Novel cancer antigens and methods

Abstract

To provide an antigenic polypeptide and a corresponding polynucleotide that are used for the treatment or prevention of cancer, and particularly used for the treatment or prevention of malignant melanoma.SOLUTION: There are disclosed particularly polypeptides and nucleic acids encoding the polypeptides, which are useful in the treatment, prevention and diagnosis of cancer, particularly malignant melanoma, especially cutaneous malignant melanoma and uveal malignant melanoma.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention is used in the treatment or prevention of cancer, in particular in the treatment or prevention of malignant melanoma (e.g., Antigenic polypeptides and corresponding polypeptides used in skin malignant melanoma or uveal malignant melanoma The present invention further relates to, inter alia, said nucleic acids and polypeptides, Loaded with and / or stimulated by these polypeptides and polynucleotides. The immune cells that recognize the polypeptides, antibodies specific to those polypeptides, and Medicines, including cells (of autologous or other origin) genetically engineered with molecules and immunogenicity Concerning the composition. [Background technology]

[0002] BACKGROUND OF THEINVENTION As part of the normal immune surveillance against pathogenic microorganisms, all cells secrete 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 peptides encode T cell receptors (TCRs) that tightly bind to the MHC I-peptide complex. This expanded T cell population can then respond to foreign antigens by stimulating the proliferation of human CD8+ T cells. 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 life of the animal, Memory CD8+ T cells can also be generated that can be re-expanded.

[0003] Expression of MHC class II molecules is usually observed in professional antigen-presenting cells such as dendritic cells (DCs) ( MHC class II molecules are normally restricted to peptides internalized from the exogenous environment. T cell adhesion molecules (CD54, CD48) and costimulatory molecules (CD40, CD80, CD In the presence of various factors, including naive CD4+ T cells, complementary TCRs react with 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 CD4+ T cells, which are the antibody-secreting counterparts of B cells. Not only does it promote differentiation into plasma cells, but it also promotes differentiation of antigen-specific CD8+ CTLs, which Thus, the immune system responds to foreign antigens in a manner that includes both short-term effector functions and long-term immunological memory. DCs help induce adaptive immune responses. They also act to translocate exogenously derived antigens (e.g., released by pathogens or tumor cells). can be delivered onto those MHC I molecules, thereby The peptide antigen cross-presentation process is carried out to stimulate naive CD8+ T cells. By providing an alternative pathway to stimulate proliferation, it contributes to the generation of immune memory.

[0004] Immunological memory (especially antigen-specific B cells / antibodies and antigen-specific CTLs) plays a key role in controlling microbial infections. Immunologic memory plays an important role in preventing and preventing diseases caused by important pathogenic microorganisms. Immunological memory has also been used to develop a number of vaccines to prevent tumor formation. Although it is known that cancer plays an important role in cancer treatment, 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 in six of all deaths worldwide.2 Of the 8.8 million cancer deaths in 2015, the most common cancers were lung cancer (1.69 million), These are 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 the patient's adaptive immune response (particularly T cell-based immune response) to the gen 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 via the ancestral germ line. HERVs are relics of integration into the genome of viruses. ) which belong to a group of endogenous retroelements characterized by the presence of Since it also includes mammalian apparent LTR retrotransposons (MaLR), they are collectively referred to as LTR elements. (Herein, we refer to them collectively as ERVs to mean all LTR elements.) ERVs constitute 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 the long terminal repeats. Several intact ERV ORFs are involved in the expression of exogenous infectious retroviruses such as HIV-1. Retroviral proteins that share characteristics with proteins encoded by viruses Such proteins can 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 processes 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 involved 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 some or all of the functional open reading frames of these genes, resulting in However, these ERV elements have lost their ability to produce infectious virus. Like other genes, they are maintained in the germline DNA, and at least some of these genes In fact, HERV-encoded proteins have the potential to be synthesized from a single molecule. Proteins that are involved in the expression of HERV-K env genes have been detected in various human cancers. Rec and Np9 are found only in malignant testicular germ cells and not in healthy cells ( Ruprecht et al., 2008, Cell Mol Life Sci 65:3366-3382). In cancers such as prostate cancer, Increased levels of HERV transcripts have been observed in comparison to healthy tissue (Wang-Johanning, , 2003, Cancer 98:187-197; Andersson et al., 1998, Int. J. Oncol 12:309-313). Moreover, overexpression of HERV-E and HERV-H has been shown to be immunosuppressive, which 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, ERV regulatory elements 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, 2011). 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 a viral vector, such as an adenoviral vector, may be In both preventive vaccination and therapeutic treatment strategies against cancer, This has been well 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. They can be used to load antigen-presenting cells (APCs), which can then be used to induce 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 cancer vaccine currently approved by the FDA.

[0011] Cancer antigens also offer a promising therapeutic opportunity by using them to generate a variety of non-vaccine therapeutic modalities. These therapies include: 1) antigen-binding biologics; 2) adoptive therapy; Cellular therapies are divided into two distinct classes.

[0012] Antigen-binding biologics usually recognize cancer cells decorated with antigens 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 be 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 cytochrome P450 motif and / or an immunostimulatory motif, both of which are useful for the therapeutic destruction of tumor cells. It promotes destruction.

[0013] Adoptive cell therapy may be based on a patient's own T cells, which are harvested and delivered to the patient via vaccination. Stimulated in vitro with chromosomal antigen preparations (including 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 could target cancer antigens. Cells (patient-derived or non-patient-derived) deliberately engineered to express an antigen-binding polypeptide that recognizes the These antigen-binding polypeptides can be based on human cells, including cells derived from a patient. These fall into the same classes as those described above for antigen-binding biologics. A phospholipid (autologous or non-autologous) 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 effective immune responses against cancer has been shown to be useful in the treatment of cancer in rodents. Promising results show improved tumor regression and better prognosis in patients with rheumatoid arthritis (Kershaw et al., 2001, Cancer Res. 61:7920-7924; Slansky et al., 2000, Immunity 13:529-538). Thus, to some extent, due to the tight 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 have in common the ability to enhance cross-reactive immune responses to HERV-K Mel tumor antigens, leading to malignant It confers protection against melanoma. WO 00 / 06598 identifies the HERV-AVL3-B tumor-associated gene 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 said disease - Patents.com The use of the 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 use of the HERV-K+ antibody to detect, prevent and treat HERV-K+ cancers. WO 2006 / 103562 discloses a method for the expression of the immunosuppressive Np9 protein from the env gene of HERV-K. The present invention also discloses a method for treating or preventing cancer caused by the protein. A pharmaceutical composition containing a nucleic acid or an antibody capable of inhibiting the activity of the protein, or an antibody against the protein It relates to an immunogenic or vaccine composition capable of inducing an immune response. WO 2007 / 109583 describes a method for treating tumors comprising the steps of: providing ... The present invention relates to a method for preventing or treating a neoplastic disease in a mammalian subject by providing a composition comprising: Compositions and methods are provided.

[0017] Humer J et al., 2006, Canc. Res., 66:1658-63, reported that endogenous leukemia-associated have identified a melanoma marker derived from a torovirus. It is used in the immunotherapy of cancer, particularly 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 that is 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 transcription products (CLTs) encoded by these CLTs. A subset of typical polypeptide sequences (i.e., open reading frames (ORFs)) are identified in the cancer It is translated within the cell and processed by components of the antigen processing machinery. , further comprising class I and class II major histocompatibility complexes (MHC class I and MHC class II) and Together with class I and class II human leukocyte antigen (HLA class I, HLA class II) molecules, tumor tissue These findings suggest that the IL-15A-binding domain is expressed on the cell surface in vivo (see Example 2). These polypeptides (herein referred to as CLT antigens) are in fact antigenic. Thus, cancer cell presentation of CLT antigens indicates that these cells are capable of recruiting cognate T cells to these antigens. It is expected that these proteins will be more susceptible to elimination by T cells that have the TCR. CLT antigen-based vaccination methods / regimens that expand T cells bearing cognate TCRs are (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 that are generated, and the T cell receptor sequence is amplified (implemented (See Example 3.) The present inventors have demonstrated that T cells specific for CLT antigens are able to regulate normal immune responses through central tolerance. It was confirmed that the T cells were not deleted from the T cell repertoire of the subjects (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). 5). Finally, qRT-PCR studies demonstrated that CLTs are more potent than 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 certain CLT antigens, is involved in the pathogenesis of melanoma. We found that it is overexpressed not only in ovarian melanomas, but also in uveal melanomas. 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 complex of transcription and splicing events driven by Because LT is expressed at high levels, and the CLT antigen polypeptide sequence is identical to that of the normal human protein Since the sequence is not that of the original antibody, it is not possible 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 the context of cancer immunotherapy. It is expected.

[0021] This CLT antigen was found in a highly expressed transcript that characterizes tumor cells. Before the Meiji Restoration, it was believed that bacteria could exist in the human body and produce protein products that stimulate the immune response. First, the CLT antigen of the present invention can be used in several forms. 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. The codons can be optimized to enhance expression of the CLT antigen, and it can be directly administered. or by inserting the encoded protein product into a vector for production in a subject as a vaccine. These can also be delivered in vivo as therapeutic or prophylactic immunotherapy against tumor cells. Third, the polynucleotides and / or polypeptides of the present invention can be used to induce a patient-derived response. These can be used to load antigen-presenting cells (APCs) that can then be administered to a subject as a vaccine. The antibody can be injected into the body to elicit a therapeutic or prophylactic immune response against tumor cells. Fourth, the polynucleotides and / or polypeptides of the invention can be administered to a subject's T cells ex vivo. and generating stimulated T cell preparations which 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 CLT antigens in complex with MHC I molecules and kills (or is able to kill) cancer cells. The compound may be further modified to enhance the expression of the polypeptide and administered to a subject as a therapeutic for treating cancer. , a chimeric version of a biological molecule that recognizes CLT antigens in complex with MHC cells. The cells may then be introduced into T cells (autologous or non-autologous) and the introduced cells may then be used in a therapeutic approach to treat cancer. These and other applications are described in more detail below. do.

[0022] Accordingly, the present invention relates to 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 the "polypeptide of the 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 present invention is directed to the immunotherapy and prevention of cancer, particularly melanoma. It is expected that the present invention will be useful in the field of embodiments. [Brief description 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 14-mer is shown (with assigned fragment ions), and the lower panel shows the spectrum. The spectral rendering shows linear pairs mapped to fragment ions. The positions of the peptide sequences are displayed. [Figure 1] Figure 1. Spectrum of peptide SEQ ID NO: 11 isolated from a tumor sample of patient Mel-3. [Diagram 2] Figure 2. Spectrum of peptide of SEQ ID NO: 12 isolated from a tumor sample of patient Mel-3. [Diagram 3] Figure 3. Spectrum of peptide of SEQ ID NO: 13 isolated from a tumor sample of patient Mel-5. [Figure 4] Figure 4. Spectrum of peptide of SEQ ID NO: 13 isolated from a tumor sample of patient Mel-16. [Diagram 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] FIG. 8. Spectrum of peptide of SEQ ID NO: 19 isolated from a tumor sample of patient Mel-3. [Figure 9] FIG. 9. Spectrum of peptide of SEQ ID NO:21 isolated from a tumor sample of patient Mel-27. [Figure 10]FIG. 10. Spectrum of peptide of SEQ ID NO:20 isolated from a tumor sample of patient Mel-27. [Figure 11] FIG. 11. Spectrum of peptide of SEQ ID NO:22 isolated from a tumor sample of patient Mel-27. [Figure 12] FIG. 12. Spectrum of peptide of SEQ ID NO:23 isolated from a tumor sample of patient Mel-27. [Figure 13] FIG. 13. Spectrum of peptide of SEQ ID NO:24 isolated from a tumor sample of patient Mel-27. [Figure 14] FIG. 14. Spectrum of peptide of SEQ ID NO:25 isolated from a tumor sample of patient Mel-16. [Figure 15] FIG. 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. [Diagram 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. [Diagram 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 14-mer is shown (with assigned fragment ions), and the lower panel shows the spectrum. The spectra show linear peptide sequences mapped to fragment ions. Shows column position. [Figure 29] FIG. 29. Spectrum of peptide of SEQ ID NO:51 isolated from a tumor sample of patient Mel-40. [Diagram 30] Figure 30. Spectrum of peptide of SEQ ID NO:51 isolated from a tumor sample of patient Mel-41. [Diagram 31]FIG. 31. Spectrum of peptide of SEQ ID NO:52 isolated from a tumor sample of patient Mel-27. [Diagram 32] FIG. 32. Spectrum of peptide of SEQ ID NO:52 isolated from a tumor sample of patient Mel-39. [Diagram 33] FIG. 33. Spectrum of peptide of SEQ ID NO:13 isolated from tumor sample of patient 2MT3. [Diagram 34] FIG. 34. Spectrum of peptide of SEQ ID NO:13 isolated from a tumor sample of patient 2MT10. [Diagram 35] FIG. 35. Spectrum of peptide of SEQ ID NO:12 isolated from tumor sample of patient 2MT3. [Diagram 36] FIG. 36. Spectrum of peptide of SEQ ID NO: 16 isolated from a tumor sample of patient 2MT4. [Figure 37] FIG. 37. Spectrum of peptide of SEQ ID NO:17 isolated from tumor sample of patient 2MT3. [Figure 38] FIG. 38. Spectrum of peptide of SEQ ID NO:53 isolated from tumor sample of patient 1MT1. [Figure 39] FIG. 39. Spectrum of peptide of SEQ ID NO:51 isolated from tumor sample of patient 2MT3. [Diagram 40] FIG. 40. Spectrum of peptide of SEQ ID NO:19 isolated from tumor sample of patient 2MT3. [Diagram 41] FIG. 41. Spectrum of peptide of SEQ ID NO:19 isolated from tumor sample of patient 2MT1. [Diagram 42] Figure 42. Spectrum of peptide of SEQ ID NO:54 isolated from a tumor sample of patient 2MT12.

[0027] For each of Figures 43 to 50, the native peptides isolated from patient tumor samples were The MS / MS spectrum (top) and the native spectrum of the synthetic peptide corresponding to the same sequence ( The alignment is shown with the bottom. [Diagram 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. [Diagram 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. [Diagram 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] FIG. 51, panels AC, show the expansion of tumor antigen-specific T cells from patient PBMC cultures in response to culture 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 melanoma patients. [Figure 53]FIG. 53 shows the response of CD8 T cells 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 response of CD8 T cells 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 the response of CD8 T cells from a normal blood donor to an HLA-A*0201-restricted peptide from CLT antigen 4 (SEQ ID NO: 76).

[0029] [Figure 56] Panels A-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] FIG. 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] FIG. 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 evaluating the 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 CLT antigen 6-derived peptide sequences. SEQ ID NO:25 is a CLT antigen 7-derived peptide sequence. SEQ ID NO:26 is a CLT antigen 8-derived peptide sequence. 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 to 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 PREFERRED EMBODIMENTS

[0035] Detailed Description of the Invention (Polypeptide) The terms "protein," "polypeptide," and "peptide" are used interchangeably herein. It is used to identify any peptide-linked amino acid chain, regardless of length, co-translational or post-translational modifications. It points 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 Later modifications, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoric acid, The term "amino acid analogue" refers to an amino acid that has the same basic chemical structure as a naturally occurring amino acid. The chemical structure of the aryl group is: Compounds that have a modified R group or modified peptide structure compared to natural amino acids. Examples include homoserine, norleucine, methionine sulfoxide, and Amino acid mimetics include methionine methylsulfonium, methionine methylsulfonium, and norleucine. They have a structure that is different 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 ribozyme.

[0037] As used herein, amino acids are referred to by their commonly known three letter symbols or by the IUPAC-IUB biochemical nomenclature committee symbols. The chemical is represented by one of the one-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 with the initial methionine residue removed; 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 invention will have a high degree of sequence identity thereto. For example, variants preferably have at least one or more sequences that are identical to the corresponding sequences in the entire length of the relevant reference sequence. at least about 80% identity, more preferably at least about 85% identity, and most preferably at least and about 90% identity (e.g., at least about 95%, at least about 98%, or at least about 99%). .

[0041] Preferably, the variant is an immunogenic variant. A variant is an immunogenic variant that is a variant of a reference sequence (i.e. At least 20%, preferably at least 50%, especially preferably at least 50%, of the activity of the sequence of which the variant is a mutant. 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 incubation of PBMCs or whole blood with 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., the proliferation of lymphocytes (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 proliferation Characterization of cell responses 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 -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 in malignant melanoma, e.g., cutaneous malignant melanoma. This would induce an immune response that Conservative substitution tables providing functionally similar amino acids are well known in the art. , and 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 would fall within one of the amino acid groupings identified below, but may Other substitutions may also be possible depending on the antigen, as long as they do not substantially affect the immunogenic properties of the antigen. The following 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, for example, 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 may occur at positions 1 to 10 (e.g. positions 1 to 5, preferably positions 1 or 2, especially positions 1 50 or less (e.g., 20 or less, particularly 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 residue to aid in expression and / or purification of the antigen of interest. Short stretches of residues (e.g., 2-6 residues) are included.

[0047] Polypeptide variants include those in which some amino acids have been deleted compared to the reference sequence. For example, such deletions may be at positions 1 to 10 (e.g., positions 1 to 5, preferably positions 1 or 2, particularly positions 1). positions), the deletions may be, for example, 50 or less (e.g., 20 or less, particularly 10 or less) at each position. Preferably, such deletions are less than 1 amino acid, more particularly less than 5 amino acid deletions. 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 reduces immunogenicity) gender may be left 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 Consecutive amino acids (e.g., at least 15 or at least 20 contiguous amino acids), particularly a small number of at least 50 contiguous amino acids, e.g., at least 100 contiguous amino acids (e.g., at least 200 consecutive amino acids). Preferably, the immunogenic fragment is a fragment of the full-length polypeptide sequence. At least 10% of the length of the queue, 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] An immunogenic fragment typically contains at least one epitope. Preferably, the immunogenic fragment comprises a T cell epitope, such as a CD4+ or CD8+ T cell epitope. The antibody comprises at least one T cell epitope selected from the group consisting of

[0051] T cell epitopes are recognized by T cells (such as CD4+ or CD8+ T cells) when bound to HLA molecules. The identification of T cell epitopes is currently being investigated. This may be achieved by epitope mapping experiments well known to those skilled in the art (see, e.g., Paul, Fu et al., J. Med. Soc. 1999, 143:1311-1323, 1999). International Immunology, 3rd ed., pp. 243-247 (1993); Beiβbarth et al., 2005, Bioinformatics ics,21(Suppl. 1):i29-i37).

[0052] As a consequence 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 The potential contribution to immune protection is quite clear.

[0053] In diverse outbred populations, such as humans, different HLA types represent populations of specific epitopes. It will be understood that this means that the content of the document may not be recognized by all members of the As a result, in order to maximize the level of recognition and magnitude of the immune response to a given polypeptide, Generally, an immunogenic fragment will contain multiple epitopes from the full length sequence (preferably multiple epitopes within the CLT antigen). It is preferable that the antibody contains 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 cellular epitopes, preferably at least two CD8+ T cell epitopes, more particularly all CD8+ Those that contain T cell epitopes, especially those associated with multiple HLA alleles, e.g. The polypeptides of SEQ ID NOs: 1 to 10 may be useful. A particular fragment of a peptide is a fragment of a peptide that contains at least one CD4+ T cell epitope, preferably at least two CD4+ T cell epitopes, more particularly 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 of the present invention, You will be able to achieve the answer.

[0055] When individual fragments of a full-length polypeptide are used, such fragments may be 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% (such as at least 90%). This response is believed 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 a year, 6 activity in 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, We used antibodies specific to immune markers such as CD8, IL2, TNF-α, IFN-γ, type 1 IFN, CD40L, and CD69. The antibody is measured by a flow cytometer (using a ELISA kit) and subsequent analysis on a flow cytometer.

[0056] In some cases, multiple fragments of a 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, at least two (3, 4, or Immunogenic fragments of the antibodies (e.g., 5) are combined and assayed in vitro in PBMC or whole blood restimulation assays (e.g., 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 and 20. The sequences ~28, 30~31, and 51~54 were found to bind to HLA class I molecules in immunopeptidome analysis. It was confirmed that the sequences of SEQ ID NOs: 18, 29 and 32 were identical to those of the immunopeptide. The tydom assay confirmed that it binds to HLA class II molecules (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 relates to 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 sequence of

[0059] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to a nucleic acid. Nucleotide monomers, particularly deoxyribonucleotide monomers or ribonucleotide monomers The term refers to a polymeric macromolecule made up of nucleotide analogs or nucleotide mers. The present invention includes nucleic acids containing modified backbone residues or linkages, which are not naturally occurring. and of non-natural origin, have similar properties to the reference nucleic acid and have similar nucleotide sequences to the reference nucleic acid. are intended to be metabolized in a manner similar to that of 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 sequence 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. Nucleic acid molecules may be synthesized by a cloning, restriction or ligation step, or by a method not found in nature. other manipulations that result in a nucleic acid molecule that is distinct from the nucleic acid molecule being synthesized (e.g., in the case of cDNA). In one embodiment, the nucleic acid of the present invention is an artificial nucleic acid. In one embodiment, the nucleic acid sequence is a cDNA sequence or a nucleic acid sequence that includes non-natural 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. This refers to a nucleic acid molecule that has a sugar moiety that is a component of the backbone. The sugar moiety is made up of the four natural bases ( Adenine (A), guanine (G), cytosine (C), thymine (T), and a ... The bases may be substituted with guanine (G), cytosine (C) and uracil (U). When used, a "corresponding RNA" refers to a RNA that has the same sequence as the reference DNA, but in which the thymine (T) in the DNA is replaced by a thymine (T) in the RNA. It is an RNA with a sequence in which the sugar moiety is replaced by uracil (U). Unnatural bases such as inosine, 7-methylguanosine, dihydrouridine and 5-methylcytidine may be linked to the natural phosphate diester between the sugar (deoxyribosyl / ribosyl) moieties. The linkages may optionally be replaced by phosphorothioate linkages. 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 The sequence includes or consists of a sequence selected from 41 to 50. Also provided are sequences A nucleic acid comprising or consisting of a variant of a sequence selected from sequence numbers 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 a nucleic acid that

[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 code for any given polypeptide. The codon 1 encodes the amino acid alanine. At every position specified, the codon modifies the encoded polypeptide. Such a nucleic acid mutation can be changed to any of the above codons without modifying the corresponding codon. , resulting in "silent" (sometimes referred to as "degenerate" or "synonymous") mutations, It is one type of conservatively modified variant. Every nucleic acid sequence disclosed herein also encompasses 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 except for UGG, which is usually the only codon for tryptophan, to make it functionally equivalent. It will be appreciated that a single molecule can be produced by using a nucleic acid sequence encoding a single polypeptide. Each silent variation of an acid is implicit in each described sequence and is intended to be within the scope of the present invention. It is provided as an embodiment.

[0063] Degenerate codon substitutions also include those in which the third position of one or more (or all) of the 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 relates to a method for the preparation of a nucleic acid sequence comprising: The nucleic acid may contain many silent variants (e.g., 1-50, 1-25, etc., particularly 1-5) compared to the reference sequence. , and more particularly one codon may be mutated).

[0065] In one embodiment, the nucleic acid of the invention is RNA. The RNA sequences provided are similar to the DNA sequences herein. corresponding to the sequence, has a ribonucleotide backbone instead of a deoxyribonucleotide backbone, They have the side chain base uracil (U) instead of thymine (T).

[0066] Thus, the nucleic acid of the present invention is an RNA equivalent of a cDNA sequence selected from SEQ ID NOs: 33 to 40 or 41 to 50. The present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: 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 a reference cDNA sequence (i.e., a deoxyribonucleotide sequence). It has a ribonucleotide backbone instead of a nucleotide backbone and a cytochrome P450 (CYP450) side chain instead of thymine (T). By "antigen" we mean an RNA sequence that contains 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 acid of the invention is a polypeptide that is optimized for expression in a human host cell. It's Don. The nucleic acid of the present invention, in the case of a DNA nucleic acid, can be transcribed and translated into a polypeptide of the present invention. and, in the case of an RNA nucleic acid, can be translated into a polypeptide of the invention.

[0068] (Polypeptides and Nucleic Acids) Preferably, the polypeptides and nucleic acids used in the present invention are isolated. A "removed" polypeptide or nucleic acid is one that has been removed from its original environment. For example, a naturally occurring The original polypeptide or nucleic acid is 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., a vector in which it 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 this is meant a sequence which has been produced and has not been synthetically modified. "Artificial" when used in reference to a polypeptide or nucleic acid sequence refers to, e.g., a naturally occurring It refers to a sequence that is not found in nature, including synthetic modifications of a sequence or non-naturally occurring sequences. .

[0070] The term "heterologous" refers to the relationship of one nucleic acid or polypeptide to another nucleic acid or polypeptide. When 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 expressed in the host organism. or a sequence not isolated, derived from, or based on a polypeptide sequence. It can also mean a column.

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

[0072] For purposes of comparing two closely related polypeptide or polynucleotide sequences, first The "% sequence identity" between the sequence of the first sequence and the second sequence may be calculated. If a polypeptide sequence shares 0% sequence identity with another polypeptide sequence, the polypeptide sequence is identical to the other polypeptide sequence. The residues in the sequence are, 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 of the nucleotide sequence, the nucleotide sequence is compared and aligned to maximize the match over the comparison window. When the amino acid residues are aligned, they are identical or a certain percentage of identical amino acid residues are selected. (i.e., 70% identity within a specified region, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity) of two or more sequences or subsequences. Preferably, the comparison is performed over the entire length of the reference sequence. It is executed in the corresponding window.

[0073] In sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequencing 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 may use the default program parameters or specify alternative parameters. and the sequence comparison algorithm performs a test relative to the reference sequence based on the program parameters. The percentage sequence identity of the sequences is calculated.

[0074] As used herein, a "comparison window" refers to a window within which one sequence and the same number of contiguous After optimally aligning the reference sequence at the position where the segments are to be compared, the sequences may be compared. Methods of sequence alignment for comparison are well known in the art. Such sequence alignments can be performed, for example, using the local homology algorithm of Smith and Waterman, 981, Adv. Appl. Math. 2:482, according to the Needleman and Wunsch homology alignment method. 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 the ePackage, Genetics Computer Group, 575 Science The algorithm was either computerized (by Dr. Madison, WI) or manually aligned. and visual inspection (see, for example, Current Protocols in Molecular Biology (eds. Ausubel et al. This can be done by the following method (see Supplementary Note 1995, Vol. 11, No. 1, pp. 1171-1175, 1995).

[0075] One example of a useful algorithm is PILEUP, which performs progressive pairwise Use alignment to create multiple sequence alignments from related sequences and compare them The relationship and percent sequence identity are shown. This is also used to create the alignment. It plots a tree or dendogram showing the clustering relationships found in the data. The progressive alignment method of Feng and Doolittle (1987, J. Mol. Evol. 35:351-3 60) simplification is used. The method used is that of Higgins and Sharp, 1989, CABIOS 5:151-15 The method is similar to that described in 3. This program is for constructing sequences with a maximum length of 5,000 nucleotides each. If the sequences are of the same structure, a maximum of 300 sequences can be aligned. The alignment procedure begins with a pairwise alignment of the two most similar sequences. Then, we create a cluster of two aligned sequences. We then divide the cluster into two clusters based on the most relevant Align adjacent sequences or clusters of aligned sequences that have high similarity. 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 the selection of specific sequences and their corresponding regions of sequence comparison. By specifying the amino acid coordinates of the sequence, and by specifying the program parameters, By using PILEUP, a reference sequence can be compared to other test sequences to determine the following: Parameters: Default gap weight (3.00), default gap length weight (0.10), and and weighted end gaps to determine percent sequence identity. , the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., 1999). 984, Nuc. Acids Res. 12:387-395).

[0076] Preferred Algorithms for Determining Percent Sequence Identity and Percent Sequence Similarity Another example is the BLAST and BLAST 2.0 algorithms, which are 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 a database sequence. A cluster of criteria that, when entered into the database, matches or satisfies some positive threshold score T. By identifying short word lengths W in the Erie sequences, high-scoring sequence pairs (HSPs) are optimized. T is called the neighborhood word score threshold (Alts 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 the cumulative alignment score. Each sequence is extended in both directions as far as possible to increase the length of the nucleotide sequence. The cumulative score in is determined by the parameters M (the reward score for a matching residue pair; always >0) and The amino acid sequence is calculated using N (the 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 reduced by X amount due to the accumulation of 1 or more negative scoring residue alignments. or the end of any sequence has been reached. For this purpose, the BLASTP program uses a default word length of 3 and an expectation (E) of 50, as well as 50 BLOSUM62 scoring matrices (Henikoff and Henikoff, 1989, Proc. N atl. Acad. Sci. USA 89:10915) Alignment (B), expectation of 10 (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, for example, Ltschul, 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 of the second sequence compared to the first sequence. The term refers to an insertion, deletion, or substitution. Two sequences may contain one, two, or more such differences. An insertion in a second sequence that is otherwise identical to the first (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, , the 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 second sequence may be generated for the purpose of comparing a first reference sequence to a second comparison sequence. In order to determine the number of additions, substitutions and / or deletions that have been made to the first sequence, the additions, substitutions and / or deletions may be ascertained. is the addition of a single residue to a first sequence (including additions to either end of the first sequence). A substitution is when 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 at the 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, for example, as described in Green and Sambrook, 2012 Molecular Cloning. ng: A Laboratory Manual, 4th ed., Cold Spring Harbour Laboratory Press They can also be obtained and manipulated using techniques that are currently available for generating polynucleotides. Alternatively, artificial gene synthesis techniques can be used to produce the desired gene (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), which is then expressed 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 products are 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 In the context of the present invention, the present invention is 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, for example, 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), To this end, 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 for promoting secretion 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 the nucleic acid of the invention , appropriate regulatory and control sequences (including promoters, termination signals, etc.) and these cells. In combination with 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 can 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, a genetic construct comprising one or more nucleic acids of the invention is When introduced into a cell, the polypeptide of the invention is produced in vivo and elicits an immune response. (e.g., DNA) can be expressed using nucleic acid expression systems, including bacterial and some viral expression systems, all of which are well known to those of skill in the art. The gene delivery system may be in any of a variety of delivery systems 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 are In one embodiment, a method for producing a cellular signaling molecule (also referred to as a "construct") is provided. Preferably, the vector is capable of transcribing a translationally active RNA molecule in a human host cell. The gene encoding the appropriate regulatory elements (such as a suitable promoter and termination signal) to enable "Translationally active RNA molecules" include nucleic acids that are capable of being converted into proteins by the translational machinery of human cells. It is an RNA molecule that can be translated into a 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") The service will provide a service called a “service meter.” In particular, the vector may be a viral vector. adeno-associated viruses (AAV) (e.g., AAV types 5 and 2), alphaviruses (e.g., vector Venezuela 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) can also be used. Well, the vector can be derived from any of the above viruses. The virus has been shown to be useful due to 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 to 200 base pairs, which are essential for the replication of viral DNA. 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 the regulation of 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 regulate DNA replication, expression of late genes, and the synthesis of host cells. It is involved in blocking (Renan, 1990). It contains most of the viral capsid protein. 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 ribosomal protein. All mRNAs transcribed from the promoter have a tripartite 5'-leader (TPL ) sequence, resulting in a mRNA that is favorable for translation. Replication-deficient adenoviruses made from the genom are particularly useful because 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 comprises: It may simply consist of a naked recombinant DNA plasmid. Science 259:1745-1749 and its review by Cohen, 1993, Science 259:16 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 in vitro introduction. The gene encoding the desired gene may be used in vivo as well. It is contemplated that DNA may also be introduced in vivo in a similar manner to express a gene product. Several delivery systems have been 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 Phase III clinical trials.

[0087] (RNA delivery) In another embodiment of the invention, an expression construct comprising one or more polynucleotide sequences is or may consist of RNA molecules 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 They can be used to deliver RNA to cells in vivo. By mimicking mRNA molecules, introduced biological molecules are translated by the host cell's translational machinery. and directly translates the polypeptide it encodes into the cell into which it is introduced. Alternatively, the RNA molecule can be designed to act as a transcription factor for 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 member of several RNA-based RNAi genomes. They share the same characteristics as the viral vectors of the serovar ... They may be further modified (e.g., by altering their sequence or by using modified nucleotides). ) and can enhance stability and translation (Schlake et al., RNA Biology 9:1319-1320). 330), and both types of RNA can be formulated, e.g., in emulsions (Brito et al., Molecular cular Therapy 2014 22:2118-2129) or in lipid nanoparticles (Kranz et al., 2006, Nature 22:2118-2129). 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 cell 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 referred to as the "composition of the invention"). The composition of the present invention preferably comprises a polypeptide, a nucleic acid or a vector of the present invention as a pharmaceutical. together with an acceptable carrier. Thus, in one embodiment, the polypeptide, nucleic acid or vector of the invention is used as a medicament. Immunogenic pharmaceutical compositions comprising the compound in association with an acceptable carrier are provided.

[0089] In another embodiment, the polypeptide, nucleic acid or vector of the invention is The vaccine composition is generally prepared by the steps of: See, for example, 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 The present invention also includes one or more (e.g., one) polypeptides of the present invention in combination with a carrier. Provided. In certain preferred embodiments of the present invention, the compositions of the present invention comprise a pharma- ceutically acceptable cysteine. 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 including.

[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 any one of the above polypeptide components. The article may comprise one or more (e.g., one) vector 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 the immune response.

[0092] (Pharmaceutical acceptable salts) The compositions of the invention comprise pharma- ceutically acceptable versions of the nucleic acids or polypeptides provided herein. It will be apparent that the present invention can also include salts thereof which are pharma- ceutically 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, Lithium, ammonium, calcium and magnesium salts.

[0093] Pharmaceutically acceptable carriers Many pharma- ceutically acceptable carriers known to those of skill 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: Parenteral, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration; Preferably, the administration is parenteral, such as intramuscular, subcutaneous or intravenous. The carrier preferably contains water and may also contain buffers for adjusting pH, stabilizers (e.g., surfactants and The composition may also include isotonicity adjusting agents (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, sodium saccharin, talcum, cellulose, glucose, sucrose, and charcoal Magnesium oxide, etc. may also be used.

[0094] Thus, the compositions of the invention may be prepared using a buffer (e.g., neutral buffered saline or phosphate buffered saline). buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrose), strun), 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, thickening agents, and / or preservatives that render 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 agent 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. Salts, saponins such as QS21, immunostimulatory oligonucleotides such as CPG, oil-in-water emulsions ( 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 composition 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 ligands The immunostimulant is selected from the group consisting of 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 developed a monoclonal antibody that blocks the interaction of immune checkpoint receptors, including CTLA4 and CTLA5. 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 a sustained / prolonged release of the compound following administration. release formulations (i.e., formulations such as capsules (e.g., polysaccharide-based), sponges, patches or gels) It may be administered in one portion.

[0098] (Storage and Packaging) The compositions of the invention may be contained in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be provided in a container having a suitable container size to maintain sterility of the formulation until use. The formulations are generally prepared as suspensions, in oily or aqueous vehicles. Alternatively, the compositions of the present invention may be stored in sterile liquid containers. In a freeze-dried state, the addition of extra liquid (such as water for injection or saline) is required only immediately prior to 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 drug may be formulated in such a way that a suitable dosage for the intended use is obtained. Factors such as drug availability, biological half-life, route of administration, product shelf life, and other pharmacological considerations. As such, 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 is about 0.1 ug to about 1000 ug of the compound per administration. The polypeptide of the invention, more typically, is administered in an amount of about 2.5 ug to about 100 ug of the polypeptide per dose. 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 ug per dose. to about 20 mg of the nucleic acid of the invention per administration, more typically from about 0.1 mg to about 10 mg of the 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 used to identify and treat cutaneous melanoma. Polypeptide sequences corresponding to expressed CLT antigens. In one embodiment, the present invention provides a polypeptide, a 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 present invention. The present invention relates to a method comprising administering a polypeptide, a nucleic acid, a vector or a composition of the invention. The present invention also relates to a method for the production of a polypeptide, nucleic acid, or nucleic acid fragment of the present invention for use in raising an immune response in humans. The present invention provides an acid, vector or composition. A polypeptide of the invention for the manufacture of a medicament for use in raising an immune response in humans. Uses of the nucleic acid, vector or composition are also provided.

[0103] Preferably, the immune response is determined by the antibody produced by the method of claim 1, comprising administering to said patient an antibody or antibody fragment thereof, comprising an antibody selected from the group consisting of SEQ ID NOs: 1 to 10 and variants thereof and an immunogen. The antibody is elevated in response to cancerous tumors expressing a 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 to 10. When expressing a polypeptide of the invention, a nucleic acid sequence ... nucleic acid sequence of the invention, a nucleic acid sequence of the nucleic acid sequence of the nucleic acid sequence of the nucleic acid sequence of the nucleic acid sequence of the nucleic acid sequence of the nucleic acid sequence of the nucleic acid sequence of the nucleic acid sequence of the nucleic acid sequence The acid, vector or composition and pharmaceutical preparations containing them are This means that it will be based on an immunogenic fragment.

[0104] Suitably, the immune response is a CD8+ T cell, CD4+ T cell and / or antibody response, particularly a CD8+ cytolytic response. These include reactive T cell responses and CD4+ helper T cell responses. Preferably, the immune response is directed against a tumor, in particular against SEQ ID NOs: 1 to 10 and variants thereof, and the immune response is directed against a tumor. The vector is induced to express sequences selected from the cytoplasmic fragment. In a suitable embodiment, 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, the cancer being a gene encoding SEQ ID NO: 1 to 10 and any of its derivatives. A method of prevention that would involve expressing a sequence selected from any one of the immunogenic fragments and variants. and administering to said human a corresponding polypeptide, nucleic acid, vector or composition of the invention. and

[0106] The present invention also relates to a method for the treatment or prevention of human cancer, comprising administering to said patient a therapeutically effective amount of said polypeptide, nucleic acid or vector of the present invention. A vector or composition, the cancer cell of which is a cancer cell 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 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 gene. Tumors expressing a sequence selected from sequence numbers 1, 3, 4, 5 and 10.

[0108] Thus, the present invention relates to polypeptides, nucleic acids, vectors for the methods or uses of the 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 is selected from the group consisting of SEQ ID NOs: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, 51 to 53, and 54 to 55. 57, 67-74, and 76-77, or consisting of the sequence selected from the above. 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 to a mammalian subject; 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 (such as simultaneous administration) or sequentially (such as prime boost An example of simultaneous administration is delivery of Simultaneous administration and simultaneous administration to opposite sites are included. 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., It may be administered within about a few hours of administration (eg, within 24 hours of a physician's visit).

[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 vaccination is performed (a "prime and boost" regimen). The disclosed polypeptide, nucleic acid or vector 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 primer and the booster are nucleic acids or vectors of the invention, and in each case Alternatively, the priming may be performed 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 herein are used to generate booster antibodies using the 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 performed after an interval of about 1 to 12 weeks, or up to 4 to 6 months. Administration may occur as frequently as every 1-6 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 acid may be used in combination with a vector and / or to treat malignant melanoma, e.g., cutaneous or bronchial Other antigenic polypeptides (or polypeptides thereof) that induce an enhanced immune response against melanoma of the vein. These other polynucleotides or vectors encoding the same can be used in combination. 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 that are specific to the patient. Patient-specific neoantigens (Lauss et al., (2017). Nature Communications, 8(1), 1738. http: / / www.nature.com / news / articles / 20170707.html) / / 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 Somatic 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 have also been used as: (i) non-specific immunostimulants / ajuva 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 It 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, 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, 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 nucleic acid sequence may be provided as a single polynucleotide comprising:

[0114] For convenience, where multiple components are present, they may be expressed as a single fusion protein or a single It is often desirable to include the nucleotide sequence of the fusion protein in 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 nucleic acid sequence is provided as a single polynucleotide such as a single polynucleotide.

[0115] (Fusion Proteins (Fusion Polypeptides)) 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 embodiments of the invention can be achieved by creating a nucleic acid construct that fuses together the corresponding sequences. The isolated polypeptide of the present invention fused to a polypeptide (hereinafter, "the combination of the present invention") The combination polypeptides of the present invention are The peptides are predicted to have the utility described herein and to have excellent immunity. Virus 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 would also provide the benefit of increasing the efficiency of construction and manufacturing of semiconductors (including semiconductor chips).

[0116] As described above in the "Combination of Antigens" section, the polypeptide of the present invention and the Combination polypeptides may also include polypeptide sequences that are not polypeptides of the invention. , may be fused to include one or more of the following: (a) other polypeptides which are melanoma-associated antigens and therefore which are to be used in the vaccine; Potentially useful proteolytic sequences (e.g., GPR143, PRAME, MAGE, etc., mentioned above) -A3 and pMel(gp100)); and (b) A polypeptide sequence capable of enhancing an immune response (ie, 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 to 14, 55 to 57, and 73 to 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 to 16, 58 to 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 includes: (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 to 14, 55 to 57, and 73 to 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 to 16, 58 to 66, 75, and 78; As well as (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 polypeptide, mutatis mutandis, as in the polypeptide of the present invention. The present invention provides nucleic acids encoding the polypeptides, as well as other aspects of the invention (vectors, compositions, cells, etc.).

[0121] (CLT antigen-binding polypeptide) An antigen-binding polypeptide that is immunospecific for a tumor-expressed antigen (the polypeptide of the present invention) The IL-1 receptor agonist (IL-1 receptor agonist) is a cytolytic agent that recruits cytolytic cells 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 used to bind to non-human animal species (e.g., rodents or camelids). The antibodies may be produced in a non-human species (e.g., modified to have a human immune system) and humanized. 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 made by growing specific antibody-producing B cells in tissue culture using hybridoma technology. Fuse with myeloma (B cell cancer) cells selected for their proliferation potential and lack of antibody chain synthesis (Kohler and Milstein, 1975, Nature 256(5517):495-497) and Nelson et al., June 2000, Mol Pathol. 53(3):111-7, incorporated herein by reference in their entireties. (the entirety 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 to 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 and recovering the cells. It can also be obtained by

[0124] Monoclonal antibodies can also be obtained by a manufacturing process that includes the following steps: : a) to a vector, in particular a phage, more particularly a filamentous bacteriophage (preferably DNA or DNA obtained from lymphocytes, particularly peripheral blood lymphocytes, of an animal previously immunized with a desired antigen Cloning steps of cDNA sequences b) transforming a prokaryotic cell with the vector under conditions allowing antibody production; , c) selecting antibodies by subjecting them to antigen affinity selection; d) recovering antibodies having the desired specificity; e) Antibody derived from B cells of patients exposed to an antigen or from animals immunized with an antigen Expressing the nucleic acid molecule encoding the polypeptide. The selected antibodies can then be produced using conventional recombinant protein production techniques (e.g., 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. It 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 capable of binding to biological toxins or cytotoxic chemicals. It may be linked to quality.

[0126] Another important class of antigen-binding polypeptides are T-cell-binding polypeptides 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 a tumor cell is TCR-based biologics (TCRs derived directly from patients or specifically engineered to recognize The antibody-binding domain (containing a specific high-affinity TCR) also attracts these immune cells to the tumor and provides a therapeutic effect. The targeting moiety recognizes a component on T cells (or another class of immune cells) that In some embodiments, the targeting moiety also acts to enhance the beneficial effects of 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 via the administration of 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 pharma- ceutical acceptable carrier. Such compositions are suitable for parenteral administration in a sterile formulation. For example, see the disclosure of pharmaceutical compositions above.

[0129] According to the present invention, there is provided a method for treating a human suffering from cancer, the method comprising the steps of: or a therapeutic method comprising expressing a sequence selected from the immunogenic fragments and variants of any one of A method for preventing a human from developing cancer, comprising the steps of: or 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 is conjugated to the antibody for use in the treatment or prevention of human cancer. An antigen-binding polypeptide of the present invention which may be used as a medicament for the treatment of a disease, The composition of the present invention is characterized in that the cancer cells are immunogenic or immunogenic fragments of SEQ ID NOs: 1 to 10 and any one of them. The antigen-binding polypeptide or composition expresses a corresponding sequence selected from Provided. Suitably, 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 the method or use of the invention is provided. 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 is selected from the group consisting of SEQ ID NOs: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, 51 to 53, and 54 to 55. 57, 67-74, and 76-77, or consisting of the sequence selected from the above. 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 a fragment thereof, etc.) is, for example, 5 to 1000 mg, for example, 25 to 500 mg For example, it may be administered in a dose of 100 to 300 mg, for example about 200 mg.

[0133] (Cell therapy to enhance 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 to the patient. The present invention may also be used in pharmaceutical compositions. or genetically engineered to express a polypeptide of the invention. The present invention provides 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 Such cells may be engineered to be antigen-presenting APCs. to enhance T cell proliferation, to improve activation and / or maintenance of T cell responses, and / or The recipient is genetically modified to be immunologically compatible (i.e., HLA haplotype-matched) with the recipient. APCs are generally isolated from 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 invention, dendritic cells or their precursors are used as APCs. Thus, in one embodiment, the APC of the present invention is a dendritic cell. APC (Banchereau and Steinman, 1998, Nature 392:245-251) and may 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.) In general, dendritic cells The vesicles were characterized by their typical shape (stellate in situ and prominent cytoplasmic projections visible in vitro). They have a number of distinct functions, including the ability to internalize, process, and present antigens efficiently, and Dendritic cells may be identified based on their ability to activate naive T cell responses. Of course, specific cell surface domains not normally found on dendritic cells in vivo or in vitro have been identified. The modified dendritic cells may be engineered to express a receptor or ligand. Apart from dendritic cells, antigen-loaded secretory vesicles (exocytic vesicles) are also contemplated by the present invention. These may be used in immunogenic compositions (Zitvogel et al., 1998, Nature 35:131-135). re Med. 4:594-600). Thus, in one embodiment, a polypeptide of the invention is loaded onto a The exosomes thus obtained are provided.

[0135] Dendritic cells and precursor cells may be derived from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood or other sources. Dendritic cells may be obtained from any suitable tissue or body fluid. For example, dendritic cells may be obtained by culturing monocytes taken from peripheral blood. Adding a combination of cytokines, such as GM-CSF, IL-4, IL-13, and / or TNFα, to the diet Alternatively, the cells may be differentiated in vitro by incubating them with 5% or more of the ... 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.

[0136] Dendritic cells are conveniently categorized as "immature" and "mature" cells, which have two well-characterized However, this nomenclature is not consistent with the classification of This should not be interpreted as excluding all possible intermediate steps in the cellular transformation. Immature dendritic cells , which are characterized as APCs with high antigen uptake and processing capacity, and which are Fcγ receptor The mature phenotype is usually associated 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 It is characterized by high expression of the -1BB gene.

[0137] The APC may also contain, for example, a polynucleotide that encodes a protein (or a portion or other variant thereof). Transfect the polypeptide into the cell surface. Such transfection may occur in vitro and then The pharmaceutical composition comprising the transfected cells can be used as described herein. Alternatively, a gene delivery 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 the vector is described, for example, in WO97 / 24447. or the method described in Mahvi et al., 1997, Immunology and Cell Biology 75:456-460. This can be done using any method commonly known in the art, such as the gene gun approach currently used. The 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), 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 incubation with a virus such as vesicular stomatitis virus (VSV). Prior to loading the polypeptide, the polypeptide is coupled to an immunological partner (e.g., a carrier). Alternatively, dendritic cells may be covalently linked to a non-conjugated immunizing molecule to provide T cell help. immunological partners of the conjugates, either individually or in the presence of the polypeptide or vector Under pressure, you may pulse.

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

[0139] In one embodiment, the antigen-presenting cells of the invention, preferably dendritic cells, are administered as medicaments. In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising the compound of formula (I) in combination with a carrier suitable for parenteral administration. It may also be a sterile composition, see, for example, the disclosure of pharmaceutical compositions above. In one embodiment, an antigen-presenting cell, preferably a dendritic cell, according to the invention for use in medicine. is provided.

[0140] Similarly, there is provided a method for treating a human suffering from cancer, the method comprising the steps of: The present invention relates to a method of treating a human being by expressing a sequence selected from any one of the immunogenic fragments and variants. A method for preventing cancer, comprising the steps of: A method of prevention which would involve expressing 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 invention, preferably Preferably, the composition comprises a dendritic cell or the antigen-presenting cell of the present invention, 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 combined with a pharma- ceutical 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, the method comprising the steps of: The present invention relates to a method of treating a human being by expressing a sequence selected from any one of the immunogenic fragments and variants. A method for preventing cancer, comprising the steps of: A method of prevention which would involve expressing 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 the like of the invention for use in the treatment or prevention of human cancer are provided. The composition of the present invention containing the exosomes, 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 sosome or composition is provided. In any one of the above embodiments, suitably the cancer is melanoma, particularly cutaneous melanoma. It is.

[0145] (Stimulated T cell therapy) APC-Mediated Generation, In Vivo or Ex Vivo, of T Cells Immunospecific for the Polypeptides of the 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 the MHC molecule (signal 1) of APC cells. The tumor-associated antigen stimulates the proliferation of T cells with immune-specific TCR for the tumor-associated antigen. This is also fine.

[0146] For successful T cell activation, the costimulatory surface molecules B7 and CD28 are required to bind to antigen-presenting cells, respectively. To achieve optimal T cell activation, the IL-1 receptor must bind to the T cell surface (signal 1). Both signals 1 and 2 are required. In contrast, the anti- Antigenic peptide stimulation (signal 1) fails to induce complete T cell activation and T cell tolerance. In addition to costimulatory molecules, there are also inhibitory molecules, such as CTLA-4 and PD-1, that mediate the proliferation and proliferation of 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 polypeptide of the present invention. It may be reintroduced into at-risk or cancer-affected patients, provided that the antigen-specific TCR The cancer cells recognize the antigens presented by the patient's MHC and express the corresponding polypeptides. Only if the compound is capable of targeting a cell and inducing its death will the compound be able to bind to the cell.

[0148] In one embodiment, the method is used to stimulate and / or expand T cells from a human suffering from cancer in vitro. and then administering the stimulated and / or expanded T cells to the human to treat the human's cancer. The present invention also provides 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, the method comprising the steps of: or one of the immunogenic fragments and variants, the method comprising: A population of leukocytes including at least T cells, optionally together with antigen-presenting cells, is removed from the human. 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 at least 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 melanoma, particularly cutaneous melanoma. It is.

[0150] In one embodiment, the antibody is selected from SEQ ID NOs: 1 to 10 and immunogenic fragments and variants thereof. The present invention relates to a method for preparing a T cell population that is cytotoxic against 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 in vitro. 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 an immunogenic fragment thereof, the T cell population SEQ ID NO: A or in the form of a polypeptide, nucleic acid or vector, or a composition containing one of the above. By this term is meant stimulation and amplification in vitro by a gene or variant thereof or an immunogenic fragment thereof.

[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 a nucleic acid molecule or vector of the invention to produce the The polypeptide will be expressed. The present invention relates to a T cell population that may be obtained by any of the above preparation methods (hereinafter referred to as the present invention). 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 are In one embodiment, a cell that is a T cell of the present invention is provided, hereinafter referred to as a T cell of the present invention. In one embodiment, the T cell population or T cells of the invention are administered in a medicament comprising ... Such compositions are suitable for parenteral administration, e.g. It may be a sterile composition. In one embodiment there is provided a T cell population or a T cell of the invention for use in medicine.

[0153] Similarly, there is provided a method for treating a human suffering from cancer, the method comprising the steps of: The present invention relates to a method of treating a human being by expressing a sequence selected from any one of the immunogenic fragments and variants. A method for preventing cancer, comprising the steps of: A method of prevention which would involve expressing a sequence selected from the immunogenic fragments and variants of the present invention. administering to said human a T cell population or T cell, or a composition comprising a T cell population or T cell of the 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, 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 above embodiments, suitably the cancer is malignant melanoma. , especially cutaneous melanoma.

[0155] In one embodiment, the preparation, method, or T cell population, T cell, antibody, or antibody for use according to the invention is A protozoan presentation cell, exosome or composition, the polypeptide of which is selected from the following: Contains the sequence: (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, 51 to 53, and 54 to 55. 57, 67-74, and 76-77, or consisting of the sequence selected from the above. and for example, the nucleic acid is selected from any one of SEQ ID NOs: 33, 35, 36, or 40; The nucleic acid sequence of the present invention 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 presenting cell, exosome or composition is provided.

[0156] (Therapy using genetically engineered immune cells) All of the above-mentioned CLT antigen-binding polypeptide derivatives form complexes with human HLA molecules. TCR or TCR mimics that recognize peptides derived from CLT antigens (Dubrovsky et al., 2016, Oncoi (see immunology) and are 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. For example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor is referred to. They may also be engineered to confer artificial specificity to particular immune effector cells. CARs may also include receptors that deliver a monoclonal antibody 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 are capable of expressing a polypeptide of the invention that is bound to HLA. The antibody will be specific for a tumor-associated antigen that is

[0158] Another approach to treating cancer in patients involves targeting 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, which is incorporated herein by reference in its entirety. (which is incorporated herein).

[0159] Such CAR T cells can be prepared by administering to a subject a cell sample comprising T cells or T cell precursors, e.g., For example, cells obtained from peripheral blood, umbilical cord blood and / or by apheresis are bound to 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 produced by transfection of cells. The cells may be integrated 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 polypeptide of the invention. For example, 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 endogenous T cell receptors and / or endogenous HLA inactivated For example, cells may be modified 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 gene may be introduced into a cell using a gene expression vector.

[0162] The cell population for CAR-expressing T cells may be enriched following transfection of the cells. For example, CAR-expressing cells can be identified by the use of an antigen bound by the CAR or a CAR-binding antibody. Alternatively, the enrichment step can be to separate (e.g., by FACS) non-expressing cells from non-expressing cells. This includes depleting T cells and depleting non-CAR expressing cells. 56+ cells can be depleted from the culture population.

[0163] The population of transfected CAR-expressing cells is then cultured in a medium that selectively enhances the proliferation of CAR-expressing T cells. They may be cultured ex vivo. Thus, CAR-expressing T cells may be expanded ex vivo. A sample of the CAR cells may be stored (or maintained in culture). For example, the sample may be They may be frozen for later expansion or analysis. CAR-expressing T cells are a promising candidate for other therapeutic approaches, such as checkpoint blockade, including PD-L1 antagonists. It may also be used in combination with a toxic agent.

[0164] In one embodiment, the antibody is adapted to express any of the above antigen-binding polypeptides on its surface. In one embodiment, a cytotoxic cell is provided which is engineered to be a T cell. Suitably, the cytotoxic cell is a T cell. 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 a pharmaceutical composition comprising a cytotoxic cell of the present invention, which is preferably a T cell. do.

[0165] A method for treating a human cancer patient, comprising: A therapeutic method for expressing a sequence selected from the immunogenic fragments and variants, or a method for preventing a human from suffering from cancer. The method for preventing cancer, the cancer being treated with SEQ ID NO: 1 to 10 and any one of the immunogenic fragments thereof, and a variant thereof, A method is provided which comprises administering to the human a cytotoxic cell, preferably a T cell, of the invention. can be.

[0166] In one embodiment, the cytotoxic cells of the invention, preferably T cells, are used in the treatment or prevention of human cancer. The cancer cells are selected from the group consisting of SEQ ID NOs: 1 to 10 and any one of the 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 can enhance their immunogenicity (e.g., the magnitude of the immune response they elicit and / or to improve coverage), or to enhance other activities (e.g., innate or adaptive immune responses or Other components may be designed to provide other aspects of activation, such as the destruction of tumor cells. It can be used in combination with

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

[0169] Checkpoint inhibitors block normal proteins on cancer cells or the T proteins that respond to them. It blocks proteins on cells, and these inhibitors help 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] Thus, one aspect of the present invention is the use of a polypeptide, a nucleic acid, a vector, an antigen-binding polypeptide, The peptide, composition, T cell, T cell population, or antigen presenting cell is administered in combination with a checkpoint inhibitor. Examples of checkpoint inhibitors include pembrolizumab, PD-1 inhibitors such as nivolumab (Opdivo) and atezolizumab (Teccent PD-L1 inhibitors such as ribavirin (Riku), avelumab (Bavencio) and durvalumab (Imfinzi), as well as and CTLA-4 inhibitors such as ipilimumab (Yervoy).

[0171] Interferons (such as alpha, beta, and gamma) are a family of proteins that the body produces in very small amounts. Interferon slows or stops cancer cell division, helping cancer cells defend themselves against the immune system. It may reduce the body's ability to defend 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] Thus, one aspect of the present invention is the use of a polypeptide, a nucleic acid, a vector, an antigen-binding polypeptide, Administration of the peptide or composition in combination with an interferon, e.g., interferon alpha Includes giving.

[0173] Also, different forms of the invention may be combined, e.g., the polypeptides, nucleic acids, 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, such as cutaneous malignant melanoma. or the polypeptides, nucleic acids, vectors, antigen-binding polypeptides, adoptive cell therapy of the present invention. In order to identify a human subject suitable for treatment with the method or composition, one or more 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: A polypeptide selected from SEQ ID NOs: 1 to 10 and any one of their immunogenic fragments or variants. A polypeptide 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 the corresponding nucleic acid expresses the and diagnosing the person as having cancer if the gene is overexpressed in the cancer cells. The present invention provides a method comprising the steps of:

[0176] The present invention relates to 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 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 which is cutaneous malignant melanoma; provide. As used herein, "overexpressed" in a cancer cell refers to a gene that is expressed at a level that is higher than the expression level of the gene in the cancer cell. This means that the level is higher than in normal cells.

[0177] The present invention relates to a method for diagnosing a person 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 of them. A polypeptide sequence selected from the group consisting of a fragment or variant thereof, or a nucleic acid encoding the polypeptide sequence. determining whether the polypeptide or the corresponding nucleic acid expresses the polypeptide; When overexpressed in the cancer cells, the human develops cutaneous melanoma or uveal melanoma. diagnosing the patient as suffering from cancer that is chromosome 10.

[0178] Overexpression is measured by 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 levels 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 in a given antibody. A human subject has undetectably low levels of the nucleic acid or polypeptide of the invention. In such cases, a diagnosis is reached by detecting a nucleic acid or polypeptide of the invention.

[0179] The present invention also provides a method of treating a human suffering from cancer, comprising: (a) the cancer cell is a cancer cell selected from the group consisting of SEQ ID NOs: 1 to 10 and any one of their immunogenic fragments or variants; A polypeptide sequence selected from the following (e.g., selected from the sequences of SEQ ID NOs: 11 to 32 and 51 to 78) or a nucleic acid encoding the polypeptide (e.g., a sequence selected from the sequences of SEQ ID NOs: 33 to 40 and 41 to 50). determining whether the gene expresses a gene encoding a nucleotide sequence selected from the group consisting of: (b) administering to said human a corresponding polypeptide, nucleic acid, vector, composition, T cell population, or a step of administering a fusion protein, a T cell, an antigen-presenting cell, an antigen-binding polypeptide or a cytotoxic cell to the subject. 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 a nucleic acid encoding the polypeptide, which allows a human to ingest the corresponding polypeptide of the invention. Polypeptides, nucleic acids, vectors, compositions, T cell populations, T cells, antigen-presenting cells, and antigen binding and may be suitable for treatment with vaccines containing polypeptides or cytotoxic cells. The use of the present invention as a biomarker for determining whether Suitably the cancer is malignant melanoma, in particular cutaneous malignant melanoma.

[0181] The present invention also provides A 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 is selected from the group consisting of SEQ ID NOs: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, 51 to 53, and 54 to 55. 57, 67-74, and 76-77, or consisting of the sequence selected from the above. 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 is provided wherein the cancer is uveal melanoma.

[0182] Suitably, the polypeptide of the present invention is SEQ ID NO: 1 to 10, or a fragment thereof, such as an immunogenic fragment ( 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 nucleic acid sequence of the present invention may be selected from any one of the fragments of the above.

[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. They may also be used in RT-PCR reactions, allowing the detection and semi-quantification of specific nucleic acids. The kit enables the generation of 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., the Targeted Green Kit) will allow detection of PCR products. (including TaqMan® probes spanning multiple exons of a target DNA) can be used to detect mRNA, e.g. For example, a kit for detecting and quantifying a transcript encoding a nucleic acid of the present invention. Assays using the ELISA kit can be set up in a multiplex format to simultaneously assay multiple nuclei in a single reaction. It will be possible to detect active DNA (i.e., DNA that has a particular epigenetic signature that indicates expression). Kits for the preparation of the pharmaceutical composition of the present invention 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 samples can also be isolated and analyzed for the presence of the nucleic acids of the invention.

[0185] The polypeptides of the invention can be detected by the method of the present invention in homogenized preparations of patient tumor samples. Detecting polypeptides using antigen-specific antibodies in an ELISA-type assay 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 the polypeptide antigen. Alternatively, the polypeptides of the present invention may be detected by immunohistochemical analysis, It involves optical microscopy of sections of patient tumor samples 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 ratio. Cells of a cancer or tumor, e.g., malignant melanoma, e.g., cutaneous malignant melanoma, are, by way of example, 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 the patient a nucleic acid or and (ii) administering to the subject a nucleic acid, polypeptide, or administering to the patient 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. ).

[0188] A method for treating human cancer, particularly malignant melanoma, e.g., cutaneous malignant melanoma, also includes administering to the subject a therapeutically effective amount of the compound of the present invention. By administering a specific nucleic acid, polypeptide, vector, cell, T cell or T cell population or composition, in which subject is produced a (and preferably the same) nucleic acid or polypeptide of the invention. The presence of has been detected.

[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 that 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. Exemplary fragments include two, three, or four of SEQ ID NOs: 11 to 14. Further exemplary fragments include SEQ ID NOs: 5 Further exemplary fragments include or consist of any one of 5-57 or 73-74. All of SEQ ID NOs: 11 to 14, 55 to 57, and 73 to 74 are included (including sequences in which duplicated sequences are not present multiple times). (Possible sequence overlaps are taken into consideration so as to avoid duplication.) Examples of nucleic acids that encode the polypeptide sequences 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 polypeptides, 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 malignant melanoma or uveal malignant 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 any one of SEQ ID NOs: 15 to 78. 16, 58-66, 75 and 78 (as much as possible, to avoid duplicated sequences being present multiple times) (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 , and in particular for the treatment of malignant melanoma, such as cutaneous malignant melanoma. 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: 17 or SEQ ID NO: 18. Suitable fragments include SEQ ID NO: 17 and SEQ ID NO: 18. Further exemplary fragments include SEQ ID NO: 53 and 67- 69. Further exemplary fragments include, but are not limited to, SEQ ID NO: 17, SEQ ID NO: 69, Further exemplary fragments include the entire sequences of SEQ ID NOs: 17-18, 53 and 67-69. (Possible sequence overlaps are taken into consideration so that duplicated sequences are not present multiple times.) Exemplary 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, T cell populations, as 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 melanoma. The present invention 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, or consist of, SEQ ID NO: 54. Further exemplary fragments include any of SEQ ID NOs: 70 to 72 and 76 to 77. Further exemplary fragments include, but are not limited to, SEQ ID NO: 19 and SEQ ID NO: 51 or Further exemplary fragments include any of SEQ ID NO: 54 and 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 have been eliminated to ensure that duplicated sequences are not present multiple times). An example of a nucleic acid encoding the polypeptide sequence is SEQ ID NO: 35 or SEQ ID NO: The corresponding nucleic acid (e.g., DNA or RNA) of T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells and The nucleic acid (e.g., DNA or RNA), T cell, T cell population, cell, Cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes are useful in the treatment of cancer, particularly malignant black blood cells. 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 includes or consists of any one of SEQ ID NOs: 20 to 22. Exemplary nucleic acids encoding the peptide sequences include, or are set forth in 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 cell, T cell population, cytotoxic cell, antigen-binding polypeptide, The peptides, antigen-presenting cells and exosomes are useful in treating cancer, particularly malignant melanoma, e.g., cutaneous melanoma. The method may be used to treat melanoma, ocular ulcer, 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. Exemplary nucleic acids encoding the peptide sequences include or are 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 cell, T cell population, cytotoxic cell, antigen-binding polypeptide, The peptides, antigen-presenting cells and exosomes are useful in treating cancer, particularly malignant melanoma, e.g., cutaneous melanoma. The present invention 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 cells The nucleic acid (e.g., (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen presenting The expressing cells and exosomes 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 cells The nucleic acid (e.g., (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen presenting The expressing cells and exosomes 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. Exemplary nucleic acids encoding the peptide sequences include or are 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 is a nucleic acid that is capable of expressing a specific targeting targeting gene. (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, anti- The present invention relates to a method for the treatment of cancer, particularly melanoma, such as cutaneous melanoma, by using 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 includes or consists of any one of SEQ ID NOs: 30 to 32. Exemplary nucleic acids encoding the peptide sequences include or are 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 cell, T cell population, cytotoxic cell, antigen-binding polypeptide, The peptides, antigen-presenting cells and exosomes are useful in treating cancer, particularly malignant melanoma, e.g., cutaneous melanoma. The method may be used to treat melanoma, ocular ulcer, or uveal melanoma. Related diagnostic methods are also provided. do. EXAMPLES

[0200] (Example) (Example 1 - CLT specific) The aim is to identify cancer-specific transcripts that consist entirely or partially of LTR elements. That was the case. As a first step, we will perform de novo assembly of comprehensive transcriptomes across cancer types. To achieve this, we used genome-wide data obtained from The Cancer Genome Atlas (TCGA) Consortium. The patients were 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) adapter for use with quality (Q20) trimming and length filtering ( Both reads were ≥35 nucleotide pairs), and khmer (v2.0) (Crusoe et al., 2015, F10 00Res., 4:900) with kmer normalization (k= 20) was performed. Reads were analyzed using STAR (2.5.2b) with the same settings as those used for the entire TCGA. The genome was then 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 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 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 clean the assembly. Contigs were quasi-mapped and those with expression levels of <0.1 transcripts per million (TPM) were removed. The remaining ones were analyzed using GMAP (v161107) (Wu et al., 2005, Bioinf., 21:1859-1875). The sequence was then mapped to GRCh38 and had 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 3D-merger algorithm (Otech., 28:511-515). The process was specifically designed to allow for the evaluation of repetitive elements, so that monoexons The completeness and quality of the transcript assemblies 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. The splice site is within a two-nucleotide window of opportunity for transcription. This process tested for the presence of 1,001,931 We identified 1,006 transcripts, of which 771,006 were spliced ​​and 230,925 were monoexos. The results showed that the antibody was soluble in water.

[0201] Separately, the assembled contigs were overlaid with genomic repeat annotations. Transcripts containing LTR elements were identified. LTR and non-LTR elements were analyzed using the amplified PCR method as described above. The following was annotated (Attig et al., 2017, Front. In Microbiol., 8:2489). We then used hidden Markov models (HMMs) representing known human repeat families (Dfam 2.0). Library v150923), RepeatMasker Open-3.0 (Smit, A., R. Hubley and P. Green, http: / / www.smit.com / article / 20130231327.html). tp: / / www.repeatmasker.org,1996-2010) was used to annotate GRCh38, which hmmer (Wheeler et al., 2013, Bioinform., 29:2487-2489). CAN is 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 that contained 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 GTEx (Th e Genotype-Tissue Expression Consortium, 2015, Science, 348:648-60) , and healthy tissue-matched controls for all cancer types). If detected in any sample at >1 TPM, the gene was considered to be specifically expressed in the cancer. A cancer was considered cancer-specific if it met the following criteria: (i) 24 samples for each cancer type (ii) expressed at <10 TPM in >90% of all healthy tissue samples; (iii) expressed in the target cancer type at least three times the median expression in any control tissue type; and (iv) in the target cancer type, the 90th of each available healthy tissue. In addition to these expression threshold criteria, transcripts are expressed at or above 3x the percentile. Selection was based on manual inspection and the identification of 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. We crossed the list of transcripts with the 5,923 transcripts that met both criteria (Cancer (termed CLT for specific LTR element-spanning transcript).

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

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

[0206] To further confirm the specificity of the cancer-specific antigens encoded by CLTs, Potential cross-reactivity with other proteins that may be expressed in the mouse was examined. For the purpose of the targeting, we compared the sequence of the predicted protein with that of any other predicted protein (over the entire length of the protein) Translated ORFs with 85% amino acid sequence identity were retained. The protein encoded by CLT, which shows >85% sequence identity with the protein, was We collated the expression patterns of the protein-coding transcripts of 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 considered. The combination of these selection criteria resulted in the selection of sufficiently unique amino acids. We generated a final list of 139 CLTs with the potential to code for proteins with amino acid sequences. .

[0207] Of these 139 CLTs, 14 were specific for cutaneous malignant melanoma (i.e., according to the above procedure). , and was confirmed to be specifically upregulated in TGCA cutaneous melanoma samples. ), and seven were specific for cutaneous melanoma and uveal melanoma (i.e., Therefore, TGCA was specifically upregulated in cutaneous and uveal melanoma samples. These four CLTs specific to cutaneous malignant melanoma are These are identified herein as having SEQ ID NOs: 34, 37, 38 and 39. These four cutaneous and uveal melanoma-specific CLTs are referred to herein 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 tides from biological samples. Affinity purification of HLA molecules, followed by elution of bound peptides from the HLA molecules and nano-purification. 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 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 genome sequences from known transcriptomes, or even entire genomes, The MS data is analyzed using a predefined list of ORFs corresponding to the open reading frames (ORFs). Although it is possible to search for However, the matching of these ultra-large sequence databases has non-linear effects that limit the identification of presented peptides. This always leads to a high false positive rate. Further technical problems (e.g., leucine mass = isoleucine 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 will be increased restrictions associated with the use of very large databases, such as databases created using In practice, immunopeptides are generated without reference to a clearly defined set of potential polypeptide sequences. It is very difficult to perform tydom analysis to identify novel antigens.

[0209] Bassani-Sternberg et al. analyzed HLA-binding peptide samples derived from 25 patients with cutaneous melanoma. We pooled MS data from the 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 predicted, these peptides included PRAME, MAGEA3, and TRPM1 (melastatin). These included peptides found within multiple tumor-associated antigens (TAA). The MS data of five of these patients were analyzed to determine the patient-specific The results of the comparison with a polypeptide list created from the mutant protein sequences of these patients revealed that Patient-specific new antigens 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) 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 The peptide sequences are concatenated into a single peptide file for each CLT, or not concatenated, and these concatenated files are The raw spectra of the PXD004894 dataset were analyzed using a single peptide file (analysis A) or a single peptide file (analysis B). The data were used to interrogate the human proteome (UniProt (Analysis A) or UniPr ot and masDB (Analysis B)) and analyzed using the Peaks™ software. Analysis was performed using the MS-DOC software (Analysis A) or Mascot software (Analysis B).

[0212] In analysis A, the results of these studies were compared with those of the 25 patients examined by Bassani-Sternberg et al. We identified 14 peptides associated with HLA class I molecules immunoprecipitated from patient tumor samples. , which could be ascribed 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 the 25 patients examined We identified 14 peptides related to the molecule that were not found in the reported proteome. We were able to attribute seven ORFs that were not identified (see Table 2). Detection of these peptides in association with HLA class I and HLA class II molecules has allowed the 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. and 2 are peptides found in the CLT antigen that were not present in the UniProt database. 1 to 32 show the representative mass spectrometry spectra of each peptide shown in Tables 1 and 2. These figures show the fragment spectra of the peptide sequences shown, which nUPLC-MS 2 in individual SKCM tumor patients (Bassani-Sternberg et al. (Images extracted from the PRIDE dataset by the PEAKS software). All fragments are represented by peptide sequences on the spectrum, and the most abundant fragment ions are located on each spectrum. In Figures 1-15 and 29-32 (Analysis A), the lower panels of the figures show the predicted spectra. The sequence annotation for the nucleotide sequence is shown in Figure 16-28 (Analysis B) on the right. The fragment ions are annotated as follows: b: N-terminal fragment ion; y : C-terminal fragment ion; -H 2 O: water loss;-NH 3 : 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 9 amplified 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 (or a 9-mer derived therefrom) binds to at least one of the supertypes tested Of these, many sequences were consistent with the HLA class I sequences examined (see Table 3). A type that is predicted to bind with high confidence (low rank score %) to a specific type within a supertype. It was.

[0214] In summary, the data presented 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 supports the identification of these C LT is translated into polypeptides (SEQ ID NOs: 1 to 10; also called CLT antigens) in tumor tissues. These are then processed by the cellular immune surveillance machinery and are 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 the peptide / HLA class II complex. Therefore, these CLT antigens and their fragments can be used to detect tumors that express these antigens. The present invention is useful in a variety of 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 mass; for peptides with multiple spectra, 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 that belong to 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 HLA class II peptides.

[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 name and cross-referenced sequence number [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 . 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, the inventors also carried out a new immunopeptidome study. Through this additional work, we also identified peptides derived from the predicted ORFs. Furthermore, we show that these CLTs are translated into CLT antigen polypeptides in tumor tissue.

[0219] We procured frozen tumor tissue from 10 patients diagnosed with malignant melanoma. ~1 g sample was homogenized, the lysate was centrifuged at high speed, and the clarified lysate was The sate was synthesized using Protein A (Protein A) covalently linked to anti-human HLA class I monoclonal antibody (W6 / 32). A) The beads were mixed. The mixture was incubated overnight at 4°C to detect the binding of antibodies to 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 isolates were isolated from the 100-kDa strain of 100-kDa serovar IgE using reversed-phase column chromatography (Ternette et al., 2018). The eluted peptides were subjected to nUPLC-MS to identify specific peptides with a given charge-to-mass ratio (m / z). The molecules are selected, isolated, fragmented, 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 evaluation, the inventors have identified CLT antigen numbers The HLA clusters for 10 malignant melanoma tumors created by the present inventors using Nos. 1, 2, 3, and 4 were Spectra from the Sequence I data set were collated (Table 4; SEQ ID NOs: 1-4) using the PEAKS™ software. Using software (v8.5 and vX, Bioinformatics Solutions Inc.), the human proteome (U 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 was 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 Immunoprecipitation 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. does not correspond to any polypeptide sequence present in the known human proteome (UniProt). The CLT antigens in our dataset were identified from 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, as outlined above, That is why.

[0222] Detection of these peptides in association with HLA class I molecules was optimized by determining the four ORFs from which they were derived. It is first translated in melanoma tissue, then 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. The characteristics of peptides found in the LT antigen are shown in Figures 33 to 42. Representative MS / MS spectra are shown. The upper panels of each figure show the MS / MS peptide fragment profiles. The files are shown with standard MS / MS annotation (b: N-terminal fragment ion; y: C-terminal fragment ion). Single ion; -H 2 O: water loss;-NH 3 : ammonia loss; [2+]: doubly charged peptide ion; pre: fragmentation Unreacted precursor peptide ions; a n-n: internal fragment ion), which is shown above, PEA The most abundant fragments 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. A rendering of the spectrum showing the column positions is shown. High- assigned to peptides in Table 4. Since the 101gP scores are consistent with these spectra, the peptide sequences discovered in these analyses are consistent with these spectra. It contains multiple fragments that match exactly 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 / ), The IgG binding profiles were evaluated using a 10-fold increase in the IgG binding profile and determined the predictive strength of their binding to HLA class I types A and B supertypes. The results of these prediction studies were used to estimate the 9-mer sequence of all peptides (or peptides contained within each complete sequence). ) were predicted to bind to at least one of the supertypes tested (Table 5 Of these, many sequences are consistent with specific types within the HLA class I supertypes examined. The peptides detected were predicted to bind to the β-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 In addition, the results are consistent with those of the tumor samples from our dataset. All peptides found were consistent with HLA tags detected in patient samples by NetMHCpan 4.0. It was predicted that the ribozyme would 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 present inventors synthesized peptides having the sequences found. The same conditions were used for the tumor samples in the data set. 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 100 nm nuclei (from our tumor tissue database - Figures 33-42). The selection of detected ion fragments corresponds to a synthetically prepared peptide with the same sequence. The m / z values ​​are shown for each of the upper and lower fragment peaks in these MS / MS spectra. Accurate alignment of the fragments was demonstrated (between the fragment ions derived from the tumor and the synthetic peptides). The small differences between the experimentally determined m / z values ​​of the 14366 and 14367 fell within the m / z tolerance range of <0.05 Daltons. The assignment of each spectrum from tumor tissue to a CLT-encoded peptide was Check the authenticity.

[0225] In summary, the peptide data presented in Table 4, Figures 33-42, and Figures 43-50 indicate that melanoma patients Provides very strong support for 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. A total of 10 normal skin, 9 normal lung and 18 normal breast tissue samples were processed to generate immunopeptidomes. The inventors extracted HLA class I data sets from these normal tissue samples. The spectra were collated, and all of the polypeptide sequences derived from CLT antigen numbers 1, 2, 3, and 4 were identified. The peptides derived from CLT antigen numbers 1, 2, 3, and 4 were identified as peptides that were expressed in 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 confirms the The CLT is translated into polypeptides (SEQ ID NOs: 1 to 4; also referred to as CLT antigens) in tumor tissue. Thus, these CLT antigens and their fragments further demonstrate that the tumor is The present invention 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 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 mass; for peptides with multiple spectra, 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 name and cross-referenced sequence number [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 . 4 : The 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 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 in accordance with 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, as well as NCI, NHGRI, NHLBI, NIDA, and NIMH) This work is based on data generated by the National Institutes of Health (NIH) and is supported by the 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 have identified a number of mutations that are present in the "mutation-associated neoantigen" (MANA) repertoire found in tumor cells of cancer patients. It has been used to identify therapeutically relevant tumor-derived epitopes present in , Cancer Discovery 2017; Le et al., Science 2017; Forde et al., NEJM 2018; Dani Lova et al., Cancer Immunol. 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 potent anti-CLT antigens in cancer patients. It can be used to identify therapeutically relevant T cell responses to the antigen.

[0233] Other assays to identify epitope-specific T cells in immune-exposed 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, the specificity of cognate T cells is brought out by activating / expanding them. What distinguishes tCR-Vβ 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) Using ELISA, individual peptides from a panel of targeted peptides derived from an antigen (or multiple antigens) can be identified. in detecting specific TCRs propagated in cells cultured with the peptide Using TCRseq applied to tumor tissue from the same patient, TCR / T cells were detected ex vivo as well. Peptide-stimulated cultures also showed increased expression of IL-1 in tumor-infiltrating lymphocytes found in cancer tissue in situ. Therefore, MANAFEST can be recognized by T cells of the patient. This is a powerful technique for identifying MANA epitopes that are useful in cancer patients and normal and tumor tissues. Functional analysis of a large number of mutant peptides detected by whole-exome sequencing of tissues It has been shown that it is possible to identify MANA peptides that are specifically related to the 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, consisted of the following steps: Step 1: A fusion protein containing epitopes that efficiently bind selected HLA class I alleles is prepared. Step 2: Select appropriate melanoma patients. The 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 the patient's 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) A PCR amplification was performed for all wells and amplified in the presence of individual CLT antigen-derived peptides ( However, there was no amplification in the presence of the control peptide or in the absence of peptide stimulation. The TCR-Vβ CDR3 sequences were then 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 that have CLT antigens. It may be determined whether the cells amplify TCRs that home to the patient's tumor, and these TCRs This study provides further evidence that T cells bearing CLT antigens 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, the 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 peptides derived from CLT antigens that amplified one or more TCRs in the EST assay. Table 7 also shows the 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 who were tested in this study and had ≥1 TCR amplification in the assay. As shown in.

[0236] FIG. 51 Panel A shows TCR amplification with MANA peptide specific to NSCLC (non-small cell lung cancer) patients. The data shown is published (Forde et al., NEJM 2018). The vertical axis represents the MANA peptides listed on the horizontal axis. Shown are wells of cells cultured in the presence of either peptide or control peptide, respectively. The prevalence of TCR-Vβ CDR3 sequences identified in wells containing MANA7 indicates that amplification in patient T cells 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. In addition, the specific amplifications observed in panels B and C were consistent with the T cell repertoire of these melanoma patients. Panel B shows that the IL-16 ... 15 HLA class IA from sources 1, 2 and 4 * 02 All wells stimulated with the peptide panel TCR detected in LMSSFSTLASL-stimulated wells of PBMCs from melanoma patient 222B The frequencies of TCR sequences are shown. Three TCR sequences were amplified. [ka] A derived from CLT antigen 2 * 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] A derived from CLT antigen 2 * 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 that these patients had ≥1 TCR amplification against CLT antigens 1–4 during the completed studies. The panels show 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 by the assay placed on top (dashed underlined or (See Examples 2 and 2.1.) Below these sequences, the peptides detected by HERVFEST are The peptides (see FIG. 51) were listed according to the number of melanoma patients in which they were detected (Table 8) and the H Shown 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 also detected in other wells Underlined bold: significant amplification of multiple TCRs, at least one of which is related to other It was also detected in

[0240] These results indicate that CLT antigens 1 to 4 are present in melanoma patients and that these CLT antigens Peptides derived from these antigens specifically elicit T cell responses in patients with malignant melanoma. These findings provide strong evidence for the role of CLT in the treatment of melanoma and identify these anti-CLTs as targets for therapeutic intervention. 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 deleted from the T cell repertoire of normal subjects (assay to show that the ELISPOT assays demonstrate that CLT antigen-specific CD8 T cells are expressed within the normal T cell repertoire of healthy individuals. may be used to indicate the presence of The tumor was not deleted by central tolerance due to the expression of cancer-specific CLT antigens in the tissue. This type of ELISPOT assay involves several steps: Step 1: CD8 T cells and CD14 monocytes are isolated from peripheral blood of normal blood donors and these cells The CD8 T cells are HLA typed and matched to the specific CLT antigens being tested. Using a magnetically labeled antibody against the CD45RO marker, naive and memory subtypes were identified. Step 2: CD14 monocytes are subjected to immunoreactions with individual or pooled CLT antigen peptides. Step 3: The expanded CD8 T cells are then co-cultured with the CD8 T cells for 14 days. These cultures were then isolated and restimulated overnight with fresh monocytes pulsed with peptide. The peptides included individual CLT antigen peptides, irrelevant control peptides, or infectious (e.g. For example, CMV, EBV, influenza, HCV) or self (e.g., Mart-1) antigens. Restimulation may include the administration of anti-interferon or anti-peptides known to elicit a response. The antibody reacts with peptides that bind to IFN-gamma antibodies. After overnight activation, cells were plated with IFNγ. The IFNγ captured on the plate was then washed away with additional anti-IFNγ antibody and a standard colorimetric dye. Dark spots are detected where IFN-γ producing cells were originally present on the plate. Data from this assay include spot count, median spot size, These include the median spot intensity and the frequency of IFNγ-producing T cells and the per cell The amount of IFNγ in the spotted erythrocytes was measured. Specific responses, measured as number or median spot size, were compared using the results for each individual sample without specific peptide. The stimulation index (SI) is derived by dividing the response 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 CLT antigen and the control antigen are Comparison of responses to antigens has demonstrated that naive subjects have a strong repertoire of CLT antigen-reactive T cells. It has been shown that the antigen-based immunogenic formulation can be used as a vaccine. Table 9 shows significant CD8 T cell responses from HLA-matched normal blood donors. The results are shown in Figures 53 to 56. Data are expressed as mean values. Statistical significance determined by one-way ANOVA with 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 against the 0201-restricted peptide. The example shown in Figure 54 is derived from CLT antigen 2 (CLT002 ​​in the figure), which is also HLA-A * Bound 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 cells from normal blood donors against 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+ CD8 T cells (panels A and C) to bundle peptides In contrast, naive CD45RO negative CD8 T cells from the same donor were significantly more potent than CLT001 and CLT00. 4 (Figure 56, panels B and D).

[0244] Table 9: CLT antigen-derived peptides inducing 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. The efficacy was assessed using HLA class I / peptide pentamer ("pentamer") staining and / or in vitro killing assays. Therefore, the assay 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 indicate the presence of a therapy-relevant T cell response against a subject.

[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 the 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 peptide binding site of an HLA class I molecule that binds to the associated CLT antigen peptide in the peptide-binding groove of the HLA molecule. The binding occurs through the coiled-coil multimerization domains of the pentamer structure. Phycoerythrin or allophycocyanin-conjugated antibody fragment specific for insulin 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 subjected to an in vitro killing assay to determine whether they can kill target cells. These assays can further test for the ability of CD8 T cell populations and A fluorescently labeled target cell population, in this case the CD8 population, which is 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 peptidase 02 Pulsed with chitin and HLA-A * 02, 03 or B * C1R cells transfected with 07, or Melanoma cell lines or patient tumor cells previously shown to express CLT / CLT antigens The peptides used to pulse T2 or C1R cells may include CLT antibodies. The original peptide or a positive control peptide is included. The target cells are carboxyfluorescein-activated Cell death may be detected by fluorescent labeling with CFSE (a cell proliferation dye). In this method, CD8 T cell-mediated apoptosis is demonstrated by the uptake of 7AAD. When target cells are killed by the cisplatin, 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. To investigate the mechanism of action of T cells from melanoma patients or healthy donors in vitro, we investigated the expression of CLT antigen-specific 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-selected cells pulsed with CLT004. The results show that the peptide-pulsed C1R-B7 target cells were killed. , evident at effector to target cell ratios of 3:1 and 1:1.

[0248] Example 6 - Mouse immunogenicity studies To demonstrate the immunogenicity of CLT antigens, mice were transfected with replication-deficient adenovirus expressing one or more CLT antigens. T cells from these mice inoculated with the viral vector 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 was introduced into mice. The mice were inoculated with IFNγ and humanely euthanized at the appropriate time points, and preparations of spleen cells were generated using murine IFNγ. The wells of a multiwell dish were derivatized with a monoclonal antibody against CLT Load 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 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 amount of DNA fragments (DNA fragments) 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 subeqeuntly The intermer region is amplified through a series of thermal cycling reactions to generate an intercalator colorimeter. The primer pairs were engineered against CLT and quantified fluorescently using SYBR Green. 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-1 974), MeWo (ATCC Reference No. HTB-65), SH-4 (ATCC Reference No. 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 at 1 × 10 6 Instant freezing of pieces The cDNA was extracted from cells and reverse transcribed to DNA. 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). Results of the qRT-PCR assay used, 3 melanoma cell lines and 4 non-melanoma 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 antibodies were targeted to different regions of CLT (SEQ ID NO: 34), which encodes CLT antigen 2 on the RNA: 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 and four non-melanoma cell lines. These target different regions of CLT (SEQ ID NO: 35), which encodes the CLT antigen 3 / 4 of Our results showed that CLTs in RNA extracted from melanoma cell lines were significantly higher than those 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 was performed to identify histopathological features of specimens. This allows visualization of the presence and expression level of a given transcript under various conditions. The assay also uses 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 using antibodies or enzymes. Visualized by a signal generated by a combination of colorimetric reactions based on RNAScope is an in situ u Hybridization-based technology, ensuring 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 that there are small Appears as red dots, with multiple dots indicating 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 were performed on representative images from each core as follows: The assessment of % of cells with positive staining for CLT probes is rounded up to the nearest 10. The estimated per cell levels of expression across a given section are: 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] The expression of each CLT was detected in tumor cores from many different patients, and for each patient analyzed We validated CLT findings from tumor-derived RNAseq data separately in each core and performed multisample analysis. We confirmed the homogeneity of expression in 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 indicates otherwise, the term "includes" " and variations such as "included" and "comprising" are intended to include any integer, step, integer combination, or combination of steps. includes any integer, step, group of integers or group of steps, but excludes any other integer, step, group of integers or group of steps. 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. 6,399,433, which is incorporated herein 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 recited above are included.

[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] TIFF2025041678000047.tif247170TIFF2025041678000048.tif98170SEQ ID NO: 35 (cDNA sequence of CLT encoding CLT antigens 3 and 4) [ka] TIFF2025041678000050.tif216170 SEQ ID NO: 36 (cDNA sequence of CLT encoding CLT antigen 5) [ka] TIFF2025041678000052.tif237170 SEQ ID NO: 37 (cDNA sequence of CLT encoding CLT antigen 6) [ka] TIFF2025041678000054.tif105170 SEQ ID NO: 38 (cDNA sequence of CLT encoding CLT antigens 7 and 8) [ka] SEQ ID NO: 39 (cDNA sequence of CLT encoding CLT antigen 9) [ka] TIFF2025041678000057.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

【Claim 1】 A novel article, method, and manufacturing method substantially described in the specification of this application.

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