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

Abstract

The present invention relates to peptides, antigen-binding proteins, nucleic acids and cells for use in immunotherapy methods.Specifically, the present invention relates to the immunotherapy of cancer, in particular the immunotherapy of fibrolamellar hepatocellular carcinoma.Furthermore, the present invention relates to tumor-associated T cell peptide epitopes and recombinant T cell receptors that can be used, for example, as active pharmaceutical ingredients in vaccine compositions to stimulate anti-tumor immune responses or can be stimulated ex vivo T cells and transferred to patients.

Description

[Technical field]

[0001] The present invention relates to peptides, antigen-binding proteins, nucleic acids and cells for use in immunotherapy methods. In particular, the present invention relates to the immunotherapy of cancer, in particular the immunotherapy of fibrolamellar hepatocellular carcinoma (FL-HCC). Furthermore, the present invention relates to tumor-associated T cell peptide epitopes and recombinant T cell receptors that can be used, for example, as active pharmaceutical ingredients in vaccine compositions to stimulate anti-tumor immune responses or that can be stimulated ex vivo T cells and transferred to patients.

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

[0003] Cancer is the second leading cause of death worldwide, accounting for an estimated 9.6 million or 1 in 6 deaths in 2018. Globally, 1 in 5 men and 1 in 6 women will develop cancer at some point in their lives, and 1 in 8 men and 1 in 11 women will die from cancer. Globally, the total number of people surviving within 5 years of a cancer diagnosis (referred to as the 5-year prevalence) is estimated to be 43.8 million. In men, lung, prostate, colorectal, gastric and liver cancers are the most common, while in women, breast, colorectal, lung, cervical and thyroid cancers are the most common.

[0004] The increasing burden of cancer is due to several factors, including population growth and ageing, and the changing predominate causes of cancer linked to social and economic development. This change in cancer causes is particularly true in settings of rapid economic growth, which has been accompanied by a shift from poverty- and infectious disease-related cancers to lifestyle-related cancers more typical of industrialized countries.

[0005] Human fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare cancer that accounts for less than 5% of all liver cancers. It is unique in that it is predominantly found in children and young adults and does not show signs of fibrosis or cirrhosis. FL-HCC commonly harbors a deletion of approximately 400 kb on chromosome 19, which results in an in-frame fusion of DnaJ heat shock protein family member B1 (DNAJB1) and protein kinase cAMP-activated catalytic subunit α (PRKACA). 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 the catalytic subunit of protein kinase A (PKA). This catalytic subunit (PKA Cα) forms an inactive tetrameric complex with PKA Cβ and two regulatory subunits and exists in the cytoplasm as the PKA holoenzyme. Upon G protein-coupled receptor activation, the catalytic subunit of PKA is activated in a cAMP-dependent manner, leading to the phosphorylation of a wide variety of cellular substrates. The crystal structure of the DNAJB1-PRKACA fusion protein shows that the catalytic, regulatory and anchor protein-binding sites are similar to those of wild-type PRKACA.

[0006] The tumorigenesis of FL-HCC is poorly understood beyond the presence of the DNAJB1-PRKACA fusion. Other than this fusion, significant recurrent mutated genes and extensive copy number alterations have been reported, but these have not been specifically linked to 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 disease.

[0007] This form of cancer is often advanced at the time of diagnosis, as symptoms do not appear until the tumor is quite large. Symptoms include vague abdominal pain, nausea, abdominal distension, fatigue, and weight loss. In addition, symptoms may include palpable hepatomegaly. Other symptoms include jaundice, ascites, fulminant hepatic failure, encephalopathy, gynecomastia (males only), lower extremity thrombophlebitis, recurrent deep vein thrombosis, anemia, and hypoglycemia. The early onset and lack of chronic liver disease suggest FL-HCC, but it is often misdiagnosed because younger patients may develop classical hepatocellular carcinoma.

[0008] Current treatment for FL-HCC includes invasive surgery. The recurrence rate of FL-HCC is very high, so two or more liver resections may be necessary. This recurrence requires regular follow-up with medical imaging (CT or MRI). As FL-HCC is a very rare disease, there is no standard chemotherapy regimen. Radiation therapy has been performed in the past, but there is little data on its use. Survival rates for FL-HCC depend largely on whether and how metastatic the cancer is, i.e., spread to lymph nodes and other organs. If distal spread (metastasis) occurs, the median survival rate drops significantly. Furthermore, the 5-year survival rate varies between 40% and 90%.

[0009] The DNAJB1-PRKACA fusion has also been detected in a variety of other gastrointestinal tumor diseases, including pancreaticobiliary eosinophilic tumors.

[0010] T cell-based 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 tumor antigen recognition and T cell-mediated cytotoxicity to eliminate cancer cells, and have only been successfully used in a few cancer types and single tumors. One of the main specific reasons for developing antigen-specific immunotherapies is the lack of suitable target structures naturally presented on the cell surface of tumor cells in a high frequency and tumor-exclusive manner 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. With regard to the presentation of tumor antigens via HLA molecules, neoepitopes resulting from tumor-specific mutations have been recently identified as the main specificity of anti-cancer T cell responses induced by immune checkpoint inhibitors, and have been suggested to be the most promising candidates for T cell immunotherapy. At the same time, it has been shown that responses to immune checkpoint inhibitors correlate with a high tumor-borne somatic mutation burden, and neoepitope-based immunotherapy has first shown promising results in individual tumor patients. However, patient / tumor specificity and intratumor heterogeneity of somatic mutations, as well as the limited number of somatic mutations that can be translated, processed, and presented as HLA-restricted neoepitopes on tumor cells, limit the broad application of tumor antigens, especially in patients with low mutation burdens.

[0011] Recently, a new source of highly immunogenic neoepitopes has been identified: fusion transcripts that are translated into products that often function as drivers of clonal tumorigenesis.T cell responses against neoepitopes derived from such fusion proteins have been detected in patients treated with immune checkpoint inhibitors and correlated with therapeutic response.

[0012] The DNAJB1-PRKACA fusion transcript is the exon 1 of the DnaJ homolog subfamily B member 1 gene (DNAJB1) linked to the cAMP-dependent protein kinase catalytic subunit alpha gene (PRKACA). In FL-HCC, the DNAJB1-PRKACA fusion transcript was detectable in 100% of patients, and all tumor cells showed expression of this fusion transcript, identifying it as an oncogenic driver in tumor pathogenesis.

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

[0014] However, none of these methods have been approved, let alone achieved widespread clinical application or efficacy, possibly because the peptides reported in the above literature are not very suitable for inducing FL-HCC-specific immune responses in patients.

[0015] Therefore, the objective of the present invention is to provide tumor-associated T cell peptide epitopes that can be used to develop improved medicines and methods for the diagnosis, prevention and treatment of cancer. More specifically, the cancer has a fusion of DnaJ heat shock protein family member B1 (DNAJB1) and protein kinase cAMP-activated catalytic subunit alpha (PRKACA) (DNAJB1-PRKACA), such as fibrolamellar hepatocellular carcinoma (FL-HCC).

[0016] The present invention fulfills these and other needs. Summary of the Invention [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 set forth in SEQ ID NOs: 1 to 5 and variant sequences having at least 88% homology to SEQ ID NOs: 1 to 5, or a pharma- ceutically acceptable salt thereof, wherein the variant sequence induces T cells that bind to major histocompatibility complex (MHC) molecules and / or cross-react with the variant sequence, and the peptide is not a full-length polypeptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present inventors have identified and characterized the peptides of the present invention as neoantigens derived from the DNAJB1-PRKACA fusion transcript, which is a tumorigenic driver of fibrolamellar hepatocellular carcinoma (FL-HCC) and many 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 therefore be appropriately administered to any subject, even humans. All peptides of SEQ ID NO:3-5 are particularly suitable for administration to subjects with the appropriate MHC type (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 have demonstrated that the neoantigens of the present invention confer the multifunctional cytotoxic CD8 T cells required for effective anti-cancer therapy. + T cells and T helper (Th)1 CD4 + We were able to demonstrate that the neoantigen of the present invention is naturally processed and presented in DNAJB1-PRKACA-expressing tumor cells by performing immunopeptidome analysis by mass spectrometry. Furthermore, within the framework of individual cure experiments, the present inventors have demonstrated that the neoantigen of the present invention is naturally processed and presented in DNAJB1-PRKACA-expressing tumor cells by inoculating a patient with FL-HCC who had repeatedly relapsed with a vaccine containing the peptide of the present invention, thereby inducing polyfunctional CD4 T cells with an activated Th1 phenotype and high TCR clonality. +It could be shown that the vaccine can induce T cells. The DNAJB1-PRKACA-specific T cell response induced by the vaccine can be maintained for a long period of time and can extend the recurrence-free survival of patients after vaccination to more than one year. Therefore, the peptide of the present invention is suitable as an active agent of a pharmaceutical composition (particularly a vaccine) for the treatment and / or prevention of cancer such as FL-HCC.

[0020] The peptides according to the invention, their HLA allotypes and their respective SEQ ID NOs are given in the table below. [Table 1]

[0021] In the event of any discrepancy between the sequences set forth in Table 1 and those set forth in the Sequence Listing, the information in the Sequence Listing shall take precedence and apply.

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

[0023] Furthermore, the term "peptide" includes salts of a series of amino acid residues that are usually linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The salts are preferably pharma- ceutically acceptable salts of the peptide, such as, for example, chloride salts or acetate salts (trifluoroacetates). It should be noted that the peptides of the present invention cannot exist as salts in vivo, and therefore, the salts of the peptides of the present invention are in a state that is significantly different from the peptides of the present invention in vivo.

[0024] Furthermore, the term "peptide" also includes "oligopeptide". As used herein, "oligopeptide" refers to a series of amino acid residues that are usually linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not critical to the present invention, so long as the correct epitope or epitopes are retained. The length of the oligopeptide is usually less than about 30 amino acid residues and more than about 15 amino acids.

[0025] Furthermore, the term "peptide" also includes "polypeptides." A "polypeptide" generally refers to a series 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 critical to the present invention, so long as the correct epitope is retained. Unlike the terms "peptide" and "oligopeptide," a "polypeptide" refers to a molecule that contains more than about 30 amino acid residues.

[0026] The "major histocompatibility complex (MHC)" is a set of cell surface proteins essential for the adaptive immune system, which recognizes foreign molecules and determines tissue compatibility in vertebrates. The main function of MHC molecules is to bind antigens derived from pathogens and present them on the cell surface for recognition by appropriate T cells. The human MHC is also called the HLA (human leukocyte antigen) complex (or simply "HLA"). The MHC gene family is divided into three subgroups: class I, class II, and class III. The complex of peptide and MHC class I molecule binds to CD8 antigens with the appropriate T cell receptor (TCR). + The complex of peptide and MHC class II molecule is recognized by CD4 receptors bearing the appropriate TCR, whereas the complex is recognized by T cells. + They are recognized by helper T cells. Both CD8- and CD4-dependent responses work together to synergistically contribute to antiviral activity, so 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 encodes the large α chain that is the component of HLA-A. Polymorphism of the HLA-A α chain is important for HLA function. This polymorphism promotes genetic diversity in the population. Each HLA has a different affinity for peptides with specific structures, so a higher HLA diversity means a greater variety of antigens can be "presented" on the cell surface.

[0027] According to the inventors, a "variant" of an amino acid sequence means that, for example, the side chains of one or two amino acid residues have been modified (e.g., by replacement with the side chains of another naturally occurring amino acid residue or otherwise), but that the peptide is still capable of binding to an MHC molecule in substantially the same manner as a peptide consisting of the amino acid sequence of SEQ ID NOs: 1 to 5. For example, the peptide may be engineered such that it at least maintains (if not improves) its ability to interact with and bind to the binding groove of an appropriate MHC molecule, such as HLA-A*02, and at least maintains (if not improves) its ability to bind to the TCR of an activated T cell.

[0028] The original (unmodified) peptides disclosed herein may be modified by substituting one or more residues at various positions in the peptide chain, and may be modified by one or more residues selected from various positions, unless otherwise stated. The substitutions are preferably located at the termini of the amino acid chain. Such substitutions may be conservative, e.g., replacing one amino acid with another of similar structure and properties, e.g., replacing a hydrophobic amino acid with another hydrophobic amino acid. To be more conservative, amino acids of the same size and chemical properties may be substituted with amino acids of similar size and chemical properties, e.g., replacing leucine with isoleucine. Studies of sequence variation in naturally occurring homologous protein families have shown that certain amino acid substitutions are often better tolerated than others, and such amino acid substitutions often correlate with similarity in size, charge, polarity and hydrophobicity between the original and substituted amino acids, thus defining them as "conservative substitutions". As used herein, a "conservative substitution" is defined as an exchange of an amino acid within one of five groups: Group 1 - small aliphatic residues with non-polar or slight polarity (Ala, Ser, Thr, Pro, GIY); Group 2 - polar negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3 - polar positively charged residues (His, Arg, Lys); Group 4 - large non-polar aliphatic residues (Met, Leu, Ile, Val, Cys); and Group 5 - large aromatic residues (Phe, Tyr, Trp). Less conservative substitutions may involve the replacement of one amino acid with another amino acid with similar properties but with a slightly different size, such as the replacement of an alanine with an isoleucine residue. Very less conservative substitutions may involve the replacement of a polar amino acid with an acidic amino acid or even a basic amino acid with an acidic amino acid.However, such "extreme" substitutions cannot be excluded as potentially ineffective, because chemical effects are not entirely predictable, and extreme substitutions may lead to unexpected new discoveries that cannot be predicted from simple chemical principles. It goes without saying that such substitutions may include structures other than common L-amino acids. That is, L-amino acids commonly found in the antigenic peptides of the present invention may be replaced with D-amino acids, and such amino acids are also included in the present disclosure. Furthermore, non-standard amino acids (i.e., amino acids other than the common amino acids that constitute natural proteins) may be used for substitution to produce the immunogens and immunogenic polypeptides of the present invention.

[0029] If substitutions at two or more positions result in a peptide with substantially the same or greater antigenic activity as defined below, the combination of substitutions will be tested to determine whether it has an additive or synergistic effect on the antigenicity of the peptide. No more than four positions in a peptide will be substituted simultaneously.

[0030] Amino acid residues that do not substantially contribute to the interaction with the T cell receptor can be modified by substituting another amino acid that does not substantially affect T cell responsiveness and does not abolish binding to the relevant MHC.

[0031] Longer (extended) peptides may be preferred. MHC class I epitopes are usually 8-11 amino acids long, but can also be generated by peptide processing from longer peptides or proteins containing the actual epitope. The residues flanking the actual epitope are preferably those that do not substantially affect the proteolytic cleavage required to expose the epitope during processing.

[0032] The peptides of the present invention can be extended by up to four amino acid residues, i.e., one, two, three or four amino acids can be added to one or both termini, with the number of amino acids added to each termini being any combination ranging from 4:0 to 0:4. Extension combinations according to the present invention are shown in Table 2. [Table 2]

[0033] The amino acids used for the extension may be those derived from the peptide of the original sequence of the protein disclosed herein, or may be other amino acids, which may improve the stability or solubility of the peptide of the invention.

[0034] Thus, the epitopes of the present invention may be identical to naturally occurring tumor-associated or tumor-specific epitopes, or may include epitopes that differ by no more than four residues compared to the reference peptide, so long as they have substantially the same antigenic activity.

[0035] In another embodiment, the peptides of the present invention are extended by adding 5 or more amino acids to one or both ends, preferably up to a total length of 30 amino acids. This extension may result in a peptide that binds to MHC class II molecules. Binding to MHC class II can be tested by methods known in the art.

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

[0037] It goes without saying that the peptides or variants of the invention have the ability to bind to human major histocompatibility complex (MHC) class I or MHC class II molecules. Binding of the peptides or variants to MHC complexes may be tested by methods known in the art.

[0038] When testing peptide-specific T cells of the invention against a substituted peptide, the substituted peptide preferably achieves a half-maximal increase in lysis over background at a peptide concentration of 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 more than one individual, preferably by T cells from at least two individuals, and more preferably by T cells from three individuals.

[0039] One skilled in the art can assess whether T cells induced by a particular 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] Such T cells can then substantially cross-react with and kill cells expressing a polypeptide comprising the native amino acid sequence of the cognate peptide as defined in the present invention. As known from the scientific literature and database information (Rammensee et al., Immunogenetics 50: 213-219 (1999); Godkin et al., Int. Immunol 9: 905-911 (1997)), certain positions of HLA-binding peptides usually 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 polar, electrophysical, hydrophobic and spatial properties of the polypeptide chain that constitutes the binding groove. Thus, a person skilled in the art can recombine the amino acid sequences shown in SEQ ID NOs: 1-5 while maintaining the known anchor residues, and determine whether such variants retain the ability to bind to MHC class I or MHC class II molecules. The variants of the present invention retain the ability to bind to the TCR of activated T cells and are therefore capable of substantially cross-reacting with and killing cells expressing a polypeptide comprising the native amino acid sequence of a cognate peptide defined in an embodiment of the present invention.

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

[0042] Thus, when the terms "homology" or "percentage of homology" are used in reference to a sequence, these terms refer to aligning the sequence to be compared (the "sequence to be compared") with a sequence described or claimed herein (the "reference sequence"), and then comparing the sequence to be compared with the sequence described or claimed herein. The percentage of homology (also known as percentage of identity) is then calculated according to the following formula: Identity (%)=100×[1-(C / R)] where C is the number of differences between the reference and the compared sequences in the alignment length between the reference and the compared sequences, and this difference is (i) each base or amino acid of the reference sequence that does not have a corresponding base or amino acid aligned to the compared sequence; (ii) each gap in the reference sequence; and (iii) each aligned base or amino acid of the reference sequence that is different from each aligned base or amino acid of the compared sequence; (iv) The alignment should start from the first position of the sequences being aligned. In the formula, R is the number of bases or amino acids of the reference sequence in the length of the alignment with the sequence to be compared, and any gaps made in the reference sequence are also counted as bases or amino acids.

[0043] If there is an alignment between the sequence to be compared and the reference sequence in which the percentage of identity calculated by the above-mentioned method is approximately equal to or greater than the specified minimum percentage of identity, the sequence to be compared has a minimum percentage of identity relative to the reference sequence, even if there is another alignment in which the percentage of identity calculated by the above-mentioned method is less than the specified 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 using the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453), which takes into account the entire length of the sequences and searches for the optimal alignment (including gaps) of two sequences, may be used. The needle program is available, for example, on 30 World Wide Web sites 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 percent identity between two polypeptides is calculated using the needle (global) program in EMBOSS with the "gap open" parameter set to 10.0 and the "gap extension" parameter set to 35-0.5 and the Blosum62 matrix.

[0045] A variant sequence having "at least 88% homology" refers to a sequence that has at least about 88% or more sequence identity over its entire length to the entire length of a reference sequence, in particular 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% sequence identity.

[0046] A protein having an amino acid sequence that has "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% identity" 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 for the adaptive immune system, which recognizes foreign molecules and determines tissue compatibility in vertebrates. The main function of MHC molecules is to bind antigens derived from pathogens and present them on the cell surface for recognition by appropriate T cells. The human MHC is also called the HLA (human leukocyte antigen) complex (or simply "HLA"). The MHC gene family is divided into three subgroups: class I, class II, and class III. The complex of peptide and MHC class I molecule binds to CD8 antigens with the appropriate T cell receptor (TCR). + The complex of peptide and MHC class II molecule is recognized by CD4 receptors bearing the appropriate TCR, whereas the complex is recognized by T cells. + They are recognized by helper T cells. Both CD8- and CD4-dependent responses work together to synergistically contribute to antiviral activity, so 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 encodes the large α chain that is the component of HLA-A. Polymorphism of the HLA-A α chain is important for HLA function. This polymorphism promotes genetic diversity in the population. Each HLA has a different affinity for peptides with specific structures, so a higher HLA diversity means a greater variety of antigens can be "presented" on the cell surface.

[0048] "Pharmaceutical" or "pharmaceutical acceptable" refers to molecular species and compositions (or salts) that do not produce adverse, allergic or other untoward reactions and do not interfere with the activity of the active pharmaceutical ingredient when appropriately administered to a mammal, particularly a human.

[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. "Full-length polypeptide" is also referred to as "derived gene / derived protein" from which the peptide of the present invention is derived. The derived protein or full-length polypeptide may be highly overexpressed in cancer compared to normal tissue, or may not be highly overexpressed in cancer compared to normal tissue. In the context of the present invention, "normal tissue" refers to 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 association. Furthermore, the peptide of the present invention itself is presented in tumor tissue. In the context of the present invention, "tumor tissue" refers to a sample obtained from a patient suffering from cancer, such as fibrolamellar hepatocellular carcinoma (FL-HCC) or other gastrointestinal cancer.

[0050] In one embodiment of the present invention, the peptide or nucleic acid of the present invention is isolated. "Isolated" means that a material is removed from the environment in which it originally exists (e.g., if it is a natural material, it means that it is removed from the natural environment). For example, a natural polynucleotide or polypeptide that exists in the body of a living animal is not isolated, but if this polynucleotide or polypeptide is separated from some or all of the materials that coexist in nature, 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] The peptides and / or nucleotides disclosed by the present invention may also be in "purified" form. "Purified" is intended to be a relative definition, not necessarily absolute purity, and may include highly purified or only partially purified preparations, as the term is understood by those skilled in the relevant art. For example, it is customary to purify individual clones isolated from a cDNA library to ensure electrophoretic homogeneity. It is expressly contemplated to purify the starting or natural material at least 10-fold, preferably 100-fold or 1,000-fold, more preferably 10,000-fold or 100,000-fold. Furthermore, the claimed polypeptides are expressly encompassed by the present invention, preferably having a purity of 99.999% by weight, or at least 99.99% or 99.9% by weight, and even more desirably 99% or more by weight.

[0052] The peptides and nucleic acids of the present invention may be in "concentrated form". As used herein, "concentrated" means that the concentration of the material is (for example) at least about 2-fold, about 5-fold, about 10-fold, about 100-fold or about 1000-fold its natural concentration, advantageously 0.01% by weight, preferably at least about 0.1% by weight. Furthermore, concentrated preparations of about 0.5%, about 1%, about 5%, about 10% or about 20% by weight are contemplated. The sequences, constructs, vectors, clones and other materials comprising the present invention may advantageously be in concentrated or isolated form.

[0053] The problem underlying the present invention can be completely solved by the disclosure herein.

[0054] In a particularly preferred embodiment of the present invention, the peptide of the present invention consists of the amino acid sequence shown in SEQ ID NO:1-5 or consists essentially of the amino acid sequence shown in SEQ ID NO:1-5.

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

[0056] However, such additional sequences may be important for efficient introduction of the peptide of the invention into cells. In one embodiment of the invention, the peptide of the invention is part of a fusion protein, for example including N-terminal or C-terminal amino acids. In another fusion, the peptide of the invention can be fused to an antibody or a functional part thereof, as described herein, more specifically to an antibody sequence to be specifically targeted by this antibody, or can be fused to an antibody specific for dendritic cells, for example as described herein.

[0057] Furthermore, the peptides of the present invention or their variants may be modified to improve their stability and / or binding to MHC molecules in order to induce 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 invention, the peptides of the invention have the ability to bind to MHC class I or MHC class II molecules, and upon binding to these MHC molecules, are capable of recognition by CD4 T cells and / or CD8 T cells.

[0059] This approach has the advantage that the peptide of the present invention can reliably exert its ability to induce an immune response (particularly, its ability to induce a T cell response).

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

[0061] This approach has the advantage that the peptide of the invention or its variants can contain any amino acid predicted to be involved in the induction of an immune response, further improving therapeutic efficacy.

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

[0063] As used herein, the term "antibody" is used in a broad sense and includes both polyclonal and monoclonal antibodies. The term "antibody" includes intact or "full-length" immunoglobulin molecules, as well as 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, so long as they exhibit any of the desired properties, i.e., specifically recognize the peptide of the present invention or its variants. If possible, the antibody of the present invention may be purchased from a commercial supplier. The antibody of the present invention may also be produced using known methods.

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

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

[0066] The "T cell receptor" (abbreviated as TCR) of the present invention refers to a heterodimeric molecule comprising an α polypeptide chain (α chain) and a β polypeptide chain (β chain), which heterodimeric receptor is capable of binding to peptide antigens presented by HLA molecules. "T cell receptor" also includes the so-called γδ TCR.

[0067] The features, properties, advantages and embodiments disclosed for the peptides and antibodies or fragments thereof of the present invention apply equally 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 complexed with a major histocompatibility complex (MHC) molecule, comprising: The antigen binding protein comprises a first polypeptide chain comprising a first variable domain comprising at least one complementarity determining region (CDR), CDR3α, and a second polypeptide chain comprising a second variable domain comprising at least one CDR, CDR3β, a) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:6, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:36, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; b) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:7, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:37, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; c) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:8, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:38, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; d) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:9, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:39, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; e) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 10, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 40, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; f) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:11, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:41, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; g) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 12, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 42, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; h) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 13, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 43, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; i) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 14, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 44, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; j) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 15, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 45, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; k) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 32, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 45, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; l) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 16, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 46, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; m) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 17, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 47, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; n) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 33, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 47, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; o) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 18, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 48, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; p) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 19, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 49, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; q) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 19, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 62, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; r) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:20, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:50, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; s) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:21, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:51, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; t) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:21, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:63, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; u) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:22, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:52, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; v) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:23, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:53, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; w) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:24, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:54, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; x) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:25, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:55, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; y) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 34, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 55, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; z) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:26, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:56, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; α) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:27, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:57, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; β) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:28, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:58, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; γ) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:29, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:59, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; δ) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 30, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 60, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; or ε) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 31, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 61, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; The CDR sequences may contain one or more amino acid mutations selected from amino acid insertions, deletions and / or substitutions. Concerning antigen-binding proteins.

[0069] In accordance with the present invention, an "antigen binding protein" as referred to herein refers to a polypeptide or binding protein capable of binding to at least one DNAJB1-PRKACA antigenic peptide, in particular the peptides provided herein.

[0070] According to the present invention, "CDR" means "complementarity determining region". 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. There are three discontinuously arranged CDRs (CDR1, CDR2 and CDR3) on the amino acid sequence of the variable domain of an antigen receptor. The CDRs of a T cell receptor (TCR) are present on two different protein chains. In humans, in 95% of T cells, the TCR is composed of an α chain and a β chain (encoded by TRA and TRB, respectively), and in 5% of T cells, the TCR is composed of a γ chain and a δ chain (γ / δ) (encoded by TRG and TRD, respectively). In the context of the antigen-binding protein of the present invention, CDR3α and CDR3β refer to the protein chains that constitute part of the complementarity determining region 3 of the α chain and the β chain of the natural TCR that corresponds to the antigen-binding protein of the present invention.

[0071] The present inventors have identified and successfully sequenced multiple TCRs capable of specifically binding to the peptides of the present invention, particularly to peptides comprising the amino acid sequence shown in SEQ ID NO: 1 or 5, from DNAJB1-PRKACA-specific T cells induced in vitro or in vivo. The features shown in a) to ε) indicate combinations of amino acid sequences of CDR3α variable chain and CDR3β variable chain of recombinant antigen-binding protein (e.g. recombinant TCR, etc.) capable of specifically binding to DNAJB1-PRKACA antigenic peptide comprising 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 set forth in Table 3 and those set forth in the Sequence Listing, the information in the Sequence Listing shall take precedence and apply.

[0074] In one embodiment of the antigen binding protein, it is understood that said CDR sequence may contain one or more amino acid mutations selected from insertion, deletion and / or substitution of amino acids. Such mutations preferably do not affect or do not significantly affect the specific binding to the DNAJB1-PRKACA antigenic peptide. More preferably, such mutations may be conservative mutations, e.g., one amino acid may be replaced by another amino acid of similar structure and properties, e.g., a hydrophobic amino acid may be replaced by another hydrophobic amino acid. Please refer to the above disclosures regarding modifications and substitutions of the peptides of the invention, which disclosures also apply mutatis mutandis to the antigen binding proteins of the invention.

[0075] In the context of this specification, "one or more" means 1, 2, 3, 4, 5, etc. amino acid mutations.

[0076] The features, properties, advantages and embodiments disclosed for the peptides of the invention apply equally to the antigen binding proteins described above.

[0077] In one embodiment of the invention, the antigen binding protein is a single chain TCR (scTCR) or a bispecific single chain antibody, and / or the first polypeptide further comprises an alpha chain constant domain or a gamma chain constant domain and the second polypeptide further comprises a beta chain constant domain or a delta chain constant domain, and / or the first variable domain is part of a TCR alpha chain or a TCR gamma chain, and / or the second variable domain is part of a TCR beta chain or a TCR delta chain, and / or the first variable domain is a TCR alpha variable domain and the second variable domain is a TCR beta variable domain, and / or the antigen binding protein comprises at least one amino acid mutation that increases stability, cell surface expression and / or pairing.

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

[0079] "Bispecific antibodies" refers to formats including, but not limited to, diabodies, Cross-Over-Dual-Variable-Domain (CODV), and / or dual variable domain (DVD) proteins. A summary of such various bispecific antibodies and methods for their production is described, for example, in Brinkmann U. and Kontermann EE MAbs. 2017 Feb-Mar; 9(2): 182-212. In particular, DVD formats are disclosed, for example, in the following scientific publications (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.Bispecific diabodies are described, for example, in 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 described in PMID:11789029.

[0080] The constant domain sequences of the antigen-binding proteins may be modified, for example, by introducing heterologous sequences (more preferably mouse sequences) that may improve expression, pairing and stability. Mutations known from the prior art (e.g., WO2018 / 104407, PCT / EP2018 / 069151, WO2011 / 044186, WO2014 / 018863, EP2432802B1 and WO2020157211A) may be introduced, for example, by substitution of undesired amino acids in the variable domains and / or introduction of disulfide bridges between the constant domains and removal of unpaired cysteines.

[0081] In one embodiment of the invention, the antigen binding protein comprises (i) one or more additional antigen-binding sites; (ii) a transmembrane region within the first polypeptide chain and / or the second polypeptide chain, which may include a cytoplasmic signaling region; (iii) diagnostic agents; (iv) a therapeutic agent; and (v)PK adjustment part The present invention further includes one or more of the following:

[0082] The "transmembrane region" may be, for example, the transmembrane domain of TCR alpha or TCR beta.

[0083] An "intracytoplasmic signaling region" is, for example, the intracellular domain of TCR alpha or TCR beta.

[0084] As used herein, a "diagnostic agent" refers to a detectable molecule or substance, such as, for example, a fluorescent molecule, a radioactive molecule, or other label known in the art that produces a signal (either directly or indirectly).

[0085] As used herein, a "therapeutic agent" refers to an agent that has a therapeutic effect. In one embodiment, such a therapeutic agent may be a cytotoxic or cytostatic agent, other anti-cancer agent, and / or an agent that induces an immune response that enhances the body's ability to fight cancer.

[0086] As used herein, a "PK modulating moiety" refers to a moiety that modulates the pharmacokinetics (PK) of an antigen binding protein described herein. Thus, a PK modulating moiety specifically modulates the in vivo half-life and distribution of an antigen binding protein disclosed herein. In one embodiment, the PK modulating moiety extends the half-life of the antigen binding protein. Examples of PK modulating moieties 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), the Fc portion of an antibody and / or unstructured polypeptides (Schellenberger et al., (2009) Nat Biotechnol. Dec; 27(12):1186-90).

[0087] In one embodiment of the invention, the antigen binding protein is a therapeutic 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 a peptide of the invention or a variant thereof, an antibody of the invention or a fragment thereof, a T cell receptor of the invention or an antigen-binding protein of the invention.

[0089] The nucleic acid encoding a particular peptide, oligopeptide or polypeptide may be a natural 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 natural polynucleotide or a stabilized form of polynucleotide, for example, a polynucleotide having a phosphorothioate backbone, and may or may not contain introns, so long as the peptide of the present invention is encoded. Of course, the polynucleotide can only encode peptides that contain natural amino acid residues linked by natural peptide bonds. Yet another aspect of the present invention provides an expression vector capable of expressing the peptide of the present invention.

[0090] As used herein, a nucleic acid molecule or nucleotide sequence "encoding" a peptide or the like refers to a nucleotide sequence that encodes a peptide that includes artificial (man-made) start and stop codons that are compatible with a biological system and sequences that are expressed by, for example, dendritic cells or other cell systems useful for producing TCRs. 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." Thus, the isolated nucleic acid molecule of the present invention may include regulatory elements, such as promoters and enhancers.

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

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

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

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

[0095] The cloning vectors or expression vectors of the present invention are useful as basic tools for producing specific antigen-binding proteins of the present invention capable of specifically binding to peptides of the present invention, in particular to peptides comprising the amino acid sequence shown in SEQ ID NO: 1 or 5. Other features shown in a) to ε) indicate combinations of nucleotide sequences encoding the CDR3α variable chain and the CDR3β variable chain of an antigen-binding protein (such as a recombinant TCR) that can be engineered to specifically bind to the DNAJB1-PRKACA antigenic peptide comprising the amino acid sequence of SEQ ID NO: 1 or 5.

[0096] In the context of this specification, "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. nucleic acid mutations.

[0097] The cloning 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 invention apply equally to the nucleic acid molecules and cloning or expression vectors described above.

[0099] Another subject of the present invention relates to a recombinant host cell, which comprises a peptide of the invention, an antibody or a fragment thereof of the invention, a T cell receptor of the invention, an antigen-binding protein of the invention or an isolated nucleic acid molecule or vector of the invention, preferably chosen from mammalian or human cells, more preferably chosen 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 invention apply equally to the aforementioned host cells.

[0101] Yet another subject of the present invention relates to an in vitro method for producing activated T cells, comprising a step of contacting in vitro T cells with antigen-bearing human MHC class I or II molecules expressed on the surface of suitable antigen-presenting cells or on the surface of an artificial structure mimicking an antigen-presenting cell, for a time sufficient to activate the T cells in an antigen-specific manner, said antigen being a peptide of the present invention.

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

[0103] The activated T cells produced by the above method selectively recognize cells that abnormally express a polypeptide comprising any of the amino acid sequences of SEQ ID NOs:1-5.

[0104] The features, properties, advantages and embodiments disclosed for the peptides of the invention apply equally to the aforementioned methods.

[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 of the invention or a variant thereof, the antibody of the invention or a fragment thereof, the T cell receptor of the invention, the antigen-binding protein of the invention, the isolated nucleic acid molecule of the invention, the vector of the invention, the recombinant host cell of the invention, the activated T cell of the invention and a labelled or labelled active ingredient, and a pharmaceutically acceptable carrier, and optionally comprising pharmaceutically acceptable excipients and / or stabilizers.

[0106] A "pharmaceutical composition" is a composition suitable for administration to humans in a medical setting. Pharmaceutical compositions are preferably sterile and preferably manufactured according to GMP guidelines.

[0107] The pharmaceutical composition of the present invention includes the peptide of the present invention in free form or in the form of a pharma- ceutically acceptable salt (see also above). In this specification, "pharmaceutically acceptable salt" refers to 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 base. For example, an acid salt is prepared by reacting the peptide of the present invention as a free base (the peptide of the present invention in its neutral form usually has a neutral -NH2 group) with a suitable acid. Acids suitable for preparing the acid salt include organic acids such as, for example, 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, salicylic acid, and the like; and inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. On the other hand, salts of bases with acidic moieties that may be present on the peptide are prepared using pharma- ceutically acceptable bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

[0108] The pharmaceutical composition of the present invention is preferably an immunotherapeutic agent such as a vaccine. The pharmaceutical composition of the present invention may be administered directly to the affected organ or the whole body of the patient via intradermal, intramuscular, subcutaneous, intraperitoneal or intravenous routes, or may be added ex vivo to cells or human cell lines obtained from the patient and then administered to the patient, or may be used in vitro to select immune cell subpopulations obtained from the patient and the selected immune cell subpopulations are then readministered 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 an immune stimulating cytokine 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. Preliminary studies have shown that mutated or non-mutated viral peptides in the form of a water-in-oil emulsion, plus XS15, when injected subcutaneously into healthy donors and individual tumor patients, induced potent CD8 in vivo responses. + T cell responses and strong Th1CD4 + It has been shown that the peptides of the present invention can induce T cell responses. The peptides of the present invention may also be used in combination with immune stimulating cytokines or administered using a suitable delivery system (e.g., liposomes). The peptides of the present invention may also be conjugated to a suitable carrier, such as keyhole limpet hemocyanin (KLH) or mannan (see WO95 / 18145 and Longenecker et al., Ann. NY Acad. Sci. 690:276-291 (1993)). Furthermore, the peptides of the present invention may be tagged, fusion proteins, or hybrid molecules. Peptides having the sequences described in the present invention are expected to stimulate CD4 T cells or CD8 T cells. However, stimulation of CD8 T cells is more efficient in the presence of help from CD4 helper T cells. Thus, for MHC class I epitopes that stimulate CD8 T cells, a fusion partner or part of a hybrid molecule may be used to stimulate CD4 T cells. +Epitopes that stimulate T cells are preferably 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 an amino acid sequence as set forth in SEQ ID NOs: 1 to 5 and at least one other peptide, the other peptide being 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 may be derived from one or more specific tumor-associated antigens (TAA) and may bind to MHC class I molecules.

[0110] In one embodiment of the pharmaceutical composition of the invention, the pharmaceutical composition of the invention comprises at least one peptide according to the invention, preferably at least two peptides according to the invention, more preferably at least three peptides according to the invention, more preferably at least four peptides according to the invention, more preferably five peptides according to the invention. A "universal" pharmaceutical composition or vaccine preferably comprises a peptide comprising the amino acid sequence of SEQ ID NO:1 and / or SEQ ID NO:2 or a variant thereof, since such peptides 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 comprising the amino acid sequence of SEQ ID NO:3 and / or SEQ ID NO:4 and / or SEQ ID NO:5 or a variant thereof.

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

[0112] Another subject of the present invention relates to a peptide or a variant thereof of the invention, an antibody or a fragment thereof of the invention, a T cell receptor of the invention, an antigen-binding protein of the invention, an isolated nucleic acid molecule of the invention, a vector of the invention, a recombinant host cell of the invention or an activated T cell of the invention for use in a medicine, preferably in the diagnosis and / or prevention and / or treatment of cancer or in the manufacture of a medicine against cancer, more preferably a cancer having a fusion of DnaJ heat shock protein family member B1 (DNAJB1) and cAMP-activated catalytic subunit alpha of protein kinase (PRKACA) (DNAJB1-PRKACA), particularly preferably fibrolamellar hepatocellular carcinoma (FL-HCC) or other cancers overexpressing a protein from which a peptide comprising SEQ ID NO: 1-5 can be obtained.

[0113] The features, properties, advantages and embodiments disclosed for the peptides of the invention apply equally to the aforementioned uses.

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

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

[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 containers may be made of various materials, such as glass and plastic. The kit of the present invention and / or its container preferably includes instructions for the reconstitution and / or use of the lyophilized formulation of the present invention, which may be attached to the container or may be associated with the container. For example, a label attached to the container may include instructions for reconstituting the lyophilized formulation to adjust it to the aforementioned peptide concentration. The label may further include instructions that the lyophilized formulation is useful for subcutaneous administration or that the lyophilized formulation is intended for subcutaneous administration.

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

[0118] Another subject of the present invention is a method for producing a personalized anti-cancer vaccine, comprising the steps of: a) identifying tumor associated peptides (TUMAPs) presented by tumor samples from individual patients; b) comparing the peptides identified in step a) with a warehouse of peptides that have been pre-screened for immunogenicity and / or over-presentation in tumors compared to normal tissues; c) selecting from the warehouse at least one peptide that matches a TUMAP identified in the patient; and d) formulating a personalized vaccine based on step c). Including, The data warehouse comprises at least one peptide of the present invention or a variant thereof.

[0119] "Personalized pharmaceutical composition" means a tailored therapy used exclusively for the treatment of a single patient, and includes actively personalized cancer vaccines and adoptive cell therapy using autologous patient tissue.

[0120] As used herein, "warehouse" refers to peptides of the present invention that have been pre-screened for immunogenicity and / or over-presentation in a particular type of tumor, specifically a cancer with a fusion of DnaJ heat shock protein family member B1 (DNAJB1) and protein kinase cAMP-activated catalytic subunit alpha (PRKACA) (DNAJB1-PRKACA). The warehouse (e.g., in the form of a database) is composed of tumor-associated peptides that are highly overexpressed in cancer cells of multiple patients with various HLA-A, HLA-B and HLA-C alleles. The warehouse includes MHC class I and MHC class II peptides. More specifically, the warehouse includes A*24, B*07 and A*02 marker peptides of MHC class I. These peptides allow quantitative comparison of the degree of T cell immunity induced by tumor-associated peptides (TUMAPs), allowing important conclusions to be drawn regarding the ability of the vaccine to induce anti-tumor responses.

[0121] In an exemplary embodiment, the identification of a peptide of the invention for inclusion in a vaccine of the invention comprises: (a) identifying tumor associated peptides (TUMAPs) presented by tumor samples from individual patients by the method described above; (b) comparing the peptides identified in step (a) with a warehouse of peptides that have been pre-screened by comparing immunogenicity and / or over-presentation in tumors with corresponding normal tissues; and (c) selecting from said warehouse at least one peptide that correlates with a tumor-associated peptide identified in said patient. It is carried out by (d) At least one newly identified peptide from step (a) may be selected and subjected to a further step of confirming its immunogenicity.

[0122] Once peptides have been selected for a personalized peptide-based pharmaceutical composition (e.g., a vaccine), the pharmaceutical composition is manufactured. The composition or vaccine of the present invention is preferably a liquid formulation of individual peptides 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 invention apply equally to the aforementioned methods.

[0124] The features mentioned above and those to be described below can be used not only in the combinations shown in the respective embodiments, but also in other combinations or alone without departing from the scope of the present invention.

[0125] The present invention will be further described with reference to the following embodiments, which describe further features, characteristics and advantages of the present invention. Furthermore, the following embodiments are for illustrative purposes only and are not intended to limit the spirit or scope of the present invention. Features described in a particular embodiment are features of the present invention and may be considered as general features that may be applied alone not only to the particular embodiment but also to any embodiment of the present invention. [Brief description of the drawings]

[0126] The present invention will now be described and explained in more detail with reference to the following examples and figures, but the present invention is not limited to these examples and figures. [Figure 1]Prediction 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) Schematic diagram outlining the DNAJB1-PRKACA fusion transcript, including exon 1 of DNAJB1 and exons 2-10 of PRKACA. (b) Prediction of HLA class II ligands derived from the fusion region of DNAJB1-PRKACA protein. Grey lines indicate ligands identified by in silico prediction methods, and black lines indicate ligands identified by mass spectrometry (MS). Solid lines indicate the nine amino acid binding core, and dashed lines indicate the extension of HLA ligands up to 15 mer. Number of alleles indicates the number of HLA alleles predicted to bind to each core sequence. (c) Prediction of HLA class I ligands of the fusion region of DNAJB1-PRKACA protein. Grey lines indicate ligands identified by in silico prediction, black boxed lines indicate refolded HLA, black lines indicate ligands identified by mass spectrometry. (d) Flow cytometric characterization of matured monocyte-derived dendritic cells (moDCs) compared to differentiated moDCs and isotype controls for the indicated cell surface markers. (e) Comparison of fragment spectra (m / z on the x-axis) of experimentally eluted peptide KREIFDRYGEEVKEFLAKAKED (PII-1) (SEQ ID NO: 1) from peptide-loaded mature moDCs ("Identified") and directly measured synthetic peptide ("Validated" inverted on the x-axis). All peptides were isotopically labeled at the 4th, 12th and 19th amino acids. (f) Representative example of functional evaluation by flow cytometry of indicated cytokines and cell surface markers in PII-1-specific CD4+ T cells from healthy volunteers after de novo priming with PII-1-loaded mature moDCs (upper panel). As a negative control, CD4+ T cells primed with PII-1 and then stimulated with a negative peptide were used (lower panel). [Diagram 2]Characterization of DNAJB1-PRKACA-derived CD8+ T cell epitopes and single-cell TCR sequencing. (a) Representative examples of flow cytometric 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 monomers. (b) Frequency of PA*24-specific CD8+ T cells compared to CD8+ T cells primed with HLA-matched negative peptide in healthy volunteers (n=5). (c) Representative examples of IFNγ and TNF production and CD107a expression of PA*24-specific CD8+ T cells stimulated with PA*24 (upper panel) or HLA-matched negative peptide (lower panel). (d) Specific lysis of PA*24-bearing autologous CD8- target cells by PA*24-specific CD8+ T cells (gray fill, dashed line (upper panel); red line (lower panel)) compared with autologous CD8- target cells bearing a negative peptide (white fill, solid line (upper panel)) at various effector:target cell ratios. PA*24-nonspecific CD8+ T cells did not lyse target cells (black line (lower panel)). Results are shown as the mean ± SD of three independent technical replicates. Evaluation was performed by paired two-tailed Student's t-test (**P<0.01; ***P<0.001). (e) PA*24-specific CD8+ T cells from two healthy volunteers (HV1, HV2) obtained by priming with aAPCs carrying HLA-A*24-PA*24 monomers were bulk-sorted by flow cytometry (left panel) and subjected to single-cell TCR sequencing. The right panel shows the physiochemical properties and amino acid sequences of the CDR3α and CDR3β regions of the most frequent T cell receptor (TCR) clones in each donor compared with the target peptide of these TCR clones, PA*24. Hydrophilicity according to the Hopp-Woods scale is shown on the y-axis, and amino acids (AA) are grouped by physiochemical properties and color-coded. [Diagram 3]Single-cell TCR sequencing of PA*24-specific CD8+ T cells. (a-c) PA*24-specific CD8+ T cells from two healthy volunteers (HVs) obtained by priming with artificial antigen-presenting cells (aAPCs) carrying HLA-A*24-PA*24 monomers were sorted by tetramer and then subjected to single-cell RNA sequencing and single-cell TCR sequencing. (a, b) Uniform Manifold Approximation and Projection (UMAP) plots showing the clonality of T cell receptor (TCR) of PA*24-specific CD8+ T cells from (a) HV1 and (b) HV2. The color code indicates the number of cells belonging to each type of expanded clone. (c) Physiochemical properties and amino acid (AA) sequence of the CDR3α / CDR3β region of the second most frequent TCR clone in HV1 are shown. Hydrophilicity according to the Hopp-Woods scale is shown on the y-axis. Amino acids are grouped according to their physiochemical properties and are color-coded. [Figure 4]Identification of naturally presented DNAJB1-PRKACA-derived HLA class I and class II ligands by mass spectrometry. (a) Schematic diagram outlining the experimental setup for expression of doxycycline (Dox)-inducible DNAJB1-PRKACA fusion gene. Hepatocellular carcinoma (HCC) cell lines were transduced with Dox-inducible DNAJB1-PRKACA plasmid or control plasmid and treated with Dox, followed by immunopeptidome analysis by mass spectrometry (MS) of either the HCC cell line itself or matured monocyte-derived dendritic cells (moDCs) from healthy volunteers (HV) incubated with lysates of the HCC cell line. (b) Immunoblots of the expression of Dox-inducible DNAJB1-PRKACA fusion protein in each HCC cell line (HLE, SMMC-7721, or HepG2) transfected with Dox-inducible DNAJB1-PRKACA plasmid (+) or control plasmid (-) in the presence or absence of 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 treatment with Dox of HCC cell lines transfected with Dox-inducible DNAJB1-PRKACA plasmid or control plasmid; (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 or not expressing 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 in HCC cell lines (n = 3) expressing the DNAJB1-PRKACA protein is shown for each sample; (f) The frequency of amino acid (AA) dispersion of HLA class II peptides on the DNAJB1-PRKACA fusion protein sequence in mature moDCs derived from healthy volunteers (n = 3) incubated with lysates of HCC cell lines expressing the DNAJB1-PRKACA protein is shown for each sample.(g, h) Fragment spectra (m / z on the x-axis) of peptides eluted in the experiment are shown. (g) EIFDRYGEEV (PA*68 / A*02; left) (SEQ ID NO: 3) extracted from SMMC-7721 cell line expressing DNAJB1-PRKACA ("Identified") or IFDRYGEEV (PC*04 / C*05; right) (SEQ ID NO: 4) extracted from HepG2 cell line expressing DNAJB1-PRKACA ("Identified") were compared with the respective synthetic peptides ("Verification" in the figure, inverted on the x-axis). (h) EVKEFLAKAKEDFLKK (PII-2) (SEQ ID NO: 2) extracted from mature moDCs from a healthy volunteer incubated with lysates of HCC cell lines expressing DNAJB1-PRKACA protein ("Identification" in the figure) were compared with the respective synthetic peptides isotope-labeled at the second amino acid ("Verification" in the figure, inverted on the x-axis). [Diagram 5] HCC cell lines expressing DNAJB1-PRKACA fusion protein are shown. (a) Flow cytometric quantification of cell surface expression of HLA class I molecules in hepatocellular carcinoma (HCC) cell lines HLE, SMMC-7721, and HepG2. (b) Immunoblot measurement of Dox-inducible DNAJB1-PRKACA fusion protein expression in monocyte-derived dendritic cells loaded with lysates of HLE HCC cell line transfected with DNAJB1-PRKACA Dox-inducible plasmid (+) or control plasmid (-). Tubulin was used as a loading control. [Figure 6]A FL-HCC patient vaccinated with a personalized DNAJB1-PRKACA-derived peptide vaccine is shown. (a) A schematic diagram of the treatment course for a fibrolamellar hepatocellular carcinoma (FL-HCC) patient treated with a DNAJB1-PRKACA-derived peptide vaccine cocktail is shown. After initial diagnosis (FD), the patient was treated with four cycles of chemotherapy (CHX) in a manner similar to that of the PHITT study (PHITT study group F). However, the tumor was found to be unresectable, so chemotherapy was temporarily suspended and liver transplantation (LTx) was performed at an early stage of the disease, 1 month (M1) after initial diagnosis (FD). Everolimus was used for immunosuppression after transplantation. The patient who underwent liver transplantation experienced four recurrences, 11 months, 15 months, 19 months, and 21 months after initial diagnosis. The tumors that occurred in the first, second, and fourth recurrences were surgically resected, and the third recurrence was treated with radiation therapy. The patient was started to be treated with olaparib, a poly ADP-ribose polymerase (PARP) inhibitor, 16 months after the initial diagnosis. At 21 and 23 months, the patient was vaccinated twice with a personalized DNAJB1-PRKACA-derived peptide vaccine containing PA*68 / A*02, PB*44, PC*04 / C*05 and PII-1. It was observed that T cell responses specific to the vaccine peptides were induced 6 weeks after the second vaccination. (b) T cell responses specific to the vaccine peptides 6 weeks after the second vaccination, as assessed by IFNγ ELISPOT assay after in vitro stimulation with the vaccine cocktail peptides (PB*44 or PII-1) and comparison with the negative peptide (neg.). (c) Longitudinal analysis of vaccine-induced T cell responses up to 10 months post-vaccination assessed 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), and (d, e) flow cytometric characterization of the indicated cytokines.Fourteen weeks after the second vaccination, (d) cytokines released from CD4+ T cells stimulated with PII-1 peptide, and (e) cytokines released from CD4+ T cells and CD8+ T cells stimulated with PB*44 peptide, compared with those released by each negative peptide (neg.). [Figure 7] Characterization of primary tumor cells and continuous immune monitoring of FL-HCC patients vaccinated with personalized DNAJB1-PRKACA-derived peptide vaccines. (a) Representative photomicrographs of FL-HCC patients at diagnosis are shown. The top left panel shows HE staining, the top right panel shows Masson's trichrome staining, the bottom left panel shows immunohistochemical staining for CK7, and the bottom right panel shows immunohistochemical staining for Hepar1. Scale bar = 500 μm, 40x. (b) Sanger sequencing of reverse transcription polymerase chain reaction (RT-PCR) products to confirm chimeric transcripts linked at the end of exon 1 of DNAJB1 and the beginning of exon 2 of PRKACA. (c) IFNγ ELISPOT assays of peptide-specific T cells from FL-HCC patients were performed after in vitro stimulation with the vaccine cocktail peptides PB*44 or PII-1 6 weeks after the first vaccination (V1M23), 6 weeks after the second vaccination (V2M24), and 45 weeks after the second vaccination (V2M33) compared to stimulation with negative peptide (neg.). [Figure 9]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 PII-1-stimulated PBMCs obtained from FL-HCC patients 31 weeks after the second vaccination of the personalized DNAJB1-PRKACA-derived peptide vaccine. (a) Uniform Manifold Approximation and Projection (UMAP) plot showing characteristic T cell clusters. (b) Heatmap of genes defining activated T cell, exhausted / late effector T cell and resting naive T cell clusters. (c) Feature plot showing gene expression of IFNG, GZMB (encoding granzyme B), TNF and CCL3 defining activated T cell clusters. (d) UMAP plot showing T cell receptor (TCR) clonality of sequenced CD4+ T cells. Color code indicates the number of cells belonging to each type of expanded clone. (e) Distribution of the 10 largest TCR clonotypes in the various cell clusters identified in PII-1-specific CD4+ T cells. (f) Physiochemical properties and amino acid sequences of the CDR3α and CDR3β regions of the most frequent TCR clones compared with the target peptide PII-1 of these TCR clones. The y-axis indicates hydrophilicity according to the Hopp-Woods scale. Amino acids (AA) are grouped according to their physicochemical properties and color-coded. [Figure 10]An exemplary sample showing the gating strategy for evaluating moDC-primed CD4+ T cells by intracellular cytokine staining (ICS) is shown. An initial gate identified lymphocytes (FSC-A vs. SSC-A), which were further gated to identify single cells (FSC-A vs. FSC-H), which were further gated to identify live cells (FSC-A vs. Aqua live / dead), and further gated 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] Illustrates the gating strategy for evaluating intracellular cytokine staining of CD8+ T cells primed with aAPC. An exemplary sample illustrates the gating strategy for intracellular cytokine staining (ICS) of CD8+ T cells primed with artificial antigen presenting cells (aAPC). The first gate identified lymphocytes (FSC-A vs. SSC-A), which were further gated to identify single cells (FSC-A vs. FSC-H), which were further gated to identify live cells (FSC-A vs. Aqua live / dead), and further gated to identify CD8+ cells (FSC-A vs. CD8-PE-Cy7). In CD8+ T cells, we analyzed the expression of IFNγ (IFNγ-PE vs. CD8-PE-Cy7), TNF (TNF-Pacific Blue vs. CD8-PE-Cy7), CD107a (CD107a-FITC vs. CD8-PE-Cy7), and TNF / IFNγ (TNF-Pacific Blue vs. IFNγ-PE). [Figure 12]Gating strategy for evaluating peptide-specific CD4+ and CD8+ T cells from FL-HCC patients vaccinated with personalized DNAJB1-PRKACA-derived peptide vaccines by intracellular cytokine staining is shown. Exemplary samples showing the gating strategy for evaluating peptide-specific CD4+ and CD8+ T cells from FL-HCC patients vaccinated with personalized DNAJB1-PRKACA-derived peptide vaccines by intracellular cytokine staining (ICS) are shown. The first gate identified lymphocytes (FSC-A vs. SSC-A), which were further gated to identify single cells (FSC-A vs. FSC-H), and further gated to identify live cells (FSC-A vs. Aqua live / dead). Cytokine production of CD4+ and CD8+ T cells (CD4-APC-Cy7 vs. CD8-PE-Cy7) was analyzed separately. In CD8+ T cells, we analyzed 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), and in CD4+ T cells, we analyzed 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). [Figure 13] 1 shows a gating strategy for evaluating PA*24 tetramer staining of CD8+ T cells. An exemplary sample shows a gating strategy for evaluating PA*24 tetramer staining of CD8+ T cells after priming with antigen presenting cells (aAPCs). The first gate identified lymphocytes (FSC-A vs. SSC-A), which were further gated to identify single cells (FSC-A vs. FSC-H), which were further gated to identify live cells (FSC-A vs. Aqua live / dead), and CD8+ / PA*24 tetramer-specific cells were analyzed (PA*24 tetramer-PE vs. CD8-PE-Cy7). [Figure 14]1 shows a gating strategy for evaluating mature moDCs. An exemplary sample shows a gating strategy for evaluating mature monocyte-derived dendritic cells (moDCs). moDCs were identified by initial gating (FSC-A vs. SSC-A) and further analyzed for 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-based 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 tumor antigen recognition and T cell-mediated cytotoxicity to eliminate cancer cells, and have only been successfully used in a few cancer types and single tumors. One of the main specific reasons for developing antigen-specific immunotherapies is the lack of suitable target structures naturally presented on the cell surface of tumor cells in a high frequency and tumor-exclusive manner 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. With regard to the presentation of tumor antigens via HLA molecules, neoepitopes resulting from tumor-specific mutations have been recently identified as the main specificity of anti-cancer T cell responses induced by immune checkpoint inhibitors, and have been suggested to be the most promising candidates for T cell immunotherapy. At the same time, responses to immune checkpoint inhibitors have been found to correlate with a high tumor-borne somatic mutation burden, 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 widespread application of tumor antigens, especially in patients with low mutation burden. In addition, fusion transcripts that are translated into products that often function as drivers of clonal tumorigenesis have recently been identified as a new source of highly immunogenic neoepitopes. T cell responses against neoepitopes derived from such fusion proteins have been detected in patients treated with immune checkpoint inhibitors and correlated with therapeutic response.

[0128] The DNAJB1-PRKACA fusion transcript is a transcript of exon 1 of the DnaJ homolog subfamily B member 1 gene (DNAJB1) linked to the cAMP-dependent protein kinase catalytic subunit alpha gene (PRKACA). This fusion transcript was first identified in fibrolamellar hepatocellular carcinoma (FL-HCC). FL-HCC is a rare but lethal tumor disease with no established treatment options other than surgical resection, and generally occurs in children and young adults with no history of primary liver disease. DNAJB1-PRKACA fusions have also been detected in various other gastrointestinal tumor diseases. In FL-HCC, DNAJB1-PRKACA fusion transcripts were detectable in 100% of patients, and expression of this fusion transcript was demonstrated in all tumor cells, identifying it as an oncogenic driver in tumor pathogenesis. Herein, we demonstrate that the DNAJB1-PRKACA fusion transcript is a major source of broadly applicable neoepitopes and are able to demonstrate for the first time its applicability in 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 + Inducing T cells The in silico prediction workflow using the NetMHCIIpan algorithm identified nine 9-amino acid (AA) long binding cores from a total of 1290 different HLA class II alleles within the 24 amino acid long fusion region of the DNAJB1-PRKACA protein (Fig. 1a, b). 83.49% of these alleles are HLA-DP combinations, 11.55% are HLA-DQ combinations, and 4.96% are HLA-DR combinations. The core sequence RYGEEVKEF (SEQ ID NO: 5), located exactly in the middle of the aforementioned fusion transcript (consisting of 5 amino acids in exon 1 (DNAJB1) and 4 amino acids in exon 2 (PRKACA)), had the highest amount of predicted HLA class II alleles (60.52% of the possible binding alleles for the entire combination of binding cores). Analysis using a combined prediction workflow of the SYFPEITHI and NetMHCpan algorithms identified 13 DNAJB1-PRKACA-derived HLA ligands for the 20 most frequent HLA class I allotypes in the European population, including 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 as an HLA class I ligand binding to the HLA-A*24:02 allele, HLA-C*04:01 allele, HLA-C*06:02 allele, and HLA-C*07:02 allele.

[0130] Monocytes obtained from healthy volunteers (HV) were induced to differentiate into mature monocyte-derived dendritic cells (moDCs; Fig. 1d), which were then transfected with the 22 amino acid long KREIFDRYGEEVKEFLAKAKED (P II-1 ) peptide (SEQ ID NO:1) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) showed that the native HLA-presented peptide derived from DNAJB1-PRKACA was presented. This mass spectrometry (MS) confirmed that the experimentally eluted PII-1 The fragment ion spectrum of P was identified by MS (Figure 1e). II-1 and a variant 12 amino acids shorter than this was predicted to bind to the HLA-DP alleles DPA1*01:03-DPB1*05:01 of each healthy volunteer. In the binding ranking of NetMHCIIpan, the binding core RYGEEVKEF (SEQ ID NO: 5) contained in the peptide containing SEQ ID NO: 1 (FIG. 1b) showed the best score of 0.74. II-1 Mature moDCs carrying CD4 + De novo priming of T cells resulted in P II-1 Pulsing with IL-14 resulted in the formation of multifunctional P cells expressing CD107a, CD154, IL-2, IFNγ, and TNF. II-1 specific CD4 + T cells were induced (Fig. 1f).

[0131] 2.2 P A*24 specific CD8 + T cell characterization and single-cell TCR sequencing Ligand RYGEEVKEF (SEQ ID NO: 5) derived from the DNAJB1-PRKACA fusion protein (P A*24 , SYFPEITHI score: 74.19%, NetMHCpan binding rank: 0.018) was refolded using HLA-A*24-P A*24 The monomer was used to generate artificial antigen-presenting cells (aAPCs) derived 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%; Fig. 2a, b). A*24 specific CD8 + T cells displayed a polyfunctional phenotype reflected by the production / expression of IFNγ, TNF and CD107a (Fig. 2c), and in vitro P A*24 Carrying autologous CD8 -The lysis of target cells at various effector:target ratios reached a maximum of 82.39% compared to nonspecific effector cells (Figure 2d). In addition, P from two healthy volunteers (HV1, HV2) bulk-sorted by flow cytometry after in vitro priming with aAPCs was significantly increased in 10 healthy volunteers (HV1, HV2) and lysed the effector cells. A*24 specific CD8 + Single-cell T cell receptor (TCR) sequencing of T cells revealed high clonality in one TCR clone that was predominant in HV2 and two TCR clones that were predominant in HV1 (Fig. 3a, b). Although there was no overlap in the V(D)J gene sequences in these three TCR clones, the physiochemical properties of the amino acids in the CDR3α and CDR3β sequences were highly similar, and the target peptide P of these TCR clones was highly similar to that of the TCR clones. A*24 , which were shown to have opposing properties in terms of hydrophilicity and chemical property groupings (Figures 2e and 3c).

[0132] 2.3 Doxycycline-inducible expression of the DNAJB1-PRKACA fusion gene results in naturally processed and presented 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 to specifically express the DNAJB1-PRKACA fusion protein (Fig. 4a, b, Fig. 5a). After expression of the DNAJB1-PRKACA fusion gene, immunopeptidome analysis of each HCC cell line by mass spectrometry detected up to 3688 HLA class I ligands, including various peptides containing any of SEQ ID NOs: 6-63 (average 2787; Fig. 4c). Twenty unique HLA class I ligands derived from the DNAJB1-PRKACA fusion protein were identified, of which two ligands had sequences spanning the aforementioned fusion region (Fig. 4d). Of the two naturally processed and presented DNAJB1-PRKACA derived neoepitopes, EIFDRYGEEV (SEQ ID NO: 3) ( 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, and EIFDRYGEEV (SEQ ID NO: 3) (P A*68 / A*02 ) binds to the HLA allotype A*68:02 of the SMMC-7721 cell line, and IFDRYGEEV (SEQ ID NO: 4) (P C*04 / C*05 ) was predicted to bind to the HLA allotype C*04:01 of the HepG2 cell line, and this result could be verified by comparison of the spectral analysis using synthetic peptides (Fig. 1c and Fig. 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 activating the expression of the DNAJB1-PRKACA fusion gene (Fig. 5b). Immunopeptidome analysis of these moDCs by mass spectrometry revealed that up to 8293 HLA class II peptides were detected (average 5956; Fig. 4f), of which 13 unique peptides were identified as derived from the DNAJB1-PRKACA fusion protein, including one peptide EVKEFLAKAKEDFLKK (SEQ ID NO: 2) (P II-2 ) had a sequence spanning the aforementioned fusion region (Fig. 4g). II-2 was predicted to bind to the HLA allele DRB1*13:02 in healthy volunteers (Fig. 1b). II-2 The peptide fragment ion spectrum experiments were verified (Figure 4h). It should be noted that in the respective negative controls, no HLA class I or HLA class II ligands were identified from these two fusion proteins.

[0133] 2.4 A personalized DNAJB1-PRKACA-derived peptide vaccine induced long-term DNAJB1-PRKACA-specific immune responses and demonstrated favorable clinical outcomes in patients with FL-HCC A personalized DNAJB1-PRKACA-derived peptide vaccine was formulated for a young patient (FL-HCC01) with histologically confirmed FL-HCC. The young patient underwent liver transplantation (LTx) early in the disease, but had recurrent multiple tumors due to chemotherapy-refractory unresectable FL-HCC (Figures 6a and 7a). For post-transplant immunosuppression, the mTOR inhibitor everolimus was administered. Due to alterations in DNA damage response (DDR) pathways (ATM and CHEK2, germline variants, and somatic deletion of BRCA2 and BAP1) and failure to achieve long-term remission, poly ADP-ribose polymerase (PARP) inhibition was initiated. Resection or radiation therapy of recurrent tumors that developed was performed. Based on the DNAJB1-PRKACA fusion gene confirmed by Sanger sequencing, the personalized vaccine was targeted to three allotype-matched short HLA class I ligands (EIFDRYGEEV (SEQ ID NO: 3) (P A*68 / A*02 ), EEVKEFLAKA (SEQ ID NO: 120) (P B*44 ), and IFDRYGEEV (SEQ ID NO: 4) (P C*04 / C*05 )) and the aforementioned long peptide KREIFDRYGEEVKEFLAKAKED (SEQ ID NO: 1) (P II-1 ) and 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 supports the activation and maturation of antigen-presenting cells and prevents rapid degradation of the vaccine peptide, thereby enabling the induction of effective and potent T cell responses (Figure 7b). This personalized DNAJB1-PRKACA-derived peptide vaccine was administered twice, 6 weeks apart. After stimulation with the vaccine cocktail peptide in vitro, analysis by IFNγ enzyme-linked immunospot (ELISPOT) assay showed that P was significantly increased 6 weeks after the second vaccination. II-1We observed that a significant peptide-targeted T cell response was induced (mean number of spots after the second vaccination was 872 compared to 1 before vaccination; Fig. 6b, c and Fig. 7c). B*44 Weak T cell responses were detected targeting P (mean number of spots after the second vaccination was 56 compared to 0 before vaccination). Longitudinal experiments using IFNγ ELISPOT assays demonstrated that P II-1 Specific T cells were maintained and showed a further increase in intensity 45 weeks after the second vaccination (mean number of spots: 1088) (Figures 6c and 7c). B*44 and P II-1 Flow cytometric evaluation of the immune response targeting the IL-11 receptor revealed that CD4+ T helper 1 (Th1) phenotype specifically expressed IFNγ and TNF. + T cells were found. B*44 CD8 against + Notably, no disease recurrence was observed in FL-HCC01 until a recent examination 15 months after the second vaccination, suggesting for the first time the clinical efficacy of vaccine-induced DNAJB1-PRKACA-specific T cell responses.

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

[0135] 3. Materials and Methods 3.1 Patients and blood samples Peripheral blood mononuclear cells (PBMCs) were isolated from FL-HCC patients by density gradient centrifugation, and PBMCs were isolated from healthy volunteers (HV) and stored at -80 °C until use in subsequent assays with T cells. Informed consent was obtained according to the Declaration of Helsinki. The study was performed in accordance with the guidelines of the local ethical 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 produced in the GMP-compliant Peptide Laboratory of the Department of Immunology at the University of Tübingen, and contains four DNAJB1-PRKACA-derived peptides (SEQ ID NOs: 1, 3, 4 and 120) and the synthetic lipopeptide TLR1 / 2 ligand XS15 (Bachem, Bubendorf, Switzerland) as an adjuvant. TM This is a peptide-based vaccine emulsified in ISA51 VG (Seppic, Paris, France). The vaccine peptides (250 μg of each peptide) and XS15 (50 μg) were combined with Montanide TM ISA51 VG was added and formulated as a 1:1 water-in-oil emulsion to obtain an injectable volume of 500 μL. The personalized vaccine was injected subcutaneously into the lower abdomen of the patient. The personalized vaccination was performed under protocol NCT05014607.

[0137] 3.3 Detection and sequencing of DNAJB1-PRKACA transcripts RNA was extracted from macrodissected 5 μm paraffin sections using a Maxwell® RSC instrument (Promega, Madison, WI, USA) and the Maxwell RSC RNA FFPE kit according to the manufacturer's instructions. Reverse transcription of RNA and polymerase chain reaction (PCR) of the DNAJB1-PRKACA breakpoint region was performed using a 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)). PCR products were run on an agarose gel to visualize the detection of the DNAJB1-PRKACA fusion protein. For sequencing, PCR products were purified (AMPure, Beckman Coulter, Brea, CA, USA), aliquots were taken, and sequencing reactions were performed in 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, Brea, CA, USA) according to the manufacturer's protocol. Sequencing reactions were purified (CleanSEQ, Beckman Coulter, Brea, CA, USA), analyzed on a GenomeLab GeXP Genetic Analysis System, and evaluated with GenomeLab GeXP software (Beckman Coulter, Brea, CA, USA).

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

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

[0140] 3.6 Doxycycline-induced expression of the DNAJB1-PRKACA fusion gene in HCC cell lines HCC cell lines, HLE, SMMC-7721, and HepG2, were cultured in Gibco's Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), penicillin, streptomycin (Merck), and plasmocin (Invivogen) at 37°C and 5% CO2 in a humidified atmosphere. The DNAJB1-PRKACA coding sequence was synthesized by Thermo Fisher and transformed with pENTR TM Cloning into vector and directional TOPO cloning (pENTR TM / D-TOPO TMThe DNAJB1-PRKACA vector was transferred into the pInducer20 (Addgene, No. 44012) destination vector using a cloning kit (Invitrogen). HEK293T cells were transfected with the helper plasmids psPAX2 and pMD2.G and the pInducer20 vector encoding DNAJB1-PRKACA or the empty vector by the calcium phosphate method to produce lentiviral particles. Each transduced HCC cell line was selected with G418 (Invivogen) for at least 2 weeks. To induce the expression of DNAJB1-PRKACA, each transduced HCC cell line was treated with 1 μg / ml doxycycline (Dox) (AppliChem) for 24 hours. To confirm DNAJB1-PRKACA expression, cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na4P2O7, 10 mM Na4V2O7, and Complete protease inhibitor cocktail (Roche)). SDS-PAGE and immunoblotting were performed as previously reported. 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. HRP-conjugated goat anti-rabbit or HRP-conjugated goat anti-mouse secondary antibodies (both Jackson ImmunoResearch) were used for visualization.

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

[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, MA, USA) and then analyzed on an online-connected Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher, Waltham, MA, USA). Samples were analyzed in triplicate. Five μL of sample, which was 20% occupied, was injected onto a 75 μm × 2 cm trapping column (Thermo Fisher, Waltham, MA, USA) at a rate of 4 μL / min for 5.75 min. Peptides were then separated on a 50 μm × 25 cm separation column (PepMap C18, Thermo Fisher, Waltham, MA, USA) at 50 °C with a flow rate of 300 nL / min, using a gradient of ACN increasing from 2.4% to 32.0% for 90 min. Eluted peptides were ionized by nanospray ionization. Next, a high-energy C-trap dissociation (HCD) method was performed at the fastest speed (3 s) to obtain a fragment spectrum with a resolution of 30,000 and a mass range restricted to 235–1151 m / z, to select peptides with positive charges between +2 and +5 to be fragmented and analyzed by the mass spectrometer.

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

[0144] 3.10 Spectral Verification Validation of the experimentally eluted peptide spectra was performed by calculating the spectral similarity with the corresponding synthetic peptides measured in the complex matrix. Spectral correlations were calculated between the MS / MS spectra of the eluted peptides and the MS / MS spectra of the synthetic peptides.

[0145] 3.11 Peptide-specific T cell proliferation and IFNγ ELISPOT assay PBMCs were pulsed with 1 μg / ml HLA class I peptide or 5 μg / ml HLA class II peptide. Each peptide with irrelevant HLA restriction was used as a negative control. Specifically, YLLPAIVHI (SEQ ID NO: 124) (derived protein: DDX5_HUMAN) was used as a negative control for HLA-A*02, and ETVITVDTKAAGKGK (SEQ ID NO: 125) (derived protein: FLNA_HUMAN) was used as a negative control for HLA class II. On days 2, 5, and 7, PBMCs were cultured for 12 days with the addition of 20 U / ml IL-2 (Novartis, Basel, Switzerland). On day 12, PBMCs stimulated with each peptide were analyzed by IFNγ enzyme-linked immunospot (ELISPOT) assay. Spots were counted using an ImmunoSpot S6 analyzer (CTL, Cleveland, OH, USA), and T cell responses were considered positive if more than 10 spots were counted per 500,000 cells and the mean number of spots was at least three times that of the negative control.

[0146] 3.12 Refolding Biotinylated HLA:peptide complexes were prepared as previously reported and tetramerized with PE-labeled streptavidin (Invitrogen) at a molar ratio of 4:1.

[0147] 3.13 Peptide-specific CD8 by aAPC + T cell induction Priming of peptide-specific cytotoxic T lymphocytes was performed using artificial antigen-presenting cells (aAPCs) as previously reported. Specifically, 800,000 streptavidin-coated microspheres (Bangs Laboratories, Fishers, IN, USA) were loaded with 200 ng of biotinylated HLA:peptide monomer and 600 ng of biotinylated anti-human CD28 monoclonal antibody (clone 9.3, produced in-house). 4.8 U / μl IL-2 (R&D Systems, Minneapolis, MN, USA) and 1.25 ng / ml IL-7 (PromoKine, Heidelberg, Germany) were added to prime CD8 + T cells were cultured. aAPC (1 × 10 6 CD8 + Four cycles of weekly stimulation with 200,000 aAPC per T cell and 5 ng / ml IL-12 (PromoKine) were performed.

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

[0149] Peptide-specific CD8 after priming with aAPCs + The frequency of T cells 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 primed with the irrelevant control peptide TYSEKTTLF (SEQ ID NO: 128) (protein of origin: MUC16_HUMAN) and stained with tetramers containing the test peptide were used. Peptide-specific CD8 + The frequency of T cells in a live single cell population is + More than 0.1% of total T cells and peptide-specific CD8 + Priming was considered successful if the frequency of T cells was at least three times higher than that of T cells. The same evaluation criteria were applied to the intracellular cytokine staining results. Samples were analyzed on a FACS Canto II cytometer (BD). The gating strategy applied to the tetramer staining analysis of the data acquired by flow cytometry is shown in Supplementary Figure 13.

[0150] 3.15 Cytotoxicity Assays Peptide-specific CD8 mAbs were assayed for their ability to induce peptide-specific target cell lysis in a flow cytometric VITAL assay. + T cells were analyzed. CD8 - Autologous target cells, P A*24 The peptide or negative peptide KYPENFFLL (SEQ ID NO: 126) (derived protein: PP1G_HUMAN) was carried and labeled with CFSE or FarRed (Life Technologies, Carlsbad, CA, USA), respectively. A*24 Specific effector cells were added at the effector-to-target ratios shown in the graph. - Specific lysis of target cells was calculated relative to control target cells.

[0151] 3.16 Peptide-specific CD4 by peptide-loaded moDCs + T cell induction To induce differentiation of monocyte-derived dendritic cells (moDCs), CD14 cells were isolated from PBMCs using magnetic activated cell sorting (MACS; Miltenyi, Bergisch Gladbach, Germany). + Cells were isolated and then cultured in a culture medium containing penicillin, streptomycin, GM-CSF, and IL-4. TM The cells were cultured in serum-free medium for hematopoietic cells at 37°C and 5% CO2 for 7 days in a humidified atmosphere. The differentiated moDCs were matured by adding LPS to the cell culture medium for 24 hours and the cell surface expression of CD80-FITC (1:40 dilution, Biolegend, Cat. No. 305206), HLA-DR-BV 711 (1:100 dilution, Biolegend, Cat. No. 307644), and CD86-BV 605 (1:400 dilution, Biolegend, Cat. No. 374214) was confirmed. The gating strategy applied to the analysis of the data obtained by flow cytometry is shown in Figure 14. CD4 + Prior to use in stimulating T cells, mature moDCs were cultured using II-1 The PBMCs of the same healthy volunteers (HVs) were incubated with the peptide for 2 h. Then, CD4+ cells were isolated from the PBMCs of the same healthy volunteers (HVs) using magnetic activated cell sorting (MACS; Miltenyi, Bergisch Gladbach, Germany). + Cells were isolated and cultured x-vivo with the addition of penicillin, streptomycin, IL-2, and IL-7. TM The cells were cultured in serum-free medium for hematopoietic cells at 37°C and 5% CO2 in a humidified atmosphere. + The cells were stimulated with peptide-loaded mature moDCs and IL-12 once a week for a total of 4 weeks. + T cell functionality was analyzed by intracellular cytokine staining.

[0152] 3.17 Loading of antigens onto mature moDCs To prepare tumor lysates, HCC cell lines transduced with DNAJB1-PRKACA-encoding plasmid or empty plasmid were treated with IFNγ and Dox for 24 h. Treated cells were harvested, washed with PBS, subjected to five freeze-thaw cycles, irradiated with 30 Gy, and sonicated for 2 min. The collected clear supernatant was then added to cell culture medium of mature moDCs and incubated for 24 h, after which 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 and statistical analyses were performed using GraphPad Prism 9.2.0 (GraphPad Software, Inc.). A P value of <0.05 was considered to indicate statistical significance.

[0154] 3.19 Single cell immune profiling Following the 10× Genomics cell preparation protocol, peptide-specific CD8 + T cells or bulk CD4 +T cells were FACS sorted, counted, and washed with 0.04% BSA / PBS. Single cells were partitioned individually into Gel Beads-in-Emulsions (GEMs) with 10× barcoded gel particles and reverse transcriptase using a Chromium Controller instrument (10× Genomics, Pleasanton, CA, USA). Single-cell gene expression and single-cell T cell receptor (VDJ) libraries were then prepared using the Chromium Next GEM Single Cell 5' Kit v2 (10× Genomics, Pleasanton, CA, USA) and the Chromium Single Cell Human TCR Amplification Kit (10× Genomics, Pleasanton, CA, USA) according to the manufacturer's instructions. These libraries were pooled and sequenced using a NextSeq 550 (Illumina, San Diego, CA, USA) with an average of 28,806, 138,975, and 3519 reads per cell. Samples were demultiplexed using bcl2fastq version 2.20.0.422 (Illumina, San Diego, CA, USA). Barcoding, alignment, VDJ annotation, and single-cell 5' gene counts were performed using Cell Ranger software version 6.0.1 (10× Genomics, Pleasanton, CA, USA). Data processing, visualization, and analysis were performed separately for each sample using scanpy and scirpy. Cells with unique gene counts <200 and no association with vdj sequences and cells with >10% mitochondrial genes were excluded from the analysis, resulting in 474 cells (FL-HCC01), 115 cells (HV1), and 3338 cells (HV2). Data were log-normalized with a scale factor of 10,000. A linear dimensionality reduction was performed taking into account only highly variable genes, and highly variable genes were defined as those with a mean minimum expression of 0.0125, a mean maximum expression of 3, and a minimum variance of 0.5. The effects of total counts and mitochondrial mass were removed.Dimensionality reduction was performed using Principal Component Analysis (PCA). The UMAP algorithm RAPIDS was run on the 10 first principal components (n_PC=10) to calculate a neighborhood graph. The Louvain algorithm RAPIDS was run for unsupervised clustering.

Claims

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

2. The peptide according to claim 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 comprises a consecutive sequence of amino acids set forth in any one of SEQ ID NOs: 1 to 5.

3. An antibody that specifically recognizes the peptide or its variant according to claim 1 or 2, in particular a soluble antibody or a membrane-bound antibody, more preferably a monoclonal antibody or a fragment thereof, preferably an antibody that specifically recognizes the peptide or its variant according to claim 1 or 2 when the peptide or its variant is bound to an MHC molecule.

4. A T cell receptor or a fragment thereof that reacts with an HLA ligand which is a peptide or a variant thereof according to claim 1 or 2, preferably a soluble T cell receptor or a membrane-bound T cell receptor, and preferably a T cell receptor that reacts with the peptide or a variant thereof according to claim 1 or 2 when the peptide or a variant thereof is bound to an MHC molecule.

5. 1. An antigen binding protein that specifically binds to at least one DNAJB1-PRKACA antigenic peptide complexed with a major histocompatibility complex (MHC) molecule, the antigen binding protein comprises a first polypeptide chain comprising a first variable domain comprising at least one complementarity determining region (CDR), CDR3α, and a second polypeptide chain comprising a second variable domain comprising at least one CDR, CDR3β; a) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:6, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:36, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; b) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:7, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:37, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; c) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:8, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:38, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; d) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:9, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:39, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; e) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 10, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 40, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; f) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:11, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:41, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:1; g) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 12, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 42, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; h) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 13, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 43, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; i) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 14, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; j) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 15, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 45, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; k) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 32, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 45, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; l) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 16, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 46, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; m) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 17, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 47, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; n) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 33, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 47, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; o) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 18, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 48, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 1; p) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 19, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 49, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; q) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 19, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 62, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; r) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:20, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:50, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; s) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:21, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:51, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; t) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:21, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:63, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; u) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:22, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:52, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; v) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:23, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:53, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; w) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:24, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:54, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; x) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:25, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:55, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; y) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 34, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 55, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; z) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:26, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:56, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; α) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:27, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:57, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; β) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:28, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:58, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; γ) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO:29, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO:59, and the DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO:5; δ) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 30, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 60, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; or ε) CDR3α comprises or consists of the amino acid sequence of SEQ ID NO: 31, CDR3β comprises or consists of the amino acid sequence of SEQ ID NO: 61, and said DNAJB1-PRKACA antigenic peptide comprises or consists of SEQ ID NO: 5; The CDR sequences may comprise one or more amino acid mutations selected from amino acid insertions, deletions and / or substitutions, Preferably, said antigen-binding protein is a single chain TCR (scTCR) or a bispecific single chain antibody, and / or Preferably, the first polypeptide further comprises an alpha chain constant domain or a gamma chain constant domain, and the second polypeptide further comprises a beta chain constant domain or a delta chain constant domain, and / or Preferably, the first variable domain is part of a TCR alpha or gamma chain, and / or Preferably, the second variable domain is part of a TCR beta or delta chain, and / or Preferably, the first variable domain is a variable domain of a TCR alpha and the second variable domain is a variable domain of a TCR beta, and / or Preferably, said antigen binding protein comprises at least one amino acid mutation that increases stability, cell surface expression and / or pairing. Antigen-binding proteins.

6. (i) one or more additional antigen-binding sites; (ii) a transmembrane region within the first polypeptide chain and / or the second polypeptide chain, which may include a cytoplasmic signaling region; (iii) diagnostic agents; (iv) a therapeutic agent; and (v) PK adjustment part and further comprising one or more of: Preferably, said antigen binding protein is a therapeutic and / or diagnostic antigen binding protein.

6. The antigen-binding protein of claim 5.

7. 10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide or a variant thereof according to claim 1 or 2, an antibody or a fragment thereof according to claim 3, a T cell receptor according to claim 4, or an antigen binding protein according to claim 5 or 6.

8. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 64-119.

9. A cloning or expression vector comprising the isolated nucleic acid molecule of claim 7 or 8.

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

11. 11. A recombinant host cell comprising a peptide or variant thereof according to claim 1 or 2, an antibody or fragment thereof according to claim 3, a T cell receptor according to claim 4, an antigen binding protein according to claim 5 or 6, an isolated nucleic acid molecule according to claim 7 or 8, or a vector according to claim 9 or 10, preferably selected from mammalian and human cells, 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-bearing human MHC class I molecule or an antigen-bearing 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 period of time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide described in claim 1 or 2 or a variant thereof.

13. Activated T cells that selectively recognize cells presenting a polypeptide comprising the peptide or its variant described in claim 1 or 2, or a polypeptide consisting of the peptide or its variant, produced by the method described in claim 12.

14. 1. A pharmaceutical composition, preferably a vaccine, comprising at least one active ingredient selected from the group consisting of the peptide or variant thereof according to claim 1 or 2, the antibody or fragment thereof according to claim 3, the T cell receptor according to claim 4, the antigen binding protein according to any one of claims 5 to 8, the isolated nucleic acid molecule according to claim 9 or 10, the vector according to claim 11 or 12, the recombinant host cell according to claim 13, the activated T cell according to claim 15 and a labelled or labelled active ingredient, and a pharmaceutically acceptable carrier, and optionally a pharmaceutically acceptable excipient and / or stabilizer.

15. A kit comprising: (a) a container containing a pharmaceutical composition comprising the peptide or variant thereof according to claim 1 or 2, the antibody or fragment thereof according to claim 3, the T cell receptor according to claim 4, the antigen binding protein according to claim 5 or 6, the isolated nucleic acid molecule according to claim 7 or 8, the vector according to claim 9 or 10, the recombinant host cell according to claim 11, or the activated T cell according to claim 13, in solution or in lyophilized form, (b) Freezing a second container containing a diluent or reconstitution solution for the dry formulation; (c) may contain at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 5; (d) optionally including instructions for (i) use of the solution, or (ii) reconstitution and / or use of the lyophilized formulation; kit.

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