peptides

Abstract

To provide peptides capable of inducing immune responses against a TGFβR2-1a frameshift mutant protein.SOLUTION: Provided is a peptide comprising a fragment of a specific sequence, the fragment comprising an amino acid sequence of the specific sequence, the peptide comprising 33 or less amino acids, the peptide having at least 70% sequence identity to the specific sequence outside the fragment, and the peptide being capable of inducing an immune response against a TGFβR2-1a frameshift mutant protein.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] The present invention provides peptides of TGBβR2 having a frameshift mutation that induces an immune response, peptide mixtures comprising peptides of TGBβR2 having a frameshift mutation that induces an immune response, T cells specific for such peptides, and T cell mixtures and T cell preparations comprising T cells specific for such peptides.The present invention also relates to pharmaceutical preparations comprising such peptides, peptide mixtures, T cells, and T cell mixtures and T cell preparations, the use of such peptides, peptide mixtures, T cells, and T cell mixtures and T cell preparations for the prevention and / or treatment of cancer, and methods for selecting peptides, peptide mixtures, T cells, T cell mixtures, and T cell preparations for the treatment of cancer. [Background technology]

[0002] DNA microsatellites are strings of repeating DNA in which a specific DNA motif (a nucleotide sequence pattern) is repeated, usually about 5 to 50 times. Microsatellite instability (MSI) is an alteration in the number of microsatellite repeats and can occur due to impaired DNA mismatch repair (MMR) enzyme activity.

[0003] MMR corrects errors that occur naturally during DNA replication, such as single-base mismatches or short insertions or deletions. When MMR activity is impaired, these naturally occurring errors are not repaired, which can lead to microsatellite instability (i.e., changes in the number of repeats) and frameshift mutations in DNA microsatellite sequences.

[0004] A frameshift mutation is the addition or deletion of one or two base pairs from a gene, resulting in a different codon at the point of mutation, and therefore the coding of a different protein. A frameshift typically results in a truncated protein sequence because the STOP codon occurs earlier than normal, and the coded protein is often defective or inactive.

[0005] In recent years, immuno-oncology is a developing field, and efforts have been focused on using the patient's own immune system to fight cancer. However, one problem is that antibodies can only bind to tumor antigens that are exposed on the surface of tumor cells. For this reason, efforts to create cancer treatments based on the body's immune system have not been as successful as expected.

[0006] TGFβR2 (SEQ ID NO: 1) is a growth factor that mediates the control of cell proliferation through interaction with TGFβ. Frameshift mutations in TGFβR2 result in biological dysfunction, which leads to uncontrolled cell proliferation and cancer progression. Single nucleotide deletions are by far the most prevalent frameshift mutations in TGFβR2, although single nucleotide additions can also occur. The amino acid sequence of TGFβR2 resulting from a single nucleotide deletion (-1a) frameshift mutation is shown in SEQ ID NO: 2.

[0007] Detection of MSI in cancers such as colorectal cancer (CRC) is performed by profiling the Bethesda panel. The Bethesda panel is a reference panel that includes five microsatellite loci, namely two mononucleotides (BAT25 and BAT26) and three dinucleotides (D5S346, D2S123, and D17S250) (Non-Patent Document 1, Non-Patent Document 2). MSI is classified as high (MSI-H) if there is instability at two or more loci, and low (MSI-L) if there is instability at one locus (Non-Patent Document 2). Microsatellites can be classified as stable (MSS) if there are no loci with instability (Non-Patent Document 2).

[0008] Approximately 15% of all CRCs are MSI-H (Non-Patent Document 1), and 99% of hereditary CRCs (hereditary nonpolyposis colorectal cancer (HNPCC)) are MSI-H (Non-Patent Document 3). Approximately 90% of MSI-H HNPCC patients have a frameshift mutation in the protein TGFβR2 (Non-Patent Document 3). People with HNPCC have somatic mutations that are thought to develop into frameshift mutations. CRC is often driven by the development of polyps, but unlike in patients without hereditary CRC, removing polyps from patients with hereditary CRC is not effective in preventing cancer.

[0009] Furthermore, approximately 22% of gastric cancers (stomach cancers) are MSI-H (Non-Patent Document 1).

[0010] Furthermore, it has been reported that frameshift mutations in TGFβR2 are found in approximately 10% of all CRCs, approximately 44% of all MSI-H cancers, and in particular, approximately 58% of MSI-H colon cancers and approximately 80% of MSI-H gastric cancers (Non-Patent Document 1).

[0011] Peptides of TGFβR2 carrying frameshift mutations have been reported to be immunogenic, although the reported results have been conflicting, as explained further below.

[0012] Patent Document 1 discloses the use of protein fragments resulting from frameshift mutations in the BAX gene and the TGFβR2 gene to treat cancer by inducing T cell immunity.

[0013] Non-Patent Document 4 reports that three peptides derived from proteins with frameshift mutations, including a peptide of TGFβR2 with a -1a frameshift mutation (referred to as FSP02 in this document: RLSSCVPVA; SEQ ID NO: 10), were able to activate specific CTLs (HLA-A2.1 restricted) in vitro. This peptide was also able to lyse the colon cancer cell line HCT116 with the corresponding frameshift mutation. However, the other two peptides of -1a frameshift TGFβR2 did not activate CTLs.

[0014] Non-Patent Document 5 reports that a peptide of TGFβR2 having a frameshift mutation (RLSSCVPVA (shown as p573 in Non-Patent Document 5); SEQ ID NO: 10 in the present specification) was able to generate a CTL line and several CTL clones. One CTL clone was able to kill an HLA-A2+ colon cancer cell line having a frameshift TGFβR2.

[0015] Non-Patent Document 6 reports a highly immunogenic peptide derived from TGFβR2 with a frameshift mutation (designated p538; SLVRLSSCVPVALMSAMTTSSSQ (SEQ ID NO: 11)) as a target for tumor-infiltrating Th cells in MSI+ tumors. This peptide was recognized by two of three patients with spontaneous MSI+ colon cancer and by all three patients with HNPCC. Several other peptides (p540: SPKCIMKEKKSLVRLSSCVPVA (SEQ ID NO: 12) and p541: PKCIMKEKKKSLVRLSSCV (SEQ ID NO: 13)) corresponding to the same frameshift mutation were also able to induce T cell responses in some patients.

[0016] However, the above studies have contradictory results, and as a result, it is unclear whether the peptide of TGFβR2 with frameshift mutation is really immunogenic. For example, the peptide FSP01 (SLVRLSSCV; SEQ ID NO: 13 in this specification) of Non-Patent Document 4 is the same as the peptide of SEQ ID NO: 428 of Patent Document 1, but Non-Patent Document 4 reports that this peptide is not immunogenic (Figure 1 of Non-Patent Document 4), while Patent Document 1 reports that this peptide is immunogenic, although only after four times of T cell stimulation (Figure 14 of Patent Document 1). Peptide FSP02 (RLSSCVPVA; SEQ ID NO: 10 herein) of Non-Patent Document 4 is the same as peptide SEQ ID NO: 439 of Patent Document 1 and peptide p573 of Non-Patent Document 5, but Non-Patent Document 4 and Non-Patent Document 5 report that this peptide is immunogenic (Figure 1 of Non-Patent Document 4; Abstract of Non-Patent Document 5 and page 472, column 1, paragraph 3), while Patent Document 1 reports that this peptide is not immunogenic even after four rounds of T cell stimulation (Figure 14 of Patent Document 1). Furthermore, Non-Patent Document 6 discloses that peptides p537 and p621 are not immunogenic, while peptides p538, p540, and p4541 are immunogenic. However, both p538 and p621 contain the sequences of peptides p523 and p573, which have been shown to be immunogenic by several studies (as described above). Thus, it is unclear why p538 is immunogenic while p621 is not. Furthermore, Patent Document 1 discloses that the peptide of SEQ ID NO: 17 can stimulate cultured T cell clones derived from patients with adenocarcinoma (FIGS. 8 and 9 of Patent Document 1), but does not induce T cell responses above background levels in T cells from healthy blood donors (FIG. 12 of Patent Document 1). The results shown in FIGS. 8 and 9 of Patent Document 1 indicate that spontaneous T cell immune responses may be induced in cancer patients, and that these T cells after culture with the peptide of SEQ ID NO: 17 can recognize the peptide of SEQ ID NO: 17.However, these results do not demonstrate that the peptide of SEQ ID NO:17 is an antigen strong enough to induce a protective immune response.

[0017] Patent document 2 discloses that peptides derived from -1a frameshifted TGFβR2 were able to induce immune responses in vitro using T cells from healthy HLA-A2.1+ donors. However, these results are limited to HLA-A2.1+ epitopes only, and no other HLA class I-restricted T cells or any HLA class II-restricted T cells were shown to be induced. Historically, vaccines consisting of only HLA class I epitopes have not been successful in treating cancer, and therefore Patent document 2 does not show that the peptides tested in this document are effective vaccines or treatments against cancer.

[0018] Thus, there is a need to provide effective vaccines and / or treatments for cancers, particularly those associated with MSI and frameshift mutations. In particular, there is a need to develop vaccines for people at risk of developing HNPCC. There is also a need to provide vaccines and / or treatments for these cancers that are cost-effective and can be used in treatments or as vaccines for as many MSI-H associated cancers as possible. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] European Patent No. 1078000 [Patent Document 2] U.S. Patent No. 8,053,552 [Non-patent literature]

[0020] [Non-Patent Document 1] Cortes-Ciriano et al., Nature Communications, 2017, vol. 8, article no. 15180

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

[0021] The present invention alleviates at least some of the above-mentioned problems, since it has been found herein that peptides comprising a fragment of TGFβR2 with a frameshift mutation can be used to induce an immune response against cancer cells, and thus are useful in the treatment and / or prevention of cancers associated with TGFβR2 with a frameshift mutation. The present invention is particularly useful in the treatment and / or prevention of cancers associated with TGFβR2 with a -1a frameshift mutation (referred to herein as "mutTGFβR2"). It has been found that a particularly useful peptide comprises a fragment of mutTGFβR2 and may include one amino acid substitution in mutTGFβR2. In particular, the peptides according to the present invention can be used to treat about 10% of all colorectal cancers, including about 90% of hereditary colorectal cancers, and about 18% of gastric cancers. The peptides of the present invention alleviate the problem of inconsistent results on the immunogenicity of peptides of mutTGFβR2, providing an effective, cost-effective vaccine and / or treatment. Furthermore, the peptides of the present invention contain multiple nested epitopes, such that they include epitopes for more than one HLA allele. This offers the advantage that the peptides can induce immune responses in patients with different HLA alleles and are therefore useful as universal treatments and / or vaccines.

[0022] In a first aspect of the present invention there is provided a peptide comprising a fragment of SEQ ID NO:8, wherein the fragment comprises at least 8 consecutive amino acids of SEQ ID NO:8 including at least one of positions 121 and 135 of SEQ ID NO:8, and wherein the peptide is capable of inducing an immune response against a TGFβR2-1a frameshift mutant protein.

[0023] Preferably, a peptide is provided comprising a fragment of SEQ ID NO:8, wherein the fragment comprises at least 12 consecutive amino acids of SEQ ID NO:8, including at least one of positions 121 and 135 of SEQ ID NO:8, and wherein the peptide is capable of inducing an immune response against a TGFβR2-1a frameshift mutant protein.

[0024] Conveniently, a fragment comprises at least 9, preferably at least 12, consecutive amino acids of SEQ ID NO:8, including at least one of positions 121 and 135 of SEQ ID NO:8.

[0025] Advantageously, the fragment comprises at least 15 consecutive amino acids of SEQ ID NO:8, including at least one of positions 121 and 135 of SEQ ID NO:8.

[0026] Conveniently, the fragment includes only one of positions 121 and 135 of SEQ ID NO:8.

[0027] Advantageously, the peptide comprises 33 or fewer amino acids.

[0028] Preferably, the peptide contains 24 or fewer amino acids.

[0029] Conveniently, the peptide comprises 20 or fewer amino acids.

[0030] Preferably, the peptide contains 17 or fewer amino acids.

[0031] Conveniently, the peptide comprises 9 or fewer amino acids.

[0032] Advantageously, the fragment comprises position 121 to position 132 of SEQ ID NO:8, or position 135 to position 146 of SEQ ID NO:8.

[0033] Preferably, the amino acid corresponding to position 121 or position 135 of SEQ ID NO:8 is a glycine.

[0034] Conveniently, the peptide has at least 70% sequence identity to SEQ ID NO:8 outside the fragment.

[0035] Advantageously, the peptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:28 or SEQ ID NO:30.

[0036] Preferably, the peptide consists solely of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:28, or SEQ ID NO:30.

[0037] In a second aspect of the present invention there is provided a peptide comprising a fragment of SEQ ID NO:2, wherein the fragment comprises positions 6 to 13 of SEQ ID NO:3, positions 7 to 22 of SEQ ID NO:3, or positions 18 to 33 of SEQ ID NO:3, the peptide comprising 40 or less amino acids, and the peptide capable of inducing an immune response against a TGFβR2-1a frameshift mutant.

[0038] Preferably, the fragment i) comprises positions 6 to 13 of SEQ ID NO: 3 and the peptide comprises 21 amino acids or less, ii) comprises positions 7 to 22 of SEQ ID NO: 3 and the peptide comprises 40 amino acids or less, or iii) comprises positions 18 to 33 of SEQ ID NO: 3 and the peptide comprises 33 amino acids or less, and the peptide is capable of inducing an immune response against a TGFβR2-1a frameshift mutant.

[0039] Conveniently, the fragment comprises positions 6 to 15 of SEQ ID NO:3, positions 2 to 22 of SEQ ID NO:3, or positions 16 to 33 of SEQ ID NO:3.

[0040] Advantageously, the peptide comprises the sequence SEQ ID NO:3.

[0041] Preferably, the peptide has at least 70% sequence identity to SEQ ID NO:3 outside the fragment.

[0042] Conveniently, the peptide comprises the sequence of SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:26.

[0043] Preferably, the peptide consists solely of SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:26.

[0044] Conveniently, the peptide comprises 33 or fewer amino acids, preferably 24 or fewer amino acids, preferably 20 or fewer amino acids, or preferably 17 or fewer amino acids.

[0045] Advantageously, the peptide i) comprises positions 6 to 13 of SEQ ID NO: 3 and comprises not more than 20 amino acids, or preferably not more than 17 amino acids, ii) comprises positions 7 to 22 of SEQ ID NO: 3 and comprises not more than 33 amino acids, preferably not more than 24 amino acids, preferably not more than 20 amino acids, or preferably not more than 17 amino acids, or iii) comprises positions 18 to 33 of SEQ ID NO: 3 and comprises not more than 24 amino acids, or preferably not more than 20 amino acids.

[0046] In a third aspect of the present invention there is provided a peptide comprising a fragment of SEQ ID NO: 2 for use in the treatment and / or prevention of cancer, wherein the fragment comprises the amino acid sequence of SEQ ID NO: 29, the peptide comprises 21 amino acids or less and the peptide is capable of inducing an immune response against a TGFβR2-1a frameshift mutant.

[0047] Preferably, the cancer is colon cancer or gastric cancer.

[0048] Advantageously, the peptide comprises 17 or fewer amino acids.

[0049] In a fourth aspect of the invention there is provided a nucleic acid molecule encoding a peptide according to the first, second or third aspect above.

[0050] In a fifth aspect of the present invention there is provided a peptide mixture comprising a first peptide and a second peptide, the first peptide and the second peptide being peptides according to the first, second or third aspect above, and wherein the first peptide is different from the second peptide.

[0051] In a sixth aspect of the present invention there is provided a vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a peptide according to the first, second or third aspect above, or a mixture of peptides according to the fifth aspect above.

[0052] In a seventh aspect of the present invention there is provided a host cell comprising a vector according to the sixth aspect above.

[0053] In an eighth aspect of the invention there is provided a T cell preparation comprising untransfected T cells specific for a peptide according to the first, second or third aspect above.

[0054] In a ninth aspect of the present invention there is provided a mixture of untransfected T cells specific for the peptide mixture of the fifth aspect above.

[0055] In a tenth aspect of the present invention there is provided a pharmaceutical composition comprising a peptide according to the first, second or third aspect as defined above, a nucleic acid molecule according to the fourth aspect as defined above, a mixture of peptides according to the fifth aspect as defined above, a vector according to the sixth aspect as defined above, a host cell according to the seventh aspect as defined above, an untransfected T cell according to the eighth aspect as defined above, or a mixture of untransfected T cells according to the ninth aspect as defined above, and a pharma- ceutically acceptable carrier, diluent or excipient.

[0056] In an eleventh aspect of the present invention there is provided a peptide according to the first or second aspect as defined above, a nucleic acid molecule according to the fourth aspect as defined above, a mixture of peptides according to the fifth aspect as defined above, a vector according to the sixth aspect as defined above, a host cell according to the seventh aspect as defined above, an untransfected T cell according to the eighth aspect as defined above, a mixture of untransfected T cells according to the ninth aspect as defined above or a pharmaceutical composition according to the tenth aspect as defined above for use in the treatment and / or prevention of cancer.

[0057] Advantageously, the cancer is colon cancer or gastric cancer.

[0058] Conveniently, the cancer comprises cancer cells which express a frameshift mutant of the TGFβR2 protein.

[0059] Preferably, the frameshift mutant of the TGFβR2 protein is a -1a frameshift mutant.

[0060] In an eleventh or twelfth aspect of the present invention there is provided a method for selecting a peptide, a nucleic acid molecule, a mixture of peptides, a vector, a host cell, a T cell, a mixture of T cells or a pharmaceutical composition for administration to a patient, comprising the steps of: i) to identify whether a cancer patient has a frameshift mutation in the TGFβR2 protein, and if so, ii) selecting a peptide according to the first, second or third aspect as described above, a nucleic acid molecule according to the fourth aspect as described above, a mixture of peptides according to the fifth aspect as described above, a vector according to the sixth aspect as described above, a host cell according to the seventh aspect as described above, an untransfected T cell according to the eighth aspect as described above, an untransfected T cell mixture according to the ninth aspect as described above, or a pharmaceutical composition according to the tenth aspect as described above; A method is provided, comprising:

[0061] In a thirteenth aspect of the present invention there is provided a method of treating cancer comprising administering to a patient in need thereof a peptide according to the first, second or third aspect as defined above, a nucleic acid molecule according to the fourth aspect as defined above, a mixture of peptides according to the fifth aspect as defined above, a vector according to the sixth aspect as defined above, a host cell according to the seventh aspect as defined above, an untransfected T cell according to the eighth aspect as defined above, a mixture of untransfected T cells according to the ninth aspect as defined above, or a pharmaceutical composition according to the tenth aspect as defined above.

[0062] In a thirteenth or fourteenth aspect of the present invention there is provided the use of a peptide according to the first, second or third aspect as defined above, a nucleic acid molecule according to the fourth aspect as defined above, a mixture of peptides according to the fifth aspect as defined above, a vector according to the sixth aspect as defined above, a host cell according to the seventh aspect as defined above, a pharmaceutical composition according to the tenth aspect as defined above comprising a peptide or a mixture of peptides, or a peptide, mixture of peptides, nucleic acid molecule, vector, host cell or pharmaceutical composition for use according to the eleventh aspect as defined above, for the preparation of an untransfected T cell according to the eighth aspect as defined above or a mixture of untransfected T cells according to the ninth aspect as defined above.

[0063] As used herein, the term "peptide" refers to a polymer of amino acid residues that is a fragment of (or has a sequence corresponding to) a longer protein. The term applies to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues are modified residues or non-naturally occurring residues, such as artificial chemical mimics of the corresponding naturally occurring amino acid. Peptides can be conjugated to other agents or moieties.

[0064] As used herein, the term "fragment" refers to a contiguous stretch of amino acids from a longer polypeptide or protein.

[0065] The percent "identity" between two sequences can be determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaeffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402) using default parameters. In particular, the BLAST algorithm can be accessed on the Internet using the URL http: / / www.ncbi.nlm.nih.gov / blast / .

[0066] As used herein, the term "immune response" refers in some embodiments to a T cell mediated immune response (i.e., T cell activation) upon recognition of a peptide. The T cell response can be an HLA-I mediated T cell response and / or an HLA-II mediated T cell response. The immune response can be any alpha beta (αβ) T cell and / or gamma delta (γδ) T cell mediated response, such that the peptide may or may not be presented to the T cell by a major histocompatibility (MHC) molecule on the surface of an antigen presenting cell.

[0067] As used herein, the term "frameshift mutant" refers to a polypeptide encoded by a nucleic acid sequence that has an addition or deletion of one or two nucleotides compared to the wild-type sequence of the nucleic acid, thereby resulting in a different codon from the point of mutation.

[0068] As used herein, the term "-1a frameshift mutant" refers to a polypeptide that results from the deletion of a single nucleotide from the wild-type nucleic acid sequence.

[0069] The term "-1a frameshift mutation" refers to an alteration in the amino acid sequence of a polypeptide, as compared to the wild-type amino acid sequence of the polypeptide, due to the deletion of a single nucleotide from the nucleic acid sequence encoding the polypeptide.

[0070] As used herein, the term "mutTGFβR2" refers to a TGFβR2 protein having a -1a frameshift mutation. The amino acid sequence of mutTGFβR2 is shown in SEQ ID NO:2.

[0071] As used herein, the term "amino acid substitution" refers to the replacement of an amino acid in a polypeptide with a different amino acid as compared to the wild-type amino acid sequence of the polypeptide.

[0072] As used herein, the term "peptide mixture" refers to two or more peptides that are mixed but not chemically bound. The mixture may exist as a dry powder, a solution, a suspension, or a colloid, and may be homogeneous or heterogeneous.

[0073] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a polymer of multiple nucleotides. Nucleic acids may include naturally occurring nucleotides, such as glycol nucleotides and threose nucleotides, or may include artificial nucleotides, such as peptide nucleotides, morpholines, and locked nucleotides.

[0074] As used herein, the term "nucleotide" refers to naturally occurring nucleotides and synthetic nucleotide analogs that are recognized by cellular enzymes.

[0075] As used herein, the term "pharmaceutical composition" means a pharmaceutical preparation suitable for administration to human or animal subjects intended for therapeutic purposes. [Brief description of the drawings]

[0076] [Figure 1] FIG. 1 shows the evolution of the consensus sequence, the peptides of the invention, and the test peptides. [Diagram 2] FIG. 1 shows a UPLC trace of freshly prepared crude fsp5 (SEQ ID NO:3). [Diagram 3] FIG. 1 shows a UPLC trace of fsp5 (SEQ ID NO: 3) purified in solution before lyophilization. [Figure 4] FIG. 1 shows a UPLC trace of purified fsp5 (SEQ ID NO: 3) after lyophilization. [Diagram 5] FIG. 1 shows an HPLC trace of crude fsp5 (SEQ ID NO: 3) after storage at room temperature for 3 days followed by reconstitution and lyophilization. [Figure 6] FIG. 1 shows a UPLC trace of purified fsp1 (SEQ ID NO: 4). [Figure 7] FIG. 1 shows a UPLC trace of purified fsp2 (SEQ ID NO:5). [Figure 8] FIG. 1 shows a UPLC trace of purified fsp3 (SEQ ID NO: 6). [Figure 9] FIG. 1 shows a UPLC trace of purified fsp4 (SEQ ID NO: 7). [Figure 10] 1 is a graph showing T cell proliferation after a single stimulation with a peptide mixture containing fsp2 (SEQ ID NO: 5) and fsp4 (SEQ ID NO: 7). [Figure 11] 1 is a graph showing T cell proliferation after a second stimulation with a peptide mixture containing fsp2 (SEQ ID NO: 5) and fsp4 (SEQ ID NO: 7). [Figure 12] FIG. 12 is another graph showing T cell proliferation in samples from donor 2, donor 3, and donor 4 shown in FIG. 11. [Figure 13] Graph showing T cell proliferation induced by fsp2 (SEQ ID NO:5), fsp6 (SEQ ID NO:26), or fsp7 (SEQ ID NO:27) after stimulation with a peptide mixture containing fsp2 (SEQ ID NO:5), fsp8 (SEQ ID NO:31), and fsp9 (SEQ ID NO:32). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] Brief Description of the Sequence Listing SEQ ID NO:1 is the full length wild type TGFβR2 protein. SEQ ID NO:2 is a full-length mutant TGFβR2 with a −1 amino acid frameshift mutation. SEQ ID NO:3 is a 33 amino acid peptide of SEQ ID NO:2, designated herein as fsp5. SEQ ID NO:4 is a peptide designated herein as fsp1. SEQ ID NO:5 is the peptide designated herein as fsp2. Free text in the sequence listing: modified peptides. SEQ ID NO:6 is the peptide designated herein as fsp3. SEQ ID NO:7 is the peptide designated herein as fsp4. Free text in the sequence listing: modified peptides. SEQ ID NO:8 is a full-length mutant TGFβR2 with a -1 amino acid frameshift mutation and with amino acid substitutions at positions 121 and 135. Free text in the sequence listing: Modified peptide; position 121 is any amino acid except cysteine; position 135 is any amino acid except cysteine. SEQ ID NO: 9 is a prior art peptide of mutTGFβR2. SEQ ID NO: 10 is a prior art peptide of mutTGFβR2. SEQ ID NO: 11 is a prior art peptide of mutTGFβR2. SEQ ID NO: 12 is a prior art peptide of mutTGFβR2. SEQ ID NO: 13 is a prior art peptide of mutTGFβR2. SEQ ID NO: 14 is a prior art peptide of mutTGFβR2. SEQ ID NO: 15 is a prior art peptide of mutTGFβR2. SEQ ID NO: 16 is the manually predicted consensus sequence of mutTGFβR2. SEQ ID NO: 17 is the epitope of mutTGFβR2 predicted by SYFPEITHI. SEQ ID NO: 18 is the epitope of mutTGFβR2 predicted by SYFPEITHI. SEQ ID NO: 19 is the epitope of mutTGFβR2 predicted by SYFPEITHI. SEQ ID NO: 20 is the epitope of mutTGFβR2 predicted by SYFPEITHI. SEQ ID NO: 21 is the epitope of mutTGFβR2 predicted by SYFPEITHI. SEQ ID NO: 22 is the epitope of mutTGFβR2 predicted by SYFPEITHI. SEQ ID NO: 23 is the epitope of mutTGFβR2 predicted by SYFPEITHI. SEQ ID NO:24 is the epitope of mutTGFβR2 predicted by SYFPEITHI. SEQ ID NO: 25 is the epitope of mutTGFβR2 predicted by SYFPEITHI. SEQ ID NO:26 is the peptide designated herein as fsp6. SEQ ID NO:27 is the peptide designated herein as fsp7. SEQ ID NO: 28 is the peptide designated herein as fsp6a. Free text in the sequence listing: modified peptides. SEQ ID NO:29 is the peptide designated herein as fsp1a. SEQ ID NO: 30 is the peptide designated herein as fsp1b. Free text in the sequence listing: modified peptides. SEQ ID NO:31 is the peptide designated herein as fsp8. SEQ ID NO:32 is the peptide designated herein as fsp9.

[0078] Detailed Description The present invention generally relates to a peptide derived from TGFβR2 having a frameshift mutation. The peptide comprises a fragment of the TGFβR2 frameshift mutant protein and can induce an immune response against the TGFβR2-1a frameshift mutant protein. In some embodiments, the TGFβR2 frameshift mutant is a -1aTGFβR2 frameshift mutant (herein referred to as "mutTGFβR2"). The amino acid sequence of the TGFβR2-1a frameshift mutant protein is shown in SEQ ID NO:2.

[0079] In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) comprises at least 8, at least 9, at least 10, at least 12, at least 14, at least 15, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, or at least 32 amino acids. In some embodiments, the fragment comprises at least 20 amino acids. In other embodiments, the fragment comprises at least 24 amino acids. In further embodiments, the fragment comprises at least 33 amino acids.

[0080] In some embodiments, the fragments contain 100 or fewer, 50 or fewer, or 40 or fewer amino acids. For example, the fragments can contain 35 or fewer, 33 or fewer, 31 or fewer, 29 or fewer, 27 or fewer, 25 or fewer, 23 or fewer, 21 or fewer, 19 or fewer, 17 or fewer, or 9 or fewer amino acids. In some embodiments, the fragments contain 33 or fewer amino acids. In other embodiments, the fragments contain 24 or fewer, 20 or fewer, 17 or fewer, or 9 or fewer amino acids.

[0081] In some embodiments, the peptide comprises at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, or at least 32 amino acids. In some embodiments, the peptide comprises at least 20 amino acids. In other embodiments, the peptide comprises at least 24 amino acids. In some embodiments, the peptide comprises at least 17 amino acids. In other embodiments, the peptide comprises at least 9 amino acids. In further embodiments, the peptide comprises at least 33 amino acids.

[0082] In some embodiments, the peptides contain 100 or fewer, 50 or fewer, or 40 or fewer amino acids. For example, the peptides can contain 35 or fewer, 33 or fewer, 31 or fewer, 29 or fewer, 27 or fewer, 25 or fewer, 23 or fewer, 21 or fewer, 19 or fewer, 17 or fewer, or 9 or fewer amino acids. In some embodiments, the peptides contain 33 or fewer amino acids. In other embodiments, the peptides contain 24 or fewer, 20 or fewer, 17 or fewer, or 9 or fewer amino acids.

[0083] Thus, in some embodiments, the peptide comprises other amino acids outside of the fragment of mutTGFβR2, however, in other embodiments, the peptide has the same length as the fragment, such that the peptide is a fragment of mutTGFβR2.

[0084] Sequence identity to mutTGFbR2 outside the fragment The peptide may have at least 70% sequence identity to mutTGFβR2 (SEQ ID NO:2) outside of the fragments of mutTGFβR2. In some embodiments, the peptide has at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:2 outside of the fragments. In some embodiments, the peptide has 100% sequence identity to SEQ ID NO:2 outside of the fragments of mutTGFβR2.

[0085] Position of the fragment within the peptide In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) begins at the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th position from the N-terminus of the peptide. In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) ends at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th position from the C-terminus of the peptide. In other embodiments, the fragment is at the C-terminus or N-terminus of the peptide.

[0086] crosslinking area In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) comprises at least one consecutive amino acid from the wild-type sequence of TGFβR2 (SEQ ID NO:1) (i.e., positions 1 to 127 of SEQ ID NO:2) and at least one consecutive amino acid from the amino acid sequence resulting from the frameshift mutation (i.e., positions 128 to 161 of SEQ ID NO:2). Thus, a fragment of mutTGFβR2 (SEQ ID NO:2) can overlap with the amino acid sequence unaffected by the frameshift mutation and the amino acid sequence resulting from the frameshift mutation. In some embodiments, the fragment comprises 1, 2, 3, 4, 5, 6, 7, or 8 consecutive amino acids from the wild-type amino acid sequence of TGFβR2 (SEQ ID NO:1). In some embodiments, at least one amino acid from the wild-type sequence of TGFβR2 (SEQ ID NO:1) is contiguous with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids from the amino acid sequence resulting from the frameshift mutation.

[0087] In some embodiments, a fragment of mutated TGFβR2 (SEQ ID NO:2) comprises positions 127 and 128 of SEQ ID NO:2, where the amino acid at position 127 of SEQ ID NO:2 is the amino acid at position 127 of wild-type TGFβR2 (SEQ ID NO:1). This corresponds to positions 8 and 9 of SEQ ID NO:3, and positions 127 and 128 of SEQ ID NO:8. In some embodiments, the fragment comprises positions 126 to 128, 125 to 128, 124 to 128, 123 to 128, 122 to 128, 121 to 128, or 120 to 128 of SEQ ID NO:2 or SEQ ID NO:8 (which correspond to positions 7 to 9, 6 to 9, 5 to 9, 4 to 9, 3 to 9, 2 to 9, or 1 to 9, respectively, of SEQ ID NO:3).

[0088] In particular, it is believed that at least two amino acid residues bridging the wild-type amino acid sequence of TGFβR2 (SEQ ID NO: 1) and the amino acid sequence resulting from the frameshift mutation provide an effective epitope and help the peptide to have good immunogenicity. In particular, in FIG. 10 and FIG. 11, it is shown that peptides containing amino acids from the wild-type sequence of TGFβR2 (SEQ ID NO: 1) (i.e., fsp1 (SEQ ID NO: 4), fsp2 (SEQ ID NO: 5), and fsp5 (SEQ ID NO: 3)) are more immunogenic than peptides not containing amino acids from the wild-type sequence of TGFβR2 (SEQ ID NO: 1) (i.e., fsp3 (SEQ ID NO: 6) and fsp4 (SEQ ID NO: 7)). In addition, it is believed that the immunogenicity of the peptide is likely to be improved when two or more amino acids from the wild-type sequence of TGFβR2 (SEQ ID NO: 1) are present consecutively with at least one amino acid from the amino acid sequence resulting from the frameshift mutation. In particular, it is believed that 5, 6, 7, or 8 amino acids from the wild-type sequence of TGFβR2 (SEQ ID NO:1) contiguous with at least one amino acid from the amino acid sequence resulting from the frameshift mutation will be particularly beneficial to the immunogenicity of the peptide.

[0089] fsp1, fsp3, fsp5, and fsp6 In some embodiments, the fragment of mutTGFβR2 (SEQ ID NO:2) comprises positions 6 to 13 of SEQ ID NO:3, positions 7 to 22 of SEQ ID NO:3, or positions 18 to 33 of SEQ ID NO:3, where the peptide comprises 40 or fewer amino acids. In some embodiments, the fragment of mutTGFβR2 (SEQ ID NO:2) comprises positions 6 to 13 of SEQ ID NO:3 and the peptide comprises 21 or fewer amino acids, or the fragment comprises positions 7 to 22 of SEQ ID NO:3 and the peptide comprises 40 or fewer amino acids, or the fragment comprises positions 18 to 33 of SEQ ID NO:3 and the peptide comprises 33 or fewer amino acids. In some embodiments, the fragment comprises positions 6 to 15, positions 6 to 17, positions 2 to 22, or positions 16 to 33 of SEQ ID NO:3. In some embodiments, the fragment comprises positions 6 to 15 of SEQ ID NO:3 and begins at the 6th position from the N-terminus of the peptide. In some embodiments, the fragment includes positions 6 to 17 of SEQ ID NO:3 and begins at the sixth position from the N-terminus of the peptide. In some embodiments, the fragment includes positions 2 to 15 of SEQ ID NO:3 and begins at the second position from the N-terminus of the peptide. In some embodiments, the fragment includes positions 2 to 22 of SEQ ID NO:3 and begins at the second position from the N-terminus of the peptide. In other embodiments, the fragment includes positions 1 to 17 or positions 1 to 24 of SEQ ID NO:3 and is the N-terminus of the peptide. In some embodiments, the fragment includes positions 16 to 33 of SEQ ID NO:3 and begins at the third position from the N-terminus of the peptide. In some embodiments, the fragment includes positions 14 to 33 of SEQ ID NO:3. In some embodiments, the fragment includes positions 14 to 33 of SEQ ID NO:3 and is the N-terminus of the peptide.

[0090] In some embodiments, the fragment comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:26. In some embodiments, the fragment consists solely of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:26. If a peptide consists solely of the amino acid sequence of SEQ ID NO:4, it is referred to herein as "fsp1." If a peptide consists solely of the amino acid sequence of SEQ ID NO:6, it is referred to herein as "fsp3." If a peptide consists solely of the amino acid sequence of SEQ ID NO:3, it is referred to herein as "fsp5." If a peptide consists solely of the amino acid sequence of SEQ ID NO:26, it is referred to herein as "fsp6."

[0091] In some embodiments, the peptide comprises a fragment consisting of only SEQ ID NO:26 and includes one or more additional amino acids at the C-terminus of the fragment. For example, the peptide can include 1, 2, 3, 4, 5, 6, or 7 additional amino acids at the C-terminus of the fragment. In some embodiments, the fragment consisting of only SEQ ID NO:26 is the N-terminus of the peptide. In some embodiments, the fragment consisting of only SEQ ID NO:26 is the N-terminus of the peptide and includes 7 additional amino acids at the C-terminus of the fragment. In another embodiment, the fragment consisting of only SEQ ID NO:26 is the N-terminus of the peptide and includes 7 additional amino acids at the C-terminus of the fragment. In some embodiments, the one or more additional amino acids have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the corresponding amino acid of SEQ ID NO:2, preferably 100% sequence identity to the corresponding amino acid of SEQ ID NO:2.

[0092] fsp2, fsp4, fsp6a, and fsp1b In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) has amino acid substitutions compared to the amino acid sequence of naturally occurring mutTGFβR2 (SEQ ID NO:2). Thus, a fragment of mutTGFβR2 (SEQ ID NO:2) can comprise at least 8 consecutive amino acids of SEQ ID NO:8, and includes at least one of positions 121 and 135 of SEQ ID NO:8. In some embodiments, a fragment can comprise at least 12 consecutive amino acids of SEQ ID NO:8, and includes at least one of positions 121 and 135 of SEQ ID NO:8. In some embodiments, a peptide includes only one of positions 121 and 135 of SEQ ID NO:8. In particular, positions 121 and 135 of SEQ ID NO:8 correspond to positions 121 and 135 of mutTGFβR2 (SEQ ID NO:2), respectively, which are both cysteine ​​residues. The amino acid substitutions at positions 121 and 135 of SEQ ID NO:8 are substitutions of cysteine ​​with any other amino acid. Thus, the amino acids at positions 121 and 135 of SEQ ID NO:8 are independently one of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the amino acid substitution is to glycine.

[0093] Amino acid substitutions at positions 121 and / or 135 of SEQ ID NO:8 from cysteine ​​to any other amino acid are useful to prevent problems in peptide production, stability, quality, and immunology. In particular, the peptides of the present invention are derived from an optimized consensus sequence (SEQ ID NO:3), as described in the Examples and shown in FIG. 1, which has been found to be difficult to synthesize due to its length. In addition, the solubility, stability, and immunogenicity of the optimized consensus sequence (SEQ ID NO:3) may be insufficient. In particular, the presence of one or more cysteine ​​residues in a peptide may lead to molecular rearrangement and / or polymerization of the peptide due to the formation of intermolecular and / or intramolecular disulfide bonds. This rearrangement and / or polymerization may reduce the immunological effectiveness of the peptide and may cause undesirable inflammatory side reactions, for example, via antibody formation and allergic reactions. Substitution of one or more cysteine ​​residues in a peptide reduces the risk of these possible problems. Thus, in some embodiments, substitution of one or more cysteine ​​residues of the optimized consensus sequence improves the ease of production, stability, quality, and immunology of the peptide, but such substitutions are not required by the invention. How peptides with amino acid substitutions relate to the optimized consensus sequence is shown in Figure 1.

[0094] Range of positions for fsp2, fsp4, fsp6a, and fsp1b In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) having an amino acid substitution includes positions 115 to 122, 116 to 123, 117 to 124, 118 to 125, 119 to 126, 120 to 127, 121 to 128, 128 to 135, 129 to 136, 130 to 137, 131 to 138, 132 to 139, 133 to 140, 134 to 141, or 135 to 142 of SEQ ID NO:8.

[0095] In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) having an amino acid substitution comprises positions 129 to 137 of SEQ ID NO:8.

[0096] In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) having an amino acid substitution includes positions 115 to 126, 116 to 127, 117 to 128, 118 to 129, 119 to 130, 120 to 131, 121 to 132, 124 to 135, 125 to 136, 126 to 137, 127 to 138, 128 to 139, 129 to 140, 130 to 141, 131 to 142, 132 to 143, 133 to 144, 134 to 145, or 135 to 146 of SEQ ID NO:8. In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) having an amino acid substitution includes positions 115 to 129, 116 to 130, 117 to 131, 118 to 132, 119 to 133, 120 to 134, 121 to 135, 122 to 136, 123 to 137, 124 to 138, 125 to 139, 126 to 140, 127 to 141, 128 to 142, 129 to 143, 130 to 144, 131 to 145, 132 to 146, 133 to 147, 134 to 148, or 135 to 149 of SEQ ID NO:8. In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) includes positions 119 to 130, 119 to 133, 120 to 131, 120 to 134, 121 to 132, 121 to 135, 133 to 144, 133 to 147, 134 to 145, 134 to 148, 135 to 146, or 135 to 149 of SEQ ID NO:8.

[0097] In some embodiments, fragments of mutTGFβR2 (SEQ ID NO: 2) having amino acid substitutions include those from positions 115 to 122, 115 to 126, 115 to 129, 116 to 123, 116 to 127, 116 to 130, 117 to 124, 117 to 128, 117 to 131, 118 to 125, 118 to 132, 118 to 134, 118 to 136, 118 to 138, 118 to 139, 119 to 140, 119 to 141, 119 to 142, 119 to 143, 119 to 144, 119 to 145, 119 to 146, 119 to 147, 119 to 148, 119 to 149, 120 ... ~position 129, position 118~position 132, position 119~position 126, position 119~position 130, position 119~position 133, position 120~position 127, position 120~position 131, position 120~position 134 , position 121~position 128, position 121~position 132, position 121~position 135, position 122~position 136, position 123~position 137, position 124~position 135, position 124~position 138, position 125~ Position 136, Position 125~Position 139, Position 126~Position 137, Position 126~Position 140, Position 127~Position 138, Position 127~Position 141, Position 128~Position 135, Position 128~Position 139, Position 128~Position 142, Position 129~Position 136, Position 129~Position 140, Position 129~Position 143, Position 130~Position 137, Position 130~Position 141, Position 130~Position 144, Position 131~Position position 138, position 131 to position 142, position 131 to position 145, position 132 to position 139, position 132 to position 143, position 132 to position 146, position 133 to position 140, position 133 to position 144, position 133 to position 147, position 134 to position 141, position 134 to position 145, position 134 to position 148, position 135 to position 142, position 135 to position 146, or position 135 to position 149.

[0098] fsp2 and fsp6a (and fsp5 with a C to G substitution) In some embodiments, the fragment of mutTGFβR2 (SEQ ID NO:2) includes position 121 but not position 135 of SEQ ID NO:8. In some embodiments, the peptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:2 outside of the fragment. In some such embodiments, the peptide has 100% sequence identity to SEQ ID NO:2 outside of the fragment. In some embodiments, the peptide includes 33 or fewer amino acids, 24 or fewer amino acids, or 17 or fewer amino acids. In some embodiments, the peptide consists of only 33 amino acids. In other embodiments, the peptide consists of only 24 amino acids. In other embodiments, the peptide consists of only 17 amino acids. In some embodiments, the fragment of mutTGFβR2 (SEQ ID NO:2) includes positions 119 to 126, 120 to 127, or 121 to 128 of SEQ ID NO:8. In some embodiments, the fragment of mutTGFβR2 (SEQ ID NO:2) comprises positions 119-130, 120-131, or 121-132 of SEQ ID NO:8. In other embodiments, the fragment comprises positions 119-133, 120-134, or 121-135 of SEQ ID NO:8. In some embodiments, the fragment consists only of positions 119-130, 119-133, 120-131, 120-134, 121-132, or 121-135 of SEQ ID NO:8. In some embodiments, the fragment of mutTGFβR2 (SEQ ID NO:2) having an amino acid substitution begins at the first, second, or third position from the N-terminus of the peptide. In some embodiments, the fragment is the N-terminus of the peptide. In some embodiments, position 121 of SEQ ID NO:8 is a glycine.

[0099] In some embodiments, the peptide comprises a fragment of mutTGFβR2 (SEQ ID NO:2) with an amino acid substitution, wherein the fragment consists of positions 119-126, 120-127, or 121-128 of SEQ ID NO:8, where position 121 of SEQ ID NO:8 is a glycine, and the peptide has 100% sequence identity to SEQ ID NO:2 outside the fragment and comprises no more than 33 amino acids. In some embodiments, the peptide comprises a fragment of mutTGFβR2 (SEQ ID NO:2) with an amino acid substitution, wherein the fragment consists of positions 119-130, 119-133, 120-131, 120-134, 121-132, or 121-135 of SEQ ID NO:8, where position 121 of SEQ ID NO:8 is a glycine, and the peptide has 100% sequence identity to SEQ ID NO:2 outside the fragment and comprises no more than 33 amino acids. In some embodiments, the fragment is the N-terminus of the peptide. In some such embodiments, the peptide consists of only 33 amino acids. In another embodiment, the peptide consists of only 24 amino acids and can consist of only the amino acid sequence of SEQ ID NO:5. A peptide consisting of only the amino acid sequence of SEQ ID NO:5 is referred to herein as "fsp2". In another embodiment, the peptide consists of only 17 amino acids and can consist of only the amino acid sequence of SEQ ID NO:28. A peptide consisting of only the amino acid sequence of SEQ ID NO:28 is referred to herein as "fsp6a".

[0100] In some embodiments, the peptide comprises a fragment consisting of only SEQ ID NO:28 and includes one or more additional amino acids at the C-terminus of the fragment. For example, the peptide can include 1, 2, 3, 4, 5, 6, or 7 additional amino acids at the C-terminus of the fragment. In some embodiments, the fragment consisting of only SEQ ID NO:28 is the N-terminus of the peptide. In some embodiments, the fragment consisting of only SEQ ID NO:28 is the N-terminus of the peptide and includes 7 additional amino acids at the C-terminus of the fragment. In another embodiment, the fragment consisting of only SEQ ID NO:28 is the N-terminus of the peptide and includes 7 additional amino acids at the C-terminus of the fragment. In some embodiments, the one or more additional amino acids have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the corresponding amino acid of SEQ ID NO:2, preferably 100% sequence identity to the corresponding amino acid of SEQ ID NO:2.

[0101] fsp4 and fsp1b In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) having an amino acid substitution includes position 135 but not position 121 of SEQ ID NO:8. In some embodiments, the peptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:2 outside the fragment. In some embodiments, the peptide includes 20 or fewer amino acids, while in other embodiments, the peptide consists of only 20 amino acids. In some embodiments, the peptide includes 9 or fewer amino acids, while in other embodiments, the peptide consists of only 9 amino acids.

[0102] In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) comprises position 129 to position 136, position 130 to position 137, position 133 to position 140, position 134 to position 141, or position 135 to position 142 of SEQ ID NO:8. In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) comprises position 129 to position 137, position 133 to position 144, position 134 to position 145, or position 135 to position 146 of SEQ ID NO:8. In some embodiments, a fragment of mutTGFβR2 (SEQ ID NO:2) having an amino acid substitution comprises position 133 to position 147, position 134 to position 148, or position 135 to position 149 of SEQ ID NO:8. In some embodiments, the fragment of mutTGFβR2 (SEQ ID NO:2) with the amino acid substitution consists only of positions 129 to 136, 130 to 137, 133 to 144, 133 to 147, 134 to 145, 134 to 148, 135 to 146, or 135 to 149 of SEQ ID NO:8. In some embodiments, the fragment of mutTGFβR2 (SEQ ID NO:2) with the amino acid substitution begins at the first, second, or third position from the N-terminus of the peptide. In some embodiments, the fragment of mutTGFβR2 (SEQ ID NO:2) with the amino acid substitution is at the N-terminus of the peptide. In some embodiments, position 135 of SEQ ID NO:8 is a glycine.

[0103] In some embodiments, a peptide comprises a fragment of mutTGFβR2 (SEQ ID NO:2) having an amino acid substitution, wherein the fragment consists only of the amino acid sequence of SEQ ID NO: 30. In some embodiments, a peptide consists of the amino acid sequence of SEQ ID NO: 30, and such peptides are referred to herein as "fsp1b."

[0104] In some embodiments, the peptide comprises a fragment of mutTGFβR2 (SEQ ID NO:2) having an amino acid substitution, wherein the fragment consists of positions 133 to 140, 133 to 144, 133 to 147, 134 to 141, 134 to 145, 134 to 148, 135 to 142, 135 to 146, or 135 to 149 of SEQ ID NO:8, where position 135 of SEQ ID NO:8 is a glycine, and the peptide has 100% sequence identity to SEQ ID NO:2 outside the fragment and comprises 20 or fewer amino acids. In some embodiments, the fragment is the N-terminus of the peptide. In some embodiments, the peptide consists of only 20 amino acids. In some embodiments, the peptide consists of the amino acid sequence of SEQ ID NO:7, and such peptides are referred to herein as "fsp4".

[0105] fsp1a The present invention also provides a peptide of mutTGFβR2 (SEQ ID NO: 2) for use in the treatment and / or prevention of cancer, wherein the peptide comprises a fragment of SEQ ID NO: 2, the fragment comprises the amino acid sequence of SEQ ID NO: 29, and the peptide comprises 21 amino acids or less.

[0106] In some embodiments, the peptide of mutTGFβR2 (SEQ ID NO:2) comprises 17 or fewer amino acids. In some embodiments, the peptide comprises 9 or fewer amino acids. In other embodiments, the peptide consists of only 21, 17, or 9 amino acids. In some embodiments, the peptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% sequence identity to SEQ ID NO:2 outside the fragment. In some embodiments, the fragment consists of only the sequence of SEQ ID NO:29.

[0107] In some embodiments, a peptide consists of the amino acid sequence of SEQ ID NO:29, and such a peptide is referred to herein as "fsp1a."

[0108] In view of the differences in immunogenicity of the amino acid sequences of SEQ ID NO:26 and SEQ ID NO:27, as well as the similarities and differences between these amino acid sequences, as described in Example 8, the amino acid sequence of SEQ ID NO:29 is believed to be immunogenic. Similarly, the amino acid sequence of SEQ ID NO:30, which is identical to SEQ ID NO:29 except for the amino acid substitution of C to G, is also believed to be immunogenic, since the C to G substitution has been shown to be immunologically acceptable.

[0109] The peptides of the invention and for use according to the invention are peptides corresponding to fragments of mutTGFβR2 (SEQ ID NO: 2) presented on the surface of cells by MHC-I or MHC-II molecules and / or peptides to which individuals normally have reactive T cells in their T cell repertoire. The peptides are capable of inducing an immune response against the TGFβR2-1a frameshift mutant protein, preferably a T cell response comprising both MHC-I restricted T cells, such as CD8+ T cells, and MHC-II restricted T cells, such as CD4+ T cells. In particular, the peptides can encompass multiple nested epitopes, such that each peptide may comprise epitopes for more than one MHC allele. This provides the advantage that the peptides can induce an immune response in patients with different MHC alleles, and are therefore useful as universal treatments and / or vaccines.

[0110] Peptide Mixture Also provided in the present invention is a mixture of the above-mentioned peptides comprising two or more different peptides, the mixture of peptides comprising a first peptide and a second peptide, wherein the first peptide and the second peptide are the above-mentioned peptides, and the first peptide is different from the second peptide. In some embodiments, the first peptide comprises a fragment of mutTGFβR2 (SEQ ID NO:2) comprising at least 8 or at least 12 contiguous amino acids of SEQ ID NO:8, including positions 6 to 13 of SEQ ID NO:3, or including positions 7 to 22 of SEQ ID NO:3, or including positions 18 to 33 of SEQ ID NO:3, or including at least one of positions 121 and 135 of SEQ ID NO:8, or comprising at least 8 contiguous amino acids of SEQ ID NO:29, and the second peptide comprises a fragment of mutTGFβR2 (SEQ ID NO:2) comprising at least 8 or at least 12 contiguous amino acids of SEQ ID NO:8, including positions 6 to 13 of SEQ ID NO:3, or including positions 7 to 22 of SEQ ID NO:3, or including positions 18 to 33 of SEQ ID NO:3, or including at least one of positions 121 and 135 of SEQ ID NO:8, or comprising at least 8 contiguous amino acids of SEQ ID NO:29, independently, wherein the second peptide is different from the first peptide. In some embodiments, the first peptide comprises a fragment of mutTGFβR2 (SEQ ID NO:2), wherein the fragment comprises at least eight consecutive amino acids of SEQ ID NO:8, including positions 6 to 13 of SEQ ID NO:3, or positions 7 to 22 of SEQ ID NO:3, or positions 18 to 33 of SEQ ID NO:3, or at least one of positions 121 and 135 of SEQ ID NO:8, and the second peptide is a peptide used for the treatment and / or prevention of the above-mentioned cancers, wherein the fragment of mutTGFβR2 (SEQ ID NO:2) comprises at least eight consecutive amino acids of SEQ ID NO:29.In some embodiments, the first peptide and the second peptide are each independently selected from peptides comprising a fragment of mutTGFβR2 (SEQ ID NO:2), wherein the fragment comprises at least eight consecutive amino acids of SEQ ID NO:8, including positions 6 to 13 of SEQ ID NO:3, positions 7 to 22 of SEQ ID NO:3, positions 18 to 33 of SEQ ID NO:3, or at least one of positions 121 and 135 of SEQ ID NO:8.

[0111] In some embodiments, the peptide mixture comprises a first peptide and a second peptide, where the first peptide comprises a fragment of SEQ ID NO:8 including position 121 but not including position 135 of SEQ ID NO:8, and the second peptide comprises a fragment of SEQ ID NO:8 including position 135 but not including position 121 of SEQ ID NO:8. In some embodiments, the first peptide comprises 33 or fewer, 24 or fewer, or 17 or fewer amino acids and has 100% sequence identity to SEQ ID NO:2 outside the fragment. In some embodiments, the fragment of SEQ ID NO:8 of the first peptide can include or consist of positions 119-126, 119-130, 119-133, 120-127, 120-131, 120-134, 121-128, 121-132, or 121-135 of SEQ ID NO:8. In some embodiments, the first peptide comprises a glycine residue at position 121 of SEQ ID NO:8. In some embodiments, the second peptide comprises 20 or fewer amino acids and has 100% sequence identity to SEQ ID NO:2 outside the fragment. In some embodiments, the second peptide's fragment of SEQ ID NO:8 comprises, and preferably consists of, positions 129-136, 130-137, 129-137, 133-140, 133-144, 133-147, 134-141, 134-145, 134-148, 135-142, 135-146, or 135-149 of SEQ ID NO:8. In some embodiments, the second peptide comprises a glycine residue at position 135 of SEQ ID NO:8.

[0112] In some embodiments, the first peptide and the second peptide independently consist solely of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. In some embodiments, the first peptide consists solely of the amino acid sequence of SEQ ID NO:5 and the second peptide consists solely of the amino acid sequence of SEQ ID NO:7.

[0113] In another embodiment, the first peptide consists of the amino acid sequence of SEQ ID NO: 5 and the second peptide consists solely of the amino acid sequence of SEQ ID NO: 7. In particular, Figure 11 shows that after two stimulations, some donors show a higher response to a mixture of SEQ ID NO: 5 and SEQ ID NO: 7 (i.e., fsp2 and fsp4) than to either SEQ ID NO: 5 alone or SEQ ID NO: 7 alone.

[0114] The peptide mixture may include one or more additional peptides in addition to the first peptide and the second peptide, which may be one or more of the peptides described above. The one or more additional peptides are different from each of the first peptide and the second peptide. When the peptide mixture includes two or more additional peptides, the additional peptides are different from each other.

[0115] The peptide mixture may contain peptides in the same or different proportions. In some embodiments, the first peptide and the second peptide are present in the mixture in the same proportion, i.e., each peptide constitutes 50% of the peptide component of the peptide mixture. In another embodiment, the proportion of the first peptide in the peptide mixture is greater than the second peptide. For example, the first peptide may constitute at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% of the peptide component of the peptide mixture. In alternative embodiments, the proportion of the second peptide in the peptide mixture is greater than the first peptide. For example, the second peptide may constitute at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% of the peptide component of the peptide mixture. In embodiments including at least one additional peptide, the peptides are present in the same proportion in the peptide component of the peptide mixture. In another embodiment, the first peptide, the second peptide, and the at least one additional peptide are present in different proportions from each other. For example, the first peptide, the second peptide, and the at least one additional peptide can each independently constitute at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 60%, at least 70%, at least 80%, or at least 90% of the peptide components of the peptide mixture.

[0116] nucleic acid In another aspect of the present invention, a nucleic acid molecule is provided that comprises a nucleotide sequence encoding a peptide or a peptide of a peptide mixture according to the disclosure above. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding at least two peptides of the peptide mixture. Also provided is a nucleic acid molecule mixture, where each nucleic acid molecule of the nucleic acid molecule mixture comprises a nucleotide sequence encoding a different peptide of a peptide mixture according to the disclosure above, such that the nucleic acid molecule mixture encodes a peptide mixture of the disclosure above. In some embodiments of the nucleic acid molecule mixture, each nucleic acid molecule comprises a nucleotide sequence encoding at least two peptides of the peptide mixture.

[0117] In some embodiments, nucleic acid molecules and mixtures of nucleic acid molecules are used to synthesize the peptides and peptide mixtures disclosed above. For example, the peptides disclosed above can be synthesized by administering nucleic acid molecules to a subject. The nucleic acid molecules are then expressed by the subject, which produces the peptides disclosed above in situ. The produced peptides then induce an immune response in the subject. In another example, nucleic acid molecules can be used to synthesize the peptides disclosed above in vitro by transforming or transfecting a host cell with a nucleic acid molecule, such that the host cell expresses the nucleic acid molecule and produces the peptides disclosed above. The peptides are then recovered or purified. In some embodiments, the peptides disclosed above are produced by chemical synthesis using methods known in the art.

[0118] T cells and T cell mixtures In another aspect of the invention, there is provided a non-transfected T cell preparation comprising non-transfected T cells specific for a peptide or peptide for use according to the above disclosure, and one or more non-transfected T cells. Further, there is provided a non-transfected T cell mixture comprising non-transfected T cells specific for each peptide in one of the peptide mixtures disclosed above.

[0119] The untransfected T cells, untransfected T cell preparations, and untransfected T cell mixtures may be ex vivo and may be generated by stimulating at least one untransfected reactive T cell ex vivo with a peptide or peptide mixture according to the disclosure above. For example, in one embodiment, the untransfected T cells are specific for a peptide comprising a fragment of SEQ ID NO:8, the fragment comprising at least 8 or at least 12 consecutive amino acids of SEQ ID NO:8, including at least one of positions 121 and 135 of SEQ ID NO:8. In another embodiment, the untransfected T cells are specific for a peptide comprising a fragment of mutTGFβR2 (SEQ ID NO:2), the fragment comprising positions 6 to 13, positions 7 to 22, or positions 18 to 33 of SEQ ID NO:3, and the peptide comprising 40 or fewer amino acids, or the fragment comprising the amino acid sequence of SEQ ID NO:29, and the peptide comprising 21 or fewer amino acids. In another embodiment, the untransfected cells are specific for a peptide comprising a fragment of mutTGFβR2 (SEQ ID NO:2), wherein the fragment comprises positions 6 to 13 of SEQ ID NO:3 and the peptide comprises 21 or fewer amino acids, or wherein the fragment comprises positions 7 to 22 of SEQ ID NO:3 and the peptide comprises 40 or fewer amino acids, or wherein the fragment comprises positions 18 to 33 of SEQ ID NO:3 and the peptide comprises 33 or fewer amino acids, or wherein the fragment comprises the amino acid sequence of SEQ ID NO:29 and the peptide comprises 21 or fewer amino acids.In another embodiment, the untransfected T cell preparation comprises one or more untransfected T cells specific for a peptide comprising a fragment of mutTGFβR2 (SEQ ID NO:2), wherein the fragment has an amino acid substitution compared to SEQ ID NO:2 and comprises at least 8 or at least 12 contiguous amino acids of SEQ ID NO:8, including at least one of positions 121 and 135 of SEQ ID NO:8, or the fragment comprises positions 6 to 13 of SEQ ID NO:3 or positions 7 to 22 of SEQ ID NO:3 and the peptide comprises 40 or fewer amino acids, or the fragment comprises positions 18 to 33 of SEQ ID NO:3 and the peptide comprises 40 or fewer amino acids, or the fragment comprises the amino acid sequence of SEQ ID NO:29 and the peptide comprises 21 or fewer amino acids. In another embodiment, the untransfected T cell preparation comprises one or more untransfected T cells specific for a peptide comprising a fragment of mutTGFβR2 (SEQ ID NO:2), wherein the fragment has an amino acid substitution compared to SEQ ID NO:2 and comprises at least 8 contiguous amino acids of SEQ ID NO:8, including at least one of positions 121 and 135 of SEQ ID NO:8, or wherein the fragment comprises positions 6 to 13 of SEQ ID NO:3 and the peptide comprises 21 or fewer amino acids, or wherein the fragment comprises positions 7 to 22 of SEQ ID NO:3 and the peptide comprises 40 or fewer amino acids, or wherein the fragment comprises positions 18 to 33 of SEQ ID NO:3 and the peptide comprises 33 or fewer amino acids, or wherein the fragment comprises the amino acid sequence of SEQ ID NO:29 and the peptide comprises 21 or fewer amino acids. In another embodiment, the untransfected T cell mixture comprises a plurality of untransfected T cells, wherein a first untransfected T cell and a second untransfected T cell are specific for a first peptide and a second peptide, respectively, of mutTGFβR2 (SEQ ID NO:2) according to the disclosure above, where the first peptide is different from the second peptide.

[0120] When the T cell receptor of any T cell disclosed herein is an αβ T cell receptor, the T cell receptor is specific for a peptide presented on an MHC molecule. When the T cell receptor of any T cell disclosed herein is a γδ T cell receptor, the T cell receptor does not necessarily require presentation of the peptide on an MHC molecule in order to recognize the peptide.

[0121] Vectors and host cells In another aspect of the present invention, there is provided a vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding the peptide or peptide mixture disclosed above. In some embodiments, the vector is a DNA vector or an RNA vector.

[0122] In a further aspect, there is provided a host cell comprising the vector described above. The host cell has been transfected or transformed with the vector such that the host cell expresses the nucleic acid molecule(s) encoded by the vector. The host cell can be any cell type that can be transfected with the vector and capable of expressing the vector. In some embodiments, the host cell is a plant cell, an animal cell, a microorganism, or a yeast cell. In some embodiments, the host cell is a dendritic cell.

[0123] Pharmaceutical Compositions Also provided is a pharmaceutical composition comprising the above-mentioned peptide, peptide mixture, untransfected T cell, untransfected T cell mixture, untransfected T cell preparation, nucleic acid molecule, vector, or host cell. Such pharmaceutical composition may also comprise at least one pharma- ceutically acceptable carrier, diluent, and / or excipient. In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients and / or adjuvants. In certain embodiments, the pharmaceutical composition may further comprise one or more ingredients pharma- ceutical active against the same disease indication. In one embodiment, the pharmaceutical composition of the present invention may further comprise one or more additional chemotherapeutic agents, one or more cancer vaccines, one or more antibodies, one or more small molecules, and / or one or more immune stimulants (e.g., cytokines). In some embodiments, the peptide, peptide mixture, untransfected T cell, untransfected T cell preparation, untransfected T cell mixture, nucleic acid, vector, host cell, or pharmaceutical composition may be used in combination with other forms of immunotherapy, including other cancer vaccines. In some embodiments, the peptide, peptide mixture, untransfected T cell, untransfected T cell preparation, untransfected T cell mixture, nucleic acid, vector, host cell, or pharmaceutical composition is used in combination with one or more cancer vaccines derived from different cancer antigens.

[0124] use The peptides, peptide mixtures, untransfected T cells, untransfected T cell preparations, untransfected T cell mixtures, nucleic acid molecules, vectors, host cells, and pharmaceutical compositions disclosed above are for use in the treatment and / or prevention of cancer, in particular cancers associated with frameshift mutations in TGFβR2, preferably -1a frameshift mutations. In particular, about 10% of all CRCs and about 44% of all MSI-H cancers have frameshift mutations in TGFβR2. The cancer can be colorectal cancer or gastric cancer. The colorectal cancer can be colon cancer or rectal cancer. The peptides, peptide mixtures, untransfected T cells, untransfected T cell preparations, untransfected T cell mixtures, nucleic acid molecules, vectors, and host cells can be used in the treatment and / or prevention of two or more of these types of cancer. In particular, the above disclosed peptides, peptide mixtures, untransfected T cells, untransfected T cell preparations, untransfected T cell mixtures, nucleic acid molecules, vectors, and host cells can be used to treat about 10% of all colon cancers (i.e., all hereditary and spontaneous CRC), including about 85.5% of all hereditary colon cancers, and about 18% of all gastric cancers. Thus, the present invention provides an effective treatment for the majority of cancers, particularly colon and gastric cancers, and more particularly hereditary colon cancers.

[0125] In some embodiments, peptides comprising a fragment of SEQ ID NO:8, where the fragment comprises at least 8 or at least 12 consecutive amino acids of SEQ ID NO:8, including position 121 of SEQ ID NO:8, and where the peptide comprises 33 or fewer amino acids, are particularly useful for treating cancer. In some embodiments, the peptide comprises a fragment of SEQ ID NO:8, where the fragment comprises at least 8 or at least 12 consecutive amino acids of SEQ ID NO:8, including position 121 of SEQ ID NO:8 but not including position 135, where the amino acid at position 121 of SEQ ID NO:8 is a glycine, and where the peptide has 100% sequence identity to SEQ ID NO:2 outside of the fragment of mutTGFβR2 (SEQ ID NO:2). In some embodiments, the peptide consists of 17, 24, or 33 amino acids, and the fragment of SEQ ID NO:8 consists of only 8, 10, 12, or 15 consecutive amino acids of SEQ ID NO:8. In some embodiments, the peptide consists of only the amino acid sequence of SEQ ID NO:5. In particular, as shown in FIG. 11, a peptide consisting only of the amino acid sequence of SEQ ID NO:5 (i.e., fsp2) was found to be particularly useful as a vaccine or treatment against cancer, since it is completely unknown to the human immune system and can induce an immune response not only against itself but also against the corresponding naturally occurring sequence (i.e., SEQ ID NO:4; fsp1). In other words, fsp2 is cross-reactive with naturally occurring mutTGFβR2. FIG. 11 shows the results of stimulating donors with a peptide mixture consisting only of SEQ ID NO:5 and SEQ ID NO:7 (i.e., fsp2 and fsp4), and for example, donor 3 showed an immune response against the peptide of SEQ ID NO:4 (i.e., fsp1; a fragment of naturally occurring mutTGFβR2) as well as SEQ ID NO:5 (i.e., fsp2).

[0126] The peptides of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 are also particularly useful as vaccines or treatments against cancer. In Figures 11 and 12, it is shown that T cells induced from PBMCs using a mixture of SEQ ID NO:5 (i.e., fsp2) and SEQ ID NO:7 (i.e., fsp4) are stimulated not only by SEQ ID NO:5 (i.e., fsp2), but also by SEQ ID NO:3 (i.e., fsp5), SEQ ID NO:4 (i.e., fsp1), and SEQ ID NO:7 (i.e., fsp4), which means that these peptides are also immunogenic and useful as vaccines or treatments against cancer. Furthermore, in Figure 13, it is shown that T cells induced from PBMCs using the peptide of SEQ ID NO:5 (i.e., fsp2) are stimulated by the peptide of SEQ ID NO:26 (i.e., fsp6), so fsp6 (SEQ ID NO:26) can also be used as a vaccine or treatment against cancer.

[0127] In view of FIG. 11, which shows that both fsp1 and fsp2 are immunogenic, FIG. 13 also shows that fsp6a (SEQ ID NO:28), which has a single C to G amino acid substitution compared to fsp6 (SEQ ID NO:26), is believed to be immunogenic. As mentioned above, fsp2 (SEQ ID NO:5) is able to induce an immune response against both itself and the corresponding naturally occurring sequence (SEQ ID NO:4, fsp1), indicating that the C to G amino acid substitution in fsp2 compared to fsp1 is immunologically acceptable. Therefore, the corresponding C to G amino acid substitution in fsp6 (as shown in fsp6a) is believed not to impair the immunogenicity of such modified peptides.

[0128] Also, in Figure 13, it is shown that the full immunogenic activity of fsp2 (SEQ ID NO:5) can be achieved by adding at least one amino acid at the C-terminus of fsp6 (SEQ ID NO:26) or fsp6a (SEQ ID NO:28) up to the full length of fsp2 (SEQ ID NO:5) (i.e. up to 7 additional amino acids at the C-terminus). Furthermore, in Figure 13, it is shown that fsp7 (SEQ ID NO:27) is too truncated at the N-terminus compared to fsp2 (SEQ ID NO:5) to be immunogenic. Therefore, in order for a peptide to be immunogenic against mutTGFβR2, it is necessary to include at least one additional amino acid at the N-terminus of fsp7 (SEQ ID NO:27).

[0129] Furthermore, as discussed above, Figure 13 shows that in light of the differences in immunogenicity of the amino acid sequences of SEQ ID NO:26 and SEQ ID NO:27, as well as the similarities and differences between these amino acid sequences, fsp1a (SEQ ID NO:29) and fsp1b (SEQ ID NO:30) are believed to be immunogenic.

[0130] The untransfected T cells, or the untransfected T cells in the T cell preparation or T cell mixture used for the treatment and / or prevention of cancer, can be of autologous or allogeneic origin, for example, xenogeneic T cells from a donor with the same or similar HLA repertoire as the patient can be administered to the patient.

[0131] The peptides, peptides for use, peptide mixtures, vectors, host cells, or pharmaceutical compositions of the invention can be administered to a subject by any suitable delivery technique known to those skilled in the art. For example, the peptides, peptide mixtures, or pharmaceutical compositions can be administered to a subject by injection in the form of a solution, in the form of liposomes, or in a dry form (e.g., coated particles, etc.), among other techniques. The host cells can be administered, for example, by blood transfusion. The vectors can be administered, for example, by subcutaneous injection or injection into a tumor. In some embodiments, the peptides, peptide mixtures, or pharmaceutical compositions can be administered, for example, once every 3 days, once a week, once a month, once every 3 months, once every 4 months, or once every 6 months, with an amount of 1 μg to 1 g of each peptide. In some embodiments, the net amount of each peptide per dose is 60 nM. For example, for intradermal injection, each peptide can be present in a volume of 0.1 ml at a concentration of 0.6 mM.

[0132] In some embodiments, the peptide or peptide mixture is administered with an adjuvant or an immune stimulant, such as GM-CSF. In embodiments using GM-CSF, the GM-CSF can be any GM-CSF, for example, glycosylated GM-CSF or non-glycosylated GM-CSF. GM-CSF is administered at a dose of 0.5 μg / m2 of body surface area. 2 ~120μg / m 2 , 1 μg / m 2 ~120μg / m 2 , 2 μg / m 2 ~115μg / m 2 , 3 μg / m 2 ~110μg / m 2 , 4 μg / m 2 ~105μg / m 2 , 5 μg / m 2 ~100μg / m 2 , 6 μg / m 2 ~95μg / m 2 , 7 μg / m 2 ~90μg / m 2 , 48 μg / m 2 ~85μg / m 2 , 9 μg / m2 ~80μg / m 2 , 10μg / m 2 ~75μg / m 2 , 11 μg / m 2 ~70μg / m 2 , 12 μg / m 2 ~65μg / m 2 , 13 μg / m 2 ~60μg / m 2 , 14 μg / m 2 ~55μg / m 2 , 15 μg / m 2 ~50μg / m 2 , 16 μg / m 2 ~45μg / m 2 , 17 μg / m 2 ~40μg / m 2 , or 18 μg / m 2 ~40μg / m 2 In some embodiments, GM-CSF is administered at a dose of 1 μg to 200 μg, 5 μg to 175 μg, 5 μg to 150 μg, 5 μg to 125 μg, 5 μg to 100 μg, 10 μg to 100 μg, 20 μg to 90 μg, 25 μg to 80 μg, 25 μg to 70 μg, 25 μg to 65 μg, or 30 μg to 60 μg per dose. In some embodiments, non-glycosylated GM-CSF is administered at a dose of 30 μg per dose. In other embodiments, glycosylated GM-CSF is administered at a dose of 60 μg per dose. In embodiments where GM-CSF is administered by intradermal injection, the dose can be 0.1 ml of a solution containing GM-CSF at a concentration of 0.3 mg / ml or 0.6 mg / ml. In some embodiments, the peptide, peptide mixture, or pharmaceutical composition can be administered, for example, once every 3 days, once every week, once every month, once every 3 months, once every 4 months, or once every 6 months in an amount of 1 μg to 1 g of each peptide.

[0133] The non-transfected T cells, non-transfected T cell mixtures, and non-transfected T cell preparations of the invention can be administered by intravenous injection and / or infusion, for example, once a month, once every two months, once every three months, once every six months, or once a year, with the amount of each non-transfected T cell specific for a peptide of the peptide mixture being 10 6 ~10 pieces 12 Preferably, the dosage is once a month for 2 to 5 months, and then less frequently thereafter.

[0134] The nucleic acids and nucleic acid mixtures of the present invention can be administered by intramuscular and / or subcutaneous injection.

[0135] Administration of the peptides, peptides for use, or peptide mixtures of the invention to a subject, or expression of the peptides or peptide mixtures by a subject, elicits an immune response to the peptides or peptide mixtures, in particular a T cell-mediated immune response. The peptides, or each peptide of the peptide mixture, can be processed by antigen-presenting cells (APCs) and presented on MHC molecules. αβ T cells are activated by T cell receptor binding to peptides presented by APCs on MHC molecules, which elicits an immune response against tumor cells bearing mutations corresponding to those present in the administered peptide(s). γδ T cells do not necessarily require antigen processing or presentation of antigens by MHC molecules.

[0136] In another aspect of the present invention, a peptide, a mixture of peptides, a non-transfected T cell, a non-transfected T cell preparation, a non-transfected T cell mixture, a nucleic acid molecule, or a pharmaceutical composition is provided for use in a method comprising diagnosing cancer and selecting an appropriate treatment. The method comprises the steps of (i) identifying whether a cancer patient has MSI-H and, if so, (ii) selecting a peptide, a mixture of peptides, a non-transfected T cell, a non-transfected T cell preparation, a non-transfected T cell mixture, a nucleic acid molecule, or a pharmaceutical composition according to the disclosure above. In some embodiments, the method further comprises in step (i) testing whether the patient has a frameshift mutation in the TGFβR2 protein and, if so, selecting a peptide, a mixture of peptides, a non-transfected T cell, a non-transfected T cell preparation, a non-transfected T cell mixture, a nucleic acid molecule, or a pharmaceutical composition according to the disclosure above. In some embodiments, the frameshift mutation is a -1a frameshift mutation. In some embodiments, the method further comprises (iii) administering to the patient the selected peptide, peptide mixture, T cell, T cell preparation, untransfected T cell mixture, nucleic acid molecule, or pharmaceutical composition. Since the peptides all correspond to the same protein, it is believed that any of the above disclosed peptides, peptide mixtures, untransfected T cells, untransfected T cell preparations, untransfected T cell mixtures, nucleic acid molecules, or pharmaceutical compositions can be used to treat cancers associated with MSI-H, in particular cancers associated with -1a frameshift mutations in TGFβR2.

[0137] In another aspect of the present invention, there is provided a method for treating and / or preventing cancer, comprising administering to a patient in need thereof a peptide, a mixture of peptides, a non-transfected T cell, a non-transfected T cell preparation, a non-transfected T cell mixture, a nucleic acid molecule, or a pharmaceutical composition according to the above disclosure. The method may comprise the steps of (i) identifying a patient with MSI-H cancer and (ii) administering to the patient a peptide, a mixture of peptides, a non-transfected T cell, a non-transfected T cell preparation, a non-transfected T cell mixture, a nucleic acid molecule, or a pharmaceutical composition according to the above disclosure. The method may further comprise the step of identifying in step (i) that the patient has a frameshift mutation in the TGFβR2 protein. In some embodiments, the frameshift mutation is a -1a frameshift mutation.

[0138] In another aspect of the invention, a kit is provided comprising a peptide, a peptide for use, a mixture of peptides, a non-transfected T cell, a non-transfected T cell mixture, a non-transfected T cell preparation, a nucleic acid molecule, a mixture of nucleic acid molecules, a vector and / or a host cell according to the disclosure above. The peptide, the peptide for use, the mixture of peptides, the non-transfected T cell, the non-transfected T cell mixture, a non-transfected T cell preparation, a nucleic acid, a mixture of nucleic acids, a vector and / or a host cell may be present in the kit itself or the peptide, the peptide for use, the mixture of peptides, the non-transfected T cell, the non-transfected T cell mixture, a non-transfected T cell preparation, a nucleic acid molecule, a mixture of nucleic acids, a vector and / or a host cell may be present as a pharmaceutical formulation. In some embodiments, the peptides, peptides for use, peptide mixtures, untransfected T cells, untransfected T cell preparations, untransfected T cell mixtures, nucleic acid molecule mixtures, vectors, and / or host cells may be contained in, for example, vials, bottles, flasks, which may be further packaged, for example, in boxes, envelopes, or bags. In some embodiments, the kits include peptide mixtures, untransfected T cell mixtures, and / or nucleic acid molecule mixtures, where the peptides, untransfected T cells, and / or nucleic acid molecules are provided in separate containers such that the peptides, untransfected T cells, and / or nucleic acid molecules are mixed by the user. EXAMPLES

[0139] Example 1 Previous studies on peptides to be used as vaccines against cancers associated with TGFbR2 with frameshift mutations have shown that at least some peptides may be immunogenic, but the results were conflicting. Therefore, consensus peptides were manually predicted based on peptides tested in previous studies.

[0140] [Table 1]

[0141] Predicted consensus sequence: SPKCIMKEKKSLVRLSSCVPVALMSAMTTSSSQ (SEQ ID NO: 16).

[0142] Example 2 An online algorithm (i.e., SYFPEITHI) was then used to predict epitopes of mutTGFβR2 for HLA class II alleles. The HLA class II alleles included in this study were HLA-DRB1 * 0101, HLA-DRB1 * 0301(DR17), HLA-DRB1 * 0401(DR4Dw4), HLA-DRB1 * 0701, HLA-DRB1 * 1101, HLA-DRB1 * The result was 1501 (DR2b).

[0143] A cutoff prediction score of 20 was used, where HLA binding strength increases with prediction score. SYFPEITHI yielded the following predicted epitopes:

[0144] [Table 2]

[0145] As a result, SYFPEITHI predicted the consensus sequence to be KCIMKEKKSLVRLSSCVPVALMSAMTTSSSQKN (SEQ ID NO: 3).

[0146] Example 3 The consensus sequence of Example 1 was compared with the consensus sequence of Example 2 to prepare an optimized consensus sequence.

[0147] [Table 3]

[0148] Example 4 The optimized consensus peptide (SEQ ID NO:3) was modified to overcome the predicted difficulties in synthetic production and use as a vaccine. In particular, the optimized consensus sequence (SEQ ID NO:3) has a length of 33 amino acids, and peptides of this length are difficult to synthetically produce with adequate quality and yield. Two cysteine ​​residues in the same peptide cause problems in peptide stability and quality due to cyclization of the peptide by the formation of intramolecular and intermolecular disulfide bonds. This cyclization of the peptide may also reduce the immunological effectiveness of the peptide, for example, by impairing effective antigen processing. This may lead to processing of unrelated T cell epitopes. In addition, cyclization of the peptide may induce undesired inflammatory side reactions, such as antibody formation or allergic reactions. In addition, the eight amino acids at the N-terminus of the optimized consensus sequence (SEQ ID NO:3) correspond to wild-type TGFβR2, so there is a risk of activating wild-type cross-reactive T cells. As a result, to overcome these problems, the optimized consensus sequence (SEQ ID NO:3) was further modified to design the peptides shown in Table 4. How the designed peptides relate to each other and to the optimized predicted consensus sequence shown in Table 3 is shown in FIG.

[0149] [Table 4]

[0150] Example 5 i) Synthesis of peptides A batch of fsp5 (SEQ ID NO:3) was prepared by solid phase peptide synthesis (SPPS) using FMOC chemistry and a Prelude synthesizer (Gyros Protein Technologies Inc., USA). The crude peptide was analyzed by UPLC and MS. The desired peptide was observed by MS, which showed a purity of about 75% in UPLC, as shown in Figure 2. UPLC system: Column; Acquity UPLC BEH C18 1.7 mm, 21 mm x 150 mm, Detection; PDA 210 nm to 500 nm, Solvent A; 0.1% TFA in water, Solvent B; 0.1% TFA in MeCN, Gradient: B 20% to 70% (2 min to 10 min, linear).

[0151] ii) Peptide solubility and purification The peptide was difficult to dissolve, and attempts were made to dissolve crude fsp5 (SEQ ID NO:3) (approximately 150 mg) in approximately 4 ml of 50% MeCN in water under gentle heating. However, no clear solution was obtained. In this situation, attempts were made to purify the crude solution by preparative HPLC (15% -> 40% MeCN in water) after filtering the crude suspension. This resulted in a large loss of material. Only small amounts of the desired peptide were obtained, and analysis by UPLC showed that the peptide was impure.

[0152] Another batch of fsp5 (SEQ ID NO:3) was prepared, dissolved in pure DMSO under gentle heating, and purified by preparative HPLC. The purity of the peptide was measured by UPLC and found to be about 90%, as shown in Figure 3. Also, only small amounts of purified fsp5 (SEQ ID NO:3) were obtained. It is important to note that the addition of even small amounts of water caused the peptide to precipitate in the crude DMSO solution, so it is highly likely that fsp5 (SEQ ID NO:3) precipitated on the column during HPLC purification.

[0153] iii) Peptide purity and stability The purity of a small amount of purified fsp5 (SEQ ID NO:3) was re-evaluated by UPLC after lyophilization, which showed a drop in purity from approximately 90% to less than 50%, as shown in Figure 4. A similar trend was observed when the purity of crude fsp5 (SEQ ID NO:3) was re-evaluated after short-term storage (3 days) at room temperature followed by lyophilization, as shown in Figure 5.

[0154] In summary, Example 5 shows that fsp5 (SEQ ID NO:3) can be synthesized but is extremely difficult to produce in sufficient quantities for any practical purpose and in the quality required for medical use, e.g., as a component of a potential cancer vaccine.

[0155] Example 6 Peptides fsp1 (SEQ ID NO: 4), fsp2 (SEQ ID NO: 5), fsp3 (SEQ ID NO: 6), and fsp4 (SEQ ID NO: 7) were synthesized using SPPS as described above and purified by HPLC. After the peptides were purified, they were lyophilized. The purity of each peptide was measured by UPLC, and the UPLC traces are shown in Figures 6 to 9. UPLC system: Column; Acquity UPLC BEH C18 1.7 mm, 21 mm x 150 mm, Detection; PDA 210 nm to 500 nm, Solvent A; 0.1% TFA in water, Solvent B; 0.1% TFA in MeCN, Gradient for fsp1 and fsp2: B 5% to 50% (0 min to 10 min, linear), Gradient for fsp3 and fsp4: B 20% to 70% (2 min to 10 min, linear). In FIG. 6 it is shown that the purity obtained for purified and lyophilized fsp1 (SEQ ID NO: 4) was 96%. In FIG. 7 it is shown that the purity obtained for purified and lyophilized fsp2 (SEQ ID NO:5) was 97%. In FIG. 8 it is shown that the purity obtained for purified and lyophilized fsp3 (SEQ ID NO:6) was 94.8%. In FIG. 9 it is shown that the purity obtained for purified and lyophilized fsp4 (SEQ ID NO: 7) was 91.9%. ESI-QTOF-MS confirmed the correct molecular weight (MW) for all four peptides (Table 5, Example 7).

[0156] In summary, Example 6 shows that peptides fsp1 (SEQ ID NO: 4), fsp2 (SEQ ID NO: 5), fsp3 (SEQ ID NO: 6), and fsp4 (SEQ ID NO: 7) can be produced without the chemical problems seen in the case of fsp5 (SEQ ID NO: 3), and therefore it is feasible to produce these shorter peptides for potential use as vaccines to induce peptide-specific T cells.

[0157] Example 7 Each peptide in Table 4 was tested for immunogenicity. i) Material

[0158] [Table 5]

[0159] Fresh buffy coats from four healthy donors were obtained from a blood bank.

[0160] ii) In vitro PBMC priming and T cell proliferation Day 1: PBMCs: Count peripheral blood mononuclear cells (PBMCs) isolated from fresh buffy coats from four healthy donors and incubate in DC medium at 15 × 10 6 Suspend at 4 x 10 cells / ml in DC medium. 6 The cells were diluted to 100 cells / ml (Table 6).

[0161] [Table 6]

[0162] In vitro stimulation: 4 x 10 per well 6Four 24-well plates were used for each donor, with each well having a volume of 1 ml.

[0163] Peptide cocktail: a solution containing peptides fsp2 and fsp4 at 10 μM each. 40 μl of the peptide solution was added to each well.

[0164] The plates were incubated in a cell incubator (37°C, 5% CO) for 14 days. 2 ) and incubated for 12 h at 4°C. IL-2 and IL-7 were added on day 3. Cells were examined daily.

[0165] Day 14 - T cell expansion: Test peptides: fsp2+fsp4 (SEQ ID NO:5 and SEQ ID NO:7), fsp1 (SEQ ID NO:4), fsp2 (SEQ ID NO:5), fsp3 (SEQ ID NO:6), fsp4 (SEQ ID NO:7), fsp5 (SEQ ID NO:3). Positive control: SEC3 Negative controls: T cells, T cells + APC (no test peptide added) Mock: DMSO in PBS

[0166] Plate setup for T cell expansion: Cells and reagents were added to wells of the plate (in triplicate) as described below in Tables 7 and 8. The total volume added to each well was 0.20 ml. T cells (PBMC): 50,000 cells / well (0.25 × 10 6 cells / ml) APC: irradiated (30 Gy, 8 min) Autologous feeder cells: 50,000 cells / well Peptides (fspx): 0.2 nmol peptide / well (each peptide), concentration: 10 μM peptide. (fsp was dissolved in DMSO before dilution to correct the concentration)

[0167] [Table 7]

[0168] [Table 8]

[0169] The plates were incubated in a cell incubator (37°C, 5% CO) for 48 h. 2 ) incubated.

[0170] Day 16 After 48 hours of incubation, 20 μl of 3H-thymidine solution (5 μCi / ml) was added to each well, and the plate was incubated for approximately 17 hours (37° C., 5% CO 2 ) incubated.

[0171] Day 17 After 17 hours, cells were harvested (Unifilter) and dried on the filter until completely dry at 45° C. After covering the bottom of the Unifilter with an adhesive cover (provided with the Unifilter), 25 μl of microscintillation fluid was added to each well, the plate was covered with a TopSeal, and 3H-thymidine incorporation was measured as counts per minute (CPM) using a microplate scintillation counter.

[0172] Breeding Results: The T cell proliferation results after in vitro priming with a cocktail of fsp2 and fsp4 peptides are shown in Figure 10 as stimulation index (SI). SI is the mean CPM (triplicate) of (T cells + APC + test peptide(s)) divided by the mean CPM (triplicate) of (T cells + APC). An SI > 2 is clearly immunogenic.

[0173] iii) In vitro restimulation of PBMCs and T cell proliferation PBMCs harvested after in vitro priming were cultured at 2 × 10 per well. 6 The individual cells were then restimulated in vitro according to the protocol described above. T cell proliferation was tested according to the protocol described above.

[0174] Breeding Results: The T cell proliferation results after the second in vitro stimulation with a cocktail of fsp2 and fsp4 peptides are shown in FIG. 11 as stimulation index (SI).

[0175] Thus, in Figures 10 and 11, it is shown that peptides fsp2 and fsp4 with amino acid substitutions are immunogenic and can activate T cells, and that the activated T cells are cross-reactive to peptides of the naturally occurring -1a frameshifted TGFβ2 protein. Also, in Figures 10 to 12, it is shown that peptides fsp1 and fsp5 of the naturally occurring -1a frameshifted TGFβ2 protein stimulated T cells derived from PMBCs and are therefore immunogenic. As a result, the modified peptide fsp2 (SEQ ID NO: 5) and peptide fsp4 (SEQ ID NO: 7) can be used to stimulate induction of T cells specific for the TGFβR2 frameshift mutant, as can the unmodified peptide fsp1 (SEQ ID NO: 4) and peptide fsp5 (SEQ ID NO: 3).

[0176] Example 8 The immunogenicity of fsp6 and fsp7 (SEQ ID NO:26 and SEQ ID NO:27, respectively) was tested using the same protocol as described in Example 7, except for the peptide mixture used to induce T cells from PMBCs obtained from fresh buffy coats from four healthy donors. The peptide mixture used to induce T cells from PMBCs in this example consisted of fsp2 (SEQ ID NO:5; 10 μM), fsp8 (SEQ ID NO:31; 10 μM), and fsp9 (SEQ ID NO:32; 10 μM). fsp8 (SEQ ID NO:31; RNRIPAVLRTEGEPLHTPSVGMRET) is a peptide of ASTE1 with a -1a frameshift mutation, and fsp9 (SEQ ID NO:32; KTILKKAGIGMCVKVSSIFFINKQK) is a peptide of TAF1β with a -1a frameshift mutation.

[0177] On day 14 after induction, T cell proliferation assays were performed as in Example 7 using fsp2 (SEQ ID NO:5), fsp6 (SEQ ID NO:26), and fsp7 (SEQ ID NO:27) as test peptides. Since fsp8 and fsp9 have amino acid sequences that are significantly different from fsp2, it is believed that only fsp2 can induce T cells that can recognize and be stimulated by fsp2, fsp6, and fsp7. In particular, as shown in FIG. 11, fsp6 and fsp7 are truncated versions of fsp2. Two more T cell stimulations were also performed, and T cell proliferation assays were performed 14 days after each stimulation.

[0178] The results of T cell proliferation after the first stimulation in vitro are shown as stimulation index (SI) in FIG. 13. In particular, FIG. 13 shows that fsp6 can activate T cells, and therefore fsp6 is immunogenic and can be used as a vaccine or treatment for cancer. In addition, FIG. 13 also shows that T cells induced by fsp2 (SEQ ID NO: 5) are cross-reactive to a peptide of naturally occurring -1a frameshift protein shorter than fsp2 (SEQ ID NO: 5), i.e., fsp6 (SEQ ID NO: 26). In addition, in view of the results shown in FIG. 11 and FIG. 12, it is considered that a peptide having the sequence of fsp6 (SEQ ID NO: 26) but the corresponding amino acid substitution in fsp2, particularly a C to G amino acid substitution similar to fsp2, is also immunogenic and useful as a vaccine or treatment for cancer. In particular, as described in Example 7, FIG. 11 shows that T cells induced by fsp2 (SEQ ID NO: 5) with a C to G amino acid substitution are cross-reactive to a peptide of naturally occurring -1a frameshift protein, and therefore the C to G amino acid substitution is immunologically acceptable. Thus, fsp6a (SEQ ID NO:28), which is identical to fsp6 (SEQ ID NO:26) except that it has the same C to G amino acid substitution as fsp2 (SEQ ID NO:5), is also believed to be immunogenic.

[0179] Furthermore, in FIG. 13, fsp6 (SEQ ID NO:26) is shown to be less immunogenic than fsp2 (SEQ ID NO:5), but both fsp6 and fsp2 are much more immunogenic than fsp7 (SEQ ID NO:27). fsp6 has the same amino acid sequence as fsp2, except that it is truncated at the C-terminus compared to fsp2 and does not have the C to G amino acid substitution. fsp7 has the same amino acid sequence as fsp2, but is truncated at the N-terminus compared to fsp2. In view of the differences in immunogenicity between these three peptides shown in FIG. 13, it is believed that adding at least one additional amino acid at the C-terminus of fsp6 up to the full length of fsp2 will provide the same immunogenic activity as fsp2. It is also believed that at least one additional amino acid at the N-terminus of fsp7 is necessary to increase its immunogenicity and for the peptide to contain an immunologically effective epitope. As a result, the 9-mer peptide LVRLSSCVP (fsp1a; SEQ ID NO: 29) is likely to be immunogenic because it contains a sequence shared by fsp6 and fsp7 and has one additional amino acid at the C-terminus compared to fsp6 and one additional amino acid at the N-terminus compared to fsp7. Furthermore, because the C to G amino acid substitution was shown to be immunologically acceptable, the 9-mer peptide (fsp1b; SEQ ID NO: 30) with a C to G amino acid substitution is also likely to be immunogenic. Section 1. A peptide comprising a fragment of SEQ ID NO:8, the fragment comprising at least 8 consecutive amino acids of SEQ ID NO:8, including at least one of positions 121 and 135 of SEQ ID NO:8, and the peptide being capable of inducing an immune response against a TGFβR2-1a frameshift mutant protein. Section 2. Item 2. The peptide according to item 1, wherein the fragment comprises at least 9, preferably at least 12, consecutive amino acids of SEQ ID NO:8, including at least one of positions 121 and 135 of SEQ ID NO:8. Section 3. Item 3. The peptide according to item 1 or 2, wherein the fragment comprises only one of positions 121 and 135 of SEQ ID NO:8. Section 4. Item 4. The peptide according to any one of Items 1 to 3, wherein the fragment comprises positions 121 to 132 of SEQ ID NO:8, positions 129 to 137 of SEQ ID NO:8, or positions 135 to 146 of SEQ ID NO:8. Section 5. Item 5. The peptide according to any one of Items 1 to 4, wherein the amino acid corresponding to position 121 or position 135 of SEQ ID NO: 8 is glycine. Section 6. The peptide according to any one of items 1 to 5, wherein the peptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:28, or SEQ ID NO:30, and preferably the peptide consists only of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:28, or SEQ ID NO:30. Section 7. 7. The peptide according to any one of items 1 to 6, wherein the peptide comprises 33 or fewer amino acids, preferably 24 or fewer amino acids, preferably 20 or fewer amino acids, preferably 17 or fewer amino acids, or preferably 9 or fewer amino acids. Section 8. A peptide comprising a fragment of SEQ ID NO:2, the fragment comprising: i) the peptide comprises positions 6 to 13 of SEQ ID NO: 3, and the peptide comprises 21 or fewer amino acids; ii) comprising positions 7 to 22 of SEQ ID NO: 3, and the peptide comprises 40 or fewer amino acids; or iii) comprising positions 18 to 33 of SEQ ID NO: 3, and the peptide comprises 33 or fewer amino acids; A peptide, which is capable of inducing an immune response against a TGFβR2-1a frameshift mutant. Section 9. Item 9. The peptide according to Item 8, wherein the fragment comprises positions 6 to 15 of SEQ ID NO: 3, positions 2 to 22 of SEQ ID NO: 3, or positions 16 to 33 of SEQ ID NO: 3, and preferably the peptide comprises the sequence of SEQ ID NO: 3. Section 10. 10. The peptide according to item 8 or 9, wherein the peptide comprises the sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 26, preferably the peptide consists only of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 26. Section 11. The peptide is i) comprising positions 6 to 13 of SEQ ID NO: 3 and comprising no more than 20 amino acids, or preferably no more than 17 amino acids; or ii) comprising positions 7 to 22 of SEQ ID NO: 3 and comprising 33 or fewer amino acids, preferably 24 or fewer amino acids, preferably 20 or fewer amino acids, or preferably 17 or fewer amino acids; or iii) The peptide according to any one of items 8 to 10, which comprises positions 18 to 33 of SEQ ID NO: 3 and comprises 24 or less amino acids, or preferably 20 or less amino acids. Section 12. A peptide comprising a fragment of SEQ ID NO:2 for use in the treatment and / or prevention of cancer, said fragment comprising the amino acid sequence of SEQ ID NO:29, said peptide comprising 21 or fewer amino acids, and said peptide being capable of inducing an immune response against TGFβR2-1a frameshift mutant, preferably said cancer being colon cancer or gastric cancer. Section 13. 13. The peptide of claim 12, wherein the peptide comprises 17 or fewer amino acids. Section 14. 14. A nucleic acid molecule encoding the peptide according to any one of items 1 to 13. Section 15. A peptide mixture comprising a first peptide and a second peptide, wherein the first peptide and the second peptide are independently a peptide according to any one of items 1 to 13, and the first peptide is different from the second peptide. Section 16. A vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding the peptide according to any one of items 1 to 13 or the peptide mixture according to item 15. Section 17. A host cell comprising the vector according to item 16. Section 18. 14. An untransfected T cell specific for the peptide according to any one of items 1 to 13. Section 19. A T cell mixture comprising non-transfected T cells specific for the peptide mixture described in item 15. Section 20. A pharmaceutical composition comprising the peptide according to any one of items 1 to 13, the nucleic acid molecule according to item 14, the peptide mixture according to item 15, the vector according to item 16, the host cell according to item 17, the untransfected T cell according to item 18, or the T cell mixture according to item 19, and a pharma- ceutically acceptable carrier, diluent, or excipient. Section 21. The peptide according to any one of Items 1 to 11, the nucleic acid molecule according to Item 14, the peptide mixture according to Item 15, the vector according to Item 16, the host cell according to Item 17, the T cell according to Item 18, the T cell mixture according to Item 19, or the pharmaceutical composition according to Item 20, for use in the treatment and / or prevention of cancer, preferably colon cancer or gastric cancer. Section 22. 1. A method for selecting a peptide, a nucleic acid molecule, a vector, a host cell, a mixture of peptides, a T cell, a mixture of T cells, or a pharmaceutical composition for administration to a patient, comprising: i) to identify whether a cancer patient has a frameshift mutation in the TGFβR2 protein, and if so, ii) selecting the peptide according to any one of items 1 to 10, the peptide according to item 12 or 13, the nucleic acid molecule according to item 14, the peptide mixture according to item 15, the vector according to item 16, the host cell according to item 17, the T cell according to item 18, the T cell mixture according to item 19, or the pharmaceutical composition according to item 20.

Claims

1. A peptide comprising a fragment of SEQ ID NO:8, the fragment comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:28, the peptide comprising 33 or fewer amino acids, the peptide having at least 70% sequence identity to SEQ ID NO:2 outside the fragment, and the peptide capable of inducing an immune response against a TGFβR2-1a frameshift mutant protein.

2. The peptide of claim 1 , wherein the peptide has 100% sequence identity to SEQ ID NO:2 outside of the fragment.

3. 3. The peptide of claim 1 or 2, wherein the peptide comprises no more than 24, preferably no more than 20, more preferably no more than 17 amino acids.

4. The peptide according to any one of claims 1 to 3, wherein the peptide consists solely of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:

28.

5. A nucleic acid molecule encoding the peptide according to any one of claims 1 to 4.

6. A peptide mixture comprising a first peptide and a second peptide, wherein the first peptide and the second peptide are independently a peptide according to any one of claims 1 to 4, and the first peptide is different from the second peptide.

7. A vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding the peptide according to any one of claims 1 to 4 or the peptide mixture according to claim 6.

8. A host cell comprising the vector of claim 7.

9. A non-transfected T cell specific for a peptide according to any one of claims 1 to 4.

10. A T cell mixture comprising non-transfected T cells specific for the peptide mixture of claim 6.

11. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, a peptide mixture according to claim 6, a vector according to claim 7, a host cell according to claim 8, a non-transfected T cell according to claim 9, or a T cell mixture according to claim 10, together with a pharma- ceutically acceptable carrier, diluent or excipient.

12. A peptide according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, a peptide mixture according to claim 6, a vector according to claim 7, a host cell according to claim 8, a T cell according to claim 9, a T cell mixture according to claim 10 or a pharmaceutical composition according to claim 11 for use in the treatment and / or prevention of cancer, preferably colon cancer or gastric cancer.

13. 1. A method for selecting a peptide, a nucleic acid molecule, a vector, a host cell, a mixture of peptides, a T cell, a mixture of T cells, or a pharmaceutical composition for administration to a patient, comprising: i) identifying whether a cancer patient has a frameshift mutation in the TGFβR2 protein, and if so, ii) selecting a peptide according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, a mixture of peptides according to claim 6, a vector according to claim 7, a host cell according to claim 8, a T cell according to claim 9, a mixture of T cells according to claim 10, or a pharmaceutical composition according to claim 11.

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