CTL antigen fusion protein for treating melanoma

Abstract

In particular, the present invention provides a fusion protein useful in the treatment and prevention of cancer, especially melanoma, and especially cutaneous melanoma and uveal melanoma. [Solution] A fusion protein is provided comprising six antigenic polypeptides (a) to (f), wherein each antigenic polypeptide (a) to (f) has an immunogenic fragment of a specific amino acid sequence or a variant thereof.

Description

[Technical Field]

[0001] (Field of invention) The present invention relates to a method for use in the treatment or prevention of cancer, particularly for melanoma (e.g., skin darkening). Fusion proteins and The present invention relates to the corresponding polynucleotide. The present invention further relates, in particular, to the fusion protein or pharmaceutical compositions and immunogenic compositions containing nucleic acids, and the medical treatment of said pharmaceutical compositions and immunogenic compositions. The use of the pharmaceutical composition and the immunogenic composition, and the therapeutic method comprising administering the pharmaceutical composition and the immunogenic composition. . [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 sexual T lymphocytes (including CTLs), but also foreign antigen-tagged cells throughout the animal's life. It can also produce memory CD8+ T cells that can be re-amplified when they appear later. .

[0003] Its expression is usually restricted to professional antigen-presenting cells (APCs) such as dendritic cells (DCs). The MHC class II molecules, which are restricted to APCs, are usually loaded with peptides taken up from the extracellular environment into the interior. 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 MHCII-peptide complex , the maturation of CD4+ T cells into effector cells (e.g., T H 1, T H 2, T H 17, T FH , T reg cells) 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 assisting in the induction of 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.

[0004] Immune memory (especially antigen-specific B cells / antibodies and antigen-specific CTLs) plays an important role in the control of microbial infections, and immune memory has been utilized to develop numerous vaccines for preventing diseases caused by important pathogenic microorganisms. Immune memory is also known to play an important role in the control of tumor formation, but few effective cancer vaccines have been developed . . ​​​​

[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 novel fusion proteins containing HERV-related antigenic sequences that can perform this function. That is the case. [Overview of the Initiative]

[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). Furthermore, qRT-PCR and RNA Scope studies have shown that CLT is a non-melanoma cell line or Compared to tissue, RNA extracted from melanoma cell lines or melanoma tumor tissue is specifically expressed. It was confirmed that this is achieved (see Example 6). The inventors have developed a unique C for vaccine delivery. A fusion protein containing the LT antigen was also produced (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. CLT antigen polypeptide sequence encoded by CLT and fusion protein containing the same It is thought that this induces an immune response against uveal melanoma cells and tumors containing them. ru.

[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 and 10), therefore, therapeutic use in the context of cancer immunotherapy It is considered suitable for this purpose.

[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, the present invention comprising CLT antigen polypeptide. A vaccine that uses a fusion protein to induce a therapeutic or prophylactic immune response against tumor cells. It can also be delivered directly to the target as a signal. Furthermore, the nucleic acid encoding the CLT antigen is The fusion protein of the present invention can be codon-optimized to enhance the expression of the encoded CLT antigen. The nucleic acid encoding the substance is administered directly, or inserted into a vector for in vivo delivery by other means. The encoded protein product enters the body and triggers a therapeutic or prophylactic immune response against tumor cells. It can be produced in the target as a vaccine that induces [the disease]. These and other uses include: This is explained in more detail below.

[0027] Therefore, the present invention relates in particular to a fusion tan comprising six antigenic polypeptides (a) to (f). A protein wherein the antigenic polypeptides (a) to (f) have the amino acid sequence: (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: 6 or its variants or SEQ ID NO: 6 or its variants piece; (d) Immunogenicity of SEQ ID NO: 7 or its variants or SEQ ID NO: 7 or its variants piece; (e) Immunogenicity of SEQ ID NO: 4 or its variants or SEQ ID NO: 4 or its variants Fragments; and (f) Immunogenicity of SEQ ID NO: 8 or its variants or SEQ ID NO: 8 or its variants piece The present invention provides a fusion protein having the following characteristics (hereinafter referred to as "the fusion protein of the present invention").

[0028] The present invention comprises a nucleic acid molecule that encodes the fusion protein of the present invention (hereinafter referred to as "the nucleic acid of the present invention"). (It also offers)

[0029] The fusion proteins and nucleic acids of the present invention, as well as related embodiments of the present invention, are described in more detail. As explained below, it is widely used in cancer immunotherapy and prevention, particularly in melanoma immunotherapy and prevention. In a broad sense, this is considered useful. [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 that amplified and pentamer-sorted CD8 T cells kill C1RB7 target cells pulsed with a peptide derived from CLT antigen 4 (SEQ ID NO: 44). [Figure 65] Figure 65 shows that amplified and pentamer-sorted CD8 T cells kill CaSki cells transfected with an open reading frame of CLT antigen 8 (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. [Figure 71] Figure 71 provides a schematic explanation of mouse immunogenicity data supporting CLT antigen fusion protein 1 (sequence number 76) and CLT antigen fusion protein 2 (sequence number 77).

[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 a linker used to construct the CLT antigen fusion protein. It is a row. 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) (Fusion protein) The term "fusion protein" refers to a protein that is bonded through peptide bonds during protein synthesis. A fusion protein refers to any protein containing at least two coupled polypeptides. These are linked together to be transcribed and translated as a single unit that produces a single protein. It can be produced by the connection of two or more genes that encode separate polypeptides.

[0040] This invention creates a nucleic acid construct by fusing sequences that encode individual polypeptides. By releasing, each polypeptide is fused into a second or further polypeptide. The present invention provides a fusion protein comprising at least six polypeptides. The protein is considered to have the usefulness described herein, and each component polypeptide Compared to [another substance], it exhibits superior immunogenicity or vaccine activity, or prophylactic or therapeutic effects. It may have advantages (including increasing the breadth and depth of the response), and in non-inbred populations. This may be of particular value. The fusion protein of the present invention is a vaccine antigen and / or vector. It also offers the advantage of increasing the efficiency of constructing and manufacturing vaccines (including nucleic acid vaccines). ru.

[0041] Therefore, the present invention is a fusion protein comprising six antigenic polypeptides (a) to (f). The antigenic polypeptides (a) to (f) have the following amino acid sequence: (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: 6 or its variants or SEQ ID NO: 6 or its variants piece; (d) Immunogenicity of SEQ ID NO: 7 or its variants or SEQ ID NO: 7 or its variants piece; (e) Immunogenicity of SEQ ID NO: 4 or its variants or SEQ ID NO: 4 or its variants Fragments; and (f) Immunogenicity of SEQ ID NO: 8 or its variants or SEQ ID NO: 8 or its variants piece The present invention provides a fusion protein having the following properties:

[0042] The fusion protein of the present invention comprises one or more antigenic polypeptides selected from (g) and (h). The antigenic polypeptide may further be included, where the antigenic polypeptide (g) and The amino acid sequence is: (g) Immunogenicity of SEQ ID NO: 3 or its variants or SEQ ID NO: 3 or its variants Fragments; and (h) Immunogenicity of SEQ ID NO: 5 or its variants or SEQ ID NO: 5 or its variants piece It holds.

[0043] In one embodiment, the fusion polypeptide comprises six antigenic polypeptides (a) to (f). In one embodiment, the fusion polypeptide comprises eight antigenic polypeptides (a) to (h). In one embodiment, the fusion polypeptide comprises seven antigenic polypeptides (a) to (g). In one embodiment, the fusion polypeptide is composed of seven antigenic polypeptides (a) to (f) and (h). Includes.

[0044] One or more antigenic polypeptides (a) to (f) (for example, antigenic polypeptides (a) to ( f) one, two, three, four, five, or all six of them are N-terminal methionine amino acid residues It may contain or consist of a sequence lacking the N-terminal. For example, antigenic polypeptide (a) has an N-terminal It may have the sequence of SEQ ID NO: 1 with the terminal methionine amino acid removed, and / or antigenic poly Peptide (b) may have the sequence of SEQ ID NO: 2, in which the N-terminal methionine amino acid is removed. and / or antigenic polypeptide (c) is a sequence number from which the N-terminal methionine amino acid has been removed. The antigenic polypeptide (d) may have the sequence of (3) and / or the N-terminal methionine amino acid The sequence of the removed sequence number 4 may be and / or antigenic polypeptide (e) may have an N-terminus It may have the sequence of SEQ ID NO: 5 with the methionine amino acid removed, and / or antigenic polyp Ptido(f) may have the sequence of SEQ ID NO: 6, in which the N-terminal methionine amino acid is removed. If present, one or more of the antigenic polypeptides (g) and (h) (for example, either one (one or both) contains or consists of a sequence lacking an N-terminal methionine amino acid residue. For example, antigenic polypeptide (g) has the N-terminal methionine amino acid removed. The sequence of sequence number 7 may be and / or the antigenic polypeptide (h) may be N-terminal methionine amine It may have the sequence of SEQ ID NO 8, in which the no acid has been removed.

[0045] Therefore, the present invention is a fusion protein comprising six antigenic polypeptides (a) to (f). The antigenic polypeptides (a) to (f) have the following amino acid sequence: (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. 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; (d) 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; (e) 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 its variant; and (f) 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 The present invention provides a fusion protein having the following properties:

[0046] The fusion protein of the present invention comprises one or more antigenic polypeptides selected from (g) and (h). The antigenic polypeptide may further be included, where the antigenic polypeptide (g) and The amino acid sequence is: (g) 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 its variant; and (h) 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. It holds.

[0047] In one embodiment, the fusion protein of the present invention has an N-terminal methionine residue from SEQ ID NO: 2. It contains an antigenic polypeptide having an amino acid sequence with the group removed. In one embodiment, this The fusion protein has an amino acid sequence obtained by removing the N-terminal methionine residue from SEQ ID NO: 6. It contains an antigenic polypeptide. In one embodiment, the fusion protein of the present invention has a sequence It contains an antigenic polypeptide having an amino acid sequence with the N-terminal methionine residue removed from number 5. In one embodiment, the fusion protein of the present invention is N-terminal methionine from SEQ ID NO: 2 Antigenic polypeptides having amino acid sequences with residues removed, from SEQ ID NO: 6 to the N-terminal methionine Antigenic polypeptide having an amino acid sequence with n residues removed, and from SEQ ID NO: 5, the N-terminal It contains an antigenic polypeptide having an amino acid sequence excluding thionine residues.

[0048] Preferably, the fusion protein of the present invention comprises six antigenic polypeptides (a) to (f), Thus, the antigenic polypeptides (a) to (f) have the following amino acid sequences: (a) Sequence ID 1; (b) Sequence ID No. 2 with the N-terminal methionine residue removed; (c) Sequence ID 6; (d) Sequence ID 7; (e) Sequence ID 4; and (f) Sequence ID 8 It holds.

[0049] Preferably, the fusion protein of the present invention comprises eight antigenic polypeptides (a) to (h), Thus, the antigenic polypeptides (a) to (h) have the following amino acid sequences: (a) Sequence ID 1; (b) Sequence ID No. 2 with the N-terminal methionine residue removed; (c) Sequence ID No. 6 with the N-terminal methionine residue removed; (d) Sequence ID 7; (e) Sequence ID 4; (f) Sequence ID 8; (g) Sequence ID 3; and (h) Sequence ID No. 5 with the N-terminal methionine residue removed. It holds.

[0050] Preferably, the fusion protein of the present invention comprises eight antigenic polypeptides (a) to (h), Thus, the antigenic polypeptides (a) to (h) have the following amino acid sequences: (a) Sequence ID 1; (b) Sequence ID No. 2 with the N-terminal methionine residue removed; (c) Sequence ID 6; (d) Sequence ID 7; (e) Sequence ID 4; (f) Sequence ID 8; (g) Sequence ID 3; and (h) Sequence ID 5 It holds.

[0051] The antigenic polypeptide of the fusion protein of the present invention is arranged in various orders from the N-terminus to the C-terminus. It can be placed. The design and sequence of polypeptides in the fusion protein of the present invention can be implemented. This is described in Example 8. In particular, the order of polypeptides in the fusion protein is important, That is, in some cases, the desired immunogenicity of the polypeptide. It can result in superior processing and presentation of the region, and in other cases, natural cancer-specific Non-natural immunogenic peptides obtained from the conjugation of target CLT antigens are released during vaccination, on the surface. It is presented on the presented class I HLA molecule, thereby inducing an undesirable T cell response. This is because it is necessary for the optimal fusion design that reduces the likelihood of obtaining it.

[0052] The fusion protein of the present invention yields a strong antigenic response to the component CLT antigen (Example 9). (See also 10), and it is thought to induce a minimal antigenic response to the junction region (implementation (See Example 8).

[0053] In one embodiment, when the fusion protein contains six antigenic polypeptides (a) to (f) The six antigenic polypeptides are arranged from N to C in the order of (a), (b), (c), (d), (e), and (f). It is placed.

[0054] 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 1, 2, 6, 7, 4, and The sequences are arranged from N to C in order of column number 8. The corresponding sequences with the N-terminal methionine deleted are , as described above, can be used as appropriate. Therefore, preferably, SEQ ID NO: 1 is N It is located at the terminal, with SEQ ID NO: 8 located at the C-terminus. Preferably, the N-terminal methionine of SEQ ID NO: 2. This is deleted. In one embodiment of the present invention, the fusion protein has the sequence of SEQ ID NO: 76 do.

[0055] In another embodiment, the fusion protein contains six antigenic polypeptides (a) to (f). In total, the six antigenic polypeptides are transferred from N to C in the order of (c), (f), (d), (b), (e), and (a). It will be placed.

[0056] In one preferred embodiment, six antigenic polypeptides are represented by SEQ ID NOs: 1-2, 4, 6-8. It has the sequence of, and sequence numbers 6, 8, 7, 2, 4, and The sequences are arranged from N to C in order of column number 1. The corresponding sequences with the N-terminal methionine deleted are , as described above, can be used as appropriate. Therefore, preferably, SEQ ID NO: 6 is N It is located at the terminal, with SEQ ID NO: 1 located at the C-terminus. Preferably, the N-terminal methionine of SEQ ID NO: 2. This is deleted. In one embodiment of the present invention, the fusion protein has the sequence of SEQ ID NO: 77 do.

[0057] In another embodiment, the fusion protein contains eight antigenic polypeptides (a) to (h). In total, the eight antigenic polypeptides are in the order of (a), (b), (g), (d), (e), (h), (c), and (f). It is arranged from N to C.

[0058] In one preferred embodiment, eight antigenic polypeptides have sequences 1 to 8. And, Array No. 1, Array No. 2, Array No. 3, Array No. 7, Array No. 4, Array No. 5, Array No. The sequence numbers 6 and 8 are arranged from N to C in that order. The corresponding sequence has the N-terminal methionine removed. The sequence can be used arbitrarily as described above. Preferably, sequence number 1 is at the end of N. It is located at the end, and sequence number 8 is located at the C-terminus. Preferably, the N-terminal methionine of sequence number 2 is Deleted. Preferably, the N-terminal methionine of sequence number 6 is deleted. Preferably, sequence number The N-terminal methionine of no. 5 is deleted. In one embodiment of the present invention, the fusion protein is It has the sequence of sequence number 78.

[0059] In another embodiment, the fusion protein contains eight antigenic polypeptides (a) to (h). In total, the eight antigenic polypeptides are in the order of (c), (g), (a), (h), (e), (f), (d), and (b). It is arranged from N to C.

[0060] In one preferred embodiment, eight antigenic polypeptides have sequences 1 to 8. And, sequence number 6, sequence number 3, sequence number 1, sequence number 5, sequence number 4, sequence number 8, sequence number The sequence is arranged from N to C in the order of sequence 7 and sequence number 2. The corresponding N-terminal methionine has been removed. The sequence can be used arbitrarily as described above. Preferably, sequence number 6 is at the end of N. It is located at the end, and sequence number 2 is located at the C-terminus. Preferably, the N-terminal methionine of sequence number 2 is Deleted. In one embodiment of the present invention, the fusion protein has the sequence of SEQ ID NO: 79 ru.

[0061] 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.

[0062] The present invention makes necessary modifications to the polypeptide of the present invention, and the aforementioned fused polypeptide The invention also provides nucleic acids that encode a vector and other embodiments of the present invention (vectors, compositions, cells, etc.).

[0063] (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.

[0064] 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].

[0065] 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.

[0066] Generally, variants of the polypeptide sequence of the fusion protein of the present invention have a high degree of It contains sequences that have a degree of sequence identity. For example, a variant has related sequences throughout its entire length. The reference sequence has preferably at least about 80% identity, 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%.

[0067] 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 (i.e.) 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.

[0068] 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.

[0069] 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.

[0070] The fusion protein of the present invention has several substitutions, for example, conserved when compared to the reference sequence. The polypeptide may contain a variant sequence having a substitution (for example, 1 to 25, e.g. (For example, 1 to 10, especially 1 to 5, and in particular, 1 amino acid residue can be modified). The number of substitutions, for example For example, the number of conservative substitutions is up to 20% of the number of residues in the reference sequence, for example, up to 10%, for example , up to 5%, for example, up to 1%. Generally, conservative substitutions are specified below. It is included in one of the amino acid classifications, but in some cases it contributes to the immunogenicity of the antigen. In some cases, other substitutions may be possible without qualitative impact. The following eight groups Each contains an amino acid that is usually a conserved substitution for the 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).

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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 following: 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.)

[0075] The immunogenic fragment of the polypeptide sequence of the fusion protein according to the present invention depends on the length of the CLT antigen and usually contains at least 9 consecutive amino acids (e.g., at least 9 or 10) derived from the full-length polypeptide sequence, e.g., at least 12 consecutive amino acids (e.g., at least 15 or at least 20 consecutive amino acids), particularly at least 50 consecutive amino acids, e.g., at least 100 consecutive amino acids (e.g., at least 200 consecutive amino acids). Preferably, the immunogenic fragment is at least 10% of the length of the full-length polypeptide sequence, e.g., at least 20%, e.g., at least 50%, e.g., at least 70% or at least 80%. Depending on the length of the CLT antigen, it usually contains at least 9 consecutive amino acids (e.g., at least 9 or 10), e.g., at least 12 consecutive amino acids (e.g., at least 15 or at least 20 consecutive amino acids), particularly at least 50 consecutive amino acids, e.g., at least 100 consecutive amino acids (e.g., at least 200 consecutive amino acids) derived from the full-length polypeptide sequence. at least 9 or 10), for example, at least 12 consecutive amino acids (for example, at least 15 or or at least 20 consecutive amino acids), particularly at least 50 consecutive amino acids, for example, at least 100 consecutive amino acids (for example, at least 200 consecutive amino acids). Preferably, the immunogenic fragment is at least 10% of the length of the full-length polypeptide sequence, for example, at least 20%, for example, at least 50%, for example, at least 70% or at least 80% .

[0076] The immunogenic fragment usually contains at least one epitope. The epitope contains B cell and T cell epitopes, and preferably the immunogenic fragment contains at least one T cell epitope of either CD4+ or CD8+ T cells.

[0077] <m The T cell epitope is a short consecutive stretch of amino acids recognized by T cells (e.g., CD4+ or CD8+ T cells) when bound to HLA molecules. The identification of T cell epitopes can be achieved by epitope mapping experiments well known to those skilled in the art (e.g., see Paul, Fundamental Immunology, 3rd Edition, 243-247 (1993); Beiβbarth et al., 2005, Bioinformatics, 21(Suppl. 1):i29-i37). The T cell epitope is a short consecutive stretch of amino acids recognized by T cells (e.g., CD4+ or CD8+ T cells) when bound to HLA molecules. The identification of T cell epitopes can be achieved by epitope mapping experiments well known to those skilled in the art (e.g., see Paul, Fundamental Immunology, 3rd Edition, 243-247 (1993); Beiβbarth et al., 2005, Bioinformatics, 21(Suppl. 1):i29-i37).

[0078] As a result of the decisive involvement of T cell responses in cancer, at least one T cell epitope ​​Fragments of the full-length polypeptides of SEQ ID NOs: 1 to 8 containing may be immunogenic and

[0079] it is readily apparent that in diverse outbred populations such as humans, different HLA types may mean that certain epitopes may not be recognized by all members of the population. As a result, in order to maximize the level of recognition and magnitude of the immune response to a polypeptide, it is generally desirable for the immunogenic

[0080] fragment to contain multiple epitopes (preferably, all epitopes within the CLT antigen) from the full-length sequence. Particular fragments of the antigenic polypeptides of SEQ ID NOs: 1 to 8 that may be useful include at least one CD8+ T cell epitope, preferably at least two CD8+ T cell epitopes, especially those containing all of the CD8+ T cell epitopes, particularly those associated with multiple HLA alleles, e.g., those associated with two, three, four, five, or more alleles). Particular fragments of the antigenic polypeptides of SEQ ID NOs: 1 to 8 that may be useful include at least one CD4+ T cell epitope, preferably at least two CD4+ T cell epitopes, especially those containing

[0081] all of the CD4+ T cell epitopes (particularly those associated with multiple HLA alleles, e.g., those associated with two, three, four, five, or more alleles). However, vaccine designers may be able to

[0081] 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 (i.e.) 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-cell markers (using antibodies specific to immune markers such as NF-α, IFNg, type 1 IFN, CD40L, and CD69) The characteristic analysis of the cellular response, followed by analysis using a flow cytometer, is performed.

[0082] 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, it provides at least 75% activity, and more particularly, at least 90% activity.

[0083] Examples of immunogenic fragments of antigenic polypeptides of SEQ ID NOs. 1-8, and therefore, the fusion of the present invention Examples of the peptide components of the protein include polypeptides that contain or consist of the sequences of SEQ ID NOs: 9 to 55. The sequences of SEQ ID NOs: 9 to 12, 18 to 19, 30, 31 to 32, and 37 to 39, 45, 4 8 to 54 have been confirmed by immunopeptidome analysis to bind to HLA class I molecules (see Example 2). The sequences of SEQ ID NOs: 13 to 17, 20 to 29, 33 to 35, 40 to 44 are predicted by NetMHC software to bind to HLA class I molecules and have been used in immunological verification assays (see Examples 3, 4, and 5).

[0084] The antigenic polypeptide component (a) of the fusion protein may contain 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 to 12. Further exemplary fragments include two, three, or four of SEQ ID NOs: 9 to 12. Further exemplary fragments include or consist of any one of SEQ ID NOs: 13 to 17. Further exemplary fragments include all of SEQ ID NOs: 9 to 17 (such that any overlapping sequences do not need to be present multiple times, and possible sequences are considered).

[0085] The antigenic polypeptide component (b) of the fusion protein may contain or consist of SEQ ID NO: 2 or a variant thereof or an immunogenic fragment of SEQ ID NO: 2 or a variant thereof. Exemplary fragments include or consist of SEQ ID NO: 18 or SEQ ID NO: 19. 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. Includes all numbers from 18 to 30 (arrays that do not require any duplicate arrays to exist multiple times). (This will be taken into consideration.)

[0086] The antigenic polypeptide component (c) of the fusion protein is SEQ ID NO: 6 or its variant or This may include or consist of immunogenic fragments of SEQ ID NO: 6 or its variants. Exemplary fragments include or consist of sequence numbers 48-51.

[0087] The antigenic polypeptide component (d) of the fusion protein is SEQ ID NO: 7 or its variant or This may include or consist of immunogenic fragments of SEQ ID NO: 7 or its variants. An exemplary fragment includes or consists of sequence number 52.

[0088] The antigenic polypeptide component (e) of the fusion protein is SEQ ID NO: 4 or its variant or This may include or consist of immunogenic fragments of SEQ ID NO: 4 or its variants. An exemplary fragment includes or consists of Sequence ID No. 36. Further exemplary fragments are: This includes or consists of Sequence ID No. 37 or Sequence ID No. 38. Further exemplary fragments are: Includes or derived from Sequence ID 39. Further exemplary fragments are Sequence IDs 40-44. It includes or consists of one of the following. Further exemplary fragments are Sequence ID No. 36 and Sequence ID No. 3 Includes either 7 or SEQ ID NO: 38. Further exemplary fragments include SEQ ID NO: 39 and SEQ ID NO: 3 Includes 7 or EQ code 38. Further exemplary fragments include all of EQ codes 36-44 (any). (Possible arrays are considered so that duplicate arrays do not need to exist multiple times.)

[0089] The antigenic polypeptide component (f) of the fusion protein is SEQ ID NO: 8 or its variant or This may include or consist of immunogenic fragments of SEQ ID NO: 8 or its variants. Exemplary fragments include or consist of sequence numbers 53-55.

[0090] The antigenic polypeptide component (g) of the fusion protein is SEQ ID NO: 3 or its variant or This may include or consist of immunogenic fragments of SEQ ID NO: 3 or its variants. An exemplary fragment includes or consists of Sequence ID No. 31. Further exemplary fragments are: Includes sequence number 31. Further exemplary fragments include any one of sequence numbers 32-35 or These consist of the following. Further exemplary fragments include Sequence IDs 31 and 32. The example fragment includes all of sequence numbers 31-35 (no duplicate sequences are required). (Possible arrays are considered to avoid any issues.)

[0091] The antigenic polypeptide component (h) of the fusion protein is SEQ ID NO: 5 or its variant or This may include or consist of immunogenic fragments of SEQ ID NO: 5 or its variants. The exemplary fragments include or consist of any one of sequence numbers 45-47.

[0092] (Linker) This invention relates to a fusion protein in which the antigenic polypeptide is linked by one or more peptide linkers. Provides a connected fusion protein. One embodiment of the present invention, the fusion of the present invention The antigenic polypeptide of a protein has one or more linkers (e.g., two, three, four, five, six). It is connected by 1 or 7 linkers. The linkers are antigenic to the fusion protein. Each polypeptide can be separated. The linker can be an "internal" one, that is, The linker is located at the N-terminus of the first polypeptide and the C-terminus of the last polypeptide in the fusion protein. It is not present at the end. In one embodiment of the present invention, one or more linkers are antigenic polypeptides It is positioned between (a) and (b), (b) and (c), (c) and (d), (d) and (e), and (e) and (f). In another embodiment, one or more linkers are antigenic polypeptides (c) and (f), (f) and (d), ( It is positioned between d) and (b), (b) and (e), and (e) and (a). In further embodiments of the present invention The linker is formed between antigenic polypeptide (a) and (b), (b) and (g), (g) and (d), (d) and (e), and (e) It is positioned between (h), (h) and (c), and (c) and (f). In yet another embodiment of the present invention The linker is formed between antigenic polypeptides (c) and (g), (g) and (a), (a) and (h), (h) and (e), and (e) and (f). It is positioned between (f) and (d), and between (d) and (b).

[0093] 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.

[0094] 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.

[0095] 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.

[0096] The fusion protein of the present invention combines separate antigenic polypeptide sequences and the resulting The reading frame acts as a single unit that produces a single protein during transcription and Six or more (for example) cDNAs that encode linkers connected to be translated and encoding It can be produced by connecting 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.

[0097] (nucleic acid) The present invention relates to isolated nucleic acids (referred to as nucleic acids of the present invention) that encode the fusion protein of the present invention. To provide (a service).

[0098] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein. nucleotide monomers, in particular deoxyribonucleotide monomers or ribonucleotide monomers This term refers to polymeric polymers made from nucleotide monomers. It comprises nucleic acids containing bodies or modified skeletal residues or bonds, and such nucleic acids are naturally occurring. It is a substance that does not exist in nature, has properties similar to a reference nucleic acid, and is a reference nucleic acid It is intended to be metabolized in a manner similar to rheotide, or to have an extended half-life within the system. It is intended that... Examples of such analogues include, but are not limited to, hos Holothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate Examples include 2-O-methylribonucleotides and peptide-nucleic acids (PNA). Preferably, "nucleic acids" The term "deoxyribonucleotide monomer" refers to the natural properties of deoxyribonucleotide monomers or ribonucleotide monomers. This refers to naturally occurring polymers. Preferably, the nucleic acid molecules of the present invention are recombinants. Recombinant A body is a nucleic acid molecule that undergoes cloning, restriction, or ligation processes, or is found in nature. Among the other steps that produce nucleic acid molecules different from those found in (for example, in the case of cDNA) It means that there is at least one product. In one embodiment, the nucleic acid of the present invention is human It is an artificial nucleic acid sequence (for example, a cDNA sequence or nucleic acid sequence with codon utilization that does not exist in nature). In one embodiment, the nucleic acid of the present invention is DNA. Alternatively, the nucleic acid of the present invention is RNA. ru.

[0099] 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.

[0100] In one embodiment, the nucleic acid of the present invention is DNA. For example, the nucleic acid is sequence numbers 56-62. and include or consist of sequences selected from 63-70. Also provided This includes or contains a variant of the sequence selected from sequence numbers 56-62 or 63-70. These nucleic acids, this variant codes for the same amino acid sequence, but the genetic code Based on degeneracy, they possess different nucleic acids.

[0101] 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.

[0102] 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).

[0103] 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).

[0104] In one embodiment, the nucleic acid of the present invention is RNA. Correspondingly, and having a ribonucleotide skeleton instead of a deoxyribonucleotide skeleton, RNA sequences having a side-chain base uracil (U) instead of thymine (T) are provided.

[0105] Therefore, the nucleic acid of the present invention is the RN of a cDNA sequence selected from SEQ ID NOs. 56-62 and 63-70. A contains or consists of equivalents, and when compared to the reference sequence, many silent May contain variations (e.g., 1 to 50, e.g., 1 to 25, especially 1 to 5, etc.) (For example, one codon can be altered.) An "RNA equivalent" is a reference cDNA sequence containing the same genetic information. Contains (i.e., a ribonucleotide skeleton instead of a deoxyribonucleotide skeleton) (Contains the same codon having a side-chain base uracil (U) instead of thymine (T)) This refers to an NA sequence.

[0106] The present invention also includes sequences that are complementary to the aforementioned cDNA and RNA sequences.

[0107] In one embodiment, the nucleic acid of the present invention is optimized for expression in human host cells. It is a codon.

[0108] In the case of the nucleic acid of the present invention, if it is a DNA nucleic acid, it is transcribed and translated into the fusion protein of the present invention. It can be translated into the fusion protein of the present invention if it is an RNA nucleic acid.

[0109] (Polypeptides and nucleic acids) Preferably, the nucleic acids used in the present invention are isolated. “Isolated” nucleic acids are It is extracted from its original environment. For example, naturally occurring nucleic acids are extracted from their natural environment. If a nucleic acid is separated from some or all of the substances it coexists with, it is considered isolated. If it is cloned in a vector that is not part of its natural environment, then isolation is necessary. It is believed that this is the case.

[0110] "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.

[0111] The term "artificial" is used in reference to polypeptides or nucleic acid sequences, for example, not to refer to natural polypeptides. This refers to sequences that are either synthetic modifications of sequences or sequences containing non-natural sequences not found in nature. do.

[0112] 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 ptyde sequence, not derived from the sequence, or based on the sequence It can also mean an array that does not contain any elements.

[0113] As described above, the fusion protein of the present invention has a variant sequence, preferably the Throughout its entire length, it exhibits at least about 80% identity with the associated reference sequence, more preferably a small degree of identity. At least about 85% identity, and most preferably at least about 90% identity (for example, Contains polypeptides that make up at least 95%, at least about 98%, or at least about 99%. It is visible.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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).

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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...)

[0122] (Production of the fusion protein of the present invention) The fusion protein of the present invention is described, for example, in the literature by Green and Sambrook, 2012 Molecular Clo Disclosed in: A Laboratory Manual, 4th edition, Cold Spring Harbour Laboratory Press. It can be obtained and manipulated using the technology available. In particular, using artificial gene synthesis, It produces polynucleotides (Nambiar et al., 1984, Science, 223:1299-1301, Sakamar and Khorana's literature, 1988, Nucl. Acids Res., 14:6361-6372, and Wells et al.'s literature, 1985, Ge ne, 34:315-323, and Grundstrom et al., 1985, Nucl. Acids Res., 13:3305-3316), Subsequently, the product can be expressed in a suitable organism to produce polynucleotides. The gene encoding the polypeptide of the fusion protein is synthesized, for example, by solid-phase DNA synthesis. It can be produced organically. The entire gene does not require a precursor template DNA and can be produced de novo. It can be synthesized. Building blocks to obtain the desired oligonucleotide The oligonucleotide chain is then sequentially cut into the growing oligonucleotide chain in the order required by the product sequence. Pulling is performed. Once chain assembly is complete, the product is released from the solid phase into the solution and deprotected. Then, collect the product. Isolate the product by high-performance liquid chromatography (HPLC) and collect the desired oligonucleotides. Nucleotides can be obtained with high purity (Verma and Eckstein, 1998, Annu. Rev.). Biochem. 67:99-134). These relatively short segments are used in various gene amplification methods. Using Mol Biol., 2012;834:93-109), it is favored for use in countless recombinant DNA-based expression systems. It can be easily assembled into suitable, longer DNA molecules. In relation to the present invention, by those skilled in the art If present, the polynucleotide encoding the polypeptide antigen of the fusion protein described in the present invention. The otide sequence can be easily incorporated into various vaccine production systems, such as those involving viral vectors. They will understand that it can be used.

[0123] To produce the fusion protein of the present invention within a microbial host (e.g., bacteria or fungi), The nucleic acid of the invention includes suitable regulatory sequences and control sequences (including promoters, termination signals, etc.) Furthermore, it contains sequences that promote the secretion of polypeptides suitable for protein production within the host. Similarly, the fusion protein of the present invention is used in eukaryotic cells (e.g., Chinese hamster ovary cells). A culture of cells or Drosophila S2 cells is prepared with a suitable regulatory sequence and control sequence (promoter). (including termination signals, etc.) and polypeptides suitable for protein production within these cells. By transduction with the nucleic acid of the present invention, which is combined with a sequence for promoting secretion, It can be produced.

[0124] The improvement in the isolation of the fusion protein of the present invention produced by recombinant means is that the protein A stretch of histidine residue toward one end (commonly known as the His tag) By adding ), it can be optionally promoted.

[0125] Fusion proteins can also be prepared synthetically.

[0126] (vector) In a further embodiment, the fusion protein of the present invention is produced in vivo and immunosuppresses A gene construct containing one or more of the nucleic acids of the present invention is used to induce an answer. It can be introduced into cells in vivo. Nucleic acids (e.g., DNA) can be used in nucleic acid expression systems, bacteria, and It may be present in any of the various delivery systems known to those skilled in the art, including several virus expression systems. For example, Rolland's paper, 1998, Crit. Rev. Therap. Drug Carrier Systems 15:143- Numerous gene transfers, including those described in 198 and the references cited therein. The techniques described are well known in this field. Some of these approaches are presented for illustrative purposes. This will be outlined below.

[0127] Therefore, vectors containing the nucleic acid molecule of the present invention (in this specification, "DNA expression constructor") A "lacto" (also called a "construct") is provided.

[0128] Preferably, the vector enables the transcription of translationally active RNA molecules within human host cells. Encode suitable regulatory elements (e.g., suitable promoters and termination signals) It contains nucleic acids. A "translationally active RNA molecule" is a protein that is translated by the human cell's translation machinery. It is an RNA molecule that can be translated into [a specific language].

[0129] Therefore, a vector containing the nucleic acid of the present invention (hereinafter referred to as "the vector of the present invention") is provided. .

[0130] In particular, the vector may be a viral vector. Viral vectors are adenoids Viruses, adeno-associated viruses (AAVs) (e.g., AAV types 5 and 2), alphaviruses (e.g., Venezuelan encephalitis virus (VEEV), Sindobis virus (SIN), Semlik Forest virus (SFV), herpesvirus, arenavirus (e.g., lymphocytic choriomeningitis virus) LCMV), measles virus, poxvirus (e.g., modified vaccinia ankara (MVA)), Lamixovirus, lentivirus, or rhabdovirus (e.g., vesicular stomatitis virus) It may also be a VSV vector, that is, the vector is one of the aforementioned viruses It can be derived from. In one embodiment of the present invention, the viral vector is adenowii It is a virus. In another embodiment of the present invention, the viral vector is a poxvirus, For example, MVA.

[0131] 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.

[0132] (Other polynucleotide delivery) An expression construct containing one or more polynucleotide sequences is simply naked recombinant DNA plus It may consist of mido. See Ulmer et al., 1993, Science 259:1745-1749 and Cohen's See the review article in Science 259:1691-1692, 1993. The transfer can be carried out, for example, by any method that physically or chemically permeates the cell membrane. Yes, it is possible. This applies especially to metastases in vitro, but it is not applicable in vivo. The same applies to the DNA encoding the target gene in vivo in the same manner. It is assumed that the DNA molecule will be transferred and the gene product will be expressed. Multiple delivery systems are used for delivery. Several products based on this technology are Approved for use in animals, while others are in Phase 2 and Phase 3 clinical trials in humans. It is currently under investigation.

[0133] (RNA delivery) 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 deliver RNA molecules in vitro or in vitro. It can be introduced into cells via a phagocytic pathway. The introduced biological molecule is then used by the host cell's translation mechanism. It's directly translated, and the encoded polypeptide is produced within the cell into which it's introduced. Uni, RNA-based constructs mimic simple messenger RNA (mRNA) molecules. It can be designed in such a way. Alternatively, RNA molecules can be processed by viral RNA-dependent RNA polymerase. By incorporating them into the structural genes for which they are introduced, they can be incorporated into the cells into which they are self It can be designed to enable amplification. In this way, self-amplifying mRNA (S This molecule, known as AM(trademark) molecule (Geall et al., 2012, PNAS, 109:14604-14609), The RNA molecule of this type shares properties with several RNA-based viral vectors. Either SAM(trademark) RNA or SAM(trademark) RNA (for example, by modifying its sequence or modified nucleic acid) (By using leotide) it can be further modified to enhance stability and translation (Schlak (e et al., RNA Biology, 9:1319-1330), both types of RNA have been formulated (e.g., Emma Lujohn (Brito et al., Molecular Therapy, 2014 22:2118-2129) or lipid nanoparticles Stability in vitro or in vivo (Kranz et al., 2006, Nature, 534:396-401) Modified (and unmodified) RNA can facilitate entry into cells. A wide variety of formulations have been tested as vaccines in animal models and in humans, using multiple RNAs. The base vaccine is being used in ongoing clinical trials.

[0134] (Pharmaceutical composition) The fusion protein, nucleic acid, and vector of the present invention are immunogenic compositions and vaccine compositions. It can be formulated for delivery in pharmaceutical compositions such as those mentioned above (hereinafter all referred to as "the composition of the present invention"). The compositions of the present invention preferably use the fusion protein, nucleic acid, or vector of the present invention as pharmaceuticals. It includes a carrier that is acceptable.

[0135] Therefore, in one embodiment, the fusion protein, nucleic acid, or vector of the present invention An immunogenic pharmaceutical composition is provided that includes a carrier that is acceptable as a pharmaceutical.

[0136] Another embodiment: A vaccine comprising a fusion protein, nucleic acid, or vector of the present invention. The composition is provided with a pharmaceutically acceptable carrier. The preparation of the pharmaceutical composition is typically, for example, For example, Powell and Newman (eds.), Vaccine Design (Subunit and Adjuvant Approaches) (V This invention is described in Accine Design (the subunit and adjuvant approach), 1995. The composition may also contain other compounds that may be biologically active or inactive. Preferably, the composition of the present invention is a sterile composition suitable for parenteral administration.

[0137] In one preferred embodiment of the present invention, one or more (e.g., one) fusion proteins of the present invention The present invention provides a pharmaceutical composition comprising a substance in combination with a carrier that is acceptable as a pharmaceutical.

[0138] In one preferred embodiment of the present invention, one or more fusion proteins of the present invention are represented by ( For example, one nucleic acid or one or more vectors of the present invention (for example, one) may be permitted as pharmaceuticals. The present invention provides compositions that include a carrier in combination with other components.

[0139] The composition of the present invention comprises one or more (e.g., one) polynucleotides and one or more (e.g., one) It may contain a fusion protein component. Alternatively, the composition may contain one or more (e.g., one) vectors and The composition may contain one or more (e.g., one) fusion protein components. Alternatively, the composition may contain one or more (e.g., For example, it may contain one vector and one or more (e.g., one) polynucleotide components. Such compositions may provide an enhanced immune response.

[0140] (Salt that is acceptable as a medicine) The compositions of the present invention are acceptable as pharmaceutically acceptable nucleic acids or fusion proteins provided herein. It will be clear that the salt obtained may contain organic bases (e.g., primary, Secondary and tertiary amines and salts of basic amino acids) and inorganic bases (e.g., sodium Pharmaceuticals containing potassium, lithium, ammonium, calcium, and magnesium salts. It can be prepared from a non-toxic base that is acceptable as such.

[0141] (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.

[0142] 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.

[0143] (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.

[0144] In the case of delivery methods using recombinant nucleic acids (e.g., DNA, RNA, viral vectors), proteins The gene encoding the base immunostimulant, the gene encoding the fusion protein of the present invention It can be easily delivered along with the child.

[0145] (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).

[0146] (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.

[0147] (Dosage) The amount of nucleic acid, fusion protein, or vector in each composition of the present invention is therapeutic or pre-treatment. It can be prepared to obtain a suitable dosage for prophylactic use. Solubility, bio Factors such as availability, biological half-life, route of administration, product shelf life, and other pharmacological properties. The scientific considerations are assumed by those skilled in the art to prepare such compositions, and therefore, Each dosage and treatment regimen may be desirable.

[0148] Typically, a composition containing a therapeutic or prophylactic effective amount is, in the composition of the present invention, per dose Approximately 0.1 ug to approximately 1000 ug of the fusion protein of the present invention, more typically, approximately 2.5 ug to approximately per dose. 100ug of fusion protein is delivered. When delivered in the form of a short synthetic long-chain peptide, administration The amount may range from 1 to 200 ug / peptide / dose. Regarding polynucleotide compositions, This typically involves approximately 10 ug to approximately 20 mg of the nucleic acid of the present invention per dose, more typically per dose Approximately 0.1 mg to 10 mg of the nucleic acid of the present invention is delivered.

[0149] (Diseases that should be treated or prevented) As described elsewhere, Sequence IDs 1-8 are overexpressed in cutaneous melanoma, and the fusion of the present invention is overexpressed in this disease. This is the polypeptide sequence corresponding to the CLT antigen of the combined protein.

[0150] In one embodiment, the present invention relates to a fusion protein and nucleic acid for use in pharmaceuticals. The present invention provides vectors or compositions.

[0151] A further aspect of the present invention is a method for inducing an immune response in a human, wherein the human, A method comprising administering a fusion protein, nucleic acid, vector, or composition of the present invention. do.

[0152] The present invention relates to a fusion protein for use in inducing an immune response in humans. We also provide proteins, nucleic acids, vectors, or compositions.

[0153] Fusion proteins, nucleic acids, vectors, or combinations used to induce an immune response against cancer in humans. The use of the product is based on the fact that the corresponding antigenic sequence (or one or more thereof) is expressed by cancer. It depends on this. Therefore, the design of fusion proteins, nucleic acids, vectors, or compositions There is a relationship between antigenic sequences that are expressed or highly likely to be expressed in cancer. Preferably, immunotherapy is used. The epidemic response is a corresponding sequence or a variant thereof selected from (a) to (f), optionally (g) and (h). It is induced in cancers that express ant or immunogenic fragments. In relation to this, "response "To do" means that the tumor, for example, sequence number A (A is one of sequence numbers 1-8) or its If a variant or immunogenic fragment is expressed (or is likely to be expressed), the present invention Fusion proteins, nucleic acids, vectors, or compositions and pharmaceuticals containing them are under Sequence ID No. A or This means it contains that variant or immunogenic fragment. It also means it contains several antigenic sequences. This refers to a larger immune response to cancer or an immune response to cancer in a wider range of patients. It could potentially be made possible.

[0154] Preferably, the immune response involves CD8+ T cells, CD4+ T cells, and / or an antibody response, particularly CD8+ cells. This includes vesicular lytic T cell responses and CD4+ helper T cell responses.

[0155] Preferably, the immune response is to a tumor, particularly a sequence selected from (a) to (f), optionally (g) and (h). Alternatively, it may be induced in tumors that overexpress these variants or immunogenic fragments.

[0156] In a preferred embodiment, the tumor is a melanoma, such as a cutaneous melanoma.

[0157] The tumor can be a primary tumor or a metastatic tumor.

[0158] A further aspect of the present invention relates to a sequence selected from Sequence IDs 1 to 8 in which cancer cells and any To treat human patients with cancer expressing any one immunogenic fragment or variant. Or, cancer is a sequence selected from sequence numbers 1-8 and any one of its immunogenic fragments. A method for preventing a person who has cancer that expresses the variant, the method for preventing the person from having the variant A method comprising administering a fusion protein, nucleic acid, vector, or composition of the present invention. .

[0159] This invention relates to cancer cells that have a corresponding sequence selected from SEQ ID NOs: 1-8 and any one thereof. This is intended for use in treating or preventing cancer in humans expressing two immunogenic fragments. The invention also provides fusion proteins, nucleic acids, vectors, or compositions.

[0160] The present invention relates to a method for treating a person suffering from cancer, wherein (a) the cancer cells are antigenic A polypeptide sequence selected from lipeptides (a) to (h), or the antigenic polypeptide, or To determine whether to express nucleic acids encoding variants or immunogenic fragments. ; and if so, (b) the human being given the corresponding fusion protein, nucleic acid, vector according to the present invention The present invention also provides a method that includes administering a composition.

[0161] 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.

[0162] The terms "prevention" and "prophylaxis" are interchangeable in this specification. It is used in a specific way.

[0163] (Treatment and vaccination system) The therapeutic regimen comprises (i) a fusion protein, nucleic acid, or vector of the present invention, and (ii) 1 of the present invention. Further fusion proteins, nucleic acids, or vectors, and / or (iii) various other therapeutic agents A therapeutically useful compound or molecule, for example, an antigen that is optionally administered simultaneously with the adjuvant. Co-delivery with further components such as sex proteins (e.g., co-administration) or sequential delivery (e.g., It can be accompanied by either prime-boost. An example of co-administration is ipsilateral co-administration. This also includes contralateral co-administration. "Simultaneous" administration is preferably delivered in the same therapeutic round. This refers to all components present. Preferably, all components are administered simultaneously (for example, DNA and tan). Simultaneous administration of protein, one component within a few minutes (for example, in the same appointment or home visit) or several It can be administered within a certain time.

[0164] "Priming" administration or first dose of the fusion protein, nucleic acid, or vector of the present invention Following this, one or more "boosting" of the fusion protein, nucleic acid, or vector of the present invention. The drug can be administered or subsequently administered ("prime and boost" method). Fusion of the present invention Proteins, nucleic acids, or vectors are used in prime-boost vaccination regimens. It is possible that both the prime and boost are the fusion proteins of the present invention. In each case, it is the same fusion protein of the present invention. Both Prime and Boost are The fusion protein of the present invention may also be used, in which case different fusion proteins of the present invention may be used. Used in the following cases. Both Prime and Boost are nucleic acids or vectors of the present invention. In each case, the nucleic acid or vector of the present invention is the same. Prime and boost Both may be nucleic acids or vectors of the present invention, in which case different nucleic acids or vectors of the present invention may be used. The vector is used in each case. Alternatively, the prime uses the nucleic acid or vector of the present invention. The boost may be performed using the fusion protein of the present invention, or p The boost is performed using the fusion protein of the present invention, and the boost is performed using the nucleic acid or vector of the present invention. It may be performed in the following manner: Typically, the first dose or "priming" dose and the second dose or The "boosting" dose is administered approximately 1 to 12 weeks later, or up to 4 to 6 months later. The "booster" dose may be administered every 1 to 6 weeks, or much later (up to several times). It may be applied (years later).

[0165] Preferably, the prime fusion protein comprises six antigenic polypeptides (a) to (f), Thus, the antigenic polypeptides (a) to (f) are used in CLT antigen fusion protein 1 (SEQ ID NO: 76). As illustrated, (a), (b), (c), (d), (e), and (f) are arranged from N to C in that order. Preferably, the boost fusion protein comprises six antigenic polypeptides (a) to (f), and here Therefore, the antigenic polypeptides (a) to (f) are used in CLT antigen fusion protein 2 (SEQ ID NO: 77), for example. As shown, (c), (f), (d), (b), (e), and (a) are arranged from N to C in that order. In short, (a) is located at the N of the prime, (f) is located at the C-terminus of the prime, and (c) is boosted (a) is present in N of the boost, and (a) is present at the C-terminus of the boost.

[0166] More preferably, the prime fusion protein comprises eight antigenic polypeptides (a) to (h). Here, the antigenic polypeptides (a) to (h) are in CLT antigen fusion protein 3 (SEQ ID NO: 78). As illustrated, the order from N to C is (a), (b), (g), (d), (e), (h), (c), and (f). They are placed in [location]. Preferably, the boost fusion protein consists of eight antigenic polypeptides (a) to (h ) comprising, where the antigenic polypeptide (a) to (h) is CLT antigen fusion protein 4 (SEQ ID NO: As illustrated in 79), the order is (c), (g), (a), (h), (e), (f), (d), and (b) It is arranged from N to C. Preferably, (a) is at the N of the prime and (f) is at the C-terminus of the prime. (c) is present in the N of the boost, and (b) is present at the C-terminus of the boost.

[0167] (Antigen combination) The fusion protein, nucleic acid, or vector of the present invention stimulates an immune response to melanoma, for example, skin One or more other antigenic polypeptides that induce melanoma or uveal melanoma (or so Can be used in combination with polynucleotides or vectors that encode the same information. These other antigenic polypeptides can be obtained from a variety of sources, and these are sufficient. Melanoma-associated antigens described in [reference], such as GPR143, PRAME, MAGE-A3, or pMel(gp100) These may include patient-specific neogenic antigens (Lauss et al. (2017), Nature). Communications, 8(1), 1738. http: / / doi.org / 10.1038 / s41467-017-01460-0), retaining type Thrombocytonic nascent antigen (Smart et al. (2018), Nature Biotechnology. http: / / doi.org / 10.103) 8 / nbt.4239), splicing variant neogenic antigen (Hoyos et al., Cancer Cell, 34(2)), 181-183. http: / / doi.org / 10.1016 / j.ccell.2018.07.008; Kahles et al. (2018), Cance r Cell, 34(2), 211-224.e6.http: / / doi.org / 10.1016 / j.ccell.2018.07.001), peptide A category known as antigens that encode T cell epitopes associated with processing disorders. Melanoma antigens contained within the Lie (TIEPPs; Gigoux, M. and Wolchok, J. (2018), JEM, 215) 2233, Marijt et al. (2018). JEM 215, 2325), or the newly discovered antigen (CL) Other types of melanoma antigens may be included, including the T antigen. Furthermore, these various sources Antigenic peptides derived from (i) nonspecific immunostimulants / adjuvant species and / or (ii) For example, to amplify the anti-melanoma-specific response induced by the co-administered antigen, The universal CD4 helper epitope is known to induce strong CD4 helper T cells. The antigens include (as polypeptides, or polynucleotides encoding these CD4 antigens) It can also be combined with (which is delivered as a vector).

[0168] We can provide nucleic acids encoding the aforementioned proteins and vectors containing them.

[0169] Formulation of different proteins, nucleic acids, or vectors in the same formulation or in separate formulations. It is possible.

[0170] 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 one or more polypeptides and one or more polynucleotide components; (4) As two or more individual polynucleotide components; (5) As a single polynucleotide encoding two or more individual polypeptide components; or (6) A single molecule encoding a fusion protein containing both (or additional) polypeptide components. As polynucleotides It can be presented.

[0171] For convenience, if several components are present, they are considered a single fusion protein or a single It is often desirable that it be included in the polynucleotide encoding the fusion protein. (See below). All components can be provided within a single fusion protein. Alternatively, All components are polynucleotides (e.g., single polynucleotides, e.g., single fusions) It can be provided as a polynucleotide that codes for a protein. [Examples]

[0172] (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.

[0173] 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 the following: Table S1) 768 data obtained from The Cancer Genome Atlas (TCGA) Consortium RNA sequencing reads derived from patient samples were used for genome guide assembly. Samples with a balanced sex distribution (excluding sex-specific samples) were processed using cutadapt(v1.13)(Marcel M. Using the reference (2011, EMBnet J., 17:3), adapter trimming and quality (Q20) Trim and length filter (both reads of the pair ≥ 35 nucleotides), and each The maximum and minimum depths are set to 200 and 3 using khmer(v2.0) (Crusoe et al., 2015). The reads were normalized using KMER (k=20) with F1000Res., 4:900. Using the same settings as before, 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), The built-in in silico depth normalization was disabled, and genome-guided assembly was performed. 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)tri After performing trimming (trimpoly in SeqClean v110222) and entropy filtering (≧0.7), Removed low-quality and artifact-containing contigs (bbduk in BBMap v36.2). Cancer type Each of the original 24 samples was processed using Salmon (v0.8.2 or v0.9.2) (Patro, R. et al., 2017, Nat.Me). Using thods, 14:417-419), a pseudo-mapping is performed on the cleaned assembly, and 0. Contigs showing transcript expression (TPM) of less than 1 per million were removed. Using GMAP (v161107) (Wu et al., 2005, Bioinf., 21:1859-1875), GRCh38 was analyzed. The data is mapped and the contiguous data is not aligned with an identity of 85% or more over 85% of its length. The g was removed from the assembly. Finally, all cancer type assemblies were put together and flattened. I then used gffread (Cufflinks v2.2.1) (Trapnell et al., 2010, Nat. Biotech., 28:511). -515) was used to integrate into the longest continuous transcript. This assembly process is repeated. Because it is specifically designed to enable rement evaluation, single exon transcripts It was retained but flagged. The integrity and quality of the transcript assembly were determined by GENCODE. Ratio of v24basic to MiTranscriptome1 (Iyer et al., 2015, Nat. Genet., 47: 199-208) The evaluation was conducted by comparison. The inventors identified a unique splice location displayed within GENCODE. Edit the list and ensure this splice site is within the trans nucleotide grace window. We tested whether it was present in the cryptome assembly. This process revealed 1,001, 931 transcripts were identified, of which 771,006 were spliced, and 230,925 were... It was from a single exon.

[0174] 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.

[0175] Using Salmon, we estimated the total transcripts (TPM) per million units for all transcripts, and for 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.

[0176] 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.

[0177] 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.

[0178] Of the 403 CLTs for cutaneous melanoma, 97 passed through these filters.

[0179] (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.

[0180] 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).

[0181] 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).

[0182] 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 fragment ions was determined (Ternette et al., 2018), and these tumor samples... MS / MS datasets corresponding to the immunopeptideome for each were created.

[0183] 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.

[0184] 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.

[0185] 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 databases. 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.

[0186] 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.

[0187] 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. .

[0188] Use NetMHCpan 4.0 prediction software (http: / / www.cbs.dtu.dk / services / NetMHCpan / ) By doing so, it is detected in association with HLA class I molecules listed in Table 1 that are 9 amino acid residues or longer. We evaluated all peptides and predicted their 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 each of them) All 9-mers included in the complete sequence are present in at least one of the tested supertypes. It was shown that binding is predicted (see Table 2). Of these, many of the sequences are detected High confidence (low rank score %) for certain types within the HLA Class I Supertype that were tested. It was predicted that they would bind. All of the detected peptides bound to the standard set of HLA types. The fact that this was expected to happen provides further validation for their detection. Furthermore, all peptides found in tumor samples derived from the inventors' dataset are also included. The present inventors found that NetMHC can bind to one of the HLA types detected in patient samples. Predicted by pan 4.0. The HLA type is associated with the peptide discovered by the inventors. Regarding the patients, according to the literature by Bassani-Sternberg et al. (2016, Nature Commun., 7: 13404) Although this has not been reported, if it has been reported, the inventors believe that this is a known HL We found a correspondence between the predicted HLA type and type A.

[0189] 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 in our database), the spectrum below shows the synthetically produced peptide of the same sequence. Corresponds to the selected m / z values ​​of the detected ion fragments in these MS / MS spectra. These figures show the above / below each of the fragment peaks. These figures show the precise alignment of the fragments. (To clarify the difference between tumor-derived fragment ions and synthetic peptide-derived fragment ions, experimentally determined) The slight difference in the resulting m / z values ​​is well within the acceptable range of m / z <0.05 Daltons. This confirms the accuracy of the assignment of each CLT-coding peptide from the ulcer tissue-derived spectrum.

[0190] 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.

[0191] 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.

[0192] 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. Chido is loaded onto HLA class I molecules, enabling the cell to become a target for cell lysis by T cells that recognize the resulting peptide / HLA class I complex. Therefore, these CLT antigens and their fragments are considered useful in various therapeutic modalities for the treatment of melanoma in patients whose tumors express these antigens. Table 1: List of peptides identified by immunopeptidome analysis of melanoma samples, with cross-reference to CLT antigen names and SEQ ID NOs [TableTableEND]] TIFF2026086469000003.tif142170 1 HLA class I peptides identified by mass spectrometry. 2 Bassani-Sternberg et al., 2016, Nature Comm., 7: 13404 (Mel-3, Mel-5, Mel-8, Mel1-6, Mel-21, Mel-27, Mel-29, Mel-30, Mel-36, Mel-39, Mel-41); our dataset (1MT1, 2MT1, 2MT3, 2MT4, 2MT10, 2MT12). 3 Calculated peptide mass. 4 5 10lgP values for peptides that gave the maximum match to the peptide / patient with multiple spectrum detection by the PEAKS™ program are shown. Values for peptides identified by analysis B performed using Mascot software are not available (na). 6 1 2 3 4 5 Deviation between observed and calculated mass; selected for peptides with multiple spectra obtained The ppm values ​​are shown. Values ​​for the peptides identified by analysis B are not available. i (na). Table 2: Peptides identified by mass spectrometry, with cross-references of CLT antigen names and sequence numbers. 18 HLA class I supertype alleles (HLA-A01:01, HLA-A02:01, HLA-A0) of length ≥ 9 residues 3:01, HLA-A11:01, HLA-A24:02, HLA-A25:01, HLA-A26:01, HLA-A68:01, HLA-B07:02, HL 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]

[0193] 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.

[0194] (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., Cancer Immunol. 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].

[0195] 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).

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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]

[0203] (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]

[0204] (Example 5 - Staining of reactive T cells with CLT antigen peptide pentamer and the peptides therefrom) (Demonstration of killing 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] (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 the intercalating dye (SYBR Green) is used. Quantitative analysis is performed using fluorescence. A primer pair is designed for CLT, and the melanoma cell line or RNA extracted from primary patient tissue was assayed. Non-melanoma cell lines were used as negative controls. The melanoma cell lines used were COLO 829 (ATCC reference number CRL-1974) and MeWo (ATCC reference number CRL-1974). C 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 TIB-8065), It included CC reference number HTB-22). All patient-derived melanoma tissue was at least stage IIC. RNA was obtained from six primary lesions and six metastases derived from patients with the disease. Extracted from and reverse transcribed into cDNA according to standard procedures. SYBR Green assay followed standard techniques. qRT-PCR analysis accompanied by output was performed on two regions of each CLT and a reference gene designed as a planar The calculation was performed using Mar. The relative quantitative value (RQ) was calculated as follows: RQ=2[Ct(reference)-Ct(target)].

[0209] 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 CL encoding CLT antigen 6 on RNA extracted from melanoma tissue samples and one non-melanoma cell line. Using two primer sets (76+77 and 78+79) targeting different regions of T (sequence number 60) The results of the qRT-PCR assay are shown. Panel F contains 12 melanoma tissue samples and 1 non-melanoma sample. Different regions of CLT (SEQ ID NO: 61), which encodes CLT antigen 7 on RNA extracted from chromoma cell lines. The results of qRT-PCR assays using two target primer sets (44+45 and 46+47) are shown. Panel G contains RNA extracted from 12 melanoma tissue samples and one non-melanoma cell line. Two primers targeting different regions of CLT (SEQ ID NO: 62), which encodes the CLT antigen 8. The results of qRT-PCR assays using samples (80-81 and 82-83) are shown. Compared to non-melanoma cells, the specificity of CLT in RNA extracted from melanoma cell lines or tissue samples Targeted expression was confirmed. Each CLT was detected in two or more cell lines or tissue samples analyzed. Expression was barely or completely undetectable in non-melanoma control cell lines.

[0210] 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.

[0211] 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

[0212] 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]

[0213] (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 done. This method involves several steps.

[0214] 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 CD14-positive monocytes, or Alternatively, dendritic cells (DCs) obtained from the monocyte fraction of the apheresis product may be used. DCs capture CD14 (for example, an anti-CD14 antibody conjugated to magnetic beads, in this case CD1). 4. Positive cells are labeled with beads and captured in a magnetic column) for positive isolation or Adhesion properties, for example, peripheral blood mononuclear cells (PBMCs) and cell cultures that allow monocyte adhesion between 4 and 48 hours. Methods such as isolation by adhesion to tissue culture plastic through incubation with a growing dish. It can be generated by DC, but is not limited to GM-CSF, IL-4, TNFα, IL-1 The use of cytokines such as β, IL-6, and prostaglandin E2 is well explained. Depending on the method, it can be generated from CD14-positive or adherent immune cell fractions. Incubation with cytokines for 2-7 days typically involves CD14+ monocytes, and then CD14... Loss of expression, and expression of DC markers such as CD11c and high levels of MHC class II, It enables differentiation into upregulated DCs. The properties of T cells for selection and / or stimulation are those of PBMCs. Monocyte-removed fraction (in the case of apheresis-derived T cells), expression of markers such as CD3, or specific Target T cell subsets, for example, but not limited to CD4, CD8, CD45RO, CD45RA, CCR7, CD62L This involves the isolation of pan-T cells using isolation techniques based on the presence or absence of markers such as CD27. obtain.

[0215] 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.

[0216] 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. This involves multiple CLT antigens to APCs such as autologous DCs produced from patient apheresis products. This may involve the introduction of (what is expected to be expressed by the patient's tumor). CLT antigen introduction Entry is by delivery of polypeptides linked to multiple CLT antigens, for example, viral This can be delivered by a culler, or as individual pooled CLT antigens, for example, via mRNA-based delivery. This could be a method. A method for delivering stabilized mature mRNA to APC (i.e., transfects (The ion) is polyethyleneimine (PEI) or calcium phosphate for nucleic acid delivery into cells. Any conventional reagent can be included. Alternatively, efficient transfection can be performed using APC. This can be achieved using lipid-based reagents for transfection. In their transfection reaction, lipid complexes, such as lipid nanoparticles (LNPs) or (mRNA) are used. Formulated in a lipid-based lipoplex (formed by simple mixing with a lipid reagent). Synthetic mRNA derived from the in vitro transcription reaction (IVT) is used. Therefore, a well-described promoter for phage T7 DNA-dependent RNA polymerase The element, followed by a cDNA encoding a highly stable mRNA 5'UTR, and the CLT antigen code cDNA encoding an optimized open reading frame (ORF), and highly stable mRNA 3'UT It releases a cDNA encoding R, a poly-A sequence of >20 nucleotides, and a functional poly-A tail. Recombinant DNA constructs containing unique restriction endonuclease sites designed for this purpose Lacto is used as a template for in vitro transcription (IVT) of mRNA encoding a suitable CLT antigen. This is possible. To create human APCs that express antigens encoding IVT mRNA, Cafri et al. Using a lipoplex method similar to that described in the literature, Nat.Comm.2019. This can be done. In short, APCs (monocytes or DCs) are plated into tissue culture flasks. This achieves 70-90% confluence. Lipid-based transfection reagents (e.g.) For example, Lipotectamine®, MessengerMAX®, or FuGENE® HD, or similar. Dilute the product appropriately in a serum-free medium such as Opti-MEM (trademark), mix, and encode the mR (mR) of the CLT antigen. Incubate with NA. This is the transfection of the CLT antigen combination to APC. This can be done using multiple CLT antigen mRNAs for the purpose of incubation. The immersion time is short (5-10 minutes) and at room temperature. The resulting mRNA-lipid complex is added to APC. 37℃ / 5%CO2 is the optimal time for presentation of the translated peptide from the mRNA molecule encoding CLT. Incubate for 16 to 72 hours, depending on the location.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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].

[0222] 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, which is known to be presented on HLA molecules (deconvoluted by mass spectrometry experiments) 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.

[0223] In conclusion, these methods can be used to "immunize" human T cells ex vivo with CLT antigen. This demonstrates that it is possible to produce immunologically reactive / cytolytic T cells. Therefore, vaccination of cancer patients with one or more CLT antigens is a therapeutic step in controlling cancer. This supports the possibility that it has value.

[0224] (Example 8 - Method for designing CLT antigen fusion protein) To facilitate the delivery of a mixture of multiple polypeptide antigens via a vectorized vaccine. The genes of the component antigens are combined to form a single ORF, and an antigenic fusion protein is created. It is highly desirable to achieve synthesis. Furthermore, directly linking the component polypeptides is not possible. Instead, these are connected by a peptide linker region: 1) Mimicking a normal human protein This reduces the potential risk of generating novel epitopes in the fused junction (safety). (Increased sexuality), and 2) CLT antigen T cell epitopes adjacent to the fusion / linker are present in tumor tissue. When expressed from individual ORFs coded as such, the process mimics their presentation. It can guarantee that it will be fused (increased effectiveness). Safe and effective fusion protein An algorithm was developed to achieve concatenation, which facilitates the design of the components. Short linkers are better suited to achieving the above objectives, so we use them in this algorithm. To use, select several Gly-based linkers, and choose some of them. This causes the protein to lose its identity with normal human proteins, and the terminals of the CLT antigen component are also affected. [ka] It also contained Lys residues to facilitate processing in the process.

[0225] 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.

[0226] To achieve the above needs, four individual fusion proteins (a vaccine of six CLT antigens) CLT antigen fusion protein 1 / CLT antigen fusion protein 2 and 8 CLT antigens for the regimen The respective settings for CLT antigen fusion protein 3 / CLT antigen fusion protein 4) for vaccine regimens Six criteria were considered in the calculation. These were applied one by one, and then sequentially as needed. Through iterative testing, we ensured that the final fusion protein candidate met all the criteria.

[0227] 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).

[0228] 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.

[0229] 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.

[0230] 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 the N-terminal methionine is also possible 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.

[0231] Fifth, one application of fusion protein cassettes is in prime / boost regeneration. Therefore, to avoid directly repeating CLT antigen binding and reducing epitope repetition, We designed a pair of fusion proteins to be used for this purpose.

[0232] 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.

[0233] (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.

[0234] (Example 9 - Antigenicity of Fusion Protein) The CLT antigen fusion protein, designed as described in Example 8, is translated and cytoplasmic. It is proteolytically processed within the sol and is associated with HLA class I molecules on the cell surface. It is thought that the cDNA construct encoding the fusion protein cassette will be presented. Example 2: Transduction into human cells, immunoprecipitation of HLA class I molecules, and discovery of CLT antigen. The MS analysis described was performed. This was done to determine if the CLT antigen fusion protein cassette was found in the tumor tissue. Maintains similar antigen-presenting properties to the previously identified component CLT antigen (as shown in Example 2). It is done to show that something was done.

[0235] 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 a fusion protein tested in the delivery of peptide epitopes derived from CLT antigens. This confirms the value of quality design.

[0236] To conduct MS-based research on fusion protein design, cultured human cells are used in CLT A suitable polII promoter for plasmid DNA encoding an antigen fusion protein cassette Transduction is performed under the control of the 5' and 3' UTRs. After expansion, the CLT antigen fusion protein cassette is coated The cultured cells are lysed, and the HLA class I peptide complex is captured by an anti-HLA class I antibody. Affinity purification is performed. Subsequently, the isolated HLA molecules and bound peptides are separated from each other. The eluted peptides are then analyzed by nUPLC-MS / MS. Subsequently, these HLA class I peptides are analyzed. MS / MS spectra obtained from down are processed using PEAKS™ software (v8.5 and vX, Bioin This can be investigated using (Formatics Solutions Inc.). For MS / MS interpretation, this software... Towea is included in the human proteome, which has polypeptides of related fusion proteins. We will evaluate all theoretical spectra of the polypeptides in parallel. Regarding these studies, Most class I HLA-binding peptides found within cells are constitutively expressed proteins. Therefore, the repertoire of analyzed sequences is related to the CLT antigen fusion protein construct. It is essential that the product contains sequences of lactose and human proteome.

[0237] The results of these studies suggest that the HLA class I repertoire of transduced cells is processed We are identifying the single and individual CLT antigen fusion protein-derived peptides presented. Using the analysis of these data, we found that the design effectively presents peptides derived from individual CLT antigens. This indicates that the tested protein fusions Cells transduced with CLT antigen fusion protein cDNA from an epitope derived from the linker region. This indicates that it is not presented efficiently.

[0238] In summary, these data show that CLT antigens derived from CLT antigen fusion protein constructs are... It can provide strong support for the translation, processing, and presentation of epitopes. However This induces T cells that recognize the CLT antigen peptide / HLA class I complex found on tumors in patients. These CLT antigens in vaccines (or other therapeutic modalities) designed to release This enables the use of composite proteins.

[0239] (Example 10: Killing of cell lines expressing fusion proteins) Anti- The immunogenicity of the original was combined with the CLT-peptide-reactive T cells described in Example 5. This can be demonstrated using an infected cell line. CL by CLT antigen-specific CD8 T cells Using cell lines transfected with T antigen fusion proteins, we investigated the effects of C in cancer patients. This indicates the presence of a therapeutically relevant T cell response to linked combinations of LT antigens.

[0240] CLT antigen fusion proteins 1, 2, 3, or 4 (SEQ ID NOs: 76-79) described in Examples 8 and 9 CaSki cells transfected with a construct encoding ) are described in Example 5. It is used as a target in the killing assay. It is derived from CLT antigens 1, 2, 3, 4, 5, 6, 7, and 8. Using HLA-pentamers, CD8 T cell lines isolated from healthy donors and melanoma patients were used. Regarding the killing ability of target cells transfected with these CLT antigen fusion proteins Test individually. Negative control cells are untransfected CaSki or unrelated cells. These are CaSki cells transfected with Instruct.

[0241] (Example 11: Mouse immunogenicity study) Immunity of individual CLT antigens within the fusion protein construct described in Example 8 To demonstrate its virulence, mice were given a priming CLT antigen fusion protein sequence (CLT antigen fusion protein). (Encodes protein 1 or CLT antigen fusion protein 3; Figures 67 and 69; SEQ ID NOs. 76 and 78) Primin Immunotherapy is administered, and boosting CLT antigen fusion protein sequences (each containing CLT antigen fusion protein) are used. Synthetic protein 2 or CLT antigen fusion protein 4 (Figures 68 and 70, SEQ ID NOs. 77 and 79) Booster immunization using MVA vectors (replica-deficient modified vaccinia ankara vectors) It can be administered.

[0242] For these studies, non-inbred mice (possessing diverse major histocompatibility class I and II molecules) Using experimental mice obtained from a population that exhibits this behavior, we aim to more accurately mimic human non-inbreeding. Furthermore, as mentioned above, this experiment involved a priming vector (CLT antigen fusion vector). Protein 1, CLT antigen fusion protein 3; Figures 67 and 69; SEQ ID NOs. 76 and 78) and Booth Ting vector (CLT antigen fusion protein 2, CLT antigen fusion protein 4; Figures 68 and 70, sequence) Using numbers 77 and SEQ ID NO: 79, fusion protein anti- Imitate the original usage.

[0243] Despite the usefulness of the design for use in humans (see Example 8), human proteo Loss of linker-derived sequences matching the M, and predicted binding to human HLA class I molecules. Many aspects of this design, including the loss / reduction of linker-derived sequences, have been tested in mouse immunotherapy models. It is not possible to test this. Nevertheless, antigen processing in mouse cells and human cells Because the mechanisms are similar, the focus of mouse research should be on the immune response to the CLT antigen itself. By doing so, these studies will be useful for the design and processing of fusion proteins. Providing information in practice (Kumanovics A, Takada T, Lindahl KF literature, Mammalian MHC genotypes) Genomic organization of the mammalian MHC. Annu Rev Immunol 2003; 21: 62 9-57. DOI: 10.1146 / annurev.immunol.21.090501.080116. Madi A, Poran A, Shifrut E Our literature shows that the mouse and human T cell receptor repertoire is conserved around the public CDR3 sequence. Clustered into a similarity network (T cell receptor repertoires of mice and humans are clustered in similarity networks around conserved public CDR3 sequence ces). eLife 2017;6:e22057. DOI: 10.7554 / eLife.22057).

[0244] To evaluate cancer-related immunogenicity in vaccinated animals, use the IFNγ ELISPOT assay. By using this method, immune cells collected from vaccinated mice can be converted into CLT antigen-specific T cells. The presence of cells will be investigated (Mennuni et al., Int. J. Cancer, 2005). In short, As described above, vaccinated mice were humanely euthanized, and the following was prepared from these animals. The spleen cells are subjected to duplicated peptides corresponding to one or more sequences of the true CLT antigen. In the presence (or absence) of [the substance], the mouse IFNγ was derivatized with a monoclonal antibody against [the substance]. The material was loaded into the wells of the Luchwell dish (see schematic diagram in Figure 71).

[0245] After a suitable incubation period (e.g., 16-24 hours), the T cells activated with the peptide were... Immobilized IFNγ secreted by cells (IFN in the presence of immobilized monoclonal antibodies) Stain with a second anti-IFNγ monoclonal antibody (selected to specifically select γ), and The presence of mouse T cells induced by a vaccine that recognizes the CLT antigen peptide loaded into the cell. Enables counting of cells / spots that exhibit specificity to the stimulated CLT antigen peptide pool. To calculate the target immune response, the number of spots was incubated in the absence of the peptide. The number of IFNγ stained spots observed in wells of spleen cells from the same animal is normalized. ru.

[0246] The data from these studies shows that the fusion protein used to vaccinate mice It shows immunogenicity of CTL antigens present in the crystalline construct, and its component CTL antigen The usefulness of these fusion proteins in priming and boosting immune responses This supports the idea of ​​sexuality.

[0247] (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] TIFF2026086469000072.tif243170 Sequence ID 58 (CDNA sequences of CLT encoding CLT antigens 3 and 4) [ka] TIFF2026086469000074.tif97170 Sequence ID 59 (CDNA sequence of CLT encoding CLT antigen 5) [ka] TIFF2026086469000076.tif146170 Sequence ID 60 (CDNA sequence of CLT encoding CLT antigen 6) [ka] TIFF2026086469000078.tif242170TIFF2026086469000079.tif63170 Sequence ID 61 (CLT cDNA sequence encoding CLT antigen 7) [ka] Sequence ID 62 (cDNA sequence of CLT encoding CLT antigen 8) [ka] TIFF2026086469000082.tif222170 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] Sequence ID 83 (codon-optimized cDNA sequence encoding CLT antigen fusion protein 4) [ka] 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. A fusion protein comprising six antigenic polypeptides (a) to (f), wherein the antigenic polypeptide Tide(a) to (f) are the amino acid sequences: (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: 6 or its variants or SEQ ID NO: 6 or its variants piece; (d) Immunogenicity of SEQ ID NO: 7 or its variants or SEQ ID NO: 7 or its variants piece; (e) Immunogenicity of SEQ ID NO: 4 or its variants or SEQ ID NO: 4 or its variants Fragments; and (f) Immunogenicity of SEQ ID NO: 8 or its variants or SEQ ID NO: 8 or its variants piece The fusion protein having the above characteristics.

2. The protein is selected from one or two further antigenic polypeptides (g) and (h). The material further comprises a genicity polypeptide, wherein the antigenic polypeptide (g) and (h) are amino acids array: (g) Immunogenicity of SEQ ID NO: 3 or its variants or SEQ ID NO: 3 or its variants Fragments; and (h) Immunogenicity of SEQ ID NO: 5 or its variants or SEQ ID NO: 5 or its variants piece A fusion protein according to claim 1, having the following characteristics.

3. The fusion protein according to claim 1, wherein the protein comprises six antigenic polypeptides (a) to (f). Quality.

4. The antigenic polypeptides (a) to (f) are arranged in the order of (a), (b), (c), (d), (e), and (f) from N to C. The fusion protein according to claim 3, which is positioned at [location].

5. The antigenic polypeptides (a) to (f) are arranged in the order of (c), (f), (d), (b), (e), and (a) from N to C. The fusion protein according to claim 3, which is positioned at [location].

6. The fusion protein according to claim 2, wherein the protein comprises eight antigenic polypeptides (a) to (h). Quality.

7. The antigenic polypeptides (a) to (h) are (a), (b), (g), (d), (e), (h), (c), and (f) The fusion protein according to claim 3, wherein the cells are arranged in order from N to C.

8. The antigenic polypeptides (a) to (h) are (c), (g), (a), (h), (e), (f), (d), and (b) The fusion protein according to claim 3, wherein the cells are arranged in order from N to C.

9. Claims 1 to 8, wherein the polypeptide is linked by one or more peptide linkers. A fusion protein as described in any one of the items.

10. The above one or more linkers, polypeptide: (i)(a) and (b), (b) and (c), (c) and (d), (d) and (e), (e) and (f); (ii)(c) and (f), (f) and (d), (d) and (b), (b) and (e), (e) and (a); (iii) (a) and (b), (b) and (g), (g) and (d), (d) and (e), (e) and (h), (h) and (c), (c) and (f); or (iv)(c) and (g), (g) and (a), (a) and (h), (h) and (e), (e) and (f), (f) and (d), (d) and (b) A fusion protein according to claim 9, positioned between the two.

11. The linker corresponds to Sequence IDs 71, 72, 73, 74, 75, and The any one of claims 9 to 10 includes or consists of a sequence selected from sequence number 84. A fusion protein as described in item 1.

12. It includes an array selected from sequence numbers 76, 77, 78, and 79, Or a fusion protein according to claim 1, comprising the sequence.

13. The aforementioned fusion protein (i) is a melanoma-associated antigen of another polypeptide; (ii) enhances the immune response. (iii) polypeptide sequences that can be strengthened (i.e., immunostimulatory sequences); and (iii) for example, antigens Universal can provide strong CD4+ which helps increase the CD8+ T cell response to epitopes. A second or further polypeptide sequence selected from polypeptide sequences containing the CD4 helper epitope A fusion protein according to any one of claims 1 to 12, which is fused to a lipeptide.

14. An isolated nucleic acid encoding a fusion protein according to any one of claims 1 to 13.

15. The nucleic acid according to claim 14, wherein the nucleic acid is DNA.

16. The nucleus according to claim 15, wherein the nucleic acid is codon-optimized for expression in human host cells. acid.

17. The nucleic acid according to claim 14, wherein the nucleic acid is RNA.

18. The nucleic acid according to any one of claims 14 to 17, wherein the nucleic acid is an artificial nucleic acid sequence.

19. A vector comprising the nucleic acid according to any one of claims 14 to 18.

20. Suitable regulatory elements for enabling the transcription of translationally active RNA molecules within human host cells The vector according to claim 19, comprising DNA encoding a gene.

21. The vector according to any one of claims 19 to 20, wherein the vector is a viral vector. 。

22. The aforementioned viral vector is adenovirus, adeno-associated virus (AAV), alphavirus Rus, herpesvirus, arenavirus, measles virus, poxvirus, paramic The vector according to claim 21 is a sovirus, lentivirus, or rhabdovirus vector Tar.

23. The vector according to claim 22, wherein the viral vector is a poxvirus (e.g., MVA) Tar.

24. The vector according to claim 22, wherein the viral vector is an adenovirus.

25. A fusion protein, nucleic acid, or vector and pharmaceutical according to any one of claims 1 to 24 An immunogenic pharmaceutical composition comprising a carrier that can be tolerated.

26. A fusion protein, nucleic acid, or vector and pharmaceutical according to any one of claims 1 to 24 A vaccine composition comprising a carrier that is acceptable.

27. The composition or vaccine further comprises one or more immunostimulants, according to claims 25 and 26. The composition described in any one of the items.

28. 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 27, selected from ferron.

29. The composition or vaccine is a sterile composition suitable for parenteral administration, according to any of claims 25 to 28. The composition described in any one of the items.

30. A fusion protein, nucleic acid, etc., according to any one of claims 1 to 29, for use in pharmaceuticals. A cult, or composition.

31. A method for inducing an immune response in a human, wherein the human is described in any one of claims 1 to 29. The method comprising administering a fusion protein, nucleic acid, vector, or composition thereof.

32. The immune response is a sequence selected from antigenic polypeptides (a) to (f), optionally (g) and (h). The method according to claim 31, which is induced against cancer expressing [the specified expression].

33. A fusion according to any one of claims 1 to 29 for use in inducing an immune response in humans. Synthetic proteins, nucleic acids, vectors, or compositions.

34. The immune response is a sequence selected from antigenic polypeptides (a) to (f), optionally (g) and (h). A fusion protein, nucleic acid, vector, or This is a composition.

35. Cancer cells express sequences selected from antigenic polypeptides (a) to (h) in cancer patients. To treat human patients or to ensure that the cancer expresses a sequence selected from polypeptides (a) to (h) A method for preventing a person who has cancer, wherein the person is given any one of claims 1 to 29. The method comprising administering the fusion protein, nucleic acid, vector, or composition described in the section. 。

36. The cancer cells express sequences selected from polypeptides (a) to (h) in humans. A fusion protein according to any one of claims 1 to 29, for use in treatment or prevention. A substance, nucleic acid, vector, or composition.

37. The cancer is a melanoma, for example, a cutaneous melanoma or a uveal melanoma, in particular a cutaneous melanoma. , a method for use or a fusion protein, nucleic acid, according to any one of claims 32, 34 to 36 A vector or composition.

38. A method of treating a person suffering from cancer, (a) The cancer cells are polypeptide sequences selected from polypeptides (a) to (h) or the polypeptide A step of determining whether or not the nucleic acid encoding the cydo is expressed; and if so, (b) The human being given the corresponding fusion protein, nucleic acid, or vector according to any one of claims 1 to 29. - or a step of administering the composition. The method including:

Images