Antigen pool

Abstract

This invention provides an antigen pool useful in the treatment of cancer, particularly melanoma, and especially cutaneous melanoma and uveal melanoma. [Solution] An antigen pool is provided for use in ex vivo stimulation and / or amplification of T cells derived from a person with cancer, wherein each antigen exists in the form of a polypeptide and / or a nucleic acid encoding the polypeptide, and the different antigens exist as separate polypeptides or nucleic acids and / or as a fusion protein or a portion of a nucleic acid encoding a fusion protein.

Description

[Technical Field]

[0001] (Field of Invention) The present invention relates to an antigen pool comprising two or more different antigens. Such an antigen pool is Equation of T cells derived from humans with cancer (e.g., cutaneous melanoma or uveal melanoma) This invention is intended for use in vivo stimulation and / or amplification. The present invention further relates to An immunogenic pharmaceutical composition comprising an antigen pool and a pharmaceutically acceptable carrier, against cancer cells Process for preparing a population of cytotoxic T cells, antigen pool, and pharmaceutical composition thereof. Immunotherapy involving loading substances and / or stimulation by antigen pools and their pharmaceutical compositions Epidemic cells and exosomes, their medical uses, and the antigen pool and immunogenic pharmaceutical composition. The present invention relates to a therapeutic method that includes administering substances, immune cells, and exosomes. [Background technology]

[0002] (Background of the invention) As part of normal immune surveillance against pathogenic microorganisms, all cells are responsible for intracellular proteins It degrades the major histocompatibility complex (MHC) class I molecules expressed on the surface of all cells. It produces peptides that are loaded into the host cell. Most of these peptides originate from the host cell. Most of the time, it is recognized as self and remains unrecognized by the adaptive immune system. However, external The non-self peptide binds tightly to the T cell receptor (TCR) of the MHC I-peptide complex. This can stimulate the expansion of naive CD8+ T cells that encode this expanded T cell population. The group can eliminate foreign antigen-tagged cells. Effector CD8+ T cells (cytotoxicity) not only (including cytotoxic T lymphocytes, CTLs), but also memory CD8+ T cells that can be re-amplified when foreign antigen-tagged cells appear later in the life of the animal can also be produced .

[0003] The expression of MHC class II molecules, which is usually restricted to professional antigen-presenting cells (APCs) such as dendritic cells (DCs), is usually loaded with peptides taken up from the extracellular environment into the interior along with them. In the presence of various factors including T cell adhesion molecules (CD54, CD48) and costimulatory molecules (CD40, CD80, CD86 ), when the complementary TCR derived from naive CD4+ T cells binds to the MHC II-peptide complex, the maturation of CD4+ T cells into effector cells (e.g., T ) is induced. These effector CD4+ T cells not only promote the differentiation of B cells into antibody-secreting plasma cells, but also promote the differentiation of antigen-specific CD8+ CTLs, thereby , T H 2, T H 17, T FH , T r eg cells) is induced. These effector CD4+ T cells can help induce an adaptive immune response to foreign antigens, including both short-term effector functions and long-term immune memory. DCs can contribute to the generation of immune memory by delivering exogenous-derived antigens (e.g., peptides or proteins released from pathogens or tumor cells onto their MHC I molecules, thereby performing the cross-presentation process of peptide antigens and providing an alternative pathway to stimulate the expansion of naive CD8+ T cells . Antigen (e.g., pathogen or tumor cell peptides or proteins released from) peptide antigens, and stimulate the expansion of naive CD8+ T cells by providing an alternative pathway, can contribute to the generation of immune memory.

[0004] Immune memory (especially antigen-specific B cells / antibodies and antigen-specific CTLs) is important in the control of microbial infections Immunological memory plays a crucial role in preventing diseases caused by important pathogenic microorganisms. It is used to develop numerous vaccines for prevention. Immunological memory controls tumorigenesis. Although it is known to play an important role in cancer, very few effective cancer vaccines have been developed. It hasn't been done.

[0005] Cancer is the second leading cause of death, accounting for nearly one-sixth of all deaths worldwide. Of the 8.8 million cancer-related deaths in the past five years, lung cancer was the most deadly (1.69 million). (788,000 cases), liver cancer (788,000 cases), colorectal cancer (774,000 cases), stomach cancer (754,000 cases), and breast cancer (571,000 cases) The number of cases was [number missing]. The economic impact of cancer in 2010 was estimated at US$1.16 trillion, and new [number missing] The number of cases is expected to increase by approximately 70% over the next 20 years (World Health Organization, Cancer Facts (World Health Organization) d Health Organization Cancer Facts), 2017).

[0006] Current treatments for cutaneous melanoma vary and depend heavily on the location of the tumor and the stage of the disease. The main treatment for non-metastatic melanoma is surgery to remove the tumor and surrounding tissue. Late-stage melanoma This may require treatment including lymph node dissection, radiation therapy, or chemotherapy. PD-1 Immunotherapy checkpoints, including the use of antibodies targeting negative immunomodulatory factors such as PD-L1 and CTLA4. Intake blockade strategies have recently revolutionized the treatment of various malignancies, including melanoma (Ribas). , A. and Wolchok, JD (2018) Science, 359:1350-1355). Checkpoint blocking. The extraordinary value of the therapy, its clinical benefits, and the patient's adaptive immune response to the patient's own cancer antigens. The well-recognized association with the answer (particularly the T cell-based immune response) is that effective cancer vaccines This reactivated the search for tin, vaccine modalities, and cancer vaccine antigens.

[0007] Human endogenous retroviruses (HERVs) are exogenous infectious retroviruses that infect the ancestral germline. This is a remnant of integration. HERV utilizes the presence of long terminal repeats (LTRs) adjacent to the viral genome. It belongs to a group of endogenous retroelements that are characteristic of mammals. This also includes the LTR retrotransposon (MaLR), and therefore, it is collectively referred to as an LTR element. It is known (here, we refer to all LTR elements collectively as ERV). ERV is a pycnoid. They constitute a significant portion (8%) of the mammal genome and are divided into approximately 100 families based on sequence homology. They can be grouped together. Many ERV sequences are adjacent to the LTR with gag, pro, pol, and e A defective plow that shares a prototype retroviral genome structure consisting of the nv gene It codes for viruses. Some intact ERV ORFs are exogenous infectious viruses such as HIV-1. It produces retroviral proteins that share characteristics with proteins encoded by Russ. Such proteins may act as antigens that induce a strong immune response. Yes (Hurst and Magiorkinis, 2015, J. Gen. Virol 96:1207-1218), by ERV The encoded polypeptide is involved in the selection process of T cells and B cell receptors, as well as in the central and peripheral systems. This suggests that sexual tolerance can be avoided. Immune reactivity to ERV products is sensitive ERV products can occur spontaneously in infections or cancers, and are the cause of several autoimmune diseases. This is considered to be related (Kassiotis and Stoye, 2016, Nat. Rev. Immunol.). 16:207-219).

[0008] Due to the accumulation of mutation and recombination events in evolution, most ERV-derived sequences are... Losing some or all of the functional open reading frames of those genes, therefore , it has lost its ability to produce infectious viruses. However, these ERV elements Genes, like other genes, are maintained in germline DNA, and less of those genes It also has the potential to produce proteins from several sources. In fact, HERV encodes The protein has been detected in various human cancers. For example, the splice of the HERV-K env gene. The subvariants Rec and Np9 are found only in malignant testicular germ cells and not in healthy cells. No (Ruprecht et al., 2008, Cell Mol Life Sci 65:3366-3382). Compared to healthy tissue. Elevated levels of HERV transcripts have also been observed in cancers such as prostate cancer (Wang-Johannin References by g, 2003, Cancer 98:187-197; References by Andersson et al., 1998, Int. J. Oncol, 12:309 -313). Furthermore, overexpression of HERV-E and HERV-H has been shown to be immunosuppressive. This, too, may contribute to the development of cancer (Mangeney et al., 2001, J. Gen. Virol. 82:2515-25). 18). However, the actual mechanisms by which HERVs may contribute to the development or pathogenicity of cancer remain unclear. It is unclear.

[0009] In addition to deregulating the expression of surrounding adjacent host genes, ERV regulatory elements The activation and translocation to new genomic sites are novel metamorphisms, some of which may be oncogenic. This may lead to the production of photoproducts (Babaian and Mager, Mob. DNA, 2016, Lock). (See the following publication, PNAS, 2014, 111:3534-3543).

[0010] A wide range of vaccine modalities are publicly known. One well-documented approach is: Antigenic poly This includes directly delivering peptides to the target, or delivering polynucleotides to their polynucleotides. The vector is designed to express the immunogenic polypeptide encoded by rheotide in vivo. It can be administered to the target by a viral vector, for example, an adenovirus vector. The use of antigens is important in both prophylactic vaccination strategies and therapeutic treatment strategies for cancer. It has been thoroughly explored for delivery (Wold et al., Current Gene Therapy, 2013, Ade novirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy, 13:421-43 3) Using immunogenic peptides, polypeptides, or polynucleotides encoding immunogenic peptides Furthermore, patient-derived antigen-presenting cells (APCs) can be loaded, and then these can be used to treat the condition. It can be injected into the target as a vaccine that induces a therapeutic or prophylactic immune response. An example of this approach is Provenge, the only anti-cancer vaccine currently approved by the FDA. That is the case.

[0011] Cancer antigens can be used to create various non-vaccine therapeutic modalities, thereby treating cancer These therapies can also be used for the treatment and prevention of [unspecified disease]. These therapies include 1) antigen-binding biological agents, and 2) adoption. Cell therapy: It is classified into two different classes.

[0012] Antigen-binding biologics typically recognize cancer cells modified with antigens and promote their destruction. It consists of valency-modified polypeptides. The antigen-binding components of these biological products are, but are not limited to, TC. R, high affinity TCR, and TCR mimics produced by various technologies (monoclonal antibody technology) These may consist of TCR-based biologics, including those based on (and others). The cytolytic portion of this type of polyvalent biological product contains cytotoxic chemicals, biotoxins, and immune cells. A system comprising targeting motifs and / or immunostimulatory motifs that promote targeting and activation. These may also be the case, and all of them promote the therapeutic destruction of tumor cells.

[0013] Adoptive cell therapy involves removing cells and ex vivo stimulating them with vaccine antigen preparations (fine cells). (T cells cultured with other factors, including cellular and cell-free components, in or out of the presence of other factors) It may be based on the individual's own T cells (Yossef et al., JCI Insight. October 4, 2018; 3(1) 9). pii:122467. doi:10.1172 / jci.insight.122467). Alternatively, adoptive cell therapy is cancer antigen Cells intentionally modified to express antigen-binding polypeptides that recognize (patient-derived or These antigen-binding polypeptides can be based on cells derived from non-patients. This falls into the same class as antigen-binding biological products described above. Therefore, Genetically engineered (autologous or non-autologous) to express cancer antigen-binding polypeptides. Lymphocytes can be administered to patients as adoptive cell therapy to treat their cancer.

[0014] The use of ERV-derived antigens to induce an effective immune response against cancer is a mouse model of cancer. The study showed promising results, promoting tumor regression and leading to a more favorable prognosis (Kershaw et al.'s paper). References, 2001, Cancer Res. 61:7920-7924; Slansky et al., 2000, Immunity 13:529-538) Therefore, HERV antigen-centered immunotherapy trials are being planned in humans (Sacha et al.) References, 2012, J.Immunol 189:1467-1479), one example is the specific and rigorous control of tumor-specific ERV antigens. Progress is limited due to the circumstances.

[0015] WO 2005 / 099750 is used when inducing a cross-reactive immune response to the HERV-K Mel tumor antigen. Existing vaccines against common infectious pathogens that provide protection against melanoma The anchor sequence is being identified.

[0016] Issue WO 00 / 06598 identifies the HERV-AVL3-B tumor-associated gene that is preferentially expressed in melanoma, and Furthermore, this relates to methods and products for diagnosing and treating conditions characterized by the expression of the gene.

[0017] WO 2006 / 119527 is an antigenic poly(I) derived from melanoma-associated endogenous retrovirus (MERV). Peptides, and their use for the detection and diagnosis of melanoma and for prognosis determination of the disease. The use of antigenic polypeptides as anti-cancer vaccines has also been disclosed.

[0018] WO 2007 / 137279 is, for example, a HERV-K+ conjugated antibody for preventing or inhibiting cancer cell proliferation. The present invention discloses methods and compositions for detecting, preventing, and treating HERV-K+ cancer using [specific technology / method]. .

[0019] WO 2006 / 103562 expresses the immunosuppressive Np9 protein derived from the env gene of HERV-K. This invention discloses a method for treating or preventing cancer. This invention inhibits the activity of the protein. A pharmaceutical composition containing nucleic acids or antibodies that can cause harm, or an immune response to such proteins. This invention relates to an immunogen or vaccine composition that can induce a response.

[0020] WO 2007 / 109583 contains a concentrated population of immune cells that respond to HERV-E antigens on tumor cells. By providing a composition, for preventing or treating neobiotic diseases in mammals The present invention provides compositions and methods.

[0021] Humer J et al., 2006, Canc. Res., 66:1658-63, described endogenous retroviral syndrome associated with melanoma. We are identifying melanoma markers derived from Russ.

[0022] Used in immunotherapy for cancer, particularly melanoma, especially cutaneous melanoma and uveal melanoma. It is necessary to identify a novel antigen pool containing HERV-associated antigenic sequences that can be used. ru. [Overview of the project]

[0023] (Summary of the invention) Surprisingly, the inventors have found that the LTR element is included in or adjacent to the LTR element. It originates from the genome sequence and is found at high levels in cutaneous melanoma cells, but is not detected in normal healthy tissue. We discovered specific RNA transcripts that are either not present or present at very low levels (Example 1). (See reference). Such transcripts are referred to herein as cancer-specific LTR-element spanning transcripts. This is referred to as CLT. Furthermore, the inventors have identified the potential properties encoded by these CLTs. A subset of lipeptide sequences (i.e., open reading frames (ORFs)) is found in cancer cells. Translated in cells, processed by components of the antigen processing apparatus, class Major histocompatibility complexes of Class I and Class II (MHC Class I and MHC Class II) and Class I and In association with human leukocyte antigen (HLA class I, HLA class II) molecules, they are found in tumor tissue. We showed that it is presented on the cell surface (see Example 2). These findings suggest that these poly The peptide (referred to herein as the CLT antigen) was shown to be substantially antigenic. Therefore, cancer cell presentation of CLT antigen is due to these cells displaying homologous T cells against the CLT antigen. It is thought that these cells become more susceptible to elimination by T cells possessing cytoplasmic receptors (TCRs), and these homogeneous TCRs CLT antigen-based vaccination methods / regimes that amplify T cells are used in cancer cells (and so This induces an immune response against tumors (including melanoma, particularly cutaneous melanoma). It is possible. T cells derived from melanoma cells are, in fact, derived from the CLT antigen disclosed herein. It is reactive to peptides, amplifies T cells, and amplifies the T cell receptor sequence. (See Example 3). The inventors have shown that T cells specific to the CLT antigen can be controlled by central tolerance to normal counteracting the CLT antigen. It was confirmed that the cells were not eliminated from the elephant T cell repertoire (see Example 4). Healthy donor T The presence and killing activity of CLT antigen-specific T cells in ex vivo cultures of cells were determined. (See Example 5). Finally, qRT-PCR studies showed that CLT cells were blacker compared to non-melanoma cell lines. It was confirmed that the RNA extracted from tumor cell lines was specifically expressed (see Example 6). The inventors also produced a fusion protein containing a unique CLT antigen (Example 8).

[0024] The inventors also surprisingly found that CLT encoding a specific CLT antigen is used in cutaneous melanoma. We discovered that it is not only overexpressed in other areas, but also in uveal melanoma. The CLT antigen polypeptide sequence encoded by CLT is used in uveal melanoma cells and to It is thought to induce an immune response against tumors containing it.

[0025] CLT and CLT antigens can be easily obtained from known tumor genome sequences found in cancer genome atlases. It is not a canonical sequence that can be driven. This CLT is driven by a transcriptional regulatory sequence of ERV origin. CLT is a transcript produced by complex transcription and splicing events. Because it is expressed in the protein, and the CLT antigen polypeptide sequence is not the sequence of a normal human protein. Therefore, they can trigger a strong and specific immune response (which has not actually been proven). As shown (see Examples 3-5), therefore, it is favored for therapeutic use in the context of cancer immunotherapy. It is considered appropriate.

[0026] It was previously known that it exists in humans, produces protein products, and stimulates immune responses. The CLT antigen, which had not been previously identified, was found in a highly expressed transcript characteristic of tumor cells. It can be used in several forms. For example, CLT antigen polypeptide can be used against tumor cells. It can also be delivered directly to the target as a vaccine that induces a therapeutic or prophylactic immune response. Furthermore, nucleic acids that can be codon-optimized to enhance the expression of the encoded CLT antigen. It can be administered directly, or the encoded protein product can be used as a therapeutic agent against tumor cells. To produce in vivo vaccines that induce a therapeutic or prophylactic immune response in the target population. It can be inserted into a delivery vector.

[0027] Each antigen exists in the form of a polypeptide and / or nucleic acid encoding a polypeptide. Using the antigen pool of the present invention, which contains the above different antigens, patient-derived antigen-presenting cells (APCs) It can then be loaded, and then used for therapeutic or prophylactic immunity against tumor cells. It can be injected into the target as a vaccine to induce a response. Furthermore, each antigen is poly Two or more different antigens existing in the form of nucleic acids encoding peptides and / or polypeptides The antigen pool of the present invention is used for ex vivo stimulation of target T cells to treat cancer. It is possible to produce stimulated T-cell preparations that can be administered to the target as therapy. These and other uses are described in more detail below.

[0028] Therefore, the present invention relates in particular to each antigen being a polypeptide and / or the polypeptide It exists in the form of nucleic acids that encode a drug, and different antigens exist as separate polypeptides, nucleic acids, and fusion molecules. Present in the antigen pool as a composite protein and / or nucleic acid encoding the fusion protein. An antigen pool comprising two or more different antigens, wherein the two or more different antigens (a) Immunogenicity of SEQ ID NO: 1 or its variants or SEQ ID NO: 1 or its variants piece; (b) Immunogenicity of SEQ ID NO: 2 or its variants or SEQ ID NO: 2 or its variants piece; (c) Immunogenicity of SEQ ID NO: 3 or its variants or SEQ ID NO: 3 or its variants piece; (d) Immunogenicity of SEQ ID NO: 4 or its variants or SEQ ID NO: 4 or its variants piece; (e) Immunogenicity of SEQ ID NO: 5 or its variants or SEQ ID NO: 5 or its variants piece; (f) Immunogenicity of SEQ ID NO: 6 or its variants or SEQ ID NO: 6 or its variants piece; (g) Immunogenicity of SEQ ID NO: 7 or its variants or SEQ ID NO: 7 or its variants Fragments; and (h) Immunogenicity of SEQ ID NO: 8 or its variants or SEQ ID NO: 8 or its variants piece An antigen pool having polypeptide sequences selected from: (hereinafter referred to as "this") It provides what is called the "antigen pool of inventions."

[0029] The antigen pool of the present invention and related embodiments of the present invention are described in more detail below. It is broadly useful in the immunotherapy and prevention of cancer, particularly in the immunotherapy and prevention of melanoma. It is expected that this will be the case. [Brief explanation of the drawing]

[0030] (Drawing description) Figures 1-38 each show the extracted MS / MS spectrum of peptides obtained from patient tumor samples (divided into sections). (along with the assigned fragment ions) and the linear peptide sequence mapped to the fragment ions The panel below or a similar table showing a spectral rendering indicating the position It indicates one of the following. [Figure 1] Figure 1. Spectrum of the peptide of Sequence ID No. 9 obtained from a tumor sample of patient Mel-3. [Figure 2] Figure 2. Spectrum of the peptide of Sequence ID No. 10 obtained from a tumor sample of patient Mel-3. [Figure 3] Figure 3. Spectrum of the peptide of SEQ ID NO: 10 obtained from a tumor sample of patient Mel-3. [Figure 4] Figure 4. Spectrum of the peptide of Sequence ID No. 10 obtained from a tumor sample of patient 2MT3. [Figure 5] Figure 5. Spectrum of the peptide of Sequence ID No. 11 obtained from a tumor sample of patient Mel-5. [Figure 6] Figure 6. Spectrum of the peptide of Sequence ID No. 11 obtained from a tumor sample of patient Mel-16. [Figure 7] Figure 7. Spectrum of the peptide of Sequence ID No. 11 obtained from a tumor sample of patient Mel-16. [Figure 8] Figure 8. Spectrum of the peptide of Sequence ID No. 11 obtained from a tumor sample of patient 2MT3. [Figure 9] Figure 9. Spectrum of the peptide of Sequence ID No. 11 obtained from a tumor sample of patient 2MT10. [Figure 10] Figure 10. Spectrum of the peptide of SEQ ID NO: 12 obtained from a tumor sample of patient Mel-5. [Figure 11] Figure 11. Spectrum of peptide SEQ ID NO: 18 obtained from a tumor sample of patient Mel-26. [Figure 12] Figure 12. Spectrum of peptide SEQ ID NO: 19 obtained from a tumor sample of patient Mel-20. [Figure 13] Figure 13. Spectrum of peptide SEQ ID NO: 19 obtained from a tumor sample of patient Mel-20. [Figure 14] Figure 14. Spectrum of peptide SEQ ID NO: 19 obtained from a tumor sample of patient 2MT4. [Figure 15] Figure 15. Spectrum of peptide SEQ ID NO: 31 obtained from a tumor sample of patient Mel-35. [Figure 16] Figure 16. Spectrum of peptide SEQ ID NO: 31 obtained from a tumor sample of patient 2MT3. [Figure 17] Figure 17. Spectrum of peptide SEQ ID NO: 32 obtained from a tumor sample of patient 1MT1. [Figure 18] Figure 18. Spectrum of peptide SEQ ID NO: 36 obtained from a tumor sample of patient Mel-3. [Figure 19] Figure 19. Spectrum of peptide SEQ ID NO: 36 obtained from a tumor sample of patient Mel-3. [Figure 20] Figure 20. Spectrum of peptide SEQ ID NO: 36 obtained from a tumor sample of patient 2MT3. [Figure 21] Figure 21. Spectrum of peptide SEQ ID NO: 36 obtained from a tumor sample of patient 2MT1. [Figure 22] Figure 22. Spectrum of peptide SEQ ID NO: 37 obtained from a tumor sample of patient Mel-40. [Figure 23] Figure 23. Spectrum of peptide SEQ ID NO: 37 obtained from a tumor sample of patient Mel-41. [Figure 24] Figure 24. Spectrum of peptide SEQ ID NO: 37 obtained from a tumor sample of patient 2MT3. [Figure 25] Figure 25. Spectrum of peptide SEQ ID NO: 38 obtained from a tumor sample of patient Mel-27. [Figure 26] Figure 26. Spectrum of peptide SEQ ID NO: 38 obtained from a tumor sample of patient Mel-39. [Figure 27] Figure 27. Spectrum of peptide SEQ ID NO: 39 obtained from a tumor sample of patient 2MT12. [Figure 28] Figure 28. Spectrum of peptide SEQ ID NO: 45 obtained from a tumor sample of patient Mel-29. [Figure 29] Figure 29. Spectrum of peptide SEQ ID NO: 48 obtained from a tumor sample of patient Mel-41. [Figure 30] Figure 30. Spectrum of peptide SEQ ID NO: 49 obtained from a tumor sample of patient Mel-41. [Figure 31]Figure 31. Spectrum of peptide SEQ ID NO: 50 obtained from a tumor sample of patient Mel-41. [Figure 32] Figure 32. Spectrum of peptide SEQ ID NO: 51 obtained from a tumor sample of patient Mel-41. [Figure 33] Figure 33. Spectrum of peptide SEQ ID NO: 52 obtained from a tumor sample of patient Mel-21. [Figure 34] Figure 34. Spectrum of peptide SEQ ID NO: 52 obtained from a tumor sample of patient 2MT3. [Figure 35] Figure 35. Spectrum of peptide SEQ ID NO: 53 obtained from a tumor sample of patient Mel-27. [Figure 36] Figure 36. Spectrum of peptide SEQ ID NO: 54 obtained from a tumor sample of patient Mel-27. [Figure 37] Figure 37. Spectra of peptide SEQ ID NO: 54 obtained from a tumor sample of patient 2MT4. Figures 38-53 show the alignment of the native MS / MS spectrum of the peptide obtained from the patient tumor sample (top) with the native spectrum of the synthetic peptide corresponding to the same sequence (bottom). [Figure 38] Figure 38 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptide-metabolism analysis of patient 2MT3, which is attributed to Sequence ID No. 10. [Figure 39] Figure 39 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptide-medication analysis of patient 2MT3, which is attributed to Sequence ID No. 11. [Figure 40] Figure 40 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptide-metabolism analysis of patient 2MT4, which is attributed to Sequence ID No. 19. [Figure 41] Figure 41 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptide-metabolism analysis of patient 2MT3, which is attributed to Sequence ID No. 31. [Figure 42] Figure 42 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptidemometry analysis of patient 1MT1, which is attributed to Sequence ID No. 32. [Figure 43]Figure 43 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptidemometry analysis of patient 2MT3, which is attributed to Sequence ID No. 36. [Figure 44] Figure 44 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptidemometry analysis of patient 2MT3, which is attributed to Sequence ID No. 37. [Figure 45] Figure 45 shows the mass spectrometry spectrum of a peptide fragment obtained from immunopeptidemometry analysis of patient 2MT12, which is associated with sequence number 39. [Figure 46] Figure 46 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptide-metabolism analysis of patient Mel-29, which is associated with Sequence ID No. 45. [Figure 47] Figure 47 shows the mass spectrometry spectrum of a peptide fragment obtained from immunopeptide-metabolism analysis of patient Mel-41, which is associated with Sequence ID No. 48. [Figure 48] Figure 48 shows the mass spectrometry spectrum of a peptide fragment obtained from immunopeptide-metabolism analysis of patient Mel-41, which is associated with Sequence ID No. 49. [Figure 49] Figure 49 shows the mass spectrometry spectrum of a peptide fragment obtained from immunopeptide-metabolism analysis of patient Mel-41, which is associated with Sequence ID No. 50. [Figure 50] Figure 50 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptidemometry analysis of patient Mel-41, which is associated with Sequence ID No. 51. [Figure 51] Figure 51 shows the mass spectrometry spectrum of a peptide fragment obtained from immunopeptide-metabolism analysis of patient Mel-21, which is associated with Sequence ID No. 52. [Figure 52] Figure 52 shows the mass spectrometry spectrum of the peptide fragment obtained from immunopeptidemometry analysis of patient Mel-27, which is associated with Sequence ID No. 53. [Figure 53] Figure 53 shows the mass spectrometry spectrum of a peptide fragment obtained from immunopeptide-metabolism analysis of patient Mel-27, which is associated with Sequence ID No. 54. [Figure 54]Panels A-C of Figure 54 show the amplification of tumor antigen-specific T cells from patient PBMC cultures in response to culture with specific tumor antigen-derived peptides. [Figure 55] Panels A-D of Figure 55 provide a summary of CLT antigen-derived peptides (SEQ ID NOs: 11, 13-15, 19-29, 33-35, 40-42) that were able to amplify specific TCR-possessing T cells derived from PBMCs of melanoma patients. [Figure 56] Figure 56 shows the CD8 T cell response from a normal blood donor to the HLA-A*02:01-restricted peptide (SEQ ID NO: 16) derived from CLT antigen 1. [Figure 57] Figure 57 shows the CD8 T cell response from a normal blood donor to the HLA-A*02:01-restricted peptide (SEQ ID NO: 30) derived from CLT antigen 2. [Figure 58] Figure 58 shows the CD8 T cell response from a normal blood donor to the HLA-A*02:01-restricted peptide (SEQ ID NO: 43) derived from CLT antigen 4. [Figure 59] Figure 59 shows the CD8 T cell response from a normal blood donor to the HLA-A*03:01-restricted peptide (SEQ ID NO: 47) derived from CLT antigen 5. [Figure 60] Figure 60 shows the CD8 T cell response from a normal blood donor to the HLA-B*07:02-restricted peptide (SEQ ID NO: 50) derived from CLT antigen 6. [Figure 61] Figure 61 shows the CD8 T cell response from a normal blood donor to the HLA-A*03:01-restricted peptide (SEQ ID NO: 52) derived from CLT antigen 7. [Figure 62] Figure 62 shows the CD8 T cell response from a normal blood donor to the HLA-A*02:01-restricted peptide (SEQ ID NO: 55) derived from CLT antigen 8. [Figure 63]Panels A-D of Figure 63 show the responsiveness of memory CD45RO-positive CD8 T cells to HLA-B*07:02-restricted peptides (SEQ ID NOs. 17 and 44) ​​derived from CLT antigen 1 and CLT antigen 4, respectively, compared to naive CD45RO-negative CD8 T cells from the same donor. [Figure 64] Figure 64 shows, in an enlarged view, pentamer-sorted CD8 T cells killing C1RB7 target cells pulsed with a peptide derived from CLT antigen 4 (SEQ ID NO: 44). [Figure 65] Figure 65 shows, magnified and sorted with pentamers, that kill CaSki cells transfected with an open reading frame of CLT antigen 008 (SEQ ID NO: 8). [Figure 66] Panels A-G of Figure 66 show the results of qRT-PCR assays to confirm the transcription of CLTs encoding CLT antigen 1 (SEQ ID NO: 56), CLTs encoding CLT antigen 2 (SEQ ID NO: 57), CLTs encoding CLT antigens 3 and 4 (SEQ ID NO: 58), CLTs encoding CLT antigen 5 (SEQ ID NO: 59), CLTs encoding CLT antigen 6 (SEQ ID NO: 60), CLTs encoding CLT antigen 7 (SEQ ID NO: 61), and CLTs encoding CLT antigen 8 (SEQ ID NO: 62) in melanoma cancer cell lines or primary tissue samples. [Figure 67] Figure 67 schematically shows the structure of CLT antigen fusion protein 1 (SEQ ID NO: 76), the linker sequence between CLT antigens, and the possible HLA binding of linker-derived epitopes. FP = fusion protein. [Figure 68] Figure 68 schematically shows the structure of CLT antigen fusion protein 2 (SEQ ID NO: 77), the linker sequence between CLT antigens, and the possible HLA binding of linker-derived epitopes. FP = fusion protein. [Figure 69] Figure 69 schematically shows the structure of CLT antigen fusion protein 3 (SEQ ID NO: 78), the linker sequence between CLT antigens, and the possible HLA binding of linker-derived epitopes. FP = fusion protein. [Figure 70]Figure 70 schematically shows the structure of CLT antigen fusion protein 4 (SEQ ID NO: 79), the linker sequence between CLT antigens, and the possible HLA binding of linker-derived epitopes. FP = fusion protein.

[0031] (Explanation of the array) Sequence ID 1 is the polypeptide sequence of CLT antigen 1. Sequence ID 2 is the polypeptide sequence of CLT antigen 2. Sequence ID 3 is the polypeptide sequence of CLT antigen 3. Sequence ID 4 is the polypeptide sequence of CLT antigen 4. Sequence ID 5 is the polypeptide sequence of CLT antigen 5.

[0032] Sequence ID 6 is the polypeptide sequence of CLT antigen 6. Sequence ID 7 is the polypeptide sequence of CLT antigen 7. Sequence ID 8 is the polypeptide sequence of CLT antigen 8. Sequence IDs 9-17 are peptide sequences derived from CLT antigen 1. Sequence IDs 18-30 are peptide sequences derived from CLT antigen 2.

[0033] Sequence IDs 31-35 are peptide sequences derived from CLT antigen 3. Sequence IDs 36-44 are peptide sequences derived from CLT antigen 4. Sequence IDs 45-47 are peptide sequences derived from CLT antigen 5. Sequence IDs 48-51 are peptide sequences derived from CLT antigen 6. Sequence ID 52 is a peptide sequence derived from CLT antigen 7.

[0034] Sequence IDs 53-55 are peptide sequences derived from CLT antigen 8. Sequence ID 56 is the cDNA sequence of CLT encoding CLT antigen 1. Sequence ID 57 is the cDNA sequence of CLT encoding CLT antigen 2. Sequence ID 58 is the cDNA sequence of CLT encoding CLT antigens 3 and 4. Sequence ID 59 is the cDNA sequence of CLT encoding CLT antigen 5.

[0035] Sequence ID 60 is the cDNA sequence of CLT encoding CLT antigen 6. Sequence ID 61 is the cDNA sequence of CLT encoding CLT antigen 7. Sequence ID 62 is the cDNA sequence of CLT encoding CLT antigen 8. Sequence ID 63 is a cDNA sequence encoding CLT antigen 1. Sequence ID 64 is a cDNA sequence that encodes CLT antigen 2.

[0036] Sequence ID 65 is a cDNA sequence that encodes CLT antigen 3. Sequence ID 66 is a cDNA sequence that encodes CLT antigen 4. Sequence ID 67 is a cDNA sequence that encodes CLT antigen 5. Sequence ID 68 is a cDNA sequence that encodes CLT antigen 6. Sequence ID 69 is a cDNA sequence that encodes CLT antigen 7.

[0037] Sequence ID 70 is a cDNA sequence that encodes CLT antigen 8. Sequence IDs 71-75 are linker sequences used to construct the CLT antigen fusion protein. That is the case. Sequence ID 76 is the polypeptide sequence of CLT antigen fusion protein 1. Sequence ID 77 is the polypeptide sequence of CLT antigen fusion protein 2. Sequence ID 78 is the polypeptide sequence of CLT antigen fusion protein 3. Sequence ID 79 is the polypeptide sequence of CLT antigen fusion protein 4.

[0038] Sequence ID 80 is a cDNA sequence encoding CLT antigen fusion protein 1. Sequence ID 81 is a cDNA sequence encoding CLT antigen fusion protein 2. Sequence ID 82 is a cDNA sequence encoding CLT antigen fusion protein 3. Sequence ID 83 is a cDNA sequence encoding CLT antigen fusion protein 4. Sequence ID 84 is the linker sequence used to construct the CLT antigen fusion protein. ru. Sequence numbers 85-87 are the TCR VB CDR3 AA sequences shown in Figure 54. [Modes for carrying out the invention]

[0039] (Detailed description of the invention) (Antigen pool) An "antigen pool" is two or more antigens in the form of polypeptides and / or nucleic acids (for example) It is a pool containing 2, 3, 4, 5, 6, 7, or 8 antigens. A pool is a pool of separate polypeptides or nucleic acids, or a pool of separate polypeptides and nucleic acids. It could be.

[0040] Polypeptides and / or nucleic acids in the antigen pool are, respectively, fusion proteins and / or fusion proteins. It can exist as a nucleic acid portion that codes for a fusion protein. (Term: "fusion protein") Through protein synthesis, at least two peptide bonds connect together This refers to any protein containing polypeptides. Fusion proteins produce a single protein. Separate polypeptides connected to be transcribed and translated as a single unit It can be generated by the connection of two or more genes that code for it.

[0041] Therefore, the present invention relates to each antigen encoding a polypeptide and / or the polypeptide. They exist in the form of nucleic acids, and different antigens exist as separate polypeptides or nucleic acids. / or as a fusion protein or as part of the nucleic acid encoding the fusion protein in an antigen pool. An antigen pool containing two or more different antigens present therein, where the two or more different antigens (a) Immunogenicity of SEQ ID NO: 1 or its variants or SEQ ID NO: 1 or its variants piece; (b) Immunogenicity of SEQ ID NO: 2 or its variants or SEQ ID NO: 2 or its variants piece; (c) Immunogenicity of SEQ ID NO: 3 or its variants or SEQ ID NO: 3 or its variants piece; (d) Immunogenicity of SEQ ID NO: 4 or its variants or SEQ ID NO: 4 or its variants piece; (e) Immunogenicity of SEQ ID NO: 5 or its variants or SEQ ID NO: 5 or its variants piece; (f) Immunogenicity of SEQ ID NO: 6 or its variants or SEQ ID NO: 6 or its variants piece; (g) Immunogenicity of SEQ ID NO: 7 or its variants or SEQ ID NO: 7 or its variants Fragments; and (h) Immunogenicity of SEQ ID NO: 8 or its variants or SEQ ID NO: 8 or its variants piece Provides an antigen pool having polypeptide sequences selected from:

[0042] The antigen pool of the present invention comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more different antigens. The antigen pool of the present invention may contain the following antigens. Preferably, the antigen pool of the present invention contains two or more antigens. The antigen pool may contain two, three, four, five, six, seven, or eight different antigens. In a preferred embodiment of the invention, the antigen pool comprises six different antigens. In a preferred embodiment, the antigen pool comprises eight different antigens.

[0043] "Two different antigens" means, for example, that antigen (a) is SEQ ID NO: 1 or a variant thereof or A polypeptide having the sequence of an immunogenic fragment of SEQ ID NO: 1 or its variant, or The polypeptide is represented by a polynucleotide, and antigen (b) is also represented by sequence number 2. Alternatively, the sequence of its variant or SEQ ID NO: 2 or an immunogenic fragment of its variant may be present. A polypeptide, or a polynucleotide encoding the polypeptide. It will be understood that this refers to a combination of antigens (a) and (b). Each antigen has multiple components. The components (for example, multiple polypeptides or polynucleotides or combinations thereof) This can be expressed as follows. The same applies to any combination of two or more antigens.

[0044] In an embodiment of the present invention, the antigen pool comprises two different antigens.

[0045] In embodiments of the present invention, the antigen pool comprises three different antigens.

[0046] In embodiments of the present invention, the antigen pool comprises four different antigens.

[0047] In embodiments of the present invention, the antigen pool comprises five different antigens.

[0048] In embodiments of the present invention, the antigen pool comprises six different antigens.

[0049] In embodiments of the present invention, the antigen pool comprises seven different antigens.

[0050] In embodiments of the present invention, the antigen pool comprises eight different antigens.

[0051] In one embodiment, the antigen pool is a polypeptide sequence: (a) Sequence ID No. 1 or its variant polypeptides having immunogenic fragments of the riant or SEQ ID NO: 1 or its variants ( Encodes a fusion protein (or any fusion protein form) or polypeptide (or any fusion protein form). It contains polynucleotides.

[0052] In one embodiment, the antigen pool is a polypeptide sequence: (b) Sequence ID No. 2 or its variant polypeptides having immunogenic fragments of riant or SEQ ID NO: 2 or its variants ( Encodes a fusion protein (or any fusion protein form) or polypeptide (or any fusion protein form). It contains polynucleotides.

[0053] In one embodiment, the antigen pool is a polypeptide sequence: (c) Sequence ID No. 3 or its variant Polypeptides having immunogenic fragments of riant or SEQ ID NO: 3 or its variants ( Encodes a fusion protein (or any fusion protein form) or polypeptide (or any fusion protein form). It contains polynucleotides.

[0054] In one embodiment, the antigen pool is a polypeptide sequence: (d) Sequence ID No. 4 or its variant polypeptides having immunogenic fragments of riant or SEQ ID NO: 4 or its variants ( Encodes a fusion protein (or any fusion protein form) or polypeptide (or any fusion protein form). It contains polynucleotides.

[0055] In one embodiment, the antigen pool is a polypeptide sequence: (e) Sequence ID No. 5 or its variant Polypeptides having immunogenic fragments of the riant or SEQ ID NO: 5 or its variants ( Encodes a fusion protein (or any fusion protein form) or polypeptide (or any fusion protein form). It contains polynucleotides.

[0056] In one embodiment, the antigen pool is a polypeptide sequence: (f) Sequence ID No. 6 or its variant polypeptides having immunogenic fragments of riant or SEQ ID NO: 6 or its variants ( Encodes a fusion protein (or any fusion protein form) or polypeptide (or any fusion protein form). It contains polynucleotides.

[0057] In one embodiment, the antigen pool is a polypeptide sequence: (g) Sequence ID No. 7 or its variant Polypeptides having immunogenic fragments of the riant or SEQ ID NO: 7 or its variants ( Encodes a fusion protein (or any fusion protein form) or polypeptide (or any fusion protein form). It contains polynucleotides.

[0058] In one embodiment, the antigen pool is a polypeptide sequence: (h) SEQ ID NO: 8 or its variant polypeptides having immunogenic fragments of riant or SEQ ID NO: 8 or its variants ( Encodes a fusion protein (or any fusion protein form) or polypeptide (or any fusion protein form). It contains polynucleotides.

[0059] In embodiments of the present invention, the antigen pool comprises six different antigens, Preferably, it has polypeptide sequences of (a), (b), (d), (f), (g), and (h). The antigen pool of the present invention comprises six different antigens, where the antigens are (a), (b), and (d) It has polypeptide sequences (f), (g)~(h).

[0060] In embodiments of the present invention, the antigen pool comprises eight different antigens, Preferably, the polypeptide sequence has (a) to (h). Therefore, the antigen pool of the present invention is , comprising eight different antigens, wherein the antigen has the polypeptide sequences (a) to (h).

[0061] In embodiments of the present invention, each of the different antigens is in the form of a separate polypeptide (that is, It exists (but not as part of the fusion protein).

[0062] In embodiments of the present invention, each of the different antigens exists as a portion of the fusion protein. ru.

[0063] In embodiments of the present invention, each of the different antigens is in the form of a separate nucleic acid (i.e., fused It exists (not as a polynucleotide that codes for a composite protein).

[0064] In embodiments of the present invention, each of the different antigens is a nucleic acid encoding a fusion protein It exists as a part.

[0065] (polypeptide) The terms "protein," "polypeptide," and "peptide" are used herein. They are interchangeable and can be used with any peptide bond, regardless of length, cotranslation, or posttranslational modification. This refers to the anoacid chain.

[0066] The term "amino acid," naturally occurring amino acids, and naturally occurring amino acids This refers to either an amino acid analog or an amino acid mimetic that functions in a similar manner to the amino acid analog. The naturally occurring amino acids include 20 L-amino acids encoded by the genetic code, as well as... Amino acids modified in this way, for example, hydroxyproline, γ-carboxyglutamic acid, and It is O-phosphoserine. The term "amino acid analog" refers to naturally occurring amino acids and They share the same basic chemical structure, namely, hydrogen, carboxyl group, amino group, and R group. It has an α-carbon, but compared to natural amino acids, it has a modified R group or modified peptide This refers to compounds that have a cytoskeleton. Examples include homoserine, norleucine, and methionine. Examples include phosphates, methionine methylsulfonium, and norleucine. These imitation compounds have a structure different from the general chemical structure of amino acids, but they are naturally occurring amino acids. This refers to chemical compounds that function in a manner similar to acids. Preferably, amino acids are naturally occurring. amino acids or amino acid analogs, in particular naturally occurring amino acids, especially those related to the genetic code It is one of the 20 L-amino acids encoded by [the enzyme].

[0067] In this specification, amino acids are referred to by their commonly known three-letter symbols, or by I Recommended by the UPAC-IUB Biochemical Nomenclature Commission It may be represented by either letters or symbols. Nucleotides are similar in that way. It may be represented by a commonly accepted single-character code.

[0068] Generally, variants of antigenic polypeptide sequences present in the antigen pool of the present invention are It includes sequences that have a high degree of sequence identity with respect to it. For example, the variant has a sequence that has a high degree of sequence identity with respect to its entire length. Therefore, the associated reference sequence preferably has at least about 80% identity, more preferably, At least about 85% identity, and most preferably at least about 90% identity (for example, It has at least about 95%, at least about 98%, or at least about 99%.

[0069] Preferably, the variant is an immunogenic variant. The variant is a polypeptide In vitro restimulation assays using PBMCs or whole blood as antigens (e.g., from a few hours to up to one year) For example, in restimulation (up to 6 months, 1 day to 1 month, or 1 to 2 weeks), the reference sequence (that is, The variant is at least 20% of the activity of the sequence in which it is a variant, preferably a small amount. It elicits a response of at least 50%, and especially at least 75% (for example, at least 90%). It is thought to be an immunogenic variant, and in this assay, for example, lymphocyte proliferation (e.g.) For example, cell activation mediated by T cell proliferation, and (measured by ELISA, etc.) in the culture supernatant. ) Production of cytokines (e.g., IFN-γ), or intracellular and extracellular staining (e.g., CD3, CD4, Antibodies specific to immunomarkers such as CD8, IL2, TNF-α, IFNg, type 1 IFN, CD40L, and CD69 are used. The characteristics of the T cell response were analyzed using (a specific method), followed by analysis using a flow cytometer. It can be done.

[0070] The variant may be, for example, a conservatively modified variant. A "decorated variant" is one in which the modification involves the substitution of an amino acid with a functionally similar amino acid. This results in residue substitutions / deletions / additions that do not substantially affect the biological function of the variant. Typically, such biological functions of variants include melanoma, for example, skin darkening. This will induce an immune response against the cancer antigens of chromoma.

[0071] Conservative substitution tables that provide functionally similar amino acids are well known in the art. Ants may contain homologs of polypeptides found in other species.

[0072] The antigens present in the antigen pool of the present invention are subject to several substitutions, for example, when compared to a reference sequence. When this occurs, the polypeptide may contain a variant sequence having a conservative substitution (for example) (1 to 25, for example, 1 to 10, especially 1 to 5, and in particular, one amino acid residue may be modified). The number of substitutions, for example, the number of conservative substitutions, is up to 20% of the number of residues in the reference sequence, for example, up to It could be 10%, for example, up to 5%, for example, up to 1%. In general, conservative substitutions are as follows: It is included in one of the amino acid classifications designated as such, but in some cases, it can affect the immune response to the antigen. In some cases, other substitutions may be possible without substantially affecting the virulence properties. The following eight examples: Each of these groups contains amino acids that are typically conserved substitutions for each other: 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's work, Proteins 1984).

[0073] Preferably, such substitution does not alter the immunological structure of the epitope (for example, the substitution The exchange does not occur within the epitope region mapped to the primary sequence, therefore, the antigen It does not significantly affect immunogenicity.

[0074] Polypeptide variants are those in which additional amino acids have been inserted compared to the reference sequence. This also includes, for example, such insertions at 1 to 10 positions (e.g., 1 to 5 positions, preferably, It can occur in one or two positions, in particular one position, and for example, 50 at each position This includes the addition of the following amino acids (e.g., 20 or fewer, especially 10 or fewer, and more particularly 5 or fewer). This is possible. Preferably, such insertion does not occur in the epitope region, so the antigen It does not significantly affect immunogenicity. One example of an insert is the expression of the target antigen. and / or short stretches of histidine residues (e.g., 2-6 residues) to aid in purification It can be listed.

[0075] Polypeptide variants include those in which amino acids are deleted compared to the reference sequence. For example, such deletions may occur at 1 to 10 locations (e.g., 1 to 5, preferably 1 or 2, in particular) It can occur at one position, and for example, at each position there may be 50 or fewer amino acids (for example) It may contain 20 or fewer deletions, especially 10 or fewer, and particularly 5 or fewer. Such deletions do not occur in epitope regions, and therefore are not prominent in the immunogenic properties of the antigen. It will not have any effect.

[0076] Those skilled in the art will know that certain protein variants can be substituted, deleted, and added (or any of these). It is likely that you are aware that this may include combinations of the above. For example, substitution / deletion / addition may be the desired It enhances (or has a neutral effect on) binding to patient HLA molecules and increases immunogenicity (or immunogenicity). (This may preserve the disease-causing properties.)

[0077] The immunogenic fragments of antigenic polypeptide sequences present in the antigen pool according to the present invention are CLT Depending on the length of the antigen, typically at least nine consecutive amino acids derived from the full-length polypeptide sequence. For example, at least 9 or 10), for example, at least 12 consecutive amino acids (for example, at least (at least 15 or 20 consecutive amino acids), in particular, at least 50 consecutive amino acids For example, at least 100 consecutive amino acids (for example, at least 200 consecutive amino acids) Includes. Preferably, the immunogenic fragment is at least 10% of the length of the full-length polypeptide sequence, e.g. For example, at least 20%, for instance, at least 50%, for instance, at least 70%, or less It is also 80%.

[0078] Immunogenic fragments typically contain at least one epitope. Epitopes are found in B cells and The T cell epitope is preferably a CD4+ or CD8+ T cell epitope. It contains at least one T cell epitope.

[0079] T cell epitopes, when bound to HLA molecules, affect T cells (e.g., CD4+ or CD8+ T cells). Therefore, it is a short, continuous stretch of recognized amino acids. Identification of T cell epitopes is This can be achieved by epitope mapping experiments, which are well known to those skilled in the art (for example) Paul's work, Fundamental Immunology, 3rd edition, 243-247 (1993); Beiβbarth et al.'s work, See Bioinformatics, 21(Suppl. 1):i29-i37, 2005.

[0080] As a result of the decisive involvement of T cell responses in cancer, at least one T cell epitope The full-length polypeptide fragments of SEQ ID NOs. 1-8 containing this substance may be immunogenic, and It is readily apparent that it may contribute to immunoprotection.

[0081] In diverse non-inbred populations such as humans, different HLA types are associated with specific epitopes. It will be understood that this means it may not be recognized by everyone in the group. As a result, in order to maximize the level of recognition and scale of the immune response to polypeptides, The epidemiogenic fragment consists of multiple epitopes derived from the full-length sequence (preferably all epitopes within the CLT antigen). It is generally desirable that it contains )

[0082] Certain fragments of the polypeptide sequence numbers 1-8 that may be useful contain at least one CD8+ T Cellular epitopes, preferably at least two CD8+ T cell epitopes, in particular all C Those containing D8+ T cell epitopes, particularly those associated with multiple HLA alleles, for example, (Includes those associated with two, three, four, five, or more alleles). May be useful. The specific fragments of the polypeptide sequence numbers 1-8 contain at least one CD4+ T cell epitope. Preferably, at least two CD4+ T cell epitopes, in particular all CD4+ T cell epitopes. Those containing tope (especially those associated with multiple HLA alleles, e.g., two, three, four) It includes (or is associated with five or more alleles). However, vaccine design Experts have combined exogenous CD4+ T cell epitopes with CD8+ T cell epitopes, and CD8+ This will allow us to achieve the desired response to T cell epitopes.

[0083] When individual fragments of a full-length polypeptide are used, such fragments make up the polypeptide. In vitro restimulation assays using PBMCs or whole blood as antigens (e.g., from a few hours to up to one year) For example, in restimulation (up to 6 months, 1 day to 1 month, or 1 to 2 weeks), the reference sequence (that is, The fragment (the sequence) has at least 20%, preferably at least 50% of its activity. In particular, if it elicits a response of at least 75% (for example, at least 90%), the immune response It is thought to be virulent, and in this assay, for example, lymphocyte proliferation (e.g., T cells) Cell activation via proliferation, cytokines (measured by ELISA, etc.) in the culture supernatant Production of ions (e.g., IFN-γ), or intracellular and extracellular staining (e.g., CD3, CD4, CD8, IL2, T T (using antibodies specific to immune markers such as NF-α, IFN-γ, type 1 IFN, CD40L, and CD69) The characteristics of the cellular response are analyzed, followed by analysis using a flow cytometer.

[0084] In some cases, multiple fragments of the full-length polypeptide (which may or may not overlap) (This may or may not apply to the entire length of the sequence) and the same as the entire length of the sequence itself It is also possible to obtain biological responses such as the above. For example, at least two (e.g., three, four) Immunogenic fragments (or 5) combined for in vitro restimulation assays of PBMCs or whole blood (For example, in T cell proliferation and / or IFN-γ production assays) at least 50% of the reference sequence , preferably provides at least 75%, especially at least 90% activity.

[0085] Examples of immunogenic fragments of the antigenic polypeptides of SEQ ID NOs: 1-8, and thus examples of the component peptides of the fusion proteins of the present invention, include polypeptides comprising or consisting of the sequences of SEQ ID NOs: 9-55. The sequences of SEQ ID NOs: 9-12, 18-19, 30, 31-32, and 37-39, 45, 48-54 have been confirmed from immunopeptidome analysis to bind to HLA class I molecules (see Example 2). The sequences of SEQ ID NOs: 13-17, 20-29, 33-35, 40-44 are predicted by NetMHC software to bind to HLA class I molecules and were used in immunological verification assays (see Examples 3, 4 and 5).

[0086] The antigenic polypeptide (a) present in the antigen pool of the present invention may comprise or consist of SEQ ID NO: 1 or a variant thereof or an immunogenic fragment of SEQ ID NO: 1 or a variant thereof. Exemplary fragments include or consist of any one of SEQ ID NOs: 9-12. Further exemplary fragments include two, three, or four of SEQ ID NOs: 9-12. Further exemplary fragments include or consist of any one of SEQ ID NOs: 13-17. Further exemplary fragments include all of SEQ ID NOs: 9-17 (where possible sequences are considered such that any overlapping sequences do not occur more than once).

[0087] The antigenic polypeptide (b) present in the antigen pool of the present invention may comprise or consist of SEQ ID NO: 2 or a variant thereof or an immunogenic fragment of SEQ ID NO: 2 or a variant thereof. or these ​​​​​​​​​​​​​​​​​​​​​​​​It may consist of. Exemplary fragments include or are derived from SEQ ID NO: 18 or SEQ ID NO: 19. It consists of. Further exemplary fragments include SEQ ID NO: 18 and SEQ ID NO: 19. Further exemplary fragments include or consist of any one of SEQ ID NOs: 20 to 30. Further exemplary fragments include all of SEQ ID NOs: 18 to 30 (possible sequences are considered so that any overlapping sequences do not need to exist multiple times).

[0088] The antigenic polypeptide (c) present in the antigen pool of the present invention may include or consist of SEQ ID NO: 3 or its variant or an immunogenic fragment of SEQ ID NO: 3 or its variant. Exemplary fragments include or consist of SEQ ID NO: 31. Further exemplary fragments include SEQ ID NO: 31. Further exemplary fragments include or consist of any one of SEQ ID NOs: 32 to 35. Further exemplary fragments include SEQ ID NO: 31 and SEQ ID NO: 32. Further exemplary fragments include all of SEQ ID NOs: 31 to 35 (possible sequences are considered so that any overlapping sequences do not need to exist multiple times).

[0089] The antigenic polypeptide (d) present in the antigen pool of the present invention may include or consist of SEQ ID NO: 4 or its variant or an immunogenic fragment of SEQ ID NO: 4 or its variant. Exemplary fragments include or consist of SEQ ID NO: 36. Further exemplary fragments include or consist of SEQ ID NO: 37 or SEQ ID NO: 38. Further exemplary fragments include or consist of SEQ ID NO: 39. Further exemplary fragments include or consist of any one of SEQ ID NOs: 40 to 44. Further exemplary fragments include SEQ ID NO: 36 and includes either SEQ ID NO: 37 or SEQ ID NO: 38. Further exemplary fragments include SEQ ID NO: 39 and includes either SEQ ID NO: 37 or SEQ ID NO: 38. Further exemplary fragments include SEQ ID NO: 36 Includes all ~44 (considers possible arrays so that no duplicate arrays exist multiple times). (It will be done).

[0090] The antigenic polypeptide (e) present in the antigen pool of the present invention is SEQ ID NO: 5 or its variant This may include immunogenic fragments of the riant or SEQ ID NO: 5 or its variants, or these It may consist of. An exemplary fragment may include or consist of any one of sequence numbers 45-47. Yes.

[0091] The antigenic polypeptide (f) present in the antigen pool of the present invention is SEQ ID NO: 6 or its variant This may include immunogenic fragments of the riant or SEQ ID NO: 6 or its variants, or these It may consist of. Exemplary fragments include or consist of sequence numbers 48-51.

[0092] The antigenic polypeptide (g) present in the antigen pool of the present invention is SEQ ID NO: 7 or its variant This may include immunogenic fragments of the riant or SEQ ID NO: 7 or its variants, or these It may consist of. An exemplary fragment includes or consists of Sequence ID No. 52.

[0093] The antigenic polypeptide (h) present in the antigen pool of the present invention is SEQ ID NO: 8 or its variant This may include immunogenic fragments of the riant or SEQ ID NO: 8 or its variants, or these It may consist of. An exemplary fragment includes or consists of sequence numbers 53-55.

[0094] (nucleic acid) Antigenic polypeptide sequences and / or fusion proteins present in the antigen pool of the present invention are provided with nucleic acids (referred to as the nucleic acids of the present invention) encoding them.

[0095] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to polymeric macromolecules made up of nucleotide monomers, particularly deoxyribonucleotide monomers or ribonucleotide monomers. This term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are both naturally occurring and non-naturally occurring, have properties similar to those of reference nucleic acids, and are intended to be metabolized in a similar manner to reference nucleotides or have an extended half-life in the system. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methylribonucleotide, peptide-nucleic acid (PNA). Preferably, the term "nucleic acid" refers to a naturally occurring polymer of deoxyribonucleotide monomers or ribonucleotide monomers. Preferably, the nucleic acid molecules of the present invention are recombinant. A recombinant is meant to be a product of at least one of the cloning, restriction or ligation steps, or other procedures that result in a nucleic acid molecule different from the nucleic acid molecule found in nature (e.g., in the case of cDNA). In certain embodiments, the nucleic acids of the present invention are artificial nucleic acid sequences (e.g., cDNA sequences or nucleic acid sequences with non-naturally occurring codon usage). In one embodiment, the nucleic acids of the present invention are DNA. Alternatively, the nucleic acids of the present invention are RNA . In one embodiment, the nucleic acids of the present invention are human artificial nucleic acid sequences (e.g., cDNA sequences or nucleic acid sequences with non-naturally occurring codon usage). In one embodiment, the nucleic acids of the present invention are DNA. Alternatively, the nucleic acids of the present invention are RNA .

[0096] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are deoxyribosyl and ribose, respectively. This refers to nucleic acid molecules that have a sugar portion as their core component. This sugar portion consists of four natural bases (DNA). Adenine (A), guanine (G), cytosine (C), thymine (T) in RNA, and adenine (A) in RNA, It may be linked to a base that is guanine (G), cytosine (C), or uracil (U). When used in detail, "corresponding RNA" means that thymine (T) in DNA corresponds to uracil (U) in RNA. Aside from being substituted, it is RNA with the same sequence as the reference DNA. The sugar portion is In addition, inosine, xanthosine, 7-methylguanosine, dihydrouridine, and 5-methyl sine It may be linked to unnatural bases such as thidine. Between sugar (deoxyribosyl / ribosyl) moieties Even if the natural phosphate diester bond is arbitrarily replaced with a phosphorothioate bond Good. Preferably, the nucleic acid of the present invention has a deoxyribonucleotide having a phosphate diester bond between sugar moieties. It consists of native bases bonded to a bosyl or ribosyl sugar skeleton.

[0097] The nucleic acid of the present invention may be DNA. For example, the nucleic acid may be selected from SEQ ID NOs. 56-62 and 63-70. It contains or consists of the selected sequences. Also provided are sequence numbers 56~ nucleic acids that contain or consist of a sequence variant selected from 62 or 63-70. This variant codes for the same amino acid sequence, but based on the degenerate nature of the genetic code, They have different nucleic acids.

[0098] Therefore, due to the degeneracy of the genetic code, a number of nucleic acids that are different but functionally identical are It can encode any given polypeptide. For example, codons GCA, GCC, GCG. , and GCU all encode the amino acid alanine. Therefore, alanine is in the codon Therefore, at all the positions defined, the codon alters the encoded polypeptide. Without causing any change, it can be modified to any of the corresponding codons listed. Nucleic acid variations are referred to as "silent" ("degenerate" or "synonymous"). This also yields a variant, which is a type of conservatively modified variant. All nucleic acid sequences disclosed herein that encode lipeptides also include all of the nucleic acids This enables silent variations. Those skilled in the art can determine each codon in nucleic acids (usually AUG is the only codon for methionine and usually the only codon for tryptophan. The ability to modify (excluding the codon UGG) to produce a functionally identical molecule. They will recognize that. Therefore, each silent nucleic acid that encodes a polypeptide Variations are potential within each of the described sequences and are provided as embodiments of the present invention. It is being done.

[0099] Degenerate codon substitution occurs when the third position of one or more selected (or all) codons is mixed. This is achieved by generating sequences that are substituted with bases and / or deoxyinosine residues. It is also possible (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al.) References, 1985, J. Biol. Chem. 260:2605-2608; Rossolini et al., 1994, Mol. Cell. Prob (Espers 8:91-98).

[0100] This product contains or consists of sequences selected from sequence numbers 56-62 and 63-70. Bright nucleic acids may contain many silent variations when compared to a reference sequence. For example, 1 to 50 codons, for example, 1 to 25 codons, especially 1 to 5 codons, and in particular 1 codon, are modified. obtain).

[0101] In embodiments of the present invention, the nucleic acid is RNA, for example, mRNA. Provided herein Corresponding to the DNA sequence, and instead of a deoxyribonucleotide backbone, a ribonucleotide backbone An RNA sequence is provided which has a grammatical structure and has a side-chain base uracil (U) instead of thymine (T). .

[0102] Therefore, the nucleic acid of the present invention is a cDNA sequence selected from SEQ ID NOs. 56-62 or 63-70. It contains or consists of RNA equivalents and, when compared to the reference sequence, many thyre It may contain variations (for example, 1 to 50 pieces, for example, 1 to 25 pieces, especially 1 to 5 pieces). In particular, one codon may be altered. An "RNA equivalent" is a genetically modified version of the reference cDNA sequence. Information is contained (i.e., ribonucleotides instead of the deoxyribonucleotide skeleton). It has a skeleton and contains the same codon, but with a side-chain base uracil (U) instead of thymine (T). (ru) This refers to an RNA sequence.

[0103] The present invention also includes sequences that are complementary to the aforementioned cDNA and RNA sequences.

[0104] Nucleic acids can be codons optimized for expression within human host cells.

[0105] In the case of nucleic acids, DNA nucleic acids are transcribed and translated into the polypeptides of the present invention, and in the case of RNA nucleic acids... The mixture may be translated into the polypeptide of the present invention.

[0106] (Polypeptides and nucleic acids) Preferably, the polypeptides and nucleic acids used in the present invention are isolated. Polypeptides or nucleic acids are extracted from their original environment. For example, Naturally occurring polypeptides or nucleic acids are derived from some or all of the substances that coexist with them in their natural system. If separated, it is isolated. Nucleic acids, for example, are part of their natural environment. If cloned within a vector, it is considered isolated.

[0107] "Naturally occurring" means, when used in reference to polypeptides or nucleic acid sequences, in nature. This refers to sequences found in and that are not synthetically modified.

[0108] When used in relation to polypeptides or nucleic acid sequences, "artificial" refers to, for example, natural polypeptides. This refers to sequences that are either synthetic modifications of sequences or sequences containing non-natural sequences not found in nature. do.

[0109] The term "heterogeneous" refers to the relationship between one nucleic acid or polypeptide and another nucleic acid or polypeptide. When used in relation to relationships, two or more sequences are not found in nature in the same relationship to each other. This indicates that a "heterogeneous" sequence is a naturally occurring nucleic acid or polypeptide found in the host organism. Not isolated from the ptydo sequence, not derived from the sequence, or based on the sequence It can also mean an array that does not contain any elements.

[0110] As described above, variants of antigenic polypeptide sequences are related throughout their entire length. Preferably, at least about 80% identity with the reference sequence, more preferably at least about 85% identity, and most preferably at least about 90% identity (for example, at least about 90%). It has 5%, at least about 98%, or at least about 99%.

[0111] To compare two closely related polypeptide or polynucleotide sequences, first The "sequence identity %" between the first sequence and the second sequence can be calculated over its entire length. If they share 100% sequence identity, then polypeptide sequences are different from other polypeptide sequences. They are said to be the same or identical. The residues in the sequence are, from left to right, that is, polypeptides. Numbers are assigned from the N-terminus to the C-terminus. The terms "identical" or "percentage of identity" are used. The word represents the maximum match across the comparison window in the context of two or more polypeptide sequences. When these are found, compared, and aligned, are they the same, or have the same percentage of a certain proportion? That is, 70% identity across a specific domain, and arbitrarily, 75%, 80%, 85%, 90%, 95%, Refers to two or more sequences or subsequences having amino acid residues that are 98% or 99% identical. In this case, the comparison is performed across a window corresponding to the entire length of the reference sequence.

[0112] In sequence comparison, one sequence acts as the reference sequence compared to the test sequence. When using Gorhythm, enter the test sequence and reference sequence into the computer, and if necessary Specify the sub-array coordinates and the array algorithm program parameters. You can use the program parameters, or specify alternative parameters. This can be done. Next, the array comparison algorithm uses the program parameters to determine the reference distribution. Calculate the sequence identity percentage of the test sequence for each column.

[0113] As used herein, the "comparison window" refers to a reference array of the same number of consecutive positions. This refers to a segment that allows comparison of these two arrays after the columns have been optimally aligned. The sequence alignment method is well known in the art. For comparison, the optimal sequence alignment... Relations are, for example, the local homology algorithm of Smith and Waterman, 1981, Adv. Ap. According to pl. Math. 2:482, or the Needleman and Wunsch homology alignment algorithm, According to 1970, J. Mol. Biol. 48:443, or the Pearson and Lipman similarity search method, 1988, Proc. According to Nat'l. Acad. Sci. USA 85:2444, or these algorithms by computer (W GAP, BESTFIT, FASTA, and TFASTA, Genetics, included in the isconsin Genetics Software Package This can be done by the Computer Group, 575 Science, Dr., Madison, WI), or by manual alignment. Tests and visual inspections (for example, Current Protocols in Molecular Biology (Ausubel et al., eds., 1999) This can be done by referring to the 5th year supplement.

[0114] One example of a useful algorithm is PILEUP. PILEUP is a progressive pairwise ally. Using a spectrometry tool, multiple sequence alignments are created from related sequences, and relationships and sequence identity are determined. This shows the percentage of sex. This is the clustering used to create the alignment. Plot a tree or dendrogram showing the relationships between the groups. PILEUP is a progressive model of Feng and Doolittle. A simplified version of the alignment method (1987, J. Mol. Evol. 35:351-360) will be used. The method is similar to the method described in the literature by Higgins and Sharp, 1989, CABIOS 5:151-153. This program uses up to 300 nucleotides, each with a maximum length of 5,000 nucleotides or amino acids. Multiple arrays can be aligned. This multiple array alignment procedure is one of the two most common types. Starting with pairwise alignment of similar sequences, clusters of two aligned sequences Create. Then, this cluster is the next most relevant or aligned sequence cluster. Align the raster. 2 array clusters are pairwise arranged into 2 individual arrays. Alignment is achieved by simple extension of the alignment. The final alignment is achieved by a series of progressive extensions. This is achieved by wise alignment. This program is used for the sequence comparison region. By specifying a specific sequence and its amino acid coordinates, as well as program parameters This is executed by specifying it. By using PILEUP, the reference sequence can be used with other test sequences. Compared to the column, the following parameters: default gap weight (3.00), default gap The percentage of sequence identity relationship is determined using top length weighting (0.10) and weighted end gap. PILEUP is a GCG sequence analysis software package, for example, version 7.0 (Devere This can be obtained from the literature by aux et al. (1984, Nuc. Acids Res. 12:387-395).

[0115] Suitable algorithms for determining sequence identity percentage and sequence similarity percentage Another example is the BLAST and BLAST 2.0 algorithms, which were developed by Altschul et al., respectively. The literature includes 1977, Nuc. Acids Res. 25:3389-3402 and the literature by Altschul et al., 1990, J. Mol. Bi It is described in ol. 215:403-410. The software for performing BLAST analysis is Nation From the NIH Center for Biotechnology Information website (www.ncbi.nlm.nih.gov / ) It is publicly available. This algorithm uses words of the same length in a database array and When sorted, if it matches or satisfies a threshold score T of several positive values, By identifying short words of length W in the Ellie sequence, high-scoring sequence pairs (HSPs) can be identified. This involves initial identification. T is called the neighbor word score threshold (according to Altschul et al.). (Above). These first neighboring word hits will find longer HSPs that contain them. It acts as a seed to initiate a search. Word hits increase the cumulative alignment score. It is stretched in both directions along each array as much as possible. The cumulative score is nucle For Otid sequences, the parameters M (reward score for matching residue pairs; always > 0) and N (matching residues) are used. Penalty score for non-performing residues; always calculated using <0). In the case of amino acid sequences, The cumulative score is calculated using a word hit matrix. The extension of the alignment score occurs when the cumulative alignment score falls by X amount from its maximum achieved value; the cumulative score If A becomes 0 or less due to the accumulation of one or more negative scoring residue alignments; or The program terminates when it reaches the end of either sequence. In the case of amino acid sequences, the BLASTP program... The default values ​​are a word length of 3 and an expected value (E) of 10, as well as a BLOSUM62 score of 50. (Henikoff and Henikoff's Literature, 1989, Proc. Natl. Acad. Sci. USA 89) (See 10915) Alignment (B), expected value of 10 (E), M=5, N=-4, and comparison of both strands are used. Yes, they are.

[0116] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (e.g., Karlin and Altschu). (See reference 1, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). BLAST algorithm One measure of similarity provided by the system is the minimum sum probability (P(N)), which is the probability of two things being equal to two things. This provides an index of the probability that a match between nucleotide sequences or amino acid sequences occurs by chance.

[0117] The "difference" between sequences is the insertion of a single residue at the position of the second sequence compared to the first sequence. This refers to insertion, deletion, or substitution. Two sequences can have one, two, or more such differences. It may contain i. Otherwise, the second sequence is identical to the first sequence (100% sequence identity). Insertions, deletions, or substitutions in a sequence result in a decrease in sequence identity. For example, identical sequences If the sequence is 9 residues long, a single substitution in the second sequence results in 88.9% sequence identity. If the same sequence is 17 amino acid residues long, then two substitutions in the second sequence constitute 88.2% of the sequence. It brings about identity.

[0118] Alternatively, a second sequence is generated in order to compare the first reference sequence with the second comparison sequence. Therefore, the number of additions, substitutions, and / or deletions made to the first sequence can be determined. Addition is the addition of one residue to the first sequence (either end of the first sequence). (Including addition to). A substitution is the substitution of one residue in the first sequence with one different residue. A deletion is the deletion of one residue from the first sequence (either end of the first sequence). (Including the absence of...)

[0119] (Fusion protein (fusion polypeptide)) The term "fusion protein" is also used in connection with peptide bonds via protein synthesis. Refers to any protein containing at least two connected polypeptides. Proteins are transcribed and translated as a single unit that produces a single protein. Produced by the connection of two or more genes encoding separate polypeptides It is possible.

[0120] This invention relates to two or more different antigens present in the antigen pool as part of a fusion protein. It provides an antigen pool containing different antigens. The fusion protein is useful as described herein. It is thought to possess superior immunogenicity or prophylactic properties compared to individual component polypeptides. It may have the advantage of therapeutic effects (including increasing the breadth and depth of the response), and is not near It can be particularly valuable in mating groups.

[0121] Antigenic polypeptides present in the antigen pool fusion protein are distributed from the N-terminus to the C-terminus. They can be arranged in various orders. The design and order of polypeptides in a fusion protein are This is described in Example 8. In particular, the order of polypeptides in the fusion protein is important. However, if such an order exists, the desired immunogenicity of the polypeptide may be It can result in superior processing and presentation of the cydoplasmic region, and in other cases, natural Non-natural immunogenic peptides obtained from the conjugation of cancer-specific CLT antigens during vaccination It is presented on surface-presented class I HLA molecules, thereby preventing undesirable T cell responses. This is because it is necessary for the optimal fusion design to reduce the likelihood of triggering it.

[0122] The fusion protein elicits a strong antigenic response to the component CLT antigen (Examples 9 and 10). (See reference), it is thought to induce a minimal antigenic response to the junction region (see Example 8). see).

[0123] Sequences (a) to (h) can all be arranged with the first N-terminal methionine removed. However, That is, one or more of the antigenic polypeptides are polypeptides lacking an N-terminal methionine. It may contain arrays.

[0124] In one embodiment, the fusion protein consists of six antigenic polypeptides (a), (b), (d), (f) If it includes (g), and (h).

[0125] In one embodiment, the fusion protein of the present invention has a sequence without an N-terminal methionine residue. It may contain an antigenic polypeptide having polypeptide sequence number 2. Therefore, the fusion protein of the present invention has the amino acid sequence of SEQ ID NO: 6 without the N-terminal methionine residue. It contains an antigenic polypeptide having. In one embodiment, the fusion protein of the present invention is , an antigenic polypeptide having the amino acid sequence of SEQ ID NO: 5 without an N-terminal methionine residue Includes. In one embodiment, the fusion protein of the present invention, without an N-terminal methionine residue Antigenic polypeptide having the amino acid sequence of column number 2, sequence lacking an N-terminal methionine residue An antigenic polypeptide having amino acid sequence number 6, and a polypeptide lacking an N-terminal methionine residue. It contains an antigenic polypeptide having the amino acid sequence of column number 5.

[0126] The fusion protein contains six different antigens (where the antigens are (a), (b), (d), (f), (g), and If it contains polypeptide sequences (a), (b), (d), (f), (g) , and (h) are polypeptide sequences: (a) Sequence ID No. 1 having an N-terminal methionine residue or not having an N-terminal methionine residue or its variant, or SEQ ID NO: 1, or an immunogenic fragment of that variant; (b) Sequence ID No. 2 which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant, or SEQ ID NO: 2, or an immunogenic fragment of that variant; (d) Sequence ID No. 4, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant or SEQ ID NO: 4 or an immunogenic fragment of that variant; (f) Sequence ID No. 6, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant or SEQ ID NO: 6 or an immunogenic fragment of that variant; (g) Sequence ID 7, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant or SEQ ID NO: 7 or an immunogenic fragment of that variant; (h) Sequence ID No. 8, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant or SEQ ID NO: 8 or an immunogenic fragment of that variant It may have.

[0127] In one embodiment, the six antigenic polypeptides are (a), (b), (f), (g), (d), and The elements are arranged from N to C in the order (h).

[0128] In one preferred embodiment, six antigenic polypeptides are represented by SEQ ID NOs: 1-2, 4, 6-8. It has an array of sequence numbers 1, 2, 6, 7, 4, and sequence number The sequences are arranged from N to C in the order of number 8. The corresponding sequence with the N-terminal methionine deleted is shown above. It can be used as is, as explained there.

[0129] For example, the fusion protein of the present invention comprises six antigenic polypeptides (a), (b), (d), (f), ( g), and (h), wherein the antigenic polypeptide (a), (b), (d), (f), (g), and (h) The amino acid sequence is: (a) Sequence ID 1; (b) Sequence ID No. 2 without the N-terminal methionine residue; (d) Sequence ID 4; (f) Sequence ID 6; (g) Sequence ID 7; and (h) Sequence ID 8 It holds.

[0130] Therefore, preferably, SEQ ID NO: 1 is located at the N-terminus and SEQ ID NO: 8 is located at the C-terminus. In this case, the N-terminal methionine of SEQ ID NO: 2 is deleted. In this embodiment of the present invention, fusion The protein has the sequence of sequence number 76.

[0131] In another embodiment, the fusion protein is composed of six antigenic polypeptides (a), (b), (d), (f If (f), (h), and (h) are included, the six antigenic polypeptides are (f), (h), (g), (b), and (d) The elements are arranged from N to C in the order of (a) and (a).

[0132] In one preferred embodiment, six antigenic polypeptides are represented by SEQ ID NOs: 1-2, 4, 6-8. It has the sequence, and sequence numbers 6, 8, 7, 2, 4, and sequence number The sequences are arranged from N to C in the order of No. 1. The corresponding sequence with the N-terminal methionine deleted is shown above. It can be used as is, as described in [reference]. Therefore, preferably, SEQ ID NO: 6 is at the N-terminus. It is present, and sequence number 1 is at the C-terminus. Preferably, the N-terminal methionine of sequence number 2 is removed. It is removed. In this embodiment of the present invention, the fusion protein has the sequence of SEQ ID NO: 77.

[0133] In another embodiment, the fusion protein contains eight antigenic polypeptides (a) to (h). .

[0134] The fusion protein contains eight different antigens (where the antigens are polypeptide sequences (a) to (h)). If it contains (has), polypeptides (a) to (h) are polypeptide sequences: (a) Sequence ID No. 1 having an N-terminal methionine residue or not having an N-terminal methionine residue or its variant, or SEQ ID NO: 1, or an immunogenic fragment of that variant; (b) Sequence ID No. 2 which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant, or SEQ ID NO: 2, or an immunogenic fragment of that variant; (c) Sequence ID No. 3, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant, or SEQ ID NO: 3, or an immunogenic fragment of that variant; (d) Sequence ID No. 4, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant or SEQ ID NO: 4 or an immunogenic fragment of that variant; (e) Sequence ID No. 5, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant, or SEQ ID NO: 5, or an immunogenic fragment of that variant; (f) Sequence ID No. 6, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant or SEQ ID NO: 6 or an immunogenic fragment of that variant; (g) Sequence ID 7, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant or SEQ ID NO: 7 or an immunogenic fragment of that variant; (h) Sequence ID No. 8, which has an N-terminal methionine residue or does not have an N-terminal methionine residue. or its variant or SEQ ID NO: 8 or an immunogenic fragment of that variant It may have.

[0135] In one embodiment, the eight antigenic polypeptides are (a), (b), (c), (g), (d), (e) They are arranged from N to C in the order of (f), and (h).

[0136] In one preferred embodiment, eight antigenic polypeptides have sequences 1 to 8. and, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 The sequence is arranged from N to C in the order of , and sequence number 8. The corresponding sequence is one in which the N-terminal methionine has been deleted. The array can be used arbitrarily, as described above.

[0137] For example, the fusion protein of the present invention comprises eight antigenic polypeptides (a) to (h), and here The antigenic polypeptides (a) to (h) have the following amino acid sequences: (a) Sequence ID 1; (b) Sequence ID No. 2 without the N-terminal methionine residue; (c) Sequence ID 3; (d) Sequence ID 4; (e) Sequence ID No. 5 without the N-terminal methionine residue; (f) Sequence ID No. 6 without the N-terminal methionine residue; (g) Sequence ID 7; and (h) Sequence ID 8 It holds.

[0138] Preferably, SEQ ID NO: 1 is located at the N-terminus and SEQ ID NO: 8 is located at the C-terminus. Preferably, The N-terminal methionine in sequence number 2 is removed. Preferably, the N-terminal methionine in sequence number 6 is removed. It is removed. Preferably, the N-terminal methionine of SEQ ID NO: 5 is deleted. In embodiments of the present invention The fusion protein has the sequence of sequence number 78.

[0139] In another embodiment, the fusion protein contains eight antigenic polypeptides (a) to (h). In total, the eight antigenic polypeptides are in the order of (f), (c), (a), (e), (d), (h), (g), and (b). It is arranged from N to C.

[0140] In one preferred embodiment, eight antigenic polypeptides have sequences 1 to 8. And, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 7 The sequence is arranged from N to C in the order of , and sequence number 2. The corresponding sequence is one in which the N-terminal methionine has been deleted. The array can be used arbitrarily, as described above.

[0141] For example, the fusion protein of the present invention comprises eight antigenic polypeptides (a) to (h), and here The antigenic polypeptides (a) to (h) have the following amino acid sequences: (a) Sequence ID 1; (b) Sequence ID No. 2 without the N-terminal methionine residue; (c) Sequence ID 3; (d) Sequence ID 4; (e) Sequence ID 5; (f) Sequence ID 6; (g) Sequence ID 7; and (h) Sequence ID 8 It holds.

[0142] Preferably, sequence number 6 is located at the N-terminus and sequence number 2 is located at the C-terminus. Preferably, The N-terminal methionine in column number 2 is deleted. In this embodiment of the present invention, the fusion protein It has the sequence of sequence number 79.

[0143] The fusion protein of the present invention comprises (i) another polypeptide that is a melanoma-associated antigen; and (ii) an immune response. (iii) polypeptide sequences that can enhance (i.e., immunostimulatory sequences); and (iii) for example, It can provide strong CD4+ cells that help increase the CD8+ T cell response to antigen epitopes. A second or further polypeptide sequence selected from polypeptide sequences containing the universal CD4 helper epitope It can be fused with polypeptides.

[0144] An example of a fusion polypeptide is one in which two or more sequences (e.g., two, three) are selected from the sequences (a) to (h). Includes an array of 4, 5, 6, 7, or 8 elements.

[0145] The present invention also provides nucleic acids encoding the aforementioned fusion protein.

[0146] (Linker) The present invention provides an antigen pool comprising two or more different antigens, wherein two or more different antigens The antigen pool is the portion of the nucleic acid encoding the fusion protein and / or the fusion protein. It may be present in the fusion protein or the nucleic acid encoding the fusion protein. In this case, two or more different antigens are located between antigenic polypeptide sequences, with one or more peptides positioned between them. They are connected together by a drinker or spacer. They are present in the antigen pool of the present invention. The antigenic polypeptide sequence has one or more linkers (e.g., two, three, four, five, six, or seven). They can be connected together by two linkers. The linkers are present in the antigen pool of the present invention. Each of the antigenic polypeptide sequences can be separated. The linker can be an "internal" one. In other words, the linker connects the N-terminus and the last polypeptide of the first polypeptide of the fusion protein. It is not present at the C-terminus of the lipeptide. Therefore, in embodiments of the present invention, two or more different If the antigen is present in the fusion protein or the nucleic acid encoding the fusion protein, then 2 The different antigens above are one or more peptide linkers positioned between antigenic polypeptide sequences. They are connected together by -.

[0147] One or more linkers are located between (a) and (b), (b) and (f), (f) and (g), (g) and (d), and (d) and (h). It can be attached. In another embodiment of the present invention, one or more linkers are (f) and (h), (h) and (g ), (g) and (b), (b) and (d), and (d) and (a) are positioned between these. Further embodiments of the present invention In this case, the linker is (a) and (b), (b) and (c), (c) and (g), (g) and (d), (d) and (e), (e) and (f), It is positioned between (f) and (h). In a further embodiment of the present invention, the linker is (a) It is positioned between (b), (b) and (d), (d) and (f), (f) and (g), (g), and (h). In further embodiments, the linker is (f) and (c), (c) and (a), (a) and (e), (e) and (d) It is positioned between (d) and (h), (h) and (g), and (g) and (b).

[0148] A linker is a cDNA that encodes a linker peptide sequence, or an encoded peptide. It is also acceptable. The linker is connected to the C-terminus of the antigenic polypeptide containing the linker sequence and the next antigenic This creates a single construct that is inserted between the N-terminuses of polypeptides, thereby enabling fusion. By linking the antigenic polypeptides of the combined protein together, each fusion of the present invention is achieved. They are positioned between the individual antigens of the protein. Individual linkers used in fusion proteins - may have the same sequence, or the linker may have a different sequence. In one embodiment of the present invention, the linker is an array selected from sequence numbers 71-75 and 84. It includes or consists of the sequence.

[0149] In this embodiment of the present invention, the fusion protein comprises a sequence selected from SEQ ID NOs: 76-79 It includes or consists of the said arrangement.

[0150] The linker may be a glycine-based linker, which has a length of 3 to 6 amino acids. The connector may also contain lysine (see Sequence IDs 71-75 and 84). Linker of the present invention This reduces the risk of introducing unwanted immunogenic epitopes, including the linker itself. ; This is an unwanted epidermal fusion produced by the direct fusion of individual antigenic polypeptides. It also interferes with taupe.

[0151] The fusion protein is formed from separate antigenic polypeptide sequences and the resulting openly The ding frame is transcribed and translated as a single unit that produces a single protein. Three or more (e.g., 3, 4, ...) of cDNA encoding linkers connected in such a way as to encode linkers. It can be produced by connecting 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 genes. The nucleic acid encoding the fusion protein contains a sequence selected from sequence numbers 80-83 or This sequence may consist of the above.

[0152] (Production of polypeptides according to the present invention) The antigenic polypeptide sequences present in the antigen pool of the present invention include, for example, Green and Samb. Rook's paper, 2012 Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbour. It can be obtained and manipulated using the techniques disclosed in our Laboratory Press. Using artificial gene synthesis, polynucleotides are produced (Nambiar et al., 1984, Sc ience, 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. Acids Res., 13:3305-3316), and then expressed in a suitable organism to produce polynucleotides. It can generate antigenic polypeptides present in the antigen pool of the present invention. Genes can be produced synthetically, for example, by solid-phase DNA synthesis. The entire gene is It can be synthesized de novo without requiring precursor template DNA. To obtain Otid, place the building blocks in the order required by the arrangement of the products. Then, the oligonucleotides are sequentially coupled to the growing oligonucleotide chain. Once the chain assembly is complete... The product is then liberated from the solid phase into the solution, deprotected, and collected. The product is then processed using high-performance liquid chromatography. By isolation using HPLC, the desired oligonucleotide can be obtained with high purity (V (Erma and Eckstein, 1998, Annu. Rev. Biochem. 67:99-134). These relatively short The segments were analyzed using various gene amplification methods (Methods Mol Biol., 2012;834:93-109) without Suitable for use in multiple recombinant DNA-based expression systems, it can be easily assembled into longer DNA molecules. In relation to the present invention, those skilled in the art will know that the polypeptide antigen described in the present invention is... The polynucleotide sequence encoding this is used in various vaccines, including, for example, viral vectors. You will understand that it can be easily used in production systems.

[0153] polypeptides present in the antigen pool of the present invention within a microbial host (e.g., bacteria or fungi). To produce this, nucleic acids have suitable regulatory sequences and control sequences (promoter, termination signal). (including, etc.) and to promote polypeptide secretion suitable for protein production within the host. The sequence includes the following. Similarly, polypeptides present in the antigen pool of the present invention are eukaryotic cells (e.g. For example, a culture of Chinese hamster ovary cells or Drosophila S2 cells is suitable. Regulatory sequences and control sequences (including promoters, termination signals, etc.) within cells This book combines sequences that promote polypeptide secretion suitable for protein production. It can be produced by transduction using the nucleic acid of the invention.

[0154] Isolation of polypeptides present in the antigen pool of the present invention produced by recombinant means The improvement involves stretching the histidine residue toward one end of the polypeptide (generally, H This can be optionally promoted by adding the tag known as the `is` tag.

[0155] Polypeptides can also be prepared synthetically.

[0156] (vector) The nuclei of the antigen pool of the present invention so that polypeptides encoded by nucleic acids are produced. Ex vivo introduction of a gene construct containing one or more acids into cells. This can be achieved. Nucleic acids (for example, DNA) can be used in nucleic acid expression systems, bacteria, and some viral expression systems. This may be present in any of the various delivery systems known to those skilled in the art. For example, Rolland's literature, 1 998, Crit. Rev. Therap. Drug Carrier Systems 15:143-198 and the citations therein Numerous gene delivery techniques, including those described in the references, are prevalent in this field. It is knowledge. Some of these approaches are outlined below for illustrative purposes.

[0157] The vector is suitable for enabling the transcription of translationally active RNA molecules within human host cells. It comprises nucleic acids that encode regulatory elements (e.g., suitable promoters and termination signals). To obtain. "Translationally active RNA molecules" are translated into proteins by the translation machinery of human cells. It is an RNA molecule that can be translated.

[0158] The vector may be a viral vector. Viral vectors include adenoviruses. Adeno-associated viruses (AAVs) (e.g., AAV types 5 and 2), alphaviruses (e.g., Bene) Zuehne encephalitis virus (VEEV), Sindbisvirus (SIN), Semlik Forest virus (SFV) ), herpesviruses, arenaviruses (e.g., lymphocytic choriomeningitis virus (LCMV)) measles virus, poxvirus (e.g., modified vaccinia ankara (MVA)), paramyx Soviruses, lentiviruses, or rhabdoviruses (e.g., vesicular stomatitis virus (VSV)) ) A vector may also be used, that is, the vector may originate from one of the aforementioned viruses. It is possible. The viral vector is an adenovirus. The viral vector is a po Xviruses, for example, MVA.

[0159] Adenoviruses are known for their intermediate genome size, ease of manipulation, high titer, and broad target cell range. Due to its range and high infectivity, it is particularly suitable for use as a gene transfer vector. The ends of the viral genome are cis elements, which are necessary for the replication and packaging of viral DNA. It contains a certain 100-200 base pair reverse repeat (ITR). Early (E) and late (L) regions of the genome. The region contains different transcription units that are divided by the initiation of viral DNA replication. Region E1 The regions (E1A and E1B) are involved in regulating the transcription of the viral genome and several cellular genes. It encodes protein. Expression of the E2 region (E2A and E2B) is related to the protein used for viral DNA replication. These proteins lead to the synthesis of DNA, late-stage gene expression, and host cells. It is involved in blockade (Renan, 1990). It contains the majority of the viral capsid protein. The product of a late gene is a single primary transcript produced by the major late promoter (MLP). It is expressed only after significant processing. MLP is particularly efficient in the later stages of infection, All mRNA transcribed from the promoter consists of a 5'-leader (TPL) sequence consisting of three parts. Because it possesses, it becomes mRNA that is favorable for translation. If one or more of the initial genes are deleted The replication-deleted adenovirus produced from the viral genome has limited replication. Furthermore, the possibility of pathogenicity spreading within vaccinated hosts and vaccinated hosts It is particularly useful because the possibility of contact is low.

[0160] (Other vehicles and methods for introducing polynucleotides into cells) An expression construct containing one or more nucleic acid sequences is simply derived from a naked recombinant DNA plasmid. It may be. Ulmer et al., 1993, Science 259:1745-1749 and Cohen, 1993, S See the review in Science 259:1691-1692. The transfer of this construct is, for example, This can be carried out by any method that physically or chemically permeates the cell membrane. This is particularly applicable to metastases treated with ex vivo therapy.

[0161] (Methods for introducing or delivering RNA into cells) Expression constructs containing one or more polynucleotide sequences are derived from naked recombinant DNA RNA. It may also consist of offspring (Ulmer et al., 2012, Vaccine 30:4414-4418). DNA-based development Regarding the current construct, various methods are used to introduce RNA molecules into cells ex vivo. It can be introduced. The introduced biological molecule is directly translated by the host cell's translation mechanism. The RNA peptide is then introduced so that the encoded polypeptide is produced within the cell into which it is introduced. The construct of the casing is designed to mimic a simple messenger RNA (mRNA) molecule. It is possible to use the RNA molecule for viral RNA-dependent RNA polymerase. By incorporating them into structural genes, they can self-amplify within the cells into which they are introduced. It can be designed to enable this. In this way, self-amplifying mRNA (SAM(trademark)) This type of RNA is known as the molecule (Geall et al., 2012, PNAS, 109:14604-14609). The molecule shares properties with several RNA-based viral vectors. mRNA-based RNA or This involves either SAM(trademark)RNA (for example, by altering its sequence or by modifying nucleotides). (By using) it can be further modified to enhance stability and translation (Schlake et al.'s text) (Reference, RNA Biology, 9:1319-1330), and formulations of both types of RNA (e.g., emulsion) In (Brito et al., Molecular Therapy, 2014 22:2118-2129) or in lipid nanoparticles (Kran z et al., 2006, Nature, 534:396-401)), ex vivo stability and / or invasion of cells This can promote the entry of nucleic acids into nanoparticles. Therefore, in one embodiment of the present invention, nucleic acids are nanoparticles The nanoparticles are formulated in the body. Preferably, the nanoparticles are lipid-based nanoparticles, for example, cations. These are sex liposomes. A wide variety of modified (and unmodified) RNA formulations are used in animals. Several RNA-based vaccines are being tested as vaccines in models and humans, and are currently in development. It is being used in clinical trials.

[0162] (Pharmaceutical composition) The antigen pool of the present invention is a pharmaceutical composition such as an immunogenic composition (hereinafter referred to as "the composition of the present invention"). It can be formulated for delivery within the body. The compositions of the present invention are preferably the antigen pool of the present invention. It contains a carrier that is acceptable as a pharmaceutical.

[0163] Therefore, in one embodiment, the antigen pool of the present invention may be a carrier that is pharmaceutically acceptable. An immunogenic pharmaceutical composition containing the following is provided.

[0164] In one preferred embodiment of the present invention, two or more (for example, two, three, four, five, six, seven) It contains an antigen pool containing 8 different antigens, where each antigen is in the form of a polypeptide. In a state where different antigens exist, in combination with a pharmaceutically acceptable carrier, separate polyps The present invention provides an immunogenic pharmaceutical composition that exists as a ptide in the antigen pool.

[0165] In one preferred embodiment of the present invention, two or more (for example, two, three, four, five, six, seven) It includes an antigen pool containing 8 different antigens, where each antigen is the polypeptide The coding nucleic acid exists in a nucleic acid form, and different antigens are combined with a pharmaceutically acceptable carrier. Therefore, the immunogenic pharmaceutical composition of the present invention exists in the antigen pool as separate polypeptides. It will be provided.

[0166] In one preferred embodiment of the present invention, two or more (for example, two, three, four, five, six, seven) It contains an antigen pool containing 8 different antigens, where each antigen is in the form of a polypeptide. In a state in which different antigens exist, in combination with a pharmaceutically acceptable carrier, a fusion protein The present invention provides an immunogenic pharmaceutical composition in which the qualitative component is present in the antigen pool.

[0167] A preferred embodiment of the present invention is an antigen comprising two or more (e.g., two) different antigens. The pool includes, where each antigen exists in the form of a nucleic acid encoding the polypeptide, Furthermore, different antigens, in combination with a pharmaceutically acceptable carrier, encode a fusion protein. The present invention provides an immunogenic pharmaceutical composition that exists in an antigen pool as a nucleic acid.

[0168] In one embodiment, two or more (for example, two, three, four, five, six, seven, eight) different It comprises an antigen pool containing antigens, where each antigen is a nucleic acid encoding the polypeptide. Different antigens, existing in various forms, can be combined with pharmaceutically acceptable carriers to form separate polymers. Peptides, nucleic acids, fusion proteins, and nucleic acids encoding said fusion proteins as antigens The present invention provides an immunogenic pharmaceutical composition present in a liquid. Such a composition is increased It can provide a strengthened immune response.

[0169] (Salt that is acceptable as a medicine) The composition of the present invention relates to nucleic acids, polypeptides, or fusion proteins provided herein. It is clear that it may contain salts that are acceptable as medicine. Such salts are organic bases. (For example, salts of primary, secondary, and tertiary amines and basic amino acids) and inorganic bases (e.g.) For example, sodium, potassium, lithium, ammonium, calcium and magnesium It can be prepared from pharmaceutically acceptable non-toxic bases, including salts.

[0170] (A carrier that is acceptable as a medicine) Many pharmacopoeia-acceptable carriers known to those skilled in the art can be used in the composition of the present invention. It is possible, but the optimal type of carrier used will vary depending on the mode of administration. Composition of the present invention These include, for example, parenteral, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration. Administration, preferably parenterally, including intramuscular, subcutaneous, or intravenous administration, any preferred method. It can be formulated for various administration modes. For parenteral administration, the carrier is preferably , containing water, and a buffer for pH control, stabilizers, such as surfactants and amino acids, and Furthermore, it may contain isotonic modifiers, such as salts and sugars. The composition is dilute at the time of use. If intended to be provided in a lyophilized form for dispensing, the formulation may contain a lyophilized protective agent. For example, it may contain sugars such as trehalose. For oral administration, the above-mentioned Either a body or a solid carrier, for example, mannitol, lactose, starch, stearin Magnesium phosphate, sodium saccharin, talcum, cellulose, glucose, sucrose Sodium and magnesium carbonate can be used.

[0171] Therefore, the composition of the present invention is a buffer (for example, neutral buffered saline or ri (Sodium-buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, if (e.g., dextran), mannitol, protein, polypeptide, or glycine) The amino acids, antioxidants, bacteriostatic agents, chelating agents such as EDTA or glutathione, and preparations are used. Solutes, suspensions, thickeners, and / or solutes that make the blood of the cypient isotonic, hypotonic, or weakly hypertonic. It may contain preservatives. Alternatively, the composition of the present invention may be formulated as a freeze-dried product. It is possible.

[0172] (immune stimulant) The composition of the present invention may also contain one or more immunostimulants. Immunostimulants are exogenous Any substance that enhances or strengthens the immune response (antibody-mediated and / or cell-mediated) to sexual antigens. It can be of quality. In relation to vaccine formulations, it is often called an adjuvant. Examples of immunostimulants include aluminum hydroxide gel (alum) or aluminum phosphate. Aluminum salts such as um, saponins including QS21, and immunostimulatory oligonucleotides such as CPG. Hydroxide, oil-in-water emulsion (for example, when the oil is squalene), aminoalkylglucosa Minido 4-phosphate, lipopolysaccharides or their derivatives, for example, 3-de-O-acylated monophosphoric acid Lipid A (3D-MPL®), and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR Examples include ligand 9, IL-12, and interferon. Therefore, the composition of the present invention One or more immunostimulants are preferably aluminum salts, saponins, and immunostimulant oligonucleotides. Cleotide, oil-in-water emulsion, aminoalkylglucosaminid 4-phosphate, lipopolysaccharide and its derivatives, and other TLR4 ligands, TLR7 ligands, TLR8 ligands, and TLR9 ligands Selected from the available options. As immune stimulants, substances that specifically interact with other immune components are selected. Interaction of clonal antibodies, such as PD-1 and CTLA4, with immune checkpoint receptors. One example is a monoclonal antibody that blocks the function of the drug.

[0173] In the case of delivery methods using recombinant nucleic acids (e.g., DNA, RNA, viral vectors), proteins The gene encoding the base immunostimulator is a polypeptide present in the antigen pool of the present invention. It can be easily delivered together with the gene that codes for cide.

[0174] (Sustained release) The compositions described herein provide a sustained release of the compound after administration. Sex-releasing formulations (i.e., capsules, sponges, patches, etc., composed of polysaccharides) It can be administered as part of a formulation (such as a gel).

[0175] (Storage and packaging) The composition of the present invention is contained in sealed ampoules or vials, or in other containers, in unit doses or multiple doses. It can be served in a container. Such a container is preferably one that keeps the formulation in place until use. It is sealed to maintain sterility. Generally, the preparation is a suspension in an oily or aqueous vehicle. It can be stored as a solution or emulsion. Alternatively, the composition of the present invention can be used Free It can be stored in a dry state.

[0176] (Dosage) The amount of nucleic acid, polypeptide, or fusion protein in each composition of the present invention is determined by the therapeutic use It can be prepared to obtain a suitable dosage for use. Solubility, BioAbility Factors such as vaxity, biological half-life, route of administration, product shelf life, and other pharmacological findings. The issues to be discussed are assumed by those skilled in the art to prepare such compositions, and therefore various factors The dosage and treatment regimen may be optimal.

[0177] Typically, a composition containing a therapeutically effective amount is about 0.1 ug to about 1 dose per administration in the composition of the present invention. 000ug of polypeptide or fusion protein, more typically about 2.5ug to about 100ug per dose. Deliver a polypeptide or fusion protein in the form of a short synthetic long-chain peptide. In such cases, the dosage may range from 1 to 200 ug / peptide / dose. Polynucleotide composition In this regard, this typically involves about 10 ug to about 20 mg of nuclei per dose in the antigen pool of the present invention. Acids, more typically, deliver approximately 0.1 mg to 10 mg of nucleic acid per dose.

[0178] (Stimulated T-cell therapy) Autologous or non-autologous T cells are extracted from a subject, for example, from peripheral blood, umbilical cord blood, and / or via ethereal cells. Tumor-related molecules isolated by cis and loaded onto MHC molecules (signal 1) of APC cells Stimulation in the presence of linked antigens induces the proliferation of T cells that have an immune-specific TCR to this antigen. It is possible.

[0179] Good T cell activation is associated with the co-stimulatory surface molecule B7 on antigen-presenting cells and T cells, respectively. CD28 binding is required (signal 2). To achieve optimal T cell activation, signal 1 is used. Both signal 1 and signal 2 are required. Conversely, antigenic peptide in the absence of co-stimulation (signal 2) T-cell stimulation (signal 1) failed to induce complete T-cell activation, resulting in T-cell tolerance. This can occur as a result. In addition to costimulatory molecules, CTLs induce signals and inhibit T cell activation. Inhibitory molecules such as A-4 and PD-1 also exist.

[0180] Therefore, if an antigen-specific TCR recognizes the antigen presented by the patient's MHC, then self Alternatively, non-autologous T cells are stimulated and expanded in the presence of the antigen pool of the present invention, and the cancer cells are identified as belonging to the present invention. Those who are at risk of cancer expressing the corresponding polypeptide in the antigen pool or those who have said cancer It can be transferred back to the infected patient, in which case the antigen-specific TCR is the corresponding port. The drug targets cancer cells expressing the lipeptide and induces their death.

[0181] Therefore, in an embodiment of the present invention, T cells derived from a person suffering from cancer are used. Use in cuvivo stimulation and / or amplification, said stimulation and for the treatment of said cancer in said human The antigen pool of the present invention or for the subsequent reintroduction of amplified T cells into the human. A composition is provided.

[0182] In a further embodiment of the present invention, cancer cells are polypeptides selected from (a) to (h). A method for treating human cancer expressing the sequence, which optionally includes antigen-presenting cells, and at least This also involves collecting a population of white blood cells, including T cells, from a person, and the antigen pool or composition of the present invention. Stimulating and / or amplifying the T cells in the presence of, and at least stimulating and / or This method includes reintroducing some or all of the leukocytes, including amplified T cells, into the human. The law is provided.

[0183] In any one of the embodiments described above, preferably, cancer is melanoma, particularly skin cancer. It is melanoma.

[0184] In another embodiment of the present invention, a polypeptide sequence selected from (a) to (h) is expressed. A process for preparing a population of T cells that are cytotoxic to cancer cells, wherein (i) (ii) obtaining T cells containing antigen-presenting cells from cancer patients and (ii) the antigen of the present invention A process is proposed that includes ex vivo stimulation and amplification by a pool or composition. The antigen pool is provided. The antigen pool is selected from (a) to (h) that cancer cells express. It may contain d.

[0185] In this context, "corresponding" means that cancer cells, for example, sequence number A (A is sequence number 1-8) If a T cell population expresses one of the following (or a variant or immunogenic fragment thereof), , polypeptides, nucleic acids, or fusion proteins, or one of the aforementioned Extraction by the form of the composition SEQ ID NO: A or a variant thereof, or by immunogenic fragments thereof. This means that it is stimulated and amplified in vivo.

[0186] For example, in such a process, the culture and expansion are carried out in the presence of dendritic cells. The dendritic cells are transfected with nucleic acid molecules and polypeptides of the antigen pool of the present invention. It expresses.

[0187] In embodiments of the present invention, the T cell population obtainable by the process described above (hereinafter referred to as "this invention") A population of T cells from the beginning of the world is provided.

[0188] In a further embodiment of the present invention, T cells stimulated with the antigen pool or composition of the present invention A cell (hereinafter referred to as a T cell in this invention) is provided.

[0189] In further embodiments of the present invention, the T cell population or T cells of the present invention may be used as a pharmaceutical product. A pharmaceutical composition is provided which contains a suitable carrier. Such a composition is, for example, non-eco It may be a sterile composition suitable for oral administration.

[0190] In another embodiment of the present invention, a population of T cells or T cells of the present invention for use in pharmaceuticals It will be provided.

[0191] Cancer cells express polypeptide sequences selected from (a) to (h), and cancer cells express (a) to (h) A method for treating cancer patients who express a polypeptide sequence selected from the following: And, in the human, the T cell population or T cells of the present invention or the T cell population or T cells of the present invention A method comprising administering a composition containing cells is also provided as an embodiment of the present invention.

[0192] A further embodiment of the present invention, for use in treating human cancer A T cell population, the T cells of the present invention, or a composition containing the T cell population or T cells of the present invention A composition is provided in which the cancer cells express a polypeptide sequence selected from (a) to (h). It can be done.

[0193] In any one of the embodiments described above, preferably, cancer is melanoma, particularly skin cancer. It is melanoma.

[0194] The use of T cell populations, T cells, or compositions to induce an immune response against cancer in humans is, The corresponding antigenic sequences (or one or more of them) in the antigen pool are expressed by cancer. It depends on the design of the antigen pool and the likelihood of cancer manifesting. There is a correlation between highly antigenic sequences. In this context, "corresponding" means that cancer, for example, , Sequence ID A (where A is one of Sequence IDs 1-8) or its variant or immunogenicity If a fragment is expressed (or is likely to be expressed), the pool is Sequence ID A or its variant. The t or immunogenic fragment (optionally in the form of a fusion protein and as a protein or nucleic acid) It means to include. Therefore, antigen pool, fusion protein, nucleic acid, vector, or There is a correlation between the design of the composition and the antigenic sequences that cancer expresses or is likely to express. The inclusion of certain antigen sequences in the antigen pool may lead to a greater immune response or broader coverage against cancer. This could potentially enable an immune response against cancer in a range of patients.

[0195] (Cell therapy to promote antigen presentation in vivo) By utilizing one of various cell delivery vehicles in a pharmaceutical composition, antigen-specific immune responses are achieved. Production can be promoted. Therefore, the present invention relates to the antigen pool of the present invention or the present invention Isolated antigen-presenting cells modified by ex vivo loading of the composition (hereinafter referred to as " The present invention provides cells that are referred to as "APCs" (Apnea-Cellulose Cells). These cells may be modified to become more efficient APCs. Antigen-presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes, and other cells. Such cells increase their ability to present antigens, activate the T cell response and / or To improve maintenance and / or immunological compatibility with the receiver (i.e., HLA haplogroup It is possible to genetically modify the gene to match the gene, but this is not always necessary. It can usually be isolated from any of the various biological fluids and organs, and is self- or homogeneous. These can be cells of the same lineage, the same lineage, or different species.

[0196] A preferred embodiment of the present invention uses dendritic cells or their precursors as APCs. Therefore, in one embodiment, the APC of the present invention is a dendritic cell. A dendritic cell is extremely It is a powerful APC (Banchereau and Steinman, 1998, Nature, 392:245-251), and prevents It has been shown to be effective as a physiological adjuvant for inducing targeted or therapeutic immunity. (See Timmerman and Levy, 1999, Ann. Rev. Med. 50: 507-529). Generally, dendritic The cell has a typical shape (star-shaped in situ, and with prominent cytoplasmic projections in vitro). Dendritic structures are visible, and they have the ability to efficiently take up, process, and present antigens. It can be identified based on its strength and its ability to activate naive T cell responses. These dendritic cells are, naturally, unique and not typically found in in vivo or ex vivo dendritic cells. It is possible to modify the cell surface receptor or ligand to express such a modification. Dendritic cells are assumed in this invention. Instead of dendritic cells, antigens are loaded... These secretory vesicles (called exosomes) can be used in immunogenic compositions (Zitvo See Gel et al., 1998, Nature Med. 4:594-600. Therefore, in one embodiment, polypeptides prepared from cells loaded with the antigen pool or composition of the present invention, Exosomes loaded with nucleic acids or fusion proteins are provided.

[0197] Dendritic cells and their precursors are obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood, or any other source. It can be obtained from suitable tissues or fluids. For example, dendritic cells produce cytokines, For example, a combination of GM-CSF, IL-4, IL-13, and / or TNFα is used in monocytes collected from peripheral blood. By adding it to the culture, it can be differentiated ex vivo. Alternatively, peripheral blood, CD34-positive cells collected from umbilical cord blood or bone marrow induce differentiation, maturation, and proliferation of dendritic cells. Leading GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and / or other compounds By adding it to the culture medium combination, it can be differentiated into dendritic cells.

[0198] Dendritic cells are conveniently classified into "immature" cells and "mature" cells, which leads to two types This makes it possible to easily distinguish well-characterized phenotypes. However, this life Nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Mature dendritic cells exhibit high antigen uptake, correlated with high expression of Fcγ receptors and mannose receptors. It is characterized as an APC with processing ability. The mature phenotype is usually these marks Characterized by lower expression of Kerr, but also class I and class II MHCs, adhesion molecules (e.g., CD54) T cell activity of co-stimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB), as well as CD11, CD40, CD80, CD86, and 4-1BB. It is characterized by the high expression of cell surface molecules involved in chemical transformation.

[0199] APC is genetically modified so that polypeptides are expressed on the cell surface, for example, protein A polynucleotide that codes for a molecule (or a part of it or other variants) and is transfected. It can also be performed. Such transfections are performed ex vivo. This can be done, and then a pharmaceutical composition containing such transfected cells is used. Alternatively, gene delivery devices targeting dendritic cells or other antigen-presenting cells can be used. The drug can be administered to the patient, resulting in transfection occurring in vivo. Example For example, in vivo and ex vivo transfection of dendritic cells is typically performed according to WO 97 / 24447. The method described in issue number, or the literature by Mahvi et al., 1997, Immunology and Cell Biology 75: Any method known in the art, such as the gene gun approach described in 456-460. This can be carried out using dendritic cells or progenitor cells. Antigen loading of dendritic cells is performed using dendritic cells or progenitor cells. expressing polypeptides, DNA (e.g., plasmid vectors), or RNA; or antigens. Recombinant bacteria or viruses (e.g., adenovirus, adeno-associated virus (AAV) (e.g., A AV types 5 and 2), alphaviruses (e.g., Venezuelan encephalitis virus (VEEV), syncytial virus) Dobisvirus (SIN), Semlik Forest Virus (SFV), Herpesvirus, Arenavirus (For example, lymphocytic choriomeningitis virus (LCMV), measles virus, poxvirus (example) For example, modified vaccinia ankara (MVA) or fowlpox, paramyxovirus, lentwin Incubate with S or rhabdovirus (e.g., vesicular stomatitis virus (VSV)). This can be achieved by: Before polypeptide loading, polypeptide This is covalently linked to an immunological partner (e.g., a carrier molecule) that provides assistance to T cells. It can be jugated. Alternatively, dendritic cells can be separated or in the presence of antigenic polypeptides. It can be pulsed with an unconjugated immunological partner.

[0200] This invention encodes a specially designed short chemically synthesized epitope of polypeptide antigens. It provides delivery of the fragment to antigen-presenting cells. Those skilled in the art can also use it as a long synthetic peptide (SLP). These known types of molecules stimulate (or load) cells in vitro. (Gornati et al., 2018, Front.Imm, 9:1484) or polypeptide antigens in vivo As a method for introducing it into original-presenting cells (Melief and van der Burg, 2008, Nat Rev Cance) (r, 8:351-60), the present invention provides a therapeutic platform for using the antigenic polypeptide. They are likely aware that they are providing it.

[0201] In one embodiment, the antigen-presenting cells of the present invention, preferably dendritic cells, can be used as pharmaceuticals. A pharmaceutical composition is provided which contains a suitable carrier. Such a composition is for parenteral administration. This can be a suitable sterilization composition. See, for example, the disclosure of the pharmaceutical composition described above. .

[0202] In one embodiment, the antigen of the present invention, preferably a dendritic cell, is used in pharmaceuticals. The presenting cells are provided.

[0203] Cancer cells express sequences selected from (a) to (h), where cancer cells express sequences selected from (a) to (h). A method for treating a person suffering from cancer who expresses a selected polypeptide sequence, In humans, preferably the antigen-presenting cells of the present invention, which are dendritic cells, or the antigen-presenting cells of the present invention A method is also provided which includes administering a composition containing the above.

[0204] In one embodiment, cancer cells express a corresponding sequence selected from (a) to (h). The antigen-presenting cells of the present invention, preferably dendritic cells, for use in treating cancer, Alternatively, a composition comprising the antigen-presenting cells of the present invention is provided.

[0205] Antigen-presenting cells or compositions administered to humans, or used to induce an immune response in humans. The antigen-presenting cells or compositions to be used are determined by the sequence expressed by the cancer. Therefore, the design of fusion proteins, nucleic acids, vectors, or compositions and the sequences in which cancer is expressed. There is a relationship between them.

[0206] In one embodiment, the exosomes of the present invention are included together with a pharmaceutically acceptable carrier. A pharmaceutical composition is provided. Such a composition is a sterile composition suitable for parenteral administration. It is possible to do so. For example, see the disclosure of the pharmaceutical composition above. The composition is an immunostimulant. The quality may be arbitrarily included - see the disclosure of immunostimulants above.

[0207] In one embodiment, the exosomes of the present invention for use in pharmaceuticals are provided.

[0208] Cancer cells express polypeptide sequences selected from (a) to (h), where cancer cells express (a A method for treating a person suffering from cancer who expresses a sequence selected from (h), By administering the exosomes of the present invention or a composition containing the exosomes of the present invention to a human being... Methods including the above are also provided.

[0209] In one embodiment, cancer cells express a corresponding sequence selected from (a) to (h). Exosomes or the exosomes of the present invention for use in treating cancer A composition containing mu is provided.

[0210] In any one of the embodiments described above, preferably, cancer is melanoma, particularly skin cancer. It is melanoma.

[0211] (Diseases that should be treated) As described elsewhere, Sequence IDs 1-8 are CLT antigens that are overexpressed in cutaneous melanoma. This is the corresponding polypeptide sequence.

[0212] The immune response is the corresponding polypeptide sequence or its immunogenic fragment selected from (a) to (h). Alternatively, it can be induced in cancers that express the variant. In relation to this, "Corresponding" means that the tumor corresponds to, for example, sequence number A (A is the cyst of sequence numbers 1-8 or 1-10). It expresses (or has the potential to express) one of the following: or a variant or immunogenic fragment thereof. If the antigen pool of the present invention has high efficacy, polypeptides, nucleic acids, fusion proteins, and this This means that the pharmaceuticals containing these are based on Sequence ID No. A or its variants or immunogenic fragments. do.

[0213] The immune response involves CD8+ T cells, CD4+ T cells, and / or antibody responses, particularly CD8+ cytolytic T cells. This may include vesicular responses and CD4+ helper T cell responses.

[0214] The immune response is directed towards tumors, particularly tumors that express polypeptide sequences selected from (a) to (h). It can be triggered in response.

[0215] In a preferred embodiment, the tumor is a melanoma, such as a cutaneous melanoma.

[0216] The tumor can be a primary tumor or a metastatic tumor.

[0217] In one embodiment of the present invention, cancer cells are selected from the polypeptide sequences of (a) to (h). or a method for treating human cancer expressing the immunogenic fragment or variant thereof, Even if not present, a population of white blood cells including T cells, along with antigen-presenting cells, is arbitrarily collected from the person concerned. To stimulate and / or amplify the T cells in the presence of the antigen pool or composition of the present invention. to, and to include at least some or all of the leukocytes, including stimulated and / or amplified T cells. A method is provided which includes reintroducing the substance into the human.

[0218] In a further embodiment of the present invention, cancer cells are selected from (a) to (h) for a sequence or the An immunogenic fragment or variant is expressed, and here, cancer cells are selected from (a) to (h). Treating human patients with cancer who express the sequence or its immunogenic fragment or variant. A method of treatment, wherein the human is given the T cell population of the present invention, T cells, antigen-presenting cells, and exoskeletons. A method is provided which includes administering a substance or composition.

[0219] In yet another embodiment of the present invention, cancer cells are selected from (a) to (h) The cells express a sequence or an immunogenic fragment or variant thereof, where cancer cells (a)~(h) Cancer expressing polypeptide sequences selected from or their immunogenic fragments or variants. A method for treating a person suffering from a disease, wherein the person is given a T cell population according to the present invention, T cells A method is provided which includes administering antigen-presenting cells, exosomes, or a composition.

[0220] In another embodiment of the present invention, cancer cells are selected from (a) to (h) in corresponding sequences or For use in treating human cancers that express the immunogenic fragment or variant. The present invention provides a T cell population, T cells, antigen-presenting cells, exosomes, or compositions.

[0221] Another embodiment of the present invention is a method for treating a person suffering from cancer, (a) The cancer cells are selected from (a) to (h) and are polypeptide sequences or immunogenic fragments thereof. (b) the process of determining whether the variant is expressed; and if so, the process of determining whether the human is Polypeptides, nucleic acids, antigen pools, compositions, T cell populations, T cells, antigen-presenting cells according to the invention A method is provided which includes the step of administering, or exosomes.

[0222] In any one of the embodiments described above, preferably, cancer is melanoma, particularly skin cancer. It is melanoma.

[0223] The transcripts corresponding to Sequence ID Nos. 14 and 20 were also overexpressed in uveal melanoma. Results and In an alternative embodiment, the tumor is a uveal melanoma and / or the tumor is The sequence selected from Sequence IDs 1, 3, and 4 is expressed. Therefore, the fusion protein of the present invention Therefore, this substance may be suitable for patients with uveal carcinoma.

[0224] (Antigen combination) The antigen pool, T cell population, T cells, antigen-presenting cells, exosomes, or compositions of the present invention , other immune responses that trigger an immune response against melanoma, such as cutaneous melanoma or uveal melanoma. It can be used in combination with immunogenic antigens. These other immunogenic antigens are diverse. These can be obtained from various sources, including well-documented melanoma-associated antigens, for example, This may include GPR143, PRAME, MAGE-A3, or pMel(gp100). Alternatively, these may include patient-specific Heterogenous antigens (Lauss et al. (2017), Nature Communications, 8(1), 1738. http: / / doi.o rg / 10.1038 / s41467-017-01460-0), retaining intron nascent antigen (Smart et al. (2018), Na Nature Biotechnology (http: / / doi.org / 10.1038 / nbt.4239), new splicing variants Live antigen (Hoyos et al., Cancer Cell, 34(2), 181-183. http: / / doi.org / 10.1016 / j.ccell) .2018.07.008; Kahles et al. (2018), Cancer Cell, 34(2), 211-224.e6.http: / / doi.or g / 10.1016 / j.ccell.2018.07.001), T cell epithelium associated with impaired peptide processing Melanoma antigens (TIEPPs; Gigoux, M) belong to a category known as antigens that code for the melanoma cell. . and Wolchok, J. (2018), JEM, 215, 2233, Marijt et al. (2018). JEM 215, 23 25) or other types of melanoma antimicrobial agents, including newly discovered antigens (including CLT antigens) The source may be included. Furthermore, antigenic peptides derived from these various sources are (i) nonspecific (ii) immunostimulants / adjuvant species and / or (ii) for example, induced by co-administered antigens It is known to induce strong CD4 helper T cells to amplify the anti-melanoma specific response. Antigens (as polypeptides, or these) that contain the universal CD4 helper epitope are present. (delivered as a polynucleotide or vector encoding the CD4 antigen) in combination with It is also possible to do so.

[0225] Different antigens that exist in the form of polypeptides and / or nucleic acids encoding polypeptides are They can be formulated in the same formulation or in separate formulations. Different antigens can be formulated in separate polyp A polypeptide, nucleic acid, or polypeptide is fused to a second or further polypeptide. It can be provided as a composite protein and / or as a nucleic acid encoding the fusion protein. ru.

[0226] More generally, when two or more components are used in combination, those components are, for example: (1) As two or more individual and / or separate antigenic polypeptide components; (2) As a fusion protein containing both (or further) polypeptide components; (3) As two or more polypeptides and two 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 single molecule encoding a fusion protein containing both (or additional) polypeptide components. As polynucleotides It can be presented.

[0227] For convenience, if several components are present, they are considered a single fusion protein or a single It is contained in the polynucleotide encoding the fusion protein. In one embodiment of the present invention All components are provided as polypeptides (for example, in a single fusion protein). In an alternative embodiment of the present invention, all components are polynucleotides (e.g., single Polynucleotides (for example, polynucleotides that encode a single fusion protein) and It will be provided as is.

[0228] (Combination therapy) A method for treating cancer according to the present invention is a combination of other therapies, in particular checkpoint inhibitors and It can be administered in combination with interferon.

[0229] Adoptive cell therapy (APC and T cell-based) enhances its immunogenicity, for example , to enhance the magnitude and / or width of the induced immune response, or to enhance other activities (e.g.) (Provides activation of other aspects of the innate or adoptive immune response, or destruction of tumor cells) It can be used in combination with other ingredients designed for this purpose.

[0230] Therefore, the composition of the present invention (i.e., immunogenic composition or pharmaceutical composition) or several A kit of such a composition includes the antigen pool, T cell population, T cells, and antigen presentation according to the present invention. Cells or exosomes combined with a pharmaceutically acceptable carrier; and (i) one or more Further immunogenic polypeptides or immunostimulatory polypeptides (e.g., interferon, I) L-12, checkpoint blocking molecules, or nucleic acids that encode such molecules, or similar (ii) vectors containing nucleic acids, (ii) translation and / or presentation of polypeptide products that are the subject of the present invention Small molecules that enhance (e.g., HDAC inhibitors or the epigenetic properties of cancer cells) Other drugs that modify the yl) or biologics (polypeptides, or such that code It can include nucleic acids, or delivered as a vector containing such nucleic acids. ru.

[0231] Chemistry interferes with normal proteins on cancer cells or proteins on T cells that respond to them. Blockpoint inhibitors attempt to overcome one of cancer's main defenses against immune system attacks. Therefore, these inhibitors are particularly important drugs to combine with CLT antigen-based therapy. It could be a class.

[0232] Therefore, the antigen pool, immunogenic composition or pharmaceutical composition of the present invention, T cells, T cells Populations, antigen-presenting cells, or exosomes are administered in combination with checkpoint inhibitors. It is possible. An example of a checkpoint inhibitor is a PD-1 inhibitor, for example, pembroliz Mab (Keytruda) and nivolumab (Opdivo), PD-L1 inhibitors, such as atezolizumab (Tecent) Examples include riq), avelumab (Bavencio), durvalumab (Imfinzi), and CTLA-4 inhibitors. For example, ipilimumab (Yervoy) is selected.

[0233] Interferons (e.g., α, β, and γ) are substances that the body produces in very small amounts. It is a family of proteins. Interferons slow down or stop the division of cancer cells. , reducing the ability of cancer cells to defend themselves from the immune system, and / or multiple sides of the adoptive immune system It can enhance the surface. Interferon is usually administered as a subcutaneous injection, for example, in the thigh. It is administered to the area or abdomen.

[0234] Therefore, the antigen pool, immunogenic composition or pharmaceutical composition of the present invention, T cells, T cells Populations, antigen-presenting cells, or exosomes are interferon, for example, interferon It can be administered in combination with α.

[0235] Different forms of the present invention can also be combined, for example, the antigen pool of the present invention It can be combined with APCs, T cells, T cell populations, or exosomes (discussed below). (It can be done).

[0236] One or more of the present invention can also be combined with conventional anti-cancer chemotherapy and / or radiation therapy. ru. [Examples]

[0237] (Examples) (Example 1 - CLT Identification) The objective is to identify cancer-specific transcripts that consist entirely or partially of LTR elements. That was the case.

[0238] As the first step, the inventors developed a comprehensive pan-cancer transcriptome using de novoasene. To achieve this, a wide variety of cancer types (32 cancer types (primary melanoma 31)) were studied. And 24 sex-balanced samples derived from each of malignant melanoma1); Table S1) representative cancers 768 patient data obtained from the Cancer Genome Atlas (TCGA) consortium. RNA sequencing reads derived from the sample were used in the genome guide assembly. Another balanced sample (excluding sex-specific samples) was processed using cutadapt(v1.13) (Marcel M.'s Using the reference (2011, EMBnet J., 17:3), adapter trimming and quality (Q20) After trimming and length filtering (both reads of the pair ≥ 35 nucleotides), each , khmer(v2.0) (Crusoe et al., 2015, F1) to set the maximum and minimum depths to 200 and 3. Using 000Res., 4:900), KMER normalized (k=20). Reads used throughout TCGA Using the same settings as above, STAR (2.5.2b) was used to map to GRCh38, and Trinity (v2.2.0 (Trinity, Grabherr, MG et al., 2011, Nat. Biotechnol., 29:644-52) We disabled the built-in in silico depth normalization and performed genome-guided assembly. The majority of the assembly process was completed within 256GB of RAM on a 32-core HPC node, and failed. The process was rerun using a 1.5TB RAM node. The resulting contigs were poly(A) trimmed. Filtering (trimpoly in SeqClean v110222) and entropy filtering (≧0.7) are performed to reduce Removed quality and artifact contigs (bbduk in BBMap v36.2). By cancer type Then, the original 24 samples were treated with Salmon (v0.8.2 or v0.9.2) (Patro, R. et al., 2017, Nat. Meth). Using ods, 14:417-419), pseudo-mapping was performed on the cleaned assembly, and 0.1 Contigs showing less than 1 million transcript (TPM) expression were removed. The remaining contigs were removed. Using GMAP (v161107) (Wu et al., 2005, Bioinf., 21:1859-1875), for GRCh38 Mapping and contigs that are not aligned with 85% or more identity over 85% or more of their length It was removed from the assembly. Finally, all cancer type assemblies were flattened together. The following is a reference to gffread (Cufflinks v2.2.1) (Trapnell et al., 2010, Nat. Biotech., 28:511-5) Using 15), the longest continuous transcript was integrated. This assembly process involves repeated elements. Because it is specifically designed to enable evaluation of the ment, the single-exon transcript is Retained but flagged. The integrity and quality of the transcript assembly are as per GENCODE v2. Comparison with 4basic and MiTranscriptome1 (Iyer et al., 2015, Nat. Genet., 47: 199-208) The inventors evaluated the unique splice locations displayed in GENCODE. Edit the list and check if this splice site is within the 2-nucleotide grace window. We tested whether they were present in the lipome assembly. This process resulted in 1,001,93 One transcript was identified, of which 771,006 were spliced, and 230,925 were single-transcribed. It belonged to one exon.

[0239] Separately, the assembled contigs are annotated using genome repeat sequence annotation. By superimposing them, transcripts containing LTR elements were identified. LTR elements and non-LT The R element was annotated as previously described (Attig et al., 2017, F (ront. In Microbiol., 8:2489). Briefly speaking, known human repeat families (Dfam 2.0 Using Hidden Markov Models (HMMs) representing Ibrary v150923, nhmmer (Wheeler The RepeatMasker Open-3.0 (Smi) was constructed from the literature (2013, Bioinform., 29:2487-2489) by the same author. Using t, A., R. Hubley, and P. Green (http: / / www.repeatmasker.org, 1996-2010), G RCh38 was annotated. HMM-based scanning was compared to BLAST-based methods. This improves the accuracy of annotations (Hubley et al., 2016, Nuc.Acid.Res., 44:81-89). RepeatMasker annotates the LTR and internal regions separately, resulting in a tabular output. The forces were analyzed, and adjacent annotations for the same element were merged. The process yielded 181,967 transcriptions containing one or more complete or partial LTR elements. Products were obtained.

[0240] Using Salmon, the total number of transcripts (TPM) per million units was estimated for all transcripts, and each cancer Expression within the type was obtained from 811 healthy tissue samples (from TCGA if available, and separately from GT) Ex (The Genotype-Tissue Expression Consortium) From 2015, Science, 348:648-60), healthy tissue-matched controls for all cancer types This was compared to barrel expression. If the transcript was detected in any sample at a TPM greater than 1, A substance was considered cancer-specific if it was thought to be expressed in cancer and met the following criteria: i) Expressed in 6 or more of the 24 samples of each cancer type; ii) Expressed in 90% of all healthy tissue samples iii, expressed at less than 10 TPM in % or more; iii, in any of the target cancer types It is expressed at more than three times the median expression level in the irradiated tissue type; and iv, the target cancer type odor And it is expressed at more than three times the 90th percentile of each healthy tissue (if available). It can be done.

[0241] Subsequently, a list of cancer-specific transcripts containing complete or partial LTR elements is created. Cross-referenced with the list of transcripts, 5,923 transcripts that meet all criteria (cancer-specific LTR elements) A list of menth-spanning transcripts (referred to as CLTs) was created.

[0242] Further curation was performed on CLT 403, which is specifically expressed in melanoma, to correct misdiagnosis. This corresponds to the assembly of potentially assembled contigs and cellular genes. Contigs were excluded. Further manual evaluation was performed to determine the splicing pattern. This was confirmed by RNA sequencing reads derived from melanoma. To discard any CLT with a median expression level exceeding 1 TPM in any normal GTEx tissue, They were then selected.

[0243] Of the 403 CLTs for cutaneous melanoma, 97 passed through these filters.

[0244] (Example 2 - Immunopeptide analysis) Mass spectrometry (MS)-based immunopeptidemic analysis is related to HLA molecules and cell surface This is a powerful technique that enables the direct identification of specific peptides (pHLA) presented. The method involves the affinity purification of pHLA from biological samples such as cells or tissues by capturing anti-HLA antibodies. Then, the isolated HLA molecules and the bound peptides were separated from each other and subjected to mass spectrometry. By coupled nano-high-performance liquid chromatography (nUPLC-MS), the eluted particles were obtained. The plutidot was analyzed (Freudenmann et al., 2018, Immunology 154(3):331-345). Mass spectrometry. In the analysis, a specific peptide with a specified charge-to-mass ratio (m / z) is selected, isolated, and fragmented. Subsequently, the fragments were subjected to a second round of mass spectrometry (MS / MS) to determine their m / z values. Then, the fragmentation spectrum (MS / MS) was examined to select the type of fragment ion that produced the detected fragment ions. The amino acid sequence of the peptide can be accurately identified.

[0245] MS / MS spectral interpretation and subsequent peptide sequence identification are performed using experimental data and a reference database. It depends on the agreement between the theoretical spectrum derived from the peptide sequence observed in the sample and the actual spectrum. All open readies derived from known transcriptomes or even the entire genome. By using a predefined list corresponding to the ORF, MS data can be processed. It is possible to search for it (Nesvizhskii et al., 2014, Nat. Methods 11:1114-1125). Matching these extremely large sequence databases limits the identification of the presented peptides. This leads to a very high false detection rate (FDR). Further technical problems (e.g., the mass of leucine = (mass of isoleucine), and theoretical problems (e.g., peptide splicing (Liepe et al. literature)). According to Science 354(6310):354-358, 2016, a known transcriptome or genome Restrictions related to the use of very large databases, such as databases created from the entire database. Therefore, in practice, a set of clearly defined potential polypeptide sequences To perform accurate immunopeptide analysis without referring to references and to identify novel antigens. This is extremely difficult (Li et al., 2016, BMC Genomics 17(Suppl 13):1031).

[0246] Bassani-Sternberg et al. (Bassani-Sternberg et al., 2016, Nature Commun., 7: 13404; Database link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894) is 2 MS / MS data collected from HLA-binding peptide samples derived from five patients with cutaneous melanoma were used in the study. These were compared against polypeptide sequences reported for the entire topotone. Analysis revealed tens of thousands of peptides that matched known human proteins. As described, these peptides contain PRAME, MAGEA3, and TRPM1 (melastatin), The study included peptides found within several tumor-associated antigens (TAAs).

[0247] The inventors obtained frozen tumor tissue from six patients diagnosed with melanoma. (0.05-1g) The sample was homogenized, the lysate was rapidly centrifuged, and the clarified lysate was used to produce anti-human HL A mixture of protein A (ProA) beads covalently bound to a class I monoclonal antibody (W6 / 32) The mixture was incubated overnight at 4°C to improve the binding of HLA class I molecules to the antibody. (Ternette et al., 2018 Proteomics 18, 1700465). By using 10% acetic acid... Then, the HLA class I binding peptide was eluted from the antibody, followed by reverse-phase column chromatography. Using this method, peptides were separated from other high-molecular-weight components (Ternette et al., 2018). The prepared eluted peptides were subjected to nUPLC-MS to obtain specific peptides with a specified charge-to-mass ratio (m / z). Selected, isolated, and fragmented within the mass spectrometer, and subjected to a second round of mass spectrometry (MS / MS), The m / z of the obtained fragments was determined (Ternette et al., 2018), and each of these tumor samples was then analyzed. We created an MS / MS dataset corresponding to the immunopeptideome.

[0248] By applying detailed knowledge of immunopeptidomics evaluation, the inventors have identified 25 black individuals. Spectra from the PXD004894 HLA class I dataset for chromoma patients (Bassani-Stern The present inventors prepared the CLT-derived ORF (from Example 1) using the literature by berg et al., 2016. The spectra of HLA-class I datasets from six melanoma patients were matched. The analysis was performed: Analysis A: 23Am derived from a subset of approximately one dozen CLTs identified in Example 1. Concatenate predicted ORFs exceeding 0 acid residues to form a single polypeptide file for each CLT. These linked ORF polypeptides are then processed using PEAKS™ software (v8.5, Bioinfo By using (rmatics Solutions Inc.), human proteome (UniProt database) Along with all the polypeptides found within, PXD004894 HLA class I was found in 25 melanoma patients. The data set was matched. Analysis B: 23Am derived from a subset of approximately one dozen CLTs identified in Example 1. Mascot software generates polypeptide files consisting of each predicted ORF exceeding 0 acid residues. By using this, the human proteome (UniProt and masDB databases) can be found Along with all polypeptides, the PXD004894 HLA class I dataset was added to 25 melanoma patients. I compared them. • Analysis C: All predictions derived from the 97 CLTs identified in Example 1 for amino acid residues of length 10 or longer ORF uses PEAKS™ software (v8.5 and vX, Bioinformatics Solutions Inc.). As a result, along with all the polypeptides found in the human proteome (UniProt), 25 individuals PXD004894 HLA class I dataset of melanoma patients and H of 6 melanoma patients of the inventors The data was matched against the LA Class I dataset.

[0249] Most class I HLA-binding peptides found within cells are constitutively expressed proteins. Because it is of quality, simultaneous matching of these databases with the UniProt proteome is the first This helps to ensure that the assignment of CLT ORF sequences to MS / MS spectra by Meisha et al. is correct. PEAKS software, like other MS / MS matching software, uses probability values ​​(-10 lgP; see Table 1). These values ​​were assigned to their respective spectral assignments, and the assignments were quantified.

[0250] The results of these studies correspond to the amino acid sequence of CLT-derived ORFs and to known human proteo Bassani-S does not support polypeptide sequences present in the UniProt and / or masDB database. Tumor samples from 25 patients examined by Ternberg et al. and the inventors' dataset >50 HLA class I molecules associated with immunoprecipitated HLA class I molecules from six melanoma patient samples. I identified Do.

[0251] Further manual adjustment of peptide spectra assigned by PEAKS software Using this revision, the data was mapped to eight CLT-derived ORFs, and as a result, the CLT antigen (Table 1; sequence number) We confirmed the spectral assignment to the peptides defined as numbers 1-8.

[0252] The detection of these peptides associated with HLA class I molecules is based on the eight ORFs from which they originate. It is translated within melanoma tissue, processed via the HLA class I pathway, and finally, HLA class This supports the idea that it is presented to the immune system as a complex with the S1 molecule. Table 1 shows that in CLT antigen The observed peptide characteristics are shown. Figures 1-37 show each of the peptides listed in Table 1. These figures show representative MS / MS spectra. 2 Depending on the individual patient SKCM Tumor (PEAKS™ software) is used to obtain data from the inventors' internal dataset or (Detected in images extracted from the Bassani-Sternberg et al. dataset stored in PRIDE) The displayed peptide sequence fragment spectra are shown. All detected fragments are spectral It is shown in the peptide sequence above the s, and the most abundant fragment ion is each s It is assigned within the vector. In Figures 1-2, 4-6, 8-9, 11-12, and 14-37, the figure The panel below shows the peptide sequences assigned to the MS / MS spectra, while the same The data is shown in tabular form on the right side of Figures 3, 7, 10, 13, and 19. The fragment ions are The following annotations are used: b: N-terminal fragment ion; y: C-terminal fragment ion; -H2O : water loss; -NH3: ammonia loss; [2+]: bicharged peptide ion; pre: unfragmented Precursor peptide ions. Consistent with the high -10 lgP scores assigned to the peptides in Table 1. These spectra show the peptide sequences (sequence number) discovered by the inventors in these analyses. It contains numerous fragments that precisely match numbers 9-12, 18-19, 31-32, 36-39, 45, 48-54. .

[0253] Use NetMHCpan 4.0 prediction software (http: / / www.cbs.dtu.dk / services / NetMHCpan / ) By doing so, all HLA class I members in Table 1, which are 9 amino acid residues or longer, were detected. Evaluating the peptides, their predicted effects on HLA class I type A and B supertypes The strength of the bond was determined. From the results of these predictive studies, 17 peptides (or their complete forms) All 9-mers in the sequence bind to at least one of the tested supertypes. This was shown to be predictable (see Table 2). Of these, many of the sequences were considered. A high level of confidence (low rank score %) is achieved in a specific type within the HLA Class I Supertype. It was predicted that all detected peptides would bind to the standard set of HLA types. The fact that this was expected provides further validation for their detection. Furthermore, all peptides found in tumor samples derived from the inventors' dataset are NetMHCpan can bind to one of the HLA types detected by the inventors in patient samples. Predicted by 4.0. The HLA type is all associated with the peptide discovered by the inventors. Regarding the patients, the literature by Bassani-Sternberg et al. (2016, Nature Commun., 7: 13404) is reported. Although not officially reported, if this is reported, the inventors believe that known HLA We found a match between the HLA type predicted for Ip.

[0254] Further assignment of tumor tissue-derived MS spectra to peptide sequences discovered by the present inventors To ensure certainty, peptides having these discovered sequences were synthesized and the original research was conducted. nUPLC-MS using the same conditions applied to tumor samples in the study 2 Provided for (Bassani-Sternberg et al.) Reference, 2016, Nature Commun., 7: 13404; data from the inventors). Selected peptides A comparison of the spectra is shown in Figures 39-54. In each figure, the upper spectrum represents the tumor. Corresponding to ulcer samples (PRIDE database (Bassani-Sternberg et al., 2016, Nature Commun.)). , 7: 13404; Database link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD0 (04894 or our database), the spectrum below shows the synthetically produced peptide of the same sequence. This corresponds to the selected m / z values ​​of the detected ion fragments in these MS / MS spectra. These figures show the above / below peaks of each fragment. This clarifies (the experimental determination of the relationship between tumor-derived fragment ions and synthetic peptide-derived fragment ions). Slight differences in m / z values ​​are well within the acceptable range of m / z <0.05 Daltons, tumor This confirms the accuracy of the assignment of tissue-derived spectra to each CLT-coding peptide.

[0255] In summary, the data shown in Tables 1 and 2 and Figures 1-53 indicate the relationship between melanoma patients and This provides very strong evidence regarding the translation, processing, and presentation of the corresponding CLT antigen. It is.

[0256] To further support the cancer specificity of these CLTs, the inventors have examined 37 normal tissue samples (10 It processes normal skin tissue, 9 normal lung tissues, and 18 normal breast tissues, and immunopeptides. Prepared for cydome analysis. The inventors have used HLA-class I dendrocytes derived from these normal tissue samples. The spectra of the datasets are matched, and Peaks™ software (V8.5 and X) is used to analyze human data. Along with all polypeptides found in the proteome (UniProt), CLT antigens 1, 2, 3, and 4, We searched for all possible peptide sequences derived from polypeptide sequences 5, 6, 7, and 8. Peptides derived from CLT antigens 1, 2, 3, 4, 5, 6, 7, and 8 were found in a set of normal tissue samples (Table 3). It was not detected within the sample, providing further evidence that CLT has cancer-specific expression.

[0257] In summary: Identifying immunopeptide-dominant peptides derived from predicted ORFs is crucial for these CLs. This indicates that T is translated into polypeptides (SEQ ID NOs: 1-8; referred to as CLT antigens) within tumor tissue. These are then processed by the cellular immune surveillance system, and the component peptides are processed. The cytoplasm is loaded onto HLA class I molecules, and the cell then processes the resulting peptide / HLA This makes it possible to target T cells that recognize the class I complex for cell lysis. These CLT antigens and their fragments are black in patients whose tumors express these antigens. It is considered useful in various therapeutic modalities for the treatment of tumors. Table 1: Cross-reference of CLT antigen name and sequence number for immunopeptide-based analysis of melanoma samples. Therefore, the list of identified peptides [Table 1] TIFF2026122961000003.tif249170TIFF2026122961000004.tif158170 1 HLA class I peptides identified by mass spectrometry. 2 Bassani-Sternberg et al., 2016, Nature Comm., 第7巻, 13404号 (Mel-3、Mel-5、Mel-8、M el-16、Mel-21、Mel-27、Mel-29、Mel-30、Mel-36、Mel-39、Mel-41); 本発明者らのデー タセット(1MT1、2MT1、2MT3、2MT4、2MT10、2MT12). 3 Calculated peptide mass. 4 PEAKS (商標) program - 10lgP values for peptides that achieved the maximum match of peptides / patients with multiple spectrum detections are shown. Values for peptides identified by analysis B performed using Mascot software are not available (na). Values for peptides identified by analysis B performed using Mascot software are not available (na). 5 Number of spectra in which the peptide was detected. 6 Deviation between the observed mass and the calculated mass; selected ppm values for peptides with multiple spectra are shown. Values for peptides identified by analysis B are not available (na). not available (na). Table 2: Peptides identified by mass spectrometry (length > 9 residues) with cross-reference of CLT antigen names and sequence numbers for 18 HLA class I supertype alleles (HLA-A01:01, HLA-A02:01, HLA-A0 (長さ>9残基)の18のHLAクラスIスーパータイプアレル(HLA-A01:01、HLA-A02:01、HLA-A0 3:01、HLA-A11:01、HLA-A24:02、HLA-A25:01、HLA-A26:01、HLA-A68:01、HLA-B07:02、HL It should be noted that there seems to be some inconsistent or unclear information in the original text, especially in parts like "Mel-3、Mel-5、Mel-8、M" where the full meaning is not entirely clear. This translation attempts to make sense of the text as best as possible based on the given rules.A-B08:01, HLA-B15:01, HLA-B18:01, HLA-B27:05, HLA-B35:01, HLA-B35:03, HLA-B40:01 Predicted NetMHCpan4.0 binding to HLA-B40:02 and HLA-B51:01. [Table 2] 1 Predicted matched to HLA Class I Supertype with rank score of ≤5.0% Combine. 2 The number of HLA Class I supertypes predicted to be combined with a rank score of ≤5.0% was 18. 3 The number of 18 HLA class I supertypes predicted to combine with a rank score of ≤2.0%. 4 The number of 18 HLA class I supertypes predicted to combine with a rank score of ≤0.5%. 5 Bassani-Sternberg et al., 2016, Nature Comm., 7: 13404 (Mel-3, Mel-8, Mel-16, Mel-21, Mel-27, Mel-29, Mel-30, Mel-36, Mel-39, Mel-41); Dataset of the inventors (1MT1, 2MT1, 2MT3, 2MT4, 2MT10, 2MT12). Table 3. Number of peptides derived from CLT antigens 1-8 in a set of normal tissue samples. [Table 3]

[0258] The results shown in Examples 1 and 2 of this specification, in whole or in part, indicate cancer. Nomu Atlas (TCGA) Research Network (http: / / cancergenome.nih.gov / ); and genotype- Tissue Expression (GTEx) Project (Common Fund of the Office of the Director of the National Institutes of Health) By the office of the Director of the National Institutes of Health, as well as NCI, Data created by (supported by NHGRI, NHLBI, NIDA, NIMH, and NINDS) It is based on Ta.

[0259] (Example 3 - HERVFEST) The Functional Enhancement of Specific T Cells (FEST) technology is based on the detection of patient T cells that respond to MANA epitopes. Therefore, among the "muta-associated neoantigen" (MANA) repertoire found in tumor cells of cancer patients... It is used to identify existing treatment-related tumor-derived epitopes (Anagnostou et al.) References: Cancer Discovery 2017; Le et al., Science 2017; Forde et al., NEJM 2018; (Danilova et al., CancerImmunol. Res. 2018). Examples 1 and 2 (Tables 1-3, Figures 1-53) explain the process. The application of FEST technology to CLT antigens discovered using the revealed method is to CLT antigens in cancer patients. It can be used to confirm the treatment-related T cell response to [the condition].

[0260] Other assays for identifying epitope-specific T cells in immune-exposed subjects (e.g.) Similar to ELISPOT, the "FEST" technology includes antigen-presenting cells and suitable antigenic peptides. By activating / expanding congeneral T cells in a cusvivo culture, their specificity is enhanced. The technology involves the T cell receptor (TCR) DNA sequence present in these amplified cultures (specifically : Using next-generation sequencing (TCRseq) targeting the TCR-Vβ CDR3 region, a single antigen A target peptide derived from (or multiple antigens) is cultured together with individual peptides derived from a panel of target peptides. In terms of detecting specific TCRs that are enlarged in the cells, it differs from other immunological assays. This differs from the previous method. Using the application of TCRseq to tumor tissue from the same patient, ex vivo peptide stimulation was performed. TCR / T cells detected in the cultured material are tumor-infiltrating phosphorus cells found in cancer tissue in situ. It can also indicate whether it is present within Pacyl cells. Therefore, MANAFEST is derived from cancer patients. Numerous sudden mutations were found by whole exome sequencing of normal and tumor tissue. This allows for the identification of functionally related MANA peptides in heteropeptides using patient T cells. It has been proven to be a powerful technique for identifying MANA epitopes that are recognized by (L e et al., Science 2017; Forde et al., NEJM 2018; Danilova et al., Cancer Immuno (nol. Res. 2018; Smith et al., J Immunother Cancer 2019).

[0261] The application of the MANAFEST method (Danilova et al., Cancer Immunol. Res. 2018) to CLT antigen is as follows: The procedure was carried out as described above. This method, which the inventors refer to as HERVFEST, consists of the following steps: Stage 1: Predicted to contain an epitope that efficiently binds to the selected HLA class I allele. The peptide was identified in the CLT antigen. Step 2: PBMCs from suitable melanoma patients were HLA class I Each type was matched to the peptide library selected in Step 1. Step 3: These patients The derived PBMCs were separated into T cell fractions and non-T cell fractions. The non-T cells were added to the patient's T cells and then returned to the patient. Then, it is divided into 20-50 wells (each containing 250,000 T cells), and various... T cell growth factor and individual CLT antigen-derived synthetic peptides (selected in step 1 / 2) for 10 days The cells were allowed to grow in between. Step 4: TCRseq (sequencing of TCR-Vβ CDR3 sequence) was performed on all wells. The procedure was carried out and amplified in the presence of individual CLT antigen-derived peptides (however, the control peptide was used). We identified the TCR-Vβ CDR3 sequence (which was not amplified in the presence or absence of peptide stimulation). Therefore, the presence of amplified TCR-Vβ CDR3 sequences in individual wells of the assay indicates that A CLT antigen-derived peptide that induced an immune response in melanoma patients was identified. Step 5: TCRseq The procedure was performed on a tumor sample, and T cells with TCRs amplified by CLT antigen were found in the patient's tumor. To determine whether to masturbate, T cells with these TCRs are affected by CLT antigens within the patient's tumor. This could also provide further evidence that it recognizes the peptide.

[0262] The HERVFEST assay was performed using peptides derived from CLT antigens 1-4 (SEQ ID NOs: 1-4). The panel of peptides used in these studies (see Step 1 above) is from the inventors. It strongly binds to eight commonly found HLA class I types in patient tumor samples available for analysis. This was based on NetMHC prediction of CLT antigen-derived peptides that are predicted to be present. Table 4 shows CLT antigen-derived peptides in which one or more TCRs were amplified. The HLA class I type of the CLT antigen peptide tested using patient PBMC-derived cultures is also shown. Yes, those PBMCs were tested in these studies, and patients who amplified one or more TCRs in this assay... Table 5 shows the HLA Class I types.

[0263] Panel A in Figure 54 shows publicly available data demonstrating TCR amplification by NSCLC patient-specific MANA peptides. This is shown (Forde et al., NEJM 2018). The vertical axis is the MANA peptide listed on the horizontal axis. This is the expressed TCR-Vβ for each well of cells cultured in the presence of the control peptide. This indicates the prevalence of CDR3 AA. Amplification in wells containing MANA7 indicates the patient's T cell repertoire. This shows that Tree contains T cells that respond to this peptide. Panels B and C in Figure 54 Two black animals incubated in the presence of the displayed CLT antigen peptide and control peptide. This shows representative TCR amplification data from PBMCs derived from chromoma patients. Similar to Panel A, Panel A The specific amplification observed in B and C indicates that the T cell repertoire of these melanoma patients is specific to C. This shows that it contains T cells that react with LT antigen-derived peptides. Panel B shows CLT antigens 1 and 2. , and 15 class I HLA-A derived from 4 * All wells stimulated with the 02 peptide panel The frequency of TCRs detected in LMSSFSTLASL-stimulated wells of PBMCs derived from melanoma patient 222B. This shows that three TCR sequences were amplified. [ka] This is HLA-A derived from CLT antigen 2. * It is an O2-binding peptide. Panel C contains CLT antigens 1, 2, and 4. 15 Class I HLA-A * 02 peptide, and 24 claustrophobic antigens derived from CLT antigens 1, 2, 3, and 4 S I HLA-A * In all wells stimulated with the 03 peptide panel, the peptides were derived from melanoma patient 224B. This shows the frequency of TCRs detected in MVACRIKTFR-stimulated wells of PBMCs. One TCR The column was amplified. [ka] This is HLA-A derived from CLT antigen 2. * It is an O3-linked peptide.

[0264] The control peptides / conditions used in these experiments are as follows: CEF=CMV, EBV, and A mixture of influenza peptides; SL9, TV9, and QK1 = HIV-1 control peptides; peptides し = Culture in the absence of peptides; baseline = T cells before culture.

[0265] Figure 55 shows CLT antigen 1 amplified in one or more TCRs in a completed study using these patients. This shows a summary of all CLT antigen peptides for ~4. Each panel contains an immunopeptide. The amino acid sequences of CLT antigens 1-4, obtained by superimposing peptides detected by dome analysis, are shown. These are (indicated by dashed underlined text or bold text; see Example 2). Below, the peptides detected by HERVFEST (see Figure 54) are listed for melanoma patients in whom they were detected. The identification number (Table 5) and the targeted HLA class I type are displayed together.

[0266] The characteristics of each HERVFEST detection method are defined as follows: • Standard characters: Significant amplification of a single TCR • Bold text: Significant amplification of multiple TCRs • Underlined italics: Significant amplification of a single TCR detected in other wells • Underlined and bold: Striking signs of multiple TCRs where at least one of them was detected in other wells Amplification.

[0267] These results indicate that CLT antigens 1-4 are present in melanoma patients and that these CLT antigens are derived from Strong evidence that these peptides induce specific T cell responses in these melanoma patients This provides evidence supporting the value of these CLT antigens as targets for therapeutic interventions to treat melanoma. I'm wearing it. Table 4: CLT antigen-derived peptides that amplified one or more TCRs in the HERVFEST assay. [Table 4] Table 5: Characterization of melanoma patient PBMCs used in the HERVFEST assay [Table 5]

[0268] (Example 4 - High affinity T cells specific to CLT antigen were deleted from the normal target T cell repertoire) (An assay to show that it is not the case.) Using the ELISPOT assay, CLT antigen-specific CD8 T cells were found to be different from the normal T cell repertoire of healthy individuals. —It is present in these patients, and therefore cancer-specific CLT anti-inflammatory drugs are present in the naive and thymic tissue of these patients. It may be shown that the deletion is not present due to central tolerance resulting from the expression of the original gene. The T assay involves several steps. Step 1: CD8 T cells and CD14 monocytes are collected from a normal blood donor. These cells are isolated from peripheral blood and typed with HLA class I to test specific cells. The CLT antigen is matched. CD8 T cells are magnetically labeled antibodies against the memory marker CD45RO. This allows for further subdivision into naive subtypes and memory subtypes. Step 2: Pulse CD14 monocytes with individual or pooled CLT antigen peptides for 3 hours, then Step 3: Co-culture with CD8 T cells for 14 days. Step 4: Isolate the proliferated CD8 T cells from these cultures. Then, restimulate overnight with fresh monocytes pulsed with peptides. These peptides include; individual CLT Antigen peptides, unrelated control peptides, or infectious agents (e.g., CMV, EBV, influenza). It is known to elicit a strong response to HCV or self-antigens (e.g., MART-1). It may contain peptides. Restimulation is performed with an anti-interferon-gamma (IFNγ) antibody. This is performed on coated plates. This antibody is used on peptide-stimulated T cells. It captures the IFNγ that is secreted. After activation overnight, the cells are washed off the plate and the plate is purged. IFNγ captured by the rate is detected with further anti-IFNγ antibodies and standard chromogenic dyes. If gamma-producing cells were present on the plate from the beginning, black spots will remain. The data obtained from the assay include the number of spots, median spot size, and spot strength. The median degree is included. These are measures of the frequency of IFNγ-producing T cells and the amount of IFNγ per cell. Furthermore, the magnitude of the response to the CLT antigen is measured by the number of spots or the number of spots. The specific response, measured at the median, was compared against monocytes without the specific peptide. It can be derived from the stimulus index (SI), which is obtained by dividing by the response. The valuation standard is derived by multiplying the stimulation index of the number of spots by the stimulation index of the spot intensity. In this way, by comparing the response to the CLT antigen with the response to the control antigen, Naïve The target group can be expanded by vaccination with CLT antigen-based immunogenic agents. It can be shown that it contains a robust repertoire of LT antigen-reactive T cells. Table 6 shows HLA ma CLT antigen-derived peptides that induced a significant CD8 T cell response from a controlled normal blood donor. A list is provided. The results are shown in Figures 56-63. The horizontal bars represent the mean of the data. M+t shows no peptide, negative control (monocytes and T cells). CEF shows positive control. The results show a mixture of 23 CMV, EBV, and influenza peptides. Statistical significance is not shown. This was measured using the one-way Anova ANOVA of the Kruskal-Wallis test, with Dunn correction applied to repeated measures. Corrected. Figure 56 shows HLA-A derived from CLT antigen 1 (CLT001 in the figure). * 02:01 Positive response to restrictive peptides This shows a significant CD8 T cell response from a regular blood donor. The example shown in Figure 57 is from the same blood donor. HLA-A * Peptides derived from CLT antigen 2 (CLT002 ​​in the figure) constrained by 02:01 This shows the CD8 response from a normal donor. Figure 58 shows the HLA response from CLT antigen 4 (CLT004 in the figure). -A * 02:01 showed a significant CD8 T cell response from normal blood donors to the restrictive peptide. Figure 59 shows HLA-A derived from CLT antigen 5 (CLT005 in the figure). * 03:01 - Normal blood against restraint peptides This shows a significant CD8 T cell response from the fluid donor. Figure 60 shows the response of CLT antigen 6 (CLT006 in the figure). Next HLA-B * 07:02 - Significant CD8 T cell response from normal blood donors to restraining peptides Figure 61 shows HLA-A derived from CLT antigen 7 (CLT007 in the figure). *03:01 - Against restrictive peptides This shows a significant CD8 T cell response from a normal blood donor. Figure 62 shows CLT antigen 8 (CL in the figure). HLA-A derived from T008) * 02:01 - Significant CD8 T cells from normal blood donors against restrictive peptides The response is shown. Figure 63 shows CLT antigens 1 and 4 in memory CD45RO-positive CD8 T cells (in the figure). HLA-B derived from CLT001 and CLT004 * This shows a lack of response to the 0702 restrictive peptide (P Nell A and C). In contrast, naive CD45RO-negative CD8 T cells from the same donor were CLT001 and CL The cells responded significantly to peptides derived from both T004 cells (Figure 63, panels B and D). Table 6: CLT antigen-derived antigens that induced a significant CD8 T cell response from HLA-matched normal blood donors. peptide [Table 6]

[0269] (Example 5 - Staining of reactive T cells with CLT antigen peptide pentamer and the peptides therefrom) (Killing of pulsed or CLT-expressing target cells) The presence and activity of CLT antigen-specific circulating CD8 T cells in healthy donors and melanoma patients are HLA class I / peptide pentamer ("pentamer") staining and / or in vitro sterilization assay Measurement can be performed using (i). Therefore, this will be explained in Examples 1 and 2 (Tables 1-3, Figures 1-53). Using the application of these methods to the CLT antigen discovered using the methods described above, in cancer patients This can demonstrate the existence of a treatment-related T cell response to the CLT antigen.

[0270] For these studies, CD8 T cells isolated from the blood of healthy donors or patients were used in various ways. Such culture methods include, for example, microbeads coated with anti-CD3 and anti-CD28 + interloyalty The cells are grown using Kin-2. Subsequently, the grown cells are subjected to the peptide bond groove of the HLA molecule. CLT peptide pentamer consisting of an HLA class I molecule bound to a linked CLT antigen peptide. Using tamers, it is possible to stain for the specific CLT antigen reactivity of the T cell receptor. The binding is specific to the coiled-coil polymerization domain of the pentamer structure. Measured by detection using antibody fragments conjugated with phosphorus or allophycocyanin. In addition to pentamer staining, memory marker CD45RO and lysosomal release marker are also used. Further surface markers such as CD107a can be investigated. Pentamer-positive and specific surface Using the association with markers, the number and phenotype of pentamer-reactive T cell populations (memory vs. naive) It is possible to infer both the stem and the trunk.

[0271] Pentamer-stained cells are sorted and purified using a fluorescence-activated cell sorter (FACS). It is also possible to do so. Subsequently, the selected cells are used in an in vitro killing assay to kill target cells. Its ability to inflict damage can be further tested. These assays use CD8 T cells. The population includes a population of fluorescently labeled target cells. In this case, the CD8 population is CLT antigen specific. Those that are selected for their pentamer and are known to induce a potent lethal response such as Mart-1. These are any CD8 T cells specific to the positive control antigen being tested. Target cells include HLA-A * T2 cells pulsed with peptide expressing 02, HLA-A *02, 03, or is B * C1R cells transfected with 07, peptide pulsed, or expressing CLT / CLT antigen. It has been previously shown that melanoma cell lines, patient tumor cells, or CLT open leaded cells can be used. This may include cell lines such as CaSki transfected with a smear frame. T2 or C1R cells. The peptide used to pulse the signal may contain either a CLT antigen peptide or a positive control peptide. It is included. Target cell death is indicated by the uptake of 7AAD. Thus, CD8 T cells Therefore, when killed by apoptosis, the target cells acquire red fluorescence. Therefore, the application of this killing assay to pentamer-selected CLT antigen-specific CD8 T cells Using this method, CLT antigen-specific T cells in ex vivo cultures of melanoma patients or healthy donor T cells. The cytotoxic activity of cells can be counted.

[0272] Figure 64 shows the peptide derived from CLT antigen 4. [ka] The upper panel shows HLA pentamer staining of healthy donor CD8 T cells. The lower panel shows the staining of HLA pentamers by [method / tool ​​name]. This involves antigen-specific killing of peptide-pulsed C1R.B7 target cells by these CD8 T cells. This indicates that the negative control for the in vitro killing assay is human cytomegalovirus (HCMV). This includes unrelated peptides and peptide-free cells derived from [the specified source]. Figure 65 shows pentamer-positive cells. Fluorescence-activated cell selection and beads coated with anti-CD3 and anti-CD28 + IL-2 are used. The peptide is derived from CLT antigen 8 after 14 days of proliferation. [ka] This shows HLA pentamer staining of healthy donor CD8 T cells. The panel on the right shows these This shows very weak antigen-specific killing of peptide-pulsed A2 target cells by CD8 T cells. However, the efficacy of CaSki cells transfected with the open reading frame of CLT antigen 8 It shows effective antigen-specific killing. The negative control for this in vitro killing assay is Peptide. Includes unrelated T2 cells without cytoplasm and untransfected CaSki cells. Born.

[0273] (Example 6 - Assay for verifying 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 used to extract from a given biological sample. This is a widely used technique for determining the amount of a specific transcript present in a given RNA. A specific nucleic acid primer sequence is designed for the target transcript, and then between the primers... The region is amplified through a series of thermal cycle reactions, and by using an insertion dye (SYBR Green), fireflies are created. Quantitative analysis is performed using light. Primer pairs are designed for CLT and applied to melanoma cell lines or primary patient tissue. The assay was performed on RNA extracted from the cells. Non-melanoma cell lines were used as negative controls. The melanoma cell lines used included COLO 829 (ATCC reference number CRL-1974) and MeWo (ATCC reference number HTB). -65), SH-4 (ATCC reference number CRL-7724), and the control cell line HepG2 (hepatocellular carcinoma, ATCC reference number HB-8065), Jurkat (T-cell leukemia, ATCC reference number TIB152), and MCF7 (adenocarcinoma, ATCC reference number HT B-22) was included. All patient-derived melanoma tissues were from patients with at least stage IIC disease. RNA was obtained from six primary lesions and six metastases originating from the individual. RNA was extracted from each sample. Reverse transcription to cDNA was performed according to a standard procedure. qR with SYBR Green detection following a standard technique. T-PCR analysis was performed using primers designed for two regions of each CLT and a reference gene. The following was done. The relative quantitative value (RQ) was calculated as follows: RQ=2[Ct(reference)-Ct(target)] .

[0274] The results of these experiments are shown in Figure 66. Panel A consists of three melanoma cell lines and four Different CLT (SEQ ID NO: 56) encoding CLT antigen 1 on RNA extracted from non-melanoma cell lines Results of qRT-PCR assays using two primer sets (1+2 and 3+4) targeting the region. Panel B shows RNA extracted from three melanoma cell lines and four non-melanoma cell lines. Two primers targeting different regions of CLT (SEQ ID NO: 57) encoding CLT antigen 2. The results of qRT-PCR assays using the (5+6 and 7+8) are shown. Panel C shows three black CLT (distributed) encoding CLT antigen 3 / 4 on RNA extracted from tumor cell lines and four non-melanoma cell lines qRT using two primer sets (9+10 and 11+12) targeting different regions of column number 58) -The results of the PCR assay are shown. Panel D shows three melanoma cell lines and four non-melanoma cells. A single plastin targeting CLT (SEQ ID NO: 59), which encodes CLT antigen 5 on RNA extracted from the cell strain. The results of a qRT-PCR assay using the lymer set (88+89) are shown. Panel E contains 12 CLT (distributed) qR using two primer sets (76+77 and 78+79) targeting different regions of column number 60) The results of the T-PCR assay are shown. Panel F contains 12 melanoma tissue samples and 1 non-melanoma sample. This method targets different regions of CLT (SEQ ID NO: 61), which encodes CLT antigen 7, on RNA extracted from cell lines. The results of qRT-PCR assays using two primer sets (44+45 and 46+47) are shown. Panel G contains CLT antigen 8 on RNA extracted from 12 melanoma tissue samples and one non-melanoma cell line. Two primer sets (80-81 and 80) target different regions of the CLT (Sequence ID 62) that encodes [the specified gene]. The results of the qRT-PCR assay using (82-83) are shown. From these results, non-melanoma cells Compared to melanoma cells, specific expression of CLT was confirmed in RNA extracted from melanoma cell lines or tissues. Each CLT was detected in two or more cell lines or tissue samples analyzed, and in non-melanoma control cells. In the strains, expression was hardly detected or not detected at all.

[0275] b) RNAScope validation of CLT expression in in situ melanoma cells Transcript expression analysis using in situ hybridization (ISH) revealed that a given It is now possible to visualize the presence and expression levels of transcripts under the histopathological conditions of the specimen. Conventional RNA ISH assays are generated by a combination of antibody or enzyme-based colorimetric reactions. Oligon specific to short stretches of desired RNA sequences, visualized by a signal. This involves the recognition of native RNA molecules in situ using a creotide probe. (RNAScop) e ensures the specificity of the generated signal and enables highly sensitive single-molecule visualization of the target transcript. A recently developed in situ system with more advanced probe chemistry that enables transformation. This is a hybridization-based technique (Wang et al., 2012 J Mol Diagn. 14(1):2 2-29). Positive staining of transcript molecules appears as small red dots in a given cell, and multiple dots are present. The dot indicates the existence of multiple transcripts.

[0276] RNAScope probes were designed for CLT and 12 formalin-fixed paraffin-embedded black skin samples. Assays were performed on tumor core sections. Expression signal scoring was performed using representative samples from each core. On the image, the following was done: • Estimated cell percentage based on positive staining of CLT probe is rounded up to the nearest 10. The estimated levels per cell expression across a given section are as follows: •0 = No dyeing • 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

[0277] The expression of each CLT was detected across several different patient tumor cores and independently, This study validates the detection of CLT from tumor-derived RNA-seq data and examines the presence of CLT within tumor tissue across specific samples. We confirmed the uniformity of expression and identified at least one C in the core of each patient analyzed. The presence of LT was emphasized. Table 10 - RNAScope scoring in melanoma patient tissue cores [Table 7]

[0278] (Example 7 - Ex vivo stimulation of T cells using a pool of CLT antigen or CLT antigen fusion protein) By stimulating T cells derived from healthy donors or patients with a given cancer in vitro, A T cell clone that recognizes a specific CLT antigen is activated, and then rapidly proliferated to produce a large number of cells. It is possible to generate CLT-reactive T cells, and in that case, the resulting antitumor activity can be observed. It can be incorporated. In the case of cancer patients, such methods can be developed as anti-cancer treatments. This method involves several steps.

[0279] A) Isolation of relevant patient immune cells Not only must T cells derived from a donor (a healthy person or a cancer patient) be isolated, but autoantibodies must also be isolated. Original presenting cells (APCs) may be required. The source of immune cells is obtained by blood collection or apheresis. It can be obtained from peripheral blood. Alternatively, T cells can be obtained from a fresh biopsy or from the excision of the patient's tumor. APCs can be isolated from tumor-infiltrating lymphocytes (TILs). APCs are differentiated antigen group (CD) 14 positive. Monocytes, or alternatively, dendritic cells (DCs) obtained from the monocyte fraction of apheresis products. It is also acceptable. DCs can capture CD14 (for example, an anti-CD14 antibody conjugated to magnetic beads). In this case, CD14-positive cells are labeled with beads and captured in a magnetic column. Sex isolation or its adhesion properties, for example, peripheral blood mononuclear cells for 4-48 hours that enable monocyte adhesion. Adhesion of PBMC to tissue culture plastic through incubation of cell culture dish It can be produced by methods such as isolation. DC is not limited to GM-CSF, IL-4 The use of cytokines such as TNFα, IL-1β, IL-6, and prostaglandin E2 is fully explained. It can be produced from CD14-positive or adherent immune cell fractions by the methods described. Incubation with such cytokines for 2-7 days from CD14+ monocytes Typically, CD14 expression is lost, and for example, high levels of MHC class II and other DC markers. It enables differentiation into DCs in which Kerr expression is upregulated. Sex of T cells for selection and / or stimulation. The quality is such that the monocyte-removed fraction of PBMCs (in the case of apheresis-derived T cells) shows the emission of markers such as CD3. Current or specific T cell subsets, for example, but not limited to CD4, CD8, CD45RO, CD45RA Pan-T cells using isolation techniques based on the presence or absence of markers such as CCR7, CD62L, and CD27. Cellular isolation is possible.

[0280] B) Selection of CLT antigen-recognizing T cells A method can be used to select T cells before stimulation by APC. Tetramers and pentamers for labeling T cells expressing a TCR that recognizes a given pHLA. This includes peptide-HLA(pHLA) polymer approaches such as dextramers or analogues. pHLA, as described in Example 2, is used in mass spectrometry (MS) experiments and / or predictive algorithms. Based on the data, it is determined that the peptide is predicted to bind to a specific HLA allotype. The polymer is determined by fluorescence activation sorting or by conjugation of the polymer into magnetic beads. It can possess tags such as phycoerythrin (PE), which can be isolated via PE antibodies. Alternatively, antibodies against the tag can be directly conjugated onto magnetic beads. To isolate different T cells that recognize different pHLA derived from CLT antigens, multimers are also used. Alternatively, they can be generated with different tags, or different polymers can be combined into magnetic beads. It can be jugated.

[0281] C) Stimulation of T cells Enhance or introduce new (memory) T cell responses from cancer patients to CLT antigens. EVE) To stimulate the T cell response, the patient's T cells are subjected to class I and class II HLA complexes. In relation to this, APCs that display peptides derived from the CLT antigen on their surface can be exposed. For example, multiple CLT antigens (expected to be expressed by the patient's tumor) in a melanoma patient A It can be exogenously delivered to the PC to cause the CLT antigen-derived peptide to be presented on the surface HLA complex. The introduction of multiple CLT antigens is by delivery of linked polypeptides or by individual CLT antigens. The primary method could be, for example, a delivery method based on pooled mRNA. Alternatively, it could be APC. The HLA molecule shown above can load exogenous synthetic peptides derived from the CLT antigen. The method for delivering stabilized mature mRNA to the APC (i.e., transfection) is to the cell Conventional methods for nucleic acid delivery into the body, such as polyethyleneimine (PEI) or calcium phosphate. It can contain drugs. Alternatively, efficient transfection can transfer to APCs. This can be achieved using lipid-based reagents for the transformation. In infection reactions, lipid complexes, such as lipid nanoparticles (LNPs) or (mRNA and lipid reagents) are used. Inv Synthetic mRNA derived from the intravitreal transcription reaction (IVT) is used. To produce these mRNAs, A well-described promoter element for T7 DNA-dependent RNA polymerase and This is followed by a cDNA encoding a highly stable mRNA 5'UTR, and codon optimization of the CLT antigen. cDNA encoding the reading frame (ORF), and highly stable mRNA encoding the 3'UTR. Designed to release cDNA, a poly-A sequence of >20 nucleotides, and a functional poly-A tail. A recombinant DNA construct containing a unique restriction endonuclease site was created. It can be used as an IVT template for mRNA encoding a suitable CLT antigen. In order to create a human APC that expresses the antigen described in Cafri et al.'s paper, Nat.Comm.2019, A lipoplex method similar to the one described can be used. In short, APCs (monocytes or DCs) are plated into tissue culture flasks, and 70-90% confluence is added. Achieves a result. Lipid-based transfection reagents (e.g., Lipotectamine®) MessengerMAX (trademark), or FuGENE (registered trademark) HD, or similar products, use Opti-MEM (trademark) or similar materials. Dilute appropriately in serum medium, mix, and incubate with mRNA encoding the CLT antigen. This involves multiple CLT antigen mRNAs for transfection of APC with a combination of CLT antigens. This can be done using the following method. The incubation time between mRNA and lipid reagents should be short (5-10 minutes). The temperature is room temperature. The resulting mRNA-lipid complex is added to APC, and the CLT is coated at 37°C / 5%CO2. Depending on the optimal timing for the presentation of the translated peptide from the mRNA molecule, the incubation period is 16-72 hours. Cube it.

[0282] The delivery of CLT antigen to APC by the described method is to enable CLT antigen in the cytoplasm of APC. This should result in the expression of antigen polypeptides, which in turn will lead to Class I and Class II HL Cellular processing of peptide fragments from the polypeptide for presentation on molecule A This will result in the selection of T cells (as described in (b)), or apheresis (either T cells derived from a TIL source or unselected T cells) have CLT antigen-derived peptides on their cell surface. When co-cultured with APCs expressing the tide-HLA complex, TCRs with specificity for a given pHLA are produced. The T cells possessed engage with the pHLA complex in addition to APC-derived co-stimulatory molecules and signals. This stimulates the activation, differentiation, and proliferation of engaged T cells. This results in, for example, transfecting IVT mRNA encoding the CLT antigen to APC using the method described above. After successful isolation, the isolated autologous CD3+ T cells are placed in a cytokine-containing medium (for example) (In the basic culture medium used, supplemented with IL-6 and IL-12 or other cytokines) Surplus T cells are co-cultured with APCs in a ratio of 10 T cells to 1 APC (10:1). Cells are co-cultured for as little as one night or up to one week, but typically 18-48 hours, to form T cells. This stimulates the cells, and then, if necessary, can enrich the T cells before amplification.

[0283] D) Enrichment of stimulated T cells T cells stimulated by APCs expressing the CLT antigen are, if necessary, pre-amplification steps. It can be further concentrated. Markers of T cell activation (e.g., CD137, CD107a, CD69) OX40, or other surface markers associated with the activation state) or functional responses of T cells (e.g.) Then, select T cells that secrete cytokines such as TNFα or IFNγ, and define the T cell population as CLT. Cells that may be antigen-specific can be enriched. Such enrichment methods include FACS. Cell sorting by means of, for example, an antibody against CD137 conjugated on magnetic beads. Alternatively, it may include a bead-based capture method using an analogue. Multiple enrichment strategies are possible. Simultaneous (e.g., cells that are double-positive for CD137 and CD69) or sequential (e.g., cells that are positive for CD137) It can be used in either of the following ways (select, and then select CD137+ cells that are positive for CD69). Such positive selection is unlikely to be stimulated by APCs expressing the CLT antigen. The cells should be removed.

[0284] E) Rapid expansion of stimulated T cells After stimulation of T cells by APCs into which CLT antigen has been introduced, bulk or concentrated (as above) (See (D)) T cells, with potential modifications for optimization, as described in the literature (e.g.) For example, rapidly expanding using the method based on the literature by Jin et al. (J Immunother, 2012), >10 8 This method can achieve a total cell count of a certain number. In such a method, cytokines such as IL-2 and Stimulating antibodies such as anti-CD3, as well as potential irradiated autologous cells ("feeders") derived from PBMCs. Cells (called cells) are used. Alternatively, stimulating antibodies against CD3 and CD28 are used. The use of Dar cells can be avoided. This process is carried out using a special gas-permeable flask. For example, using a G-Rex flask or a sealed growth system (e.g., a WAVE bioreactor) Furthermore, it can be automated or enhanced. Significant T cell proliferation (100-1000 times) occurs at the start. Depending on the number of T cells, this can be achieved in as little as 7 to 14 days.

[0285] F) Expansion of T cells for evidence of CLT antigen immunogenicity The ex vivo autostimulation process recognizes target cells (including tumor cells) that express the CLT antigen. To demonstrate the expansion of T cells, we used a specific CLT antigen peptide-HLA(pHLA) complex corresponding to the T cell. Using a multimer, the presence of T cells that are responsive to a specific CLT antigen (pHLA) can be detected. This can be done. Multiple pHLAs derived from combinations of CLT antigens are used with different labels, and This can demonstrate the recognition of multiple CLT antigens by subvivo-stimulated T cells.

[0286] The functional assay is ex vivo, responding to target cells that present peptides derived from the CLT antigen. It also demonstrates the capabilities of immunized T cells, which can be achieved through various approaches. First, a cytokine release assay is performed to compare ex vivo-stimulated T cells and target cells. T cell activation can be tested through co-culture with (e.g., IFNγ ELISpot assay). Alternatively, T cell-mediated killing of target cells can be performed using cytotoxic assays, for example, co-culture with T cells. A FACS-based method for evaluating cell death in targeted cells (e.g., by 7-AAD measurement), Other methods, for example, monitoring markers of apoptosis in target cells or special The impedance (electrical measure of cell viability) of adherent target cells plated on a specific surface. It can be measured using a method that measures [something].

[0287] Target cells for such assays can be generated using various methods. For example, classify suitable human cells having HLA that matches the APC used for ex vivo stimulation. CLT antigen, known to be presented on HLA molecules (deconvoluted by mass spectrometry), When pulsed (see Example 2), it can be pulsed with peptides derived from the device. Furthermore, it is possible to evaluate tumor cell lines that match the HLA type of APC. Finally, primary tumor Cervical cells (especially those derived from the same patient donor from which the starting T cells and APCs used in this process were obtained) It is possible to evaluate tumor cells.

[0288] In conclusion, using these methods, a) human T cells were converted ex vivo using autologous APC, a) The ability to "immunize" with CLT antigens, b) Immunized T cells and unimmunized T cells c) It can potentially concentrate more than, rapidly expand immunized T cells by several pairs It is possible to produce several times more total cells, and d) rapidly expanded immunized T cells retain the ability to recognize target cells that express the same HLA and CLT antigens as those that have been immunized. This can be shown. These data were obtained by applying one or more CLT antigens to cancer patients. This supports the possibility that the cuvivo stimulation protocol has therapeutic value in controlling cancer.

[0289] (Example 8 - Method for designing CLT antigen fusion protein) One way to facilitate the delivery of a mixture of multiple polypeptide antigens is to use the O2O This is achieved by combining RFs into a single ORF, leading to the synthesis of antigenic fusion proteins. Furthermore, instead of directly linking the component polypeptides, these are linked using peptide linkers. - Connected by region: 1) Novel fusion junctions mimicking normal human proteins 1) Reduce the potential risk of generating epitopes (increased safety), and 2) Fusion / Phosphorus The CLT antigen T cell epitope adjacent to the Kerr is an individual ORF encoded by the tumor tissue. When expressed, it is guaranteed that it will be processed in a manner that mimics its presentation. (Increased efficacy) is possible. To facilitate the design of safe and effective fusion proteins. An algorithm was developed to achieve the linking of the above. A simple, short linker achieves the above objective. These are used in this algorithm because they are excellent at achieving this goal. Selecting multiple glycerides, some of which are normal human proteins It eliminates the identity with the component CLT antigen, and the terminal [ka] It also contained Lys residues to facilitate processing in the process.

[0290] For this embodiment, the algorithm is applied to CLT antigens 1, 2, 4, 6, 7, and 8 (CLT antigen fusion protein) Two different CLT antigens: CLT 1 and 2 (CLT antigen 1-8, CLT antigen fusion proteins 3 and 4) This was applied to the set.

[0291] To achieve the above requirements, four individual fusion proteins (antigenic stings of six CLT antigens) CLT antigen fusion protein 1 / CLT antigen fusion protein 2 and the antigenicity of 8 CLT antigens for stimulation In the design of each of the CLT antigen fusion proteins 3 / 4 for stimulation, Six criteria were considered. These were applied one by one, and then iterated sequentially as necessary. This ensured that the final fusion protein candidate met all the criteria.

[0292] Firstly, it was implemented using Standalone Blast ver2.9.0 (AltSchul et al., J.Mol.Biol.1990). When determined by the blastp search performed, any portion of the linker peptide contains 9- To prevent mer peptides from being identical to the human proteome, fusion protein formulations I designed the columns. For completeness, I used this blastp search on the Ensemble database (www.ensem). Three proteome subdatabases extracted from bl.org; SwissProt human proteome Trembl Ensembl Human up000005640 Proteome, and Trembl Whole Human Proteome (2019 This was implemented against (created on August 14th).

[0293] Secondly, a 9-mer peptide containing any portion of the linker peptide is NetMHCpan 4.0 (And MHC Class I supertypes (see below) according to the literature by reatta and Nielsen, Bioinformatics 2016. (Reference) To prevent it from becoming a strong binder (rank ≤ 0.5) as predicted, Fusion Tan We designed a protein sequence. A fusion protein encoding the CLT antigen suitable for use in melanoma therapy. For quality production, the following MHC HLA class I types are important for the target population of melanoma patients. Part type: HLA-A * 01:01, HLA-A * 02:01, HLA-A * 03:01, HLA-A * 11:01, HLA-A * 24:02, HLA -A * 25:01, HLA-A *26:01, HLA-A * 68:01, HLA-B 07:02, HLA-B * 08:01, HLA-B * 18:01, HLA-B * 27:05, HLA-B * 35:01, HLA-B * 35:03, HLA-B * 40:01, HLA-B * 40:02, HLA-B 51:01, HLA-C * 07: 01, HLA-C * This is a crucial driver that brings about the 07:02 option.

[0294] Thirdly, the CLT antigen (actually) was found to have an HLA-binding peptide that precisely aligns with its C-terminus. (See Example 2) The C-terminal anchor residue (when expressed in tumor tissue, usually a stop codon) (released by) so that it is produced similarly in the context of fusion proteins To prioritize the C-terminal positioning of the assisting fusion protein design, the fusion protein A chromosome sequence was designed. When the C-terminal configuration is not possible for such a CLT antigen, phosphorus The Kerr sequence was performed on a NetChop 3.1 server (Nielsen et al., Immunogenetics 2005). Further optimization based on roteasome cleavage site prediction, and the real (produced by stop codons) The linker sequence that is thought to produce the C-terminus found in the (formed) CTA antigen polypeptide I selected it.

[0295] Fourth, the weak binder (rank) of the selected MHC Class I Super Type (see above) All 9-mers containing any portion of the linker peptide predicted to have a score of ≤2.0 The peptide sequence is modified, and the linker is adjusted, or in some cases, N- By removing terminal methionine from the CLT antigen component, binding is eliminated or reduced. A fusion protein sequence was designed. Methionine is located at the N-terminal position of the MHC class I binding peptide. It is rarely seen in this location (Abelin et al., Immunity 2017; Alvarez et al., Molec Cellular & Cellular Proteomics 2019), cleavage of N-terminal methionine in selected MHC class I This strategy was adopted to eliminate the 9-mer, which was predicted to bind weakly to the molecule. HLA Class I type (HLA-A * 02:01, HLA-A * 03:01, HLA-B * It is a weak binder (07:02). Prioritizing the loss / reduction of the predicted peptide sequence for modification. Where possible, the same procedure was followed. By using this, you can weaken all other selected supertypes (see above). We achieved the disappearance / reduction of the peptide sequence predicted to be the binder.

[0296] Fifth, one application of fusion proteins is in prime / boost antigenic stimulation regimens. Therefore, to avoid directly repeating CLT antigen binding and reducing epitope repetition We designed a pair of fusion protein designs to be used for this purpose.

[0297] Sixth, we improved the fusion protein pair to prime construct and boost construct The predicted weak binding epitopes that were repeated between tracts were removed (CLT antigen fusion protein) This was performed for components 3 and 4. This is achieved by removing the N-terminal methionine residue. (See the arguments above regarding N-terminal methionine removal), or another linker This was achieved either by using (see above) or by other means.

[0298] (Implementation of CLT antigen design) The results of the above fusion protein design strategies are schematically shown in Figures 67-70. The construct (CLT antigen fusion protein 1 (SEQ ID NO: 76), CLT antigen fusion protein 2 (SEQ ID NO: 76) Number 77), CLT antigen fusion protein 3 (SEQ ID NO: 78), CLT antigen fusion protein 4 (SEQ ID NO: 79) That was the case.

[0299] (Example 9 - Antigenicity of a pool of CLT antigens or CLT antigen fusion proteins) As described in Examples 1-6, the individual CLT antigens discovered and verified, as well as in Example 8. The CLT antigen fusion protein, designed as described, is translated and enters the cytosol. It is processed proteolytically and presented on the cell surface in association with HLA class I molecules. It is thought that this is due to cDNA constructs or fusion proteins encoding individual CLT antigens. A pool (2 or more) of cDNA constructs encoding a cassette is transduced into human cells, H Immunoprecipitation of LA class I molecules was performed, and the discovery of the CLT antigen was performed using MS analysis as described in Example 2. This involves multiple exogenously inserted individual CLT antigens and / or CLT antigen fusion proteins. The crystalline cassette exhibits similar antigen-presenting properties to the previously identified component CLT antigen within tumor tissue (Examples) This is done to show that they maintained (as shown in 2).

[0300] MS-based immunopeptide analysis directly identifies specific peptides associated with HLA class I molecules. It is a powerful technology that enables precise detection. However, it lacks the ability to detect individual peptides. This is influenced by its biophysical properties, which affects the proteins present within the cell. Reactivity is limited by HLA alleles expressed in the cell lines used in these studies. Therefore, this method allows for the detection of previously identified HLA-binding peptides in tissue or cell samples. The probability of discovering everything is low. Nevertheless, the detection of HLA class I binding peptides is still... Partley is either a pool of constructs encoding individual CLT antigens or CLT antigens Is it as a part of the fusion protein design that will be tested in the delivery of the derived peptide epitope? One of these methods confirms the value of combining multiple CLT antigens.

[0301] Regarding the design of individual CLT antigens and / or CLT antigen fusion proteins inserted into multiple exogenous sources. To conduct MS-based research, cultured human cells are subjected to a pool of individual CLT antigens or CLT antigens. The proto-fusion protein cassette is controlled under the control of a suitable polII promoter and the 5' and 3'UTRs. Transduction is performed using plasmid DNA. After expansion, the cultured cells are lysed and HLA class I-peptide is applied. The tide complex is purified by affinity capture with an anti-HLA class I antibody. Subsequently, the isolated HLA fraction is purified. The molecule and the bound peptide were separated from each other, and the eluted peptide was analyzed by nUPLC-MS / MS. Analyze the results. Then, the MS / MS spectra obtained from these HLA class I pulldowns are processed using PEA. By using KS™ software (v8.5 and vX, Bioinformatics Solutions Inc.) To investigate, for the interpretation of MS / MS, this software will look at the relevant individual CLT antigen or CLT antibody. All polypeptides contained in the human proteome that have the polypeptide of the proto-fusion protein The theoretical spectra of these will be evaluated in parallel. For these studies, the c Since most of the HLA-binding peptides in LA are derived from constitutively expressed proteins, analysis is performed. The repertoire of sequences includes related CLT antigens and human proteome sequences. It is essential.

[0302] The results of these studies suggest that the HLA class I repertoire of transduced cells is processed The single and individual CLT antigen-derived peptides presented are identified. The solution of these data Using analysis, the individual cDNAs are used as a mixture and / or as parts of the CLT antigen fusion protein design. The combination of multiple CLT antigens leads to the effective presentation of peptides derived from CLT antigens. This demonstrates that the linker region of the tested protein fusion is Epitopes originating from the region efficiently deliver fusion protein cDNA in transduced cells. This indicates that it is not shown.

[0303] In summary, these data are based on CLT antigens derived from multiple CLT antigens used in combination. This can provide strong support for the translation, processing, and presentation of epitopes. , stimulates T cells that recognize the CLT antigen peptide / HLA class I complex found on tumors in patients. We support the development of therapeutic modalities designed to expand and broaden the scope of treatment.

[0304] (Example 10: Killing of cell lines expressing CLT antigen) Both are open reading frames of multiple individual CLT antigens or CLT antigen fusion proteins. The immunogenicity of antigens obtained from cells expressing multiple CLT antigens derived from a pool of microorganisms is actually Transfected CLT-peptide-reactive T cells in combination with the CLT-peptide-reactive T cells described in Example 5 This can be demonstrated using cell lines. Multiple CLT antigens can be detected by CLT antigen-specific CD8 T cells. Using the killing of infected cell lines, the treatment of CLT antigen combinations in cancer patients This indicates the presence of a therapeutically related T cell response.

[0305] CaSki cells transfected with the construct described in Example 8 To be used as the target of the killing assay described in 5. CLT antigens 1, 2, 3, 4, 5, 6, 7, Related HLA pentamers derived from 8 were isolated from healthy donors and melanoma patients. CD8 T cell lines are transfected with multiple CLT antigens or CLT antigen fusion proteins. The killing ability of these transfected target cells is then individually tested. Negative results are obtained. Irradiated cells are transfected with untransfected CaSki or unrelated constructs. These are fected CaSki cells.

[0306] (Sequence Listing) Sequence ID 1 (Polypeptide sequence of CLT antigen 1) [ka] Sequence ID 2 (Potential polypeptide sequence of CLT antigen 2) [ka] Sequence ID 3 (Potential polypeptide sequence of CLT antigen 3) [ka] Sequence ID 4 (CLT antigen 4 polypeptide sequence) [ka] Sequence ID 5 (Polypeptide sequence of CLT antigen 5) [ka] Sequence ID 6 (Polypeptide sequence of CLT antigen 6) [ka] Sequence ID 7 (polypeptide sequence of CLT antigen 7) [ka] Sequence ID 8 (polypeptide sequence of CLT antigen 8) [ka] Sequence ID 9 (peptide sequence derived from CLT antigen 1) [ka] Sequence ID 10 (peptide sequence derived from CLT antigen 1) [ka] Sequence ID 11 (peptide sequence derived from CLT antigen 1) [ka] Sequence ID 12 (peptide sequence derived from CLT antigen 1) [ka] Sequence ID 13 (peptide sequence derived from CLT antigen 1) [ka] Sequence ID 14 (peptide sequence derived from CLT antigen 1) [ka] Sequence ID 15 (peptide sequence derived from CLT antigen 1) [ka] Sequence ID 16 (peptide sequence derived from CLT antigen 1) [ka] Sequence ID 17 (peptide sequence derived from CLT antigen 1) [ka] Sequence ID 18 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 19 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 20 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 21 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 22 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 23 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 24 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 25 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 26 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 27 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 28 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 29 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 30 (peptide sequence derived from CLT antigen 2) [ka] Sequence ID 31 (peptide sequence derived from CLT antigen 3) [ka] Sequence ID 32 (peptide sequence derived from CLT antigen 3) [ka] Sequence ID 33 (peptide sequence derived from CLT antigen 3) [ka] Sequence ID 34 (peptide sequence derived from CLT antigen 3) [ka] Sequence ID 35 (peptide sequence derived from CLT antigen 3) [ka] Sequence ID 36 (peptide sequence derived from CLT antigen 4) [ka] Sequence ID 37 (peptide sequence derived from CLT antigen 4) [ka] Sequence ID 38 (peptide sequence derived from CLT antigen 4) [ka] Sequence ID 39 (peptide sequence derived from CLT antigen 4) [ka] Sequence ID 40 (peptide sequence derived from CLT antigen 4) [ka] Sequence ID 41 (peptide sequence derived from CLT antigen 4) [ka] Sequence ID 42 (peptide sequence derived from CLT antigen 4) [ka] Sequence ID 43 (peptide sequence derived from CLT antigen 4) [ka] Sequence ID 44 (peptide sequence derived from CLT antigen 4) [ka] Sequence ID 45 (peptide sequence derived from CLT antigen 5) [ka] Sequence ID 46 (peptide sequence derived from CLT antigen 5) [ka] Sequence ID 47 (peptide sequence derived from CLT antigen 5) [ka] Sequence ID 48 (peptide sequence derived from CLT antigen 6) [ka] Sequence ID 49 (peptide sequence derived from CLT antigen 6) [ka] Sequence ID 50 (peptide sequence derived from CLT antigen 6) [ka] Sequence ID 51 (peptide sequence derived from CLT antigen 6) [ka] Sequence ID 52 (peptide sequence derived from CLT antigen 7) [ka] Sequence ID 53 (peptide sequence derived from CLT antigen 8) [ka] Sequence ID 54 (peptide sequence derived from CLT antigen 8) [ka] Sequence ID 55 (peptide sequence derived from CLT antigen 8) [ka] Sequence ID 56 (cDNA sequence of CLT encoding CLT antigen 1) [ka] Sequence ID 57 (cDNA sequence of CLT encoding CLT antigen 2) [ka] TIFF2026122961000073.tif142170 Sequence ID 58 (CDNA sequences of CLT encoding CLT antigens 3 and 4) [ka] TIFF2026122961000075.tif255170 Sequence ID 59 (cDNA sequence of CLT encoding CLT antigen 5) [ka] TIFF2026122961000077.tif82170 Sequence ID 60 (CDNA sequence of CLT encoding CLT antigen 6) [ka] TIFF2026122961000079.tif248170TIFF2026122961000080.tif23170 Sequence ID 61 (cDNA sequence of CLT encoding CLT antigen 7) [ka] TIFF2026122961000082.tif44170 Sequence ID 62 (CDNA sequence of CLT encoding CLT antigen 8) [ka] TIFF2026122961000084.tif229170 Sequence ID 63 (cDNA sequence encoding CLT antigen 1) [ka] Sequence ID 64 (cDNA sequence encoding CLT antigen 2) [ka] Sequence ID 65 (cDNA sequence encoding CLT antigen 3) [ka] Sequence ID 66 (cDNA sequence encoding CLT antigen 4) [ka] Sequence ID 67 (cDNA sequence encoding CLT antigen 5) [ka] Sequence ID 68 (cDNA sequence encoding CLT antigen 6) [ka] Sequence ID 69 (cDNA sequence encoding CLT antigen 7) [ka] Sequence ID 70 (cDNA sequence encoding CLT antigen 8) [ka] Sequence ID 71 (linker sequence used in CLT antigen fusion proteins 1, 2, 3, and 4) [ka] Sequence ID 72 (linker sequence used in CLT antigen fusion proteins 1, 2, and 4) [ka] Sequence ID 73 (linker sequence used in CLT antigen fusion proteins 1 and 3) [ka] Sequence ID 74 (linker sequence used in CLT antigen fusion proteins 1, 3, and 4) [ka] Sequence ID 75 (linker sequence used in CLT antigen fusion proteins 1, 2, 3, and 4) [ka] Sequence ID 76 (Potential sequence of CLT antigen fusion protein 1) [ka] Sequence ID 77 (Potential sequence of CLT antigen fusion protein 2) [ka] Sequence ID 78 (Potential sequence of CLT antigen fusion protein 3) [ka] Sequence ID 79 (Polypeptide sequence of CLT antigen fusion protein 4) [ka] Sequence ID 80 (codon-optimized cDNA sequence encoding CLT antigen fusion protein 1) [ka] Sequence ID 81 (codon-optimized cDNA sequence encoding CLT antigen fusion protein 2) [ka] Sequence ID 82 (codon-optimized cDNA sequence encoding CLT antigen fusion protein 3) [ka] TIFF2026122961000105.tif126170 Sequence ID 83 (Codon-optimized cDNA sequence encoding CLT antigen fusion protein 4) [ka] TIFF2026122961000107.tif155170 Sequence ID 84 (Linker sequence used in CLT antigen fusion protein 3) [ka] Sequence ID 85 (TCR VB CDR3 AA sequence) [ka] Sequence ID 86 (TCR VB CDR3 AA sequence) [ka] Sequence ID 87 (TCR VB CDR3 AA sequence) [ka]

Claims

1. An antigen pool comprising two or more different antigens, wherein each antigen is a polypeptide and / or The nucleic acid exists in the form of a polypeptide, and the different antigens are separate polypeptides. or as nucleic acid and / or as a fusion protein or nucleic acid encoding a fusion protein It exists in the antigen pool as a part, and here, the two or more different antigens (a) Immunogenicity of SEQ ID NO: 1 or its variants or SEQ ID NO: 1 or its variants piece; (b) Immunogenicity of SEQ ID NO: 2 or its variants or SEQ ID NO: 2 or its variants piece; (c) Immunogenicity of SEQ ID NO: 3 or its variants or SEQ ID NO: 3 or its variants piece; (d) Immunogenicity of SEQ ID NO: 4 or its variants or SEQ ID NO: 4 or its variants piece; (e) Immunogenicity of SEQ ID NO: 5 or its variants or SEQ ID NO: 5 or its variants piece; (f) Immunogenicity of SEQ ID NO: 6 or its variants or SEQ ID NO: 6 or its variants piece; (g) Immunogenicity of SEQ ID NO: 7 or its variants or SEQ ID NO: 7 or its variants Fragments; and (h) Immunogenicity of SEQ ID NO: 8 or its variants or SEQ ID NO: 8 or its variants piece The antigen pool having polypeptide sequences selected from:

2. The two or more different antigens mentioned above are present in the fusion protein or the nucleic acid encoding the fusion protein. When present, one or more peptide linkers positioned between antigen polypeptide sequences The antigen pool according to claim 1, which is connected to the antigen pool.

3. The linker includes an array selected from sequence numbers 71, 72, 73, 74, 75, or 84. The antigen pool according to claim 2, wherein the sequence is the sequence.

4. The antigen pool comprises six different antigens, where the antigens are (a), (b), (d), (f), ( The antigen pool according to any one of claims 1 to 3, having polypeptide sequences of g) and (h). 。

5. The antigen pool comprises eight different antigens, where the antigen is a polypeptide of (a) to (h). An antigen pool according to any one of claims 1 to 4, having a sequence.

6. A nucleic acid antigen pool, where the nucleic acid is RNA, for example, messenger RNA. An antigen pool according to any one of claims 1 to 5.

7. The antigen pool according to any one of claims 1 to 6, wherein the nucleic acid is formulated in nanoparticles.

8. Claim that the nanoparticles are lipid-based nanoparticles, for example, cationic liposomes. The antigen pool described in 7.

9. Immunotherapy comprising an antigen pool and a pharmaceutically acceptable carrier according to any one of claims 1 to 8 A highly toxic pharmaceutical composition.

10. The composition according to claim 9, further comprising one or more immunostimulants.

11. The aforementioned immune stimulating substance is an aluminum salt, a saponin, an immune stimulating oligonucleotide, and water Oil-type emulsion, aminoalkylglucosaminid 4-phosphate, lipopolysaccharide and its Derivatives, as well as other TLR4 ligands, TLR7 ligands, TLF9 ligands, IL-12, and inter - The composition according to claim 10, selected from ferrons.

12. Use in ex vivo stimulation and / or amplification of T cells derived from cancer patients For the treatment of the cancer in the human, the stimulated and / or amplified T cells in the human An antigen pool or composition according to any one of claims 1 to 11 for subsequent reintroduction.

13. Therapy for human cancer in which the cancer cells express a polypeptide sequence selected from (a) to (h) A method comprising optionally including a population of white blood cells, including at least T cells, together with antigen-presenting cells. To be collected from, in the presence of the antigen pool or composition according to any one of claims 1 to 11 To stimulate and / or amplify the T cells, and to reduce some or all of the leukocytes, The method comprising reintroducing stimulated and / or amplified T cells into the human.

14. Claim 12 or Claim, the cancer is a melanoma, for example, a cutaneous melanoma or a uveal melanoma. An antigen pool or composition for use or method as described in item 13.

15. A population of cytotoxic T cells against cancer cells expressing sequences selected from (a) to (h) A process for preparation, wherein (i) T cells are optionally obtained from cancer patients together with antigen-presenting cells. (ii) the T cell population is an antigen pool or composition according to any one of claims 1 to 11. The process comprising ex vivo stimulation and amplification thereof.

16. A T cell population obtainable by the process described in claim 15.

17. T cells stimulated with the antigen pool or composition according to any one of claims 1 to 11.

18. Ex vivo loading of an antigen pool or composition according to any one of claims 1 to 11 Therefore, modified antigen-presenting cells.

19. The antigen-presenting cell according to claim 18, which is a monocyte or a cell derived from a monocyte, for example, a dendritic cell. 。

20. The antigen pool or composition according to any one of claims 1 to 11 is loaded into cells or Exosomes loaded with polypeptides or nucleic acids prepared from there.

21. A T cell population, T cells, antigen-presenting cells, or exoskeleton according to any one of claims 16 to 20. A pharmaceutical composition comprising mu together with a carrier that is acceptable as a pharmaceutical.

22. A T cell population, T cells, antigen preparation according to any one of claims 16 to 20, for use in pharmaceuticals. Shower cells, or exosomes.

23. The cancer cells express a sequence selected from (a) to (h), where the cancer cells express a sequence selected from (a) to (h) A method for treating people with cancer that involves expressing a polypeptide sequence selected from [a specific set of cells]. In this case, the human being is given a T cell population, T cells, antigen-presenting cells, according to any one of claims 16 to 21. The method comprising administering exosomes or a composition.

24. The aforementioned cancer cells express sequences selected from (a) to (h), used in the treatment of cancer in humans. A T cell population, T cells, antigen-presenting cells, etc., according to any one of claims 16 to 21, for the purpose of Sosomes, or compositions.

25. Claims 15, 23, and The cancer is a melanoma, for example, a cutaneous melanoma or a uveal melanoma. A process, method, or use for T cell population, T cells, antigen preparation as described in any one of paragraphs 24. Showing cells, exosomes, or compositions.

26. A method of treating a person suffering from cancer, (a) Determine whether the cancer cells express a polypeptide sequence selected from (a) to (h). The process of; and if so, (b) The human being given the polypeptide, nucleic acid, antigen, according to any one of claims 1 to 11 and 16 to 21. A process of administering a drug, composition, T cell population, T cells, antigen-presenting cells, or exosomes. The method including:

27. The method according to claim 26, wherein the cancer is melanoma, for example, cutaneous melanoma or uveal melanoma. Law or use.

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