during embryonic development can recognize AFP. Short term T cell lines were generated from PBMCs isolated from 10 healthy donors (6 males and 4 females) and 15 HCC patients (12 males and 3 females) and the frequency of GM-CSF producing AFP_46-55 T cells were analyzed in short term T cell culture using an intracellular cytokine assay for GM-CSF. AFP_46-55 -specific CD4+ T cells were detected in all healthy donors and

HCC patients (Figure 7A). A higher frequency of AFP_46-55 CD4+ T cells were detected in HCC patients than healthy controls (p<0.01) (Figure 7A), suggesting that these cells are expanded in vivo in response to the tumour.

We have shown that tumour embolisation reduce tumor burden, improve survival and expands AFP-specific ThI cells in vivo¹. However, the effect of this treatment on the shift of ThI/ Treg balance was unknown. Trans-arterial chemoembolisation (TACE) or embolisation (TAE) are the most widely used treatments for unresectable HCC and involves the injection of coils or particles into the hepatic artery with or without a chemotherapeutic agent respectively, leading to obstruction of the hepatic artery branch supplying the tumor. This procedure produces necrosis of the well-vascularised tumor and a reduction in tumor burden. We analyzed circulating AFP_46-55 CD4⁺ T cell responses after short term in-vitro expansion before and 3 months after the treatment in 5 consecutive treatment-naϊve patients undergoing chemo/embolisation. AFP_46-55 CD4+ T cells were detected in all 5 patients and the responses were ranging from 0.2% to 11% of CD4+ T cells (Figure 7B). In HCC03, the frequency OfAFP_46-55 CD4+ T cells was reduced from 8% of CD4+ T cells before TAE (serum AFP=2625 ng/ ml) to 1.5% after TAE (serum AFP=I 640 ng/ml). In HCC04, the response was reduced from 11% of CD4+ T cells before TACE (serum AFP=7 ng/ml) to 7% after the treatment (serum AFP=IO ng/ml) (Figure 7B).

Ex vivo detection ofAFP46-55 specific TGF β producing cells The frequency of peptide specific TGFβ releasing cells was analyzed using

ELISPOT assays for TGFβ. PBMCs isolated from four healthy donors (HD-I, HD-2, HD-3 and HD-4) were stimulated with AFP_46-55 or AFP_364-373 for 18 hours and the frequency of peptide specific TGFβ producing cells was analyzed ex vivo. BMPCs from three out of four patients (HD-11, HD-2 and HD-3) responded to AFP_46-55 and released TGFβ (Figure 8). AFP364.373 peptide did not stimulate TGFβ production above the background (cells cultured in medium only) (data not shown).

Discussion

The presence of high frequencies of CD4+ CD25+ regulatory T cells in peripheral blood and tumour tissue of HCC have been reported, however there is no information about their antigen specificity. Previously tumour specific human CD4+ regulatory T cells and their ligand were described by Wang et α/⁶'⁷. These cells were generated from TILs of patients with melanoma and upon antigen recognition produced IFN-γ, IL- 10 and GM-CSF but not TGF-β. In this study we have shown that antigen specific TGF-β producing regulatory T cells with the ability to suppress T cell proliferation can be generated from CD4⁺ T cells of all cancer patients and healthy donors. A unique cytokine profile produced by AFP_46-55 CD4+ T cells suggest that these cells differ from typical antigen-induced regulatory T cells that produce IL-IO . In some studies, clones derived from mice that have been orally tolerized with low antigen dose primarily produced TGF-β, and these cells termed Th3 cells. Regulatory T cells that exclusively produce TGF-β have not been observed in other models. To our knowledge, this is the first report describing peptide epitope-specific TGF-β producing regulatory T cells in man. The expanded AFP_46-55 CD4+ T cells suppressed T cell proliferation in a cell contact dependent mechanism, similar to naturally occurring CD4+ CD25+ regulatory T cells.

Contact dependent inhibitory function of AFP-CD4 T cells in in vitro assay may not exclude the effects of secreted cytokines such as TGF-β by these cells in modulation of immune responses in vivo. Immunosuppressive pathways mediated by TGF-β may obscure immune surveillance mechanisms, resulting in failure to recognize or respond adequately to self, foreign, or tumor-associated antigens. Another cytokine produced at high levels by AFP46-55 CD4+ T cells is GM-CSF. The role of GM-CSF in modulation of T cell responses in man is not fully understood. However, in animal models, it has been shown that the administration of GM-CSF expands FOXP3+ regulatory

CD4+CD25+ T cells and suppresses autoimmune diseases. This suppression is believed to be through activation and generation of regulatory dendritic cells. In support of the physiologic relevance of the identified epitopes we show that purified AFP can be recognized by AFP_46-55 CD4⁺ T cells. Furthermore, we have shown that HLA class II matched and mismatched EBV B cell lines (protein or peptide pulsed) but not HCC cell line (peptide pulsed or non-pulsed HepG2 cells) are recognized by AFP_46-55 Treg (unpublished data). This result indicates that processing and presentation of this epitope by antigen-presenting cells can take place via the exogenous pathway, rather than by direct recognition of tumour, as HCCs do not express MHC class II molecules on the cell surface.

Although AFP₄₆.₅₅ CD4+ T cells were detected after short term T cell culture in both cancer patients and healthy donors, the frequency of these cells in cancer patients was significantly higher than that in healthy donors. This may suggest that these cells are expanded in vivo in response to the tumour antigen. In support of this notion, we have shown that embolisation of tumour (removal of tumour burden) significantly reduces the frequencies of AFP-specific CD4+ regulatory T cells. In contrast, this treatment result in the activation and expansion of AFP-specific CD4+ T cells (ThI type)². A reduction in tumour burden/regulatory factors by embolisation may explain in part the observed concomitant expansion of pre-existing AFP-specific ThI and reduction of AFP-specific regulatory T cells. This may suggest that TACE/ TAE improve survival by reduction of tumour burden and balancing the immune responses in favor of ThI type response.

AFP is an oncofetal antigen and has intrinsic immunoregulatory properties and recombinant AFP (MM-093) is currently in a pilot clinical study for patients with autoimmune disease with the aim to suppress self reactive immune responses (www.merrimackpharma.com). The administration of the intact antigen would avoid having to select specific epitopes to suit MHC-disparate individuals. This is not the case for the AFP -derived epitope identified in this study as the response to this epitope can be detected in all individuals tested. In this study, donors and patients (30 in total) were not selected based on their HLA haploytpes and determination of HLA class II haplotypes from some patients showed that AFP_46-55 T cell responses is detectable in patients with completely different HLA class II haplotypes (unpublished data).

Furthermore, AFP₄₆.₅₅ CD4+ T cells recognized HLA class II mismatch peptide-pulsed EBV- B cells and produce GM-CSF (unpublished data), suggesting that AFP_46-55 is a promiscuous epitope and its recognition is not restricted to one HLA class II haplotype. One of the disadvantages of using intact antigen is the activation of pathogenic B and T cells⁹. In fact, several B cell, CTLs and CD4+ ThI epitopes within AFP sequence have been reported¹' ¹⁰' ' ' . Therefore, AFP-derived peptide therapy with the aim to activate Treg cells and suppress immunopathology provides a safer and inexpensive approach. In support of this approach, improved clinical responses have been reported in patients with autoimmune diseases and allergy undergoing peptide immunotherapy. It is believed that the up-regulation of regulatory cytokines such as TGF-β and IL-IO play a major role in limiting allergic and autoimmune pathology⁹.

Taken together, we have identified and characterized self-antigen specific CD4+ T cells from HCC patients and healthy donors. Their cytokine profile, phenotypic and functional characteristics suggest that these cells are antigen-specific Treg cells and recognize an AFP peptide as a natural ligand. Here we show that tumour may stimulate the expansion of self antigen-specific Treg cells and the removal of antigen source diminishes the expansion of these cells in vivo. These results will be instrumental in the development of peptide-based immunotherapy for treatment of cancer as well as autoimmune disease.

Claims

1. A peptide comprising a fragment of the human alpha- fetoprotein molecule of SEQ ID NO: 1, wherein said peptide comprises:

(a) amino acids 46-55 of SEQ ID NO: 1

(b) a variant of (a) in which one, two or three amino acids are deleted; or

(c) a variant of (a) comprising one, two, three or four amino acid substitutions from the sequence of (a); wherein said peptide is capable of activating T cells that produce TGF-β and/or GM-

CSF, and wherein said peptide does not comprise a B cell epitope, a CTL epitope or a ThI epitope.

2. A peptide according to claim 1 which is capable of activating NK cells that produce TGF-β and/or GM-CSF.

3. A peptide according to claim 1 or 2 comprising

(a) amino acids 46-55 of SEQ ID NO: 1 ;

(b) amino acids 47-55 of SEQ ID NO: 1 ;

(c) amino acids 44-57 of SEQ ID NO: 1 ; or

(d) amino acids 42-55 of SEQ ID NO: 1.

4. A peptide according to claim 1 or 2 that consists essentially of

(a) amino acids 46-55 of SEQ ID NO: 1 ;

(b) amino acids 47-55 of SEQ ID NO: 1 ;

(c) amino acids 44-57 of SEQ ID NO: 1 ; or

(d) amino acids 42-55 of SEQ ID NO: 1.

5. A vector capable of expressing a peptide according to any one of claims 1 to 4.

6. An isolated antigen presenting cell which presents at its surface a peptide according to any one of claims 1 to 4.

7. An antigen presenting cell according to claim 6, wherein said peptide is presented on an MHC class II molecule.

8. A method of producing regulatory T cells capable of inhibiting T cell proliferation, the method comprising

(a) contacting a sample from an individual with a peptide according to any one of claims 1 to 4, wherein said sample comprises antigen presenting cells and T cells; or

(b) contacting a sample from an individual with an antigen presenting cell according to claim 6 or 7, wherein said sample comprises T cells.

9. An isolated regulatory T cell obtainable by the method of claim 8.

10. A pharmaceutical formulation comprising a peptide according to any one of claims 1 to 4, a vector according to claim 5, an antigen presenting cell according to claim 6 or 7 or a T cell according to claim 9 and a pharmaceutically acceptable carrier.

11. A method of inhibiting T cell proliferation comprising contacting a population of T cells with an antigen presenting cell according to claim 6 or 7 or a regulatory T cell according to claim 9.

12. A method of inhibiting T cell proliferation comprising:

(b) contacting a sample from an individual with an antigen presenting cell according to claim 6 or 7, wherein said sample comprises T cells; and contacting a population of T cells with said sample of (a) or (b)

13. A method according to claim 12, wherein said sample further comprises NK cells.

14. A method according to claim 11, 12 or 13, wherein said contacting step(s) are carried out in vitro or ex vivo.

15. A method of treating or preventing a hypersensitivity reaction or of preventing or decreasing an immune response comprising administering to a patient in need theieof a peptide according to any one of claims 1 to 4, a vector according to claim 5, an antigen presenting cell according to claim 6 or 7 or a regulatory T cell according to claim 9.

16. A method according to claim 15, wherein said hypersensitivity reaction is selected from an autoimmune disease, an allergy, a delayed type hypersensitivity reaction and transplant rejection.

17. A peptide according to any one of claims 1 to 4, a vector according to claim 5, an antigen presenting cell according to claim 6 or 7 or a regulatory T cell according to claim 9 for use in treating or preventing a hypersensitivity reaction or of preventing or decreasing an immune response.

18. A peptide, vector or cell according to claim 17, wherein said hypersensitivity reaction is selected from an autoimmune disease, an allergy, a delayed type hypersensitivity reaction and transplant rejection.