Novel cancer antigens and methods

Abstract

Antigenic polypeptides and corresponding polynucleotides for use in the treatment or prevention of cancer, particularly in the treatment or prevention of malignant melanoma, are provided. [Solution] Provided are isolated polypeptides having specific amino acid sequences, and the isolated polypeptides fused to a further polypeptide selected from (i) one or more other polypeptides, (ii) other polypeptides that are melanoma-associated antigens, (iii) polypeptide sequences that can enhance immune responses, and (iv) polypeptide sequences that can provide strong CD4+ help to increase CD8+ T cell responses to antigen epitopes.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention is used in the treatment or prevention of cancer, and in particular in the treatment or prevention of malignant melanoma (e.g., Antigenic polypeptides and corresponding polypeptides used in cutaneous malignant melanoma or uveal malignant melanoma The present invention further relates to, inter alia, the nucleic acids and polypeptides described above, loaded with and / or stimulated by these polypeptides and polynucleotides. Immune cells specific for those polypeptides, as well as antibodies that recognize the polypeptides. Medicines containing cells (autologous or otherwise derived) genetically engineered with molecules and immunogenicity It relates to a composition. [Background technology]

[0002] BACKGROUND OF THE INVENTION As part of normal immune surveillance against pathogenic microorganisms, all cells produce intracellular proteins on major histocompatibility complex (MHC) class I molecules expressed on the surface of all cells. Most of these peptides originate from the host cell. are recognized as self and remain unrecognized by the adaptive immune system. The peptide encodes a T cell receptor (TCR) that tightly binds to the MHC I-peptide complex. This expanded T cell population can stimulate the proliferation of foreign antigens. Effector CD8+ T cells (including cytotoxic T lymphocytes, CTLs) that can eliminate the original tagged cells and, further, when the foreign antigen-tagged cells appear later in the animal's life, Memory CD8+ T cells can also be generated that can be re-expanded.

[0003] Expression of MHC class II molecules is usually observed on professional antigen-presenting cells such as dendritic cells (DCs) ( MHC class II molecules are normally restricted to antigen-binding proteins (APCs), and MHC class II molecules are normally restricted to antigen-binding proteins (APCs) internalized from the exogenous environment. loaded with peptides. T cell adhesion molecules (CD54, CD48) and costimulatory molecules (CD40, CD80, CD In the presence of various factors, including 86), complementary TCRs from naive CD4+ T cells recognize MHCII-peptides. Upon binding to the complex, CD4+ T cell effector cells (e.g., T H 1. T H 2. T H 17, T FH , T r eg These effector CD4+ T cells are induced to mature into antibody-secreting B cells. It not only promotes differentiation into plasma cells but also promotes differentiation of antigen-specific CD8+ CTLs, which Thus, immune responses to foreign antigens, including both short-term effector functions and long-term immunological memory, are essential. DCs help induce adaptive immune responses. They also act to transduce exogenously derived antigens (e.g., released by pathogens or tumor cells). can deliver a target molecule (such as a peptide or protein) onto those MHC I molecules, thereby Cross-presentation of peptide antigens is carried out to stimulate naive CD8+ T cells. By providing an alternative pathway for stimulating proliferation, it contributes to the generation of immunological memory.

[0004] Immunological memory (especially antigen-specific B cells / antibodies and antigen-specific CTLs) plays a key role in controlling microbial infections. immunological memory plays an important role in preventing diseases caused by important pathogenic microorganisms. Immunological memory has also been used to control tumorigenesis. Although it is known that cancer plays an important role in cancer prevention, few effective cancer vaccines have been developed. do not have.

[0005] Cancer is the second most common cause of morbidity and accounts for nearly one-sixth of all deaths worldwide.2 Of the 8.8 million cancer deaths in 2015, the most deadly cancers were lung cancer (1.69 million), Liver cancer (788,000), colorectal cancer (774,000), stomach cancer (754,000) and breast cancer (571,000). The economic impact of cancer in 2010 was estimated at US$1.16 trillion, with the number of new cases expected to grow by 2050. It is expected to increase by approximately 70% over a 20-year period (World Health Organization, Cancer Facts, 2017).

[0006] Current treatments for cutaneous melanoma are diverse and depend largely on the location of the tumor and the stage of the disease. The primary treatment for non-metastatic melanoma is surgery to remove the tumor and surrounding tissue. Late-stage melanoma may require treatment including lymph node dissection, radiation therapy, or chemotherapy. Immune checkpoint blockade strategies target negative immune regulators such as PD-1 / PD-L1 and CTLA4. These include the use of targeted antibodies and have recently been used to treat a variety of malignancies, including melanoma. (Ribas, A. and Wolchok, J.D., (2018) Science, 359:1350-135 5) The extraordinary value of checkpoint inhibitor therapy and its clinical benefits and patients' own cancer control. The well-known association of a patient's adaptive immune response (especially T cell-based immune responses) with the antigen is This has stimulated the search for effective cancer vaccines, vaccine modalities, and cancer vaccine antigens.

[0007] Human endogenous retroviruses (HERVs) are exogenous infectious retroviruses that are transmitted through the ancestral germline. HERVs are relics of integration into the long terminal repeats (LTRs) flanking the viral genome. ) belongs to a group of endogenous retroelements characterized by the presence of Since it also contains apparent mammalian LTR retrotransposons (MaLR), they are collectively referred to as LTR elements. (Hereinafter, all LTR elements are collectively referred to as ERVs.) ERVs comprise a significant proportion (8%) of the mammalian genome and are classified into approximately 100 families based on sequence homology. Many ERV sequences encode defective proviruses, which These are prototype retroviruses consisting of gag, pro, pol, and env genes flanked by LTRs. Some intact ERV ORFs are required for the transmission of exogenous infectious retroviruses such as HIV-1. Retroviral proteins that share characteristics with proteins encoded by viruses Such proteins may act as antigens to induce strong immune responses. (Hurst and Magiorkinis, 2015, J. Gen. Virol 96:1207-1218), which Polypeptides encoded by ERVs are involved in the T cell and B cell receptor selection process and These results suggest that ERV products can circumvent central and peripheral tolerance. Immune reactivity against ERVs can occur naturally in infections and cancers, and ERV products are expressed in several It has been implicated in the pathogenesis of autoimmune diseases (Kassiotis and Stoye, 2016, Nat Rev. Immunol. 16:207-219).

[0008] Due to the accumulation of mutations and recombination events during evolution, most ERV-derived sequences loss of functional open reading frames of some or all of these genes, resulting in However, these ERV elements have lost their ability to produce infectious virus. Like other genes, they are maintained in germline DNA, and at least some of these genes In fact, HERV-encoded proteins have the potential to produce proteins from the Proteins have been detected in various human cancers. For example, splice variants of the HERV-K env gene Rec and Np9, which are found only in malignant testicular germ cells, are not found in healthy cells ( Ruprecht et al., 2008, Cell Mol Life Sci 65:3366-3382). In cancers such as prostate cancer, Elevated levels of HERV transcripts have been observed in tissues compared to healthy tissues (Wang-Johanning, 2004). , 2003, Cancer 98:187-197; Andersson et al., 1998, Int. J. Oncol 12:309-313). Furthermore, overexpression of HERV-E and HERV-H has been shown to be immunosuppressive, and these also , which could contribute to cancer progression (Mangeney et al., 2001, J. Gen. Virol. 82 However, the actual mechanisms by which HERVs can contribute to cancer progression or pathogenesis remain unclear. The mechanism is still unknown.

[0009] In addition to deregulating the expression of surrounding adjacent host genes, Activation and translocation to new genomic sites can lead to the generation of novel transcripts, Some of them may be carcinogenic (Babaian and Mager, Mob. DNA, 2009). 16, Lock et al., PNAS, 2014, 111:3534-3543).

[0010] A wide range of vaccine modalities are known. One well-described approach is immunotherapy. The antigenic polypeptide is delivered to a subject to enhance immune responses (including B cell and T cell responses) and stimulate immune memory. Alternatively, the polynucleotide may be delivered by direct delivery of the polypeptide. The vector is used to express the immunogenic polypeptide encoded by the peptide in vivo. The use of viral vectors, such as adenoviral vectors, can be In both preventive vaccination strategies and therapeutic treatment strategies against cancer, This has been extensively explored for the purpose of ors for Gene Therapy, Vaccination and Cancer Gene Therapy, 13:421-433). immunogenicity Peptides, polypeptides, or polynucleotides encoding them may also be used to administer patient-derived antibodies. can be used to load antigen-presenting cells (APCs), which then elicit a therapeutic or prophylactic immune response. Alternatively, the vaccine can be injected into the subject as a vaccine to induce Pr It is the only anti-cancer vaccine currently approved by the FDA.

[0011] Cancer antigens also offer a promising therapeutic approach by using them to create a variety of non-vaccine therapeutic modalities. These therapies include: 1) antigen-binding biologics; 2) adoptive tumor suppressors; Cellular therapies fall into two distinct classes.

[0012] Antigen-binding biologics typically recognize antigen-decorated cancer cells and promote their destruction. These biologics are composed of multivalent polypeptides engineered to bind to the antigen. The component may comprise a TCR-based biologic, which may comprise a TCR, a high affinity TCR, and TCR mimetics produced by various technologies (including those based on monoclonal antibody technology). The cytolytic components of these types of multivalent biologics include, but are not limited to, , cytotoxic chemicals, biotoxins, targeting motifs that facilitate immune cell targeting and activation. The antibody may comprise a phospholipid and / or an immunostimulatory motif, both of which are useful for therapeutic destruction of tumor cells. It promotes destruction.

[0013] Adoptive cell therapy may be based on a patient's own T cells, which are extracted and administered as a vaccine. stimulated in vitro with a chromosomal antigen preparation (other factors containing cellular and acellular components) (JCI Insight. October 2018, 4;3(19 pii:122467. doi:10.1172 / jci.insight.122467). Alternatively, adoptive cell therapy can target cancer antigens. Cells (patient-derived or non-patient-derived) deliberately engineered to express an antigen-binding polypeptide that recognizes the target gene. These antigen-binding polypeptides can be based on human cells, including cells derived from the patient. These fall into the same class as those described above for antigen-binding biologics. A phospholipid (autologous or non-autologous) gene that has been genetically engineered to express an antigen-binding polypeptide. The cells can also be administered to patients as adoptive cell therapy to treat their cancer.

[0014] The use of ERV-derived antigens to mount an effective immune response against cancer has been shown to be useful in cancer rodents. Promising results in promoting tumor regression and leading to more favorable prognosis in patients with rheumatoid arthritis (Kershaw et al., 2001, Cancer Res. 61:7920-7924; Slansky et al., 2000, Immunity 13:529-538). Therefore, due to the severe restriction of identified tumor-specific ERV antigens, HERV antigen-centered immunotherapy trials despite limited research progress has been studied in humans (Sacha et al., 2012, J. Immunol 189:1467-1479).

[0015] WO 2005 / 099750 identifies anchor sequences for existing vaccines against infectious pathogens. These commonly enhance cross-reactive immune responses to HERV-K Mel tumor antigens, leading to malignant progression. It confers protection against melanoma. WO 00 / 06598 identifies the HERV-AVL3-B tumor-associated gene that is preferentially expressed in malignant melanoma. and methods and products for diagnosing and treating conditions characterized by expression of the gene. Regarding. WO 2006 / 119527 discloses an antigenic polypeptide derived from melanoma-associated endogenous retrovirus (MERV). and its use for the detection and diagnosis of malignant melanoma and for the prognosis of this disease - Patents.com The use of antigenic polypeptides as anti-cancer vaccines is also disclosed. do.

[0016] WO 2007 / 137279 describes, for example, HERV-K+ binding antibodies for preventing or inhibiting cancer cell proliferation. The present invention discloses methods and compositions for the detection, prevention and treatment of HERV-K+ cancers using the method. WO 2006 / 103562 discloses a method for the production of HERV-K-derived immunosuppressive Np9 protein from the env gene. The present invention also discloses a method for treating or preventing cancer caused by the protein. A pharmaceutical composition containing a nucleic acid or antibody capable of inhibiting the activity of the protein, or an antibody against the protein It relates to immunogenic or vaccine compositions capable of inducing an immune response. WO 2007 / 109583 describes a method for treating tumors containing an enriched population of immune cells that reacts to HERV-E antigens on tumor cells. and a method for preventing or treating neoplastic disease in a mammalian subject by providing a composition comprising the compound of formula (I) and (II). Compositions and methods are provided.

[0017] Humer J et al., 2006, Canc. Res., 66:1658-63, reported that endogenous levels of leukocyte antigen (LEA) associated with malignant melanoma have identified a melanoma marker derived from a retrovirus. It is used in the immunotherapy of cancer, especially malignant melanoma, more particularly cutaneous malignant melanoma and uveal malignant melanoma. There is a need to identify additional HERV-associated antigenic sequences that can be used. Summary of the Invention

[0018] (Summary of the Invention) The inventors have surprisingly found that the LTR element is a nucleotide sequence that contains or is adjacent to the LTR element. It is derived from a genomic sequence found at high levels in cutaneous melanoma cells but not in normal, healthy tissue. We found certain RNA transcripts that were either undetectable or found at very low levels in tissues ( (See Example 1.) Such transcripts are referred to herein as cancer-specific LTR-element spanning transcripts. Furthermore, the present inventors have identified the latent transcripts encoded by these CLTs. A subset of the polypeptide sequences (i.e., open reading frames (ORFs)) are identified in cancer It is translated intracellularly and processed by components of the antigen processing machinery. , furthermore, class I and class II major histocompatibility complexes (MHC class I and MHC class II) and along with class I and class II human leukocyte antigen (HLA class I, HLA class II) molecules in tumor tissue These findings suggest that the IL-16 receptor agonist is expressed on the surface of cells identified within the IL-16 receptor (see Example 2). These polypeptides (herein referred to as CLT antigens) are antigenic in nature. Thus, cancer cell presentation of CLT antigens allows these cells to recruit cognate T cells to these CLT antigens. It is expected that these proteins will be more susceptible to elimination by T cells with the TCR. CLT antigen-based vaccination methods / regimens that expand T cells bearing cognate TCRs are useful for the treatment of cancer cells. (and tumors containing same), in particular malignant melanoma, more particularly cutaneous malignant melanoma Indeed, T cells from melanoma subjects are expected to elicit responses as described herein. The T cells react to the CLT antigen-derived peptides and amplify the T cell receptor sequence (implemented). (See Example 3.) The present inventors have demonstrated that T cells specific for CLT antigens can induce normal immune responses through central tolerance. It was confirmed that the T cells were not deleted from the subject's T cell repertoire (see Example 4). The presence and killing activity of CLT antigen-specific T cells in ex vivo cultures of T cells was determined (see Example 1). Finally, qRT-PCR studies demonstrated that CLTs significantly increased the expression of leukocyte-specific leukocytes in melanoma compared with non-melanoma cell lines. It was confirmed to be specifically expressed in RNA extracted from chromatin cell lines (see Example 7). .

[0019] The present inventors have also surprisingly found that CLT, which encodes a certain CLT antigen, is involved in the development of melanoma. We found that it is not only overexpressed in uveal melanoma but also in uveal malignant melanoma. The CLT antigen polypeptide sequences encoded by these CLTs were found to be involved in uveal melanoma. It is expected that this will elicit an immune response against the cells and the tumors that contain them.

[0020] The CLT and CLT antigens that are the subject of the present invention are derived from known tumor genomes found in The Cancer Genome Atlas. This CLT is not a canonical sequence that can be easily derived from the ERV-derived transcriptional regulatory sequence. C is a transcription product resulting from a complex of transcription and splicing events driven by Because LT is expressed at high levels, and the CLT antigen polypeptide sequence is similar to that of the normal human protein Since the sequence is not that of the original antibody, it is unlikely to induce a strong and specific immune response (as has been established in practice, see Example 3). ~5) and may therefore be suitable for therapeutic use in cancer immunotherapy settings. It is expected.

[0021] This CLT antigen, found in a highly expressed transcript that characterizes tumor cells, is Before the Meiji Restoration, it was believed that bacteria existed in the human body to produce protein products and stimulate the immune response. First, the CLT antigen of the present invention can be used in several ways. The polypeptides can be used as vaccines to elicit therapeutic or prophylactic immune responses against tumor cells. Second, the nucleic acids of the present invention can be delivered directly to a subject. Codons can be optimized to enhance expression of the CLT antigen, allowing for direct administration. and inserting the encoded protein product into a vector as a vaccine to be produced in a subject. These can also be delivered in vivo to induce therapeutic or prophylactic immunity against tumor cells. Third, the polynucleotides and / or polypeptides of the present invention can be administered to patients. can be used to load antigen-presenting cells (APCs), which can then be administered to a subject as a vaccine. can be injected intravenously to elicit a therapeutic or prophylactic immune response against tumor cells. Fourth, the polynucleotides and / or polypeptides of the present invention can be administered to a subject's T cells ex vivo. and generating stimulated T cell preparations that can be used to treat cancer. Fifth, a biological molecule such as a T cell receptor (TCR) or a TCR mimetic can be administered to a subject. recognizes the CLT antigen in complex with the MHC I molecule and kills (or kills) cancer cells. The compound may be further modified to enhance the expression of the compound (promoting the growth of the compound) and administered to a subject as a cancer treatment therapy. , a chimeric version of a biological molecule that recognizes CLT antigens in complex with MHC cells. The cells may be transduced into T cells (autologous or non-autologous), and the transduced cells may be used in cancer treatment therapy. These and other applications are described in more detail below. do.

[0022] Accordingly, the present invention provides, inter alia, an isolated polypeptide comprising a sequence selected from: (a) any one of SEQ ID NOs: 1 to 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); (hereinafter referred to as "polypeptide of the present invention"). The present invention also relates to nucleic acid molecules encoding the polypeptides of the invention (hereinafter "nucleic acids of the invention"). ").

[0023] The polypeptides of the invention and the nucleic acids of the invention, as well as related aspects of the invention, are described in more detail below. As described below, the immunotherapy and prevention of cancer, particularly melanoma, It is expected that the present invention will be useful in the field of embodiments. [Brief explanation of the drawings]

[0024] DESCRIPTION OF THE DRAWINGS For each of Figures 1-15, the top panel shows peptides isolated from patient tumor samples. The extracted MS / MS spectrum of the α-amino acid is shown (with assigned fragment ions), and the bottom panel shows the spectrum. The graph shows a spectral rendering of the linear pair mapped to the fragment ions. The position of the peptide sequence is displayed. [Figure 1] Figure 1. Spectrum of peptide SEQ ID NO: 11 isolated from a tumor sample of patient Mel-3. [Figure 2] Figure 2. Spectrum of peptide SEQ ID NO: 12 isolated from a tumor sample of patient Mel-3. [Figure 3] Figure 3. Spectrum of peptide SEQ ID NO: 13 isolated from a tumor sample of patient Mel-5. [Figure 4] Figure 4. Spectrum of peptide SEQ ID NO: 13 isolated from a tumor sample of patient Mel-16. [Figure 5] Figure 5. Spectrum of peptide of SEQ ID NO: 15 isolated from a tumor sample of patient Mel-26. [Figure 6] Figure 6. Spectrum of peptide of SEQ ID NO: 16 isolated from a tumor sample of patient Mel-20. [Figure 7] Figure 7. Spectrum of peptide of SEQ ID NO: 17 isolated from a tumor sample of patient Mel-35. [Figure 8] Figure 8. Spectrum of peptide of SEQ ID NO: 19 isolated from a tumor sample of patient Mel-3. [Figure 9] Figure 9. Spectrum of peptide of SEQ ID NO: 21 isolated from a tumor sample of patient Mel-27. [Figure 10]Figure 10. Spectrum of peptide of SEQ ID NO: 20 isolated from a tumor sample of patient Mel-27. [Figure 11] Figure 11. Spectrum of peptide of SEQ ID NO: 22 isolated from a tumor sample of patient Mel-27. [Figure 12] Figure 12. Spectrum of peptide of SEQ ID NO: 23 isolated from a tumor sample of patient Mel-27. [Figure 13] Figure 13. Spectrum of peptide of SEQ ID NO: 24 isolated from a tumor sample of patient Mel-27. [Figure 14] Figure 14. Spectrum of peptide of SEQ ID NO: 25 isolated from a tumor sample of patient Mel-16. [Figure 15] Figure 15. Spectrum of peptide of SEQ ID NO: 26 isolated from a tumor sample of patient Mel-41.

[0025] [Figure 16] FIG. 16 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-15, the fragment being assigned SEQ ID NO:27. [Figure 17] FIG. 17 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-10, the fragment being assigned SEQ ID NO:29. [Figure 18] FIG. 18 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-5, the fragment being assigned SEQ ID NO:14. [Figure 19] FIG. 19 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-4, the fragment being assigned SEQ ID NO:21. [Figure 20] FIG. 20 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-18, the fragment being assigned SEQ ID NO: 31. [Figure 21] FIG. 21 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-16, the fragment being assigned SEQ ID NO:13. [Figure 22]FIG. 22 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-3, the fragment being assigned SEQ ID NO:19. [Figure 23] FIG. 23 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-6, the fragment being assigned SEQ ID NO:28. [Figure 24] FIG. 24 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-18, the fragment being assigned SEQ ID NO:18. [Figure 25] FIG. 25 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-4, the fragment being assigned SEQ ID NO:30. [Figure 26] FIG. 26 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-4, the fragment being assigned SEQ ID NO:20. [Figure 27] FIG. 27 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-20, the fragment being assigned SEQ ID NO:16. [Figure 28] FIG. 28 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient Mel-3, the fragment being assigned SEQ ID NO:12.

[0026] For each of Figures 29-42, the top panel shows peptides isolated from patient tumor samples. The extracted MS / MS spectrum of the α-amino acid is shown (with assigned fragment ions), and the bottom panel shows the spectrum. The graph shows a spectral rendering of the linear peptide sequence mapped to fragment ions. Shows the column position. [Figure 29] Figure 29. Spectrum of the peptide of SEQ ID NO: 51 isolated from a tumor sample of patient Mel-40. [Figure 30] Figure 30. Spectrum of the peptide of SEQ ID NO: 51 isolated from a tumor sample of patient Mel-41. [Figure 31]Figure 31. Spectrum of peptide of SEQ ID NO: 52 isolated from a tumor sample of patient Mel-27. [Figure 32] Figure 32. Spectrum of peptide of SEQ ID NO: 52 isolated from a tumor sample of patient Mel-39. [Figure 33] Figure 33. Spectrum of peptide of SEQ ID NO: 13 isolated from tumor sample of patient 2MT3. [Figure 34] Figure 34. Spectrum of peptide of SEQ ID NO: 13 isolated from tumor sample of patient 2MT10. [Figure 35] Figure 35. Spectrum of peptide of SEQ ID NO: 12 isolated from tumor sample of patient 2MT3. [Figure 36] Figure 36. Spectrum of peptide of SEQ ID NO: 16 isolated from tumor sample of patient 2MT4. [Figure 37] Figure 37. Spectrum of peptide of SEQ ID NO: 17 isolated from tumor sample of patient 2MT3. [Figure 38] Figure 38. Spectrum of peptide of SEQ ID NO: 53 isolated from tumor sample of patient 1MT1. [Figure 39] Figure 39. Spectrum of peptide of SEQ ID NO: 51 isolated from tumor sample of patient 2MT3. [Figure 40] Figure 40. Spectrum of peptide of SEQ ID NO: 19 isolated from tumor sample of patient 2MT3. [Figure 41] Figure 41. Spectrum of peptide of SEQ ID NO: 19 isolated from tumor sample of patient 2MT1. [Figure 42] Figure 42. Spectrum of peptide of SEQ ID NO: 54 isolated from tumor sample of patient 2MT12.

[0027] For each of Figures 43-50, the native peptides isolated from patient tumor samples were analyzed. The native MS / MS spectrum (top) and the native spectrum of the synthetic peptide corresponding to the same sequence ( The alignment is shown with the bottom. [Figure 43] FIG. 43 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient 2MT3, the fragment being assigned SEQ ID NO:13. [Figure 44] FIG. 44 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient 2MT3, the fragment being assigned SEQ ID NO:12. [Figure 45] FIG. 45 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient 2MT4, the fragment being assigned SEQ ID NO:16. [Figure 46] FIG. 46 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient 2MT3, the fragment being assigned SEQ ID NO:17. [Figure 47] FIG. 47 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient 1MT1, the fragment being assigned SEQ ID NO:53. [Figure 48] FIG. 48 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient 2MT3, the fragment being assigned SEQ ID NO:51. [Figure 49] FIG. 49 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient 2MT3, the fragment being assigned SEQ ID NO:19. [Figure 50] FIG. 50 shows the mass spectrometry spectrum of a peptide fragment obtained in the immunopeptidome analysis of patient 2MT12, the fragment being assigned SEQ ID NO:54.

[0028] [Figure 51] Figure 51, panels AC, shows the expansion of tumor antigen-specific T cells from patient PBMC cultures in response to incubation with specific tumor antigen-derived peptides. [Figure 52] Panels A to D of Figure 52 show a summary of CLT antigen-derived peptides (SEQ ID NO: 55 to SEQ ID NO: 72) that were able to expand T cells having specific TCRs from PBMCs of malignant melanoma patients. [Figure 53]FIG. 53 shows the CD8 T cell response from a normal blood donor to an HLA-A*0201-restricted peptide from CLT antigen 1 (SEQ ID NO: 73). [Figure 54] FIG. 54 shows the CD8 T cell response from a normal blood donor to an HLA-A*0201-restricted peptide from CLT antigen 2 (SEQ ID NO: 75). [Figure 55] FIG. 55 shows CD8 T cell responses from normal blood donors to an HLA-A*0201-restricted peptide from CLT antigen 4 (SEQ ID NO: 76).

[0029] [Figure 56] Panels A to D of Figure 56 show the responsiveness of memory CD45RO-positive CD8 T cells to HLA-B*0702-restricted peptides (SEQ ID NOs: 74 and 77) from CLT antigen 1 and CLT antigen 4, respectively, compared with naive CD45RO-negative CD8 T cells from the same blood donor. [Figure 57] Figure 57 shows HLA pentamer staining of normal CD8 T cells specific for CLT antigen 1-derived peptide (SEQ ID NO: 73), CLT antigen 2-derived peptide (SEQ ID NO: 78) and CLT antigen 4-derived peptide (SEQ ID NO: 77). [Figure 58] Figure 58 shows that expanded, pentamer-selected CD8 T cells kill C1RB7 target cells pulsed with a CLT antigen 4-derived peptide (SEQ ID NO: 77). [Figure 59] Panels A-C of Figure 59 show the results of qRT-PCR assays assessing transcription of CLT (sequence number 33), encoding CLT antigen 1, CLT (sequence number 34), encoding CLT antigen 2, and CLT (sequence number 35), encoding CLT antigens 3 and 4, in melanoma cancer cell lines.

[0030] (Array description) SEQ ID NO: 1 is the polypeptide sequence of CLT antigen 1. SEQ ID NO:2 is the polypeptide sequence of CLT antigen 2. SEQ ID NO:3 is the polypeptide sequence of CLT antigen 3. SEQ ID NO: 4 is the polypeptide sequence of CLT antigen 4. SEQ ID NO: 5 is the polypeptide sequence of CLT antigen 5. SEQ ID NO: 6 is the polypeptide sequence of CLT antigen 6. SEQ ID NO: 7 is the polypeptide sequence of CLT antigen 7. SEQ ID NO: 8 is the polypeptide sequence of CLT antigen 8. SEQ ID NO: 9 is the polypeptide sequence of CLT antigen 9. SEQ ID NO: 10 is the polypeptide sequence of CLT antigen 10.

[0031] SEQ ID NOs: 11 to 14 are peptide sequences derived from CLT antigen 1. SEQ ID NOs: 15 and 16 are CLT antigen 2-derived peptide sequences. SEQ ID NOs: 17 and 18 are CLT antigen 3-derived peptide sequences. SEQ ID NO: 19 is a CLT antigen 4-derived peptide sequence. SEQ ID NOs: 20 to 22 are peptide sequences derived from CLT antigen 5. SEQ ID NOs: 23 and 24 are peptide sequences derived from CLT antigen 6. SEQ ID NO: 25 is a CLT antigen 7-derived peptide sequence. SEQ ID NO: 26 is a peptide sequence derived from CLT antigen 8. SEQ ID NOs: 27 to 29 are peptide sequences derived from CLT antigen 9. SEQ ID NOs: 30 to 32 are peptide sequences derived from CLT antigen 10.

[0032] SEQ ID NO: 33 is the cDNA sequence of CLT encoding CLT antigen 1. SEQ ID NO: 34 is the cDNA sequence of CLT encoding CLT antigen 2. SEQ ID NO: 35 is the cDNA sequence of CLT encoding CLT antigens 3 and 4. SEQ ID NO: 36 is the cDNA sequence of CLT encoding CLT antigen 5. SEQ ID NO: 37 is the cDNA sequence of CLT encoding CLT antigen 6. SEQ ID NO: 38 is the cDNA sequence of CLT encoding CLT antigens 7 and 8. SEQ ID NO: 39 is the cDNA sequence of CLT encoding CLT antigen 9. SEQ ID NO: 40 is the cDNA sequence of CLT encoding CLT antigen 10.

[0033] SEQ ID NO: 41 is the cDNA sequence encoding CLT antigen 1. SEQ ID NO: 42 is the cDNA sequence encoding CLT antigen 2. SEQ ID NO: 43 is the cDNA sequence encoding CLT antigen 3. SEQ ID NO: 44 is the cDNA sequence encoding CLT antigen 4. SEQ ID NO: 45 is the cDNA sequence encoding CLT antigen 5. SEQ ID NO: 46 is the cDNA sequence encoding CLT antigen 6. SEQ ID NO: 47 is the cDNA sequence encoding CLT antigen 7. SEQ ID NO: 48 is the cDNA sequence encoding CLT antigen 8. SEQ ID NO: 49 is the cDNA sequence encoding CLT antigen 9. SEQ ID NO: 50 is the cDNA sequence encoding CLT antigen 10.

[0034] SEQ ID NOs: 51 and 52 are peptide sequences derived from CLT antigen 4. SEQ ID NO: 53 is a CLT antigen 3-derived peptide sequence. SEQ ID NO: 54 is a CLT antigen 4-derived peptide sequence. SEQ ID NOs: 55 to 57 are peptide sequences derived from CLT antigen 1. SEQ ID NOs: 58 to 66 are peptide sequences derived from CLT antigen 2. SEQ ID NOs: 67 to 69 are peptide sequences derived from CLT antigen 3. SEQ ID NOs: 70 to 72 are peptide sequences derived from CLT antigen 4. SEQ ID NOs: 73 to 74 are peptide sequences derived from CLT antigen 1. SEQ ID NO: 75 is a CLT antigen 2-derived peptide sequence. SEQ ID NOs: 76 to 77 are peptide sequences derived from CLT antigen 4. SEQ ID NO: 78 is a CLT antigen 2-derived peptide sequence. DETAILED DESCRIPTION OF THE INVENTION

[0035] (Detailed Description of the Invention) (polypeptide) The terms "protein," "polypeptide," and "peptide" are used interchangeably herein. Any peptide-linked amino acid chain, regardless of length, co-translational or post-translational modifications, is used. It refers to either one.

[0036] The term "amino acid" refers to naturally occurring amino acids as well as amino acids that are similar to naturally occurring amino acids. It refers to any one of amino acid analogs and amino acid mimetics that function according to the formula: The amino acids are the 20 L-amino acids encoded by the genetic code and the amino acids Later modified, e.g., hydroxyproline, γ-carboxyglutamic acid, and O-phosphoric acid. The term "amino acid analog" refers to an amino acid that has the same basic chemical structure as a naturally occurring amino acid. The chemical structure of the α-carbon bonded to hydrogen, carboxyl group, amino group, and R group. Compounds that have a modified R group or modified peptide structure compared to natural amino acids. Examples include homoserine, norleucine, methionine sulfoxide, Amino acid mimetics include methionine methylsulfonium, methionine methylsulfonium, and norleucine. They have a structure that differs from the general chemical structure of amino acids, but are similar to naturally occurring amino acids. Preferably, the amino acid is a naturally occurring amino acid or are amino acid analogs, particularly naturally occurring amino acids, and more particularly amino acids encoded by the genetic code. It is one of the 20 L-amino acids encoded by the ribonucleotides.

[0037] Amino acids are referred to herein by their commonly known three letter symbols or by the IUPAC-IUB Biochemical Nomenclature Committee symbols. The chemical is represented by one of the single-letter symbols recommended by the Biochemical Nomenclature Commission. Nucleotides are also represented by commonly accepted single-letter codes. This may be the case.

[0038] Accordingly, the present invention provides an isolated polypeptide comprising a sequence selected from: (a) any one of SEQ ID NOs: 1 to 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a).

[0039] The present invention also provides an isolated polypeptide comprising a sequence selected from: (a) any one of SEQ ID NOs: 1 to 10 excluding the first methionine residue; and , (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a).

[0040] Generally, variants of the polypeptide sequences of the present invention will have a high degree of sequence identity thereto. For example, variants preferably have at least one sequence that is at least 100% identical to the full length of the relevant reference sequence. at least about 80% identity, more preferably at least about 85% identity, and most preferably at least and have about 90% identity (at least about 95%, at least about 98%, or at least about 99%, etc.) .

[0041] Preferably, the variant is an immunogenic variant. A variant is a variant of a reference sequence (i.e., At least 20%, preferably at least 50%, especially of the activity of the sequence of which the mutant is a A variant that elicits a response that is at least 75% (e.g., at least 90%) is considered to be an immunogenic variant. This response can be measured, for example, by in vitro immunoprecipitation of PBMCs or whole blood using the polypeptide as an antigen. Restimulation assays (e.g., restimulation over periods ranging from a few hours up to a year, 6 months or less, 1 day or In the period from 1 month to 1-2 weeks, etc., lymphocyte proliferation (e.g., T cell proliferation) is mediated by Activation, production of cytokines (e.g., IFN-γ) in the culture supernatant (measured by ELISA, etc.), or T cell Characterization of the cellular response was performed using intracellular and extracellular staining (e.g., CD3, CD4, CD8, IL2, TNF-α, IFN g, using antibodies specific for immune markers such as type 1 IFN, CD40L, and CD69) and subsequent flow This is measured by analysis on a cytometer.

[0042] A variant can be, for example, a conservatively modified variant. The modification may be a substitution of an amino acid with a functionally similar amino acid or a modification of the biology of the mutant. These modifications result in substitutions / deletions / additions of residues that do not substantially affect the target protein function. The biological function of such variants is to target cancer antigens of malignant melanoma, e.g., cutaneous malignant melanoma. This will induce an immune response that Conservative substitution tables providing functionally similar amino acids are well known in the art. , may also include homologs of polypeptides found in other species.

[0043] Variants of the polypeptides of the invention may contain a number of substitutions, e.g., conservative substitutions, compared to the reference sequence. Substitutions (e.g., 1 to 25, 1 to 10, etc., particularly 1 to 5, and more particularly 1 amino acid residue may be changed) The number of substitutions, e.g., conservative substitutions, may be up to the number of residues in the reference sequence. 20%, for example up to 10%, for example up to 5%, for example up to 1%. Suitable substitutions will fall within one of the amino acid groupings identified below, but may Other substitutions may also be possible, provided they do not substantially affect the immunogenic properties of the antigen. The next eight groups each contain amino acids that are usually conservative substitutions for one another: .

[0044] 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, e.g., Creighton, Proteins 1984).

[0045] Preferably, such substitutions do not alter the immunological structure of the epitope (e.g., (The substitutions do not occur within the epitope region mapped to the primary sequence), therefore, It does not significantly affect the immunogenic properties of the original.

[0046] Polypeptide variants also include those in which additional amino acids are inserted compared to a reference sequence. For example, such insertions can be made at positions 1 to 10 (e.g., positions 1 to 5, preferably positions 1 or 2, especially positions 1). ) and the insertions may occur at, for example, 50 or fewer (e.g., 20 or fewer, especially It may also include the addition of up to 10 amino acids, more particularly up to 5 amino acids. The insertion does not occur in the epitope region and therefore does not significantly affect the immunogenic properties of the antigen. An example of an insertion would be a histidine to aid in expression and / or purification of the antigen of interest. A short stretch of residues (e.g., 2-6 residues) is included.

[0047] Polypeptide variants include those in which some amino acids have been deleted compared to the reference sequence. For example, such deletions may occur at positions 1 to 10 (e.g., positions 1 to 5, preferably positions 1 or 2, particularly positions 1). positions), and the deletions may occur, for example, at 50 or fewer (e.g., 20 or fewer, particularly 10 or fewer) positions. The amino acid sequence may also include deletions of up to 10 amino acids, more particularly up to 5 amino acids. The deletion does not occur in the epitope region and therefore does not significantly affect the immunogenic properties of the antigen. stomach.

[0048] Those skilled in the art will appreciate that particular protein variants may include substitutions, deletions, and additions (or any combination thereof). For example, substitutions / deletions / additions may be made to the desired patient's H Enhances binding to LA molecules (or has a neutral effect) and enhances immunogenicity (or inhibits immunogenicity) (Gender may remain unchanged).

[0049] The immunogenic fragments of the present invention are typically fragments of the full-length polypeptide sequence depending on the length of the CLT antigen. is at least 9 (e.g., at least 9 or 10) consecutive amino acids, e.g., at least 12 Contiguous amino acids (e.g., at least 15 or at least 20 contiguous amino acids), particularly a small number of amino acids. at least 50 consecutive amino acids, e.g., at least 100 consecutive amino acids (e.g., at least Preferably, the immunogenic fragment contains a fragment of the full-length polypeptide sequence, such as a fragment of the full-length polypeptide sequence (e.g., 200 consecutive amino acids). At least 10% of the length of the column, e.g. at least 20%, e.g. at least 50%, e.g. at least It would be at least 70%, or at least 80%, etc.

[0050] Immunogenic fragments typically contain at least one epitope. Epitopes are those that stimulate B-cell and Preferably, the immunogenic fragment comprises a T cell epitope, such as a CD4+ or CD8+ T cell epitope. and at least one T cell epitope of

[0051] T cell epitopes are expressed by T cells (such as CD4+ or CD8+ T cells) when bound to HLA molecules. T cell epitopes are short, consecutive stretches of amino acids that are recognized by the T cell. This may be achieved by epitope mapping experiments well known to those skilled in the art (see, e.g., Paul, Fu National Immunology, 3rd ed., pp. 243-247 (1993); Beißbarth et al., 2005, Bioinformatics ics,21(Suppl. 1):i29-i37).

[0052] As a result of the crucial involvement of T cell responses in cancer, at least one T cell epitope Fragments of the full-length polypeptides of SEQ ID NOs: 1-10, including: The potential contribution to immune protection is quite clear.

[0053] In diverse outbred populations, such as humans, different HLA types represent populations where a particular epitope is present. It will be understood that this means that the information may not be recognized by all members of the As a result, to maximize the level of recognition and magnitude of the immune response to a given polypeptide, Generally, immunogenic fragments contain multiple epitopes from the full-length sequence (preferably multiple epitopes within the CLT antigen). It is desirable to include all epitopes.

[0054] Particular fragments of the polypeptides of SEQ ID NOs: 1-10 that may be useful include those that bind to at least one CD8+ T cell epitopes, preferably at least two CD8+ T cell epitopes, more particularly all CD8+ those containing T cell epitopes, especially those associated with multiple HLA alleles, e.g., 2, 3, Polypeptides of SEQ ID NOS: 1-10 that may be useful include those associated with 4, 5 or more alleles. A particular fragment of a peptide comprises at least one CD4+ T cell epitope, preferably at least two CD4+ T cell epitopes, more particularly those containing all CD4+ T cell epitopes (especially those containing multiple H those associated with LA alleles, e.g., those associated with 2, 3, 4, 5 or more alleles) However, those skilled in the art of vaccine design may incorporate exogenous CD4+ T cell epitopes into the vaccines of the present invention. In combination with the CD8+ T cell epitopes, the desired response to the CD8+ T cell epitopes of the present invention You will be able to achieve the answer.

[0055] When individual fragments of a full-length polypeptide are used, such individual fragments may be selected from the reference sequence ( That is, the fragment has at least 20%, preferably at least 10%, of the activity of the sequence of which it is a fragment. An antibody is considered immunogenic if it elicits a response that is at least 50%, particularly at least 75% (e.g., at least 90%). This response is thought to be due to, for example, PBMC or In vitro restimulation assays of whole blood (e.g., restimulation over periods ranging from a few hours up to 1 year, 6 activity on lymphocyte proliferation (e.g., T cell proliferation) Activation of cells through cell proliferation, production of cytokines (e.g., IFN-γ) in the culture supernatant (ELISA) A, etc.), or characterization of T cell responses using intracellular and extracellular staining (e.g., CD3, CD4, Antibodies specific to immune markers such as CD8, IL2, TNF-α, IFN-γ, type 1 IFN, CD40L, and CD69 were used. (used for ELISA) and subsequent analysis on a flow cytometer.

[0056] In some cases, multiple fragments of the full-length polypeptide (which may or may not overlap, (which may or may not span the entire full-length sequence) to the full-length sequence itself For example, a combination of at least two (3, 4, or Immunogenic fragments of the antibodies (e.g., 5) are combined and tested in an in vitro restimulation assay (e.g., PBMC or whole blood). Preferably, the sequence matches at least 50% of the reference sequence in a T cell proliferation and / or IFN-γ production assay. Preferably it provides at least 75%, more particularly at least 90% activity.

[0057] Examples of immunogenic fragments of the polypeptides of SEQ ID NOs: 1 to 10, and therefore examples of peptides of the invention, are: The present invention also includes polypeptides comprising or consisting of the sequences of SEQ ID NOs: 11 to 32. Examples of immunogenic fragments of the polypeptides numbered 1 to 4, and therefore examples of peptides of the invention, are SEQ ID NOs: SEQ ID NOs: 11 to 17, 19 The sequences ~28, 30~31, and 51~54 were found to bind to HLA class I molecules in immunopeptidome analysis. The sequences of SEQ ID NOs: 18, 29 and 32 were confirmed to be identical to those of the immunopeptides (see Examples 2 and 2.1). The sequence was confirmed to bind to HLA class II molecules by thymome analysis (see Example 2). The sequences numbered 55 to 78 are predicted to bind to HLA class I molecules by the NetMHC software. and used in immunological validation assays (see Examples 3, 4 and 5).

[0058] (nucleic acid) The present invention provides isolated nucleic acids encoding the polypeptides of the invention (referred to as nucleic acids of the invention). For example, the nucleic acid of the present invention includes a sequence selected from SEQ ID NOs: 33 to 40 or 41 to 50. It consists of a bell or an array thereof.

[0059] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to a nucleic acid. Nucleotide monomers, especially deoxyribonucleotide monomers or ribonucleotide monomers The term refers to a polymeric macromolecule made from known nucleotide analogs or The term "nucleic acid" includes nucleic acids containing modified backbone residues or linkages, which are naturally occurring. and of non-natural origin, have similar properties to the reference nucleic acid, and are similar to the reference nucleotide or intended to have a long half-life in the body. Examples of such analogs include, but are not limited to: , phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates These include ribonucleotides, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). The term "nucleic acid" refers to a naturally occurring nucleic acid of deoxyribonucleotide or ribonucleotide monomers. The term "recombinant" refers to a polymer of origin. Preferably, the nucleic acid molecules of the present invention are recombinant. Recombinant refers to a polymer of origin. Nucleic acid molecules may be synthesized by cloning, restriction or ligation steps, or by other methods not found in nature. other manipulations that result in nucleic acid molecules that are distinct from the nucleic acid molecules being synthesized (e.g., in the case of cDNA). In one embodiment, the nucleic acid of the present invention is an artificial nucleic acid. sequences (e.g., cDNA sequences or nucleic acid sequences containing unnatural codon usage). Alternatively, the nucleic acid of the present invention is DNA. Alternatively, the nucleic acid of the present invention is RNA.

[0060] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are deoxyribosyl and ribosyl, respectively. It refers to a nucleic acid molecule that has a sugar backbone that is made up of the four natural bases (the bases in DNA). Adenine (A), guanine (G), cytosine (C), thymine (T), and adenine (A), guanine (G) in RNA It may be attached to the bases guanine (G), cytosine (C) and uracil (U). When used, a "corresponding RNA" is a DNA sequence that has the same sequence as the reference DNA, but where the thymine (T) in the DNA is replaced by the RNA It is an RNA with a sequence in which uracil (U) is substituted in the sugar moiety. Unnatural bases such as inosine, 7-methylguanosine, dihydrouridine, and 5-methylcytidine may be linked to a natural phosphate diester between the sugar (deoxyribosyl / ribosyl) moieties The linkages may optionally be replaced by phosphorothioate linkages. Preferably, the nucleic acids of the present invention comprise: Attached to a deoxyribosyl or ribosyl sugar backbone with phosphodiester bonds between the sugar moieties It consists of natural bases.

[0061] In one embodiment, the nucleic acid of the present invention is DNA. For example, the nucleic acid is a sequence represented by SEQ ID NO: 33 to 40 or Also provided are sequences comprising or consisting of sequences selected from 41 to 50. A nucleic acid comprising or consisting of a variant of a sequence selected from sequences 33 to 40 or 41 to 50. Thus, the variants encode the same amino acid sequence but have different sequences based on the degeneracy of the genetic code. It has nucleic acid.

[0062] Thus, due to the degeneracy of the genetic code, a large number of different but functionally identical nucleic acids are possible. For example, the codons GCA, GCC, GCG, and GCU can all be used to encode any given polypeptide. The codon 1 encodes the amino acid alanine. At every position specified, the codon modifies the encoded polypeptide. Such nucleic acid mutations can be made to any of the above codons without altering the corresponding codon. , resulting in "silent" (sometimes called "degenerate" or "synonymous") variants, It is one type of conservatively modified variant. Every nucleic acid sequence disclosed herein also contemplates every silent variation of the nucleic acid. Those skilled in the art will recognize each codon in a nucleic acid (usually AUG, which is the only codon for methionine, and and UGG, which is usually the only codon for tryptophan, are modified to produce a functionally equivalent It will be appreciated that a single molecule can be produced. Each silent variation is implicit in each described sequence and is a feature of the present invention. It is provided as an embodiment.

[0063] Degenerate codon substitutions also occur when the third position of one or more (or all) selected codons is replaced with a mixed base. and / or by generating sequences substituted with deoxyinosine residues. (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8:91-98) .

[0064] The present invention provides a method for the preparation of a nucleic acid sequence comprising or consisting of a sequence selected from SEQ ID NOs: 33 to 40 or 41 to 50. Nucleic acids contain many silent variants (e.g., 1-50, 1-25, etc., particularly 1-5) compared to a reference sequence. , and more particularly, one codon may be mutated).

[0065] In one embodiment, the nucleic acids of the invention are RNA. The RNA sequences provided are not intended to be construed as limiting the scope of the invention as defined by the appended claims. corresponding to the sequence, has a ribonucleotide backbone instead of a deoxyribonucleotide backbone, and has the side chain base uracil (U) instead of thymine (T).

[0066] Thus, the nucleic acids of the present invention are RNA equivalents of the cDNA sequences selected from SEQ ID NOs: 33-40 or 41-50. and have many silent mutations ( For example, 1 to 50, 1 to 25, etc., particularly 1 to 5, and more particularly 1 codon may be mutated). An "RNA equivalent" is a sequence that contains the same genetic information as the reference cDNA sequence (i.e., deoxyribonucleotides). It has a ribonucleotide backbone instead of a nucleotide backbone and a side chain instead of thymine (T). The term "RNA sequence" refers to an RNA sequence containing identical codons with the base uracil (U).

[0067] The present invention also includes sequences complementary to the above cDNA and RNA sequences. In one embodiment, the nucleic acids of the invention are cloned into vectors optimized for expression in human host cells. It's Don. The nucleic acids of the present invention, in the case of DNA nucleic acids, can be transcribed and translated into the polypeptides of the present invention. and, in the case of an RNA nucleic acid, can be translated into a polypeptide of the present invention.

[0068] (Polypeptides and Nucleic Acids) Preferably, the polypeptides and nucleic acids used in the present invention are isolated. A "naturally occurring" polypeptide or nucleic acid is one that has been removed from its original environment. The original polypeptide or nucleic acid may be separated from some or all of the materials with which it coexists in natural systems. Nucleic acid is isolated when it is separated from the natural environment, e.g., when it is in a vector that is not part of its natural environment. A gene is considered isolated when it is cloned into a vector.

[0069] "Naturally occurring," when used in reference to a polypeptide or nucleic acid sequence, means a polypeptide that is not found in nature. By "synthetic" is meant a sequence that has not been synthetically modified. "Artificial" when used in reference to a polypeptide or nucleic acid sequence refers to, for example, a naturally occurring refers to a sequence not found in nature, including synthetic modifications of a sequence or non-naturally occurring sequences .

[0070] The term "heterologous" refers to the relationship between one nucleic acid or polypeptide and another nucleic acid or polypeptide. As used in the context of, two or more sequences are not found in the same relationship to each other in nature. "Heterologous" sequences also refer to sequences that are not naturally occurring nucleic acids found in the host organism. or a sequence not isolated from, derived from, or based on a polypeptide sequence. It can also mean a column.

[0071] As mentioned above, polypeptide variants preferably have at least one amino acid sequence that is at least 100% identical to the full length of the relevant reference sequence. At least about 80% identity, more preferably at least about 85% identity, and most preferably at least have at least about 90% identity (e.g., at least about 95%, at least about 98%, or at least about 99%) do.

[0072] For purposes of comparing two closely related polypeptide or polynucleotide sequences, first The "% sequence identity" between a sequence of interest and a second sequence may be calculated. If a polypeptide sequence shares 0% sequence identity, then the polypeptide sequence is identical to the other polypeptide sequence. The residues in the sequence are listed from left to right, i.e., from the N-terminus of the polypeptide to the C The term "identical" or percent "identity" refers to the degree to which two or more polypeptides In the context of the sequence, the sequences are compared and aligned to maximize the match over the comparison window. When the amino acid residues are identical, or a specific percentage of identical amino acid residues are (i.e., 70% identity within the specified region, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity) refers to two or more sequences or subsequences. Preferably, the comparison is over the entire length of the reference sequence. It is executed on the corresponding window.

[0073] For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a computer algorithm, the test and reference sequences are input into a computer and, if necessary, Specify the sub-array coordinates and the array algorithm program parameters. You can use the default program parameters or specify alternative parameters. and the sequence comparison algorithm compares the sequences against a reference sequence based on the program parameters. The percentage sequence identity of the sequences is calculated.

[0074] As used herein, a "comparison window" is a window within which one sequence and the same number of consecutive After optimally aligning the reference sequence at the position where the segments are to be compared, these sequences may be compared. Methods of sequence alignment for comparison are well known in the art. Suitable sequence alignments can be performed using, for example, the local homology algorithm of Smith and Waterman, 981, Adv. Appl. Math. 2:482, according to Needleman and Wunsch homology alignment. algorithm, 1970, J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, 198 8, Proc. Nat'l. Acad. Sci. USA 85:2444. GAP, BESTFIT, FASTA, and TFASTA in e Package, Genetics Computer Group, 575 Science by computerized implementation of the algorithm of (Dr. Madison, WI) or by manual alignment. and visual inspection (see, e.g., Current Protocols in Molecular Biology (eds. Ausubel et al.) This can be done by the following method (see the 1995 supplement).

[0075] One example of a useful algorithm is PILEUP, which is a progressive pairwise Use alignment to create multiple sequence alignments from groups of related sequences and compare them. The relationship and percent sequence identity are shown. This is also used to create the alignment. Plots a tree or dendogram showing the clustering relationships found in the dataset. , Feng and Doolittle's progressive alignment method (1987, J. Mol. Evol. 35:351-3 60) is used. The method used is that of Higgins and Sharp, 1989, CABIOS 5:151-15 This program is similar to the method described in [3]. The program is for sequences of up to 5,000 nucleotides in length each. If the sequences are of the same sequence or amino acid, up to 300 sequences can be aligned. The alignment procedure begins with a pairwise alignment of the two most similar sequences. This cluster is then divided into two clusters based on the most relevant Align adjacent sequences or clusters of aligned sequences that have high relatedness. Clusters are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is a series of progressive pairwise alignments. This program allows you to select specific sequences and their corresponding regions of sequence comparison. By specifying the amino acid coordinates of By using PILEUP, a reference sequence can be compared to other test sequences to determine the following patterns: Parameters: Default gap weight (3.00), default gap length weight (0.10), and and weighted end gaps to determine percent sequence identity. , GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., 1 984, Nuc. Acids Res. 12:387-395).

[0076] Suitable Algorithms for Determining Percent Sequence Identity and Percent Sequence Similarity Another example is the BLAST and BLAST 2.0 algorithms, described in Altschul et al., 1999, respectively. 77, Nuc. Acids Res. 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol. 215: Software for performing BLAST analyses is available from the National Center for Publicly available via the website for Biotechnology Information (www.ncbi.nlm.nih.gov / ) This algorithm aligns words of the same length in the database sequence. If checked in, the criteria match or meet some positive threshold score T. High-scoring sequence pairs (HSPs) are optimized by identifying short word lengths W in the array. T is called the neighborhood word score threshold (Altsc These initial neighborhood word hits can be used to find longer HSPs containing them. They act as seeds for initiating discovery searches. The word hits are added to a cumulative alignment score. The nucleotide sequence is extended in both directions along each sequence as far as possible to increase the length of the sequence. The cumulative score in is determined by the parameters M (reward score for a matching residue pair; always > 0) and and N (penalty score for mismatched residues; always <0). For each direction, the cumulative score is calculated using a scoring matrix. The word hit extension stops when the cumulative alignment score reaches its maximum achieved value. The cumulative score is increased by X amount due to the accumulation of 1 or more negatively scoring residue alignments. or when the end of either sequence is reached. For this purpose, the BLASTP program uses a default word length of 3 and an expectation (E) of 50. 50 BLOSUM62 scoring matrices (Henikoff and Henikoff, 1989, Proc. N atl. Acad. Sci. USA 89:10915) Alignment (B), 10 expectation (E), M=5, N=-4 , as well as a comparison of both strands.

[0077] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and A (See Iltschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by algorithms is the minimum total probability (P(N)), which is 2 It provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance.

[0078] A "difference" between sequences is the insertion of one residue at one position in the second sequence compared to the first sequence. The two sequences may contain one, two or more such differences. An insertion in a second sequence that is otherwise identical to the first sequence (100% sequence identity), Deletions or substitutions result in a decrease in the percent sequence identity. For example, if the identical sequences are 9 residues long, , substitution of 1 in the second sequence results in 88.9% sequence identity. In the long case, two substitutions in the second sequence result in 88.2% sequence identity.

[0079] Alternatively, a first reference sequence may be generated to generate a second sequence for the purpose of comparing the first reference sequence to a second comparison sequence. The number of additions, substitutions and / or deletions made to the first sequence may be ascertained. is the addition of a single residue to the first sequence (including additions to either end of the first sequence). A substitution is one in which a residue in a first sequence is replaced with a different residue. A deletion is the deletion of one residue from the first sequence (either end of the first sequence). (including deletions at the ends).

[0080] (Production of Polypeptides of the Invention) The polypeptides of the present invention can be prepared by, for example, the methods described in Green and Sambrook, 2012 Molecular Cloning. ng: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press They can also be obtained and manipulated using techniques currently available, particularly those for generating polynucleotides. Artificial gene synthesis techniques can also be used to achieve this (Nambiar et al., 1984, Science e, 223:1299-1301; Sakamar and Khorana, 1988, Nucl. Acids Res., 14:6361-6372 , Wells et al., 1985, Gene, 34:315-323, and Grundstrom et al., 1985, Nucl. Aci ds Res., 13:3305-3316), followed by expression in a suitable organism to produce the polynucleotide. The gene encoding the polypeptide of the present invention may be synthesized, for example, by solid-phase DNA synthesis. Entire genes can be produced synthetically without the need for precursor template DNA. To obtain the desired oligonucleotide, the building blocks may be synthesized by Sequentially ligated to the growing oligonucleotide chain in the order required by the product sequence. Once chain assembly is complete, the product is released from the solid phase into solution, deprotected, and collected. The product was isolated by high performance liquid chromatography (HPLC) to obtain the desired oligonucleotide. It can also be obtained in high purity (Verma and Eckstein, 1998, Annu. Rev. Biochem. 67:99- 134). These relatively short segments can be amplified by various gene amplification methods (Methods Mol Biol., 2012 ;834:93-109) can be easily assembled into longer DNA molecules, which can be counted. Suitable for use in an unlimited number of recombinant DNA-based expression systems. The polynucleotide sequence encoding the polypeptide antigen according to the present invention is e.g. Understand that it can be easily used in various vaccine production systems, including virus vectors. It would be.

[0081] For the purpose of producing the polypeptides of the invention in microbial hosts (e.g., bacterial or fungal), For this purpose, the nucleic acid of the present invention may be provided with suitable regulatory and control sequences (promoters, termination signals, etc.). ), as well as a polypeptide suitable for protein production in a host. Similarly, the polypeptides of the present invention may be expressed in eukaryotic cells (e.g., Chinese A culture of cells (hamster ovary cells or Drosophila S2 cells) containing a nucleic acid of the invention , appropriate regulatory and control sequences (including promoters, termination signals, etc.) and these cells and a sequence for promoting polypeptide secretion suitable for protein production within the and could be generated by transduction.

[0082] Improved isolation of the polypeptides of the invention produced by recombinant techniques may optionally be achieved by isolating the polypeptides. A stretch of histidine residues towards one end of the peptide (commonly known as a His tag) This could be facilitated by adding Polypeptides may also be prepared synthetically.

[0083] (vector) In additional embodiments, genetic constructs comprising one or more nucleic acids of the invention are When introduced into cells, the polypeptide of the present invention is produced in vivo and elicits an immune response. (e.g., DNA) can be expressed in a variety of nucleic acid expression systems, including bacterial and some viral expression systems, all of which are well known to those skilled in the art. It may be in any of a variety of delivery systems known in the art. Numerous gene delivery techniques are well known in the art. See, e.g., Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems 15:143-198. and the references cited therein. Some of these are outlined below for illustrative purposes.

[0084] In accordance with the above, vectors (also referred to herein as "DNA expression constructs") containing the nucleic acid molecules of the present invention may be used. Also referred to as "constructs" are provided. Preferably, the vector is capable of transcribing an RNA molecule that is translationally active in a human host cell. The gene encoding the appropriate regulatory elements (such as a suitable promoter and termination signal) to enable the expression of the gene. "Translationally active RNA molecules" include nucleic acids that can be converted into proteins by the translational machinery of human cells. It is an RNA molecule that can be translated into protein.

[0085] In accordance with the above, a vector comprising a nucleic acid of the present invention (hereinafter referred to as a "vector of the present invention") A service called a "Tar" is provided. In particular, the vector may be a viral vector. adeno-associated viruses (AAV) (e.g., AAV types 5 and 2), alphaviruses (e.g., vector viruses), Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SIN), Semliki Forest virus (SF) V), herpesviruses, arenaviruses (e.g., lymphocytic choriomeningitis virus (LCMV) ), measles virus, poxvirus (modified vaccinia virus (MVA), etc.), paramyxovirus vectors, such as vesicular stomatitis virus (VSV), lentivirus, or rhabdovirus vectors. That is, the vector can be derived from any of the above viruses. The virus is characterized by its medium genome size, ease of manipulation, high titer, wide target cell range, and Due to their high infectivity, they are particularly suitable for use as gene transfer vectors. Both ends of the genome contain inverted repeats (ITRs) of 100–200 base pairs, which are essential for viral DNA replication. and cis elements necessary for packaging. Early (E) and late (L) regions of the genome The E1 region contains different transcription units that are divided by the initiation of viral DNA replication. The regions (E1A and E1B) are involved in regulating the transcription of the viral genome and some cellular genes. Expression of the E2 region (E2A and E2B) encodes proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell function. It is involved in blocking (Renan, 1990). It contains most of the viral capsid proteins. The products of late genes, including the major late promoter (MLP), are expressed as a single primary transcript. MLP is expressed only after significant processing of the protein. All mRNAs transcribed from the promoter contain a tripartite 5'-leader (TPL ) sequence, resulting in mRNA that is favorable for translation. Replication-deficient adenoviruses made from the genus Virus are particularly useful because they These are replication-restricted and have the potential for pathogenic spread within vaccinated hosts. and because there is less chance of contact with vaccinated hosts.

[0086] (Other Polynucleotide Delivery) In certain embodiments of the invention, an expression construct comprising one or more polynucleotide sequences is It may simply consist of a naked recombinant DNA plasmid. Science 259:1745-1749 and its review by Cohen, 1993, Science 259:16 91-1692. Introduction of these constructs can be achieved, for example, by physically or chemically modifying the cell membrane. This can be achieved by any method of transfection, particularly in vitro transfection. The method may be applied to in vivo use as well as to in vivo use. It is contemplated that DNA can also be introduced in vivo in a similar manner to express a gene product. Several delivery systems are used to deliver DNA molecules to animal models and humans. Several products based on this technology have been approved for use in animals and have been shown to be effective in humans. Others are in Phase II and III clinical trials.

[0087] (RNA delivery) In another embodiment of the invention, an expression construct comprising one or more polynucleotide sequences is It may also consist of an RNA molecule derived from recombinant DNA (Ulmer et al., 2012, Vaccine 30:4 For DNA-based expression constructs, various methods are available for expressing RNA molecules in vitro or RNA-based constructs can be used for in vivo delivery into cells. mimicking mRNA molecules so that the introduced biological molecule is translated by the host cell's translational machinery. is directly translated by the nucleotide sequence of the nucleotide sequence, producing the polypeptide it encodes in the cell into which it is introduced. Alternatively, the RNA molecule can be designed to be capable of binding to the viral RNA-dependent RNA polymerase. By incorporating them into their structural genes, they self-amplify within the cells into which they are introduced. In this way, self-amplifying mRNA (SAM™) molecules (Geall et al., This type of RNA molecule, known as a ribosomal RNA (RIRNA), is a type of RNA molecule that is expressed in several RNA-based RNAs. share properties with other viral vectors. They can be either mRNA-based or SAM™ RNA. They may be further modified (e.g., by altering their sequence or by using modified nucleotides). ), which can enhance stability and translation (Schlake et al., RNA Biology 9:1319-1 330), and both types of RNA can be formulated (e.g., in emulsions (Brito et al., Mol. cular Therapy, 2014 22:2118-2129) or in lipid nanoparticles (Kranz et al., 2006, Nature 534:396-401) to improve in vitro or in vivo stability and / or cell entry. Myriad formulations of modified (and unmodified) RNA have been shown to promote cellular proliferation in animal models and humans. It has been tested as a vaccine, with several RNA-based vaccines in ongoing clinical trials. It is used.

[0088] (Pharmaceutical composition) The polypeptides, nucleic acids and vectors of the present invention can be used in immunogenic and vaccine compositions, etc. may be formulated for delivery in a pharmaceutical composition (hereinafter all "compositions of the invention"). The composition of the present invention preferably contains the polypeptide, nucleic acid or vector of the present invention as a pharmaceutical. together with a suitably acceptable carrier. Thus, in one embodiment, the polypeptide, nucleic acid or vector of the invention can be used as a medicament. Immunogenic pharmaceutical compositions comprising the compound together with an acceptable carrier are provided.

[0089] In another embodiment, the polypeptide, nucleic acid or vector of the invention is prepared in a pharmaceutically acceptable form. The preparation of pharmaceutical compositions generally comprises the steps of: See, e.g., Powell and Newman (eds.), Vaccine Design (the subunit and adjuvant approach), 199 The compositions of the present invention may also contain other compounds, which may be biologically active or inactive. Preferably, the compositions of the present invention are sterile compositions suitable for parenteral administration. be.

[0090] In certain preferred embodiments of the present invention, the pharmaceutical compositions of the present invention are and / or a carrier, such as a carrier containing one or more (e.g., one) polypeptides of the present invention. Provided. In certain preferred embodiments of the present invention, the compositions of the present invention comprise pharmaceutically acceptable carriers. One or more (e.g., one) nucleic acids of the invention in combination with a carrier or one or more (e.g., one) nucleic acids of the invention The vectors of the invention are provided as containing vectors.

[0091] In one embodiment, the compositions of the invention comprise one or more (e.g., one) polynucleotides and one or more Alternatively, the composition may comprise one or more of the above polypeptide components. The product may comprise one or more (e.g., one) vectors and one or more (e.g., one) polypeptide components. Alternatively, the composition may comprise one or more (e.g., one) vector and one It may also contain more than one (e.g., one) polynucleotide component. The composition may be provided to enhance an immune response.

[0092] (Pharmaceutically acceptable salts) The compositions of the invention may be prepared by administering to a subject a pharmaceutical acceptable carrier or a recipient thereof a nucleic acid or polypeptide as provided herein. It will be apparent that the present invention may also include salts thereof which are pharmaceutically acceptable. They can be prepared from non-toxic bases, including organic bases (e.g., primary, secondary and and salts of tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, salts of lithium, ammonium, calcium and magnesium).

[0093] Pharmaceutically acceptable carriers Many pharmaceutically acceptable carriers known to those skilled in the art may be used in the compositions of the present invention. Although it is possible to use a carrier in a pharmaceutical composition, the most suitable type of carrier used will vary depending on the mode of administration. The compositions of the present invention may be formulated for any suitable mode of administration, including, for example: administration via parenteral, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular routes; Preferably, parenteral administration is used, such as intramuscular, subcutaneous or intravenous administration. The carrier preferably contains water and may contain pH adjusting buffers, stabilizers (e.g., surfactants and The composition may also include an isotonic acid, an amino acid, and an isotonicity adjusting agent (e.g., salts and sugars). If intended to be provided in a lyophilized form to be diluted at the time of use, the formulation may contain a lyoprotectant, e.g. For oral administration, the carriers or Any of the solid carriers, mannitol, lactose, starch, magnesium stearate um, saccharin sodium, talcum, cellulose, glucose, sucrose, and charcoal Magnesium oxide, etc. can also be used.

[0094] Thus, the compositions of the present invention may be prepared in a buffer (e.g., neutral buffered saline or phosphate buffered saline). buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextrose), strane), mannitol, proteins, polypeptides, or amino acids such as glycine , antioxidants, bacteriostatic agents, chelating agents such as EDTA or glutathione, and preparations Contains solutes, suspending agents, thickeners, and / or preservatives that make the solution isotonic, hypotonic, or slightly hypertonic with blood Alternatively, the compositions of the present invention may be formulated as a lyophilizate.

[0095] (immunostimulant) The compositions of the present invention may also include one or more immunostimulants. Any substance that enhances or strengthens the immune response (antibody-mediated and / or cell-mediated) to an antigen. Immunostimulants are often called adjuvants in the context of vaccine formulations, and Examples include aluminum hydroxide gel (alum) or aluminum phosphate. Salt, saponin such as QS21, immunostimulatory oligonucleotide such as CPG, oil-in-water emulsion ( For example, if the oil is squalene), aminoalkyl glucosaminide 4-phosphate, lipopolysaccharide or a derivative thereof (e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPL®) and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and In view of the above, the one or more immunostimulants of the compositions of the present invention are preferably , aluminum salts, saponins, immunostimulatory oligonucleotides, oil-in-water emulsions, Aminoalkyl glucosaminide 4-phosphate, lipopolysaccharides and their derivatives, and other TLR4 receptors The immunostimulant is selected from a ligand, a TLR7 ligand, a TLR8 ligand, and a TLR9 ligand. Also, monoclonal antibodies that specifically interact with other immune components, such as PD-1 and We have developed a monoclonal antibody that blocks the interaction of immune checkpoint receptors, including CTLA4 and IL-1. It can also include.

[0096] For recombinant nucleic acid delivery methods (e.g., DNA, RNA, viral vectors), proteins The gene encoding the base immunostimulant is the gene encoding the polypeptide of the present invention. They may be readily delivered together.

[0097] (sustained release) The compositions described herein are intended to provide sustained / prolonged release of the compound following administration. release formulations (i.e., formulations such as capsules (e.g., polysaccharides), sponges, patches or gels) It may also be administered in portions.

[0098] (Storage and packaging) The compositions of the present invention may be packaged in unit-dose or multi-dose containers such as sealed ampoules or vials. Such containers may be provided in a container suitable for maintaining sterility of the formulation until use. The formulations are generally prepared as suspensions, in oily or aqueous vehicles, or as emulsions. Alternatively, the compositions of the present invention may be stored in sterile liquid containers. A freeze-dried product that requires the addition of an additional solution (such as water for injection or saline) only immediately before use. You can also save it in.

[0099] (Dosage) The amount of nucleic acid, polypeptide or vector in each composition of the invention may vary depending on the therapeutic or prophylactic use. The dosage may be formulated in such a way that a suitable dosage for the patient is obtained. factors such as bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations. Therefore, considerations will be taken into account by those skilled in the art of formulating such compositions. , various dosages and treatment regimens may be desirable.

[0100] Typically, a composition containing a therapeutically or prophylactically effective amount will contain about 0.1 μg to about 1000 μg of the compound per administration. Polypeptides of the invention, more typically from about 2.5 ug to about 100 ug of polypeptide per dose, are administered. When delivered in the form of short synthetic long-chain peptides, the dosage is 1-200ug / peptide / dose. For polynucleotide compositions, these are typically in the range of about 10 μg per dose. to about 20 mg of a nucleic acid of the invention, more typically about 0.1 mg to about 10 mg of a nucleic acid of the invention per administration Deliver.

[0101] (Diseases to be treated or prevented) As described elsewhere in the specification, SEQ ID NOS: 1-10 are over-expressed in cutaneous malignant melanoma. The polypeptide sequence corresponding to the expressed CLT antigen. In one embodiment, the present invention provides a polypeptide, nucleic acid, or a polypeptide of the present invention for use in medicine. A vector or composition is provided.

[0102] A further aspect of the invention is a method of increasing an immune response in a human, comprising administering to the human the The present invention also relates to methods comprising administering a polypeptide, nucleic acid, vector or composition of the invention. The present invention also provides a method for the production of a polypeptide of the invention, a nucleic acid sequence, or a polypeptide of the invention for use in raising an immune response in humans. The present invention provides an acid, vector or composition. Polypeptides of the invention for the manufacture of a medicament for use in raising an immune response in humans Uses of the nucleic acids, vectors or compositions are also provided.

[0103] Preferably, the immune response is mediated by the antibody of SEQ ID NOs: 1 to 10 and variants of any one thereof and an immunogen. The antibody is elevated in cancerous tumors expressing the corresponding sequence selected from the antibody fragment. "Corresponding" in this context means that the tumor corresponds to, for example, SEQ ID NO: A (where A is one of SEQ ID NOs: 1-10). When expressing a polypeptide of the invention, a nucleic acid fragment thereof, or a variant or immunogenic fragment thereof, The acid, vector or composition and the pharmaceutical preparation containing the same are not limited to SEQ ID NO: A or variants or variants thereof. This means that it will be based on immunogenic fragments.

[0104] Suitably, the immune response is a CD8+ T cell, CD4+ T cell and / or antibody response, particularly a CD8+ cytolytic response. These include immunosuppressive T cell responses and CD4+ helper T cell responses. Preferably, the immune response is directed against the tumor, in particular against SEQ ID NOS: 1 to 10 and variants thereof, and the immune response is directed against the tumor. The vector is then induced to express sequences selected from the protease fragment. In suitable embodiments, the tumor is a melanoma tumor, for example a cutaneous melanoma tumor. The tumor can be a primary tumor or a metastatic tumor.

[0105] A further aspect of the present invention is a method for treating a human cancer patient, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10. and therapeutic methods expressing sequences selected from any one of the immunogenic fragments and variants thereof. or a method for preventing a human from developing cancer, wherein the cancer is a cancer of the type selected from SEQ ID NOS: 1 to 10 and the like. A prophylactic method would involve expressing a sequence selected from any one of the immunogenic fragments and variants. and administering to the person the corresponding polypeptide, nucleic acid, vector or composition of the invention. and

[0106] The present invention also provides a method for the treatment or prevention of human cancer, comprising administering to a subject a polypeptide, nucleic acid, vector, or combination thereof ... for use in the treatment or prevention of human cancer, a vector or composition, the cancer cells of which are selected from the group consisting of SEQ ID NOS: 1 to 10 and any one of the immunogenic groups thereof; A polypeptide, nucleic acid, vector or fragment expressing a corresponding sequence selected from the fragments. A composition is provided.

[0107] The transcripts corresponding to SEQ ID NOs: 33, 35, 36 and 40 are also overexpressed in uveal melanoma. As a result, in another embodiment, the tumor is a uveal melanoma tumor and / or a genotype. Tumors expressing sequences selected from sequence numbers 1, 3, 4, 5 and 10.

[0108] Thus, the present invention provides polypeptides, nucleic acids, vectors for the methods or uses of the present invention. or a composition, the polypeptide comprising a sequence selected from: (a) any one of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide may be any of SEQ ID NOs: 11-14, 17-18, 19, 20-22, 30-32, 51-53, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64 57, 67 to 74, and 76 to 77, or consisting of the sequence selected from the sequence and for example, the nucleic acid is selected from any one of SEQ ID NOs: 33, 35, 36, or 40. or comprising a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50, and The cancer is uveal melanoma. The terms "prevention" and "prophylaxis" are used interchangeably herein.

[0109] (Treatment and vaccination regimens) The therapeutic regimen comprises (i) administering a polypeptide, nucleic acid, or vector of the invention; (ii) administering one or more a further polypeptide, nucleic acid or vector of the invention and / or (iii) optionally an adjuvant Various other therapeutically useful compounds or molecules, such as antigenic proteins, may be administered simultaneously with the and additional components such as, at the same time (e.g., simultaneous administration) or sequentially (e.g., prime boost An example of simultaneous administration is delivery of This includes simultaneous administration and simultaneous administration at opposite sites. It relates to all components being delivered during the same treatment session. Preferably, all components are administered simultaneously. (e.g., simultaneous administration of both DNA and protein), but one component may be administered within minutes (e.g., within the same appointment). It may be administered within 24 hours (approximately 24 hours before or at the time of a doctor's visit) or within a few hours.

[0110] In some embodiments, the "priming" of a polypeptide, nucleic acid or vector of the invention " or first administration followed by one or more "boosters" of the polypeptides, nucleic acids or vectors of the invention. In one embodiment, a "prime and boost" or booster immunization is performed (a "prime and boost" strategy). The polypeptide, nucleic acid or vector of interest may be administered in a single prime-boost vaccination regimen. In one embodiment, both the prime and boost are used with a polypeptide of the invention. and in each case the same polypeptide of the invention. Both the gene and the booster are nucleic acids or vectors of the invention, and in each case the same nucleic acid or vector of the invention Alternatively, the primer may be prepared using a nucleic acid or vector of the present invention. In this case, the boost may be performed using a polypeptide of the present invention, and the prime may be performed using a polypeptide of the present invention. The polypeptides described above are used to generate booster antibodies, and the booster antibodies are generated using nucleic acids or vectors of the present invention. Typically, a first or "priming" administration and a second or "boost" administration are administered. The administration is given after an interval of about 1 to 12 weeks, or up to 4 to 6 months. Doses may be given as frequently as every 1 to 6 weeks, or later (up to several years later). Good too.

[0111] (Combination of antigens) A polypeptide, nucleic acid or vector of the invention may be synthesized in combination with one or more other polypeptides or vectors of the invention. The nucleic acids may be used in combination with vectors and / or to treat malignant melanoma, e.g., cutaneous or bronchial Other antigenic polypeptides (or polypeptides thereof) that elicit an enhanced immune response against uveal malignant melanoma These other polynucleotides or vectors encoding the same can be used in combination. The antigenic polypeptides may be derived from a variety of sources, including GPR143, PRAME, M They may also include well-described melanoma-associated antigens such as AGE-A3 or pMel (gp100). Alternatively, they may contain other species of melanoma antigens, including those specific to the patient. Patient-specific neoantigens (Lauss et al., (2017). Nature Communications, 8(1), 1738. http: / / www.lauss.com / ). / / doi.org / 10.1038 / s41467-017-01460-0), retained intron neoantigens (Smart et al. , (2018). Nature Biotechnology. http: / / doi.org / 10.1038 / nbt.4239), splice variants Body neoantigens (Hoyos et al., Cancer Cell, 34(2), 181-183. http: / / doi.org / 10.1016 / j.cc ell.2018.07.008;Kahles et al. (2018), Cancer Cell, 34(2),211-224.e6. http: / / d oi.org / 10.1016 / j.ccell.2018.07.001), T cells associated with impaired peptide processing Melanoma antigens (TIEs) belong to a category known as epitope-encoding antigens. PPs; Gigoux, M. and Wolchok, J. (2018), JEM, 215, 2233; Marijt et al. (2018 ), JEM 215, 2325), or new antigens that may be discovered (including CLT antigens). Antigenic peptides derived from these various sources also include (i) nonspecific immunostimulants / Ajuba and / or (ii) a potent antibody that contains, for example, a universal CD4 helper epitope. Antigens (as polypeptides or as CD4 antigens) known to elicit CD4 helper T cells in combination with a polynucleotide or vector encoding the antigen can also amplify the anti-melanoma specific response elicited by co-administered antigens. I guess so.

[0112] The different polypeptides, nucleic acids or vectors may be formulated in the same formulation or in separate formulations. Alternatively, the polypeptide may be a polypeptide of the invention, in which the polypeptide of the invention is a second or further polypeptide. It may also be provided as a fusion protein fused to a polypeptide (see below). A nucleic acid may be provided that encodes the fusion protein.

[0113] More generally, when two or more components are used in combination, the components For example: (1) as two or more individual antigenic polypeptide components; (2) as a fusion protein containing both (or additional) polypeptide components; (3) as one or more polypeptide and one or more polynucleotide components; (4) as two or more individual polynucleotide components; (5) as a single polynucleotide encoding two or more individual polypeptide components; or (6) A fusion protein encoding both (or additional) polypeptide components. The sequences may be provided as a single polynucleotide.

[0114] For convenience, when multiple components are present, they may be combined into a single fusion protein or a single It is often desirable for the fusion protein to be contained within a polynucleotide encoding the fusion protein. In one embodiment of the invention, all components are polypeptides. In another embodiment of the invention, the entire All components may be polynucleotides (e.g., those encoding a single fusion protein). The fragment is provided as a single polynucleotide such as a fragment of the fragment of the

[0115] (Fusion Protein (Fusion Polypeptide)) As an embodiment of the above discussion of antigen combinations, the present invention also provides a method for producing a combination of antigens encoding individual antigens. The second or further nucleic acid constructs of the invention can be prepared by fusing together sequences that correspond to the nucleic acid sequences of the invention. The isolated polypeptide of the present invention fused to a polypeptide (hereinafter "combination of the present invention") The combination polypeptides of the present invention are and are predicted to have the utility described herein for polypeptides that have excellent immune responses. virulence or vaccine activity, or prophylactic or therapeutic effect (increasing the breadth and depth of response) This may have advantages, including: Fusion of the polypeptides of the invention may also be used as vaccine antigens and / or vectored vaccines (nucleic acid vaccines). This will also provide benefits that increase the efficiency of construction and manufacturing of the casing (including the casing).

[0116] As described above in the "Combination of Antigens" section, the polypeptides of the present invention and the Combination polypeptides may also be polypeptide sequences that are not polypeptides of the invention. , may be fused to include one or more of the following: (a) other polypeptides that are melanoma-associated antigens and therefore are not immunogenic in the vaccine; Potentially useful protease sequences (e.g., GPR143, PRAME, MAGE, etc., as mentioned above) -A3 and pMel(gp100)); and (b) A polypeptide sequence capable of enhancing an immune response (i.e., an immunostimulatory sequence). (c) For example, it contains a universal CD4 helper epitope and provides strong CD4 help. Polypeptide sequences capable of enhancing CD8+ T cell responses to CLT antigen epitopes Column.

[0117] Exemplary fusion polypeptides include two or more sequences selected from SEQ ID NOs: 1, 2, 3, and 4 (e.g., , 2, 3 or 4); or, for each such sequence, a variant of that sequence, or It includes immunogenic fragments of the sequence.

[0118] One exemplary fusion polypeptide comprises: (i) a sequence selected from: (a) the sequence of SEQ ID NO: 1; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a), for example, selected from SEQ ID NOs: 11-14, 55-57, and 73-74; Piece; (ii) a sequence selected from: (a) the sequence of SEQ ID NO: 2; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a), for example, selected from SEQ ID NOs: 15-16, 58-66, 75, and 78; Piece; (iii) a sequence selected from: (a) the sequence of SEQ ID NO: 3; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a), for example, selected from SEQ ID NOs: 17-18, 53, and 67-69; and (iv) a sequence selected from: (a) the sequence of SEQ ID NO: 4; and (b) a variant of the sequence of (a); and (c) an immunoglobulin having a sequence of (a), for example, selected from SEQ ID NOs: 19, 51 to 52, 54, 70 to 72, and 76 to 77; Epidemiogenic fragment. For example, the fusion polypeptide comprises the sequences of SEQ ID NOs: 1, 2, 3 and 4.

[0119] Another exemplary fusion polypeptide comprises: (i) a sequence selected from: (a) the sequence of SEQ ID NO: 1; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a), for example, selected from SEQ ID NOs: 11-14, 55-57, and 73-74; Piece; (ii) a sequence selected from: (a) the sequence of SEQ ID NO: 2; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a), for example, selected from SEQ ID NOs: 15-16, 58-66, 75, and 78; one piece; and (iii) a sequence selected from: (a) the sequence of SEQ ID NO: 4; and (b) a variant of the sequence of (a); and (c) an immunoglobulin having a sequence of (a), for example, selected from SEQ ID NOs: 19, 51 to 52, 54, 70 to 72, and 76 to 77; Epidemiogenic fragment. For example, the fusion polypeptide comprises the sequences of SEQ ID NOs: 1, 2 and 4.

[0120] The present invention also provides the fusion polypeptides, mutatis mutandis, as well as the polypeptides of the present invention. Nucleic acids encoding the genes, as well as other aspects of the invention (vectors, compositions, cells, etc.) are provided.

[0121] (CLT antigen-binding polypeptide) An antigen-binding polypeptide that is immunospecific for a tumor-expressed antigen (the polypeptide of the present invention) The cytolytic cells are recruited to antigen-modified tumor cells to mediate their destruction. Such antigen-binding polypeptides could be designed to recruit cytolytic cells. One of the mechanisms is known as antibody-dependent cell-mediated cytotoxicity (ADCC). The invention provides antigen-binding polypeptides immunospecific for the polypeptides of the invention. Monoclonal antibodies and fragments thereof, such as domain antibodies, Fab fragments, Fv fragments, and VHH fragments Antigen-binding polypeptides, including antibodies, can be expressed in non-human animal species (e.g., rodents or camelids). They may be produced in a non-human species (e.g., modified to have a human immune system) and humanized, or may be produced in a non-human species (e.g., modified to have a human immune system). It may also be produced in genetically modified rodents.

[0122] Antigen-binding polypeptides can be produced by methods well known to those skilled in the art. Clonal antibodies are produced using hybridoma technology to grow specific antibody-producing B cells in tissue culture. Fuse with myeloma (B-cell cancer) cells selected for their proliferative potential and lack of antibody chain synthesis (Kohler and Milstein, 1975, Nature 256(5517):495-497) and Nelson et al., 2000 (June), Mol Pathol. 53(3):111-7, incorporated herein by reference in their entirety. (The body of which is incorporated herein.)

[0123] Monoclonal antibodies against a desired antigen can be prepared, for example: a) Lymphocytes obtained from the peripheral blood of animals (including humans) that have been previously immunized / exposed with the desired antigen. immortalizing the lymphocytes with immortal cells, preferably myeloma cells, to form hybridomas; b) Cultivating the resulting immortalized cells (hybridomas) to produce antibodies with the desired specificity recovering the cells; It can also be obtained by

[0124] Monoclonal antibodies can also be obtained by a manufacturing process comprising the steps of: : a) vectors, particularly phages, more particularly filamentous bacteriophages (preferably DNA obtained from lymphocytes, particularly peripheral blood lymphocytes, of an animal previously immunized with a desired antigen, or Cloning steps of cDNA sequences b) transforming a prokaryotic cell with the vector under conditions that allow antibody production , c) selecting antibodies by subjecting them to antigen affinity selection; d) recovering antibodies with the desired specificity; e) Antibodies obtained from B cells of patients exposed to an antigen or from animals immunized with an antigen and expressing the nucleic acid molecule encoding the vector. The selected antibodies can then be purified using conventional recombinant protein production techniques (e.g., genetic It may also be produced from engineered CHO cells.

[0125] The present invention provides isolated antigen-binding polypeptides immunospecific for the polypeptides of the invention. Preferably, the antigen-binding polypeptide is a monoclonal antibody or a fragment thereof. is. In certain embodiments, the antigen-binding polypeptide is conjugated to a cytotoxic moiety. Examples of cytotoxic moieties include the Fc domain of an antibody, which binds to Fc receptor-bearing cells to promote ADCC. Alternatively, the antigen-binding polypeptide may be a biological toxin or a cytotoxic chemical. It may be linked to quality.

[0126] Another important class of antigen-binding polypeptides is T-cell receptors that bind to HLA-presented fragments of the antigens of the invention. In this embodiment, the CLT antigen (or its derivatives) on the surface of tumor cells is TCR-based biologics (TCRs derived directly from patients or specifically engineered to recognize TCRs) The immune cells (containing a specific high-affinity TCR) also attract these immune cells to the tumor, providing a therapeutic effect. It contains a targeting moiety that recognizes a component on T cells (or another class of immune cells) that In some embodiments, the targeting moiety also acts as a beneficial agent for redirected immune cells. It may also stimulate various activities (including cytolytic activity).

[0127] Thus, in one embodiment, the antigen-binding polypeptide is a polypeptide of the invention; or or a portion thereof. The polypeptide is a T cell receptor. In one embodiment, the antigen-binding polypeptides of the invention are administered to a subject by cytotoxic cells or other The antibody may also be linked to another polypeptide capable of binding to an immune component of the antibody.

[0128] In one embodiment, the antigen-binding polypeptide is for use in medicine. In one embodiment, the antigen-binding polypeptides of the invention are administered in a pharmaceutically acceptable carrier. Such compositions are sterile and suitable for parenteral administration. See, for example, the disclosure of pharmaceutical compositions above.

[0129] According to the present invention, there is provided a method for treating a human suffering from cancer, wherein the cancer cells are selected from SEQ ID NOS: 1 to 10 and the like. or a therapeutic method comprising expressing a sequence selected from the immunogenic fragments and variants of any one of A method for preventing cancer in humans, wherein the cancer cells are those of SEQ ID NOS: 1 to 10 and any of the a prophylactic method which would express a sequence selected from one of the immunogenic fragments and variants, administering an antigen-binding polypeptide of the invention or a composition comprising the antigen-binding polypeptide to the human The method includes administering

[0130] In one embodiment, a cytotoxic moiety conjugated to a cytotoxic moiety for use in the treatment or prevention of human cancer is provided. an antigen-binding polypeptide of the present invention which may be used in a method for the preparation of a compound of the present invention; The present invention relates to a composition of matter, wherein the cancer cells are selected from SEQ ID NOS: 1 to 10 and any one of immunogenic fragments thereof. the antigen-binding polypeptide or composition expressing a corresponding sequence selected from Provided. Preferably, in any of the above embodiments, the cancer is malignant melanoma, particularly cutaneous malignant melanoma. do.

[0131] In one embodiment, an antigen-binding polypeptide or a polypeptide for a method or use of the invention is or a composition, wherein the polypeptide comprises a sequence selected from: (a) any one of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide may be any of SEQ ID NOs: 11-14, 17-18, 19, 20-22, 30-32, 51-53, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64 57, 67 to 74, and 76 to 77, or consisting of the sequence selected from the sequence and for example, the nucleic acid is selected from any one of SEQ ID NOs: 33, 35, 36, or 40. or comprising a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50, and The cancer is uveal melanoma. Alternatively, compositions are provided.

[0132] The antigen-binding polypeptide (antibody or fragment thereof, etc.) is, for example, 5 to 1000 mg, for example, 25 to 500 mg For example, it may be administered at a dose of 100 to 300 mg, for example about 200 mg.

[0133] (Cell therapy that enhances antigen presentation in vivo) To promote the generation of an antigen-specific immune response, any of a variety of cell delivery vehicles can be administered intravenously. The invention therefore provides a method for the ex vivo loading of a polypeptide of the invention into a pharmaceutical composition. or genetically engineered to express a polypeptide of the invention. and providing an isolated antigen-presenting cell (hereinafter referred to as "APC of the present invention"). Antigen-presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes, and Other cells that may be engineered to become antigen-presenting cells. to enhance T cell proliferation, to improve activation and / or maintenance of T cell responses, and / or is genetically modified to be immunologically compatible with the recipient (i.e., HLA haplotype-matched). APCs are generally found in various body fluids and organs. The cells can be isolated from any of the following and can be autologous, allogeneic, syngeneic or xenogeneic.

[0134] In certain preferred embodiments of the present invention, dendritic cells or their precursors are used as APCs. Thus, in one embodiment, the APCs of the present invention are dendritic cells. Dendritic cells are highly potent APC (Banchereau and Steinman, 1998, Nature 392:245-251) and can be used prophylactically or therapeutically. It has been shown to be effective as a physiological adjuvant for eliciting therapeutic immunity (T (See Immerman and Levy, 1999, Ann. Rev. Med. 50:507-529.) Generally, dendritic cells The vacuoles have a typical shape (stellate in situ and prominent cytoplasmic projections visible in vitro). They have dendrites, the ability to efficiently take up, process, and present antigens, and Dendritic cells may also be identified based on their ability to activate naive T cell responses. Of course, specific cell surface molecules not normally found on dendritic cells in vivo or ex vivo are The modified dendritic cells may be engineered to express a receptor or ligand. Apart from dendritic cells, antigen-loaded secretory vesicles (exocytospores) are also contemplated by the present invention. These may be used in immunogenic compositions (Zitvogel et al., 1998, Nature re Med. 4:594-600). Thus, in one embodiment, a medicament is loaded with a polypeptide of the invention. The exosomes are provided.

[0135] Dendritic cells and precursor cells can be derived from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood, or other sources. They may be obtained from any suitable tissue or body fluid. For example, dendritic cells may be obtained from a culture of monocytes taken from peripheral blood. Adding a combination of cytokines, such as GM-CSF, IL-4, IL-13, and / or TNFα, to the culture Alternatively, cells collected from peripheral blood, umbilical cord blood, or bone marrow may be differentiated in vitro. CD34-positive cells were treated with GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and / or is added to the culture medium in combination with other compounds that induce differentiation, maturation and proliferation of dendritic cells. The cells may be differentiated into dendritic cells by this method.

[0136] Dendritic cells are conveniently categorized as "immature" and "mature" cells, with two well-characterized However, this nomenclature is not suitable for the This should not be interpreted as excluding all possible intermediate steps in the process of dendritic cell proliferation. , which are characterized as APCs with high antigen uptake and processing capacity, and which have Fcγ receptors. The mature phenotype is usually correlated with high expression of the mannose receptor and the ribosomal receptor. Although expression of CAR is low, cell surface molecules involved in T cell activation, such as class I and class II MHC , adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4 -1BB).

[0137] The APC may also contain, for example, a polynucleotide encoding a protein (or a portion or other variant thereof). Transfect the cells with a nucleotide and genetically modify the polypeptide so that it is expressed on the cell surface. Such transfection may occur in vitro and then The pharmaceutical composition comprising the transfected cells is used as described herein. Alternatively, a gene transfer vehicle that targets dendritic cells or other antigen-presenting cells may be used. They may be administered to a patient, resulting in transfection occurring in vivo. In vivo and ex vivo transfection of . or the method described in Mahvi et al., 1997, Immunology and Cell Biology 75:456-460. This can be done using any method generally known in the art, such as the gene gun approach currently used. Antigen loading into dendritic cells may be carried out by injecting dendritic cells or precursor cells with a polypeptide, DNA, or the like. A (e.g., a plasmid vector) or RNA; or a recombinant bacterium or virus expressing the antigen. viruses (e.g., adenoviruses, adeno-associated viruses (AAV) (e.g., AAV types 5 and 2), Alphaviruses (e.g., Venezuelan equine encephalitis virus (VEEV) and Sindbis virus (SI N), Semliki Forest virus (SFV), herpesviruses, arenaviruses (e.g., lymphocytic choriomeningitis virus (LCMV), measles virus, poxvirus (modified vaccinia virus), (MVA) or fowlpox), paramyxovirus, lentivirus, or rhabdovirus (water This may be achieved by incubating the cells with a virus such as vesicular stomatitis virus (VSV). Before loading the polypeptide, the polypeptide is introduced into an immunological partner (e.g., a carrier). Alternatively, dendritic cells may be covalently linked to non-conjugated antigens (e.g., ribonucleotides) to provide T cell help. immunological partners, either individually or in the presence of a polypeptide or vector Under, you may pulse.

[0138] The present invention provides a method for the preparation of specifically designed, short, chemically synthesized epitope constructs of polypeptide antigens. The encoded fragments are provided for delivery to antigen-presenting cells. Those skilled in the art will appreciate that such molecules are The antigenic polypeptides of the present invention may be used to infect, known as synthetic long peptides (SLPs). A therapeutic platform for stimulating (or loading) cells in vitro (Gornati et al. , 2018, Front. Imm, 9:1484), or in vivo, polypeptide antigens are delivered to antigen-presenting cells. As a method of introduction (Melief and van der Burg et al., 2008, Nat Rev Cancer, 8:351-6 0) will appreciate that we provide

[0139] In one embodiment, the antigen-presenting cells of the present invention, preferably dendritic cells, are administered in a pharmaceutically acceptable carrier. Such compositions are suitable for parenteral administration. The compositions may also be sterile compositions, see, for example, the disclosure of pharmaceutical compositions above. In one embodiment, an antigen-presenting cell, preferably a dendritic cell, of the invention for use in medicine. is provided.

[0140] Similarly, there is provided a method for treating a human suffering from cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and any of the above. and a method of treating or administering to a human a subject the therapeutically effective amount of a human subject expressing a sequence selected from any one of the immunogenic fragments and variants. A method for preventing cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and any one of them. A prophylactic method which would express a sequence selected from the immunogenic fragments and variants of the present invention. The antigen-presenting cells, preferably dendritic cells, or a composition comprising the antigen-presenting cells of the present invention are administered to the subject. The method includes administering to a subject.

[0141] In one embodiment, the antigen-presenting cells of the present invention, preferably human cancer cells, are used for the treatment or prevention of human cancer. Preferably, a composition comprising dendritic cells or the antigen-presenting cells of the present invention, wherein the cancer cells are A vector expressing a corresponding sequence selected from sequence numbers 1 to 10 and any one of the immunogenic fragments thereof. The present invention provides the antigen-presenting cell or a composition thereof.

[0142] In one embodiment, the exosomes of the present invention are contained in a pharmaceutical composition comprising the exosomes of the present invention together with a pharmaceutically acceptable carrier. Such compositions may be sterile compositions suitable for parenteral administration. See, for example, the disclosure of pharmaceutical compositions above. The compositions may optionally contain an immunostimulant. See the immunostimulant disclosure above. In one embodiment, there is provided an exosome of the invention for use in medicine.

[0143] Similarly, there is provided a method for treating a human suffering from cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and any of the above. and a method of treating or administering to a human a subject the therapeutically effective amount of a human subject expressing a sequence selected from any one of the immunogenic fragments and variants. A method for preventing cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and any one of them. A prophylactic method which would express a sequence selected from the immunogenic fragments and variants of the present invention. administering to the human an exosome or a composition comprising the exosome of the present invention. The law is provided.

[0144] In one embodiment, the exosomes or IgG4-dependent ... The composition of the present invention containing the exosome, wherein the cancer cells are those of SEQ ID NOs: 1 to 10 and their The exogenous vector expresses a corresponding sequence selected from any one of the immunogenic fragments. A lysosome or composition is provided. In any one of the above embodiments, suitably the cancer is malignant melanoma, particularly cutaneous malignant melanoma. is.

[0145] (Stimulated T cell therapy) APC-mediated generation, in vivo or ex vivo, of T cells immunospecific for the polypeptides of the present invention In addition, autologous or non-autologous T cells may also be obtained from a subject, e.g., from peripheral blood, umbilical cord blood, and and / or tumor cells isolated by apheresis and loaded onto MHC molecules (signal 1) of APC cells. Stimulation in the presence of a tumor-associated antigen induces proliferation of T cells bearing TCRs immunospecific for this antigen. That's fine.

[0146] For successful T cell activation, the costimulatory surface molecules B7 and CD28 are required to bind to antigen-presenting cells, respectively. It is necessary for the IL-1 receptor to bind to the IL-1 receptor on the T cell surface (signal 2). To achieve optimal T cell activation, Both signals 1 and 2 are required. In contrast, the anti- Page 11 ... Antigenic peptide stimulation (signal 1) fails to induce full T cell activation and induces T cell tolerance. In addition to costimulatory molecules, there are also inhibitory molecules such as CTLA-4 and PD-1, which act to inhibit T Induce signals to prevent cell activation.

[0147] Thus, autologous or non-autologous T cells are stimulated in the presence of a polypeptide of the invention, The cancer cells express the corresponding polypeptides of the present invention. It may be reintroduced into at-risk or cancer-affected patients, provided that their antigen-specific TCR The cancer cells recognize the antigens presented by the patient's MHC and express the corresponding polypeptides. Only if it targets the cell and will induce its death.

[0148] In one embodiment, the method is used to stimulate and / or expand T cells from cancer-affected humans in vitro. and then administering the stimulated and / or expanded T cells to the human to treat the human's cancer. and a polypeptide, nucleic acid, vector or composition of the invention for reintroduction into a mammal. .

[0149] The present invention relates to a method for treating human cancer, wherein the cancer cells are selected from SEQ ID NOS: 1 to 10 and any of the and expressing a sequence selected from one of the immunogenic fragments and variants, the method comprising: A population of leukocytes containing at least T cells, optionally together with antigen-presenting cells, is removed from the individual. and infecting the T cells with the presence of a corresponding polypeptide, nucleic acid, vector or composition of the invention. and stimulating and / or amplifying the stimulated and / or amplified T and reintroducing some or all of the white blood cells, including the cells, into the human. do. In any one of the above embodiments, suitably the cancer is malignant melanoma, particularly cutaneous malignant melanoma. is.

[0150] In one embodiment, the antibody is selected from SEQ ID NOs: 1 to 10 and immunogenic fragments and variants of any one thereof. and a method for preparing a T cell population that is cytotoxic to cancer cells expressing a sequence selected from the above. (a) obtaining T cells and antigen-presenting cells from a cancer patient; and (ii) treating the T cells ex vivo. Stimulating and amplifying the population with the corresponding polypeptide, nucleic acid, vector or composition of the invention. A method is provided that includes: "Corresponding" in this context means that the cancer cells correspond to, for example, SEQ ID NO: A (where A is SEQ ID NO: 1 to 10) or a variant or immunogenic fragment thereof, the T cell population SEQ ID NO: A or SEQ ID NO: B in the form of a polypeptide, nucleic acid or vector, or a composition containing one of the foregoing This means that the antibody or variant or immunogenic fragment thereof is stimulated and amplified in vitro.

[0151] For example, in such a preparation process, the culture and expansion are carried out in the presence of dendritic cells. The dendritic cells are transfected with the nucleic acid molecules or vectors of the present invention to produce the antibodies of the present invention. The polypeptide will be expressed. The present invention relates to a T cell population (hereinafter referred to as the present invention) that may be obtained by any of the above preparation methods. The present invention provides a T cell population of the present invention.

[0152] In one embodiment, T cells stimulated with a polypeptide, nucleic acid, vector or composition of the invention In one embodiment, a cell (hereinafter referred to as a T cell of the present invention) is provided. In one embodiment, the T cell population or T cells of the invention are administered in a pharmaceutically acceptable carrier. Such compositions are suitable for parenteral administration, for example. It may be a sterile composition. In one embodiment there is provided a T cell population or T cell of the invention for use in medicine.

[0153] Similarly, there is provided a method for treating a human suffering from cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and any of the above. and a method of treating or administering to a human a subject the therapeutically effective amount of a human subject expressing a sequence selected from any one of the immunogenic fragments and variants. A method for preventing cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and any one of them. A prophylactic method which would express a sequence selected from the immunogenic fragments and variants of the present invention. a T cell population or T cells or a composition comprising the T cell population or T cells of the present invention, The method includes administering

[0154] In one embodiment, the T cell population of the invention for use in the treatment or prevention of human cancer, or a composition comprising the T cell population or T cell of the present invention, wherein the cancer cell is A vector expressing a corresponding sequence selected from sequence numbers 1 to 10 and any one of the immunogenic fragments thereof. The T cell population of the present invention, the T cell of the present invention, or the T cell population or T cell of the present invention In any one of the foregoing embodiments, suitably the cancer is malignant melanoma. , especially cutaneous malignant melanoma.

[0155] In one embodiment, the preparation, method, or T cell population, T cell, anti- A prototypic presentation cell, exosome, or composition, the polypeptide of which is selected from the following: Contains the following array: (a) any one of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide may be any of SEQ ID NOs: 11-14, 17-18, 19, 20-22, 30-32, 51-53, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64 57, 67 to 74, and 76 to 77, or consisting of the sequence selected from the sequence and for example, the nucleic acid is selected from any one of SEQ ID NOs: 33, 35, 36 or 40, or comprises a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50, or consists of a column; and The cancer is uveal melanoma. Presenting cells, exosomes or compositions are provided.

[0156] (Therapy using genetically engineered immune cells) All of the above-mentioned derivatives of CLT antigen-binding polypeptides form complexes with human HLA molecules. TCR or TCR mimics that recognize peptides derived from CLT antigens (Dubrovsky et al., 2016, Oncoi and (see immunology) to be expressed on the surface of T cells (autologous or non-autologous). may be engineered, which can then be administered as adoptive T cell therapy for the treatment of cancer.

[0157] These derivatives fall into the category of "chimeric antigen receptors (CARs)" and are referred to herein as such. When used herein, it refers to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor. They may also be engineered to confer artificial specificity to particular immune effector cells. CARs may also include receptors that deliver monoclonal antibodies to T cells, for example, for use in adoptive cell therapy. It may also be used to confer specificity to clonal antibodies, thereby allowing the production of a large number of specific T The CAR can be used to generate cells that express a polypeptide of the invention that binds to HLA. The antibody will be specific for a tumor-associated antigen that is

[0158] Another approach to treating cancer in patients is to target antigens expressed on tumor cells. The goal is to genetically modify T cells to target the immune system through the expression of chimeric antigen receptors (CARs). This technique is described in Wendell and June, 2017, Cell, 168:724-740 (in its entirety). (which is incorporated herein).

[0159] Such CAR T cells can be prepared by administering to a subject a cell sample containing T cells or T cell precursors, e.g., For example, cells obtained from peripheral blood, umbilical cord blood and / or by apheresis are used to bind HLA. A chimeric T cell receptor (CAR) encoding a chimeric T cell receptor (CAR) that is immunospecific for the polypeptide of the present invention to which it is linked. Such nucleic acids may be prepared by transfection of cells. The cells may be incorporated into the genome and an effective amount of the cells may be administered to the subject to A T cell response may be provided against cells expressing the polypeptides of the invention. A cell sample may be obtained from the subject.

[0160] The cells used to generate the CAR-expressing T cells can be autologous or non-autologous. It is understood that Transgenic CAR-expressing T cells express inactivated endogenous T cell receptors and / or endogenous HLA. For example, cells may be transfected to eliminate expression of endogenous α / β T cell receptors (TCRs). You may operate it.

[0161] Methods for transfecting cells are well known in the art, including electroporation, Highly efficient transfection methods can also be used. For example, CAR constructs The nucleic acid or vector of the invention expressing the The vector may be introduced into cells using a vector.

[0162] The cell population for CAR-expressing T cells may be enriched after transfection of the cells. For example, CAR-expressing cells can be expressed by the antigen bound by the CAR or by the use of a CAR-binding antibody. Alternatively, the non-expressing cells can be sorted (e.g., by FACS) from the non-expressing cells. This includes depleting T cells and depleting cells that do not express CAR. 56+ cells can be depleted from the culture population.

[0163] The population of transfected CAR-expressing cells is grown in a medium that selectively enhances the proliferation of CAR-expressing T cells. They may also be cultured in vitro, and thus CAR-expressing T cells may be expanded in vitro. A sample of CAR cells may be stored (or maintained in culture). For example, the sample may be They may be cryopreserved for later expansion or analysis. CAR-expressing T cells are compatible with other therapies, such as checkpoint blockade, including PD-L1 antagonists. It may also be used in combination with an anti-inflammatory agent.

[0164] In one embodiment, a mammalian cell line is adapted to express any of the above antigen-binding polypeptides on its surface. In one embodiment, engineered cytotoxic cells are provided. Suitably, the cytotoxic cells are T cells. In one embodiment, a pharmaceutical composition comprising the antigen-binding polypeptide on its surface for use in medicine. Cytotoxic cells, preferably T cells, engineered to express either do. The present invention provides pharmaceutical compositions comprising the cytotoxic cells of the present invention, which are preferably T cells. do.

[0165] A method for treating a human cancer patient, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and any one of SEQ ID NOS: 1 to 10. A therapeutic method for expressing a sequence selected from the immunogenic fragments and variants, or a method for preventing humans from developing cancer. A method for preventing cancer, the cancer being treated with SEQ ID NOs: 1 to 10 and any one of immunogenic fragments thereof, and a variant thereof, Methods are provided which comprise administering to the human cytotoxic cells, preferably T cells, of the invention. can be.

[0166] In one embodiment, the cytotoxic cells of the invention, preferably T cells, are used to treat or prevent human cancer. The cancer cells are selected from the group consisting of SEQ ID NOs: 1 to 10 and any one of immunogenic fragments thereof. The present invention relates to a method for expressing a corresponding sequence selected from the group consisting of:

[0167] (combination therapy) The cancer treatment methods of the present invention may be used in combination with other therapies, particularly checkpoint inhibitors and interferon. This may be performed in combination with Polypeptides, nucleic acids, vectors, antigen-binding polypeptides, and (APC and T cell-based) Adoptive cell therapy may enhance their immunogenicity (e.g., the magnitude of the immune response elicited and / or to improve coverage) or to enhance other activities (e.g., the innate or adaptive immune response or Other components designed to provide other aspects of activation, such as destruction of tumor cells, It can be used in combination with

[0168] Thus, the present invention provides a composition (i.e., an immunogenic, vaccine, or pharmaceutical composition) of the present invention. or a polypeptide, nucleic acid, or vector of the invention in a pharmaceutically acceptable carrier. and (i) one or more additional immunogenic or immunostimulatory polypeptides (e.g., interferon, IL-12, checkpoint inhibitor molecules or nucleic acids encoding the same, or (ii) a small molecule (e.g., an HDAC inhibitor or a vector containing a nucleic acid of a cancer cell); other drugs that modify the protein) or biologics (polypeptides or nucleic acids encoding them, or delivered as a vector containing the nucleic acid thereof), and a kit of several compositions comprising: To provide.

[0169] Checkpoint inhibitors target normal proteins on cancer cells or the T proteins that respond to them. These inhibitors block proteins on cells, helping to protect cancer cells from immune system attacks. In particular, it is combined with CLT antigen-based therapy to try to overcome one of the main defenses of It may be an important class of drugs.

[0170] Therefore, one aspect of the present invention is a method for producing a polypeptide, a nucleic acid, a vector, an antigen-binding polypeptide, or a polypeptide of the present invention. Peptides, compositions, T cells, T cell populations, or antigen-presenting cells, in combination with checkpoint inhibitors Examples of checkpoint inhibitors include pembrolizumab, PD-1 inhibitors such as (Keytruda) and nivolumab (Opdivo), and atezolizumab (Tecent PD-L1 inhibitors such as ribavirin (Riq), avelumab (Bavencio), and durvalumab (Imfinzi), as well as and CTLA-4 inhibitors such as ipilimumab (Yervoy).

[0171] Interferons (alpha, beta, and gamma) are a family of proteins that the body produces in very small amounts. Interferon slows or stops cancer cell division, preventing cancer cells from defending themselves against the immune system. may reduce the body's ability to protect itself and / or enhance multiple aspects of the adaptive immune system Interferon is usually administered as a subcutaneous injection, for example in the thigh or abdomen.

[0172] Therefore, one aspect of the present invention is a method for producing a polypeptide, a nucleic acid, a vector, an antigen-binding polypeptide, or a polypeptide of the present invention. Administration of the peptide or composition in combination with interferon, e.g., interferon alpha Includes giving.

[0173] Also, different aspects of the present invention may be combined, for example, the polypeptides, nucleic acids, etc. of the present invention. and vectors may be combined with the APCs, T cells or T cell populations of the invention (described below). (It is being done). One or more modalities of the present invention may also be used in combination with conventional anti-cancer chemotherapy and / or radiation therapy. Good too.

[0174] (diagnosis) In another aspect, the present invention provides a method for diagnosing cancer, particularly malignant melanoma, e.g., cutaneous malignant melanoma. or the polypeptides, nucleic acids, vectors, antigen-binding polypeptides, adoptive cell therapy To identify a human subject suitable for treatment with the method or composition, one or more of the methods Methods of using the polypeptides or nucleic acids of the invention are provided.

[0175] Accordingly, the present invention provides a method for diagnosing whether a human is suffering from cancer, comprising: determining whether the cancer cells are A polypeptide selected from SEQ ID NOs: 1 to 10 and any one of their immunogenic fragments or variants. a peptide sequence (e.g., selected from the sequences of SEQ ID NOs: 11 to 32 and 51 to 78); or a polypeptide thereof Nucleic acids encoding the code sequences (for example, sequences selected from SEQ ID NOs: 33 to 40 and SEQ ID NOs: 41 to 50) determining whether the polypeptide or corresponding nucleic acid is expressed in the and diagnosing the person as suffering from cancer if the gene is overexpressed in the cancer cells. The present invention provides a method comprising the steps of:

[0176] The present invention provides a method for diagnosing a human suffering from cancer, which is cutaneous malignant melanoma, comprising: A polypeptide selected from sequence numbers 2, 6, 7, 8 and 9, and immunogenic fragments or variants thereof. determining whether the gene sequence or nucleic acid encoding the polypeptide sequence is expressed; and when the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cell, diagnosing the human as suffering from cancer that is cutaneous malignant melanoma; provide. As used herein, "overexpressed" in a cancer cell refers to the level of expression in the cancer cell. This means that the level is higher than that in normal cells.

[0177] The present invention provides a method for diagnosing a human suffering from cancer, which is cutaneous malignant melanoma or uveal malignant melanoma. wherein the cancer cells are selected from SEQ ID NOs: 1, 3, 4, 5 and 10, and immunogenic fragments of any one thereof. A polypeptide sequence selected from the fragments or variants thereof, or a sequence encoding the polypeptide sequence. determining whether the polypeptide or corresponding nucleic acid is expressed; When overexpressed in the cancer cells, the person develops cutaneous malignant melanoma or uveal malignant melanoma. diagnosing a patient suffering from cancer that is chromosome 1.

[0178] Overexpression is measured by the expression of the nucleoside analogs of the present invention in control human subjects known to be cancer-free. Overexpression can be determined by reference to the level of the acid or polypeptide. Nucleic acids or polypeptides of the invention are significantly higher in test subjects than in control subjects. The term "detectable" refers to a level (e.g., 30%, 50%, 100%, or 500% higher) that is detected. and human subjects have undetectably low levels of the nucleic acids or polypeptides of the invention. In such cases, detection of the nucleic acids or polypeptides of the invention leads to a diagnosis.

[0179] The present invention also provides a method for treating a human suffering from cancer, comprising: (a) The cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and immunogenic fragments or variants thereof. a polypeptide sequence selected from (e.g., selected from the sequences of SEQ ID NOs: 11 to 32 and 51 to 78) or nucleic acids encoding the polypeptides (e.g., sequences of SEQ ID NOs: 33 to 40 and 41 to 50) determining whether the gene expresses a gene selected from the group consisting of: (b) administering to said human the corresponding polypeptides, nucleic acids, vectors, compositions, T cell populations, or the like of the present invention; a step of administering a fusion protein, a T cell, an antigen-presenting cell, an antigen-binding polypeptide, or a cytotoxic cell The present invention provides a method including:

[0180] Similarly, a polypeptide isolated from a tumor of a human suffering from cancer, comprising a sequence selected from the following: Use of: (a) any one of SEQ ID NOs: 1 to 10; or (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); or the use of nucleic acids encoding the polypeptides, such that the human is able to derive the corresponding polypeptides of the invention. polypeptides, nucleic acids, vectors, compositions, T cell populations, T cells, antigen-presenting cells, antigen binding polypeptides or cytotoxic cells, may be suitable for treatment with vaccines. and use of the compound as a biomarker for determining whether the compound is a marker for apoptosis or inflammatory bowel disease. Suitably the cancer is malignant melanoma, particularly cutaneous malignant melanoma.

[0181] The present invention also provides The polypeptide comprises a sequence selected from: (a) any one of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide may be any of SEQ ID NOs: 11-14, 17-18, 19, 20-22, 30-32, 51-53, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64 57, 67 to 74, and 76 to 77, or consisting of the sequence selected from the sequence and for example, the nucleic acid is selected from any one of SEQ ID NOs: 33, 35, 36, or 40. or comprising a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50, and The method or use of the present invention provides wherein the cancer is uveal melanoma.

[0182] Preferably, the polypeptide of the present invention is a fragment thereof, such as SEQ ID NO: 1 to 10, or an immunogenic fragment thereof ( For example, it has a sequence selected from the sequences of SEQ ID NOs: 11 to 32 and 51 to 78. Preferably, the nucleic acid of the present invention is SEQ ID NO: 33 to 40 or 41 to 50, or an immunogenic fragment thereof, etc. The fragments of the present invention may have or contain a sequence selected from any one of the following:

[0183] Kits for detecting the presence of nucleic acids are well known. A kit containing at least two hybridizing oligonucleotides is used for real-time PCR. It may also be used in RT-PCR reactions, allowing for the detection and semi-quantification of specific nucleic acids. The kit generates a fluorescent signal as a result of Förster resonance energy transfer (FRET). (e.g., TaqMan® kits), or by binding of double-stranded DNA (e.g., SYBR® kits). Some kits (e.g., standard Green kits) will allow for detection of PCR products. probes spanning multiple exons of target DNA) are used to detect mRNA, e.g. For example, certain kits allow for the detection and quantification of transcripts encoding the nucleic acids of the present invention. Assays using nucleases can be set up in a multiplex format to simultaneously measure multiple nucleases in a single reaction. It will be possible to detect active DNA (i.e., DNA with specific epigenetic features that indicate expression). Kits for the preparation of the compounds may also be used. Additional components that may be present in such kits include: Components include diagnostic reagents or reporters that facilitate detection of the nucleic acids of the invention. .

[0184] The nucleic acids of the invention may also be detected in a liquid biopsy using a blood sample from the patient. Such a procedure provides a non-invasive alternative to surgical biopsy. Plasma from the animals can also be isolated and analyzed for the presence of the nucleic acids of the invention.

[0185] The polypeptides of the invention can be obtained by the preparation of the invention in homogenized tumor samples from patients. Detected using antigen-specific antibodies in an ELISA-type assay to detect polypeptides of interest. Alternatively, the polypeptides of the present invention may be detected by immunohistochemical analysis. It involves staining sections of patient tumor samples with appropriately labeled antibody preparations. Examination using a light microscope is used to identify the presence of polypeptide antigens. Alternatively, the polypeptides of the present invention may be detected by immunohistochemical analysis, It involves optical microscopy of patient tumor sample sections stained with appropriately labeled antibody preparations. Microscopic examination is used to identify the presence of polypeptide antigens.

[0186] The polypeptides of the present invention also have the ability to stimulate T cells to respond to the polypeptides. This may be detected by determining whether the gene can increase the sex. Cells of a cancer or tumor, e.g., malignant melanoma, e.g., cutaneous malignant melanoma, are, by way of example, cancer, e.g., For example, it may be obtained from a biopsy of a malignant melanoma, for example a cutaneous malignant melanoma.

[0187] A method for treating human cancer, particularly malignant melanoma, e.g., cutaneous malignant melanoma, comprises: (i) administering to a subject a nucleic acid or (ii) detecting the presence of a polypeptide of the invention; and (iii) administering to the subject a nucleic acid, polypeptide of the invention. administering a vaccine, vector, cell, T cell or T cell population or composition (and preferably Preferably, the same nucleic acid or polypeptide as that detected, or a fragment thereof, is administered. ) is included.

[0188] A method for treating human cancer, particularly malignant melanoma, e.g., cutaneous malignant melanoma, also includes administering to the subject a therapeutic agent according to the present invention. administering a specific nucleic acid, polypeptide, vector, cell, T cell or T cell population or composition in which the subject (and preferably the same) nucleic acid or polypeptide of the invention is The presence of

[0189] In particular, the cancer to be diagnosed and possibly treated is malignant melanoma, e.g., cutaneous malignant melanoma. It is chromoma. A polypeptide of the present invention having SEQ ID NO: 1, 3, 4, 5 or 10 or a fragment thereof was detected. In this case, the cancer may be cutaneous melanoma or uveal melanoma.

[0190] SPECIAL EMBODIMENTS In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 1. Exemplary fragments include or consist of any one of SEQ ID NOs: 11 to 14. Suitable fragments include two, three, or four of SEQ ID NOs: 11-14. Further exemplary fragments include SEQ ID NOs: 5 Further exemplary fragments include or consist of any one of 5-57 or 73-74. and all of SEQ ID NOs: 11 to 14, 55 to 57, and 73 to 74 (without multiple overlapping sequences). (Possible sequence overlaps are taken into consideration so that the sequence is consistent.) Examples of nucleic acids that encode the polypeptide sequence are: The reference comprises or consists of SEQ ID NO: 33 or SEQ ID NO: 41. Acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic (cytocotic) cells, antigen-binding proteins The nucleic acid (e.g., DNA or RNA) is a polypeptide, an antigen-presenting cell, or an exosome. A) T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exocytic cells. The lysosomes are useful for the treatment of cancer, particularly malignant melanoma, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0191] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:2. Exemplary fragments include or consist of SEQ ID NO: 15 or SEQ ID NO: 16. Suitable fragments include SEQ ID NO: 15 and SEQ ID NO: 16. Further exemplary fragments include SEQ ID NOs: 58-66, 75 and 78. Further exemplary fragments include those of SEQ ID NOs: 15 to 78. 16, 58-66, 75 and 78 (to the extent possible, avoid multiple occurrences of duplicated sequences) (Such sequence overlaps are taken into consideration.) An example of a nucleic acid encoding the polypeptide sequence is SEQ ID NO:3. 4 or SEQ ID NO: 42. or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, anti- The nucleic acid (e.g., DNA or RNA), T cells, T cells, and exosomes are provided. Cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes are involved in cancer , and in particular for the treatment of malignant melanoma, e.g., cutaneous malignant melanoma. Related Diagnostic Methods is also provided.

[0192] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 3. Exemplary fragments include or consist of SEQ ID NO: 17 or SEQ ID NO: 18. Further exemplary fragments include SEQ ID NO: 18. Suitable fragments include SEQ ID NO: 17 and SEQ ID NO: 18. Further exemplary fragments include SEQ ID NOs: 53 and 67 69. Further exemplary fragments include SEQ ID NO: 17, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, S Further exemplary fragments include the entire sequences of SEQ ID NOS: 17-18, 53 and 67-69. (Possible sequence overlaps are taken into account so that duplicated sequences are not present more than once.) Examples of nucleic acids encoding the polypeptide sequences include SEQ ID NO:35 or SEQ ID NO:43 or consisting of the corresponding nucleic acids (e.g., DNA or RNA), T cells, and T cell populations listed above. , cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes The nucleic acid (e.g., DNA or RNA), T cell, T cell population, cytotoxic cell, anti- The antigen-binding polypeptides, antigen-presenting cells and exosomes are useful in treating cancer, particularly malignant melanoma, e.g., cutaneous It may also be used to treat cutaneous melanoma or uveal melanoma. Related diagnostic methods are also , provided.

[0193] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 4. An exemplary fragment comprises or consists of SEQ ID NO: 19. Further exemplary fragments include SEQ ID NO: 51 or SEQ ID NO: 52. Further exemplary fragments include SEQ ID NO: 54. Further exemplary fragments include any of SEQ ID NOs: 70-72 and 76-77. Further exemplary fragments include SEQ ID NO: 19 and SEQ ID NO: 51 or Further exemplary fragments include SEQ ID NO: 54 and any of SEQ ID NO: 51 or Further exemplary fragments include any of SEQ ID NO: 19, 51-52, 54, 70-72, and and 76-77 (possible sequence overlaps were determined to ensure that duplicated sequences were not present multiple times). An example of a nucleic acid encoding the polypeptide sequence is SEQ ID NO: 35 or SEQ ID NO: 36. The corresponding nucleic acid (e.g., DNA or RNA), T, cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells and and exosomes. The nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cells, Cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes are useful in the treatment of cancer, especially malignant black It may also be used to treat melanoma, such as cutaneous melanoma or uveal melanoma. Diagnostic methods are also provided.

[0194] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 5. An exemplary fragment comprises or consists of any one of SEQ ID NOs: 20 to 22. Examples of nucleic acids encoding the peptide sequences include or include SEQ ID NO: 36 or SEQ ID NO: 45. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxicity Cytocotic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes are provided. The nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, Peptides, antigen-presenting cells and exosomes are useful in the treatment of cancer, particularly malignant melanoma, e.g., cutaneous melanoma. The method may be used to treat uveal melanoma or uveal melanoma. Related diagnostic methods are also provided. do.

[0195] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 6. An exemplary fragment comprises or consists of SEQ ID NO: 23 or SEQ ID NO: 24. Examples of nucleic acids encoding the peptide sequences include or include SEQ ID NO: 37 or SEQ ID NO: 46. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxicity Cytocotic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes are provided. The nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, Peptides, antigen-presenting cells and exosomes are useful in the treatment of cancer, particularly malignant melanoma, e.g., cutaneous melanoma. The compounds may also be used to treat tumors. Related diagnostic methods are also provided.

[0196] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 7. An exemplary fragment comprises or consists of SEQ ID NO: 25. Exemplary nucleic acids include or consist of SEQ ID NO: 38 or SEQ ID NO: 47. , corresponding nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytotoxic (cytocotic) cells , antigen-binding polypeptides, antigen-presenting cells, and exosomes are provided. (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen presenting The exosomes and cells are used to treat cancer, particularly malignant melanoma, e.g., cutaneous malignant melanoma. Related diagnostic methods are also provided.

[0197] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 8. An exemplary fragment comprises or consists of SEQ ID NO: 26. Exemplary nucleic acids include or consist of SEQ ID NO: 38 or SEQ ID NO: 48. , corresponding nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytotoxic (cytocotic) cells , antigen-binding polypeptides, antigen-presenting cells, and exosomes are provided. (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen presenting The exosomes and cells are used to treat cancer, particularly malignant melanoma, e.g., cutaneous malignant melanoma. Related diagnostic methods are also provided.

[0198] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 9. An exemplary fragment comprises or consists of any one of SEQ ID NOs: 27 to 29. Examples of nucleic acids encoding the peptide sequences include or include SEQ ID NO: 39 or SEQ ID NO: 49. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxicity The nucleic acid includes a sex cell, an antigen-binding polypeptide, an antigen-presenting cell, and an exosome. (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, anti- The present invention relates to the treatment of cancer, particularly malignant melanoma, such as cutaneous malignant melanoma, by using exosomes and exosomes. Related diagnostic methods are also provided.

[0199] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 10. An exemplary fragment comprises or consists of any one of SEQ ID NOs: 30 to 32. Exemplary nucleic acids encoding the peptide sequences include or include SEQ ID NO: 40 or SEQ ID NO: 50. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxicity Cytocotic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes are provided. The nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, Peptides, antigen-presenting cells and exosomes are useful in the treatment of cancer, particularly malignant melanoma, e.g., cutaneous melanoma. The method may be used to treat uveal melanoma or uveal melanoma. Related diagnostic methods are also provided. do. [Example]

[0200] (Example) (Example 1 - CLT specific) The goal is to identify cancer-specific transcripts that consist entirely or partially of LTR elements. That was the case. As a first step, we will conduct de novo assembly of comprehensive transcriptomes across cancer types. To achieve this, we obtained and analyzed the genome sequences from The Cancer Genome Atlas (TCGA) Consortium. The tumors were derived from a wide variety of cancer types (32 cancer types (31 primary and 1 metastatic melanoma)). , R from 768 patient samples, representing 24 gender-balanced samples; Table S1). NA sequencing reads were used for genome-guided assembly. The samples (excluding sex-specific tissues) were analyzed using cutadapt (v1.13) (Marcel M, 2011, EMBnet J ., 17:3) is an adapter for use with quality (Q20) trimming and length filtering ( Both reads of 35 or more nucleotide pairs were also analyzed using khmer (v2.0) (Crusoe et al., 2015, F10 00Res., 4:900) with kmer normalization (k = 0.00) with maximum and minimum depths of 200 and 3, respectively. 20) was performed. Reads were analyzed using STAR (2.5.2b) with the same settings as those used throughout TCGA. The sequence was mapped to GRCh38 and analyzed using Trinity (v2.2.0) (Grabherr, MG, et al., 2011, Nat. Biotechnol., 29:644-52) to disable the built-in in silico depth normalization. The majority of the assembly process was performed on a 32-core HPC node. The process completed within 256GB RAM on the node, and the failed process was rerun using the 1.5TB RAM node. The resulting contigs were poly(A) trimmed (using trimpoly in SeqClean v110222) and encoded. Tropic filtering (≥0.7) was used to remove low-quality and artificial contigs (bbd in BBMap v36.2). For each cancer type, the original 24 samples were analyzed using Salmon (v0.8.2 or v0.9.2) (Patro, R., (2017, Nat. Methods, 14:417-419) to the cleaned assembly. Contigs were quasi-mapped and expressed at transcripts per million (TPM) <0.1 were removed. The remaining ones were analyzed using GMAP(v161107) (Wu et al., 2005, Bioinf., 21:1859-1875). and map to GRCh38, with 85% or more identity over 85% or more of its length. Contigs that did not match were removed from the assembly. Finally, all cancer types were combined. The assembly was flattened and analyzed using gffread (Cufflinks v2.2.1) (Trapnell et al., 2010, Nat. Biology). The longest continuous transcript was merged using the fusion of the nucleotide sequences. The process was specifically designed to allow for the evaluation of repetitive elements, so monoexons The completeness and quality of the transcript assembly were verified using GE NCODE v24basic and MiTranscriptome1 (Iyer et al., 2015, Nat. Genet., 47:199-208) A list of unique splice sites represented by GENCODE was compiled. and the splice site is within a two-nucleotide window. This process tested for the presence of 1,001,931 identified 771,006 transcripts, of which 230,925 were spliced ​​and 771,006 were monoexonin- The results showed that the compound was soluble in water.

[0201] Separately, the assembled contigs were overlaid with genome repeat annotations. Transcripts containing LTR elements were identified. LTR and non-LTR elements were analyzed using the A. Annotated (Attig et al., 2017, Front. In Microbiol., 8:2489). Then, a hidden Markov model (HMM) representing known human repeat families was used (Dfam 2.0 Lab). Library v150923), RepeatMasker Open-3.0 (Smit, A., R. Hubley and P. Green, ref., ht tp: / / www.repeatmasker.org,1996-2010) was used to annotate GRCh38, which is HMM-based scalars were constructed using the HMM algorithm (Wheeler et al., 2013, Bioinform., 29:2487-2489). Compared to BLAST-based methods (Hubley et al., 2016, Nuc. Acid. Res., 44:81-89), RepeatMasker can annotate LTRs and internal regions separately. To parse the tabular output, adjacent annotations for the same element are This process merged all 18 sequences containing one or more complete or partial LTR elements. It yielded 1,967 transcripts.

[0202] Salmon was used to estimate transcripts per million (TPM) abundance for all transcripts and within each cancer type. Expression of Th was analyzed in 811 healthy tissue samples (from TCGA when available, otherwise from GTEx (Th e Genotype-Tissue Expression Consortium, 2015, Science, 348:648-60) Expression was compared to that in healthy tissue-matched controls for all cancer types. If detected in any sample at greater than 1 TPM, the gene is considered to be specifically expressed in cancer. A cancer was considered cancer-specific if it was not present and the following criteria were met: (i) 24 samples of each cancer type; (ii) expressed at <10 TPM in >90% of all healthy tissue samples; (iii) expressed in the target cancer type at 3-fold or greater than the median expression in any control tissue type and (iv) in the target cancer type, expressed in 90% of each available healthy tissue. In addition to these expression threshold criteria, transcripts are expressed at or above three times the percentile. Selection was based on manual inspection and included potential misassembled contigs or their 3' nontranslated sequences. Transcripts with LTR elements within the untranslated region (UTR) were excluded. If this was not possible, transcripts corresponding to both strands were considered.

[0203] The list of cancer-specific transcripts was then analyzed for transcripts containing complete or partial LTR elements. This was crossed with the list of transcripts to produce a list of 5,923 transcripts that met both criteria (cancer Specific LTR element-spanning transcripts (termed CLT).

[0204] To identify CLTs with the potential to encode proteins, ) Based on the length and compatibility of the score, an ORF prediction algorithm is performed. HMMs are then used in Ensembl C. The training was performed using hexamers derived from DS sequences and on ORFs of 300 or more nucleotides. The sense hexamer score exceeded the antisense score. We identified 885 CLTs that have the potential to encode proteins at least 99 amino acids long.

[0205] To identify unique protein sequences potentially encoded by CLTs, a selection The sequence translated from the largest ORF of the CLT was analyzed using tblastn (BLAST+v2.3.0) software. All 210+ nucleotides from the entire transcript assembly were extracted without soft-masking. The query was searched against the ORFs translated from the original. No hits or E value > 10 -5 Hits Only the ant CLT was maintained.

[0206] To further ensure the specificity of the cancer-specific antigens encoded by CLTs, Potential cross-reactivity with other proteins that may be expressed in the human genome was examined. For the purpose of the target, we compared it with one of the other predicted proteins (over the entire length of the protein) Translated ORFs with 85% amino acid sequence identity were retained. For proteins encoded by CLT that show >85% sequence identity with the protein, We collated the expression patterns of transcripts encoding these proteins. Each CLT was selected if its product was expressed in a cancer-specific manner (based on the above criteria). These were retained on the candidate list. If additional transcripts are expressed in healthy tissues, they will be included. The combination of these selection criteria resulted in a sufficiently unique amino acid sequence. We generated a final list of 139 CLTs that potentially encode proteins with amino acid sequences. .

[0207] Of these 139 CLTs, 14 were specific for cutaneous malignant melanoma (i.e., according to the methodology TGCA was specifically upregulated in cutaneous melanoma samples. ), and 7 were specific for cutaneous melanoma and uveal melanoma (i.e., Therefore, TGCA specifically upregulates cutaneous melanoma samples and uveal melanoma samples. These four CLTs specific to cutaneous malignant melanoma are , which are identified herein as having SEQ ID NOs: 34, 37, 38 and 39. These four cutaneous melanoma- and uveal melanoma-specific CLTs are herein referred to as: They are identified as having SEQ ID NOs: 33, 35, 36 and 40.

[0208] Example 2 - Immunopeptidome Analysis Immunopeptidome analysis identifies specific peptides associated with HLA molecules in cells or tissues. This is a powerful technique that allows the direct detection of nucleotides from biological samples. Affinity purification of HLA molecules, followed by elution of bound peptides from the HLA molecules and nano-ultrafiltration. High-performance liquid chromatography-mass spectrometry (nUPLC-MS 2 ) consisting of peptide evaluation by (Freu Denmann et al., 2018, Immunology 154(3):331-345). The analytical (MS) spectrum identifies short peptides bound to HLA class I and HLA class II molecules. The software used for spectrum interpretation and sequence identification The software relies on the availability of a predefined list of protein sequences for spectral matching. All open data from known transcriptomes, or even entire genomes, Analyze MS data using a predefined list of ORFs corresponding to the open reading frames (ORFs). Although it is possible to search for it (Nesvizhskii et al., 2014, Nat. Methods 11:1114-1125) However, the matching of these ultra-large sequence databases has non-linearity that limits the identification of presented peptides. This always leads to a high false positive rate. Further technical issues (e.g., leucine mass = isoleucine mass) mass), and theoretical issues (e.g., peptide splicing (Liepe et al., 2016, Science e 354(6310):354-358)) from known transcriptomes or whole genomes. There are increasing limitations associated with the use of very large databases, such as databases created using In practice, immunopeptides are generated without reference to a well-defined set of potential polypeptide sequences. It is very difficult to perform tydome analysis to identify novel antigens.

[0209] Bassani-Sternberg et al. analyzed HLA-binding peptide samples derived from 25 patients with cutaneous melanoma. We analyzed the MS data collected from the pool of reported polypeptides for the entire human proteome. (Bassani-Sternberg et al., 2016, Nature Commun., 7:13404). Their analysis revealed hundreds of thousands of peptides that matched known human proteins. As expected, these peptides include PRAME, MAGEA3, and TRPM1 (melastatin). These included peptides found within multiple tumor-associated antigens (TAA). Among them, the MS data of five patients were analyzed to identify patient-specific When compared with a polypeptide list created from the mutant protein sequences of these patients, Patient-specific neoantigens presented on HLA class I and HLA class II molecules have been identified .

[0210] Many of the predicted polypeptide sequences (ORFs) derived from the 139 CLTs described in Example 1 are not contained within the human proteome. Applying detailed knowledge of immunopeptidome assessment By doing so, the inventors were able to use the RAW data file (database) of Bassani-Sternberg et al. Link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894) for this series of new The sequences were compared with novel potential CLT antigen sequences.

[0211] To perform this analysis, peptides from all possible ORFs encoded by each CLT were extracted. The peptide sequences may or may not be concatenated into a single peptide file for each CLT, and these concatenated files may be used. A single peptide file (Analysis A) or a single peptide file (Analysis B) was added to the raw spectra in the PXD004894 dataset. The data were used to interrogate the human proteome (UniProt (Analysis A) or UniPr The results were then aligned with all polypeptides found in the .ot and masDB (Analysis B) and analyzed using the Peaks™ software. Analysis was performed using either the software (Analysis A) or Mascot software (Analysis B).

[0212] In analysis A, the results of these studies were compared with the 25 patients examined by Bassani-Sternberg et al. We identified 14 peptides associated with HLA class I molecules immunoprecipitated from tumor samples of patients. , which can be attributed to eight ORFs that were not found in the reported proteome. In analysis B, the results of these studies were examined by Bassani-Sternberg et al. HLA class I or HLA class II molecules immunoprecipitated from tumor samples of 25 patients examined We identified 14 peptides related to the molecule that were not found in the reported proteome. The HLA clusters from the patients cited were attributed to seven ORFs that were not identified (see Table 2). Detection of these peptides in association with HLA class I and HLA class II molecules allows identification of the peptides from which they originate. The ORFs (Tables 1 and 2, SEQ ID NOs: 1 to 10) are translated in melanoma tissue and express HLA class I or HLA It can be confirmed that the antigen is presented to the immune system in a complex with class II molecules. The polypeptides encoded by these ORFs were defined as CLT antigens. Table 1 and 2 are peptides found in the CLT antigen that were not present in the UniProt database. 1 to 32 show the typical mass spectrometry spectra of each peptide shown in Tables 1 and 2. These figures show the fragment spectra of the indicated peptide sequences, which nUPLC-MS 2 detected in individual SKCM tumor patients by (Bassani-Sternberg et al. (Images extracted from the PRIDE dataset by the PEAKS software). All fragments are represented by peptide sequences on the spectrum, with the most abundant fragment ions in each spectrum. In Figures 1-15, 29-32 (Analysis A), the bottom panels of the figures show the predicted spectra. The sequence annotation to the nucleotide sequence is shown in Figures 16-28 (Analysis B), while similar data are presented on the right side of Figures 16-28 (Analysis B). The fragment ions are annotated as follows: b: N-terminal fragment ion; y: : C-terminal fragment ion; -H2O: water loss; -NH3: ammonia loss; [2+]: doubly charged peptide ion; pr e: Unfragmented precursor peptide ion.

[0213] From Tables 1 and 2, the number of peptides detected in association with HLA class I was evaluated. The predicted strength of binding to the supertypes was determined. Specifically, the nine antisense sequences cited in Table 3 were used. All HLA class I-associated peptides of length ≥ 1 amino acid were predicted using the NetMHC 4.0 prediction software (htt p: / / www.cbs.dtu.dk / services / NetMHC / ) and HLA class I types A and B The results of these prediction studies showed that all 11 peptides bind to the ATPase inhibitors. (or a 9-mer derived therefrom) binds to at least one of the supertypes tested. Among these, many sequences were found to be related to the HLA class I sequences tested (see Table 3). Predicted to bind with high confidence (low rank score %) to a specific type within a supertype It was.

[0214] In summary, the data shown in Tables 1 to 3 and Figures 1 to 32 indicate that, within melanoma patients, This provides very strong support that the corresponding CLT antigen is presented. In summary, the identification of immunopeptidome peptides derived from predicted ORFs indicates that these C LT is translated into polypeptides (SEQ ID NOS: 1 to 10; also referred to as CLT antigens) in tumor tissues. These are then processed by the cellular immune surveillance machinery and identified as HLA class I antibodies. The resulting peptide / HLA class I complex or The cells are targeted for cytolysis by T cells that recognize peptide / HLA class II complexes. Therefore, these CLT antigens and their fragments can be used to detect tumors that express these antigens. It is useful in various therapeutic modalities for the treatment of malignant melanoma in patients expressing It is predicted that there will be.

[0215] (Table 1: Peptides identified by immunopeptidome analysis of SKCM tumor samples (Analysis A) (List of CLT antigen names and cross-referenced sequence numbers) [Table 1] 1 : Peptides identified by mass spectrometry. All peptides are HLA class I peptides. 2 Bassani-Sternberg et al., 2016, Nature Comm., 7:13404. 3 : Calculated peptide mass. 4 : Peaks™ program area of ​​mass spectrum; peptides for which multiple spectra were obtained For, indicate the selected area value. 5 : Number of spectra in which the peptide was detected. 6 Deviation between observed and calculated masses; for peptides for which multiple spectra were obtained, The selected ppm value is shown.

[0216] (Table 2: Peptides identified by immunopeptidome analysis of SKCM tumor samples (Analysis B) (List of CLT antigen names and cross-referenced sequence numbers) [Table 2] 1 : Peptides identified by mass spectrometry. * indicates peptides (HLA class II) All peptides except for ) are HLA class I peptides. 2 Bassani-Sternberg et al., 2016, Nature Comm., 7:13404. 3 : Calculated peptide mass. 4 : Number of spectra in which the peptide was detected. 5 : Difference between observed and calculated mass; for peptides with multiple spectra, The selected delta mass values ​​are shown. * indicates an HLA class II peptide.

[0217] Table 3: Mass spectrometry-identified peptides (length ≥ 9 residues) in 12 HLA class I supertypes Type alleles (HLA-A0101, HLA-A0201, HLA-A0301, HLA-A2402, HLA-A2601, HLA-B0702 , HLA-B0801, HLA-B1501, HLA-B2705, HLA-B3901, HLA-B4001, HLA-B5801) NetMHC 4.0 binding, and CLT antigen names and cross-referenced sequence numbers [Table 3] 1 : Predicted binding to matched HLA class I supertypes at any rank score . 2 : HLA class I supertypes predicted to bind with a rank score of <5.1% (weak binding) Fraction. 3 : HLA class I supertypes predicted to bind with a rank score of <2.1% (stronger binding) Fraction of pu. 4 Bassani-Sternberg et al., 2016, Nature Comm., 7:13404.

[0218] Example 2.1 - Additional Immunopeptidome Analysis In addition to the analysis described in Example 2, we also conducted a new immunopeptidome study. Through this additional work, we also identified peptides derived from predicted ORFs. Furthermore, we show that these CLTs are translated into CLT antigen polypeptides in tumor tissues.

[0219] We procured frozen tumor tissue from 10 patients diagnosed with malignant melanoma. ~1 g of sample was homogenized, the lysate was centrifuged at high speed, and the clarified lysate was The sate was prepared by covalently binding Protein A (Pro) to an anti-human HLA class I monoclonal antibody (W6 / 32). A) The mixture was mixed with the beads. The mixture was incubated overnight at 4°C to detect the antibodies to the HLA class I molecules. (Ternette et al., 2018, Proteomics 18, 1700465). HLA class I binding The peptides were eluted from the antibody using 10% acetic acid, which was then separated from other high molecular weight components. The purified β-glucan was isolated from the β-glucan by reversed-phase column chromatography (Ternette et al., 2018). The purified eluted peptides were subjected to nUPLC-MS to identify specific peptides with a given charge-to-mass ratio (m / z). The fragments are selected, isolated, and fragmented in a mass spectrometer, and subjected to MS / MS to reveal the m / z of the resulting fragment ions. (Ternette et al., 2018) to the immunopeptidome of each of these tumor samples. The corresponding MS / MS data sets were generated.

[0220] By applying detailed knowledge of immunopeptidome assessment, we have identified CLT antigen numbers HLA clusters for 10 malignant melanoma tumors prepared by the present inventors using Nos. 1, 2, 3, and 4. The spectra of the Seq I data set were collated (Table 4; SEQ ID NOS: 1-4), which were analyzed using the PEAKS™ software. The human proteome (U) was analyzed using software (v8.5 and vX, Bioinformatics Solutions Inc.). The search was performed by aligning (for each CLT) all polypeptide sequences found in the The majority of class I HLA-binding peptides found in cells are constitutively expressed proteins. Because the data are derived from proteins, the simultaneous matching of these databases with the UniProt proteome is a key step in the invention. This helps to confirm that our assignment of CLT ORF sequences to MS / MS spectra is correct.

[0221] The results of these studies identified eight individual peptides (Table 4; SEQ ID NOS: 1-4), which was immunoprecipitated from tumor samples from 10 melanoma patient samples procured by the present inventors. These peptides correspond to the amino acid sequence of the CLT-derived ORF. and does not correspond to any polypeptide sequence present in the known human proteome (UniProt). The CLT antigens identified in our dataset were SEQ ID NOS: 1 to 4 (Table 4). Of the eight peptides, two were from tumor samples from patients examined by Bassani-Sternberg et al. These results were related to HLA class I molecules immunoprecipitated from the pool (see Example 2 and Tables 1 and 2). In addition to the 10 individual peptides (from the same CLT antigen SEQ ID NOs: 1-4) outlined above. That is why.

[0222] The detection of these peptides in association with HLA class I molecules was best achieved by identifying the four ORFs from which they were derived. First, it is translated in melanoma tissue, processed through the HLA class I pathway, and finally This confirms that C is presented to the immune system in a complex with HLA class I molecules. Figures 33 to 42 show the characteristics of peptides found in the LT antigen. Representative MS / MS spectra are shown. The upper panels of each figure show the MS / MS peptide fragment profiles. The file is shown with standard MS / MS annotation (b: N-terminal fragment ion; y: C-terminal fragment). Single ion; -H2O: water loss; -NH3: ammonia loss; [2+]: doubly charged peptide ion; pre: fragmentation Unreacted precursor peptide ions; a n -n: internal fragment ion), which was shown above, PEA The most abundant fragment images in the images extracted from the inventors' dataset by the KS software The bottom panel of each figure shows the linear peptide sequence mapped to the fragment ions. Rendering of the spectrum showing column positions assigned to the peptides in Table 4. These spectra are consistent with the peptide sequences discovered in these analyses, as they are consistent with the 101gP scores. It contains a number of fragments that exactly match the sequence (SEQ ID NOs: 12, 13, 16, 17, 19, 51, 53 and 54).

[0223] All peptides detected in association with HLA class I in Table 4 that are at least 9 AA (amino acids) in length using the NetMHCpan 4.0 prediction software (http: / / www.cbs.dtu.dk / services / NetMHCpan / ) to determine their predicted strength of binding to HLA class I type A and B supertypes. The results of these prediction studies were used to estimate the 9-mer sequence of all peptides (or 9-mers contained within each complete sequence). ) were predicted to bind to at least one of the supertypes tested (Table 5 Of these, many sequences are specific to specific types within the HLA class I supertypes examined. The detected peptides were predicted to bind to the peptide with high confidence (low rank score %). The fact that all were predicted to bind to HLA types predicted to be present in the patient population Furthermore, the results of the present study are consistent with those of the previous study. All peptides found were identical to HLA types detected in patient samples by NetMHCpan 4.0. It was predicted to bind to one of the types.

[0224] Further assignment of MS spectra from tumor tissue to peptide sequences discovered in Example 2.1 To provide certainty, the inventors synthesized peptides having these discovered sequences and The same conditions as those applied to the tumor samples in the data were used for nUPLC-MS. 2 Select The spectra of the peptides are compared in Figures 43 to 50. In each figure, the upper spectrum is the tumor sample. The spectra below correspond to the 1000 sigma-like molecules (from our tumor tissue database - Figures 33-42). The selection of detected ion fragments corresponds to synthetically prepared peptides with the same sequence. The m / z values ​​are shown for each upper / lower fragment peak in these MS / MS spectra. The precise alignment of the fragments was demonstrated (between tumor-derived and synthetic peptide-derived fragment ions). The small differences between the experimentally determined m / z values ​​of the nucleotides fall within the m / z tolerance range of <0.05 Daltons. (These are the results of the assignment of each spectrum from tumor tissue to a CLT-encoded peptide.) Check the authenticity.

[0225] In summary, the peptide data presented in Table 4, Figures 33-42, and Figures 43-50 demonstrate that melanoma patients provides very strong support for the translation, processing, and presentation of the corresponding CLT antigen in humans. do.

[0226] To further confirm the cancer specificity of these CLTs, we performed a multicenter study of 37 normal tissue samples. The samples (10 normal skin, 9 normal lung, and 18 normal breast tissues) were processed and immunopeptidome We extracted HLA class I data sets from these normal tissue samples. The spectra were collated and all of the polypeptide sequences of CLT antigen numbers 1, 2, 3 and 4 were derived. The peptides derived from CLT antigen numbers 1, 2, 3, and 4 were identified as peptides of the normal tissue. was not detected in the set of tissue samples (Table 6), further confirming that CLT exhibits cancer-specific expression. was provided.

[0227] In summary, this additional immunopeptidome identification derived from predicted ORFs reveals that these The CLT is translated into polypeptides (SEQ ID NOS: 1 to 4; also referred to as CLT antigens) in tumor tissue. Therefore, these CLT antigens and their fragments further demonstrate that the tumor expresses these antigens. It is useful in a variety of therapeutic modalities for the treatment of malignant melanoma in patients expressing It is predicted that this will happen.

[0228] Table 4: Peptides identified by additional immunopeptidome analysis of melanoma tumor samples. List of CLT antigens and their cross-referenced sequence numbers [Table 4] ND - Not determined. 1 : HLA class I peptides identified by mass spectrometry. 2 : Our dataset (1MT1, 1MT2, 1MT3, 2MT1, 2MT2, 2MT3, 2MT4, 2MT9, 2MT10, 2MT12). 3 : Calculated peptide mass. 4 : Peaks™ program area of ​​mass spectrum; peptides for which multiple spectra were obtained For, indicate the selected area value. 5 : Number of spectra in which the peptide was detected. 6 Deviation between observed and calculated masses; for peptides for which multiple spectra were obtained, The selected ppm value is shown.

[0229] Table 5: Mass spectrometry-identified peptides (length ≥ 9 residues) in 12 HLA class I supertypes Type alleles (HLA-A0101, HLA-A0201, HLA-A0301, HLA-A2402, HLA-A2601, HLA-B0702 , HLA-B0801, HLA-B1501, HLA-B2705, HLA-B3901, HLA-B4001, HLA-B5801) NetMHC 4.0 binding, and CLT antigen names and cross-referenced sequence numbers [Table 5] 1 : Predicted binding to matched HLA class I supertypes at any rank score . 2 : HLA class I supertypes predicted to bind with a rank score of <5.1% (weak binding) Fraction. 3 : HLA class I supertypes predicted to bind with a rank score of <2.1% (stronger binding) Fraction of pu. 4 : Inventors' database (1MT1, 1MT2, 1MT3, 2MT1, 2MT2, 2MT3, 2MT4, 2MT9, 2MT10, 2MT12).

[0230] Table 6: Number of peptides derived from CLT antigens 1 to 4 in the normal tissue sample set [Table 6]

[0231] The results presented in Examples 1, 2 and 2.1 herein may be used in whole or in part as described in The Cancer Genome Atlas (TCGA) Research Network (http: / / cancergenome.nih.gov / ) and Genotype-Tissue Expression (GTEx) Projects (funded by the Office of the Director of the National Institutes of Health Common Fund and other funding sources from NCI, NHGRI, NHLBI, NIDA, and NIMH) and supported by NINDS).

[0232] Example 3 - HERVFEST The Functional Expansion of Specific T Cells (FEST) technology is based on the detection of patient T cells that react to MANA epitopes. Based on this, we investigated the presence of mutation-associated neoantigens (MANA) in the repertoire of tumor cells from cancer patients. It has been used to identify therapeutically relevant tumor-derived epitopes present in tumors (Anagnostou et al., 2013). Contribution, Cancer Discovery 2017; Le et al., Science 2017; Forde et al., NEJM 2018; Dani Ilova et al., CancerImmunol. Res. 2018). In Examples 1, 2, and 2.1 (Tables 1-6, Figures 1-50), Application of the FEST technique to the CLT antigens discovered using the described method has demonstrated the potential for the detection of CLT antigens in cancer patients. It can be used to identify therapeutically relevant T cell responses to antigens.

[0233] Other assays to identify epitope-specific T cells in immunized subjects (e.g., Similar to ELISA (e.g., ELISPOT), the "FEST" technique involves the use of antigen-presenting cells and suitable antigenic peptides. In vitro culture, cognate T cells are activated and expanded to reveal their specificity. What distinguishes this assay from other immunological assays is that these amplified cultures (specifically, TCR-Vβ Next-generation sequencing of T cell receptor (TCR) DNA sequences present within the CDR3 region (TCRseq) Utilizing genomic DNA sequencing, individual peptides from a panel of target peptides derived from an antigen (or multiple antigens) can be identified. in detecting specific TCRs propagated in cells cultured with the peptide TCR / T cells detected ex vivo using TCRseq applied to tumor tissue from the same patient. Peptide-stimulated cultures also showed increased activity in tumor-infiltrating lymphocytes found in cancer tissue in situ. Therefore, MANAFEST can be recognized by the patient's T cells. This is a powerful technique for identifying MANA epitopes, and it has been shown to be effective in detecting MANA epitopes in normal and tumor tissues from cancer patients. Functional analysis of the large number of mutant peptides detected by whole-exome sequencing of tissues It has been demonstrated that it is possible to identify functionally relevant MANA peptides (Le et al., Science 2017; Fo Rde et al., NEJM 2018; Danilova et al., Cancer Immunol. Res. 2018; Smith et al. , J Immunother Cancer 2019).

[0234] The application of the MANAFEST method (Danilova et al., Cancer Immunol. Res. 2018) to CLT antigens: The method, referred to herein as HERVFEST, was carried out as follows: Step 1: Construct a hybridoma containing epitopes that efficiently bind selected HLA class I alleles. Step 2: Select appropriate melanoma patients. These PBMCs were then mixed with the peptide library selected in step 1 according to their HLA class I type. Step 3: PBMCs from these patients were separated into T cell and non-T cell fractions. The non-T cells were repopulated with patient T cells and then split into 20-50 wells (250,000 T cells per culture). various T cell growth factors and synthetic peptides derived from individual CLT antigens (selected in step 1 / 2) Step 4: TCRseq (sequencing of TCR-Vβ CDR3 sequences) ) was performed for all wells and amplified in the presence of individual CLT antigen-derived peptides ( However, no amplification occurred in the presence of the control peptide or in the absence of peptide stimulation. The TCR-Vβ CDR3 sequences were identified. The presence of the -Vβ CDR3 sequence identified CLT antigen-derived peptides that elicited immune responses in melanoma patients. Step 5: Similarly, TCRseq is performed on tumor samples to identify T cells bearing CLT antigens. It may be determined whether the cells amplify TCRs that home to the patient's tumor, and these TCRs This study provides additional evidence that T cells bearing the CLT antigen recognize peptides derived from CLT antigens in patients' tumors. do.

[0235] HERVFEST assays were performed with peptides derived from CLT antigens 1 to 4 (SEQ ID NOs: 1 to 4). The peptide panel used in the study (see step 1 above) was a NetMHC predictor of CLT antigen-derived peptides. Based on the measurements, these peptides were identified in patient tumor samples available for our analysis. These HERVFs were predicted to bind strongly to the eight HLA class I types commonly found in humans. Table 7 shows the CLT antigen-derived peptides that amplified one or more TCRs in the EST assay. Table 7 also shows the results of each patient's The HLA class I types of the CLT antigen peptides tested in the PBMC-derived cultures are also shown. Table 8 lists the HLA class I types of patients tested in this study who had ≥1 TCR amplification in the assay. Shown below.

[0236] Figure 51 Panel A shows TCR amplification with MANA peptide specific to NSCLC (non-small cell lung cancer) patients. The published data are shown (Forde et al., NEJM 2018). The vertical axis represents the number of MANA peptides listed on the horizontal axis. The plots are shown for wells of cells cultured in the presence of either peptide or control peptide, respectively. The prevalence of TCR-Vβ CDR3 sequences identified in the MANA7-containing wells was shown. Panels B and C of Figure 51 show that the repertoire contains T cells that respond to this peptide. , incubated in the presence of the indicated CLT antigen peptides and control peptides. Representative TCR amplification data from PBMCs from two melanoma patients are shown. Same as panel A. Furthermore, the specific amplification observed in panels B and C is consistent with the T cell repertoire of these melanoma patients. Panel B shows that the IL-16 IgG1-positive cells contain T cells that react with specific CLT antigen-derived peptides. 15 HLA class IA from HLA 1, 2 and 4 * All wells stimulated with the 02 peptide panel TCR detected in LMSSFSTLASL-stimulated wells of PBMCs from melanoma patient 222B The frequencies of three TCR sequences are shown. [ka] is a CLT antigen 2-derived A * Panel C shows 15 peptides derived from CLT antigens 1, 2, and 4. HLA class IA * 02 peptide, and 24 HLA class IA from CLT antigens 1, 2, 3, and 4 * 03 In all wells stimulated with the peptide panel, MVAC of PBMCs from melanoma patient 224B was observed. The frequency of TCRs detected in RIKTFR-stimulated wells is shown. One TCR sequence was amplified. [ka] is a CLT antigen 2-derived A * 03 binding peptide.

[0237] Control peptides / conditions used in these experiments were as follows: CEF = CMV, EB V, and a mixture of influenza peptides; SL9, TV9, and QK1 = HIV-1 control peptides ; No peptide = cultured in the absence of peptide; Baseline = T cells before culture.

[0238] Figure 52 shows the results of the CT1-4 antigens that amplified one or more TCRs during the completed studies in these patients. Each panel shows a summary of all CLT antigen peptides identified by immunopeptidome analysis. The amino acid sequences of CLT antigens 1 to 4 are shown with the peptides detected at the top (dashed underlined or (See Examples 2 and 2.1). Below these sequences, the peptides detected by HERVFEST are listed. The peptides (see Figure 51) were listed according to the number of melanoma patients in which they were detected (Table 8) and the target H Displayed together with LA Class I type.

[0239] The characteristics of each HERVFEST detection are defined as follows: Non-decorative characters: significant amplification of a single TCR Bold: significant amplification of multiple TCRs · Underlined italics: significant amplification of a single TCR detected in other wells Bold underlined text: significant amplification of multiple TCRs, at least one of which is related to other It was also detected within the

[0240] These results indicate that CLT antigens 1 to 4 are present in patients with malignant melanoma, and that these CLT antigens Peptides derived from these genes specifically elicit T cell responses in patients with malignant melanoma. These findings provide strong evidence for the role of CLTs in melanoma and identify these CLT antibodies as targets for therapeutic intervention for the treatment of melanoma. It confirms the value of the original.

[0241] Table 7: CLT antigen-derived peptides that amplified one or more TCRs in the HERVFEST assay [Table 7]

[0242] Table 8: Characteristics of melanoma patient PBMCs used in the HERVFEST assay [Table 8]

[0243] Example 4 - High-affinity T cells specific for CLT antigens are removed from the T cell repertoire of normal subjects (assay to show that the ELISPOT assays demonstrate that CLT antigen-specific CD8 T cells are present within the normal T cell repertoire of healthy individuals. may be used to indicate the presence of naive and thymic The tumor-specific CLT antigen expression in the tissues was not eliminated by central tolerance. This type of ELISPOT assay involves several steps. Step 1: CD8 T cells and CD14 monocytes are isolated from the peripheral blood of normal blood donors. The CD8 T cells are HLA typed and matched to the specific CD8 T antigens being tested. Using a magnetically labeled antibody against the CD45RO receptor, we identified naive and memory subtypes. Step 2: CD14 monocytes can be further subdivided into individual or pooled CLT antigen peptides. Step 3: The expanded CD8 T cells were then co-cultured with the CD8 T cells for 14 days. These cultures are isolated and restimulated overnight with fresh monocytes pulsed with peptide. Peptides include individual CLT antigen peptides, irrelevant control peptides, or infectious (e.g., Antigens that are potent against antigens such as CMV, EBV, influenza, and HCV) or self (e.g., Mart-1) Restimulation may include peptides known to elicit a response. The assay is performed on plates coated with an antibody against IFN-gamma (IFNγ). After overnight activation, cells were plated and then incubated with IFNγ to capture IFNγ secreted by activated T cells. The IFNγ captured on the plate was then washed away from the plate and the IFNγ was then adsorbed onto the plate using additional anti-IFNγ antibodies and a standard colorimetric dye. Dark spots are detected where IFNγ-producing cells were originally present on the plate. The data obtained from this assay include the number of spots, median spot size, These include the median spot intensity and the frequency of IFNγ-producing T cells per cell. Furthermore, the magnitude of the response to CLT antigen was measured using spot Specific responses, measured as number or median spot size, were compared between the 2000 and 2001 samples without the specific peptide. Derived from the stimulation index (SI), which is the response divided by the background response to monocytes The evaluation criteria for stimulation intensity are the stimulation index of the number of spots multiplied by the stimulation index of the spot intensity. By this method, the response to the CLT antigen and the control antigen Comparison of responses to antigens has shown that naive subjects have a strong repertoire of CLT antigen-reactive T cells. It can be shown that the vaccine contains CLT antigen-based immunogenic formulations. Table 9 shows significant CD8 T cell responses from HLA-matched normal blood donors. The results are shown in Figures 53 to 56. The horizontal bars indicate the number of CLT antigen-derived peptides that induced the desired response. Data are expressed as mean values. Statistical significance was determined by one-way analysis of variance (Kruskal-Wallis test). The results were measured using the method described above, and repeated measurements were corrected using the Dunn correction. HLA-A from * We demonstrated significant CD8 T cell responses from normal blood donors to the 0201-restricted peptide. The example shown in Figure 54 is derived from CLT antigen 2 (CLT002 ​​in the figure), and is similar to HLA-A. * Confined by 0201 Figure 55 shows the CD8 response from a normal donor to the bundled peptide. HLA-A from CLT004 in the figure * Significant CD8 T responses from normal blood donors to the 0201-restricted peptide Figure 56 shows the cellular response from HLA-B antigens 1 and 4 (CLT001 and CLT004 in the figure). * 0702 Lack of response in memory CD45RO-positive CD8 T cells (panels A and C) to bundle peptides In contrast, naive CD45RO-negative CD8 T cells from the same donor were significantly different from CLT001 and CLT00. 4 (Figure 56, panels B and D).

[0244] Table 9: CLT antigen-derived peptides that induce significant CD8 T cell responses from HLA-matched normal blood donors Chid) [Table 9]

[0245] Example 5 - Staining of reactive T cells with CLT antigen peptide pentamers Presence and activity of circulating CD8 T cells specific for CLT antigens in healthy donors and patients with malignant melanoma. Efficacy was assessed using HLA class I / peptide pentamer ("pentamer") staining and / or in vitro killing assays. Therefore, the assays described in Examples 1, 2 and 2.1 (Tables 1 to 6, Figures 1 to 50) can be used to measure the Application of these techniques to CLT antigens discovered using the methods described above will provide insight into the role of CLT antigens in cancer patients. It can be used to demonstrate the presence of a therapeutically relevant T cell response to a tumor.

[0246] For these studies, CD8 T cells isolated from the blood of healthy donors or patients were cultured in various Various culture methods, such as anti-CD3 and anti-CD28 coated microbeads and interferon The cells were then grown using Ikin-2. The grown cells were then grown using CLT peptide pentamer. The presence or absence of specific CLT antigen reactivity of the T cell receptor can be stained, and the pentamer - is a molecule of HLA class I that binds to the relevant CLT antigen peptide in the peptide-binding groove of the HLA molecule The binding occurs through the coiled-coil multimerization domain of the pentameric structure. Phycoerythrin- or allophycocyanin-conjugated antibody fragments specific for erythrin In addition to this pentamer staining, memory markers are also used. Surface markers such as CD45RO and the lysosomal release marker CD107a can also be interrogated. Linking pentamer positivity with specific surface markers identifies a pentamer-reactive T cell population It can be used to infer both the number and state (memory vs. naive / stem) of

[0247] Pentamer-stained cells were also sorted and purified using a fluorescence-activated cell sorter (FACS). The selected cells may then be tested in an in vitro killing assay to determine whether they can kill target cells. These assays can further test for the CD8 T cell population and its ability to A fluorescently labeled target cell population, in this case a CD8 population, specific for the CLT antigen, or pentamer-selected CD8 T cells that induce a potent killing response, such as Mart-1. or specific for a positive control antigen known to be The target cells in the study were pulsed with peptides and expressed HLA-A * T2 cells expressing 02, peptidase Pulsed with tide and HLA-A * 02, 03 or B * C1R cells transfected with 07, or Malignant melanoma cell lines previously shown to express CLT / CLT antigens or patient tumor cells The peptides used to pulse T2 or C1R cells may include CLT antibodies. The original peptide or a positive control peptide is included. Cell proliferation may be fluorescently labeled with CFSE (a cell proliferation dye), and cell death may be detected by This technique involves CD8 T cell-mediated apoptosis, as demonstrated by the uptake of 7AAD. When target cells are killed by cis, they acquire red fluorescence and become red / green double positive. Therefore, we applied this killing assay to pentamer-selected CLT antigen-specific CD8 T cells. The aim is to develop CLT antigen-specific T cells in ex vivo cultures of melanoma patients or healthy donor T cells. It can be used to enumerate the cytotoxic activity of cells. Figure 57 shows the cytotoxic activity of CLT antigens 1, 2 and 4 (C in the figure). HLA pentamers of healthy donor CD8 T cells with peptides derived from LT001, CLT002, and CLT004 Figure 58 shows staining of expanded CLT004 pentamer-sorted cells pulsed with CLT004. The significant killing of peptide-pulsed C1R-B7 cells was observed. This is evident when the effector to target cell ratio is 3:1 and 1:1.

[0248] Example 6 - Mouse Immunogenicity Studies To demonstrate the immunogenicity of CLT antigens, mice were transfected with replication-deficient adenoviruses expressing one or more CLT antigens. T cells obtained from these mice inoculated with viral vectors were analyzed by IFNγ ELISPOT assay (M The presence of CLT antigen-specific T cells was examined using the method described in Ennuni et al., Int. J. Cancer, 2005. Briefly, a recombinant adenovirus expressing the CLT antigen is introduced into mice. The mice were inoculated with IFNγ and humanely euthanized at the appropriate time points. Spleen cell preparations were then injected into the mouse spleen. The wells of a multiwell dish were derivatized with a monoclonal antibody against CLT. Loading is performed in the presence (or absence) of overlapping peptides corresponding to the antigen. After an appropriate time, Immobilized IFNγ was stained with different monoclonal antibodies, allowing the number of cells / spots to be counted. These are then compared to the total number of cells loaded in the well to provide a quantitative readout of CLT antigen-reactive T cells. It will be possible to read the data.

[0249] Example 7 - Verification assay of CLT expression in malignant melanoma cells a) qRT-PCR validation of CLT expression in melanoma cell lines Quantitative real-time polymerase chain reaction (qRT-PCR) is a method for determining the number of nucleotides in a given biological sample. It is a widely used technique for determining the amount of specific transcripts present in extracted RNA. Specific nucleic acid primer sequences are designed for the transcript of interest and then subsequently purified. The intermer region is amplified through a series of thermal cycling reactions, and the intercalator color is Quantification was performed fluorescently using SYBR Green. Primer pairs were engineered against CLT and Assays were performed on RNA extracted from melanoma cell lines. Non-melanoma cell lines were used as negative controls. Specifically, the malignant melanoma cell line COLO 829 (ATCC reference number CRL-1) was used as a control. 974), MeWo (ATCC reference number HTB-65), SH-4 (ATCC reference number CRL-7724) and control cell lines HepG2 (hepatocellular carcinoma, ATCC reference number HB-8065), Jurkat (T-cell leukemia, ATCC reference number TIB152) ) and MCF7 (adenocarcinoma, ATCC reference number HTB-22) were grown in vitro and RNA was added to 1 × 10 6 Instant freezing of pieces The cDNA was extracted from the cells and reverse transcribed to cDNA. qRT-PCR analysis with SYBR Green detection was performed according to standard techniques. Analysis was performed using primers designed for two regions of each CLT and a reference gene. Relative quantification (RQ) was calculated as follows: RQ=2[Ct(reference)-Ct(target)] .

[0250] The results of these experiments are shown in Figure 59. Panel A shows the PCR product using two primer sets (1+2 and 3+4). The results of the qRT-PCR assay used were from three melanoma cell lines and four non-melanoma cells. Targeting different regions of CLT (SEQ ID NO: 33), which encodes CLT antigen 1 on RNA extracted from cell lines Panel B shows the qRT-PCR using two primer sets (5+6 and 7+8). R assay results, extracted from three melanoma cell lines and four non-melanoma cell lines. The following RNAs were targeted to different regions of CLT (SEQ ID NO: 34) encoding CLT antigen 2. Panel C shows the results of a qRT-PCR assay using two primer sets (9+10 and 11+12). The results were obtained on RNA extracted from three melanoma cell lines and four non-melanoma cell lines. These target different regions of CLT (SEQ ID NO: 35), which encodes the CLT antigen 3 / 4 of the The results of

[14] showed that CLT in RNA extracted from melanoma cell lines was significantly higher than that in non-melanoma cells. This CLT was specifically expressed in each of the melanoma cell lines tested. It was served.

[0251] b) RNAScope validation of CLT expression in melanoma cells in situ In situ hybridization (ISH) transcript expression analysis is performed using histopathological specimens. This allows visualization of the presence and expression level of a given transcript under certain conditions. Assays also use oligonucleotide probes specific for short stretches of the desired RNA sequence. This involves the in situ recognition of native RNA molecules, which can be achieved by antibody or enzyme-based methods. visualized by a signal generated by a combination of colorimetric reactions based on RNAScope is an in situ probe with recently developed, more advanced probe chemistries. u Hybridization-based technology, ensuring the specificity of the signal generated, and allows for sensitive, single-molecule visualization of target transcripts (Wang et al. , 2012 J Mol Diagn. 14(1):22-29). Positive staining of transcript molecules indicates the presence of small molecules within a given cell. Appears as red dots, multiple dots indicate the presence of multiple transcripts.

[0252] RNAScope probes were designed against CLT and 12 formalin-fixed, paraffin-embedded Sections of cutaneous melanoma tumor cores were assayed. Expression signals were scored as follows: and performed on a representative image from each core as follows: The evaluation of % of cells with positive staining for the CLT probe is rounded up to the nearest 10. The estimated per cell level of expression across a given section is: 0 = no staining · 1 = 1-2 dots per cell 2 = 2-6 dots per cell 3 = 6-10 dots per cell · 4 = more than 10 dots per cell.

[0253] Expression of each CLT was detected in tumor cores from many different patients, and for each patient analyzed Validate CLT findings from tumor-derived RNAseq data in separate cores and perform multi-sample analysis. We confirmed the uniformity of expression within tumor tissue across the pool and further confirmed the presence of at least one CLT. Highlighted.

[0254] Table 10 - RNAScope Scoring in Melanoma Patient Tissue Cores [Table 10]

[0255] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprises" " and variations such as "included" and "comprises" include the listed integers, steps, integers, etc. includes any integer, step, group of integers or group of steps, but excludes any other integer, step, group of integers or group of steps. This will be understood to mean that it is not

[0256] All patents, patent applications and literature references cited throughout this specification are hereby incorporated by reference. No. 6,239,999, filed on Dec. 1, 2003, which is hereby incorporated in its entirety. The present invention relates to preferred and more preferred groups, and preferred and more preferred groups, and all combinations of the groups of embodiments listed above. The present application provides the following aspects of the invention. (Aspect 1) An isolated polypeptide comprising a sequence selected from: (c) any one of SEQ ID NOs: 1 to 10; and (d) a variant of the sequence of (a); and (c) Immunogenic fragment of the sequence in (a). (Aspect 2) A sequence selected from any one of SEQ ID NOs: 11 to 32 and 51 to 78, or a sequence selected from the sequence 2. The isolated peptide of embodiment 1, (Aspect 3) 3. The isolated polypeptide of embodiment 1 or embodiment 2, comprising: (ii) one or more other polypeptides according to embodiment 1 or embodiment 2; (iii) other polypeptides capable of enhancing an immune response (i.e., immunostimulatory sequences). ), and (iv) provide strong CD4+ help that augments CD8+ T cell responses to antigen epitopes. a polypeptide sequence (e.g., containing a universal CD4 helper epitope) that can be used to the isolated polypeptide fused to a further polypeptide selected from: (Aspect 4) An isolated nucleic acid encoding the polypeptide according to any one of aspects 1 to 3. (Aspect 5) 5. The nucleic acid of embodiment 4, which is DNA. (Aspect 6) A sequence selected from any one of SEQ ID NOs: 33 to 40 and 41 to 50, or a sequence selected from the sequence 6. The nucleic acid according to embodiment 5. (Aspect 7) 7. The nucleic acid of embodiment 6, which is codon optimized for expression in a human host cell. (Aspect 8) 5. The nucleic acid of embodiment 4, which is RNA. (Aspect 9) The nucleic acid of embodiment 4, 5, 7 or 8, which is an artificial nucleic acid sequence. (Aspect 10) A vector comprising the nucleic acid according to any one of aspects 4 to 9. (Aspect 11) Suitable regulatory elements to allow transcription of translationally active RNA molecules in human host cells. 11. The vector of embodiment 10, comprising DNA encoding a ment. (Aspect 12) 12. The vector of embodiment 10 or embodiment 11, which is a viral vector. (Aspect 13) Adenovirus vectors, adeno-associated viruses (AAV), alphaviruses, herpes simplex viruses viruses, arenaviruses, measles viruses, poxviruses, paramyxoviruses, 13. The vector of embodiment 12, which is a tivirus and rhabdovirus vector. (Aspect 14) The polypeptide, nucleic acid, or vector according to any one of aspects 1 to 13 is used in a pharmaceutical composition comprising: An immunogenic pharmaceutical composition comprising the compound of formula (I) and a carrier capable of administering the compound of formula (I) to a mammal. (Aspect 15) The polypeptide, nucleic acid, or vector according to any one of aspects 1 to 13 is used in a pharmaceutical composition comprising: A vaccine composition comprising the vaccine composition together with a carrier capable of administering the vaccine. (Aspect 16) 16. The composition of embodiment 14 or embodiment 15, comprising one or more immunostimulants. (Aspect 17) The immunostimulant comprises an aluminum salt, a saponin, an immunostimulatory oligonucleotide, and a water Oil emulsion, aminoalkyl glucosaminide 4-phosphate, lipopolysaccharide and its derivatives and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and 17. The composition of embodiment 16, selected from: interferon. (Aspect 18) Aspect 18. The composition of any one of aspects 14 to 17, which is a sterile composition suitable for parenteral administration. (Aspect 19) A polypeptide, nucleic acid or vector according to any one of aspects 1 to 18 for use in medicine. - or composition. (Aspect 20) 20. A method of increasing an immune response in a human, comprising administering to said human a therapeutic agent according to any one of aspects 1 to 18. The method comprises administering the polypeptide, nucleic acid, vector or composition described above. (Aspect 21) The immune response is generated from SEQ ID NOs: 1 to 10 and any one of variants and immunogenic fragments thereof. 21. The method of embodiment 20, wherein the expression level is elevated in cancerous tumors that express the selected sequence. (Aspect 22) The polypeptide according to any one of aspects 1 to 18 for use in increasing an immune response in humans. peptide, nucleic acid, vector or composition. (Aspect 23) The immune response is generated from SEQ ID NOs: 1 to 10 and immunogenic fragments or variants thereof. 23. The method of claim 22, wherein the expression of the selected corresponding sequence is elevated in cancerous tumors expressing the selected corresponding sequence. A polypeptide, nucleic acid, vector or composition of the invention. (Aspect 24) A method for treating a human cancer patient, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and any one of the immunoglobulins. A therapeutic method for expressing a sequence selected from the epitoxigenic fragments and variants, or a method for preventing humans from developing cancer. a method for preventing cancer, the cancer being a cancer comprising the antibody of SEQ ID NO: 1 to 10, and immunogenic fragments and variants thereof; 19. A method of preventing the expression of a sequence selected from a variant, comprising the steps of: and administering to said human a corresponding polypeptide, nucleic acid, vector or composition as described above. , the method. (Aspect 25) A polypeptide according to any one of aspects 1 to 18 for use in treating or preventing human cancer. , nucleic acid, vector or composition, wherein the cancer cell is a cell selected from SEQ ID NOs: 1 to 10 and any one thereof the polypeptide, nucleic acid, or the like, which expresses a corresponding sequence selected from the immunogenic fragments. , vector or composition. (Aspect 26) For use in the ex vivo stimulation and / or expansion of T cells derived from cancer-affected humans. The stimulated and / or expanded T cells are then reintroduced into the human to treat the human's cancer. 19. The polypeptide, nucleic acid, vector or composition according to any one of aspects 1 to 18, which is introduced . (Aspect 27) A method for treating human cancer, wherein the cancer cells are composed of SEQ ID NOS: 1 to 10 and any one of the immunogens thereof. and expressing a sequence selected from the group consisting of a human recombinant human recombinant fragment and a human recombinant human variant. and extracting a leukocyte population containing at least T cells together with antigen-presenting cells; The presence of a corresponding polypeptide, nucleic acid, vector or composition according to any one of aspects 1 to 18. and stimulating and / or amplifying some or all of the leukocytes in the presence of a leukocyte antigen. and reintroducing at least the stimulated and / or expanded T cells into the human. (Aspect 28) any one of aspects 21 and 23-27, wherein the cancer is malignant melanoma, e.g., cutaneous malignant melanoma; A method according to, or a polypeptide, nucleic acid, vector or composition for use in, (Aspect 29) A sequence selected from SEQ ID NOs: 1 to 10 and any one of immunogenic fragments and variants thereof is generated. A method for preparing a T cell population that is cytotoxic to expressing cancer cells, comprising: (a) injecting T cells into a target cell; (ii) obtaining a population of T cells, optionally together with antigen-presenting cells, from a cancer patient; and (iii) treating the population of T cells ex vivo with A method for stimulating a subject with a corresponding polypeptide, nucleic acid, vector or composition according to any one of aspects 1 to 18. amplified and stimulated. (Aspect 30) A T cell population obtainable from the method of embodiment 29. (Aspect 31) Stimulated with the polypeptide, nucleic acid, vector or composition according to any one of aspects 1 to 18 T-cells. (Aspect 32) The polypeptide, nucleic acid, vector or composition according to any one of aspects 1 to 18 is administered to a living body. and (iii) expressing a polypeptide according to any one of aspects 1 to 3 by external loading and modification. Antigen-presenting cells genetically engineered to (Aspect 33) 33. The antigen-presenting cell of embodiment 32, which is a dendritic cell. (Aspect 34) A method for treating a patient, comprising: loading a polypeptide, nucleic acid, vector, or composition according to any one of aspects 1 to 18; or genetically engineered to express the polypeptide according to any one of aspects 1 to 3. Exosomes loaded with polypeptides produced by cells. (Aspect 35) The T cell population, T cell, antigen-presenting cell, or exosome according to any one of aspects 30 to 34. A pharmaceutical composition comprising the compound of formula (I) in combination with a pharmaceutically acceptable carrier. (Aspect 36) A T cell population, T cells, antigen-presenting T cells or the like according to any one of aspects 30 to 34 for use in medicine. Indicated cells or exosomes. (Aspect 37) A method for treating a human suffering from cancer, wherein the cancer cells are selected from SEQ ID NOS: 1 to 10 and any one of the immunoglobulins thereof. A therapeutic method for expressing a sequence selected from the epitoxigenic fragments and variants, or a method for preventing humans from developing cancer. The method for preventing cancer, wherein the cancer cells are infected with SEQ ID NOs: 1 to 10 and any one of immunogenic fragments thereof, 36. A method of preventing a disease, comprising expressing a sequence selected from the group consisting of a nucleotide sequence ... Administering the T cell population, T cells, antigen-presenting cells, exosomes, or composition described in the above item to the human. The method, comprising: (Aspect 38) The T cell population according to any one of aspects 30 to 35 for use in the treatment or prevention of human cancer. T cells, antigen-presenting cells, exosomes or compositions, wherein the cancer cells are selected from the group consisting of SEQ ID NOs: 1 to 10 and The T expresses a corresponding sequence selected from any one of the immunogenic fragments thereof. A cell population, a T cell, an antigen-presenting cell, an exosome or a composition. (Aspect 39) any one of aspects 29, 37, and 38, wherein the cancer is malignant melanoma, e.g., cutaneous malignant melanoma; A method for preparing, using or administering a T cell population, T cell, antigen-presenting cell, exocytosis or the like, as described in sosome or composition. (Aspect 40) An isolated antibody which is immunospecific for the polypeptide of any one of aspects 1 to 3. Antigen-binding polypeptides. (Aspect 41) 41. The antigen-binding polypeptide of embodiment 40, which is a monoclonal antibody or fragment thereof. (Aspect 42) 42. An antigen-binding polypeptide according to embodiment 40 or embodiment 41, which is conjugated to a cytotoxic moiety. (Aspect 43) 43. An antigen-binding polypeptide according to any one of aspects 40 to 42 for use in medicine. (Aspect 44) The antigen-binding polypeptide of any one of aspects 40 to 42 can be administered in a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the compound of formula (I) together with a carrier. (Aspect 45) A method for treating a human suffering from cancer, wherein the cancer cells are selected from SEQ ID NOS: 1 to 10 and any one of the immunoglobulins thereof. A therapeutic method for expressing a sequence selected from the epitoxigenic fragments and variants, or a method for preventing humans from developing cancer. The method for preventing cancer, wherein the cancer cells are infected with SEQ ID NOs: 1 to 10 and any one of immunogenic fragments thereof, 42. A method of preventing a disease in which a sequence selected from the group consisting of a steroid hormone and a steroid hormone-modifying agent is expressed, comprising administering to a subject a disease ... administering to said human an antigen-binding polypeptide or composition of any one of claims 1 to said human. method. (Aspect 46) The antigen according to any one of aspects 40 to 42 and 44 for use in the treatment or prevention of human cancer. The binding polypeptide or composition, wherein the cancer cell is a The antigen-binding polypeptide expresses a corresponding sequence selected from the immunogenic fragments. or composition. (Aspect 47) 47. The method of claim 45 or 46, wherein the cancer is malignant melanoma, such as cutaneous malignant melanoma. Antigen-binding polypeptide or composition. (Aspect 48) An HLA-binding polypeptide that is a polypeptide according to any one of aspects 1 to 3 or a part thereof. An isolated antigen-binding polypeptide immunospecific for (Aspect 49) 49. The antigen-binding polypeptide of embodiment 48, which is a T-cell receptor or a fragment thereof. (Aspect 50) to another polypeptide capable of binding to cytotoxic cells or other immune components within the subject 50. The antigen-binding polypeptide of embodiment 48 or embodiment 49, wherein the polypeptide is linked to (Aspect 51) and expressing the antigen-binding polypeptide of any one of aspects 48 to 50 on its surface. Cytotoxic cells engineered to (Aspect 52) 52. The cytotoxic cell according to embodiment 51, which is a T cell. (Aspect 53) 53. A cytotoxic cell according to embodiment 51 or embodiment 52 for use in medicine. (Aspect 54) 53. A pharmaceutical composition comprising a cell according to embodiment 51 or embodiment 52. (Aspect 55) A method for treating a human cancer patient, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 10 and any one of the immunoglobulins. A therapeutic method for expressing a sequence selected from the epitoxigenic fragments and variants, or a method for preventing humans from developing cancer. a method for preventing cancer, the cancer being a cancer comprising the antibody of SEQ ID NO: 1 to 10, and immunogenic fragments and variants thereof; 53. A method of preventing a cell of embodiment 51 or embodiment 52 which would express a sequence selected from the variants. administering to said human cells. (Aspect 56) A cytotoxic cell according to aspect 51 or aspect 52 for use in the treatment or prevention of human cancer. Therefore, the cancer cells are selected from the corresponding immunogenic fragments of SEQ ID NOs: 1 to 10 and any one of them. The cytotoxic cell as described above, wherein the cell expresses a sequence (Aspect 57) 1. A method for diagnosing whether a human is afflicted with cancer, comprising: The cancer cells are selected from SEQ ID NOs: 1 to 10 and any one of their immunogenic fragments or variants. determining whether the polypeptide sequence or nucleic acid encoding the polypeptide sequence is expressed and if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cell. if so, diagnosing said human as suffering from cancer. (Aspect 58) A method for diagnosing a human suffering from cutaneous malignant melanoma cancer, comprising: 7, 8 and 9, and immunogenic fragments or variants thereof; determining whether the polypeptide expresses a nucleic acid encoding the polypeptide sequence; If the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, the human develops a skin malignancy. diagnosing the patient as suffering from melanoma cancer. (Aspect 59) 1. A method for diagnosing a human suffering from cancer, the cancer being cutaneous malignant melanoma or uveal malignant melanoma, comprising: The cancer cells are selected from the group consisting of SEQ ID NOs: 1, 3, 4, 5 and 10, and immunogenic fragments or variants thereof. A method for expressing a polypeptide sequence selected from a variety of polypeptides or a nucleic acid encoding said polypeptide sequence. determining whether the polypeptide or corresponding nucleic acid is overexpressed in the cancer cells; and If the expression of the IL-11 gene is positive, the person is afflicted with a cancer that is cutaneous malignant melanoma or uveal malignant melanoma. and diagnosing the condition as being caused by the disease. (Aspect 60) 1. A method of treating a human suffering from cancer, comprising: (a) The cancer cells are selected from SEQ ID NOS: 1 to 10 and immunogenic fragments or variants thereof. or a nucleic acid encoding the polypeptide (e.g., SEQ ID NO: 3). determining whether the gene expresses a sequence selected from sequences 3 to 40 and 41 to 50; If it is expressed, (b) administering to the human a compound according to any one of aspects 1 to 18, 30 to 35, 40 to 42, 44, 50, 51, and 53. corresponding polypeptides, nucleic acids, vectors, compositions, T cell populations, T cells, antigen-presenting cells administering an exosome, an antigen-binding polypeptide, or a cytotoxic cell; The method comprising: (Aspect 61) Use of a polypeptide isolated from a tumor of a human suffering from cancer, the polypeptide comprising a sequence selected from: (a) any one of SEQ ID NOs: 1 to 10; or (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); or the use of a nucleic acid encoding the polypeptide, wherein the human is a human according to any one of aspects 1 to 18, 30 to 35. 40 to 42, 44, 51, 52 and 54, the corresponding polypeptide, nucleic acid, vector vector, composition, T cell population, T cell, antigen-presenting cell, exosome, antigen-binding polypeptide To determine whether a patient would be suitable for treatment with a vaccine containing a steroid or cytotoxic cell Use as a biomarker for (Aspect 62) 62. The method or method according to claim 60 or 61, wherein the cancer is malignant melanoma, such as cutaneous malignant melanoma. is used. (Aspect 63) A method according to any one of aspects 21 and 23 to 27, or a polypeptide or nucleic acid for use therein. An acid, vector or composition, wherein the polypeptide comprises a sequence selected from: (a) any one of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide is selected from the group consisting of SEQ ID NOs: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, and 51 to 57. , 67 to 74, and 76 to 77, or consisting of the sequence; and for example, the nucleic acid is selected from any one of SEQ ID NOs: 33, 35, 36, or 40; comprises a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50, or consists of a column; and The cancer is uveal melanoma. tar or composition. (Aspect 64) 46. ​​An antigen-binding polypeptide or a method according to claim 45, or a method for use according to claim 46. A composition, wherein the polypeptide comprises a sequence selected from: (a) any one of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide is selected from the group consisting of SEQ ID NOs: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, and 51 to 57. , 67 to 74, and 76 to 77, or consisting of the sequence; and for example, the nucleic acid is selected from any one of SEQ ID NOs: 33, 35, 36, or 40; comprises a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50, or consists of a column; and The cancer is uveal melanoma. Composition. (Aspect 65) A method for preparing, a method or a population of T cells for use according to any one of aspects 29, 37 and 38. , T cells, antigen-presenting cells, exosomes or compositions, wherein the polypeptide is Contains a sequence selected from: (a) any one of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide is selected from the group consisting of SEQ ID NOs: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, and 51 to 57. , 67 to 74, and 76 to 77, or consisting of the sequence; and for example, the nucleic acid is selected from any one of SEQ ID NOs: 33, 34, 36, or 40; comprises a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50, or consists of a column; and The cancer is uveal melanoma. A cell, an antigen-presenting cell, an exosome, or a composition. (Aspect 66) 62. The method or use according to embodiment 60 or embodiment 61, wherein the polypeptide is selected from Contains arrays: (a) any one of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide is selected from the group consisting of SEQ ID NOs: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, and 51 to 57. , 67 to 74, and 76 to 77, or consisting of the sequence; and for example, the nucleic acid is selected from any one of SEQ ID NOs: 33, 35, 36, or 40; comprises a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50, or consists of a column; and The method or use as described above, wherein the cancer is uveal melanoma. (Aspect 67) Two or more (e.g., 2, 3, or 4) sequences selected from the sequences of SEQ ID NOs: 1, 2, 3, and 4; or For each of the sequences, a variant of the sequence or an immunogenic fragment of the sequence is included in embodiment 3. The fusion polypeptide according to claim 1. (Aspect 68) 68. The fusion polypeptide of embodiment 67, comprising: (i) a sequence selected from: (a) the sequence of SEQ ID NO: 1; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); (ii) a sequence selected from: (a) the sequence of SEQ ID NO: 2; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); (iii) a sequence selected from: (a) the sequence of SEQ ID NO: 3; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); and (iv) a sequence selected from: (a) the sequence of SEQ ID NO: 4; and (b) a variant of the sequence of (a); and (c) Immunogenic fragment of the sequence in (a). (Aspect 69) 69. The fusion polypeptide of embodiment 68, comprising the sequences of SEQ ID NOs: 1, 2, 3 and 4. (Aspect 70) 69. The fusion polypeptide of embodiment 68, comprising: (i) a sequence selected from: (a) the sequence of SEQ ID NO: 1; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); (ii) a sequence selected from: (a) the sequence of SEQ ID NO: 2; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); and (iii) a sequence selected from: (a) the sequence of SEQ ID NO: 4; and (b) a variant of the sequence of (a); and (c) Immunogenic fragment of the sequence in (a). (Aspect 71) 71. The fusion polypeptide of embodiment 70, comprising the sequences of SEQ ID NOs: 1, 2 and 4. (Aspect 72) 72. An isolated nucleic acid encoding the fusion polypeptide of any one of embodiments 67 to 71. (Aspect 73) 73. The nucleic acid of embodiment 72, which is DNA. (Aspect 74) A vector comprising the nucleic acid of embodiment 73. (Aspect 75) Suitable regulatory elements to allow transcription of translationally active RNA molecules in human host cells. 74. The vector of embodiment 73, comprising DNA encoding a ment. (Aspect 76) 76. The vector of embodiment 74 or embodiment 75, which is a viral vector.

[0257] (Sequence Listing) SEQ ID NO: 1 (polypeptide sequence of CLT antigen 1) [ka] SEQ ID NO: 2 (polypeptide sequence of CLT antigen 2) [ka] SEQ ID NO: 3 (polypeptide sequence of CLT antigen 3) [ka] SEQ ID NO: 4 (polypeptide sequence of CLT antigen 4) [ka] SEQ ID NO: 5 (polypeptide sequence of CLT antigen 5) [ka] SEQ ID NO: 6 (polypeptide sequence of CLT antigen 6) [ka] SEQ ID NO: 7 (polypeptide sequence of CLT antigen 7) [ka] SEQ ID NO: 8 (polypeptide sequence of CLT antigen 8) [ka] SEQ ID NO: 9 (polypeptide sequence of CLT antigen 9) [ka] SEQ ID NO: 10 (polypeptide sequence of CLT antigen 10) [ka] SEQ ID NO: 11 (CLT antigen 1-derived peptide sequence) [ka] SEQ ID NO: 12 (CLT antigen 1-derived peptide sequence) [ka] SEQ ID NO: 13 (CLT antigen 1-derived peptide sequence) [ka] SEQ ID NO: 14 (CLT antigen 1-derived peptide sequence) [ka] SEQ ID NO: 15 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 16 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 17 (CLT antigen 3-derived peptide sequence) [ka] SEQ ID NO: 18 (CLT antigen 3-derived peptide sequence) [ka] SEQ ID NO: 19 (CLT antigen 4-derived peptide sequence) [ka] SEQ ID NO: 20 (CLT antigen 5-derived peptide sequence) [ka] SEQ ID NO: 21 (CLT antigen 5-derived peptide sequence) [ka] SEQ ID NO: 22 (CLT antigen 5-derived peptide sequence) [ka] SEQ ID NO: 23 (CLT antigen 6-derived peptide sequence) [ka] SEQ ID NO: 24 (CLT antigen 6-derived peptide sequence) [ka] SEQ ID NO: 25 (CLT antigen 7-derived peptide sequence) [ka] SEQ ID NO: 26 (CLT antigen 8-derived peptide sequence) [ka] SEQ ID NO: 27 (CLT antigen 9-derived peptide sequence) [ka] SEQ ID NO: 28 (CLT antigen 9-derived peptide sequence) [ka] SEQ ID NO: 29 (CLT antigen 9-derived peptide sequence) [ka] SEQ ID NO: 30 (CLT antigen 10-derived peptide sequence) [ka] SEQ ID NO: 31 (CLT antigen 10-derived peptide sequence) [ka] SEQ ID NO: 32 (CLT antigen 10-derived peptide sequence) [ka] SEQ ID NO: 33 (cDNA sequence of CLT encoding CLT antigen 1) [ka] SEQ ID NO: 34 (cDNA sequence of CLT encoding CLT antigen 2) [ka] TIFF0007762132000047.tif247170TIFF0007762132000048.tif98170SEQ ID NO: 35 (CLT cDNA sequence encoding CLT antigens 3 and 4) [ka] TIFF0007762132000050.tif216170 SEQ ID NO: 36 (cDNA sequence of CLT encoding CLT antigen 5) [ka] TIFF0007762132000052.tif237170 SEQ ID NO: 37 (cDNA sequence of CLT encoding CLT antigen 6) [ka] TIFF0007762132000054.tif105170 SEQ ID NO: 38 (CLT cDNA sequence encoding CLT antigens 7 and 8) [ka] SEQ ID NO: 39 (cDNA sequence of CLT encoding CLT antigen 9) [ka] TIFF0007762132000057.tif178170 SEQ ID NO: 40 (cDNA sequence of CLT encoding CLT antigen 10) [ka] SEQ ID NO: 41 (cDNA sequence encoding CLT antigen 1) [ka] SEQ ID NO: 42 (cDNA sequence encoding CLT antigen 2) [ka] SEQ ID NO: 43 (cDNA sequence encoding CLT antigen 3) [ka] SEQ ID NO: 44 (cDNA sequence encoding CLT antigen 4) [ka] SEQ ID NO: 45 (cDNA sequence encoding CLT antigen 5) [ka] SEQ ID NO: 46 (cDNA sequence encoding CLT antigen 6) [ka] SEQ ID NO: 47 (cDNA sequence encoding CLT antigen 7) [ka] SEQ ID NO: 48 (cDNA sequence encoding CLT antigen 8) [ka] SEQ ID NO: 49 (cDNA sequence encoding CLT antigen 9) [ka] SEQ ID NO: 50 (cDNA sequence encoding CLT antigen 10) [ka] SEQ ID NO: 51 (CLT antigen 4-derived peptide sequence) [ka] SEQ ID NO: 52 (CLT antigen 4-derived peptide sequence) [ka] SEQ ID NO: 53 (CLT antigen 3-derived peptide sequence) [ka] SEQ ID NO: 54 (CLT antigen 4-derived peptide sequence) [ka] SEQ ID NO: 55 (CLT antigen 1-derived peptide sequence) [ka] SEQ ID NO: 56 (CLT antigen 1-derived peptide sequence) [ka] SEQ ID NO: 57 (CLT antigen 1-derived peptide sequence) [ka] SEQ ID NO: 58 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 59 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 60 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 61 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 62 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 63 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 64 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 65 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 66 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 67 (CLT antigen 3-derived peptide sequence) [ka] SEQ ID NO: 68 (CLT antigen 3-derived peptide sequence) [ka] SEQ ID NO: 69 (CLT antigen 3-derived peptide sequence) [ka] SEQ ID NO: 70 (CLT antigen 4-derived peptide sequence) [ka] SEQ ID NO: 71 (CLT antigen 4-derived peptide sequence) [ka] SEQ ID NO: 72 (CLT antigen 4-derived peptide sequence) [ka] SEQ ID NO: 73 (CLT antigen 1-derived peptide sequence) [ka] SEQ ID NO: 74 (CLT antigen 1-derived peptide sequence) [ka] SEQ ID NO: 75 (CLT antigen 2-derived peptide sequence) [ka] SEQ ID NO: 76 (CLT antigen 4-derived peptide sequence) [ka] SEQ ID NO: 77 (CLT antigen 4-derived peptide sequence) [ka] SEQ ID NO: 78 (CLT antigen 2-derived peptide sequence) [ka]

Claims

1. An isolated polypeptide comprising a sequence selected from: (a) any one of SEQ ID NOs: 2 to 4; and (b) a variant of the sequence of (a), which is at least 90% identical to any one of SEQ ID NOS: 2-4 and which elicits a specific immune response thereagainst; and (c) An immunogenic fragment of the sequence of (a), wherein the immunogenic fragment consists of a sequence selected from any one of SEQ ID NOs: 15-19, 51-54, 58-72 and 75-78.

2. 2. The isolated polypeptide of claim 1, comprising a sequence selected from any one of SEQ ID NOs: 2-4, 15-19, 51-54, 58-72 and 75-78.

3. 3. The isolated polypeptide of claim 1 or claim 2, wherein the isolated polypeptide is fused to a second polypeptide or to one or more other polypeptides comprising a sequence selected from the group consisting of: (i) a sequence of SEQ ID NO: 1 to 10, and any one of its variants, which are at least 90% identical thereto and which elicit a specific immune response thereagainst, and immunogenic fragments thereof, wherein the immunogenic fragments consist of a sequence selected from any one of SEQ ID NO: 11 to 32 and SEQ ID NO: 51 to 78; (ii) another polypeptide which is a melanoma-associated antigen; (iii) a polypeptide sequence capable of enhancing an immune response (i.e., an immunostimulatory sequence); and (iv) a polypeptide sequence comprising a universal CD4 helper epitope capable of providing strong CD4+ help to increase CD8+ T cell responses to the antigen epitope.

4. An isolated nucleic acid encoding the polypeptide of any one of claims 1 to 3.

5. A vector comprising the nucleic acid of claim 4.

6. An immunogenic pharmaceutical composition comprising the polypeptide, nucleic acid or vector according to any one of claims 1 to 5.

7. 7. The composition of claim 6 for use in raising an immune response in humans, wherein the immune response is raised against cancerous tumors expressing a corresponding sequence selected from SEQ ID NOs: 2 to 4, any one of variants thereof which are at least 90% identical thereto and which elicit a specific immune response thereagainst, and immunogenic fragments thereof, wherein the immunogenic fragments consist of a sequence selected from any one of SEQ ID NOs: 15 to 19, 51 to 54, 58 to 72 and 75 to 78.

8. 7. The composition of claim 6, for use in the ex vivo stimulation and / or expansion of T cells from a human suffering from cancer, the stimulated and / or expanded T cells then being reintroduced into the human to treat the cancer in the human.

9. A method for preparing a T cell population that is cytotoxic against cancer cells expressing a sequence selected from SEQ ID NOs: 2 to 4 and any one of their variants, which are at least 90% identical thereto and which elicit a specific immune response against said variants, and immunogenic fragments thereof, which consist of a sequence selected from any one of SEQ ID NOs: 15 to 19, 51 to 54, 58 to 72 and 75 to 78, the method comprising: (i) stimulating and amplifying a T cell population obtained from a cancer patient with the corresponding polypeptide, nucleic acid, vector or composition described in any one of claims 1 to 6 in vitro.

10. A T cell population that is specifically cytotoxic to cancer cells that express the polypeptide of claim 1.

11. A T cell that is specifically cytotoxic to cancer cells that express the polypeptide of claim 1.

12. An antigen-presenting cell modified by ex vivo loading with a polypeptide, nucleic acid, vector or composition described in any one of claims 1 to 6, or genetically engineered to express a polypeptide described in any one of claims 1 to 3.

13. An exosome loaded with the polypeptide, nucleic acid, vector or composition of any one of claims 1 to 6, or loaded with a polypeptide produced from a cell genetically engineered to express the polypeptide of any one of claims 1 to 3.

14. 4. An isolated antigen-binding polypeptide that is immunospecific for a polypeptide of any one of claims 1 to 3, wherein the antigen-binding polypeptide is an antibody or antigen-binding fragment thereof.

15. A pharmaceutical composition comprising the T cell population, T cells, antigen-presenting cells, exosomes, or antigen-binding polypeptide of any one of claims 10 to 14.

16. 4. An isolated antigen-binding polypeptide immunospecific for an HLA-binding polypeptide which is a polypeptide of any one of claims 1 to 3, wherein the antigen-binding polypeptide is an antibody or antigen-binding fragment thereof.

17. A cytotoxic cell genetically engineered to express the antigen-binding polypeptide of claim 16 on its surface.

18. 20. A pharmaceutical composition comprising the polypeptide, nucleic acid, vector, T cell population, T cell, antigen-presenting cell, exosome, antigen-binding polypeptide, cytotoxic cell, or composition of any one of claims 1 to 6, 10 to 14, and 17 for use in medicine.

19. A pharmaceutical composition comprising the cells of claim 17.

20. 18. A pharmaceutical composition comprising the polypeptide, nucleic acid, vector, T cell population, T cell, antigen-presenting cell, exosome, antigen-binding polypeptide, cytotoxic cell, or composition of any one of claims 1 to 6, 10 to 15, and 17 for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NOs: 2 to 4 and immunogenic fragments thereof, wherein the immunogenic fragment consists of a sequence selected from any one of SEQ ID NOs: 15 to 19, 51 to 54, 58 to 72, and 75 to 78.

21. Use of a polypeptide isolated from a tumor of a human suffering from cancer, the polypeptide comprising a sequence selected from: (a) any one of SEQ ID NOs: 2 to 4; or (b) a variant of the sequence of (a), which is at least 90% identical to any one of SEQ ID NOS: 2-4 and which elicits a specific immune response thereagainst; and (c) an immunogenic fragment of the sequence of (a), wherein the immunogenic fragment consists of a sequence selected from any one of SEQ ID NOs: 15-19, 51-54, 58-72, and 75-78; Or the use of a nucleic acid encoding said polypeptide, 20. Use as a biomarker to obtain data for determining whether the person would be suitable for treatment with a vaccine comprising the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cell, antigen-presenting cell, exosome, antigen-binding polypeptide or cytotoxic cell of any one of claims 1 to 6, 10 to 15, 17 and 19.

22. 4. The fusion polypeptide of claim 3, comprising two or more sequences selected from the sequences of SEQ ID NOs: 1, 2, 3 and 4; or a variant of each of said sequences, said variant being at least 90% identical to any one of SEQ ID NOs: 1, 2, 3 or 4 and eliciting a specific immune response thereagainst, or an immunogenic fragment thereof, said immunogenic fragment consisting of a sequence selected from any one of SEQ ID NOs: 11 to 19 and 51 to 78 and eliciting a specific immune response thereagainst.

23. 23. An isolated nucleic acid encoding the fusion polypeptide of claim 22, wherein the nucleic acid is DNA.

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