Peptides and antigen-binding proteins for use in immunotherapy for fibrous hepatocellular carcinoma (FL-HCC) and other cancers.

Abstract

The present invention provides peptides, antigen-binding proteins, nucleic acids, and cells for use in immunotherapy methods. [Solution] The present invention provides a peptide comprising an amino acid sequence selected from the group consisting of amino acid sequences shown in a plurality of specific sequences and variant sequences having at least 88% homology with the plurality of specific sequences, or a pharmaceutically acceptable salt thereof, wherein the variant sequence induces T cells that bind to and / or cross-react with major histocompatibility complex (MHC) molecules, and the peptide is not a full-length polypeptide.

Description

Technical Field

[0001] The present invention relates to peptides, antigen-binding proteins, nucleic acids and cells for use in immunotherapy methods. Specifically, the present invention relates to cancer immunotherapy, particularly immunotherapy for fibrolamellar hepatocellular carcinoma (FL-HCC). Further, the present invention can be used, for example, as an active pharmaceutical ingredient of a vaccine composition that stimulates an antitumor immune response, or relates to tumor-associated T cell peptide epitopes and recombinant T cell receptors that can stimulate ex vivo T cells and transfer them to a patient.

[0002] The present invention relates to the field of molecular biology, and more specifically, to the field of molecular immunology.

Background Art

[0003] Cancer is the second leading cause of death worldwide, accounting for an estimated 9.6 million deaths, or one in six, in 2018. Worldwide, one in five men and one in six women will develop cancer at some point in their lives, and one in eight men and one in eleven women will die from cancer. Worldwide, the total number of people surviving within five years after cancer diagnosis (referred to as the 5-year prevalence rate) is estimated to be 43.8 million. In men, lung cancer, prostate cancer, colorectal cancer, gastric cancer and liver cancer are most commonly seen, and in women, breast cancer, colorectal cancer, lung cancer, cervical cancer and thyroid cancer are most commonly seen.

[0004] The increasing burden of cancer is due to several factors such as population growth and aging, as well as the main causes of cancer that change in relation to social and economic development. The change in the causes of cancer is particularly applicable in situations where rapid economic growth is seen, and with rapid economic growth, there is a shift from cancers related to poverty and infectious diseases to cancers related to lifestyle habits more typical in industrialized countries.

[0005] Human fibrous lamellar hepatocellular carcinoma (FL-HCC) is a rare cancer accounting for less than 5% of all liver cancers. It is primarily seen in children and young adults and is unique in that it does not show signs of fibrosis or cirrhosis. FL-HCC universally has a deletion of approximately 400 kb on chromosome 19, which leads to the formation of an in-frame fusion of DNAJ heat shock protein family member B1 (DNAJB1) and the cAMP-activating catalytic subunit α (PRKACA) of protein kinase. DNAJB1 encodes a subunit of the heat shock factor 46 (HSP40) complex. HSP40 activates the ATPase of HSP70 and acts as a molecular chaperone that can be induced by various environmental stresses. PRKACA encodes a catalytic subunit of protein kinase A (PKA). This catalytic subunit (PKA Cα), together with PKA Cβ and two regulatory subunits, forms an inactive tetrameric complex and exists in the cytoplasm as the PKA holoenzyme. When the G protein-coupled receptor is activated, the catalytic subunit of PKA is activated in a cAMP-dependent manner, leading to phosphorylation of various types of cellular substrates. The crystal structure of the DNAJB1-PRKACA fusion protein shows that the catalytic site, regulatory subunit binding site, and anchor protein binding site are similar to those of wild-type PRKACA.

[0006] Little is known about tumorigenesis in FL-HCC, other than the presence of a DNAJB1-PRKACA fusion. While other reports have indicated significant recurrent mutations and widespread copy number variations, these have not been associated with any known oncogenes or tumor suppressors. Unlike liver cancer in the elderly, FL-HCC is not associated with known disease risk factors such as alcoholism, chronic hepatitis infection, or liver fluke infection.

[0007] FL-HCC is a type of cancer that often presents with no symptoms until the tumor is quite large, meaning it is frequently advanced by the time it is diagnosed. Symptoms include vague abdominal pain, nausea, abdominal distension, fatigue, and weight loss. Additionally, palpable hepatomegaly may be present. Other symptoms include jaundice, ascites, fulminant hepatic failure, encephalopathy, gynecomastia (males only), thrombophlebitis of the lower extremities, recurrent deep vein thrombosis, anemia, and hypoglycemia. While early onset and the absence of chronic liver disease suggest FL-HCC, misdiagnosis is common because classical hepatocellular carcinoma can also occur in younger patients.

[0008] Currently, invasive surgery is the only treatment for FL-HCC. Because the recurrence rate of FL-HCC is very high, two or more liver resections may be necessary. Due to these recurrences, regular follow-up with medical imaging (CT or MRI) is required. Because FL-HCC is a very rare disease, there is no standard chemotherapy regimen. While radiotherapy has been used in the past, there is limited data on its use. The survival rate for FL-HCC depends heavily on the presence and extent of cancer metastasis, i.e., its spread to lymph nodes and other organs. If distal spread (metastasis) occurs, the median survival rate drops significantly. The 5-year survival rate also fluctuates between 40% and 90%.

[0009] The DNAJB1-PRKACA fusion has also been detected in various other gastrointestinal tumors, such as pancreaticobiliary eosinophilic tumors.

[0010] T-cell immunotherapies, including immune checkpoint inhibitors (ICIs), CAR-T cells, adoptive T-cell transfer, and vaccination, have achieved breakthroughs in the treatment of malignant diseases. However, these therapies rely on the recognition of tumor antigens and the elimination of cancer cells through T-cell-mediated cytotoxicity, and are only effectively used in a limited number of cancer patients and single tumors. One of the main specific reasons for developing antigen-specific immunotherapies is the lack of suitable target structures that are naturally and exclusively presented on the surface of tumor cells at high frequency and recognized by the immune system. Tumor antigens are presented by HLA-independent surface molecules or by intracellular protein-derived T-cell epitopes presented by HLA class I or HLA class II molecules. Regarding HLA molecule-mediated presentation of tumor antigens, neoepitopes resulting from tumor-specific mutations have recently been identified, as neoepitopes exhibiting primary specificity in anti-cancer T-cell responses induced by immune checkpoint inhibitors, suggesting them to be the most promising candidates for T-cell immunotherapy. Simultaneously, responses to immune checkpoint inhibitors have been shown to correlate with high somatic mutational loads caused by tumors, and neoepitope-based immunotherapy has shown promising results for the first time in individual tumor patients. However, patient / tumor specificity and intratumoral heterogeneity of somatic mutations, as well as the limited number of somatic mutations that are translated, processed, and presented as HLA-restricted neoepitopes on tumor cells, limit the broad application of tumor antigens, especially in patients with low mutational loads.

[0011] Furthermore, in recent years, fusion transcripts that translate into products that often function as clonal tumorigenetic drivers have been identified as a new source of highly immunogenic neoepitopes. T cell responses to neoepitopes derived from such fusion proteins have been detected in patients treated with immune checkpoint inhibitors, and a correlation with treatment response has been observed.

[0012] The DNAJB1-PRKACA fusion transcript is formed by linking exon 1 (DNAJB1) of the DnaJ homolog subfamily B member 1 gene to the cAMP-dependent protein kinase catalytic subunit α gene (PRKACA). In FL-HCC, the DNAJB1-PRKACA fusion transcript was detectable in 100% of patients, and expression of this fusion transcript was shown in all tumor cells, leading to its identification as a tumorigenetic driver in the pathogenesis of tumors.

[0013] Immunotherapy for FL-HCC using peptides is disclosed in WO2020 / 257575 and WO2021 / 138582.

[0014] However, none of these methods have been approved, nor have they achieved widespread clinical application or efficacy. One possible reason for this is that the peptides reported in the aforementioned literature are not very suitable for inducing FL-HCC-specific immune responses in patients.

[0015] Therefore, a fundamental objective of the present invention is to provide tumor-associated T-cell peptide epitopes that can be used in the development of improved pharmaceuticals and methods for the diagnosis, prevention, and treatment of cancer. More specifically, the cancers are those having a fusion of DNAJ heat shock protein family member B1 (DNAJB1) and the protein kinase cAMP-activating catalytic subunit α (PRKACA) (DNAJB1-PRKACA), such as fibrous lamellar hepatocellular carcinoma (FL-HCC).

[0016] This invention satisfies these and other needs. [Overview of the Initiative] [Means for solving the problem]

[0017] The present invention provides a peptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 1-5 and variant sequences having at least 88% homology to SEQ ID NOs: 1-5, or a pharmaceutically acceptable salt thereof, wherein the variant sequence induces T cells that bind to and / or cross-react with major histocompatibility complex (MHC) molecules, and the peptide is not a full-length polypeptide. [Modes for carrying out the invention]

[0018] The inventors identified and characterized the peptides of the present invention as neoantigens derived from DNAJB1-PRKACA fusion transcripts, which are tumorigenetic drivers in fibrous lamellar hepatocellular carcinoma (FL-HCC) and numerous other gastrointestinal tumor diseases. The peptide of SEQ ID NO: 1 and / or the peptide of SEQ ID NO: 2 have a universal MHC type (human: HLA allotype class II) and can be appropriately administered to any target, including humans. The peptides of SEQ ID NOs: 3 to 5 are all particularly suitable for administration to targets with appropriate MHC types (human: SEQ ID NO: 3 = HLA allotype A*68:02; SEQ ID NO: 4 = HLA allotype C*04:01; C*05:01; SEQ ID NO: 5 = HLA allotype A*24:02).

[0019] The inventors of the present invention have found that the neoantigen of the present invention is a multifunctional cytotoxic CD8, which is necessary for effective anticancer therapy. + T cells and T helper (Th)1 CD4 + We were able to demonstrate that it induces T cells. Furthermore, by performing immunopeptide-metabolism analysis by mass spectrometry, we were able to prove that the neoantigen of the present invention is presented through natural processing in DNAJB1-PRKACA-expressing tumor cells. In addition, within the framework of individual curative experiments, the inventors found that by inoculating FL-HCC patients who experienced repeated relapses with a vaccine containing the peptide of the present invention, they were able to induce an activated Th1 phenotype and a multifunctional CD4 with high TCR clonality. +We were able to demonstrate that T cells can be induced. The DNAJB1-PRKACA-specific T cell response induced by the vaccine is maintained over a long period and can extend the relapse-free survival period of patients after vaccination to more than one year. Therefore, the peptide of the present invention is suitable as an active agent in pharmaceutical compositions (especially vaccines) for the treatment and / or prevention of cancers such as FL-HCC.

[0020] The peptide, HLA allotype, and their respective sequence numbers according to the present invention are shown in the table below. [Table 1]

[0021] In the event of any discrepancy between the sequences shown in Table 1 and the sequences shown in the sequence listing, the information in the sequence listing shall take precedence and shall apply.

[0022] In this specification, "peptide" usually refers to a sequence of amino acid residues linked together by peptide bonds between the α-amino groups and carbonyl groups of adjacent amino acids. Peptides are preferably 7 to 12 amino acid long, more preferably 8 to 11 amino acid long, but may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 amino acid long, or longer.

[0023] Furthermore, the term "peptide" typically includes a salt of a sequence of amino acid residues linked together by peptide bonds between the α-amino groups and carbonyl groups of adjacent amino acids. This salt is preferably a pharmaceutically acceptable salt of the peptide, such as a chloride salt or an acetate salt (trifluoroacetate). It should be noted that since the peptide of the present invention cannot exist as a salt in vivo, the salt of the peptide of the present invention is in a significantly different state from the peptide of the present invention in vivo.

[0024] Furthermore, the term “peptide” also includes “oligopeptides.” In this specification, “oligopeptide” typically refers to a sequence of amino acid residues linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not important in this invention, as long as one or more exact epitopes are preserved. The length of an oligopeptide is typically less than about 30 amino acid residues and longer than about 15 amino acid residues.

[0025] Furthermore, the term "peptide" also includes "polypeptide." A "polypeptide" typically refers to a sequence of amino acid residues linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not important in this invention, as long as the precise epitopes are preserved. Unlike the terms "peptide" and "oligopeptide," a "polypeptide" refers to a molecule containing more than approximately 30 amino acid residues.

[0026] The major histocompatibility complex (MHC) is a set of cell surface proteins essential to the adaptive immune system, recognizing foreign molecules and determining histocompatibility in vertebrates. The main function of MHC molecules is to bind to antigens derived from pathogens, present them on the cell surface, and allow them to be recognized by appropriate T cells. Human MHC is also called the HLA (human leukocyte antigen) complex (or simply "HLA"). The MHC gene family is classified into three subgroups: Class I, Class II, and Class III. The complex, consisting of peptides and MHC Class I molecules, has an appropriate T cell receptor (TCR) for CD8 + While recognized by T cells, the complex consisting of a peptide and an MHC class II molecule is recognized by CD4 cells with the appropriate TCR. + It is recognized by helper T cells. Since both CD8-dependent and CD4-dependent responses work together synergistically to contribute to antiviral activity, the identification and characterization of viral antigens and their corresponding T cell receptors are important for the development of viral immunotherapies such as vaccines and cell therapies. The HLA-A gene is located on the short arm of chromosome 6 and codes for the large α chain that is a component of HLA-A. Polymorphism of the α chain of HLA-A is important for HLA function. This polymorphism promotes genetic diversity in the population. Since each HLA has a different affinity for peptides of a specific structure, higher HLA diversity means that there are more types of antigens that can be "presented" on the cell surface.

[0027] According to the present inventors, a "variant" of an amino acid sequence means that, for example, the side chains of one or two amino acid residues are modified (for example, by substitution with the side chains of another native amino acid residue or other side chains), but the peptide can still bind to an MHC molecule in substantially the same manner as the peptide consisting of the amino acid sequence of SEQ ID NOs: 1-5. For example, the peptide may be recombinant such that its ability to interact with and bind to a suitable MHC molecule such as HLA-A*02 is at least maintained (if not improved), and its ability to bind to the TCR of activated T cells is at least maintained (if not improved).

[0028] The original (unmodified) peptides disclosed herein can be recombined by substituting one or more residues at various sites within the peptide chain, unless otherwise specified, or by selecting one or more residues from various sites. The substitution is preferably located at the end of the amino acid chain. Such substitutions may be conservative; for example, one amino acid may be substituted with another amino acid of similar structure and properties, such as a hydrophobic amino acid being substituted with another hydrophobic amino acid. For even greater conservation, substitutions may be made between amino acids of the same size and chemical properties, or between amino acids of similar size and chemical properties, such as leucine being substituted with isoleucine. Studies of sequence variations in naturally homologous protein families have shown that certain amino acid substitutions are often more tolerable than others, and such substitutions often correlate with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and the substituted amino acid; for this reason, they are defined as "conservative substitutions." In this specification, "conservative substitution" is defined as the exchange of an amino acid within one of five groups: Group 1 - small nonpolar or slightly polar aliphatic residues (Ala, Ser, Thr, Pro, GIY); Group 2 - polar negative-charged residues and their amides (Asp, Asn, Glu, Gln); Group 3 - polar positive-charged residues (His, Arg, Lys); Group 4 - large nonpolar aliphatic residues (Met, Leu, Ile, Val, Cys); and Group 5 - large aromatic residues (Phe, Tyr, Trp). Substitutions with low conservation may involve one amino acid being replaced by another amino acid with similar properties but slightly different size, such as the substitution of alanine with an isoleucine residue. Substitutions with very low conservation may involve polar amino acids being replaced by acidic amino acids, or even basic amino acids being replaced by acidic amino acids.However, such "extreme" substitutions cannot be dismissed on the grounds that they may be ineffective, because chemical reactions cannot be fully predicted, and extreme substitutions can sometimes lead to accidental new discoveries that cannot be predicted from simple chemical principles. Furthermore, it goes without saying that such substitutions may include structures other than common L-amino acids. In other words, L-amino acids commonly found in the antigenic peptides of the present invention may be substituted with D-amino acids, and such amino acids are also included in this disclosure. Moreover, non-standard amino acids (i.e., amino acids other than the common amino acids that make up natural proteins) may be used for substitution to produce the immunogens and immunogenic polypeptides of the present invention.

[0029] If a peptide with substantially equivalent or higher antigenic activity than that defined below is obtained by substitution of two or more positions, a test will be conducted to investigate whether the combination of substitutions produced an additive or synergistic effect on the peptide's antigenicity. The number of positions simultaneously substituted within the peptide should be no more than four.

[0030] Amino acid residues that do not substantially contribute to the interaction with the T cell receptor can be recombined by substituting them with other amino acids that do not substantially affect T cell responsiveness and do not cause the loss of binding to the relevant MHC.

[0031] Long (extended) peptides may be preferable. MHC class I epitopes are typically 8-11 amino acids long, but they can also be produced by peptide processing from longer peptides or proteins containing the actual epitope. It is preferable that the residues adjacent to the actual epitope do not substantially affect the proteolytic cleavage required to expose the epitope during the processing process.

[0032] The peptide of the present invention can be elongated by up to 4 amino acid residues, meaning that 1, 2, 3, or 4 amino acids can be added to one or both ends, and the number of amino acids added to each end can be any combination in the range of 4:0 to 0:4. The elongation combinations according to the present invention are shown in Table 2. [Table 2]

[0033] The amino acids used for elongation may be derived from the original sequence peptide of the protein disclosed herein, or they may be other amino acids. This elongation can improve the stability or solubility of the peptide of the present invention.

[0034] Therefore, the epitopes of the present invention may be the same as natural tumor-associated epitopes or natural tumor-specific epitopes, and may include epitopes that differ from the reference peptide by four or fewer residues, as long as they have substantially the same antigenic activity.

[0035] In another embodiment, the peptide of the present invention is elongated by the addition of five or more amino acids to one or both ends, and its total length is preferably up to 30 amino acids. This elongation may yield a peptide that binds to MHC class II molecules. Binding to MHC class II can be tested by methods known in the art.

[0036] Accordingly, the present invention provides MHC class I epitope peptides and their variants, the total length of which the peptide or variant is 8 to 100 amino acids, preferably 8 to 30 amino acids, most preferably 8 to 14 amino acids, i.e., most preferably 8, 9, 10, 11, 12, 13, or 14 amino acids, and if the peptide is an extended class II molecule-binding peptide, its total length may be 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids.

[0037] It goes without saying that the peptide or variant of the present invention has the ability to bind to human major histocompatibility complex (MHC) class I molecules or MHC class II molecules. The binding of the peptide or variant to the MHC complex may be tested by methods known in the art.

[0038] When testing peptide-specific T cells of the present invention against a substituted peptide, the peptide concentration at which half of the maximum increase in solubility relative to the background is achieved by the substituted peptide is preferably about 1 mM or less, preferably about 1 μM or less, more preferably about 1 nM or less, even more preferably about 100 pM or less, and most preferably about 10 pM or less. The substituted peptide is preferably recognized by T cells from two or more individuals, preferably by T cells from at least two individuals, and more preferably by T cells from three individuals.

[0039] Those skilled in the art can evaluate whether T cells induced by a specific peptide variant can cross-react with the original peptide itself (Appay et al., 2006; Colombetti et al., Eur. J. Immunol. 36: 1805-1814 (2006); Fong et al., Proc. Natl. Acad. Sci. USA 98: 8809-8814 (2001); Zaremba et al., Cancer Res. 57: 4570-4577 (1997)).

[0040] Next, such T cells can substantially cross-react with and kill cells expressing polypeptides containing the native amino acid sequence of the cognitive peptide as defined in the embodiments of the present invention. As can be seen from scientific literature and database information (Rammensee et al., Immunogenetics 50: 213-219 (1999); Godkin et al., Int. Immunol 9: 905-911 (1997)), specific positions in HLA-binding peptides typically contain anchor residues that form a core sequence that fits into the binding motif of the HLA receptor, and these anchor residues are determined by the polarity, electrophysical properties, hydrophobicity, and spatial properties of the polypeptide chain constituting the binding groove. Therefore, those skilled in the art can recombine the amino acid sequences shown in SEQ ID NOs: 1-5 while maintaining known anchor residues and verify whether such variants retain the ability to bind to MHC class I or MHC class II molecules. Because the variants of the present invention retain the ability to bind to the TCR of activated T cells, they can substantially cross-react with and kill cells expressing polypeptides containing the native amino acid sequence of a cognitive peptide as defined in aspects of the present invention.

[0041] In this invention, "homology" refers to the degree of identity between two amino acid sequences (i.e., peptide sequences or polypeptide sequences). This "homology" is determined by aligning and comparing the two sequences under optimal conditions for comparison. Such sequence homology can be calculated by creating an alignment, for example, using the ClustalW algorithm. Generally available sequence analysis software, more specifically Vector NTI, GENETYX, or other tools, are provided through public databases.

[0042] Therefore, when the terms “homologousity” or “percentage of homology” are used in relation to sequences, these terms mean that the sequence being compared ("comparison sequence") is aligned with the sequence described herein or in the claims ("reference sequence"), and then compared with the sequence described herein or in the claims. The homology (also known as the percentage of identity) is then determined according to the following formula: Identity (%)=100×[1-(C / R)] In the formula, C is the number of differences between the reference sequence and the comparison sequence in the alignment length between the reference sequence and the comparison sequence, and this difference is (i) Each base or amino acid in the reference sequence that does not have a corresponding base or amino acid that is aligned to the sequence to be compared, (ii) Each gap in the reference sequence, and (iii) Composed of each aligned base or amino acid of a reference sequence that is different from the aligned base or amino acid of the sequence being compared, (iv) Alignment must start from the first element of the sequence being aligned. In the formula, R represents the number of bases or amino acids in the reference sequence relative to the alignment length with the sequence being compared. Gaps created in the reference sequence are also counted as bases or amino acids.

[0043] If there is an alignment between the comparison sequence and the reference sequence in which the percentage of identity calculated by the method described above is approximately equal to or greater than the minimum value of the predetermined percentage of identity, then the comparison sequence has the minimum value of the predetermined percentage of identity with respect to the reference sequence, even if there is another alignment in which the percentage of identity calculated by the method described above is less than the predetermined percentage of identity.

[0044] Methods for comparing the identity / homology of two or more sequences are known in the art. For example, the "needle" program (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453), which uses the Needleman-Wunsch global alignment algorithm, can be used to find the optimal alignment (including gaps) of two sequences, taking the entire sequence length into consideration. The needle program is available on, for example, 30 worldwide websites and is described in more detail in the following publication (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). According to this disclosure, the degree of identity between two polypeptides is calculated using the EMBOSS needle(global) program and the Blosum62 matrix, with the "gap open" parameter set to 10.0 and the "gap extension" parameter set to 35-0.5.

[0045] A variant sequence having "at least 88% homology" refers to a sequence that, in its entire length, has at least approximately 88% or more sequence identity with the entire length of the reference sequence, in particular at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% sequence identity.

[0046] A protein having an amino acid sequence that is "at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical" to a reference sequence may contain amino acid mutations such as deletions, insertions, and / or substitutions compared to the reference sequence.

[0047] The major histocompatibility complex (MHC) is a set of cell surface proteins essential to the adaptive immune system, recognizing foreign molecules and determining histocompatibility in vertebrates. The main function of MHC molecules is to bind to antigens derived from pathogens, present them on the cell surface, and allow them to be recognized by appropriate T cells. Human MHC is also called the HLA (human leukocyte antigen) complex (or simply "HLA"). The MHC gene family is classified into three subgroups: Class I, Class II, and Class III. The complex, consisting of peptides and MHC Class I molecules, has an appropriate T cell receptor (TCR) for CD8 + While recognized by T cells, the complex consisting of a peptide and an MHC class II molecule is recognized by CD4 cells with the appropriate TCR. + It is recognized by helper T cells. Since both CD8-dependent and CD4-dependent responses work together synergistically to contribute to antiviral activity, the identification and characterization of viral antigens and their corresponding T cell receptors are important for the development of viral immunotherapies such as vaccines and cell therapies. The HLA-A gene is located on the short arm of chromosome 6 and codes for the large α chain that is a component of HLA-A. Polymorphism of the α chain of HLA-A is important for HLA function. This polymorphism promotes genetic diversity in the population. Since each HLA has a different affinity for peptides of a specific structure, higher HLA diversity means that there are more types of antigens that can be "presented" on the cell surface.

[0048] "Pharmacologically" or "pharmaceutically acceptable" means a molecular species and composition (or salt) that, when administered appropriately to mammals (especially humans), does not cause adverse effects, allergic reactions, or other undesirable reactions, and does not interfere with the activity of the active pharmaceutical ingredient.

[0049] According to the present invention, “full-length polypeptide” refers to the original protein from which the peptide of the present invention (e.g., DNAJB1-PRKACA fusion transcript) is derived. The “full-length polypeptide” is also called the “derived gene / derived protein” from which the peptide of the present invention is derived. The derived protein or full-length polypeptide may or may not be highly overexpressed in cancer compared to normal tissue. In relation to the present invention, “normal tissue” means healthy peripheral blood mononuclear cells (PBMCs) or other normal tissue cells from which the gene from which the peptide of the present invention is derived shows high tumor relevance. Furthermore, the peptide of the present invention itself is presented in tumor tissue. In relation to the present invention, “tumor tissue” means a sample obtained from a patient suffering from cancer such as fibrous lamellar hepatocellular carcinoma (FL-HCC) or other gastrointestinal cancers.

[0050] In one embodiment of the present invention, the peptide or nucleic acid of the present invention is isolated. "Isolated" means that a material has been removed from the environment in which it originally existed (for example, if it is a natural material, it means it has been removed from the natural environment). For example, a natural polynucleotide or polypeptide present in the body of a living animal is not isolated, but if this polynucleotide or polypeptide is separated from a part or all of the material that coexists with it in nature, then this polynucleotide or polypeptide is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition is isolated in that it is not part of the natural environment.

[0051] Furthermore, the peptides and / or nucleotides disclosed in this invention may be in a “purified” form. “Purified” is intended to be a relative definition and does not necessarily have to be absolute purity, and may include highly purified or partially purified preparations, as the term is understood by those skilled in the art in the relevant field. For example, it is customary to purify individual clones isolated from a cDNA library to ensure uniformity in electrophoresis. It is explicitly assumed that the starting material or natural material is purified at least 10-fold, preferably 100-fold or 1,000-fold, and more preferably 10,000-fold or 100,000-fold. Furthermore, polypeptides described in the claims having a purity of preferably 99.999% by weight, or at least 99.99% by weight or 99.9% by weight, and more preferably 99% by weight or more, are explicitly included in this invention.

[0052] The peptides and nucleic acids of the present invention may be in a “concentrated form.” In this specification, “concentrated” means that the concentration of the material is at least about 2 times, about 5 times, about 10 times, about 100 times, or about 1000 times its natural concentration, advantageously 0.01% by weight, and preferably at least about 0.1% by weight. Furthermore, concentrated preparations of about 0.5% by weight, about 1% by weight, about 5% by weight, about 10% by weight, or about 20% by weight are envisioned. Sequences, constructs, vectors, clones, and other materials comprising the present invention may be advantageous in a concentrated or isolated form.

[0053] The fundamental problems underlying the present invention can be completely resolved by the disclosure herein.

[0054] In one particularly preferred embodiment of the present invention, the peptide of the present invention consists of or substantially consists of the amino acid sequences shown in SEQ ID NOs: 1 to 5.

[0055] "Substantially comprising" means that the peptide of the present invention includes, in addition to the sequence or variant thereof shown in any of SEQ ID NOs: 1 to 5, an additional amino acid sequence at the N-terminus and / or C-terminus that is not necessarily required for the formation of the peptide portion that functions as an epitope of the MHC molecular epitope.

[0056] However, such additional sequences may be important for efficiently introducing the peptide of the present invention into cells. In one embodiment of the present invention, the peptide of the present invention is, for example, part of a fusion protein containing an N-terminal amino acid or a C-terminal amino acid. In another fusion, the peptide of the present invention can be fused to an antibody or its functional portion, as described herein, more specifically, to the sequence of an antibody to become a specific target of that antibody, or to a dendritic cell-specific antibody, as described herein, for example.

[0057] Furthermore, the peptide or variant of the present invention may be modified to improve stability and / or binding to MHC molecules for the purpose of inducing a stronger immune response. Methods for optimizing such peptide sequences are well known in the art and include, for example, the introduction of reverse peptide bonds or non-peptide bonds.

[0058] In one embodiment of the present invention, the peptide has the ability to bind to MHC class I molecules or MHC class II molecules, and when bound to these MHC molecules, it becomes recognizable by CD4 T cells and / or CD8 T cells.

[0059] This method has the advantage of ensuring that the immune response-inducing ability (particularly the T-cell response-inducing ability) of the peptide of the present invention is reliably exhibited.

[0060] In another embodiment of the present invention, the amino acid sequence of the peptide or a variant of the present invention includes a continuous sequence of amino acids shown in any of SEQ ID NOs: 1 to 5.

[0061] This method has the advantage that the peptide or variant of the present invention can contain any amino acid that is expected to be involved in inducing an immune response. This further improves therapeutic efficacy.

[0062] Another subject of the present invention relates to an antibody that specifically recognizes the peptide or variant of the present invention, particularly a soluble antibody or a membrane-bound antibody, more preferably a monoclonal antibody or a fragment thereof, which preferably specifically recognizes the peptide or variant of the present invention when the peptide or variant is bound to an MHC molecule.

[0063] In this specification, the term “antibody” is used in a broad sense and includes both polyclonal and monoclonal antibodies. The term “antibody” includes not only intact immunoglobulin molecules and “full-length” immunoglobulin molecules, but also fragments (e.g., CDRs, Fv fragments, Fab fragments, and Fc fragments) or polymers of such immunoglobulin molecules, as well as humanized forms of such immunoglobulin molecules, insofar as they exhibit any of the desired properties, i.e., specifically recognize the peptide or variant of the present invention. If possible, the antibodies of the present invention may be purchased from a distributor. Alternatively, the antibodies of the present invention may be manufactured using known methods.

[0064] The features, properties, advantages, and embodiments disclosed for the peptides of the present invention also apply to the aforementioned antibodies and their fragments.

[0065] Another subject of the present invention relates to a T cell receptor or fragment thereof that reacts to an HLA ligand which is the peptide or a variant of the present invention, preferably a soluble T cell receptor or a membrane-bound T cell receptor, and preferably a T cell receptor that reacts to the peptide or a variant of the present invention when the peptide or a variant of the present invention is bound to an MHC molecule.

[0066] The "T cell receptor" (abbreviated as TCR) of the present invention refers to a heterodimer molecule containing an α-polypeptide chain (α-chain) and a β-polypeptide chain (β-chain), and this heterodimer receptor can bind to peptide antigens presented by HLA molecules. The "T cell receptor" also includes so-called γδTCRs.

[0067] The features, properties, advantages, and embodiments disclosed for the peptides and antibodies or fragments thereof of the present invention also apply to the aforementioned T cell receptors.

[0068] Another subject of the present invention is an antigen-binding protein that specifically binds to at least one DNAJB1-PRKACA antigenic peptide that forms a complex with a major histocompatibility complex (MHC) molecule, The antigen-binding protein comprises a first polypeptide chain containing a first variable domain including CDR3α as at least one complementarity-determining region (CDR), and a second polypeptide chain containing a second variable domain including CDR3β as at least one CDR. a) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 6, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 36, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; b) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 7, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 37, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; c) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 8, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 38, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; d) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 9, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 39, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; e) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 10, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 40, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; f) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 11, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 41, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; g) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 12, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 42, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; h) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 13, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 43, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; i) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 14, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 44, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; j) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 15, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 45, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; k) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 32, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 45, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; l) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 16, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 46, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; m) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 17, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 47, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; n) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 33, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 47, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; o) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 18, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 48, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; p) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 19, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 49, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; q) Whether CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 19, whether CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 62, and whether the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; r) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 20, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 50, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; s) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 21, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 51, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; t) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 21, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 63, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; u) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 22, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 52, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; v) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 23, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 53, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; w) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 24, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 54, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; x) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 25, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 55, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; y) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 34, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 55, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; z) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 26, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 56, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; α) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 27, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 57, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; β) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 28, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 58, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; γ) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 29, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 59, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; δ)CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 30, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 60, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; or ε)CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 31, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 61, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; The CDR sequence may contain one or more amino acid mutations selected from amino acid insertions, deletions, and / or substitutions. Regarding antigen-binding proteins.

[0069] According to the present invention, the “antigen-binding protein” as described herein refers to at least one DNAJB1-PRKACA antigenic peptide, in particular a polypeptide or binding protein that can bind to the peptide provided herein.

[0070] According to the present invention, "CDR" means "complementarity-determining region." A CDR is a part of the variable chain of an immunoglobulin (antibody) produced by a B cell, or a part of the variable chain of a T cell receptor produced by a T cell, and binds to a specific antigen. Three discontinuously arranged CDRs (CDR1, CDR2, and CDR3) exist on the amino acid sequence of the variable domain of the antigen receptor. The CDR of the T cell receptor (TCR) exists on two different protein chains. In humans, in 95% of T cells, the TCR consists of an α chain and a β chain (encoded by TRA and TRB, respectively), and in 5% of T cells, the TCR consists of a γ chain and a δ chain (γ / δ) (encoded by TRG and TRD, respectively). With respect to the antigen-binding protein of the present invention, CDR3α and CDR3β represent protein chains that constitute part of the complementarity-determining region 3 of the α chain and β chain of the native TCR corresponding to the antigen-binding protein of the present invention.

[0071] The inventors have identified and successfully sequenced several TCRs from DNAJB1-PRKACA-specific T cells induced in vitro or in vivo that can specifically bind to the peptide of the present invention, particularly the peptide containing the amino acid sequence shown in SEQ ID NO: 1 or 5. The features shown in a) to ε) indicate combinations of amino acid sequences of the CDR3α variable chain and CDR3β variable chain of recombinant antigen-binding proteins (e.g., recombinant TCRs) that can specifically bind to the DNAJB1-PRKACA antigenic peptide containing the amino acid sequence of SEQ ID NO: 1 or 5.

[0072] The antigen-binding proteins provided by the present inventors are listed in Table 3 below. [Table 3-1] [Table 3-2] [Table 3-3]

[0073] In the event of any discrepancy between the sequences shown in Table 3 and those shown in the sequence listing, the information in the sequence listing shall take precedence and shall apply.

[0074] In one embodiment of the antigen-binding protein, the CDR sequence is interpreted as containing one or more amino acid mutations selected from amino acid insertions, deletions, and / or substitutions. Preferably, such mutations do not affect, or have a significant effect on, the specific binding to the DNAJB1-PRKACA antigenic peptide. More preferably, such mutations are conservative mutations, for example, one amino acid being replaced by another amino acid of similar structure and properties, or, for example, a hydrophobic amino acid being replaced by another hydrophobic amino acid. Refer to the aforementioned disclosures relating to peptide modification and substitution of the present invention, which also apply mutatis mutandis to the antigen-binding protein of the present invention.

[0075] In the context of this specification, "one or more" means one, two, three, four, five, or any other amino acid mutation.

[0076] The features, properties, advantages, and embodiments disclosed for the peptides of the present invention are also applicable to the aforementioned antigen-binding proteins.

[0077] In one embodiment of the present invention, the antigen-binding protein is a single-chain TCR (scTCR) or a bispecific single-chain antibody, and / or the first polypeptide further comprises a constant domain of an α chain or a constant domain of a γ chain, the second polypeptide further comprises a constant domain of a β chain or a constant domain of a δ chain, and / or the first variable domain is part of a TCRα chain or a TCRγ chain, and / or the second variable domain is part of a TCRβ chain or a TCRδ chain, and / or the first variable domain is a variable domain of TCRα, the second variable domain is a variable domain of TCRβ, and / or the antigen-binding protein contains at least one amino acid mutation that increases stability, cell surface expression and / or pairing.

[0078] In this specification, "single-chain TCR (scTCR)" refers to a protein in which the variable domains of a TCR, such as Vα and Vβ, or Vδ and Vγ, are arranged on a single polypeptide chain. Typically, these variable domains are separated by a linker, which usually contains 5 to 20 amino acids, for example, 5 to 15 amino acids.

[0079] "Bispecific antibodies" refer to formats including, but are not limited to, diabody, cross-over-dual-variable-domain (CODV), and / or bivariable-domain (DVD) proteins. An overview of these various bispecific antibodies and their manufacturing methods is described, for example, in Brinkmann U. and Kontermann EE MAbs. 2017 Feb-Mar; 9(2): 182-212. In particular, the DVD format is disclosed in the following scientific publications, for example: (Wu C et al. Nat Biotechnol 2007; 25:1290-7; PMID:17934452; Wu C. et al. MAbs 2009; 1:339-47; Lacy SE et al. MAbs 2015; 7:605-19; PMID:25764208; Craig RB et al. PLoS One 2012; 7:e46778; 25 PMID:23056448; Piccione EC et al. MAbs 2015). CODV is disclosed, for example, in Onuoha SC et al. Arthritis Rheumatol. 2015 Oct; 67(10):2661-72, and, for example, in WO2012 / 135345 and WO2016 / 116626.Examples of bispecific diaboves include Holliger P et al. Protein Eng 1996; 9:299-305; PMID:8736497; Atwell JL et al. Mol Immunol 1996; 33:1301-12; PMID:9171890; Kontermann RE, Nat Biotechnol 1997; 15:629-31; 30 PMID:9219263; Kontermann RE et al. Immunotechnology 1997; 3:137-44; PMID:9237098; Cochlovius B et al. Cancer Res 2000; 60:4336-41; PMID:10969772; and DeNardo DG et al. Cancer Biother Radiopharm 2001; 16:525-35; It is listed under PMID:11789029.

[0080] The constant domain sequence of the antigen-binding protein may be recombined, for example, by introducing a heterologous sequence (more preferably a mouse sequence) that can improve expression, pairing, and stability. Known mutations from the prior art (e.g., WO2018 / 104407, PCT / EP2018 / 069151, WO2011 / 044186, WO2014 / 018863, EP2432802B1, and WO2020157211A) may be introduced, for example, by substituting undesirable amino acids in the variable domain and / or introducing disulfide crosslinks between constant domains and removing unpaired cysteine.

[0081] In one embodiment of the present invention, the antigen-binding protein is (i) One or more additional antigen-binding sites; (ii) Transmembrane regions within the first polypeptide chain and / or the second polypeptide chain, which may include an intracellular signaling region; (iii) Diagnostic agents; (iv) Therapeutic agents; and (v)PK adjustment part It further includes one or more of the following.

[0082] The "transmembrane region" may be, for example, the transmembrane domain of TCRα or TCRβ.

[0083] The "cytoplasmic signaling region" is, for example, the intracellular domain of TCRα or TCRβ.

[0084] In this specification, “diagnostic agent” means a detectable molecule or detectable substance, such as a fluorescent molecule, a radioactive molecule, or any other label known in the art that generates a signal (directly or indirectly).

[0085] In this specification, “therapeutic agent” means a drug having a therapeutic effect. In one embodiment, such therapeutic agent may be a cytotoxic agent or a cell proliferation inhibitor, other anticancer agents, and / or a drug that induces an immune response that enhances the body’s ability to fight cancer.

[0086] In this specification, “PK modulating portion” refers to the portion that modulates the pharmacokinetics (PK) of the antigen-binding protein described herein. Therefore, the PK modulating portion modulates, in particular, the in vivo half-life and distribution of the antigen-binding protein disclosed herein. In one embodiment, the PK modulating portion extends the half-life of the antigen-binding protein. Examples of PK regulatory regions include, but are not limited to, PEG (Dozier et al., 10 (2015) Int J Mol Sci. Oct 28; 16(10):25831-64 and Jevsevar et al., (2010) Biotechnol J.Jan; 5(1):113-28), PASylation (Schlapschy et al., (2013) Protein Eng Des Sel. Aug;26(8):489-501), albumin (Dennis et al., (2002) J Biol Chem. Sep 20; 277(38):35035-43), and the Fc portion of antibodies and / or non-structural polypeptides (Schellenberger et al., (2009) Nat Biotechnol. Dec; 27(12):1186-90).

[0087] In one embodiment of the present invention, the antigen-binding protein is a therapeutic antigen-binding protein and / or a diagnostic antigen-binding protein.

[0088] Another subject of the present invention is an isolated nucleic acid molecule comprising a nucleotide sequence encoding the peptide or variant thereof of the present invention, the antibody or fragment thereof of the present invention, the T cell receptor of the present invention, or the antigen-binding protein of the present invention.

[0089] The nucleic acid encoding a particular peptide, oligopeptide, or polypeptide may be a native nucleic acid or a synthetically constructed nucleic acid. The nucleic acid (e.g., polynucleotide) may be, for example, DNA, cDNA, PNA, RNA, or a combination thereof, may be single-stranded and / or double-stranded, may be a native polynucleotide or a stabilized form of polynucleotide, may be, for example, a polynucleotide having a phosphorothioate skeleton, may contain or may not contain introns, as long as the peptide of the present invention is encoded. Naturally, a polynucleotide can only encode peptides containing native amino acid residues linked by native peptide bonds. Yet another aspect of the present invention provides an expression vector capable of expressing the peptide of the present invention.

[0090] In this specification, a nucleic acid molecule or nucleotide sequence that "encodes" a peptide or the like refers to a nucleotide sequence encoding a peptide that includes artificial (man-made) start and stop codons suitable for a biological system, and a sequence expressed by, for example, dendritic cells or another cell system useful for TCR production. The coding sequence is controlled by a nucleic acid region (e.g., DNA) that is involved in the binding of RNA polymerase to initiate transcription, such as a "promoter." Therefore, the isolated nucleic acid molecule of the present invention may contain regulatory factors such as promoters and enhancers.

[0091] "Isolated" has the meaning defined above.

[0092] In one embodiment of the present invention, the isolated nucleic acid molecule of the present invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 64 to 119.

[0093] Another subject of the present invention relates to a cloning vector or expression vector comprising an isolated nucleic acid molecule of the present invention.

[0094] Another subject of the present invention is a cloning vector or expression vector encoding an antigen-binding protein that specifically binds to at least one DNAJB1-PRKACA antigenic peptide that forms a complex with a major histocompatibility complex (MHC) molecule, The molecule includes, under the control of a promoter, a first nucleotide sequence encoding a first variable domain containing CDR3α as at least one complementarity-determining region (CDR), and a second nucleotide sequence encoding a second variable domain containing CDR3β as at least one CDR. a) The first nucleotide sequence includes or consists of SEQ ID NO: 64, the second nucleotide sequence includes or consists of SEQ ID NO: 94, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; b) The first nucleotide sequence includes or consists of SEQ ID NO: 65, the second nucleotide sequence includes or consists of SEQ ID NO: 95, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; c) The first nucleotide sequence includes or consists of SEQ ID NO: 66, the second nucleotide sequence includes or consists of SEQ ID NO: 96, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; d) The first nucleotide sequence includes or consists of SEQ ID NO: 67, the second nucleotide sequence includes or consists of SEQ ID NO: 97, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; e) The first nucleotide sequence includes or consists of SEQ ID NO: 68, the second nucleotide sequence includes or consists of SEQ ID NO: 98, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; f) The first nucleotide sequence includes or consists of SEQ ID NO: 69, the second nucleotide sequence includes or consists of SEQ ID NO: 99, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; g) The first nucleotide sequence includes or consists of SEQ ID NO: 70, the second nucleotide sequence includes or consists of SEQ ID NO: 100, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; h) The first nucleotide sequence includes or consists of SEQ ID NO: 71, the second nucleotide sequence includes or consists of SEQ ID NO: 101, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; i) The first nucleotide sequence includes or consists of SEQ ID NO: 72, the second nucleotide sequence includes or consists of SEQ ID NO: 102, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; j) The first nucleotide sequence includes or consists of SEQ ID NO: 73, the second nucleotide sequence includes or consists of SEQ ID NO: 103, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; k) The first nucleotide sequence includes or consists of SEQ ID NO: 90, the second nucleotide sequence includes or consists of SEQ ID NO: 103, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; l) The first nucleotide sequence includes or consists of SEQ ID NO: 74, the second nucleotide sequence includes or consists of SEQ ID NO: 104, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; m) The first nucleotide sequence includes or consists of SEQ ID NO: 75, the second nucleotide sequence includes or consists of SEQ ID NO: 105, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; n) The first nucleotide sequence includes or consists of SEQ ID NO: 91, the second nucleotide sequence includes or consists of SEQ ID NO: 105, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; o) The first nucleotide sequence includes or consists of SEQ ID NO: 76, the second nucleotide sequence includes or consists of SEQ ID NO: 106, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; p) The first nucleotide sequence includes or consists of SEQ ID NO: 77, the second nucleotide sequence includes or consists of SEQ ID NO: 107, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; q) The first nucleotide sequence includes or consists of SEQ ID NO: 77, the second nucleotide sequence includes or consists of SEQ ID NO: 120, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; r) The first nucleotide sequence includes or consists of SEQ ID NO: 78, the second nucleotide sequence includes or consists of SEQ ID NO: 108, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; s) The first nucleotide sequence includes or consists of SEQ ID NO: 79, the second nucleotide sequence includes or consists of SEQ ID NO: 109, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; t) The first nucleotide sequence includes or consists of SEQ ID NO: 79, the second nucleotide sequence includes or consists of SEQ ID NO: 121, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; u) The first nucleotide sequence includes or consists of SEQ ID NO: 80, the second nucleotide sequence includes or consists of SEQ ID NO: 110, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; v) The first nucleotide sequence includes or consists of SEQ ID NO: 81, the second nucleotide sequence includes or consists of SEQ ID NO: 111, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; w) The first nucleotide sequence includes or consists of SEQ ID NO: 82, the second nucleotide sequence includes or consists of SEQ ID NO: 112, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; x) The first nucleotide sequence includes or consists of SEQ ID NO: 83, the second nucleotide sequence includes or consists of SEQ ID NO: 113, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; y) The first nucleotide sequence includes or consists of SEQ ID NO: 84, the second nucleotide sequence includes or consists of SEQ ID NO: 114, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; z) The first nucleotide sequence includes or consists of SEQ ID NO: 93, the second nucleotide sequence includes or consists of SEQ ID NO: 114, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; α) The first nucleotide sequence includes or consists of SEQ ID NO: 85, the second nucleotide sequence includes or consists of SEQ ID NO: 115, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; β) The first nucleotide sequence includes or consists of SEQ ID NO: 86, the second nucleotide sequence includes or consists of SEQ ID NO: 116, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; γ) The first nucleotide sequence includes or consists of SEQ ID NO: 87, the second nucleotide sequence includes or consists of SEQ ID NO: 117, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; δ) The first nucleotide sequence includes or consists of SEQ ID NO: 88, the second nucleotide sequence includes or consists of SEQ ID NO: 118, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; or ε) The first nucleotide sequence includes or consists of SEQ ID NO: 89, the second nucleotide sequence includes or consists of SEQ ID NO: 119, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; The first nucleotide sequence and / or the second nucleotide sequence may contain one or more nucleic acid mutations selected from nucleotide insertions, deletions, and / or substitutions. Regarding cloning vectors or expression vectors.

[0095] The cloning vector or expression vector of the present invention is useful as a basic tool for producing specific antigen-binding proteins of the present invention that can specifically bind to the peptides of the present invention, particularly peptides containing the amino acid sequence shown in SEQ ID NO: 1 or 5. Further features, as shown in a) to ε), represent combinations of nucleotide sequences encoding the CDR3α and CDR3β variable chains of an antigen-binding protein (e.g., recombinant TCR) that is recombinant to specifically bind to the DNAJB1-PRKACA antigenic peptide containing the amino acid sequence of SEQ ID NO: 1 or 5.

[0096] In the context of this specification, "one or more" means one, two, three, four, five, six, seven, eight, nine, ten, and so on, nucleic acid mutations.

[0097] The cloning vectors or expression vectors provided by the present inventors are listed in Table 3 above.

[0098] The features, properties, advantages, and embodiments disclosed for the peptides of the present invention also apply to the aforementioned nucleic acid molecules and cloning or expression vectors.

[0099] Another subject of the present invention relates to recombinant host cells comprising the peptide of the present invention, the antibody or fragment thereof, the T cell receptor of the present invention, the antigen-binding protein of the present invention, or an isolated nucleic acid molecule or vector of the present invention, preferably selected from mammalian or human cells, and more preferably selected from antigen-presenting cells such as dendritic cells, T cells or NK cells.

[0100] The features, properties, advantages, and embodiments disclosed for the peptides of the present invention are also applicable to the aforementioned host cells.

[0101] A further subject of the present invention relates to an in vitro method for producing activated T cells, comprising the step of contacting T cells in vitro with an antigen-carrying human MHC class I molecule or an antigen-carrying human MHC class II molecule expressed on the surface of a suitable antigen-presenting cell or on the surface of an artificial structure that mimics an antigen-presenting cell for a time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is the peptide of the present invention.

[0102] Activated T cells targeting the peptide of the present invention are useful in therapeutic methods. Therefore, a further aspect of the present invention provides activated T cells obtained by the method of the present invention.

[0103] The activated T cells produced by the above method selectively recognize cells that abnormally express polypeptides containing the amino acid sequences of SEQ ID NOs. 1 to 5.

[0104] The features, properties, advantages, and embodiments disclosed for the peptides of the present invention also apply to the methods described above.

[0105] Another subject of the present invention relates to a pharmaceutical composition comprising at least one active ingredient selected from the group consisting of the peptide or variant thereof of the present invention, the antibody or fragment thereof of the present invention, the T cell receptor of the present invention, the antigen-binding protein of the present invention, the isolated nucleic acid molecule of the present invention, the vector of the present invention, the recombinant host cell of the present invention, the activated T cell of the present invention, and a labeled or labeled active ingredient, and a pharmaceutically acceptable carrier, which may also comprise pharmaceutically acceptable excipients and / or stabilizers.

[0106] A "pharmaceutical composition" is a composition suitable for administration to humans in a medical setting. The pharmaceutical composition is preferably sterilized and preferably manufactured in accordance with GMP guidelines.

[0107] The pharmaceutical compositions of the present invention comprise the peptide of the present invention in free form or in the form of a pharmaceutically acceptable salt (see also above). In this specification, “pharmaceutically acceptable salt” means a derivative of the peptide of the present disclosure modified by forming a salt of the peptide of the present disclosure with an acid or a base. For example, a salt with an acid is prepared by reacting the peptide of the present invention as a free base (the neutral form of the peptide of the present invention typically has a neutral -NH2 group) with a suitable acid. Suitable acids for preparing salts with acids include, for example, organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid; and, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. On the other hand, salts of acidic moieties that may be present on the peptide and bases are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, and trimethylamine.

[0108] The pharmaceutical composition of the present invention is preferably an immunotherapy agent such as a vaccine. The pharmaceutical composition of the present invention may be administered directly to the diseased organ or the whole body of a patient via an intradermal, intramuscular, subcutaneous, intraperitoneal, or intravenous route, or it may be administered to the patient after being ex vivo added to cells obtained from the patient or a human cell line, or it may be used in vitro to select an immune cell subpopulation obtained from a patient, and the selected immune cell subpopulation may be re-administered to the patient. When administering the nucleic acid of the present invention to cells in vitro, it may be useful to transfect the cells to co-express immunostimulatory cytokines such as interleukin-2.

[0109] The peptides of the present invention may be substantially pure. The peptides of the present invention may be combined with an immunostimulatory adjuvant such as the TLR1 / 2 ligand XS15. In preliminary studies, when XS15 was added to mutant or non-mutant viral peptides in the form of a water-in-oil emulsion and subcutaneously injected into healthy donors and individual cancer patients, it was shown that a strong CD8 + T cell response and a strong Th1 CD4 + T cell response could be induced. Also, the peptides of the present invention may be used in combination with immunostimulatory cytokines or administered using a suitable delivery system (e.g., liposomes). Further, the peptides of the present invention can also be conjugated to a suitable carrier such as keyhole limpet hemocyanin (KLH) or mannan (see WO95 / 18145 and Longenecker et al., Ann. N.Y. Acad. Sci. 690:276-291 (1993)). Moreover, the peptides of the present invention may be tagged, may be fusion proteins, or may be hybrid molecules. Peptides having the sequences described in the present invention are expected to stimulate CD4 T cells or CD8 T cells. However, the stimulation of CD8 T cells is more efficient in the presence of assistance by CD4 helper T cells. Therefore, for MHC class I epitopes that stimulate CD8 T cells, due to a fusion partner or part of a hybrid molecule, CD4 +Epitopes that stimulate T cells are suitably provided. Epitopes that stimulate CD4 T cells or CD8 T cells are well known in the art, including those identified in the present invention. In one embodiment, the vaccine of the present invention comprises at least one peptide having the amino acid sequence shown in SEQ ID NOs: 1 to 5, and at least one other peptide, which is preferably 2 to 50 peptides, more preferably 2 to 25 peptides, more preferably 2 to 20 peptides, and most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 peptides. These peptides are derived from one or more specific tumor-associated antigens (TAAs) and may be bound to MHC class I molecules.

[0110] In one embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention comprises at least one peptide according to the present invention, preferably comprising at least two peptides according to the present invention, more preferably comprising at least three peptides according to the present invention, more preferably comprising at least four peptides according to the present invention, and more preferably comprising five peptides according to the present invention. A “universal” pharmaceutical composition or vaccine preferably comprises a peptide or variant thereof comprising the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, for the reason that such a peptide can be administered to an individual regardless of the individual’s MHC or HLA allotype. A pharmaceutical composition administered to an individual with MHC or HLA allotype A*68:02 (SEQ ID NO: 3), C*04:01;C*05:01 (SEQ ID NO: 4), or A*24:02 (SEQ ID NO: 5) preferably comprises a peptide or variant thereof comprising the amino acid sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4 and / or SEQ ID NO: 5.

[0111] The pharmaceutical composition of the present invention may contain, as the sole active ingredient, the peptide of the present invention, the antibody or fragment thereof, the T cell receptor of the present invention, the antigen-binding protein of the present invention, the nucleic acid molecule or expression vector of the present invention, the recombinant host cell of the present invention, or the activated T lymphocyte of the present invention. However, in another embodiment, the pharmaceutical composition of the present invention may contain additional active ingredients such as small molecules (e.g., ibrutinib and / or ideasilib and / or vencrexta).

[0112] Another subject of the present invention relates to peptides or variants thereof, antibodies or fragments thereof, T cell receptors thereof, antigen-binding proteins thereof, isolated nucleic acid molecules thereof, vectors thereof, recombinant host cells thereof, or activated T cells thereof, for use in pharmaceuticals. The use is preferably in the diagnosis and / or prevention and / or treatment of cancer, or in the manufacture of pharmaceuticals for cancer, and the cancer is more preferably a cancer having a fusion of DnaJ heat shock protein family member B1 (DNAJB1) and the cAMP-activating catalytic subunit α of protein kinase (PRKACA) (DNAJB1-PRKACA), and is particularly preferably fibrous lamellar hepatocellular carcinoma (FL-HCC), or other cancers that overexpress proteins from which peptides including SEQ ID NOs. 1-5 are obtained.

[0113] The features, properties, advantages, and embodiments disclosed for the peptides of the present invention also apply to the uses described above.

[0114] Another subject of the present invention is a kit, (a) A container comprising a pharmaceutical composition containing, in solution or lyophilized form, a peptide or variant thereof of the present invention, an antibody or fragment thereof, a T cell receptor of the present invention, an antigen-binding protein of the present invention, an isolated nucleic acid molecule of the present invention, a vector of the present invention, a recombinant host cell of the present invention, or an activated T cell of the present invention, (b) A second container may include a diluent or reconstitution solution for a lyophilized preparation, (c) It may contain at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 5. (d) (i) instructions relating to the use of the solution or (ii) instructions relating to the reconstitution and / or use of the lyophilized preparation, Regarding the kit.

[0115] The kit of the present invention may further comprise one or more of (iii) buffer, (iv) diluent, (v) filter, (vi) needle, and (v) syringe. The container is preferably a bottle, vial, syringe, or test tube, and may be a multipurpose container. The pharmaceutical composition is preferably freeze-dried.

[0116] The kit of the present invention preferably includes the lyophilized formulation of the present invention in a suitable container and instructions for its reconstitution and / or use. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., dual-chamber syringes), and test tubes. The container may be made of various materials such as glass or plastic. The kit of the present invention and / or its container preferably includes instructions describing the reconstitution and / or use of the lyophilized formulation of the present invention, which may be affixed to the container or provided with the container. For example, a label affixed to the container may indicate that the lyophilized formulation is to be reconstituted to the aforementioned peptide concentration. The label may further indicate that the lyophilized formulation is useful for subcutaneous administration or that the lyophilized formulation is for subcutaneous administration.

[0117] The features, properties, advantages, and embodiments disclosed for the peptides of the present invention also apply to the aforementioned kits.

[0118] Another subject of the present invention is a method for producing a personalized anti-cancer vaccine, a) A step of identifying tumor-associated peptides (TUMAPs) presented in tumor samples derived from individual patients; b) A step of comparing a warehouse of pre-screened peptides with the peptides identified in step a) by comparing their immunogenicity and / or overpresentation in tumors with those in normal tissues; c) the step of selecting from the warehouse at least one peptide that matches TUMAP identified in the patient; and d) A process for formulating personalized vaccines based on step c). Includes, The present invention relates to a method wherein the data warehouse comprises at least one peptide or variant thereof.

[0119] "Personalized pharmaceutical compositions" refer to tailored treatments specifically for a single patient, and include actively personalized cancer vaccines and adoptive cell therapies using the patient's own tissue.

[0120] In this specification, “warehouse” refers to the peptides of the present invention that have been pre-screened for immunogenicity and / or overexpression in a specific type of tumor, which is specifically a cancer having a fusion of the DnaJ heat shock protein family member B1 (DNAJB1) and the protein kinase cAMP-activating catalytic subunit α (PRKACA) (DNAJB1-PRKACA). This warehouse (e.g., in the form of a database) consists of tumor-associated peptides highly overexpressed in cancer cells of multiple patients having various HLA-A, HLA-B, and HLA-C alleles. This warehouse includes MHC class I peptides and MHC class II peptides. More specifically, this warehouse includes MHC class I A*24 marker peptide, B*07 marker peptide, and A*02 marker peptide. Since these peptides allow for quantitative comparison of the degree of T-cell immunity induced by tumor-associated peptides (TUMAPs), it is possible to draw important conclusions regarding the antitumor response-inducing ability of vaccines.

[0121] In an exemplary embodiment, the identification of the peptide of the present invention contained in the vaccine of the present invention is (a) The step of identifying tumor-associated peptides (TUMAPs) presented in tumor samples derived from individual patients using the method described above; (b) A step of comparing the immunogenicity and / or overpresentation in tumors with a warehouse of pre-screened peptides compared with the corresponding normal tissues, and the peptides identified in step (a); (c) The step of selecting from the warehouse at least one peptide that correlates with the tumor-associated peptide identified in the patient. It was done by (d) A further step may be performed in which at least one peptide newly identified in step (a) is selected and its immunogenicity is confirmed.

[0122] Once peptides for a personalized peptide-based pharmaceutical composition (e.g., a vaccine) have been selected, the pharmaceutical composition is manufactured. The composition or vaccine of the present invention is preferably a liquid formulation in which the individual peptides are dissolved in 20-40% DMSO, preferably about 30-35% DMSO (e.g., about 33% DMSO).

[0123] The features, properties, advantages, and embodiments disclosed for the peptides of the present invention also apply to the methods described above.

[0124] The features described above and those described later are not limited to the combinations shown in each embodiment, but can also be used in other combinations or individually without departing from the scope of the present invention.

[0125] The present invention will be described in more detail by reference to the following embodiments. These embodiments describe further features, characteristics, and advantages of the present invention. Furthermore, the following embodiments are for illustrative purposes only and do not limit the spirit or scope of the present invention. Features described in specific embodiments are features of the present invention and may be considered general features that can be applied independently not only in specific embodiments but also in any embodiment of the present invention. [Brief explanation of the drawing]

[0126] The present invention will be described and explained in more detail with reference to the following embodiments and drawings, but the present invention is not limited to these embodiments and drawings. [Figure 1]This paper presents predictions of HLA class I and HLA class II ligands derived from the DNAJB1-PRKACA fusion gene, and characterization of CD4+ T cell epitopes derived from DNAJB1-PRKACA. (a) A schematic diagram showing an overview of the DNAJB1-PRKACA fusion transcript containing exon 1 of DNAJB1 and exons 2-10 of PRKACA. (b) Predictions of HLA class II ligands derived from the fusion region of the DNAJB1-PRKACA protein. Gray lines show ligands identified by in silico prediction, and black lines show ligands identified by mass spectrometry (MS). Solid lines show 9 amino acid-binding cores, and dashed lines show HLA ligand extensions of up to 15mer. The number of alleles indicates the number of HLA alleles predicted to bind to each core sequence. (c) Predictions of HLA class I ligands from the fusion region of the DNAJB1-PRKACA protein. The gray lines represent ligands identified by in silico prediction, the lines enclosed in black frames represent refolded HLA, and the black lines represent ligands identified by mass spectrometry. (d) For the cell surface markers shown in the graph, the characteristics of mature monocyte-derived dendritic cells (moDCs) compared with differentiated moDCs and isotype controls by flow cytometry are shown. (e) A comparison of the fragment spectra (m / z on the X-axis) of the peptide KREIFDRYGEEVKEFLAKAKED(PII-1) (SEQ ID NO: 1) (labeled "Identification" in the figure) experimentally eluted from mature moDCs carrying the peptide, and the directly measured synthetic peptide (labeled "Verification" in the figure, inverted on the X-axis) is shown. In both peptides, the 4th, 12th, and 19th amino acids were isotoped. (f) A representative example of functional evaluation is shown in the upper panel, where cytokines and cell surface markers were evaluated by flow cytometry in PII-1-specific CD4+ T cells derived from healthy volunteers after de novo priming with PII-1-carrying mature moDCs. CD4+ T cells that were primed with PII-1 and then stimulated with a negative peptide were used as a negative control (lower panel). [Figure 2]This paper describes the characterization of DNAJB1-PRKACA-derived CD8+ T cell epitopes and single-cell TCR sequencing. (a) A representative example of flow cytometry characterization of PA*24-specific CD8+ T cells from healthy volunteers obtained by priming with in vitro artificial antigen-presenting cells (aAPCs) carrying HLA-A*24-PA*24 monomer is shown. (b) The frequency of PA*24-specific CD8+ T cells compared with CD8+ T cells primed with HLA-matched negative peptides is shown in healthy volunteers (n=5). (c) Representative examples of IFNγ and TNF production and CD107a expression in PA*24-stimulated PA*24-specific CD8+ T cells (upper panel) or PA*24-specific CD8+ T cells stimulated with HLA-matched negative peptides (lower panel) are shown. (d) Various effector cells:Specific cell lysis of PA*24-carrying autologous CD8-target cells by PA*24-specific CD8+ T cells compared to autologous CD8-target cells carrying negative peptides (white fill, solid line (upper panel)) in various effector cell:target cell ratios (gray fill, dashed line (upper panel); red line (lower panel)). PA*24 nonspecific CD8+ T cells did not lyse target cells (black line (lower panel)). Results are shown as mean ± SD of three independent technical replicates. Paired Student's two-tailed t-tests were used for evaluation (**P<0.01; ***P<0.001). (e) PA*24-specific CD8+ T cells derived from two healthy volunteers (HV1, HV2) obtained by priming with aAPC carrying the HLA-A*24-PA*24 monomer were bulk sorted by flow cytometry (left panel), and single-cell TCR sequencing was performed. The right panel shows the results of comparing the physiological and chemical properties and amino acid sequences of the CDR3α and CDR3β regions of the most frequently occurring T cell receptor (TCR) clones in each donor with PA*24, the target peptide of these TCR clones. Hydrophilicity on the Hopp-Woods scale is shown on the y axis, and amino acids (AA) are grouped by physiological and chemical properties and shown with color codes. [Figure 3]This shows single-cell TCR sequencing of PA*24-specific CD8+ T cells. (a~c) PA*24-specific CD8+ T cells derived from two healthy volunteers (HV) obtained by priming with artificial antigen-presenting cells (aAPC) carrying HLA-A*24-PA*24 monomer were sorted by tetramer, and then single-cell RNA sequencing and single-cell TCR sequencing were performed. (a, b) Uniform Manifold Approximation and Projection (UMAP) plots showing the clonality of the T cell receptor (TCR) of PA*24-specific CD8+ T cells from (a) HV1 and (b) HV2 are shown. The color code indicates the number of cells belonging to each type of clone that proliferated. (c) The physiological and chemical properties and amino acid (AA) sequence of the CDR3α / CDR3β region of the second most frequent TCR clone in HV1 are shown. Hydrophilicity on the Hopp-Woods scale is shown on the y axis. Amino acids are grouped according to their physiological and chemical properties and shown with color codes. [Figure 4]This paper shows the mass spectrometry identification of naturally presented DNAJB1-PRKACA-derived HLA class I and class II ligands. (a) A schematic diagram is shown outlining the experimental setup for the expression of the doxycycline (Dox)-induced DNAJB1-PRKACA fusion gene. Hepatocellular carcinoma (HCC) cell lines were transduced with the Dox-induced DNAJB1-PRKACA plasmid or a control plasmid, treated with Dox, and then the HCC cell lines themselves were subjected to immunopeptide analysis by mass spectrometry (MS), or mature monocyte-derived dendritic cells (moDCs) from healthy volunteers (HV) incubated with lysates of these HCC cell lines were subjected to immunopeptide analysis by mass spectrometry (MS). (b) Immunoblots showing the expression of the Dox-inducible DNAJB1-PRKACA fusion protein in each HCC cell line (HLE, SMMC-7721, or HepG2) into which the Dox-inducible DNAJB1-PRKACA plasmid (+) or control plasmid (-) was introduced, with or without treatment with Dox. GAPDH was used as a loading control. (c, e) (c) HLA class I and HLA class II peptides identified by mass spectrometry after HCC cell lines into which the Dox-inducible DNAJB1-PRKACA plasmid or control plasmid was introduced were treated with Dox; (e) HLA class I and HLA class II peptides identified by mass spectrometry in mature moDCs from healthy volunteers incubated with lysates of HCC cell lines expressing the DNAJB1-PRKACA protein or lysates of HCC cell lines not expressing the DNAJB1-PRKACA protein. (d, f) (d) The frequency of amino acid (AA) dispersion of HLA class I ligands on the DNAJB1-PRKACA fusion protein sequence is shown for each sample in HCC cell lines expressing DNAJB1-PRKACA protein (n = 3); (f) The frequency of amino acid (AA) dispersion of HLA class II peptides on the DNAJB1-PRKACA fusion protein sequence is shown for each sample in mature moDCs derived from healthy volunteers (n=3) incubated with lysates of HCC cell lines expressing DNAJB1-PRKACA protein.(g, h) Fragment spectra (m / z on the X axis) of the peptides eluted in the experiment are shown. (g) EIFDRYGEEV (PA*68 / A*02; left) (SEQ ID NO: 3) ("Identified") extracted from the SMMC-7721 cell line expressing DNAJB1-PRKACA, or IFDRYGEEV (PC*04 / C*05; right) (SEQ ID NO: 4) ("Identified") extracted from the HepG2 cell line expressing DNAJB1-PRKACA, were compared with their respective synthetic peptides ("Validation" in the figure, inverted on the X axis). (h) EVKEFLAKAKEDFLKK (PII-2) (SEQ ID NO: 2) ("Identified") extracted from mature moDCs from healthy volunteers incubated with lysates of HCC cell lines expressing DNAJB1-PRKACA protein, were compared with their respective synthetic peptides with the second amino acid isotope labeled ("Validation" in the figure, inverted on the X axis). [Figure 5] This shows HCC cell lines expressing the DNAJB1-PRKACA fusion protein. (a) Flow cytometry quantification of HLA class I molecule cell surface expression in the hepatocellular carcinoma (HCC) cell lines HLE, SMMC-7721, and HepG2. (b) Immunoblot measurement of Dox-induced DNAJB1-PRKACA fusion protein expression when HCC cell line HLE, into which the DNAJB1-PRKACA Dox-induced plasmid (+) or control plasmid (-) was introduced, was treated with doxycycline (Dox), and monocyte-derived dendritic cells were loaded with the lysate of these cells. Tubulin was used as a loading control. [Figure 6]This shows a patient with FL-HCC who received a personalized DNAJB1-PRKACA-derived peptide vaccine. (a) This shows a schematic diagram of the treatment course when treating a patient with fibrous hepatocellular carcinoma (FL-HCC) with a DNAJB1-PRKACA-derived peptide vaccine cocktail. After initial diagnosis (FD), the patient was treated with four cycles of chemotherapy (CHX) in the same manner as in the PHITT trial (PHITT trial group F), but because the tumor was found to be unresectable, chemotherapy was temporarily interrupted, and an early-stage liver transplant (LTx) was performed one month after initial diagnosis (FD) (M1). Everolimus was used for immunosuppression after transplantation. This patient who received a liver transplant experienced four relapses at 11, 15, 19, and 21 months after initial diagnosis. Tumors that occurred in the first, second, and fourth relapses were surgically resected, and the third relapse was treated with radiotherapy. Treatment with olaparib (a poly-ADP-ribose polymerase (PARP) inhibitor) was initiated 16 months after the initial diagnosis. At 21 and 23 months, patients received two doses of a personalized DNAJB1-PRKACA-derived peptide vaccine containing PA*68 / A*02, PB*44, PC*04 / C*05, and PII-1. Six weeks after the second vaccination, a T-cell response specific to this vaccine peptide was observed. (b) The vaccine peptide-specific T-cell response six weeks after the second vaccination is shown, assessed by measuring with an IFNγ ELISPOT assay after in vitro stimulation with the vaccine cocktail peptide (PB*44 or PII-1) and comparing with the negative peptide (neg.). (c) A longitudinal analysis of vaccine-induced T cell responses up to 10 months after vaccination, evaluated by IFNγ ELISPOT assay after in vitro stimulation with vaccine cocktail peptides (PA*68 / A*02, PB*44, PC*04 / C*05 or PII-1). (d, e) Flow cytometry characterization of cytokines shown in the graphs.The results of comparing cytokines released from CD4+ T cells stimulated with (d) PII-1 peptide and from CD4+ T cells and CD8+ T cells stimulated with (e) PB*44 peptide 14 weeks after the second vaccination with cytokines released from each negative peptide (neg.) are shown. [Figure 7] This shows the characterization of primary tumor cells and continuous immunomonitoring of FL-HCC patients vaccinated with a personalized DNAJB1-PRKACA-derived peptide vaccine. (a) Representative micrographs of FL-HCC patients at the time of diagnosis are shown. The upper left panel shows HE staining, the upper right panel shows Masson's trichrome staining, the lower left panel shows CK7 immunohistochemical staining, and the lower right panel shows Hepar1 immunohistochemical staining. Scale bar = 500 μm, 40x magnification. (b) Sanger sequencing of reverse transcription polymerase chain reaction (RT-PCR) products to confirm the chimeric transcript linked at the end of exon 1 of DNAJB1 and the beginning of exon 2 of PRKACA is shown. (c) Six weeks after the first vaccination (V1M23), six weeks after the second vaccination (V2M24), and 45 weeks after the second vaccination (V2M33), peptide-specific T cells derived from FL-HCC patients were stimulated in vitro with the vaccine cocktail peptide PB*44 or PII-1, followed by an IFNγELISPOT assay and compared with stimulation with the negative peptide (neg.). [Figure 9]This shows single-cell RNA sequencing of vaccine-induced PII-1-specific CD4+ T cells. (a-e) Single-cell RNA sequencing analysis of CD4+ T cells sorted from PBMCs obtained from FL-HCC patients stimulated with PII-1 31 weeks after the second dose of a personalized DNAJB1-PRKACA-derived peptide vaccine is shown. (a) Uniform Manifold Approximation and Projection (UMAP) plots showing characteristic T cell clusters are shown. (b) Heatmaps of genes defining each cluster of activated T cells, exhausted / late effector T cells, and quiescent naive T cells are shown. (c) Feature plots showing gene expression of IFNG, GZMB (encoding granzyme B), TNF, and CCL3 that define activated T cell clusters are shown. (d) UMAP plots showing the clonality of the T cell receptor (TCR) of the sequenced CD4+ T cells are shown. Color codes indicate the number of cells belonging to each type of clone that proliferated. (e) Shows the distribution of the 10 largest TCR clone types in various cell clusters identified in PII-1 specific CD4+ T cells. (f) Shows the results of comparing the physiological and chemical properties and amino acid sequences of the CDR3α and CDR3β regions of the most frequent TCR clones with PII-1, the target peptide of these TCR clones. The y-axis indicates hydrophilicity on the Hopp-Woods scale. Amino acids (AA) are grouped by physiological and chemical properties and shown in color code. [Figure 10]This paper describes a gating strategy for evaluating CD4+ T cells primed with moDCs using intracellular cytokine staining. An exemplary sample is shown illustrating the gating strategy for evaluating CD4+ T cells primed with monocyte-derived dendritic cells (moDCs) using intracellular cytokine staining (ICS). The first gating identifies lymphocytes (FSC-A vs. SSC-A), followed by further gating to identify single cells (FSC-A vs. FSC-H), then further gating to identify living cells (FSC-A vs. Aqua live / dead), and finally gating to identify CD4+ cells (FSC-A vs. CD4-APC-Cy7). In CD4+ T cells, we analyzed the expression of CD107a (FSC-A vs. CD107a-FITC), CD154 (FSC-A vs. CD154-APC), IL-2 (FSC-A vs. PE-Cy7), IFNγ (FSC-A vs. IFNγ-PE), and TNF (FSC-A vs. TNF-Pacific Blue). [Figure 11] This paper describes a gating strategy for evaluating CD8+ T cells primed with aAPCs by intracellular cytokine staining. An exemplary sample is shown demonstrating a gating strategy for CD8+ T cells primed with artificial antigen-presenting cells (aAPCs) by intracellular cytokine staining (ICS). The first gating identifies lymphocytes (FSC-A vs. SSC-A), followed by further gating to identify single cells (FSC-A vs. FSC-H), further gating to identify living cells (FSC-A vs. Aqua live / dead), and finally gating to identify CD8+ cells (FSC-A vs. CD8-PE-Cy7). In CD8+ T cells, we analyzed IFNγ expression (IFNγ-PE vs. CD8-PE-Cy7), TNF expression (TNF-Pacific Blue vs. CD8-PE-Cy7), CD107a expression (CD107a-FITC vs. CD8-PE-Cy7), and TNF / IFNγ expression (TNF-Pacific Blue vs. IFNγ-PE). [Figure 12]This paper describes a gating strategy for evaluating peptide-specific CD4+ T cells and CD8+ T cells obtained from FL-HCC patients vaccinated with a personalized DNAJB1-PRKACA peptide vaccine using intracellular cytokine staining. Exemplary samples demonstrating the gating strategy for evaluating peptide-specific CD4+ T cells and CD8+ T cells obtained from FL-HCC patients vaccinated with a personalized DNAJB1-PRKACA peptide vaccine using intracellular cytokine staining (ICS) are shown. Lymphocytes were identified by initial gating (FSC-A vs. SSC-A), further gating was used to identify single cells (FSC-A vs. FSC-H), and further gating was used to identify live cells (FSC-A vs. Aqua live / dead). Cytokine production from CD4+ T cells and CD8+ T cells (CD4-APC-Cy7 vs. CD8-PE-Cy7) was analyzed separately. In CD8+ T cells, IFNγ expression (IFNγ-PE vs. CD8-PE-Cy7), TNF expression (TNF-Pacific Blue vs. CD8-PE-Cy7), and TNF / IFNγ expression (TNF-Pacific Blue vs. IFNγ-PE) were analyzed. In CD4+ T cells, IFNγ expression (IFNγ-PE vs. CD4-APC-Cy7), TNF expression (TNF-Pacific Blue vs. CD4-APC-Cy7), and TNF / IFNγ expression (TNF-Pacific Blue vs. IFNγ-PE) were analyzed. [Figure 13] This paper describes a gating strategy for evaluating PA*24 tetramer staining of CD8+ T cells. An exemplary sample is shown demonstrating a gating strategy for evaluating PA*24 tetramer staining of CD8+ T cells after priming with antigen-presenting cells (aAPCs). Lymphocytes were identified by initial gating (FSC-A vs. SSC-A), further gating was used to identify single cells (FSC-A vs. FSC-H), and further gating was used to identify live cells (FSC-A vs. Aqua live / dead). CD8+ / PA*24 tetramer-specific cells were then analyzed (PA*24 tetramer-PE vs. CD8-PE-Cy7). [Figure 14]This paper presents a gate strategy for evaluating mature moDCs. An exemplary sample is shown demonstrating a gate strategy for evaluating mature monocyte-derived dendritic cells (moDCs). The first gate identified moDCs (FSC-A vs. SSC-A), and further analysis was performed on the cell surface expression of CD80 (CD80-FITC vs. count), HLA-DR (HLA-DR-BV 711 vs. count), and CD86 (CD86-BV 605 vs. count). [Examples]

[0127] 1. Introduction T-cell immunotherapies, including immune checkpoint inhibitors (ICIs), CAR-T cells, adoptive T-cell transfer, and vaccination, have achieved breakthroughs in the treatment of malignant diseases. However, these therapies rely on the recognition of tumor antigens and the elimination of cancer cells through T-cell-mediated cytotoxicity, and are only effectively used in a limited number of cancer patients and single tumors. One of the main specific reasons for developing antigen-specific immunotherapies is the lack of suitable target structures that are naturally and exclusively presented on the surface of tumor cells at high frequency and recognized by the immune system. Tumor antigens are presented by HLA-independent surface molecules or by intracellular protein-derived T-cell epitopes presented by HLA class I or HLA class II molecules. Regarding HLA molecule-mediated presentation of tumor antigens, neoepitopes resulting from tumor-specific mutations have recently been identified, as neoepitopes exhibiting primary specificity in anti-cancer T-cell responses induced by immune checkpoint inhibitors, suggesting them to be the most promising candidates for T-cell immunotherapy. Simultaneously, responses to immune checkpoint inhibitors have been observed to correlate with high somatic mutational loads caused by tumors, and neoepitope-based immunotherapy has shown promising results for the first time in individual tumor patients. However, patient / tumor specificity and intratumoral heterogeneity of somatic mutations, as well as the limited number of somatic mutations that are translated, processed, and presented on tumor cells as HLA-restricted neoepitopes, limit the broad application of tumor antigens, especially in patients with low mutational loads. Furthermore, in recent years, fusion transcripts that are translated into products that often function as clonal tumorigenetic drivers have been identified as a new source of highly immunogenic neoepitopes. T-cell responses to neoepitopes derived from such fusion proteins have been detected in patients treated with immune checkpoint inhibitors, and a correlation with treatment response has been observed.

[0128] The DNAJB1-PRKACA fusion transcript is formed by linking exon 1 (DNAJB1) of the DnaJ homolog subfamily B member 1 gene to the cAMP-dependent protein kinase catalytic subunit α gene (PRKACA). This fusion transcript was first identified in fibrous lamellar hepatocellular carcinoma (FL-HCC). FL-HCC is a rare but fatal tumor with no established treatment options other than surgical resection, and generally occurs in children and young adults with no history of primary liver disease. The DNAJB1-PRKACA fusion has also been detected in various other gastrointestinal tumors. In FL-HCC, the DNAJB1-PRKACA fusion transcript was detectable in 100% of patients, and its expression was observed in all tumor cells, leading to its identification as a tumorigenetic driver in the pathogenesis of the tumor. In this specification, the inventors demonstrate that DNAJB1-PRKACA fusion transcripts are a major source of widely applicable neoepitopes and, for the first time, have demonstrated their applicability to immunotherapy for FL-HCC patients.

[0129] 2 Results 2.1 The DNAJB1-PRKACA fusion gene is a source of HLA class I and HLA class II neoantigens, and DNAJB1-PRKACA-specific CD4 + Induces T cells An in silico prediction workflow using the NetMHCIIpan algorithm identified nine 9-amino acid (AA) binding cores from a total of 1290 different HLA class II alleles within the 24-amino acid fusion region of the DNAJB1-PRKACA protein (Figure 1a, b). Of these alleles, 83.49% were HLA-DP combinations, 11.55% were HLA-DQ combinations, and 4.96% were HLA-DR combinations. The core sequence RYGEEVKEF (SEQ ID NO: 5), located exactly in the center of the aforementioned fusion transcript (consisting of 5 amino acids from exon 1 (DNAJB1) and 4 amino acids from exon 2 (PRKACA)), had the highest predicted amount of HLA class II alleles (60.52% of the total number of binding core combinations were alleles that could potentially bind). Analysis using a predictive workflow combining the SYFPEITHI algorithm and the NetMHCpan algorithm identified 13 DNAJB1-PRKACA-derived HLA ligands for the 20 most frequent HLA class I allotypes in the European population. These included peptides containing SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 (Figure 1c). Furthermore, it is noteworthy that this HLA class II binding core RYGEEVKEF (SEQ ID NO: 5) was also predicted to be an HLA class I ligand that binds to the HLA-A*24:02 allele, HLA-C*04:01 allele, HLA-C*06:02 allele, and HLA-C*07:02.

[0130] Mature monocyte-derived dendritic cells (moDCs; Figure 1d), obtained by differentiating monocytes from healthy volunteers (HVs), were subjected to a 22-amino acid length KREIFDRYGEEVKEFLAKAKED(P) protein fusion region. II-1 When the peptide (SEQ ID NO: 1) was supported and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS), it was shown that the HLA-presenting peptide derived from DNAJB1-PRKACA is presented in its natural state. This mass spectrometry (MS) confirmed, through verification using isotope-labeled synthetic peptides, that the P eluted experimentally was found to be present.II-1 The fragment ion spectrum of was identified (Figure 1e). P was identified by MS. II-1 The variant that is 12 amino acids shorter or shorter than this was predicted to bind to the HLA-DP allele DPA1*01:03-DPB1*05:01 in each healthy volunteer, and in terms of NetMHCIIpan binding order, the binding core RYGEEVKEF (sequence number 5) (Figure 1b) contained in the peptide containing SEQ ID NO: 1 showed the best score of 0.74. II-1 In mature moDCs carrying CD4 derived from healthy volunteers, + When T cells were de novo primed, P II-1 Pulsation by this process induces multifunctional P that expresses CD107a, CD154, IL-2, IFNγ, and TNF. II-1 specific CD4 + T cells were induced (Figure 1f).

[0131] 2.2 P A*24 specific CD8 + T cell characterization and single-cell TCR sequencing The ligand RYGEEVKEF (SEQ ID NO: 5) (P) derived from the DNAJB1-PRKACA fusion protein. A*24 (SYFPEITHI score: 74.19%, NetMHCpan binding rank: 0.018) Refolding, HLA-A*24-P A*24 This was performed using monomers. Artificial antigen-presenting cells (aAPCs) using these monomers were used to analyze CD8 from healthy volunteers. + When T cells were primed, P A*24 specific CD8 + T cells were induced, and the frequency of these peptide-specific T cells reached a maximum of 15.74% (mean 4.97%; Figure 2a, b). A*24 specific CD8 + T cells exhibit a multifunctional phenotype reflected in the production / expression of IFNγ, TNF, and CD107a (Figure 2c), and in vitro P A*24 Carrying my own CD8 -Cells were specifically lysed, and the lysis of target cells at various effector:target ratios reached up to 82.39% compared to non-specific effector cells (Figure 2d). In addition, P2 cells derived from two healthy volunteers (HV1, HV2) that were primed with aAPC in vitro and then bulk sorted by flow cytometry were obtained. A*24 specific CD8 + Single-cell T cell receptor (TCR) sequencing of T cells revealed high clonality in one TCR clone that was dominant in HV2 and in two TCR clones that were dominant in HV1 (Figure 3a, b). While there was no duplication of the V(D)J gene sequence in these three TCR clones, high similarity was observed in the physiochemical properties of the amino acids in the CDR3α and CDR3β sequences. The target peptide of these TCR clones was P A*24 In comparison, it was shown that hydrophilicity and chemical properties have contradictory characteristics in terms of grouping them (Figures 2e and 3c).

[0132] 2.3 Doxycycline-induced expression of the DNAJB1-PRKACA fusion gene leads to the spontaneous processing and presentation of DNAJB1-PRKACA-specific ligands for HLA class I and HLA class II. Three hepatocellular carcinoma (HCC) cell lines expressing HLA class I molecules—HLE, SMMC-7721, and HepG2—were transduced with a doxycycline (Dox)-inducible plasmid containing the DNAJB1-PRKACA fusion gene under the control of a tetracycline-responsive promoter, allowing for the specific expression of the DNAJB1-PRKACA fusion protein (Figures 4a, b, and 5a). After expression of the DNAJB1-PRKACA fusion gene, immunopeptide-metaphorical analysis of each HCC cell line by mass spectrometry revealed up to 3688 HLA class I ligands, including various peptides containing one of sequence numbers 6-63 (average 2787; Figure 4c). Twenty unique HLA class I ligands derived from the DNAJB1-PRKACA fusion protein were identified, two of which had sequences spanning the aforementioned fusion region (Figure 4d). Of these two DNAJB1-PRKACA-derived neoepitopes presented after natural processing, EIFDRYGEEV (SEQ ID NO: 3) (P A*68 / A*02) was identified in the SMMC-7721 cell line, and IFDRYGEEV (SEQ ID NO: 4) (P C*04 / C*05 ) was identified in the HepG2 cell line, EIFDRYGEEV (SEQ ID NO: 3) (P A*68 / A*02 ) binds to HLA allotype A*68:02 of the SMMC-7721 cell line, and IFDRYGEEV (SEQ ID NO: 4) (P C*04 / C*05 It was predicted that ) would bind to HLA allotype C*04:01 of the HepG2 cell line, and this result was verified by comparative spectral analysis using synthetic peptides (Figure 1c and Figure 4e). Next, to investigate the processing and presentation of HLA class II peptides derived from the DNAJB1-PRKACA fusion protein, mature moDCs from three healthy volunteers were incubated with lysates of HLE cell lines after activation of DNAJB1-PRKACA fusion gene expression (Figure 5b). Immunopeptide-metalysis of these moDCs by mass spectrometry revealed up to 8293 HLA class II peptides (average 5956; Figure 4f), of which 13 unique peptides were identified as being derived from the DNAJB1-PRKACA fusion protein, and one of these peptides, EVKEFLAKAKEDFLKK (SEQ ID NO: 2) (P II-2 ) had a sequence that extended across the aforementioned fusion region (Figure 4g). P is a neoepitope derived from DNAJB1-PRKACA. II-2 It was predicted that it would bind to the HLA allele DRB1*13:02 in healthy volunteers (Figure 1b). Using isotope-labeled synthetic peptides, P II-2 We validated the peptide fragment ion spectroscopy (Figure 4h). Note that no HLA class I or HLA class II ligands derived from these two fusion proteins were identified in any of the negative controls.

[0133] 2.4 Personalized DNAJB1-PRKACA-derived peptide vaccines induced long-term DNAJB1-PRKACA-specific immune responses and demonstrated favorable clinical outcomes in FL-HCC patients. A personalized DNAJB1-PRKACA-derived peptide vaccine was formulated for a young patient (FL-HCC01) with histologically confirmed FL-HCC. This young patient had undergone a liver transplant (LTx) early in the disease but had recurrent multiple tumors due to unresectable FL-HCC that was refractory to chemotherapy (Figures 6a and 7a). Everolimus, an mTOR inhibitor, was administered for post-transplant immunosuppression. Because the DNA damage response (DDR) pathway was altered (ATM and CHEK2, germline variants, and somatic deletions of BRCA2 and BAP1), and long-term remission could not be achieved, poly-ADP-ribose polymerase (PARP) inhibition was initiated. Recurrent tumors were surgically removed or radiotherapy was performed. Based on the DNAJB1-PRKACA fusion gene confirmed by Sanger sequencing, the personalized vaccine contained three allotype-matched short HLA class I ligands (EIFDRYGEEV (SEQ ID NO: 3) (P A*68 / A*02 ), EEVKEFLAKA (Sequence ID 120) (P B*44 ), and IFDRYGEEV(Sequence ID 4)(P C*04 / C*05 )) and the aforementioned long-chain peptide KREIFDRYGEEVKEFLAKAKED (SEQ ID NO: 1) (P II-1 ) was composed of the following. Furthermore, Montanide TM A novel Toll-like receptor (TLR) 1 / 2 agonist, XS15, emulsified in ISA51 VG, was added to the vaccine as an adjuvant. XS15 facilitates the activation and maturation of antigen-presenting cells and prevents the rapid degradation of the vaccine peptide, thereby enabling the induction of an effective and robust T cell response (Figure 7b). This personalized DNAJB1-PRKACA-derived peptide vaccine was administered twice, 6 weeks apart. In vitro analysis using an IFNγ enzyme-coupled immunospot (ELISPOT) assay after stimulation with the vaccine cocktail peptide revealed that P 6 weeks after the second vaccination, II-1A significant T-cell response targeting the peptide was observed (the average number of spots before vaccination was 1, compared to an average of 872 after the second vaccination; Figures 6b, c, and 7c). Furthermore, HLA class I peptide P B*44 A weak T-cell response targeting P was detected (the average number of spots before vaccination was 0, compared to an average of 56 after the second vaccination). Longitudinal experiments using the IFNγ ELISPOT assay showed that P persisted over a long period. II-1 Specific T cells were maintained, and their intensity was shown to increase further 45 weeks after the second vaccination (mean number of spots: 1088) (Figures 6c and 7c). Furthermore, P B*44 and P II-1 When the immune response targeting was evaluated by flow cytometry, a T helper 1 (Th1) phenotype CD4 specifically expressing IFNγ and TNF was identified. + T cells were discovered. B*44 CD8 + No T-cell response was observed. Notably, no disease relapse was observed in FL-HCC01 up to a recent examination conducted 15 months after the second vaccination, suggesting for the first time the clinical efficacy of a vaccine-induced DNAJB1-PRKACA-specific T-cell response.

[0134] 2.5 Single-cell RNA sequencing of vaccine-induced P II-1 specific CD4 + T cell TCR clonality has been demonstrated. Vaccine-induced HLA-DPA1*01:03-DPB1*06:01-P II-1 Responsive CD4 +T cells were grown in vitro, single-cell RNA sequencing was performed using 10× Genomics, and unsupervised clustering of the data defined three T cell clusters. Specifically, (i) activated T cells expressing cytokines and chemokines, defined by high expression of IFNG, TNF, GZMB (encoding granzyme B), CCL3, and CCL4 (Figure 5a, b, c); (ii) T cells exhibiting exhausted or late-effector profiles expressing PDCD1, LAG3, HAVCR2, and CTLA4 (Figure 8a, b, and 9a); and (iii) resting naive T cells defined by expression of SELL (encoding CD62L), CCR7, and TCF7 (Figure 8a, b, and 9b). High TCR clonality was observed in the activated T cell cluster, with cells assigned to large clones (clonality ≥ 4) accounting for 74.2% compared to the proportion in quiescent naive T cells (1.3%) and exhausted T cell clusters (31.4%) (Figures 8d and 9c). A total of 10 TCR clones were identified by clustering, of which 8 were mainly assigned to the activated T cell cluster. High physiological and chemical similarity was observed in the amino acids of the CDR3α and CDR3β sequences among these TCR clones, suggesting that the target peptide of these TCR clones is P II-1 In comparison, it was shown that hydrophilicity and chemical properties have contradictory characteristics in terms of grouping (Figures 8e, f and 9d).

[0135] 3. Materials and Methods 3.1 Patient and blood sample Peripheral blood mononuclear cells (PBMCs) were isolated from FL-HCC patients by density gradient centrifugation, and PBMCs were isolated from healthy volunteers (HV). These were stored at -80°C until use in subsequent T cell assays. Informed consent was obtained in accordance with the Declaration of Helsinki. This study was conducted in accordance with the guidelines of the local ethics committee (713 / 2018B02, 406 / 2019BO2). HLA typing of the samples was performed by the Department of Hematology and Oncology (Tübingen, Germany).

[0136] 3.2 Personalized peptide vaccines The personalized vaccine was developed and manufactured at the peptide laboratory of the Department of Immunology at the University of Tübingen, in compliance with Good Manufacturing Practice (GMP) standards for pharmaceutical manufacturing and quality control. It contains four DNAJB1-PRKACA-derived peptides (SEQ ID NOs: 1, 3, 4, and 120) and XS15 (Bachem, Bubendorf, Switzerland), a TLR1 / 2 ligand consisting of a synthetic lipopeptide, as an adjuvant, and Montanide TM This is a peptide-based vaccine emulsified in ISA51 VG (Seppic, Paris, France). It contains vaccine peptides (250 μg each) and XS15 (50 μg), along with Montanide. TM By adding ISA51 VG, a 1:1 water-in-oil emulsion was prepared to obtain a 500 μL injectable volume. This personalized vaccine was subcutaneously injected into the patient's lower abdomen. Personalized vaccination was performed according to the NCT05014607 protocol.

[0137] 3.3 Detection and sequencing of DNAJB1-PRKACA transcripts RNA was extracted from 5 μm paraffin sections macrodissected using a Maxwell® RSC instrument (Promega, Madison, Wisconsin, USA) and the Maxwell RSC RNA FFPE kit according to the manufacturer's instructions. Reverse transcription of the RNA and polymerase chain reaction (PCR) of the DNAJB1-PRKACA cleavage region were performed using the QIAGEN OneStep RT-PCR kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions (forward primer: 5'-GTTCAAGGAGATCGCTGAGG-3' (SEQ ID NO: 122), reverse primer: 5'-TTCCCGGTCTCCTTGTGTTT-3' (SEQ ID NO: 123)). To visualize the detection of the DNAJB1-PRKACA fusion protein, the PCR products were run on an agarose gel. To perform sequencing, the PCR product was purified (AMPure, Beckman Coulter, Blair, California, USA), preparative samples were collected, and a sequencing reaction was performed on a final volume of 10 μl using 1 μM each of forward and reverse primers and 2 μl of GenomeLab DTCS-Quick Start Master Mix (Beckman Coulter, Blair, California, USA) according to the manufacturer's protocol. The sequencing reaction product was purified (CleanSEQ, Beckman Coulter, Blair, California, USA), analyzed using the GenomeLab GeXP gene analysis system, and evaluated with GenomeLab GeXP software (Beckman Coulter, Blair, California, USA).

[0138] 3.4 Prediction of DNAJB1-PRKACA ligand in silico Predictions for DNAJB1-PRKACA ligands in HLA class I were performed using SYFPEITHI 1.0 and NetMHCpan 4.1 for all possible peptide sequences of 8-12 amino acid length across the fusion region, for the 20 most frequent HLA class I allotypes in the European population (tools.iedb.org). Predictions for DNAJB1-PRKACA ligands in HLA class II were performed using NetMHCIIpan 4.0 for all possible peptide sequences of 15 amino acid length across the fusion region, for all allele combinations listed.

[0139] 3.5 Quantification of HLA cell surface expression The QIFIKIT bead-based quantitative flow cytometry assay (Dako, K0078) was used according to the manufacturer's instructions to analyze HLA cell surface expression in HCC cell lines, following previous reports. Briefly, samples were stained with either pan-HLA class I specific monoclonal antibody (mAb) W6 / 32 (manufactured in-house) or IgG isotype control (BioLegend, 400202). Flow cytometry analysis was performed using a FACSCanto II analyzer (BD).

[0140] 3.6 Expression of DNAJB1-PRKACA fusion gene induced by doxycycline in HCC cell lines As HCC cell lines, HLE cell line, SMMC-7721 cell line, and HepG2 cell line were cultured in Gibco Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (FCS), penicillin, streptomycin (both from Merck), and plasmocin (Invivogen) under a humid atmosphere at 37°C and 5% CO2. The DNAJB1-PRKACA coding sequence was synthesized by commissioning Thermo Fisher and pENTR TM Cloning into a vector, directional TOPO cloning (pENTR TM / D-TOPO TMUsing a cloning kit (Invitrogen), the DNA was transferred into the pInducer20 (Addgene, No. 44012) destination vector. Lentiviral particles were produced by transfecting HEK293T cells with the helper plasmids psPAX2 and pMD2.G, along with the pInducer20 vector encoding DNAJB1-PRKACA or an empty vector, using the calcium phosphate method. Each transduced HCC cell line was selected with G418 (Invivogen) for at least two weeks. To induce DNAJB1-PRKACA expression, each transduced HCC cell line was treated with 1 μg / ml doxycycline (Dox) (AppliChem) for 24 hours. To confirm the expression of DNAJB1-PRKACA, cells were lysed in lysis buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% Triton X-100, 50mM NaF, 10mM Na4P2O7, 10mM Na4V2O7, and Complete protease inhibitor cocktail (Roche)). SDS-PAGE and immunoblotting were performed according to previously reported methods. For immunoblotting, anti-PKAα cat antibody (Santa Cruz, clone A-2), anti-GAPDH antibody (Cell Signaling, clone D16H11), and anti-tubulin antibody (Merck, clone DM1A) were used as primary antibodies. Visualization was performed using HRP-labeled goat anti-rabbit secondary antibody or HRP-labeled goat anti-mouse secondary antibody (both from Jackson ImmunoResearch).

[0141] 3.7 Isolation of HLA Ligands Using pan-HLA class I-specific monoclonal antibody W6 / 32, pan-HLA class II-specific monoclonal antibody Tu-39, and HLA-DR-specific monoclonal antibody L243 (all manufactured in-house), HLA class I and HLA class II molecules were isolated by standard immunoaffinity purification, and HLA ligands were extracted.

[0142] 3.8 Analysis of HLA ligands by liquid chromatography-tandem mass spectrometry (LC-MS / MS) Peptide samples were separated by reversed-phase liquid chromatography (nanoUHPLC, UltiMate 3000 RSLCnano, Thermo Fisher, Waltham, Massachusetts, USA) and then analyzed using an online-connected Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher, Waltham, Massachusetts, USA). The sample analysis was performed in three technical replicates. 5 μL of sample with a 20% occupancy was injected into a 75 μm × 2 cm trap column (Thermo Fisher, Waltham, Massachusetts, USA) at a rate of 4 μL / min for 5.75 minutes. Next, peptides were separated by flowing a concentration gradient from 2.4% to 32.0% ACN over 90 minutes at 50°C and a flow rate of 300 nL / min through a 50 μm × 25 cm separation column (PepMap C18, Thermo Fisher, Waltham, Massachusetts, USA). The eluted peptides were ionized by nanospray ionization. Next, high-energy C-trap dissociation (HCD) was performed at the highest speed (3 seconds) to obtain fragment spectra with a resolution of 30,000 and a mass range limited to 235–1151 m / z. Peptides with positive charges of +2 to +5 were selected and fragmented, and then analyzed by mass spectrometer.

[0143] 3.9 Data Processing Data processing was performed according to previous reports. Using Proteome Discoverer (v1.3, Thermo Fisher), the complete sequence of the DNAJB1-PRKACA fusion protein was integrated with the search results from the SequestHT search engine (University of Washington) against the human proteome (Swiss-Prot database, 20,279 investigated protein sequences, September 27, 2013). The precursor mass tolerance was set to 5 ppm, and the fragment mass tolerance was set to 0.02 Da. Oxidized methionine was accepted as a dynamic modification. The false discovery rate (FDR) (estimated using the Percolator algorithm 2.04) was limited to 5% for HLA class I-presenting peptides and 1% for HLA class II-presenting peptides. HLA class I annotation was performed using SYFPEITHI 1.0 and NetMHCpan 4.1.

[0144] 3.10 Spectral Verification The spectra of the peptides eluted experimentally were validated by calculating the spectral similarity with those of the corresponding synthetic peptides measured in the composite matrix. Spectral correlation was calculated between the MS / MS spectra of the eluted peptides and those of the synthetic peptides.

[0145] 3.11 Peptide-Specific T Cell Proliferation and IFNγ ELISPOT Assay PBMCs were pulsed with either 1 μg / ml HLA class I peptide or 5 μg / ml HLA class II peptide. Peptides independent of each HLA restriction were used as negative controls. Specifically, YLLPAIVHI (SEQ ID NO: 124) (derived protein: DDX5_HUMAN) was used as the negative control for HLA-A*02, and ETVITVDTKAAGKGK (SEQ ID NO: 125) (derived protein: FLNA_HUMAN) was used as the negative control for HLA class II. On days 2, 5, and 7, 20 U / ml IL-2 (Novartis, Basel, Switzerland) was added, and the PBMCs were cultured for 12 days. On day 12, PBMCs stimulated with each peptide were analyzed by IFNγ enzyme-coupled immunosorbent spot (ELISPOT) assay. Using an ImmunoSpot S6 analyzer (CTL, Cleveland, Ohio, USA), spots were counted, and a T cell response was considered positive if the number of counted spots per 500,000 cells exceeded 10, and the average number of spots was at least three times the average number of spots in the negative control.

[0146] 3.12 Refolding Biotinylated HLA:peptide complexes were prepared according to previous reports and tetramerized using PE-labeled streptavidin (Invitrogen) in a 4:1 molar ratio.

[0147] 3.13 Peptide-specific CD8 by aAPC + T cell induction Peptide-specific cytotoxic T lymphocyte priming was performed using artificial antigen-presenting cells (aAPCs) as previously reported. Specifically, 800,000 streptavidin-coated microspheres (Bangs Laboratories, Fishers, Indiana, USA) were loaded with 200 ng of biotinylated HLA:peptide monomer and 600 ng of biotinylated anti-human CD28 monoclonal antibody (clone 9.3, manufactured in-house). 4.8 U / μl IL-2 (R&D Systems, Minneapolis, Minnesota, USA) and 1.25 ng / ml IL-7 (PromoKine, Heidelberg, Germany) were added to target CD8. + T cells were cultured. aAPC(1×10 6 CD8 + The T cells were stimulated once a week for four cycles using 200,000 aAPCs and 5 ng / ml IL-12 (PromoKine).

[0148] 3.14 Cytokine staining and tetramer staining Peptide-specific CD4 + T cells and peptide-specific CD8 +The functionality of T cells was analyzed by intracellular cytokine staining (ICS), as previously reported. Cells were pulsed with 10 μg / ml of each peptide, and incubated for 12-16 hours with 10 μg / ml brefelzin A (Sigma-Aldrich, St. Louis, Missouri, USA) and 10 μg / ml GolgiStop (BD, Franklin Lakes, New Jersey, USA). Staining was performed using Cytofix / Cytoperm (BD), APC / Cy7 anti-human CD4 antibody (1:100 dilution, BioLegend, catalog No. 300518, RRID: AB_314086), PE / Cy7 anti-human CD8 antibody (1:400 dilution, Beckman Coulter, catalog No. 737661, RRID: AB_1575980), Pacific Blue anti-human tumor necrosis factor antibody (TNF, 1:120 dilution, BioLegend, catalog No. 502920, RRID: AB_528965), and FITC anti-human CD107a antibody (1:100 dilution, BioLegend, catalog No. 328606, RRID: The tests were performed using AB_1186036), APC anti-human IL-2 antibody (1:40 dilution, BioLegend, catalog No. 500309, RRID: AB_315096), and PE anti-human IFNγ monoclonal antibody (1:200 dilution, BioLegend, catalog No. 506507, RRID: AB_315440). PMA and ionomycin (Sigma-Aldrich) were used as positive controls. As negative control peptides with matching HLA-restrictive properties, YLLPAIVHI (SEQ ID NO: 124) (derived protein: DDX5_HUMAN) was used for HLA-A*02, KYPENFFLL (SEQ ID NO: 126) (derived protein: PP1G_HUMAN) was used for HLA-A*24, EEFGRAFSF (SEQ ID NO: 127) (derived protein: HLA-DP_HUMAN) was used for HLA-B*44, and ETVITVDTKAAGKGK (SEQ ID NO: 125) (derived protein: FLNA_HUMAN) was used for HLA class II. The gate strategies applied to the analysis of data obtained by flow cytometry are shown in Figures 10, 11, and 12.

[0149] Peptide-specific CD8 after priming with aAPC + T cell frequency was measured by staining with PE-Cy7 anti-human CD8 monoclonal antibody and HLA:peptide tetramer-PE. As a negative control, cells from the same donor were used, primed with the unrelated control peptide TYSEKTTLF (SEQ ID NO: 128) (derived protein: MUC16_HUMAN) and stained with a tetramer containing the test peptide. Peptide-specific CD8 + The frequency of T cells is related to CD8 in a living single-cell population. + Peptide-specific CD8, representing more than 0.1% of the total T cells and in negative control cells. + Priming was considered successful if the T cell frequency was at least three times higher. The same evaluation criteria were applied to the results of intracellular cytokine staining. Samples were analyzed using a FACS Canto II cytometer (BD). The gating strategy applied to tetramer staining analysis of data obtained by flow cytometry is shown in Supplementary Figure 13.

[0150] 3.15 Cytotoxic Assay In the VITAL assay using flow cytometry, the ability to induce peptide-specific target cell lysis was assessed by peptide-specific CD8 + T cells were analyzed. CD8 - In autologous target cells, P A*24 The peptide or negative peptide KYPENFFLL (SEQ ID NO: 126) (derived protein: PP1G_HUMAN) was loaded and labeled with CFSE or FarRed (Life Technologies, Carlsbad, California, USA), respectively. A*24 Specific effector cells were added in the effector-target ratio shown in the graph. CD8 cells carrying each peptide. - The specific lysis of target cells was calculated relative to control target cells.

[0151] 3.16 Peptide-specific CD4 by peptide-supported moDC + T cell induction To induce differentiation of monocyte-derived dendritic cells (moDCs), magnetically activated cell sorting (MACS; Milteny GmbH, Bergisch Gladbach, Germany) is used to extract CD14 cells from PBMCs. + Cells were isolated and then treated with penicillin, streptomycin, GM-CSF, and IL-4 in X-VIVO. TM Cells were cultured in serum-free hematopoietic cell medium at 37°C and 5% CO2 under a humid atmosphere for 7 days. Differentiated moDCs were matured by adding LPS to the cell culture medium and culturing for 24 hours. Cell surface expression of CD80-FITC (1:40 dilution, Biolegend, catalog No. 305206), HLA-DR-BV 711 (1:100 dilution, Biolegend, catalog No. 307644), and CD86-BV 605 (1:400 dilution, Biolegend, catalog No. 374214) was confirmed. The gating strategy applied to the analysis of data obtained by flow cytometry is shown in Figure 14. CD4 + Before using to stimulate T cells, mature moDCs are P II-1 The peptide was incubated for 2 hours. Next, CD4 was extracted from PBMCs of the same healthy volunteer (HV) using magnetic activated cell sorting (MACS; Milteny GmbH, Bergisch Gladbach, Germany). + Cells were isolated and treated with penicillin, streptomycin, IL-2, and IL-7 in X-VIVO. TM 15. Hematopoietic cells were cultured in serum-free medium under a humid atmosphere at 37°C and 5% CO2. + Cells were stimulated once a week for a total of four weeks with mature moDCs carrying peptides and IL-12. Peptide-specific CD4 + The functionality of T cells was analyzed using intracellular cytokine staining.

[0152] 3.17 Antigen loading onto mature moDCs To prepare tumor lysates, HCC cell lines transduced with plasmids encoding DNAJB1-PRKACA or empty plasmids were treated with IFNγ and Dox for 24 hours. The treated cells were harvested, washed with PBS, subjected to five freeze-thaw cycles, irradiated with 30 Gy of radiation, and sonicated for 2 minutes. The harvested clear supernatant was then added to the cell culture medium of mature moDCs and incubated for 24 hours. Afterward, the antigen-loaded mature moDCs were harvested and subjected to HLA immunoprecipitation.

[0153] 3.18 Software and Statistical Analysis Population coverage of HLA allotypes was calculated using the IEDB population coverage tool (www.iedb.org). All figures were created and statistical analyses were performed using GraphPad Prism 9.2.0 (GraphPad Software). A p-value < 0.05 was considered statistically significant.

[0154] 3.19 Single-cell immunoprofiling analysis Following the 10× Genomics cell preparation protocol, peptide-specific CD8 + T cells or bulk CD4 +T cells were sorted and counted using FACS and washed with 0.04% BSA / PBS. Single cells were partitioned into Gel Beads-in-Emulsion (GEM) units, each containing 10× barcoded gel particles and reverse transcriptase, using a Chromium Controller instrument (10× Genomics, Pleasanton, California, USA). Next, single-cell gene expression libraries and single-cell T cell receptor (VDJ) libraries were prepared using the Chromium Next GEM Single Cell 5' Kit v2 (10× Genomics, Pleasanton, California, USA) and the Chromium Single Cell Human TCR Amplification Kit (10× Genomics, Pleasanton, California, USA) according to the manufacturer's instructions. These libraries were pooled and sequenced using NextSeq 550 (Illumina, San Diego, California, USA) with an average of 28,806 reads, 138,975 reads, and 3,519 reads per cell. Demultiplexing of samples was performed using bcl2fastq version 2.20.0.422 (Illumina, San Diego, California, USA). Barcoding, alignment, VDJ annotation, and single-cell 5' gene counting were performed using Cell Ranger software version 6.0.1 (10× Genomics, Pleasanton, California, USA). Furthermore, data processing, visualization, and analysis were performed individually for each sample using scanpy and scirpy. By excluding cells with fewer than 200 unique gene counts and no relevance to the vdj sequence, and cells with more than 10% mitochondrial genes, 474 cells (FL-HCC01), 115 cells (HV1), and 3338 cells (HV2) were obtained. Data were log-normalized with a scale factor of 10,000. Linear dimensionality reduction was performed, taking into account only highly variable genes. Highly variable genes were defined as those with an average minimum expression level of 0.0125, an average maximum expression level of 3, and a minimum variance of 0.5. The effects of total count and mitochondrial volume were removed.Dimensionality reduction was performed using principal component analysis (PCA). The neighboring station graph was calculated by running the RAPIDS function of the UMAP algorithm on the 10 first principal components (n_PC=10). Unsupervised clustering was then performed using the RAPIDS function of the Louvain algorithm.

[0155] This invention includes the following inventions. [1] A peptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 1-5 and variant sequences having at least 88% homology to SEQ ID NOs: 1-5, or a pharmaceutically acceptable salt thereof, wherein the variant sequence induces T cells that bind to and / or cross-react with major histocompatibility complex (MHC) molecules, and the peptide is not a full-length polypeptide. [2] The peptide according to [1], wherein the peptide has the ability to bind to an MHC class I molecule or an MHC class II molecule, and when bound to the MHC molecule, is recognizable by CD4 T cells and / or CD8 T cells, and preferably the amino acid sequence of the peptide includes a continuous sequence of amino acids shown in any of SEQ ID NOs: 1 to 5. [3] An antibody that specifically recognizes the peptide or variant described in [1] or [2] above, more preferably a soluble antibody or a membrane-bound antibody, more preferably a monoclonal antibody or a fragment thereof, and preferably an antibody that specifically recognizes the peptide or variant described in [1] or [2] above when the peptide or variant is bound to an MHC molecule. [4] A T cell receptor or fragment thereof that reacts to an HLA ligand which is the peptide or a variant thereof described in [1] or [2], preferably a soluble T cell receptor or a membrane-bound T cell receptor, and preferably a T cell receptor that reacts to the peptide or a variant thereof described in [1] or [2] when the peptide or a variant thereof is bound to an MHC molecule. [5] An antigen-binding protein that specifically binds to at least one DNAJB1-PRKACA antigenic peptide that forms a complex with a major histocompatibility complex (MHC) molecule, The antigen-binding protein comprises a first polypeptide chain containing a first variable domain including CDR3α as at least one complementarity-determining region (CDR), and a second polypeptide chain containing a second variable domain including CDR3β as at least one CDR. a) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 6, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 36, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; b) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 7, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 37, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; c) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 8, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 38, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; d) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 9, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 39, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; e) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 10, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 40, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; f) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 11, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 41, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; g) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 12, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 42, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; h) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 13, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 43, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; i) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 14, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 44, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; j) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 15, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 45, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; k) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 32, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 45, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; l) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 16, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 46, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; m) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 17, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 47, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; n) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 33, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 47, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; o) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 18, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 48, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 1; p) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 19, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 49, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; q) Whether CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 19, whether CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 62, and whether the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; r) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 20, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 50, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; s) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 21, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 51, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; t) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 21, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 63, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; u) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 22, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 52, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; v) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 23, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 53, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; w) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 24, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 54, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; x) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 25, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 55, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; y) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 34, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 55, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; z) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 26, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 56, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; α) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 27, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 57, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; β) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 28, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 58, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; γ) CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 29, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 59, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; δ)CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 30, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 60, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; or ε)CDR3α contains or consists of the amino acid sequence of SEQ ID NO: 31, CDR3β contains or consists of the amino acid sequence of SEQ ID NO: 61, and the DNAJB1-PRKACA antigenic peptide contains or consists of SEQ ID NO: 5; The CDR sequence may contain one or more amino acid mutations selected from amino acid insertions, deletions, and / or substitutions. Preferably, the antigen-binding protein is a single-chain TCR (scTCR) or a bispecific single-chain antibody, and / or Preferably, the first polypeptide further comprises a constant domain of an α chain or a constant domain of a γ chain, and the second polypeptide further comprises a constant domain of a β chain or a constant domain of a δ chain, and / or Preferably, the first variable domain is part of the TCRα chain or the TCRγ chain, and / or Preferably, the second variable domain is part of the TCRβ chain or TCRδ chain, and / or Preferably, the first variable domain is the variable domain of TCRα, the second variable domain is the variable domain of TCRβ, and / or Preferably, the antigen-binding protein contains at least one amino acid mutation that increases stability, cell surface expression, and / or pairing. Antigen-binding protein. [6](i) one or more additional antigen-binding sites; (ii) Transmembrane regions within the first polypeptide chain and / or the second polypeptide chain, which may include an intracellular signaling region; (iii) Diagnostic agents; (iv) Therapeutic agents; and (v)PK adjustment part It further includes one or more of the following: Preferably, the antigen-binding protein is a therapeutic antigen-binding protein and / or a diagnostic antigen-binding protein. The antigen-binding protein described in [5] above. [7] An isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide or variant thereof as described in [1] or [2], an antibody or fragment thereof as described in [3], a T cell receptor as described in [4], or an antigen-binding protein as described in [5] or [6]. [8] An isolated nucleic acid molecule containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 64-119. [9] A cloning vector or expression vector comprising the isolated nucleic acid molecule described in [7] or [8] above.

[10] A cloning vector or expression vector encoding an antigen-binding protein that specifically binds to at least one DNAJB1-PRKACA antigenic peptide that forms a complex with a major histocompatibility complex (MHC) molecule, The molecule includes, under the control of a promoter, a first nucleotide sequence encoding a first variable domain containing CDR3α as at least one complementarity-determining region (CDR), and a second nucleotide sequence encoding a second variable domain containing CDR3β as at least one CDR. a) The first nucleotide sequence includes or consists of SEQ ID NO: 64, the second nucleotide sequence includes or consists of SEQ ID NO: 94, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; b) The first nucleotide sequence includes or consists of SEQ ID NO: 65, the second nucleotide sequence includes or consists of SEQ ID NO: 95, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; c) The first nucleotide sequence includes or consists of SEQ ID NO: 66, the second nucleotide sequence includes or consists of SEQ ID NO: 96, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; d) The first nucleotide sequence includes or consists of SEQ ID NO: 67, the second nucleotide sequence includes or consists of SEQ ID NO: 97, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; e) The first nucleotide sequence includes or consists of SEQ ID NO: 68, the second nucleotide sequence includes or consists of SEQ ID NO: 98, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; f) The first nucleotide sequence includes or consists of SEQ ID NO: 69, the second nucleotide sequence includes or consists of SEQ ID NO: 99, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; g) The first nucleotide sequence includes or consists of SEQ ID NO: 70, the second nucleotide sequence includes or consists of SEQ ID NO: 100, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; h) The first nucleotide sequence includes or consists of SEQ ID NO: 71, the second nucleotide sequence includes or consists of SEQ ID NO: 101, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; i) The first nucleotide sequence includes or consists of SEQ ID NO: 72, the second nucleotide sequence includes or consists of SEQ ID NO: 102, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; j) The first nucleotide sequence includes or consists of SEQ ID NO: 73, the second nucleotide sequence includes or consists of SEQ ID NO: 103, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; k) The first nucleotide sequence includes or consists of SEQ ID NO: 90, the second nucleotide sequence includes or consists of SEQ ID NO: 103, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; l) The first nucleotide sequence includes or consists of SEQ ID NO: 74, the second nucleotide sequence includes or consists of SEQ ID NO: 104, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; m) The first nucleotide sequence includes or consists of SEQ ID NO: 75, the second nucleotide sequence includes or consists of SEQ ID NO: 105, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; n) The first nucleotide sequence includes or consists of SEQ ID NO: 91, the second nucleotide sequence includes or consists of SEQ ID NO: 105, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; o) The first nucleotide sequence includes or consists of SEQ ID NO: 76, the second nucleotide sequence includes or consists of SEQ ID NO: 106, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 1; p) The first nucleotide sequence includes or consists of SEQ ID NO: 77, the second nucleotide sequence includes or consists of SEQ ID NO: 107, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; q) The first nucleotide sequence includes or consists of SEQ ID NO: 77, the second nucleotide sequence includes or consists of SEQ ID NO: 120, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; r) The first nucleotide sequence includes or consists of SEQ ID NO: 78, the second nucleotide sequence includes or consists of SEQ ID NO: 108, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; s) The first nucleotide sequence includes or consists of SEQ ID NO: 79, the second nucleotide sequence includes or consists of SEQ ID NO: 109, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; t) The first nucleotide sequence includes or consists of SEQ ID NO: 79, the second nucleotide sequence includes or consists of SEQ ID NO: 121, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; u) The first nucleotide sequence includes or consists of SEQ ID NO: 80, the second nucleotide sequence includes or consists of SEQ ID NO: 110, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; v) The first nucleotide sequence includes or consists of SEQ ID NO: 81, the second nucleotide sequence includes or consists of SEQ ID NO: 111, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; w) The first nucleotide sequence includes or consists of SEQ ID NO: 82, the second nucleotide sequence includes or consists of SEQ ID NO: 112, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; x) The first nucleotide sequence includes or consists of SEQ ID NO: 83, the second nucleotide sequence includes or consists of SEQ ID NO: 113, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; y) The first nucleotide sequence includes or consists of SEQ ID NO: 84, the second nucleotide sequence includes or consists of SEQ ID NO: 114, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; z) The first nucleotide sequence includes or consists of SEQ ID NO: 93, the second nucleotide sequence includes or consists of SEQ ID NO: 114, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; α) The first nucleotide sequence includes or consists of SEQ ID NO: 85, the second nucleotide sequence includes or consists of SEQ ID NO: 115, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; β) The first nucleotide sequence includes or consists of SEQ ID NO: 86, the second nucleotide sequence includes or consists of SEQ ID NO: 116, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; γ) The first nucleotide sequence includes or consists of SEQ ID NO: 87, the second nucleotide sequence includes or consists of SEQ ID NO: 117, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; δ) The first nucleotide sequence includes or consists of SEQ ID NO: 88, the second nucleotide sequence includes or consists of SEQ ID NO: 118, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; or ε) The first nucleotide sequence includes or consists of SEQ ID NO: 89, the second nucleotide sequence includes or consists of SEQ ID NO: 119, and the DNAJB1-PRKACA antigenic peptide includes or consists of SEQ ID NO: 5; The first nucleotide sequence and / or the second nucleotide sequence may contain one or more nucleic acid mutations selected from nucleotide insertions, deletions, and / or substitutions. Cloning vector or expression vector.

[11] Recombinant host cells comprising the peptide or variant thereof described in [1] or [2], the antibody or fragment thereof described in [3], the T cell receptor described in [4], the antigen-binding protein described in [5] or [6], the isolated nucleic acid molecule described in [7] or [8], or the vector described in [9] or

[10] , preferably selected from mammalian cells and human cells, and more preferably selected from antigen-presenting cells such as dendritic cells, T cells and NK cells.

[12] An in vitro method for producing activated T cells, comprising the step of contacting T cells in vitro with an antigen-carrying human MHC class I molecule or an antigen-carrying human MHC class II molecule expressed on the surface of a suitable antigen-presenting cell or on the surface of an artificial structure that mimics an antigen-presenting cell for a time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is the peptide or a variant thereof described in [1] or [2].

[13] Activated T cells that selectively recognize cells presenting a polypeptide comprising the peptide or a variant thereof described in [1] or [2], or a polypeptide comprising the peptide or a variant thereof, prepared by the method described in

[12] above.

[14] A pharmaceutical composition, preferably a vaccine, comprising at least one active ingredient selected from the group consisting of a peptide or variant thereof as described in [1] or [2], an antibody or fragment thereof as described in [3], a T cell receptor as described in [4], an antigen-binding protein as described in any one of [5] to [8], an isolated nucleic acid molecule as described in [9] or

[10] , a vector as described in

[11] or

[12] , a recombinant host cell as described in

[13] , an activated T cell as described in

[15] , and a labeled or labeled active ingredient, and a pharmaceutically acceptable carrier, which may also comprise pharmaceutically acceptable excipients and / or stabilizers.

[15] It is a kit, (a) A container comprising a pharmaceutical composition containing in solution or in a lyophilized form the peptide or variant thereof described in [1] or [2], the antibody or fragment thereof described in [3], the T cell receptor described in [4], the antigen-binding protein described in [5] or [6], the isolated nucleic acid molecule described in [7] or [8], the vector described in [9] or

[10] , the recombinant host cell described in

[11] , or the activated T cell described in

[13] , (b) A second container may include a diluent or reconstitution solution for a lyophilized preparation, (c) It may contain at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 5. (d) (i) instructions relating to the use of the solution or (ii) instructions relating to the reconstitution and / or use of the lyophilized preparation, kit.

Claims

[Claim 1] A peptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 1-5 and variant sequences having at least 88% homology to SEQ ID NOs: 1-5, or a pharmaceutically acceptable salt thereof, wherein the variant sequence induces T cells that bind to and / or cross-react with major histocompatibility complex (MHC) molecules, and the peptide is not a full-length polypeptide.

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