Novel cancer antigens and methods

Abstract

To provide antigenic polypeptides for use in the treatment or prevention of cancer, in particular for use in the treatment or prevention of melanoma (e.g., cutaneous melanoma or uveal melanoma).SOLUTION: Provided is an isolated polypeptide having a specific sequence, or an isolated polypeptide selected from a variant or immunogenic fragment of the specific sequence. The polypeptide is useful for the treatment, prevention, and diagnosis of cancer, particularly melanoma, especially cutaneous melanoma and uveal melanoma.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for the treatment or prevention of cancer, particularly melanoma (e.g., cutaneous melanoma). Antigenic polypeptides and their derivatives for use in the treatment or prevention of melanoma (melanoma or uveal melanoma) The present invention further relates, inter alia, to said nucleic acids and polypeptides. , loaded with and / or stimulated by these polypeptides and polynucleotides. The immune cells that recognize the polypeptides, the antibodies specific to those polypeptides, and the Pharmaceutical compositions containing cells (autologous or otherwise derived) genetically engineered with molecules that It relates to immunogenic compositions. [Background technology]

[0002] BACKGROUND OF THE INVENTION As part of normal immune surveillance against pathogenic microorganisms, all cells produce intracellular proteins It breaks down the ATP to produce peptides, which are then expressed on major histocompatibility complex (MHC) class I molecules. These molecules originating from the host cell are loaded onto the surface of all cells, and the molecules are expressed on the surface of all cells. Most of the peptides are recognized as self and remain unrecognized by the adaptive immune system. On the other hand, foreign (non-self) peptides can stimulate the proliferation of naive CD8+ T cells, which encodes a T cell receptor (TCR) that tightly binds the MHC I-peptide complex. The expanded T cell population is an effector CD8+ T cell population that can eliminate cells tagged with foreign antigens. T cells (including cytotoxic T lymphocytes, CTLs) can be produced, and foreign antigen-tagged cells can be generated. Memory CD8+ T cells that can be re-expanded if they appear later in the animal's life can also be produced.

[0003] Expression of MHC class II molecules is usually observed on professional antigen-presenting cells such as dendritic cells (DCs) ( APCs), and MHC class II molecules are normally internalized from the extracellular environment. Loaded with peptides. T cell adhesion molecules (CD54, CD48) and costimulatory molecules (CD40, CD80, In the presence of various factors, including CD86, complementary TCRs from naive CD4+ T cells bind to MHCII-peptides. Upon binding to the ATP-binding complex, CD4+ T cell effector cells (e.g., T H 1. T H 2. T H 17, T FH , T reg These effector CD4+ T cells are induced to mature into antibody-secreting B cells. It can promote differentiation into antigen-specific CD8+ CTLs, and This allows for the development of immune responses to foreign antigens, including both short-term effector functions and long-term immunological memory. DCs help induce adaptive immune responses by targeting exogenously derived antigens (e.g., released from pathogens or tumor cells). By delivering a peptide or protein (e.g., a peptide or protein) onto those MHC I molecules, It is capable of carrying out the cross-presentation process of peptide antigens and is expressed by naive CD8+ T cells. contribute to the generation of immune memory by providing an alternative pathway for stimulating cell proliferation. do.

[0004] Immunological memory (specifically, antigen-specific B cells / antibodies and antigen-specific CTLs) controls microbial infections Immunological memory plays an important role in preventing diseases caused by important pathogenic microorganisms. Immunological memory has also been used to develop numerous vaccines to prevent tumorigenesis. Although it is known that cancer plays an important role in the control of cancer, few effective cancer vaccines have been developed. It has not been done.

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

[0006] Current treatments for cutaneous melanoma are diverse and depend largely on the location of the tumor and the stage of the disease. The primary treatment for non-metastatic melanoma is surgery to remove the tumor and surrounding tissue. Late-stage melanoma may require treatment, including lymph node dissection, radiation therapy, or chemotherapy. Immune checkpoint blockade strategies target negative immune regulators such as PD-1 / PD-L1 and CTLA4. These include the use of antibodies that target the cytoplasm of the nucleus, which have recently been used to treat various malignancies, including melanoma. (Ribas, A. and Wolchok, J.D., (2018) Science, 359:1350-13 55) The extraordinary value of checkpoint inhibitor therapy and its clinical benefits and the patient's own cancer Well-known association with a patient's adaptive immune response to antigens (especially T cell-based immune responses) This has stimulated the search for effective cancer vaccines, vaccine modalities and cancer vaccine antigens.

[0007] Human endogenous retroviruses (HERVs) are exogenous infectious retroviruses that are transmitted through the ancestral germline. HERVs are relics of integration into the long terminal repeats (LTRs) flanking the viral genome. ) belongs to a group of endogenous retroelements characterized by the presence of Since it also contains apparent mammalian LTR retrotransposons (MaLR), they are collectively referred to as LTR elements. (Hereinafter, all LTR elements are collectively referred to as ERVs.) ERVs comprise a significant proportion (8%) of the mammalian genome and are classified into approximately 100 families based on sequence homology. Many ERV sequences encode defective proviruses, They are prototypic retroviruses consisting of gag, pro, pol, and env genes flanked by long terminal repeats. Some intact ERV ORFs are expressed in exogenous infections such as HIV-1. Retroviral proteins that share characteristics with proteins encoded by virulent retroviruses These proteins act as antigens to induce a strong immune response. (Hurst and Magiorkinis, 2015, J. Gen. Virol 96:1207-1218) It is believed that ERV-encoded polypeptides mediate the selection process of T cell and B cell receptors. These results suggest that ERV-produced viruses can circumvent the immune system and central and peripheral tolerance. Immune reactivity to ERV products can arise spontaneously in infections and cancers, and some ERV products It has been implicated as a cause of several autoimmune diseases (Kassiotis and Stoye, 201 6, Nat. Rev. Immunol. 16:207-219).

[0008] Due to the accumulation of mutations and recombination events during evolution, most ERVs have their genetic loss of some or all functional open reading frames in the offspring, resulting in infectious viruses However, these ERV elements have lost their ability to produce viruses. maintain in the germline DNA as well as in the offspring, and at least some of those genes They have the potential to produce proteins. In fact, the proteins encoded by HERVs are It has been detected in various human cancers. For example, the Re splice variant of the HERV-K env gene c and Np9 are found only in malignant testicular germ cells and not in healthy cells (Ruprecht et al. (2008, Cell Mol Life Sci 65:3366-3382). Elevated levels of HERV transcripts have been observed compared to those in the control group (Wang-Johanning 2003, C Ancer 98:187-197; Andersson et al., 1998, Int. J. Oncol 12:309-313). Overexpression of VE and HERV-H has been shown to be immunosuppressive, and these may also contribute to the progression of cancer. (Mangeney et al., 2001, J. Gen. Virol. 82:2515- 2518). However, the actual mechanisms by which HERVs can contribute to cancer progression or pathogenesis remain unclear. It is still unknown.

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

[0010] A wide range of vaccine modalities are known. One well-described approach aims to enhance immune responses (including B cell and T cell responses) and stimulate immunological memory, Alternatively, the polynucleotide may be delivered to a subject via a so that the immunogenic polypeptide encoded by the polynucleotide is expressed in vivo, It may be administered to a subject by a vector, such as a viral vector, e.g., an adenoviral vector. The use of mAb in both preventive vaccination and therapeutic treatment strategies against cancer has been shown to be It has been extensively explored for antigen delivery (Wold et al., Current Gene Therapy, 2013, " Adenovirus Vectors for Gene Therapy, Vaccination, and Cancer Gene Therapy ctors for Gene Therapy, Vaccination and Cancer Gene Therapy)” 13:421-433). immunity Patient-derived peptides, polypeptides, or polynucleotides encoding them may also be administered. can be used to load native antigen-presenting cells (APCs), which can then be used to administer therapeutic or prophylactic immunizations. The vaccine can be injected into the subject as a vaccine to elicit an immune response. An example of this approach is The vaccine is Provenge, the only anti-cancer vaccine currently approved by the FDA.

[0011] Cancer antigens also offer a valuable tool for developing a variety of non-vaccine therapeutic modalities using them. These therapies include: 1) antigen-binding biologics; 2) adoptive cell transfer; Therapies are divided into two different classes.

[0012] Antigen-binding biologics typically recognize antigen-decorated cancer cells and promote their destruction. These biologics are composed of multivalent polypeptides engineered to bind to the antigen. The components may also consist of TCR-based biologics, which can be delivered by a variety of techniques. TCRs, high affinity TCRs and TCRs (including those based on monoclonal antibody technology) Mimetics of the cytolytic components of these types of multivalent biologics include, but are not limited to: are used to administer cytotoxic chemicals, biological toxins, and targeting agents that facilitate the targeting and activation of immune cells. The agonist and / or the immunostimulatory motif may comprise a targeting motif and / or an immunostimulatory motif, both of which may be present in tumor cells. It promotes therapeutic destruction.

[0013] Adoptive cell therapy may be based on a patient's own T cells, which are extracted and administered as a vaccine. stimulated in vitro with a chromosomal antigen preparation (in the presence of other factors, including cellular and acellular components) (Yossef et al., JCI Insight. 2018, 10(1):111-114, 2018). Mon, 4;3(19) pii:122467. doi:10.1172 / jci.insight.122467). Alternatively, adoptive cell therapy , cells (patients) deliberately engineered to express an antigen-binding polypeptide that recognizes a cancer antigen These antigen-binding polypeptides can also be based on cells derived from the patient or non-patient. Peptides fall into the same classes as those described above for antigen-binding biologics. and genetically engineered (autologous or non-autologous) to express a cancer antigen-binding polypeptide. The lymphocytes may be administered to a patient as adoptive cell therapy to treat the patient's cancer. can be done.

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

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

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

[0017] Humer J et al., 2006, Canc. Res., 66:1658-63, reported that endogenous levels of leukocytes associated with melanoma We have identified a melanoma marker derived from a retrovirus. For use in immunotherapy of cancer, especially melanoma, especially cutaneous melanoma and uveal melanoma There is a need to identify additional HERV-associated antigenic sequences that can be used to treat HERV-associated diseases. Summary of the Invention

[0018] (Summary of the Invention) The present inventors have surprisingly found that the LTR element-containing gene is expressed at high levels in cutaneous melanoma cells. found in the blood, but are undetectable or found at very low levels in normal healthy tissue. We have discovered certain RNA transcripts that can be used to identify specific genes (see Example 1). The products are referred to as cancer-specific LTR-element spanning transcripts (CLTs). The potential polypeptide sequences encoded by these CLTs (i.e., open A subset of open reading frames (ORFs) are translated in cancer cells and are involved in the antigen processing machinery. It is processed by components and then converted to class I human leukocyte antigen (HLA class I) It has been shown that the IL-14-16 agonist binds to a IL-14 molecule and is presented on the surface of cells found in tumor tissue (see Example 2). These findings suggest that these polypeptides (herein referred to as CLT antigens) are in fact Therefore, cancer cell presentation of this CLT antigen indicates that these cells susceptibility to elimination by T cells bearing the cognate T cell receptor (TCR) for the CLT antigen of the It is expected that CLT antigen-based vaccines will expand T cells with these cognate TCRs. The inoculation method / regimen is to inoculate cancer cells (and tumors containing them), particularly melanoma, especially skin melanoma. It is expected that this will induce an immune response against melanoma tumors. These T cells respond to the CLT antigen-derived peptides disclosed herein (see Example 3). The authors demonstrated that T cells specific for CLT antigens induce T cell repression in normal subjects through central tolerance. Finally, qRT-PCR studies confirmed that the nucleotide sequence of ... , CLT in RNA extracted from melanoma tumor tissue compared to non-melanoma control cell lines It was confirmed that the protein is specifically expressed in the IL-11 subunit of the IL-11 subunit (see Example 5).

[0019] The present inventors have also surprisingly found that CLTs encoding certain CLT antigens are expressed in cutaneous melanoma. We found that it is not only overexpressed in uveal melanoma, but also in uveal melanoma. The CLT antigen polypeptide sequences encoded by these CLTs were expressed in uveal melanoma cells. and is expected to elicit an immune response against tumors containing the same.

[0020] The CLT and CLT antigens that are the subject of the present invention are derived from known tumor genomes found in The Cancer Genome Atlas. This CLT is not a canonical sequence that can be easily derived from the ERV-derived transcriptional regulatory sequence. It is a transcript that results from a complex transcription and splicing event driven by These CLTs are expressed at high levels, and the CLT antigen polypeptide sequence is similar to that of normal human proteins. Since the sequence is not that of a protein, it is likely to elicit a strong and specific immune response, making it a promising candidate for cancer immunotherapy. It is expected that these compounds will be suitable for therapeutic use in clinical settings.

[0021] This CLT antigen, found in a highly expressed transcript that characterizes tumor cells, is Before the Meiji Restoration, no known proteins were present in the human body and produced protein products. First, the CLT antigen polypeptide of the present invention can be used in the following forms: Direct delivery to the subject as a vaccine to elicit a therapeutic or prophylactic immune response against tumor cells Second, the nucleic acids of the present invention can enhance the expression of the CLT antigens they encode. Codons can be optimized to achieve therapeutic or prophylactic immunity against tumor cells. for direct administration, or alternatively, for in vivo delivery, as a vaccine to elicit an immune response. It can be inserted into a vector to allow production of the encoded protein product in a subject. Third, the polynucleotides and / or polypeptides of the present invention can be used to treat antigen-presenting cells derived from patients. can be used to load cells (APCs) which can then be used to deliver therapeutic or It can be injected into a subject as a vaccine to induce a preventative immune response. The polynucleotides and / or polypeptides of the present invention can be used to stimulate T cells in vitro in a subject. and stimulated T cell preparations are generated that can be used to treat cancer. Fifth, it recognizes the CLT antigen that forms a complex with the MHC I molecule and targets cancer cells. T cell receptors (TCRs) or TCR mimetics that have been further modified to kill (or promote killing of) Sixth, biological molecules such as MHC cells and complexes may be administered to a subject as a cancer treatment. A chimeric version of a biological molecule that recognizes the CLT antigen forming the complex (autologous, or non-autologous) T cells, and the transduced cells are administered to a subject as a treatment for cancer. These and other applications are described in more detail below.

[0022] Therefore, the present invention particularly provides: (a) any one of SEQ ID NOs: 1 to 8; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); An isolated polypeptide (hereinafter referred to as the "polypeptide of the invention") comprising a sequence selected from (Provide The present invention also relates to nucleic acid molecules encoding the polypeptides of the invention (hereinafter "nucleic acids of the invention"). ").

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

[0024] DESCRIPTION OF THE DRAWINGS For each of Figures 1-14, the top panel shows peptides obtained from patient tumor samples. The extracted MS / MS spectrum of the compound (with assigned fragment ions) is shown in the upper panel, and the lower panel shows the spectrum of the compound. The figure shows a rendering of the linear peptides mapped to fragment ions. The position of the chit sequence is displayed. [Figure 1] Figure 1: Spectrum of peptide SEQ ID NO: 9 obtained from a tumor sample of patient Mel-27. [Figure 2] Figure 2: Spectrum of peptide SEQ ID NO: 10 obtained from a tumor sample of patient Mel-21. [Figure 3] Figure 3: Spectrum of peptide SEQ ID NO: 11 obtained from a tumor sample of patient Mel-41. [Figure 4] Figure 4: Spectrum of peptide SEQ ID NO: 12 obtained from a tumor sample of patient Mel-41. [Figure 5] Figure 5: Spectrum of peptide SEQ ID NO: 13 obtained from a tumor sample of patient Mel-41. [Figure 6] Figure 6: Spectrum of peptide SEQ ID NO: 14 obtained from a tumor sample of patient Mel-41. [Figure 7] Figure 7: Spectrum of peptide SEQ ID NO: 15 obtained from a tumor sample of patient Mel-21. [Figure 8] Figure 8: Spectrum of peptide SEQ ID NO: 16 obtained from a tumor sample of patient Mel-21. [Figure 9] Figure 9: Spectrum of peptide SEQ ID NO: 17 obtained from a tumor sample of patient Mel-21. [Figure 10] Figure 10: Spectrum of peptide SEQ ID NO: 18 obtained from a tumor sample of patient Mel-15. [Figure 11] Figure 11: Spectrum of peptide SEQ ID NO: 19 obtained from a tumor sample of patient Mel-27. [Figure 12] Figure 12: Spectrum of peptide SEQ ID NO: 20 obtained from a tumor sample of patient Mel-27. [Figure 13]Figure 13: Spectrum of peptide SEQ ID NO: 21 obtained from a tumor sample of patient Mel-25. [Figure 14] Figure 14: Spectrum of peptide SEQ ID NO: 22 obtained from a tumor sample of patient Mel-25.

[0025] Each of Figures 15-28 shows native MS / MS of peptides obtained from patient tumor samples. Alignment of the spectrum to the native spectrum of a synthetic peptide corresponding to the same sequence Indicates the event. [Figure 15] Figure 15: Spectrum of the peptide of SEQ ID NO: 9 obtained from a tumor sample of patient Mel-27. [Figure 16] Figure 16: Spectrum of peptide SEQ ID NO: 10 obtained from a tumor sample of patient Mel-20. [Figure 17] Figure 17: Spectrum of peptide SEQ ID NO: 11 obtained from a tumor sample of patient Mel-41. [Figure 18] Figure 18: Spectrum of peptide SEQ ID NO: 12 obtained from a tumor sample of patient Mel-41. [Figure 19] Figure 19: Spectrum of peptide SEQ ID NO: 13 obtained from a tumor sample of patient Mel-41. [Figure 20] Figure 20: Spectrum of peptide SEQ ID NO: 14 obtained from a tumor sample of patient Mel-41. [Figure 21] Figure 21: Spectrum of peptide SEQ ID NO: 15 obtained from a tumor sample of patient Mel-21. [Figure 22] Figure 22: Spectrum of peptide SEQ ID NO: 16 obtained from a tumor sample of patient Mel-21. [Figure 23] Figure 23: Spectrum of peptide SEQ ID NO: 17 obtained from a tumor sample of patient Mel-21. [Figure 24] Figure 24: Spectrum of peptide SEQ ID NO: 18 obtained from a tumor sample of patient Mel-15. [Figure 25]Figure 25: Spectrum of peptide SEQ ID NO: 19 obtained from a tumor sample of patient Mel-27. [Figure 26] Figure 26: Spectrum of peptide SEQ ID NO: 20 obtained from a tumor sample of patient Mel-27. [Figure 27] Figure 27: Spectrum of peptide SEQ ID NO: 21 obtained from a tumor sample of patient Mel-25. [Figure 28] Figure 28: Spectrum of peptide SEQ ID NO: 22 obtained from a tumor sample of patient Mel-25. [Figure 29] Figure 29: Spectrum of peptide SEQ ID NO: 15 obtained from tumor sample of patient 2MT3. [Figure 30] Figure 30: Spectrum of peptide of SEQ ID NO: 20 obtained from tumor sample of patient 2MT4. [Figure 31] Figure 31: Spectrum of peptide of SEQ ID NO: 21 obtained from tumor sample of patient 2MT4.

[0026] [Figure 32] FIG. 32 shows the killing of expanded, pentamer-sorted CD8 T cells against CaSki cells transfected with the open reading frame for CLT antigen 6 (SEQ ID NO: 6). [Figure 33] FIG. 33 shows CD8 T cell responses from normal blood donors to an HLA-A *02:01-restricted peptide from CLT antigen 1 (SEQ ID NO: 9). [Figure 34] FIG. 34 shows the CD8 T cell response from a normal blood donor to an HLA-A *03:01-restricted peptide from CLT antigen 1 (SEQ ID NO: 10). [Figure 35] FIG. 35 shows CD8 T cell responses from normal blood donors to the HLA-B *07:02-restricted peptide from CLT antigen 2 (SEQ ID NO: 13). [Figure 36] FIG. 36 shows CD8 T cell responses from normal blood donors to an HLA-A*03:01-restricted peptide from CLT antigen 3 (SEQ ID NO: 15). [Figure 37]FIG. 37 shows CD8 T cell responses from normal blood donors to an HLA-A*03:01-restricted peptide from CLT antigen 5 (SEQ ID NO: 18). [Figure 38] FIG. 38 shows CD8 T cell responses from normal blood donors to an HLA-A*02:01-restricted peptide from CLT antigen 6 (SEQ ID NO: 39). [Figure 39] Panels A to C of Figure 39 show the results of qRT-PCR assays verifying the transcription of CLT encoding CLT antigen 2 (SEQ ID NO: 24), CLT encoding CLT antigen 3 (SEQ ID NO: 25), and CLT encoding CLT antigen 6 (SEQ ID NO: 28) in melanoma cancer cell lines.

[0027] (Array description) SEQ ID NO: 1 is the polypeptide sequence of CLT antigen 1. SEQ ID NO:2 is the polypeptide sequence of CLT antigen 2. SEQ ID NO:3 is the polypeptide sequence of CLT antigen 3. SEQ ID NO: 4 is the polypeptide sequence of CLT antigen 4. SEQ ID NO: 5 is the polypeptide sequence of CLT antigen 5. SEQ ID NO: 6 is the polypeptide sequence of CLT antigen 6. SEQ ID NO: 7 is the polypeptide sequence of CLT antigen 7. SEQ ID NO: 8 is the polypeptide sequence of CLT antigen 8.

[0028] SEQ ID NOs: 9 and 10 are CLT antigen 1-derived peptide sequences. SEQ ID NOs: 11 to 14 are peptide sequences derived from CLT antigen 2. SEQ ID NO: 15 is a CLT antigen 3-derived peptide sequence. SEQ ID NOs: 16 and 17 are CLT antigen 4-derived peptide sequences. SEQ ID NO: 18 is a CLT antigen 5-derived peptide sequence. SEQ ID NOs: 19 and 20 are CLT antigen 6-derived peptide sequences. SEQ ID NO: 21 is a CLT antigen 7-derived peptide sequence. SEQ ID NO: 22 is a peptide sequence derived from CLT antigen 8.

[0029] SEQ ID NO: 23 is the cDNA sequence of CLT encoding CLT antigen 1. SEQ ID NO: 24 is the cDNA sequence of CLT encoding CLT antigen 2. SEQ ID NO: 25 is the cDNA sequence of CLT encoding CLT antigen 3. SEQ ID NO: 26 is the cDNA sequence of CLT encoding CLT antigen 4. SEQ ID NO: 27 is the cDNA sequence of CLT encoding CLT antigen 5. SEQ ID NO: 28 is the cDNA sequence of CLT encoding CLT antigen 6. SEQ ID NO: 29 is the cDNA sequence of CLT encoding CLT antigen 7. SEQ ID NO: 30 is the cDNA sequence of CLT encoding CLT antigen 8.

[0030] SEQ ID NO: 31 is the cDNA sequence encoding CLT antigen 1. SEQ ID NO: 32 is the cDNA sequence encoding CLT antigen 2. SEQ ID NO: 33 is the cDNA sequence encoding CLT antigen 3. SEQ ID NO: 34 is the cDNA sequence encoding CLT antigen 4. SEQ ID NO: 35 is the cDNA sequence encoding CLT antigen 5. SEQ ID NO: 36 is the cDNA sequence encoding CLT antigen 6. SEQ ID NO: 37 is the cDNA sequence encoding CLT antigen 7. SEQ ID NO: 38 is the cDNA sequence encoding CLT antigen 8. SEQ ID NO: 39 is a CLT antigen 6-derived peptide sequence. DETAILED DESCRIPTION OF THE INVENTION

[0031] (Detailed Description of the Invention) (polypeptide) The terms "protein," "polypeptide," and "peptide" are used interchangeably herein. Any peptide-linked amino acid chain, regardless of length, co-translational or post-translational modifications, is used. It refers to either one.

[0032] The term "amino acid" refers to naturally occurring amino acids as well as amino acids that are similar to naturally occurring amino acids. It refers to any one of amino acid analogs and amino acid mimetics that function according to the formula: The amino acids are the 20 L-amino acids encoded by the genetic code and those that are later modified. amino acids such as hydroxyproline, γ-carboxyglutamic acid and O-phosphoryl The term "amino acid analog" refers to an amino acid that has the same basic chemical structure as a naturally occurring amino acid. That is, a compound having an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. but have a modified R group or a modified peptide backbone compared to natural amino acids. Examples include homoserine, norleucine, methionine sulfoxide, methyl Amino acid mimetics include methylsulfonium and norleucine. They have a different chemical structure than the general amino acids, but are formed in a manner similar to naturally occurring amino acids. Preferably, the amino acid is a naturally occurring amino acid or an amino acid analogs, especially naturally occurring amino acids, and even those encoded by the genetic code. It is one of the 20 L-amino acids.

[0033] Amino acids are referred to herein by either their commonly known three letter symbols or by their one letter symbols. The IUPAC-IUB Biochemical Nomenclature Commission recommends Nucleotides are also sometimes represented by the commonly accepted single-letter codes. It may be represented by a do.

[0034] Thus, the present invention provides: (a) any one of SEQ ID NOs: 1 to 8; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a). .

[0035] The present invention also provides: (a) any one of SEQ ID NOs: 1 to 8 excluding the first methionine residue; and To, (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a). .

[0036] Generally, variants of the polypeptide sequences of the present invention will have a high degree of sequence identity thereto. For example, variants preferably have at least one sequence that is at least 100% identical to the full length of the relevant reference sequence. at least about 80% identity, more preferably at least about 85% identity, and most preferably at least and have about 90% identity (at least about 95%, at least about 98%, or at least about 99%, etc.) .

[0037] Preferably, the variant is an immunogenic variant. A variant is a variant of a reference sequence (i.e., at least 20%, preferably at least 50%, of the activity of the sequence of which the antibody is a variant; and In particular, it is considered to be an immunogenic variant that elicits a response that is at least 75% (such as at least 90%). This response can be observed, for example, by in vivo immunohistochemistry of PBMCs or whole blood using the polypeptide as an antigen. Trans-stimulation assay (e.g., from a few hours up to 1 year, up to 6 months, etc., from 1 day to 1 month, or 1- (restimulation over a 2-week period) to stimulate lymphocyte proliferation (e.g., T cell proliferation) activation of T cells, production of cytokines (e.g., IFN-γ) in the culture supernatant (measured by ELISA, etc.), or Characterization of cellular responses was performed using intracellular and extracellular staining (e.g., CD3, CD4, CD8, IL2, TNF-α, I using antibodies specific for immune markers such as FNg, type 1 IFN, CD40L, and CD69) and subsequent Measured by analysis on a flow cytometer.

[0038] A variant can be, for example, a conservatively modified variant. The modification may be a substitution of an amino acid with a functionally similar amino acid or a modification of the biology of the mutant. These modifications result in substitutions / deletions / additions of residues that do not substantially affect the target protein function. The biological function of such variants is to inhibit the expression of melanoma, e.g., cutaneous melanoma cancer antigens. It will induce an immune response. Conservative substitution tables providing functionally similar amino acids are well known in the art. , may include homologs of polypeptides found in other species.

[0039] Variants of the polypeptides of the invention may contain a number of substitutions, e.g., conservative substitutions, compared to the reference sequence. Substitutions (e.g., 1 to 25, 1 to 10, etc., particularly 1 to 5, and particularly 1 amino acid residue) The number of substitutions, e.g., the number of conservative substitutions, may be expressed as the number of residues in the reference sequence. It may be at most 20%, such as at most 10%, such as at most 5%, such as at most 1%. Conservative substitutions will fall within one of the amino acid classes identified below, but may Other substitutions are also possible as long as they do not substantially affect the immunogenic properties of the antigen. Each group contains amino acids that are generally conservative substitutions for one another.

[0040] 1) Alanine (A), Glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, e.g., Creighton, Proteins 1984).

[0041] Preferably, such substitutions do not alter the immunological structure of the epitopes (e.g., they substitutions do not occur within the epitope region mapped to the primary sequence), therefore, does not significantly affect the immunogenic properties of

[0042] Polypeptide variants also include those in which additional amino acids are inserted compared to a reference sequence. For example, such insertions can be made at positions 1 to 10 (e.g., positions 1 to 5, preferably positions 1 or 2). , particularly at position 1), and the insertions may be, for example, up to 50 (e.g., up to 20) at each position. The amino acid sequence may include the addition of up to 10 amino acids, particularly up to 10 amino acids, and more particularly up to 5 amino acids. Such insertions do not occur in the epitope region and therefore do not significantly affect the immunogenic properties of the antigen. An example of an insert is a Histidine dehydrogenase (HDS) fragment to aid in the expression and / or purification of an antigen of interest. It contains a short stretch (e.g., 2 to 6 residues) of cysteine ​​residues.

[0043] Polypeptide variants include those in which amino acids have been deleted compared to a reference sequence, e.g. Such deletions may occur at positions 1 to 10 (e.g., positions 1 to 5, preferably positions 1 or 2, especially positions 1). positions), and the deletions may occur, for example, at 50 or fewer (e.g., 20 or fewer, particularly 10 or fewer) positions. The amino acid deletion may include a deletion of at least 1 amino acid, or even at most 5 amino acids. Preferably, such deletions does not occur in the epitope region and therefore does not significantly affect the immunogenic properties of the antigen. stomach.

[0044] Those skilled in the art will appreciate that particular protein variants may include substitutions, deletions, and additions (or any combination thereof). For example, substitutions / deletions / additions may be made to the desired patient's H Enhances binding to LA molecules (or has a neutral effect) and enhances immunogenicity (or inhibits immunogenicity) (and keep the gender unchanged).

[0045] Depending on the length of the CLT antigen, the immunogenic fragments of the present invention may be fragments of the full-length polypeptide sequence. Usually at least 9 (e.g., at least 9 or 10) consecutive amino acids, at least 12 consecutive amino acids (e.g., at least 15 or at least 20 consecutive amino acids), particularly at least At least 50 contiguous amino acids, at least 100 contiguous amino acids (e.g., at least 200 contiguous amino acids), Preferably, the immunogenic fragment will contain the full-length polypeptide sequence. At least 10% of the length of the column, e.g. at least 20%, e.g. at least 50%, e.g. at least The probability of a variance is likely to be 70% or at least 80%.

[0046] Immunogenic fragments typically contain at least one epitope. Epitopes are those that stimulate B-cell and Preferably, the immunogenic fragment comprises a T cell epitope, such as a CD4+ or CD8+ T cell epitope. and at least one T cell epitope of

[0047] T cell epitopes are molecules that bind to T cells (e.g., CD4+ or CD8+ T cells) when bound to an HLA molecule. T cell epitopes are short, consecutive stretches of amino acids that are recognized by This may be achieved by epitope mapping experiments well known to those skilled in the art (see, e.g., Paul, , Fundamental Immunology, 3rd ed., pp. 243-247 (1993); Beiβbarth et al., 2005, Bioinfo rmatics, 21(Suppl 1):i29-i37).

[0048] As a result of the crucial involvement of T cell responses in cancer, at least one T cell epitope Fragments of the full-length polypeptides of SEQ ID NOs: 1-8, including the fragments, may be immunogenic and may be useful in immunoprotection. The potential for contribution to the control of

[0049] In diverse outbred populations, such as humans, different HLA types represent populations where a particular epitope is present. It will be understood that this means that the As a result, to maximize the level of recognition and magnitude of the immune response to a given polypeptide, Therefore, immunogenic fragments generally contain multiple epitopes from the full-length sequence (preferably CLT antigens). It is desirable to include all epitopes within the

[0050] Particular fragments of the polypeptides of SEQ ID NOs: 1-8 that may be useful include fragments of at least one CD8+ T cell epitopes, preferably at least two CD8+ T cell epitopes, and even all CD8+ T those containing cellular epitopes (especially those associated with multiple HLA class I alleles, e.g., 2 , 3, 4, 5 or more alleles). Particular fragments of the polypeptides 1 to 8 contain at least one CD4+ T cell epitope, preferably Those containing at least two CD4+ T cell epitopes, and even all CD4+ T cell epitopes ( Particularly those associated with multiple HLA class II alleles, e.g., 2, 3, 4, 5 or more However, those skilled in the art of vaccine design will appreciate that exogenous C The CD4+ T cell epitopes of the present invention can be combined with the CD8+ T cell epitopes of the present invention to form the CD8+ T The desired response to the cellular epitope could be achieved.

[0051] When individual fragments of the full-length polypeptide are used, such individual fragments may be That is, at least 20%, preferably at least A substance is immunogenic if it elicits a response that is at least 50%, particularly at least 75% (such as at least 90%). This response is thought to be due to, for example, the activation of PBMC or whole-cell lymphoma cells using the polypeptide as an antigen. In vitro restimulation assays of blood (e.g., from a few hours up to 1 year, up to 6 months, etc.) activity in lymphocyte proliferation (e.g., T cell proliferation) Activation of cells through proliferation, production of cytokines (e.g., IFN-γ) in the culture supernatant (ELISA) or characterization of T cell responses by intracellular and extracellular staining (e.g., CD3, CD4, C Uses antibodies specific to immune markers such as D8, IL2, TNF-α, IFN-γ, type 1 IFN, CD40L, and CD69 ) and subsequent analysis on a flow cytometer.

[0052] In some cases, multiple fragments of the full-length polypeptide (which may or may not overlap, (which may or may not span the entire full-length sequence) to the full-length sequence itself For example, a combination of at least two (3, 4, or Immunogenic fragments of the antibodies (e.g., 5) are combined and tested in an in vitro restimulation assay (e.g., PBMC or whole blood). Preferably, the sequence matches at least 50% of the reference sequence in a T cell proliferation and / or IFN-γ production assay. Preferably, it provides at least 75% and even at least 90% activity.

[0053] Examples of immunogenic fragments of the polypeptides of SEQ ID NOs: 1 to 8, and therefore examples of peptides of the invention, are The present invention includes polypeptides comprising or consisting of the sequences of SEQ ID NOs: 9 to 22 and 39. The sequences of Nos. 9 to 22 were confirmed to bind to HLA class I molecules by immunopeptidome analysis. The sequence of SEQ ID NO: 39 was identified as an HLA class I molecule by the NetMHC software (see Example 2). and was used in an immunological validation assay (see Example 4).

[0054] (nucleic acid) The present invention provides isolated nucleic acids encoding the polypeptides of the invention (referred to as nucleic acids of the invention). For example, the nucleic acid of the present invention is a nucleic acid having a sequence selected from SEQ ID NOs: 23 to 30 or 31 to 38. or consisting of the sequence:

[0055] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to a nucleic acid. Nucleotide monomers, especially deoxyribonucleotide monomers or ribonucleotide monomers The term refers to a polymeric macromolecule made from known nucleotide analogs or includes nucleic acids containing modified backbone residues or linkages, which are naturally occurring and non-naturally occurring, which have similar properties to the reference nucleic acid and which have similar properties to the reference nucleotides. intended to be metabolized in a similar manner or to have a prolonged half-life in the system Examples of such analogs include, but are not limited to, However, phosphorothioates, phosphoramidates, methylphosphonates, chiral methyl These include phosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). The term "nucleic acid" refers to a nucleic acid consisting of deoxyribonucleotide or ribonucleotide monomers. It refers to a polymer of natural origin. Preferably, the nucleic acid molecules of the present invention are recombinant. is a sequence in which a nucleic acid molecule is isolated by a cloning, restriction or ligation step, or by natural other procedures that result in nucleic acid molecules that are distinct from those found in In one embodiment, the nucleic acid of the invention is an artificial A nucleic acid sequence (e.g., a cDNA sequence or a nucleic acid sequence containing unnatural codon usage). In some cases, the nucleic acid of the present invention is DNA. Alternatively, the nucleic acid of the present invention is RNA.

[0056] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are each composed of deoxyribosyl components. and ribosyl moieties. This sugar moiety is one of the four naturally occurring Bases (adenine (A), guanine (G), cytosine (C), thymine (T) in DNA and adenine (A) in RNA may be attached to the bases a) adenine (A), guanine (G), cytosine (C), and uracil (U). As used herein, a "corresponding RNA" is an RNA having the same sequence as the reference DNA, but differing from the DNA. It is an RNA with a sequence in which thymine (T) is replaced by uracil (U) in the RNA. The sugar moiety can also be Inosine, xanthosine, 7-methylguanosine, dihydrouridine and 5-methylcytidine The natural bases between the sugar (deoxyribosyl / ribosyl) moieties may be linked to unnatural bases such as The phosphodiester linkages may optionally be replaced by phosphorothioate linkages. In particular, the nucleic acids of the present invention may be deoxyribosyl or phosphodiester-containing nucleic acids having phosphodiester bonds between the sugar moieties. It consists of a natural base attached to a ribosyl sugar backbone.

[0057] In one embodiment, the nucleic acid of the present invention is DNA. For example, the nucleic acid may be any of SEQ ID NOs: 23 to 30 or or comprising or consisting of a sequence selected from 31 to 38. Also provided are , comprising or consisting of a variant of a sequence selected from SEQ ID NOs: 23 to 30 or 31 to 38 Nucleic acids, variants of which encode the same amino acid sequence but differ in their sequence due to the degeneracy of the genetic code. The nucleic acids have different structures.

[0058] Thus, because of the degeneracy of the genetic code, a large number of different but functionally identical nucleic acids are possible. For example, the codons GCA, GCC, GCG, and GCU can all be used to encode any given polypeptide. The codon 1 encodes the amino acid alanine. At every position designated, the codon modifies the encoded polypeptide. Such nucleic acid mutations can be modified to any of the above codons without altering the corresponding codon. , resulting in "silent" (sometimes called "degenerate" or "synonymous") variants, It is one type of conservatively modified variant. Every nucleic acid sequence disclosed herein also encompasses every possible silent variation within that nucleic acid. The skilled artisan will be able to identify each codon in a nucleic acid (usually the only codon for methionine). and UGG, which is normally the only codon for tryptophan) It will be recognized that functionally identical molecules can be produced by combining the polypeptides of 1 and 2. Each silent variation of a nucleic acid encoding a and is provided as one aspect of the present invention.

[0059] Degenerate codon substitution also refers to the substitution of one or more (or all) of the selected codons at the third position with a mixed base. This can also be achieved by generating sequences substituted with methylamino and / or deoxyinosine residues. (Batzer et al., 1991, Nucleic Acids Res. 19:5081; Ohtsuka et al., 1985 , J. Biol. Chem. 260:2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8:91- 98).

[0060] The present invention includes or consists of a sequence selected from SEQ ID NOs: 23 to 30 or 31 to 38. The nucleic acid of interest may contain many silent variants (e.g., 1-50, 1-25, etc., particularly 1- The codons may be modified in any order (up to 5, and even one codon may be modified).

[0061] As described above, the nucleic acids of the present invention contain the first codon for methionine (i.e., ATG or AUG). or consisting of a sequence selected from SEQ ID NOs: 31 to 38 or variants thereof, excluding This sometimes happens.

[0062] In one embodiment, the nucleic acids of the invention are RNA. The RNA sequences provided are those provided herein. corresponding to the DNA sequence, and It has a base sequence of uracil (U) instead of thymine (T).

[0063] Therefore, the nucleic acid of the present invention is an RNA having a cDNA sequence selected from SEQ ID NOs: 23 to 30 or 31 to 38. Contains or consists of equivalents and many silent mutations compared to the reference sequence. (for example, 1 to 50, 1 to 25, etc., particularly 1 to 5, and even 1 codon may be modified) An "RNA equivalent" is one that contains the same genetic information as a reference cDNA sequence (i.e., deoxyribonucleotides). It has a ribonucleotide backbone instead of a silibonucleotide backbone, and the side chain base is thymine. (T) refers to an RNA sequence containing the same codon but with uracil (U) instead of T.

[0064] The present invention also includes sequences complementary to the above cDNA and RNA sequences. In one embodiment, the nucleic acids of the invention are cloned into vectors optimized for expression in human host cells. It's Don. The nucleic acids of the present invention, in the case of DNA nucleic acids, can be transcribed and translated into the polypeptides of the present invention. and, in the case of an RNA nucleic acid, can be translated into a polypeptide of the present invention.

[0065] (Polypeptides and Nucleic Acids) Preferably, the polypeptides and nucleic acids used in the present invention are isolated. A "naturally occurring" polypeptide or nucleic acid is one that has been removed from its original environment. The original polypeptide or nucleic acid is separated from some or all of the materials with which it coexists in the natural system. Nucleic acid is isolated when it is isolated from a vector that is not part of its natural environment. A gene is considered isolated when it is cloned into a target.

[0066] "Naturally occurring," when used in reference to a polypeptide or nucleic acid sequence, means a polypeptide that is not found in nature. By "synthetic" is meant a sequence that has not been synthetically modified. "Artificial" when used in reference to a polypeptide or nucleic acid sequence refers to, for example, a naturally occurring refers to a sequence not found in nature, including synthetic modifications of a sequence or non-naturally occurring sequences .

[0067] The term "heterologous" refers to the relationship between one nucleic acid or polypeptide and another nucleic acid or polypeptide. As used in the context of, two or more sequences are not found in the same relationship to each other in nature. "Heterologous" sequence also refers to a nucleic acid or sequences naturally occurring in a host organism. is not isolated from, derived from, or based on a polypeptide sequence It can also mean:

[0068] As mentioned above, polypeptide variants preferably have at least one amino acid sequence that is at least 100% identical to the full length of the relevant reference sequence. At least about 80% identity, more preferably at least about 85% identity, and most preferably at least at least about 90% identical (e.g., at least about 95%, at least about 98%, or at least about 99%) It has.

[0069] For purposes of comparing two closely related polypeptide or polynucleotide sequences, first The "% sequence identity" between a sequence and a second sequence may be calculated. % sequence identity, the polypeptide sequence is identical to the other polypeptide sequence. The residues in the sequence are ordered from left to right, i.e., from the N-terminus to the C-terminus of the polypeptide. The term "identical" or percent "identity" refers to the degree to which two or more polypeptides are identical. In the context of sequences, they are compared and aligned for maximum correspondence over the comparison window. When the amino acid sequence is expressed as a sequence of 2 or more amino acids, the sequence is identical or has a specified percentage of identical amino acid residues. (i.e., 70% identity within the specified region, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% The comparison refers to two or more sequences or subsequences (of identity). Preferably, the comparison covers the entire length of the reference sequence. It runs on the window.

[0070] For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a comparison algorithm, the test and reference sequences are input into a computer and, if necessary, Specify the sub-array coordinates according to the Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then performs a comparison of the sequence of interest with respect to the reference sequence, based on the program parameters. The percentage sequence identity of the test sequences is calculated.

[0071] As used herein, a "comparison window" is a window within which one sequence and the same number of consecutive A comparison of a segment of a reference sequence at a position between the two sequences is called a comparison. This is done after the sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal sequence alignment for comparison is well known, for example, by Smith and Waterman "Local Homology Algorithm," 1981, Adv. Appl. Math. 2:482, by Needleman and W. by the search for similarity algorithm of Pears, J. Mol. Biol. 1970, 48:443 The similarity search method of Limton and Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444, These include GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Algorithm Computing at the Netics Computer Group, 575 Science Dr., Madison, WI by a gridded implementation or by manual alignment and visual inspection (e.g., "State of the Art in Molecular Biology"). "Current Protocols in Molecular Biology," edited by Ausubel et al., 1995, supplemented; This can be done by

[0072] One example of a useful algorithm is PILEUP, which is a progressive pairwise Use alignment to create multiple sequence alignments from groups of related sequences and compare them. The relationship and percent sequence identity are shown. This is also used to create the alignment. Plots a tree or dendrogram showing the clustering relationships observed. A simple version of Doolittle's progressive alignment method (1987, J. Mol. Evol. 35:351-360) The method used is that described in Higgins and Sharp, 1989, CABIOS 5:151-153. This program is similar to the method described in

[10] . A maximum of 300 sequences can be aligned by amino acid sequence. The alignment procedure begins with a pairwise alignment of the two most similar sequences and then This cluster is then sorted into the most related sequences. Align two clusters of sequences to two clusters of aligned sequences. The alignment is made by a simple extension of the pairwise alignment of the individual sequences. The alignment is achieved by a series of progressive, pairwise alignments. The program allows you to specify specific sequences and their amino acid coordinates for a region of sequence comparison. and by specifying the program parameters. UP was used to compare the reference sequence with the other test sequence using the following parameters: default GATE Gap weight (3.00), default gap length weight (0.10), and weighted end gap PILEUP is a GCG sequence analysis software that determines the percent sequence identity relationship. Software package, e.g., version 7.0 (Devereaux et al., 1984, Nuc. Acids Res. 12:387-395).

[0073] Other algorithms suitable for determining percent sequence identity and sequence similarity include: Examples of such algorithms are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., 1977 and 1980, respectively. Nuc. Acids Res. 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol. 215:403- 410. Software for performing BLAST analyses is available from the National Center for Publicly available via the Biotechnology Information website at www.ncbi.nlm.nih.gov / This algorithm aligns words of the same length in the database sequence. If the query is High-scoring sequence pairs (HSPs) are first identified by identifying short word lengths W in the sequences. T refers to the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits initiat e searches to find longer HSPs containing them. The word hits act as seeds for as long as the cumulative alignment score can be increased. The cumulative score is calculated based on the nucleotide sequence. The parameters M (reward score for a matching residue pair; always > 0) and N (reward score for mismatching residues) are For amino acid sequences, the score is calculated using a penalty score (always <0). A cumulative score is calculated using a matching matrix. Extension stops when: the cumulative alignment score drops by X amount from its maximum achieved value; The product score became 0 or less due to the accumulation of 1 or more negative scoring residue alignments; Or, if the end of either sequence is reached. For amino acid sequences, use the BLASTP program. The program was set to the defaults: word length 3, expectation (E) 10, and BLOSUM62 score. The integrating matrix (Henikoff and Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89 :10915) using an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and both strands Comparison of the two is used.

[0074] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and A (See Iltschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by algorithms is the minimum sum probability (P(N)), which is the sum of two provides an indication of the probability that a match between two nucleotide sequences or two amino acid sequences would occur by chance do.

[0075] A "difference" between sequences is a difference of one residue at one position in the second sequence compared to the first sequence. This refers to an insertion, deletion, or substitution. Two sequences may have one, two, or more such differences. An insertion in a second sequence that is otherwise identical to the first sequence (100% sequence identity). Deletions or substitutions result in a decrease in the percent sequence identity. For example, if the identical sequences are 9 residues long, , substitution of 1 in the second sequence results in 88.9% sequence identity. In the case of , two substitutions in the second sequence result in a sequence identity of 88.2%.

[0076] Alternatively, a first reference sequence may be generated to generate a second sequence for the purpose of comparing the first reference sequence to a second comparison sequence. The number of additions, substitutions and / or deletions made to the first sequence may be ascertained. An addition is the addition of one residue to the first sequence (at either end of the first sequence). A substitution is the replacement of one residue in a first sequence with a different residue. A deletion of 1 is a deletion of one residue from the first sequence (the first sequence (including deletions at either end of the

[0077] (Production of Polypeptides of the Invention) The polypeptides of the present invention can be prepared, for example, as described in Green and Sambrook, 2012, "Molecular Cloning." "Molecular Cloning: A Laboratory Manual," 4th ed., Cold Spring It can be obtained and manipulated using techniques disclosed in Harbour Laboratory Press. In particular, polynucleotides can be generated using artificial gene synthesis techniques. (Nambiar et al., 1984, Science, 223:1299-1301; Sakamar and Khorana, 1988, Nucl. Acids Res., 14:6361-6372; Wells et al., 1985, Gene, 34:315-323; and Grund Strom et al., 1985, Nucl. Acids Res., 13:3305-3316), followed by expression in a suitable organism. The gene encoding the polypeptide of the present invention may be, for example, For example, the entire gene can be synthetically produced by solid-phase DNA synthesis. To obtain the desired oligonucleotide, it may be synthesized de novo without the need for a DNA. , the building blocks are assembled into the growing oligonucleotide in the order required by the product sequence. Once chain assembly is complete, the product is released from the solid phase into solution. The product is isolated by high performance liquid chromatography (HPLC) to give the desired product. oligonucleotides can be obtained with high purity (Verma and Eckstein, 1998, An nu. Rev. Biochem. 67:99-134). These relatively short segments can be used in various gene amplification methods. (Methods Mol Biol., 2012;834:93-109) can be easily assembled into longer DNA molecules. These are suitable for use in a myriad of recombinant DNA-based expression systems. In the context of the disclosure, the skilled artisan will be able to identify polynucleotides encoding the polypeptide antigens according to the present invention. The nucleotide sequence is suitable for use in various vaccine production systems, including, for example, viral vectors. You will find it easy to use.

[0078] For purposes of producing the polypeptides of the invention in microbial hosts (e.g., bacterial or fungal), For this purpose, the nucleic acid of the present invention may be provided with suitable regulatory and control sequences (promoters, termination signals, etc.). ), as well as a polypeptide suitable for protein production in a host. Similarly, the polypeptides of the present invention may be expressed in eukaryotic cells (e.g., Chinese Cultures of Drosophila hamster ovary cells or Drosophila S2 cells were transfected with the nucleic acid of the present invention. Therefore, suitable regulatory and control sequences (including promoters, termination signals, etc.) and their In addition, the polypeptide may be secreted in a manner suitable for intracellular protein production. Therefore, it could be generated by transduction.

[0079] Improved isolation of the polypeptides of the invention produced by recombinant techniques can optionally be achieved by isolating the polypeptides. a stretch of histidine residues towards one end of the peptide (commonly known as a His tag) This would be facilitated by adding Polypeptides may also be prepared synthetically.

[0080] (vector) In additional embodiments, genetic constructs comprising one or more of the nucleic acids of the invention are When introduced into cells, the polypeptide of the present invention is produced in vivo and elicits an immune response. Nucleic acids (e.g., DNA) can be expressed in nucleic acid expression systems, including bacterial and some viral expression systems, as known to those skilled in the art. The gene delivery system may be present in any of a variety of delivery systems known to those skilled in the art. See, for example, Rolland, 1998, Crit. Rev. Therap. "Drug Carrier Systems" Airliner Systems 15:143-198, and those described in the references cited therein. Some of these approaches are outlined below for illustrative purposes.

[0081] From the above, vectors (also referred to herein as "DNA expression constructs" or "DNA expression constructs") containing the nucleic acid molecules of the present invention are A "construct" (also referred to as a "construct") is provided. Preferably, the vector is capable of transcribing an RNA molecule that is translationally active in a human host cell. The gene encoding the appropriate regulatory elements (such as a suitable promoter and termination signal) to enable the expression of the gene. "Translationally active RNA molecules" include nucleic acids that can be converted into proteins by the translational machinery of human cells. It is an RNA molecule that can be translated into protein. From the above, a vector containing the nucleic acid of the present invention (hereinafter referred to as the "vector of the present invention") ") is provided.

[0082] In particular, the vector may be a viral vector. adeno-associated viruses (AAV) (e.g., AAV types 5 and 2), alphaviruses (e.g., vector viruses), Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SIN), Semliki Forest virus (SF) V), herpesviruses, arenaviruses (e.g., lymphocytic choriomeningitis virus (LCMV) ), measles virus, poxvirus (modified vaccinia virus (MVA), etc.), paramyxovirus vectors, such as vesicular stomatitis virus (VSV), lentivirus, or rhabdovirus vectors. Well, the vector can be derived from any of the above viruses. The virus is characterized by its medium genome size, ease of manipulation, high titer, wide target cell range, and high Due to its low infectivity, this virus is particularly suitable for use as a gene transfer vector. Both ends of the genome contain inverted repeats (ITRs) of 100–200 base pairs, which form the duplex of the viral DNA. The early (E) and later (L) regions of this genome are cis-elements necessary for the synthesis and packaging of ribosomal RNA. The late (L) region contains different transcription units, which are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) regulates the transcription of the viral genome and some cellular genes. The expression of the E2 region (E2A and E2B) is required for viral DNA replication. These proteins are involved in DNA replication, expression of late genes, and and host cell shutoff (Renan, 1990). The products of most late genes are driven by a single major late promoter (MLP). It is expressed only after significant processing of the primary transcript. MLP is expressed especially efficiently late in infection. Well, all mRNAs transcribed from this promoter are 5'-tripartite. It has a leader (TPL) sequence, making it a preferred mRNA for translation. Replication-deficient adenoviruses, which are made from viral genomes with deletions in the genome, are particularly useful. , because they have limited replication in vaccinated hosts and are unable to spread pathogenically. This is because the likelihood of infection is low and the likelihood of pathogenic contact in vaccinated hosts is also low. do.

[0083] (Other Polynucleotide Delivery) In certain embodiments of the invention, an expression construct comprising one or more polynucleotide sequences is or simply consist of a naked recombinant DNA plasmid. Ulmer et al., 1999 93, Science 259:1745-1749 and its review by Cohen, 1993, Science 25 9:1691-1692. Introduction of the construct can be achieved, for example, by physically or chemically modifying the cell membrane. This can be done by any method that makes the cells permeable to the Although the gene of interest is applicable to in vivo introduction, it is equally applicable to in vivo use. Encoding DNA can also be introduced in vivo in a similar manner to express the gene product. A number of delivery systems are used to deliver DNA molecules to animal models and humans. Several products based on this technology have been approved for use in animals and are being tested in humans. There are others in Phase II and III clinical trials.

[0084] (RNA delivery) In another embodiment of the invention, an expression construct comprising one or more polynucleotide sequences is It may also consist of an integrative recombinant DNA-derived RNA molecule (Ulmer et al., 2012, Vaccine 30:4414-4418). For DNA-based expression constructs, various methods are available for in vitro expression of RNA molecules. RNA-based constructs can be used for introduction into cells either in vivo or in vivo. Mimicking messenger RNA (mRNA) molecules, the introduced biological molecule is transduced into the host cell's translational machinery. and produces the polypeptide it encodes in the cell into which it is introduced. Alternatively, the RNA molecule can be designed to act as a vector for viral RNA-dependent RNA polymerase. By incorporating them into the structural genes, they can be self-expressed in the cells into which they are introduced. It may be designed in a manner that allows it to be amplified. Thus, self-amplifying mRNA (SAM™) This type of RNA molecule, known as a ribosomal RNA molecule (Geall et al., 2012, PNAS, 109:14604-14609), It shares characteristics with some RNA-based viral vectors: mRNA-based RNA or SAM® Any of the nucleotides can be modified (e.g., by altering their sequence or by using modified nucleotides). Therefore, further modifications can be made to enhance stability and translation (Schlake et al., RNA Biol. logy, 9:1319-1330), and both types of RNA can be formulated (e.g., in emulsions (Brito Molecular Therapy 2014 22:2118-2129) or lipid nanoparticles (Kranz et al. 2006, Nature, 534:396-401) and in vitro or in vivo stability and / or Myriad formulations of modified (and unmodified) RNA may facilitate cell entry. A number of RNA-based vaccines have been tested as vaccines in models and humans. is being used in ongoing clinical trials.

[0085] (Pharmaceutical composition) The polypeptides, nucleic acids and vectors of the present invention can be used in immunogenic and vaccine compositions, etc. It may be formulated for delivery in a pharmaceutical composition (hereinafter all referred to as the "composition of the invention"). The composition of the present invention preferably contains the polypeptide, nucleic acid or vector of the present invention as a pharmaceutical. together with an acceptable carrier. Thus, in one embodiment, the polypeptide, nucleic acid or vector of the invention can be used as a medicament. Immunogenic pharmaceutical compositions comprising the compound together with an acceptable carrier are provided.

[0086] In another embodiment, the polypeptide, nucleic acid or vector of the invention is prepared in a pharmaceutically acceptable form. The preparation of pharmaceutical compositions generally comprises the steps of: See, e.g., Powell and Newman (eds.), Vaccine Design (Subunit and Adjuvant Approaches) " (Vaccine Design (the subunit and adjuvant approach)), 1995. The compositions of the invention may also contain other compounds, which may be biologically active or inactive. Preferably, the compositions of the present invention are sterile compositions suitable for parenteral administration.

[0087] In certain preferred embodiments of the present invention, the pharmaceutical compositions of the present invention are and / or a carrier, such as a carrier containing one or more (e.g., one) polypeptides of the present invention. Provided. In certain preferred embodiments of the present invention, the compositions of the present invention comprise pharmaceutically acceptable carriers. One or more (e.g., one) nucleic acids of the invention in combination with a carrier or one or more (e.g., one) nucleic acids of the invention The vectors of the invention are provided as containing vectors.

[0088] In one embodiment, the compositions of the invention comprise one or more (e.g., one) polynucleotides and one or more Alternatively, the composition may comprise one or more of the above polypeptide components. The product may comprise one or more (e.g., one) vectors and one or more (e.g., one) polypeptide components. Alternatively, the composition may comprise one or more (e.g., one) vector and one It may contain more than one (e.g., one) polynucleotide component. The composition may be provided to enhance an immune response.

[0089] (Pharmaceutically acceptable salts) The compositions of the invention may be prepared by administering to a subject a pharmaceutical acceptable carrier or a recipient thereof a nucleic acid or polypeptide as provided herein. It will be apparent that the compounds may include salts thereof which are pharmaceutically acceptable. The base can be prepared from any non-toxic base, which may be an organic base (e.g., primary, secondary and tertiary amines, and salts of basic amino acids) and inorganic bases (e.g., sodium, potassium (salts of calcium, lithium, ammonium, calcium and magnesium).

[0090] Pharmaceutically acceptable carriers Many pharmaceutically acceptable carriers known to those skilled in the art may be used in the compositions of the present invention. Although it is possible to use a carrier in a pharmaceutical composition, the most suitable type of carrier used will vary depending on the mode of administration. The compositions of the present invention may be formulated for any suitable mode of administration, including, for example: administration via parenteral, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular routes; Preferably, parenteral administration includes, for example, intramuscular, subcutaneous or intravenous administration. The carrier preferably comprises water, and may contain buffers for adjusting pH, stabilizers (e.g., surfactants), etc. and amino acids), and tonicity adjusting agents (e.g., salts and sugars). If the composition is to be provided in lyophilized form for dilution at the time of use, the formulation may contain a lyoprotectant. For oral administration, the carriers may include sugars such as trehalose. or any solid carrier, mannitol, lactose, starch, magnesium stearate sodium saccharin, talcum, cellulose, glucose, sucrose, and and magnesium carbonate, etc. can be used.

[0091] Thus, the compositions of the present invention may be prepared in a buffer (e.g., neutral buffered saline or phosphate buffered saline). buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextrose), strane), mannitol, proteins, polypeptides, or amino acids such as glycine , antioxidants, bacteriostatic agents, chelating agents such as EDTA or glutathione, and preparations Contains solutes, suspending agents, thickeners, and / or preservatives that make the solution isotonic, hypotonic, or slightly hypertonic with blood Alternatively, the compositions of the present invention may be formulated as a lyophilizate.

[0092] (immunostimulant) The compositions of the present invention may also include one or more immunostimulants. Any substance that enhances or strengthens the immune response (antibody-mediated and / or cell-mediated) to an antigen. Immunostimulants are often referred to as adjuvants in the context of vaccine formulations, Examples include aluminum hydroxide gel (alum) or aluminum phosphate. ammonium salts, saponins such as QS21, immunostimulatory oligonucleotides such as CPG, oil-in-water emulsions (e.g., when the oil is squalene), aminoalkyl glucosaminide 4-phosphate, lipopolysaccharide Sugars or their derivatives (e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPL (registered trademark)) and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and In view of the above, the one or more immunostimulants of the composition of the present invention are preferably Suitable examples include aluminum salts, saponins, immunostimulatory oligonucleotides, oil-in-water emulsions, and the like. aminoalkyl glucosaminide 4-phosphate, lipopolysaccharides and their derivatives, and The immunostimulatory agent is selected from other TLR4 ligands, TLR7 ligands, TLR8 ligands, and TLR9 ligands. Active agents also include monoclonal antibodies that interact specifically with other immune components, e.g. A monoclonal antibody that blocks the interaction of immune checkpoint receptors, including PD-1 and CTLA4 It may also include antibodies.

[0093] For recombinant nucleic acid delivery methods (e.g., DNA, RNA, viral vectors), protein-based The gene encoding the immunostimulant of the present invention is combined with the gene encoding the polypeptide of the present invention. It may be conveniently delivered to

[0094] (sustained release) The compositions described herein are sustained release formulations that result in sustained / sustained release of the compound after administration. a part of a preparation (e.g., a capsule, sponge, patch, or gel (e.g., made of a polysaccharide)) It can be administered as

[0095] (Storage and packaging) The compositions of the present invention may be packaged in unit-dose or multi-dose containers such as sealed ampoules or vials. Such containers may be hermetically sealed to ensure sterility of the formulation until use. Formulations are generally prepared as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, the compositions of the present invention may be stored in a lyophilized state. and only add a sterile liquid carrier (such as water for injection or saline) immediately before use. It may be whatever you need.

[0096] (Dosage) The amount of nucleic acid, polypeptide or vector in each composition of the invention may vary depending on the therapeutic or prophylactic use. The dosage may be formulated in such a way that a suitable dosage for the patient is obtained. factors such as lability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations. Considerations will be taken into account by those skilled in the art in preparing such compositions, and therefore various dosages may be used. Posinosity and treatment regimen will be as desired.

[0097] Typically, a composition containing a therapeutically or prophylactically effective amount will contain about 0.1 μg to about 1000 μg of the compound per administration. The polypeptides of the invention, more typically about 2.5 μg to about 100 μg of polypeptide per dose, are delivered. When delivered in the form of short synthetic long-chain peptides, the dosage is 1-200ug / peptide / dose. For polynucleotide compositions, these typically range from about 10 μg to about 10 μg per dose. Approximately 20 mg of the nucleic acid of the invention is delivered, more typically from about 0.1 mg to about 10 mg of the nucleic acid of the invention per administration. Reach.

[0098] (Diseases to be treated or prevented) As described elsewhere in the specification, SEQ ID NOS: 1-8 are identified as antibodies overexpressed in cutaneous melanoma. It is a polypeptide sequence corresponding to the CLT antigen expressed. In one embodiment, the present invention provides a polypeptide, nucleic acid, or a polypeptide of the present invention for use in medicine. A vector or composition is provided.

[0099] A further aspect of the invention is a method of increasing an immune response in a human, comprising administering to the human the method comprising administering a polypeptide, nucleic acid, vector or composition of the invention. do. The present invention also provides a method for the production of a polypeptide of the invention, a nucleic acid sequence, or a polypeptide of the invention for use in raising an immune response in humans. The present invention provides an acid, vector or composition. Polypeptides of the invention for the manufacture of a medicament for use in raising an immune response in humans Uses of the nucleic acids, vectors or compositions are also provided.

[0100] Preferably, the immune response is mediated by the immunogen SEQ ID NOs: 1 to 8 and variants of any one thereof. In this context, the expression of a gene fragment is increased in response to a cancerous tumor expressing a corresponding sequence selected from the gene fragment. "Corresponding" in this context means that the tumor corresponds to, for example, SEQ ID NO: A (where A is one of SEQ ID NOs: 1-8) or When expressing a polypeptide, nucleic acid, vector, or variant thereof, or an immunogenic fragment thereof, The vector or composition and the pharmaceutical containing them are not intended to be used in combination with SEQ ID NO: A or a variant thereof or an immunosuppressant thereof. This means that it may be based on an epidemiological fragment.

[0101] Preferably, the immune response is a CD8+ T cell response, a CD4+ T cell response and / or an antibody response, particularly a CD8 + cytolytic T cell responses and CD4+ helper T cell responses. Preferably, the immune response is directed against tumors, particularly SEQ ID NOS: 1 to 8 and variants thereof and immunogenic fragments thereof. The vector is induced to express a sequence selected from the fragment.

[0102] In a preferred embodiment, the tumor is a melanoma tumor, such as a cutaneous melanoma tumor. The tumor may be a primary tumor or a metastatic tumor.

[0103] A further aspect of the invention is a method of treating a human patient suffering from cancer, comprising administering to said patient a therapeutically effective amount of a compound selected from the group consisting of hydroxybenzoates, ... The cell is a sequence selected from SEQ ID NOs: 1 to 8 and any one of immunogenic fragments and variants thereof. or a method of preventing a human from contracting cancer, wherein the cancer expresses the sequence Expressing a sequence selected from numbers 1 to 8 and immunogenic fragments and variants of any one thereof the method, wherein the corresponding polypeptide, nucleic acid, vector or composition of the invention is administering to said human an agent.

[0104] The present invention also relates to a method for treating or preventing cancer in humans, comprising administering to a subject a polypeptide, a nucleic acid, a polypeptide of the present invention ... A vector or composition, wherein the cancer cells are selected from SEQ ID NOS: 1 to 8 and any one of them. the polypeptide, nucleic acid, vector or A composition is provided.

[0105] Transcripts corresponding to SEQ ID NOs: 23, 24, 25, 27 and 30 are also overexpressed in uveal melanoma. Thus, in another embodiment, the tumor is a uveal melanoma tumor and / or A tumor expressing a sequence selected from SEQ ID NOs: 1, 2, 3, 5 and 8.

[0106] Thus, the present invention encompasses a polypeptide having a sequence selected from: (a) any one of SEQ ID NOs: 1, 2, 3, 5, and 8; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide is selected from any one of SEQ ID NOs: 9 to 15, 18, and 22. The nucleic acid may comprise or consist of the sequence, and for example, the nucleic acid may be SEQ ID NO: 23, 24, 25, 27 and 30 or any one of SEQ ID NOs: 31, 32, 33, 35 and 38 comprising or consisting of a sequence selected from the group consisting of: The polypeptide for the method or use of the present invention, wherein the cancer is uveal melanoma. A nucleic acid, vector or composition is provided. The terms "prevention" and "prophylaxis" are used interchangeably herein.

[0107] (Treatment and vaccination regimens) The therapeutic regimen comprises (i) a polypeptide, nucleic acid, or vector of the invention; and (ii) one or more (iii) various other therapeutic agents; and a molecule or other further component, such as a compound or antigenic protein useful for Optionally, co-administration with an adjuvant may be performed at the same time (e.g., simultaneous administration) or sequentially (e.g., prime-boost administration). Examples of simultaneous administration include delivery of the same drug to the same external site. Simultaneous administration includes simultaneous administration to opposite sites and simultaneous administration to opposite opposite sites. Preferably, all components are delivered during the same treatment session. The components are administered simultaneously (e.g., both DNA and protein are administered simultaneously), but components are administered within minutes (e.g., during the same medical appointment or doctor's visit) or within hours. may be given.

[0108] In some embodiments, "priming" a polypeptide, nucleic acid, or vector of the invention or the first administration is followed by one or more "boosters" of the polypeptides, nucleic acids or vectors of the invention. In one embodiment, a "prime and boost" or booster dose is administered (a "prime and boost" regimen). The polypeptide, nucleic acid or vector of In one embodiment, both the prime and boost are polypeptides of the invention. In one embodiment, the polypeptides are Both the gene and the booster are nucleic acids or vectors of the invention, and in each case Alternatively, the primers may be prepared using the nucleic acid or vector of the present invention. The boost may be performed using a polypeptide of the present invention, and the prime may be performed using the polypeptide of the present invention. The boost is performed using the nucleic acid or vector of the invention. Typically, this involves a first or "priming" administration and a second or "boosting" administration. The next booster dose is administered after an interval of approximately 1 to 12 weeks, or up to 4 to 6 months. Doses may be given as frequently as every 1 to 6 weeks, or later (up to several years later). That's fine.

[0109] (antigen combination) A polypeptide, nucleic acid or vector of the invention may be synthesized in combination with one or more other polypeptides or vectors of the invention. or in combination with a nucleic acid or vector, and / or to treat melanoma, e.g., cutaneous melanoma Other antigenic polypeptides that elicit immune responses against melanoma or uveal melanoma polypeptide (or a polynucleotide or vector encoding it) These other antigenic polypeptides can be derived from a variety of sources. The source of these is well-described membrane proteins such as GPR143, PRAME, MAGE-A3 or pMel (gp100). They may contain melanoma-associated antigens, or they may contain melanoma antigens from other species. These include patient-specific neoantigens (Lauss et al., (2017) Nature Communications ations, 8(1), 1738. http: / / doi.org / 10.1038 / s41467-017-01460-0), retained int Ron neoantigen (Smart et al., (2018) Nature Biotechnology. http: / / doi.org / 10.1038 / nbt.4239), splice variant neoantigens (Hoyos et al., Cancer Cell, 34(2), 181-183. http: / / doi.org / 10.1016 / j.ccell.2018.07.008; Kahles et al. (2018), Cancer Cell, 34(2),211-224.e6. http: / / doi.org / 10.1016 / j.ccell.2018.07.001), damaged peptides A category known as antigens that encode T cell epitopes associated with processing Melanoma antigens belonging to the TEPPs (Tetra-Induced Epithelial Polypeptides; Gigoux, M. and Wolchok, J. (2018), JEM, 215 , 2233, Marijt et al., (2018), JEM 215, 2325), or neoantigens that may be discovered (C Furthermore, antigenic peptides derived from these various sources are also included ( i) non-specific immunostimulants / adjuvants, and / or (ii) e.g., universal CD4 receptors. It is an antigen (polypeptide) that contains a group epitope and is known to induce strong CD4 helper T cells. as peptides or as polynucleotides or vectors encoding these CD4 antigens. Anti-melanoma specific antibodies elicited by co-administered antigens in combination with It may also be possible to amplify the target response.

[0110] The different polypeptides, nucleic acids or vectors may be formulated in the same formulation or in separate formulations. Alternatively, the polypeptide may be a polypeptide of the invention, in which the polypeptide of the invention is a second or further polypeptide. It may also be provided as a fusion protein fused to a polypeptide (see below). A nucleic acid may be provided that encodes the fusion protein.

[0111] More generally, when two or more components are used in combination, the components For example, (1) as two or more individual antigenic polypeptide components, (2) as a fusion protein containing both (or additional) polypeptide components , (3) as one or more polypeptide and one or more polynucleotide components: (4) as two or more individual polynucleotide components, (5) as a single polynucleotide encoding two or more individual polypeptide components; And, or (6) Encoding a fusion protein containing both (or additional) polypeptide components. As a single polynucleotide to be encoded, could exist.

[0112] For convenience, when multiple components are present, they may be combined into a single fusion protein or a single It is often desirable for the fusion protein to be contained within a polynucleotide encoding the fusion protein. In one embodiment of the invention, all components are polypeptides. In another embodiment of the invention, the entire All components may be polynucleotides (e.g., those encoding a single fusion protein). The fragment is provided as a single polynucleotide such as a fragment of the fragment of the

[0113] (fusion protein) As an embodiment of the above discussion of antigen combinations, the present invention also provides antibodies encoding individual antigens. The second or further nucleic acid constructs of the invention can be prepared by fusing together sequences that correspond to the nucleic acid constructs of the invention. The isolated polypeptide of the present invention fused to a polypeptide (hereinafter "combination of the present invention") The combination polypeptides of the present invention are and are predicted to have the utility described herein for polypeptides that have excellent immune responses. Epidemic or vaccine activity, or prophylactic or therapeutic effect (increased breadth and depth of response) These may have advantages over conventional methods (including the use of steroids) and may be particularly valuable in outbred populations. Fusion of the polypeptides of the present invention may also be used as vaccine antigens and / or vectored vaccines (nucleic acid vaccines). It would also provide the advantage of increasing the efficiency of construction and manufacturing of polymers (including cutin).

[0114] As described above in the "Combination of Antigens" section, the polypeptides of the present invention and the Combination polypeptides may also be polypeptide sequences that are not polypeptides of the invention. , may be fused to include one or more of the following: (a) melanoma-associated antigens, and therefore useful as immunogenic sequences in vaccines; Other possible polypeptides (e.g., the above-mentioned GPR143, PRAME, MAGE-A3 and pMe l(gp100)); and (b) A polypeptide sequence capable of enhancing an immune response (i.e., an immunostimulatory sequence). (c) For example, they contain universal CD4 helper epitopes and provide strong CD4+ help. and enhance CD8+ T cell responses to CLT antigen epitopes. Peptide sequence.

[0115] The present invention also relates to nucleic acids encoding the fusion proteins and polypeptides of the present invention. Mutatis mutandis, other aspects of the invention (vectors, compositions, cells, etc.) are provided.

[0116] (CLT antigen-binding polypeptide) An antigen-binding polypeptide (polypeptide of the invention) that is immunospecific for a tumor-expressed antigen. The cytolytic cells are recruited to antigen-modified tumor cells to mediate their destruction. The mechanism of recruitment of cytolytic cells by such antigen-binding polypeptides may be One of the mechanisms is known as antibody-dependent cell-mediated cytotoxicity (ADCC). The present invention provides antigen-binding polypeptides immunospecific for the polypeptides of the invention. Monoclonal antibodies and fragments thereof, such as domain antibodies, Fab fragments, Fv fragments, and VHH fragments. Antigen-binding polypeptides, including antibodies, can be expressed in non-human animal species (e.g., rodents or camelids). They may be produced in a non-human species (e.g., modified to have a human immune system) and humanized, or may be produced in a non-human species (e.g., modified to have a human immune system). It may also be produced in genetically engineered rodents.

[0117] Antigen-binding polypeptides can be produced by methods well known to those skilled in the art. Clonal antibodies are produced by using hybridoma technology to grow specific antibody-producing B cells in tissue culture. fused with myeloma (B-cell cancer) cells selected for their proliferation potential and lack of antibody chain synthesis. (Kohler and Milstein, 1975, Nature 256(5517):495-49 7 and Nelson et al., 2000 (June), Mol Pathol. 53(3):111-7, are incorporated by reference in their entirety. (The body of which is incorporated herein.)

[0118] Monoclonal antibodies against a desired antigen can be prepared, for example: a) Lymphocytes obtained from the peripheral blood of animals (including humans) that have been previously immunized / exposed with the desired antigen. immortalizing the lymphocytes with immortal cells, preferably myeloma cells, to form hybridomas; b) The resulting immortalized cells (hybridomas) are cultured to produce antibodies with the desired specificity. harvesting viable cells; can be obtained by

[0119] Monoclonal antibodies can be obtained by a process comprising the following steps: a) vectors, particularly phages, more particularly filamentous bacteriophages, (preferably DNA or other antigens obtained from lymphocytes, particularly peripheral blood lymphocytes, of animals previously immunized with a desired antigen. Cloning step of cDNA sequence b) transforming a prokaryotic cell with the vector under conditions that allow antibody production; c) selecting antibodies by subjecting them to antigen affinity selection; d) recovering antibodies having the desired specificity; e) derived from B cells of patients exposed to the antigen or from animals immunized with the antigen in the laboratory and expressing the antibody-encoding nucleic acid molecule. The selected antibodies can then be purified using conventional recombinant protein production techniques (e.g., genetic It may be produced from engineered CHO cells.

[0120] The present invention provides isolated antigen-binding polypeptides immunospecific for the polypeptides of the invention. Preferably, the antigen-binding polypeptide is a monoclonal antibody or a fragment thereof. is. In certain embodiments, the antigen-binding polypeptide is conjugated to a cytotoxic moiety. An example of a cytotoxic moiety includes the Fc domain of an antibody, which binds to Fc receptor-bearing cells to promote ADCC. Alternatively, the antigen-binding polypeptide may be a biotoxin or a cytotoxic agent. It may be linked to a chemical substance.

[0121] Another important class of antigen-binding polypeptides is T cell polypeptides that bind to HLA-presenting fragments of the antigens of the invention. In this embodiment, the target antigen (CTL antigen or its derivatives) on the surface of a tumor cell is included. TCR-based biologics (TCRs derived directly from patients or specifically engineered TCRs) that recognize the TCR-derived TCRs. The TCRs (containing the selected high-affinity TCRs) are also present on T cells (or other types of immune cells) and mediate these immune responses. and a targeting moiety that recognizes a component that attracts immune cells to the tumor. In some embodiments, the targeting moiety may also provide a redirection effect. They may also stimulate beneficial activities (including cytolytic activity) of stimulated immune cells.

[0122] Thus, in one embodiment, the antigen-binding polypeptide is a polypeptide of the invention; or or a portion thereof. The polypeptide is a T cell receptor. In one embodiment, the antigen-binding polypeptides of the invention are administered to a subject by cytotoxic cells or other The antibody may be linked to another polypeptide capable of binding to an immune component of the antibody.

[0123] In one embodiment, the antigen-binding polypeptide is for use in medicine. In one embodiment, the antigen-binding polypeptides of the invention are administered in a pharmaceutically acceptable carrier. Such compositions are sterile and suitable for parenteral administration. See, for example, the disclosure of pharmaceutical compositions above.

[0124] According to the present invention, there is provided a method of treating a human suffering from cancer, wherein the cells of the cancer contain SEQ ID NO: Nos. 1 to 8 and immunogenic fragments and variants thereof; The method or the method for preventing a human from developing cancer, wherein the cancer cells are those of SEQ ID NO: 1. to 8, and immunogenic fragments and variants of any one thereof. The method further comprises administering to a subject an antigen-binding polypeptide of the present invention or an antigen-binding polypeptide thereof. The method comprises administering to the human a composition comprising:

[0125] In one embodiment, a compound conjugated to a cytotoxic moiety for use in the treatment or prevention of cancer in humans is provided. an antigen-binding polypeptide of the present invention, which may be A composition comprising: a cancer cell comprising SEQ ID NOs: 1 to 8 and an immunogenic fragment of any one thereof; and wherein the antigen-binding polypeptide or composition expresses a corresponding sequence selected from do. Preferably, in any of the above embodiments, the cancer is melanoma, particularly cutaneous melanoma. do.

[0126] In one embodiment, the polypeptide comprises a sequence selected from: (a) any one of SEQ ID NOs: 1, 2, 3, 5, and 8; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide is selected from any one of SEQ ID NOs: 9 to 15, 18, and 22. The nucleic acid may comprise or consist of the sequence, and for example, the nucleic acid may be SEQ ID NO: 23, 24, 25, 27 and 30 or a sequence selected from any one of SEQ ID NOs: 31, 32, 33, 35 and 38; or a sequence thereof; and the cancer is uveal melanoma, are provided antigen-binding polypeptides or compositions for use.

[0127] The antigen-binding polypeptide (antibody or fragment thereof, etc.) is, for example, 5 to 1000 mg, for example, 25 to 500 mg For example, it may be administered at a dose of 100 to 300 mg, for example about 200 mg.

[0128] (Cell therapy that enhances antigen presentation in vivo) Any of a variety of cellular delivery vehicles can be used within pharmaceutical compositions to stimulate antigen-specific immune responses. Therefore, the present invention provides a method for loading the polypeptide of the present invention ex vivo. or genetically engineered to express a polypeptide of the invention. , providing cells that are isolated antigen-presenting cells (hereinafter referred to as "APCs of the present invention"). Antigen-presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes, and Other cells that may be engineered to become APCs. Such cells do not necessarily have to be. Although not required, it is possible to enhance antigen presentation, improve activation and / or persistence of T cell responses, and and / or genetically modified to be immunocompatible (i.e., HLA haplotype-matched) with the receptor. APCs can generally be isolated from any of a variety of body fluids and organs and can be autologous. The cells may be autologous, allogeneic, syngeneic or xenogeneic.

[0129] In certain preferred embodiments of the present invention, dendritic cells or their precursors are used as APCs. Thus, in one embodiment, the APCs of the present invention are dendritic cells. Dendritic cells are highly potent APC (Banchereau and Steinman, 1998, Nature 392:245-251) and can be used prophylactically or therapeutically. It has been shown to be effective as a physiological adjuvant for eliciting therapeutic immunity (T (See Immerman and Levy, 1999, Ann. Rev. Med. 50:507-529.) Generally, dendritic cells The vacuoles have a typical shape (stellate in situ and prominent cytoplasmic projections visible in vitro). They have dendrites, the ability to efficiently take up, process and present antigens, and Dendritic cells may also be identified based on their ability to activate naive T cell responses. Of course, specific cell surface receptors not normally found on dendritic cells in vivo or ex vivo are also present. The modified dendritic cells may be engineered to express surface receptors or ligands. Instead of dendritic cells, antigen-loaded secretory vesicles (e.g., ectodermal vesicles) are also contemplated by the present invention. These vesicles, called vesicosomes, may be used in immunogenic compositions (Zitvogel et al., 1998, Na (See, e.g., J. Med. 4:594-600). Thus, in one embodiment, the polypeptides of the invention are The exosomes are provided.

[0130] Dendritic cells and precursor cells can be derived from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood, or other sources. They may be obtained from any suitable tissue or body fluid. For example, dendritic cells may be collected from peripheral blood. A combination of cytokines such as GM-CSF, IL-4, IL-13, and / or TNFα is administered to the isolated monocyte culture. Alternatively, peripheral blood, umbilical cord blood, or bone marrow may be added to differentiate the cells in vitro. CD34-positive cells collected from the marrow were treated with GM-CSF, IL-3, TNFα, CD40 ligand, LPS, and flt3 ligand. and / or other compounds that induce differentiation, maturation and proliferation of dendritic cells. By adding them to the culture medium, they may be differentiated into dendritic cells.

[0131] Dendritic cells are conveniently categorized into "immature" and "mature" cells, which allows for easy differentiation between two well-characterized phenotypes. However, This nomenclature should not be construed as excluding all possible intermediate steps in differentiation. Immature dendritic cells are characterized as APCs with high antigen uptake and processing capabilities. This correlates with high expression of Fcγ receptors and mannose receptors. These markers are usually expressed at low levels, but they are involved in T cell activation, such as class I and class II MHC. cell surface molecules that mediate cell proliferation, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD4 These cells are characterized by high expression of CD80, CD86, and 4-1BB.

[0132] APCs can also be genetically engineered to, for example, express proteins (or portions thereof or other modifications thereof). a polynucleotide encoding a variant of the polypeptide, and the polypeptide is expressed in the cells. Such transfection may be carried out in vitro. The transfected cells may then be transfected into a pharmaceutical composition as described herein. Alternatively, gene transfer vectors can be used to target dendritic cells or other antigen-presenting cells. The vehicle may be administered to a patient, allowing transfection to occur in vivo. Transfection of dendritic cells in vivo and in vitro is described, for example, in WO97 / 24447. or as described in Mahvi et al., 1997, Immunology and Cell Biology 75:456-460. This can be done using any method generally known in the art, such as the gene gun approach that has been used Antigen loading into dendritic cells may be carried out by injecting dendritic cells or precursor cells with a polypeptide, with DNA (e.g., a plasmid vector) or RNA; or with recombinant bacteria or Viruses (e.g., adenoviruses, adeno-associated viruses (AAV) (e.g., AAV types 5 and 2) , alphaviruses (e.g., Venezuelan equine encephalitis virus (VEEV)), Sindbis virus ( SIN), Semliki Forest virus (SFV), herpesviruses, arenaviruses (e.g., lymphoviruses), leukemia virus (LCMV), measles virus, poxvirus (modified vaccinia virus), varicella (MVA) or fowlpox), paramyxovirus, lentivirus, or rhabdovirus ( This may be achieved by incubation with a virus such as vesicular stomatitis virus (VSV). Prior to loading, the polypeptide is coupled to an immunological partner (e.g., a kinase inhibitor) that provides T cell help. Alternatively, dendritic cells may be covalently linked to an unconjugated immunoglobulin (e.g., a carrier molecule). Pulsed with the biological partners, either individually or in the presence of the polypeptide or vector. Good too.

[0133] The present invention provides a method for the preparation of specifically designed, short, chemically synthesized epitope constructs of polypeptide antigens. The encoded fragments are provided for delivery to antigen-presenting cells. Those skilled in the art will appreciate that such molecules are The antigenic polypeptides of the present invention may be used to generate peptides known as growing chain peptides (SLPs). For stimulating (or loading) cells in vitro (Gornati et al., 2018, Front. Immunol. 9:1484), or as a method for introducing polypeptide antigens into antigen-presenting cells in vivo ( Melief and van der Burg et al., 2008, Nat Rev Cancer, 8:351-60) therapeutic platform You will understand that we provide a form.

[0134] In one embodiment, the antigen-presenting cells of the present invention, preferably dendritic cells, are administered in a pharmaceutically acceptable carrier. Such compositions are suitable for parenteral administration. The compositions may also be sterile compositions, see, for example, the disclosure of pharmaceutical compositions above. In one embodiment, an antigen-presenting cell, preferably a dendritic cell, of the invention for use in medicine. is provided.

[0135] Similarly, there is provided a method of treating a human suffering from cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 8. and immunogenic fragments and variants thereof. or a method for preventing a human from developing cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 8 and and expressing a sequence selected from any one of the immunogenic fragments and variants thereof. The method comprises using an antigen-presenting cell of the present invention, preferably a dendritic cell, or an antigen-presenting cell thereof of the present invention. The method includes administering to the human a composition comprising the cells.

[0136] In one embodiment, the antigen-presenting cells of the invention for use in the treatment or prevention of human cancer, Preferably, a composition comprising dendritic cells or antigen-presenting cells of the present invention, expressing a corresponding sequence selected from SEQ ID NOs: 1 to 8 and any one of immunogenic fragments thereof. The antigen-presenting cell or composition thereof is provided.

[0137] In one embodiment, the exosomes of the present invention are contained in a pharmaceutical composition comprising the exosomes of the present invention together with a pharmaceutically acceptable carrier. Such compositions may be sterile compositions suitable for parenteral administration. See, for example, the disclosure of pharmaceutical compositions above. The compositions may optionally contain an immunostimulant. See the immunostimulant disclosure above. In one embodiment, there is provided an exosome of the invention for use in medicine.

[0138] Similarly, there is provided a method of treating a human suffering from cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 8. and immunogenic fragments and variants thereof. or a method for preventing a human from developing cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 8 and and expressing a sequence selected from any one of the immunogenic fragments and variants thereof. The method further comprises administering the exosomes of the present invention or a composition containing the exosomes of the present invention to the subject. The method further comprises administering to the subject a therapeutically effective amount of the compound.

[0139] In one embodiment, the exosomes or antigen-binding fragments of the present invention are provided for use in the treatment or prevention of cancer in humans. The composition of the present invention contains the exosome, and the cancer cells are SEQ ID NOS: 1 to 8 and the like. The said enzyme expresses a corresponding sequence selected from any one of the immunogenic fragments A xosome or composition is provided. In any one of the above embodiments, suitably the cancer is melanoma, particularly cutaneous melanoma. be.

[0140] (Stimulated T cell therapy) APC-mediated generation, in vivo or ex vivo, of T cells immunospecific for the polypeptides of the present invention In addition, autologous or non-autologous T cells can be obtained from a subject, e.g., from peripheral blood, umbilical cord blood, and and / or tumor cells isolated by apheresis and loaded onto MHC molecules (signal 1) of APC cells. Stimulation in the presence of a tumor-associated antigen induces proliferation of T cells bearing TCRs immunospecific for this antigen. That's fine.

[0141] For successful T cell activation, the costimulatory surface molecules B7 and CD28 are required to bind to antigen-presenting cells, respectively. It is necessary for the IL-1 receptor to bind to the IL-1 receptor on the T cell surface (signal 2). To achieve optimal T cell activation, Both signals 1 and 2 are required. In contrast, the anti- Page 11 ... Antigenic peptide stimulation (signal 1) fails to induce full T cell activation and induces T cell tolerance. In addition to costimulatory molecules, there are inhibitory molecules such as CTLA-4 and PD-1, which act to inhibit T-cell proliferation. Induce signals to inhibit cell activation.

[0142] Thus, autologous or non-autologous T cells are stimulated in the presence of a polypeptide of the invention, and the cancer cells express the corresponding polypeptide of the present invention. It may be reintroduced into patients at risk of or suffering from cancer, but only if the The antigen-specific TCR recognizes the antigen presented by the patient's MHC and synthesizes the corresponding polypeptide. Only if the compound will target cancer cells that express the gene and induce their death.

[0143] In one embodiment, the method is for the ex vivo stimulation and / or expansion of T cells from a human suffering from cancer. a polypeptide, nucleic acid, vector or composition of the invention for use, and reintroducing the stimulated and / or expanded T cells into the human to treat the human's cancer. The polypeptide, nucleic acid, vector or composition is provided for use in the treatment of a cancer.

[0144] The present invention relates to a method for treating human cancer, wherein the cancer cells are selected from SEQ ID NOS: 1 to 8 and their immunogens. and expressing a sequence selected from the group consisting of a human avian ... and removing a population of leukocytes comprising at least T cells, optionally together with antigen-presenting cells, from the patient. and then injecting the T cells into the presence of the corresponding polypeptide, nucleic acid, vector, or composition of the invention. and stimulating and / or expanding at least the stimulated and / or expanded T cells. and reintroducing some or all of the leukocytes, including the cells, into the person. provide. In any one of the above embodiments, suitably the cancer is melanoma, particularly cutaneous melanoma. be.

[0145] In one embodiment, the antibody is selected from SEQ ID NOs: 1 to 8 and immunogenic fragments and variants of any one thereof. A process for preparing a T cell population that is cytotoxic against cancer cells expressing a selected sequence. (a) obtaining T cells and antigen-presenting cells from a cancer patient; and (ii) administering the T cells and antigen-presenting cells in vitro. and stimulating the T cell population with the corresponding polypeptide, nucleic acid, vector or composition of the invention. and amplifying.

[0146] "Corresponding" in this context means that the cancer cells correspond to, for example, SEQ ID NO: A (A is SEQ ID NO: 1-8) or a variant thereof, or an immunogenic fragment thereof, The group may be in the form of a polypeptide, a nucleic acid, or a vector, or a composition comprising one of the foregoing. In vitro stimulation and amplification by No. A or its variants or immunogenic fragments thereof. means.

[0147] For example, in such a process, the culture and expansion is carried out in the presence of dendritic cells. Dendritic cells can be transfected with the nucleic acid molecules or vectors of the invention to deliver the polypeptides of the invention. The peptide will be expressed.

[0148] The present invention relates to a T cell population obtainable by any of the above processes (hereinafter referred to as the present invention). The T cell population is provided. In one embodiment, T cells stimulated with a polypeptide, nucleic acid, vector or composition of the invention In one embodiment, a cell (hereinafter referred to as a T cell of the present invention) is provided.

[0149] In one embodiment, the T cell population or T cells of the invention are administered in a pharmaceutically acceptable carrier. Such compositions are suitable for parenteral administration, for example. It may be a sterile composition. In one embodiment there is provided a T cell population or T cell of the invention for use in medicine.

[0150] Similarly, there is provided a method of treating a human suffering from cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 8. and immunogenic fragments and variants thereof. or a method for preventing a human from developing cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 8 and and expressing a sequence selected from any one of the immunogenic fragments and variants thereof. The method comprises administering to a subject a T cell population or T cell of the present invention or a T cell population or T The method includes administering to the human a composition comprising the cells.

[0151] In one embodiment, the T cell population of the invention for use in the treatment or prevention of cancer in a human, a composition comprising a T cell population or T cell of the present invention, The cells express a corresponding sequence selected from SEQ ID NOs: 1 to 8 and any one of the immunogenic fragments thereof. The T cell population of the present invention, the T cell of the present invention, or a composition comprising the T cell population or T cell of the present invention In any one of the above embodiments, suitably the cancer is melanoma, particularly It is cutaneous melanoma.

[0152] In one embodiment, the polypeptide comprises a sequence selected from: (a) any one of SEQ ID NOs: 1, 2, 3, 5, and 8; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide is selected from any one of SEQ ID NOs: 9 to 15, 18, and 22. The nucleic acid may comprise or consist of the sequence, and for example, the nucleic acid may be SEQ ID NO: 23, 24, 25, 27 and 30 or any one of SEQ ID NOs: 31, 32, 33, 35 and 38 comprising or consisting of a sequence selected from the group consisting of: The T cell for the process, method or use of the present invention, wherein the cancer is uveal melanoma. Populations, T cells, antigen-presenting cells, exosomes or compositions are provided.

[0153] (Therapy using genetically engineered immune cells) All of the above-mentioned derivatives of CLT antigen-binding polypeptides form complexes with human HLA molecules. TCR or TCR mimics that recognize peptides derived from CLT antigens (Dubrovsky et al., 2016, Oncoi (see immunology) and can be genetically engineered to be expressed on the surface of (autologous or non-autologous) T cells. The antibodies may be expressed in a manner that is then administered as an adoptive T cell therapy for the treatment of cancer.

[0154] These derivatives fall into the "chimeric antigen receptor (CAR)" category and are used herein. When used, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor It can also refer to cells engineered to confer artificial specificity to particular immune effector cells. CARs can be used to confer the specificity of monoclonal antibodies to T cells. and thereby generate large numbers of specific T cells for use in, for example, adoptive cell therapy. CAR is a polypeptide of the present invention that binds to HLA and provides cell specificity. It will be directed against tumor-associated antigens.

[0155] Another approach to treating cancer in patients is to target antigens expressed on tumor cells. The goal is to genetically modify T cells to target the immune system through the expression of chimeric antigen receptors (CARs). This technique is described in Wendell and June, 2017, Cell, 168:724-740 (in its entirety). (which is incorporated herein).

[0156] Such CAR T cells can be prepared by administering to a subject a cell sample containing T cells or T cell precursors, e.g., For example, cells obtained from peripheral blood, umbilical cord blood and / or by apheresis are used to bind HLA. A chimeric T cell receptor (CAR) encoding a chimeric T cell receptor (CAR) that is immunospecific for the polypeptide of the present invention to which it is linked. Such nucleic acids may be prepared by transfection of cells. and administering to the subject an effective amount of the cells, It is also possible to provide a T cell response against cells expressing the polypeptides of the invention. If desired, a cell sample may be obtained from the subject.

[0157] The cells used to generate the CAR-expressing T cells can be autologous or non-autologous. It is understood that Transgenic CAR-expressing T cells are engineered to target endogenous T cell receptor inactivation and / or endogenous HLA inactivation. For example, cells may be engineered to eliminate expression of endogenous α / β T cell receptors (TCRs). The operation may be performed in this manner.

[0158] Methods for transfecting cells are well known in the art, including electroporation, Highly efficient transfection methods can also be used. For example, CAR constructs The nucleic acid or vector of the invention expressing the The vector may be introduced into cells using a vector.

[0159] The cell population for CAR-expressing T cells may be enriched after transfection of the cells. For example, CAR-expressing cells can be expressed by the antigen bound by the CAR or by the use of a CAR-binding antibody. Alternatively, the non-expressing cells can be sorted (e.g., by FACS) from the non-expressing cells. This includes depleting T cells and depleting cells that do not express CAR. 56+ cells can be depleted from the culture population.

[0160] The population of transfected CAR-expressing cells is cultured in a medium that selectively enhances the proliferation of CAR-expressing T cells. They may also be cultured in vitro, thus allowing CAR-expressing T cells to be expanded in vitro.

[0161] A sample of the CAR cells may be stored (or maintained in culture). For example, the sample may be subsequently The cells may be cryopreserved for propagation or analysis. CAR-expressing T cells are compatible with other therapies, such as checkpoint blockade, including PD-L1 antagonists. It may also be used in combination with an anti-inflammatory agent.

[0162] In one embodiment, a mammalian cell line is adapted to express any of the above antigen-binding polypeptides on its surface. In one embodiment, engineered cytotoxic cells are provided. Suitably, the cytotoxic cells are T cells.

[0163] In one embodiment, a pharmaceutical composition comprising the antigen-binding polypeptide on its surface for use in medicine. Cytotoxic cells, preferably T cells, engineered to express either do. The present invention provides pharmaceutical compositions comprising the cytotoxic cells of the present invention, which are preferably T cells. do.

[0164] A method of treating a human patient suffering from cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 8 and SEQ ID NOS: 1 to 8. the method, wherein the sequence selected from any one of the immunogenic fragments and variants thereof is expressed in the Alternatively, a method for preventing a human from contracting cancer, wherein the cancer is a cancer selected from the group consisting of SEQ ID NOS: 1 to 8 and any of the above. The method will express a sequence selected from any one of the immunogenic fragments and variants. and administering to said human the cytotoxic cells, preferably T cells, of the present invention. A method is provided.

[0165] In one embodiment, the cytotoxic cells of the invention, preferably T cells, are used to treat or prevent human cancer. The cancer cells are selected from the group consisting of SEQ ID NOS: 1 to 8 and any one of them. The fragment expresses the corresponding sequence selected from the cytoplasmic fragment.

[0166] (combination therapy) The cancer treatment methods of the present invention may be used in combination with other therapies, particularly checkpoint inhibitors and interferon. This may be performed in combination with Polypeptides, nucleic acids, vectors, antigen-binding polypeptides, and (APC and T cell-based) Adoptive cell therapy may enhance their immunogenicity (e.g., the magnitude and / or potency of the immune response elicited). or improves the range of immune responses), or for other activities (e.g., the innate or adaptive immune response). other components engineered to provide other aspects of the target protein (such as activation of other aspects of the target protein, or destruction of tumor cells) It can be used in combination with components.

[0167] Thus, the present invention provides a composition (i.e., an immunogenic, vaccine, or pharmaceutical composition) of the present invention. or a kit of several of these compositions together with a pharmaceutically acceptable carrier. a polypeptide, nucleic acid, or vector of the invention, and; (i) one or more further immunogenic or or immunostimulatory polypeptides (e.g., interferon, IL-12, checkpoint inhibitors) (ii) a small molecule (e.g., a nucleic acid encoding the same, or a vector containing the nucleic acid); For example, HDAC inhibitors or other drugs that alter the epigenetic profile of cancer cells) or biologics ( The polypeptide or nucleic acid encoding it, or a vector containing the nucleic acid ) which enhances the translation and / or presentation of the polypeptide product that is the subject of the present invention. Provides services that include:

[0168] Checkpoint inhibitors target normal proteins on cancer cells or the T proteins that respond to them. These inhibitors block proteins on cells, helping to protect cancer cells from immune system attacks. to combine with CLT antigen-based therapy to try to overcome one of the main defenses of These drugs may be a particularly important class of drugs.

[0169] Therefore, one aspect of the present invention is a method for producing a polypeptide, a nucleic acid, a vector, an antigen-binding polypeptide, or a polypeptide of the present invention. Peptides, compositions, T cells, T cell populations, or antigen-presenting cells, in combination with checkpoint inhibitors Examples of checkpoint inhibitors include pembrolizumab, PD-1 inhibitors such as (Keytruda) and nivolumab (Opdivo), and atezolizumab (Tecent PD-L1 inhibitors such as ribavirin (Riq), avelumab (Bavencio), and durvalumab (Imfinzi), as well as and CTLA-4 inhibitors such as ipilimumab (Yervoy).

[0170] Interferons (alpha, beta, and gamma) are a family of proteins that the body produces in very small amounts. Interferon slows or stops cancer cell division, preventing cancer cells from defending themselves against the immune system. It may reduce the body's ability to defend itself and / or enhance multiple aspects of the adaptive immune system. Ferron is usually administered by subcutaneous injection, for example in the thigh or abdomen.

[0171] Therefore, one aspect of the present invention is a method for producing a polypeptide, a nucleic acid, a vector, an antigen-binding polypeptide, or a polypeptide of the present invention. Administration of the peptide or composition in combination with interferon, e.g., interferon alpha Includes giving.

[0172] Also, different aspects of the present invention may be combined, for example, the polypeptides, nucleic acids, etc. of the present invention. and vectors may be combined with the APCs, T cells or T cell populations of the invention (described below). (Can be). One or more modalities of the present invention may also be used in combination with conventional anti-cancer chemotherapy and / or radiation therapy. Good too.

[0173] (diagnosis) In another aspect, the present invention provides a method for diagnosing cancer, particularly melanoma, such as cutaneous melanoma. or the polypeptides, nucleic acids, vectors, antigen-binding polypeptides, adoptive cell therapy To identify a human subject suitable for treatment with the method or composition, one or more of the methods Methods of using the polypeptides or nucleic acids of the invention are provided.

[0174] Accordingly, the present invention provides a method for diagnosing a human suffering from cancer, comprising: The cell is a polypeptide selected from SEQ ID NOs: 1 to 8 and any one of immunogenic fragments or variants thereof. a peptide sequence (e.g., selected from the sequences of SEQ ID NOs: 9 to 22 and 39), or a polypeptide thereof Nucleic acid encoding a sequence (e.g., selected from the sequences of SEQ ID NOs: 23 to 30 and 31 to 38) determining whether the polypeptide or corresponding nucleic acid expresses If the gene is overexpressed in the cancer cells, the person is diagnosed as suffering from cancer. The method further comprises:

[0175] The present invention was a method for diagnosing a human suffering from cancer, which is cutaneous melanoma. and the cells of the cancer are either SEQ ID NO: 4, 6 or 7, or an immunogenic fragment or variant thereof. or expressing a polypeptide sequence selected from the group consisting of: and determining whether the polypeptide or corresponding nucleic acid is present in the cancer cell. If the gene is overexpressed in the human, the human is diagnosed as suffering from a cancer that is cutaneous melanoma. The method further comprises the step of: As used herein, "overexpressed" in a cancer cell refers to the level of expression in the cancer cell. This means that the level is higher than that in normal cells.

[0176] The present invention relates to a method for treating a human suffering from a cancer that is cutaneous melanoma or uveal melanoma. A method for diagnosing a cancer, comprising: a polypeptide sequence selected from one of the immunogenic fragments or variants, or a polypeptide sequence thereof determining whether the nucleic acid encoding the polypeptide or polypeptides is expressed; If the corresponding nucleic acid is overexpressed in the cancer cells, the person will have cutaneous melanoma or diagnosing the subject as suffering from cancer that is uveal melanoma, to provide.

[0177] Overexpression is measured by the expression of a nucleic acid or polypeptide of the invention in control human subjects known to be cancer-free. Overexpression can be determined by reference to the level of the polypeptide. The nucleic acid or polypeptide is detected at a significantly higher level (e.g., 3 or 4 times higher) in the test subject than in the control subject. The expression level of the nuclei of the present invention is shown to be 0%, 50%, 100%, or 500% higher. If the nucleic acid or polypeptide of the invention has undetectably low levels of the nucleic acid or polypeptide, A diagnosis can be made at the time of peptide detection.

[0178] The present invention also provides a method of treating a human suffering from cancer, comprising: (a) the cancer cells are selected from SEQ ID NOs: 1 to 8 and any one of their immunogenic fragments or variants; a polypeptide sequence (e.g., selected from the sequences of SEQ ID NOs: 9 to 22 and 39), or A nucleic acid encoding a polypeptide (e.g., selected from the sequences of SEQ ID NOs: 23 to 30 and 31 to 38) determining whether the gene expresses the nucleotide sequence; and if so, (b) administering to said human the corresponding polypeptides, nucleic acids, vectors, compositions, or T cell populations of the invention. administering a T cell, an antigen-presenting cell, an antigen-binding polypeptide, or a cytotoxic cell. The method includes:

[0179] Similarly, a polypeptide isolated from a tumor of a human suffering from cancer, comprising a sequence selected from the following: Use of: (a) any one of SEQ ID NOs: 1 to 8; or (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); or the use of nucleic acids encoding the polypeptides, such that the human is able to derive the corresponding polypeptides of the invention. polypeptides, nucleic acids, vectors, compositions, T cell populations, T cells, antigen-presenting cells, antigen binding polypeptides or cytotoxic cells, may be suitable for treatment with vaccines. and use of the compound as a biomarker for determining whether the compound is a marker for apoptosis or inflammatory bowel disease. Suitably the cancer is melanoma, particularly cutaneous melanoma.

[0180] The present invention also encompasses sequences wherein the polypeptide is selected from: (a) any one of SEQ ID NOs: 1, 2, 3, 5, and 8; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); For example, the polypeptide is selected from any one of SEQ ID NOs: 9 to 15, 18, and 22. The nucleic acid may comprise or consist of the sequence, and for example, the nucleic acid may be SEQ ID NO: 23, 24, 25, 27 and 30, or any one of SEQ ID NOs: 31, 32, 33, 35 and 38 and the cancer comprises or consists of a sequence selected from one of the following: The method or use of the present invention is directed to a tumor.

[0181] Preferably, the polypeptide of the present invention is SEQ ID NO: 1 to 8, or a fragment thereof, such as an immunogenic fragment thereof. The fragment has a sequence selected from the fragments (for example, selected from the sequences of SEQ ID NOs: 9 to 22 and 39). Preferably, the nucleic acid of the present invention is SEQ ID NO: 23 to 30 or 31 to 38, or an immunogenic fragment thereof, etc. The fragment has or comprises a sequence selected from any one of the fragments of

[0182] Kits for detecting the presence of nucleic acids are well known. A kit containing at least two hybridizing oligonucleotides is used for real-time PCR. It may also be used in RT-PCR reactions, allowing for the detection and semi-quantification of specific nucleic acids. The kit generates a fluorescent signal as a result of Förster resonance energy transfer (FRET). by (e.g., TaqMan® kit) or in binding of double-stranded DNA (e.g., SYBR (Registered Trademark Green Kit), will allow detection of PCR products. , including TaqMan® probes spanning multiple exons of the target DNA) , for example, to allow for the detection and quantification of the transcript-encoding nucleic acid of the present invention. Assays using ATP are set up in a multiplex format to allow simultaneous analysis of multiple ATPs in one reaction. It will detect nucleic acids that are active (i.e., DNA with specific epigenetic characteristics that indicate expression). Kits for detecting the presence of β-glucan may also be used. Additional components that may be present in such kits include: Components include diagnostic reagents or reporters that facilitate detection of the nucleic acids of the invention. do.

[0183] The nucleic acids of the invention may also be detected in a liquid biopsy using a blood sample from the patient. Such a procedure provides a non-invasive alternative to surgical biopsy. Plasma from the animals can be isolated and analyzed for the presence of nucleic acids of the invention.

[0184] The polypeptides of the present invention can be detected using antigen-specific antibodies in an ELISA-type assay. may be obtained by detecting the polypeptides of the invention in a homogenized preparation of a patient tumor sample. Alternatively, the polypeptides of the present invention can be used in immunohistochemical analysis. The tumor may be detected by staining patient tumors using appropriately labeled antibody preparations. Sections of the sample are examined using a light microscope to identify the presence of polypeptide antigens. As a further alternative, the polypeptides of the present invention can be detected by immunohistochemical analysis. may be detected by staining patient tumor samples with appropriately labeled antibody preparations. Sections of the sample are examined using a light microscope to identify the presence of polypeptide antigens. is.

[0185] The polypeptides of the present invention also have the advantage that they increase the reactivity to the polypeptides. This may be detected by determining whether or not the antibody is able to stimulate T cells.

[0186] A method for treating human cancer, particularly melanoma, e.g., cutaneous melanoma, comprises: (i) administering to a subject a nucleic acid or (ii) detecting the presence of a polypeptide of the invention; and (iii) administering to the subject a nucleic acid, polypeptide of the invention. administering a vaccine, vector, cell, T cell or T cell population or composition (preferably administering the same nucleic acid or polypeptide or a fragment thereof that was detected .

[0187] The method for treating human cancer, particularly melanoma, e.g., cutaneous melanoma, also includes (preferably (i) administering the nucleic acid or polypeptide of the present invention to a subject in which the presence of the nucleic acid or polypeptide of the present invention has been detected This includes administering a peptide, vector, cell, T cell or T cell population or composition.

[0188] In particular, the cancer to be diagnosed and possibly treated is melanoma, e.g., cutaneous melanoma. It is a theme. If a polypeptide of the present invention is detected that is SEQ ID NO: 1, 2, 3, 5 or 8 or a fragment thereof , the cancer is predicted to be cutaneous or uveal melanoma.

[0189] SPECIAL EMBODIMENTS In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 1. Exemplary fragments include or consist of any one of SEQ ID NOs: 9 and 10. Examples of nucleic acids encoding the peptide sequence include or are SEQ ID NO: 23 or SEQ ID NO: 31. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxicity (cytotoxicity) and the like are listed above. Exosomes, antigen-binding polypeptides, antigen-presenting cells, and exosomes are provided. nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides The antigen-presenting cells and exosomes are useful in the treatment of cancer, particularly melanoma, e.g., cutaneous melanoma or It may be used to treat uveal melanoma. Related diagnostic methods are also provided.

[0190] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:2. Exemplary fragments include or consist of any one of SEQ ID NOs: 11 to 14. Examples of nucleic acids encoding the peptide sequence include or are SEQ ID NO: 24 or SEQ ID NO: 32. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxicity (cytotoxicity) and the like are listed above. Exosomes, antigen-binding polypeptides, antigen-presenting cells, and exosomes are provided. nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides The antigen-presenting cells and exosomes are useful in the treatment of cancer, particularly melanoma, e.g., cutaneous melanoma or It may be used to treat uveal melanoma. Related diagnostic methods are also provided.

[0191] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:3. An exemplary fragment comprises or consists of SEQ ID NO: 15. Exemplary nucleic acids include or consist of SEQ ID NO: 25 or SEQ ID NO: 33. nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic (cytocotic) cells, antigens Binding polypeptides, antigen-presenting cells, and exosomes are provided. or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and and exosomes, which are useful in the treatment of cancer, particularly melanoma, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0192] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:4. An exemplary fragment comprises or consists of any one of SEQ ID NOs: 16 and 17. Examples of nucleic acids encoding the peptide sequences include or are SEQ ID NO:26 or SEQ ID NO:34. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxicity (c Exosomes, antigen-presenting cells, and cytocotic cells are provided. The nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, Tides, antigen-presenting cells and exosomes are useful in the treatment of cancer, particularly melanoma, e.g., cutaneous melanoma. Related diagnostic methods are also provided.

[0193] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:5. An exemplary fragment comprises or consists of SEQ ID NO: 18. Exemplary nucleic acids include or consist of SEQ ID NO: 27 or SEQ ID NO: 35. nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic (cytocotic) cells, antigens Binding polypeptides, antigen-presenting cells, and exosomes are provided. or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and and exosomes, which are useful in the treatment of cancer, particularly melanoma, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0194] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:6. Exemplary fragments include or consist of any one of SEQ ID NOs: 19, 20. An example includes or consists of SEQ ID NO: 39. Exemplary acids include or consist of SEQ ID NO: 28 or SEQ ID NO: 36. Nucleic acids (e.g. DNA or RNA), T cells, T cell populations, cytocotic cells, antigen binding Polypeptides, antigen-presenting cells, and exosomes are provided. RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and The xenosomes may be used in the treatment of cancer, particularly melanoma, such as cutaneous melanoma. Diagnostic methods for the same are also provided.

[0195] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:7. An exemplary fragment comprises or consists of SEQ ID NO: 21. Exemplary nucleic acids include or consist of SEQ ID NO: 29 or SEQ ID NO: 37. nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic (cytocotic) cells, antigens Binding polypeptides, antigen-presenting cells, and exosomes are provided. or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and The exosomes can be used to treat cancer, particularly melanoma, such as cutaneous melanoma. Related diagnostic methods are also provided.

[0196] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:8. An exemplary fragment comprises or consists of SEQ ID NO: 22. Exemplary nucleic acids include or consist of SEQ ID NO: 30 or SEQ ID NO: 38. nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides The nucleic acid (e.g., DNA or RNA), T cells, antigen-presenting cells, and exosomes are provided. cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and exosomal cells The drug is used to treat cancer, particularly melanoma, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided. [Example]

[0197] (Example) (Example 1 - CLT specific) The goal is to identify cancer-specific transcripts that consist entirely or partially of LTR elements. That was the case. First step: de novo assembly of comprehensive, cross-cancer transcriptomes To achieve this, we obtained data from The Cancer Genome Atlas (TCGA) Consortium, A wide variety of cancer types (32 cancer types (31 primary and 1 metastatic melanoma) each) were included. RNA sequences from 768 patient samples, representing 24 individually balanced samples (Table S1). The sex-balanced sequencing reads were used for genome-guided assembly. The samples (excluding sex-specific tissues) were used as adapters using cutadapt (v1.13) (Marcel M, 2014). 11, EMBnet J., 17:3) were used to quality (Q20) trim and length filter (reads were both Both are 35 or more nucleotide pairs), and khmer (v2.0) (Crusoe et al., 2015, F1000Res. , 4:900) and kmer normalization (k=20) with maximum and minimum depths of 200 and 3, respectively. The reads were analyzed using STAR (2.5.2b) with the same settings as those used for the entire TCGA. 38 and analyzed using Trinity (v2.2.0) (Trinity, Grabherr, MG et al., 2011, Nat. Biol. 2011). otechnol., 29:644-52), disabling the built-in in silico depth normalization. Genome-guided assembly was performed. Most of the assembly process was performed on a 32-core HPC node. The process was completed within 256GB RAM, and the failed process was re-run using a 1.5TB RAM node. The contigs were poly(A) trimmed (using trimpoly in SeqClean v110222) and entropy was calculated. Filtering (≥0.7) to remove low-quality and artificial contigs (bbduk in BBMap v36.2) For each cancer type, the original 24 samples were analyzed using Salmon (v0.8.2 or v0.9.2) (Patro, R., et al., Reference , 2017, Nat. Methods, 14:417-419) to clean the assembly. Contigs with expression levels <0.1 transcripts per million (TPM) were removed. The remaining ones were analyzed using GMAP(v161107) (Wu et al., 2005, Bioinf., 21:1859-1875). and mapped to GRCh38, with 85% or more identity over 85% or more of its length. Contigs that did not match were removed from the assembly. Finally, all cancer types were combined into an assembly. The assembly was flattened and analyzed using gffread (Cufflinks v2.2.1) (Trapnell et al., 2010, Nat. Biotech h., 28:511-515) to merge the longest continuous transcript. The process was specifically designed to allow for the evaluation of repetitive elements, allowing for the detection of monoexon transitions. Transcripts were retained but flagged. The completeness and quality of the transcript assemblies were verified using GENC. ODE v24basic and MiTranscriptome1 (Iyer et al., 2015, Nat. Genet., 47:199-208) A list of unique splice sites represented by GENCODE was compiled. The splice site is located within a two-nucleotide window in the transcriptome. This process resulted in 1,001,931 transcripts. We identified 771,006 spliced ​​and 230,925 monoexonic products. I got some results.

[0198] Separately, the assembled contigs were overlaid with genome repeat annotations. Transcripts containing LTR elements were identified. LTR and non-LTR elements were identified as previously described. The sequences were annotated as described in (Attig et al., 2017, Front. In Microbiol., 8:2489). Briefly, a hidden Markov model (HMM) representing known human repeat families was used ( Dfam 2.0 library v150923), RepeatMasker Open-3.0 (Smit, A., R. Hubley and P. Green GRCh38 was annotated using the literature (http: / / www.repeatmasker.org, 1996-2010). and constructed using nhmmer (Wheeler et al., 2013, Bioinform., 29:2487-2489). HMM-based Scanning was performed using a BLAST-based method (Hubley et al., 2016, Nuc. Acid. Res., 44:81-89). RepeatMasker improves annotation accuracy compared to other methods. RepeatMasker separates LTR and internal regions. To annotate, the tabular output is parsed to find adjacent rows for the same element. Annotations were merged. In this process, one or more complete or partial LTR elements were merged. We obtained 181,967 transcripts, including the nucleotide sequence.

[0199] Salmon was used to estimate transcripts per million (TPM) abundance for all transcripts and within each cancer type. Expression of Th was analyzed in 811 healthy tissue samples (from TCGA when available, otherwise from GTEx (Th e Genotype-Tissue Expression Consortium, 2015, Science, 348:648-60) Expression was compared to that in tissue-matched healthy controls (for all cancer types). A gene was considered expressed in the cancer if it was detected at >1 TPM in any sample. and were considered cancer-specific if the following criteria were met: (i) among 24 samples of each cancer type; (ii) expressed at less than 10 TPM in more than 90% of all healthy tissue samples (iii) expressed in the target cancer type at a level greater than or equal to three times the median expression in any control tissue type; and v) For the targeted cancer type, incidence is greater than or equal to 3 times the 90th percentile of each available healthy tissue. Appear.

[0200] The list of cancer-specific transcripts was then analyzed for transcripts containing complete or partial LTR elements. The list of transcripts was crossed with the target transcripts to create a list of 5,923 transcripts that met all criteria (cancer (Termed CLT for specific LTR element-spanning transcript).

[0201] Further curation was performed on 403 CLTs specifically expressed in melanoma, and misassembly was identified. Contigs that may have been cross-referenced and those corresponding to cellular gene assemblies were excluded. Additional manual evaluation was performed to determine whether the splicing patterns were distinct. Supported by original RNA sequencing reads from cancers determined to be differentially expressed The CLT was further triaged to determine whether it was in any of the normal GTEx tissues. Those with median expression greater than 1 TPM were discarded. Of 403 CLTs for cutaneous melanoma, 97 passed these filters.

[0202] Example 2 - Immunopeptidome Analysis Mass spectrometry (MS)-based immunopeptidome analysis identifies HLA molecules (HLAp) that bind to and are expressed on the cell surface. It is a powerful technique that allows the direct detection of specific peptides being displayed. This technology detects HLAp from biological samples such as cells or tissues by capturing anti-HLA antibodies. The isolated HLA molecules and bound peptides are then separated from each other. The isolated and eluted peptides were analyzed by nano-ultra-performance liquid chromatography-mass spectrometry (nUPLC-MS). (Freudenmann et al., 2018, Immunology 154(3):331-345). Specific peptides with defined mass-to-charge ratios (m / z) are selected, isolated, fragmented, and then analyzed by 2 The fragment ions are then subjected to a second mass spectrometry (MS / MS) to obtain the m / z values. Spectra (MS / MS) were collated to identify selected peptides that generated the detected fragment ions. The amino acid sequence of the protein can be accurately identified.

[0203] MS / MS spectrum interpretation and subsequent peptide sequence identification were performed using experimental data and reference data. The theoretical spectra generated from the peptide sequences contained in the database matched well with the All known transcriptomes, or even entire genomes, are dependent on MS data using a predefined list of corresponding open reading frames (ORFs). Although it is possible to search for data (Nesvizhskii et al., 2014, Nat. Methods 11:1114), -1125), matching these ultra-large sequence databases leads to very high false discovery rates (FDR). Furthermore, technical issues (e.g., leucine leucine mass = isoleucine mass), and theoretical issues (e.g., peptide splicing (Liepe et al. (2016, Science 354(6310):354-358)) is a known transcriptome or genome. Increase the limits associated with the use of very large databases, such as those created from entire systems. Thus, in effect, it refers to a well-defined set of possible polypeptide sequences. Without this, it is extremely difficult to perform accurate immunopeptidome analysis and identify novel antigens. (Li et al., 2016, BMC Genomics 17(Suppl 13):1031).

[0204] In this way, we identified 10 residues derived from the 97 cutaneous melanoma CLTs in Example 1. A database of all the predicted polypeptide sequences (ORFs) was constructed. , 2,269 ORFs were obtained, ranging in length from 10 to 207 amino acids.

[0205] Bassani-Sternberg et al. analyzed HLA-binding peptide samples derived from 25 cutaneous melanoma patients. The MS / MS data collected from the pool were compared with polypeptides reported for the entire human proteome. The DNA sequence was matched against the DNA sequence (Bassani-Sternberg et al., 2016, Nature Commun., 7:1340 4;Database link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894). These analyses revealed tens of thousands of peptides that matched known human proteins. As expected, these peptides included PRAME, MAGEA3, and TRPM1 (melastatin). These included peptides found within multiple tumor-associated antigens (TAAs).

[0206] We obtained frozen tumor tissue from two patients diagnosed with melanoma. 0.6–1 g of sample was homogenized, the lysate was centrifuged at high speed, and the clarified lysate was collected. Protein A isoform covalently linked to an anti-human HLA class I monoclonal antibody (W6 / 32) The mixture was incubated overnight at 4°C and the HLA class I molecules were isolated. Improved antibody binding (Ternette et al., 2018 Proteomics 18, 1700465). The bound peptides were eluted from the antibody using 10% acetic acid, and then the peptides were loaded onto a reversed-phase column. It was separated from other high molecular weight components using chromatography (Ternette et al., 2018). The purified eluted peptides were subjected to nUPLC-MS to identify specific peptides with defined charge-to-mass ratios (m / z). Peptides are selected, isolated, fragmented in a mass spectrometer, and subjected to a second mass analysis (MS / MS). The m / z of the resulting fragment ions was determined (Ternette et al., 2018), and these tumors An MS / MS dataset corresponding to the immunopeptidome of each sample was generated.

[0207] By applying detailed knowledge of immunopeptidome assessment, we identified 25 melanomas. PXD004894 HLA class I dataset from rhesus mammary tumor patients (Bassani-Sternberg et al., 2016) ) and 10 melanomas generated by the inventors containing CLT-derived ORFs. The spectra of tumor HLA class I datasets were collated, and these CLT-derived ORFs were identified by PEAKS ( Human protein was analyzed using the NIH (trademark) software (v8.5 and vX, Bioinformatics Solutions Inc.). All polypeptide sequences found in the UniProt genome were aligned to identify those longer than 9 AA. The majority of class I HLA-binding peptides found in cells are constitutively expressed. Because they are derived from proteins, simultaneous matching of these databases with the UniProt proteome is , to confirm that our assignment of CLT ORF sequences to MS / MS spectra was correct. PEAKS software, like other MS / MS matching software, The assignment is quantified by assigning a significance probability (p-value) to the assignment (-10lgP; see Table 1).

[0208] The results of these studies identified over 50 individual peptides, which were Tumor samples from 25 patients examined by Nberg et al. and our dataset It bound to HLA class I molecules immunoprecipitated from two melanoma patient samples in the study. These correspond to the amino acid sequences of the CLT-derived ORFs and are present in the known human proteome (UniProt). The sequences did not correspond to the polypeptide sequences.

[0209] Further manual evaluation of peptide spectra assigned by PEAKS software The spectra were then assigned to peptides mapped to eight CLT-derived ORFs. These were used to confirm the identity of the CLT antigens, which were then defined as CLT antigens (Table 1; SEQ ID NOS: 1 to 8).

[0210] The detection of these peptides that bind to HLA class I molecules was initially based on the eight ORFs from which they originated. In melanoma tissue, it is translated into This confirms that they are presented to the immune system as a complex with class I molecules. Figures 1-14 and 29-31 show the characteristics of the peptides found within the T antigen. The top panel of each figure shows a representative MS / MS spectrum generated from the peptide. The peptide fragment profile is shown with standard MS / MS annotation (b: N-terminal fragment). ion; y: C-terminal fragment ion; -H2O: water loss; -NH3: ammonia loss; [2+]: doubly charged peptide ion; pre: unfragmented precursor peptide ion; a n -n: internal fragment ion), as shown above The most abundant fragment ion peaks (PEAKS software, our internal From the dataset or PRIDE database link: https: / / www.ebi.ac.uk / pride / archive Images extracted from the Bassani-Sternberg et al. dataset stored in / projects / PXD004894 The bottom panel of each figure shows the positions of the linear peptide sequence mapped to the fragment ions. Spectral rendering showing the positions of the high-101gPs assigned to the peptides in Table 1 is shown. These spectra are consistent with the scores we found in these analyses. It contains a number of fragments that exactly match the peptide sequences (SEQ ID NOS: 9-22).

[0211] All peptides longer than 9 AA that were detected binding to HLA class I in Table 1 were included in the NetMHCpa The data were evaluated using the NetMHCpan 4.0 prediction software (http: / / www.cbs.dtu.dk / services / NetMHCpan / ). The predicted strength of binding to HLA class I type A and B supertypes was determined. The results of the study showed that all 14 peptides (or 9-mers contained in each complete sequence) tested The results showed that the nucleotide sequences were predicted to bind to at least one of the supertypes (see Table 2). Among these, many sequences were highly correlated with specific types within the HLA class I supertypes examined. All detected peptides were predicted to bind with high confidence (low rank score %). The fact that they were predicted to bind to HLA types predicted to be present in the population is a major factor in their detection. Furthermore, the Basis Although only one report by Sternberg et al. Peptides identified within the pool or discovered in tumor samples from our dataset All of the genotypes were linked to one of the HLA types reported for the patient by NetMHCpan 4.0. It was predicted that they would match.

[0212] Further assignment of tumor tissue-derived MS spectra to the peptide sequences discovered by the inventors To provide certainty, peptides with these discovered sequences were synthesized and compared with those in the original study. Using the same conditions as those applied to tumor samples (Bassani-Sternberg et al., 2016, Nature Commun., 7: 13404; our database), nUPLC-MS 2 Selected The spectra of the peptides are compared in Figures 15 to 28. In each figure, the upper spectrum is the Pride data. Bass (Bassani-Sternberg et al., 2016, Nature Commun., 7: 13404; database link) Link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894 or our data The lower spectrum corresponds to a tumor sample (from the database), and the lower spectrum corresponds to a synthetically prepared The selected m / z values ​​of the detected ion fragments correspond to the peptides. These figures show the exact alignment of the fragments. The experimentally determined m / z values ​​between tumor-derived and synthetic peptide-derived fragment ions were clearly (Slight differences are within the m / z tolerance range of <0.05 Daltons), and each spectrum from tumor tissue Confirm the authenticity of the assignments to CLT-encoded peptides in the vector.

[0213] In summary, the data presented in Tables 1 and 2, Figures 1-14 and 29-31, and Figures 15-28 Highly potent support for translation, processing, and presentation of the corresponding CLT antigens in tumor patients. Provides a route.

[0214] To further confirm the cancer specificity of these CLTs, we performed a multi-detector cytogenetic analysis of 37 normal tissue samples ( Ten normal skin, nine normal lung, and 18 normal breast tissues were processed and subjected to immunopeptidome analysis. The inventors used the HLA class I dataset from these normal tissue samples. The spectra were collated to identify all possible peptides derived from the polypeptide sequences of CLT antigens 1 to 8. These peptides derived from CLT antigens were analyzed in a set of normal tissue samples. These CLTs were not detected in the tumor-specific expression group (Table 3), further confirming that these CLTs exhibit cancer-specific expression.

[0215] In summary, the identification of peptides in the immunopeptidome derived from predicted ORFs is We have demonstrated that CLT is translated into polypeptides (SEQ ID NOS: 1 to 8; referred to as CLT antigens) in tumor tissues. These polypeptides are then processed by the cellular immune surveillance machinery and The component peptide is loaded onto HLA class I molecules, resulting in peptide / HLA class I complexes. T cells that recognize the These CLT antigens and their fragments are expressed in melanomas of patients whose tumors express these antigens. It is anticipated that these compounds will be useful in a variety of therapeutic modalities for the treatment of cancer.

[0216] Table 1: Peptides identified by immunopeptidome analysis of melanoma tumor samples List and cross-reference with CLT antigen names and sequence numbers) [Table 1] 1 : HLA class I peptides identified by mass spectrometry. 2 Bassani-Sternberg et al., 2016, Nature Comm., 7: 13404. Kit base (2MT3, 2MT4). 3 : Calculated peptide mass. 4 :PEAKS™ program - 101gP value is the peak for which a detection spectrum greater than 1 is obtained. The peptides with the highest match to the peptide / patient are shown. 5 : Number of spectra in which the peptide was detected. 6 : deviation between observed and calculated mass; the selected ppm value is therefore greater than 1. The peptides from which the following can be obtained are shown.

[0217] Table 2: Mass spectrometry-identified peptides (length ≥ 9 residues) of 18 HLA class I supertypes Type alleles (HLA-A0101, HLA-A0201, HLA-A0301, HLA-A1101, HLA-A2402, HLA-A2501 , HLA-A2601, HLA-A6801, HLA-B0702, HLA-B0801, HLA-B1501, HLA-B1801, HLA-B2705, H Predicted NetMHCpan4.0 for HLA-B3501, HLA-B3503, HLA-B4001, HLA-B4002, HLA-B5101 binding and cross-reference with CLT antigen name and sequence number) [Table 2] 1: Rank score ≦ 2.0% predicted to bind to the matched HLA class I supertype Ta. 2 : The number of 18 HLA class I supertypes predicted to bind with a rank score of ≦2.0% ( all joins). 3 : The number of 18 HLA class I supertypes predicted to bind with a rank score of ≦0.5% ( strong bond). 4 Bassani-Sternberg et al., 2016, Nature Comm., 7: 13404.

[0218] Table 3: Number of peptides derived from CLT antigens 1 to 8 in the normal tissue sample set. [Table 3]

[0219] The results presented in Examples 1 and 2 herein, in whole or in part, were supported by The Cancer Genome Atlas (TCGA) research. Research Network (http: / / cancergenome.nih.gov / ) and the Genotype-Tissue Expression (GTEx) Project Funds from the Office of the Director of the National Institutes of Health (NIH) Common Fund, as well as from NCI, NHGRI, NHLBI, NIDA, NIMH, and This work is based on data generated by the National Institutes of Health (NIH) and the National Institutes of Infectious Diseases (NINDS).

[0220] Example 3 - Assay to demonstrate T cell specificity for CLT antigens in melanoma patients (a) Staining of reactive T cells with CLT antigen peptide pentamer The presence and activity of circulating CD8 T cells specific for CLT antigens in melanoma patients is related to HLA class I / Using peptide pentamer ("pentamer") staining and / or in vitro killing assays Therefore, it can be found using the methods described in Examples 1 and 2 (Tables 1-3, Figures 1-31). Application of these techniques to selected CLT antigens will allow for the identification of therapeutically relevant T cell responses to CLT antigens in cancer patients. Can be used to indicate the existence of an answer.

[0221] For these studies, CD8 T cells isolated from patients' blood were cultured using various culture methods, e.g. For example, anti-CD3 and anti-CD28 coated microbeads and interleukin-2 were used. The expanded cells then use the CLT peptide pentamer to target their T cell receptors. It can be stained for specific CLT antigen reactivity, which is located in the peptide-binding groove of the HLA molecule. It consists of a pentamer of HLA class I molecules that bind to related CLT antigenic peptides within The binding is specific to the coiled-coil multimerization domain of the pentameric structure, phycoerythrocytosis. The antibody fragments conjugated with phospholipid or allophycocyanin are used for detection. In addition to this pentamer staining, the memory marker CD45RO and the lysosomal release marker CD45R were also stained. Surface markers such as CD107a can be used to identify specific pentamers. Positive binding to surface markers indicates the number and state (memory) of the pentamer-reactive T cell population. It can be used to infer both the naive and the stem (vs. naive / stem).

[0222] Pentamer-stained cells were also sorted and purified using a fluorescence-activated cell sorter (FACS). The selected cells may then be tested in an in vitro killing assay to determine whether they can kill target cells. These assays can further test for the ability of CD8 T cell populations and , containing a fluorescently labeled target cell population. In this case, the CD8 population is a CLT antigen-specific cell or CD 8 T cells that are pentamer-selected and known to induce potent killing responses, such as Mart-1. The target cells in these studies are specific for the positive control antigens used. Released HLA-A * T2 cells expressing HLA-A 02, peptide-pulsed, * 02, 03 or B * C1R cells transfected with 07, previously shown to express the CLT / CLT antigen melanoma cell lines, or patient tumor cells or CLT open reading frames The death of target cells can be achieved by the action of 7AAD. Thus, CD8 T cell-mediated apoptosis leads to the target When target cells are killed, they acquire red fluorescence. Therefore, pentamer-selected CLT antigens The application of such killing assays to specific CD8 T cells has been demonstrated in melanoma patients or healthy donor T cells. Can be used to demonstrate the cytotoxic activity of CLT antigen-specific T cells in ex vivo cell cultures .

[0223] Figure 32 shows the CLT antigen 6-derived peptide (peptide [ka] HLA pentamer staining of healthy donor CD8 T cells in NIH followed by fluorescent activation of pentamer-positive cells Activated cell sorting was performed using anti-CD3 and anti-CD28 coated beads and IL-2 for 14 days. The right panel shows the results of proliferation of these CD8+ T cells. Although the antigen-specific killing activity was very weak against A2 target cells, the open reading frame of CLT antigen 6 CaSki cells transfected with the IL-11 cloning frame showed effective antigen-specific killing. The negative control for this in vitro killing assay was an irrelevant control without peptide. T2 cells and untransfected CaSki cells are included.

[0224] (b) HERVfest analysis of T cell specificity in melanoma patients The functional expansion (fest) of specific T cells is a promising strategy to target tumors in patients who respond to checkpoint blockade therapy. "Mutation-associated neoantigens" found in tumor cells ( m utation- a ssociated n eo a ntigen: MANA) have been used to identify specific tumor-derived epitopes present in the genome (A Nagnostou et al., Cancer Discovery 2017; Le et al., Science 2017). Examples 1 and 2 Applying this technique to CLT antigens discovered using the methods described in (Tables 1-3, Figures 1-3). and can confirm the presence of treatment-related T cell responses against CLT antigens in cancer patients.

[0225] Other assays to identify epitope-specific T cells in immunized subjects (e.g., Similar to ELISA (e.g., ELISPOT), the "fest" technique involves the use of antigen-presenting cells and suitable antigenic peptides. Expanding cognate T cells in ex vivo cultures unlocks their specificity. What differs from the immunological assays described above is the T cell receptor (TCR) present in these expanded cultures. Next-generation sequencing of TCR (T cell receptor) mRNA (specifically, TCRseq, which targets the TCR-Vβ CDR3 region) (Standard HLA binding algorithms are used to predict the patient's HLA type.) The aim is to detect specific TCRs propagated in cells cultured with a target peptide (selected for this purpose). In tumor tissue from the same patient, taken after successful checkpoint inhibitor therapy, The application of TCRseq then allows us to identify which TCRs are detected within ex vivo peptide-stimulated cultures. This could be used to determine whether R / T cells are also present in immunosuppressive sites of cancer. In the case of MANAfest, this method involves the production of antibodies in each patient's tumor and in T cells within the patient's tumor. Used to identify specific TCRs that recognize MHC-presented neoantigenic peptides detected even in and discovered by whole-exome sequencing of normal and tumor tissue from each patient. The aim is to identify functionally relevant neo-antigenic peptides among the thousands of possible variant peptides. (Le et al., Science 2017).

[0226] The application of MANAfest technology (Anagnostou et al., 2017, Cancer Discovery) to CLT antigens has The procedure is as follows: Step 1: Select an epitope that efficiently binds the selected HLA supertype. Identify peptides within the CLT antigen that are predicted to contain the tope. Step 2: Suitable patients? These PBMCs were selected and matched by HLA type with the peptide library selected in step 1. Step 4: PBMCs from these patients are separated into T cell and non-T cell fractions. The non-T cells are irradiated (to prevent proliferation) and then reintroduced into the patient's T cells, then split into 20-50 samples. Divide the T cell growth factors and individual CLT-specific synthetic peptides (selected in step 1) into 10-10 Grow for 4 days. Step 4: TCRseq (sequencing of epitope-specific TCR-Vβ CDR3 sequences) A quantification test (quantification of cognate T cells / TCRs) was performed for all wells to identify the cognate T cells / TCRs amplified in the presence of the test peptide. These TCR specificities were identified in unexpanded / expanded T cells. The TCR is determined by comparing the data obtained from this step using TCRseq. The data allows us to determine which peptides elicited an immune response in the patient. Step 5: TCRse q was performed on tumor samples to determine which of the specifically amplified TCRs were involved in checkpoint inhibition. We determined whether T cells with this TCR homed to the tumor in patients who responded to the therapy, and whether T cells with this TCR were involved in the This study provides evidence that may contribute to the efficacy of checkpoint inhibitor therapy.

[0227] Example 4 - High affinity T cells specific for CLT antigens are isolated from the T cell repertoire of normal subjects assay to show that it has not been deleted) ELISPOT assays demonstrate that CLT antigen-specific CD8 T cells are present within the normal T cell repertoire of healthy individuals. This can be used to demonstrate the presence of thymic antibodies in the naive and thymic tissues of these patients. The expression of cancer-specific CLT antigens was not eliminated by central tolerance. This type of ELISPOT assay involves several steps. Phase 1: CD8 T cells and CD14 monocytes can be isolated from the peripheral blood of normal blood donors. The cells are HLA typed to match the specific CD8 T antigens tested. Using a magnetically labeled antibody against the marker CD45RO, we identified naive and memory subtypes. Step 2: CD14 monocytes can be further subdivided into types. Step 3: The expanded CD8 T cells were pulsed with ATP for 3 hours and then co-cultured with CD8 T cells for 14 days. These cultures are isolated and restimulated overnight with fresh monocytes pulsed with peptide. Peptides include individual CLT antigen peptides, irrelevant control peptides, or infectious (e.g., CM) peptides. V, EBV, influenza, HCV) or self (e.g., MART-1) antigens. Restimulation may include peptides known to induce immune responses. This is done on plates coated with an antibody against IFNγ. After overnight activation, cells were plated with IFNγ. The IFNγ captured on the plate was then analyzed by further anti-IFNγ antibody and standard colorimetric analysis. The IFNγ-producing cells were detected with a dark stain in the areas where they were originally present on the plate. The data obtained from this assay include the number of spots, the size of the spots, and These include the median and median spot intensities, which indicate the frequency and cell density of IFNγ-producing T cells. is a measure of the amount of IFNγ per 1000 cells / mL. And further, a measure of the magnitude of the response to CLT antigens is The specific response, measured as the number of spots or median spot size, was calculated based on the specific peptide Stimulation index (SI) is the response to monocytes divided by the background response to monocytes containing no The evaluation criteria for stimulation intensity can be calculated by multiplying the stimulation index of the number of spots by the spot intensity. This method is used to calculate the response to CLT antigens and Comparison of responses to both the control and naive subjects revealed a potent increase in CLT antigen-reactive T cells. It can be shown that the CLT antigen-based immunogenic formulation contains a diverse repertoire of Table 4 shows significant CD8 A list of CLT antigen-derived peptides that induced T cell responses is provided, and the results are shown in Figures 33 to 38. The horizontal bars in the figure represent the average data. M+T indicates the negative control (monocytes and T cells) without peptide. CEF indicates positive control (a mixture of 23 CMV, EBV, and influenza peptides). Statistical significance was measured using one-way analysis of variance with Kruskal-Wallis test and repeated measures. Correction for the determination was performed using Dunn's correction. Figure 33 shows the results of the CLT antigen 1 (CLT in the figure) from a normal blood donor. Figure 34 shows a significant CD8 T cell response to HLA-A *02:01-restricted peptides derived from the positive control group. Significant activity against HLA-A*0301-restricted peptides derived from CLT antigen 1 (CLT001 in the figure) from normal blood donors. Figure 35 shows a CD8 T cell response derived from CLT antigen 2 (CLT002 ​​in the figure) from a normal blood donor. Figure 36 shows significant CD8 T cell responses to the LA-B*0702 restricted peptide. A significant CD8 T cell response to an HLA-A*0301-restricted peptide derived from CLT antigen 3 (CLT003 in the figure) was observed. Figure 37 shows the HLA-A*0301 restriction antibody from a normal blood donor, derived from CLT antigen 5 (CLT005 in the figure). Figure 38 shows a significant CD8 T cell response to the peptide. A significant CD8 T cell response to an HLA-A*0201-restricted peptide derived from CLT006 in the figure is shown.

[0228] Table 4: CLT antigen-derived CD8 T cell responses from HLA-matched normal blood donors (Peptide) [Table 4]

[0229] Example 5 - Validation assay in melanoma cells a) qRT-PCR validation of CLT expression in melanoma cell lines Quantitative real-time polymerase chain reaction (qRT-PCR) is a method for determining the number of nucleotides in a given biological sample. It is a widely used technique for determining the amount of specific transcripts present in extracted RNA. Specific nucleic acid primer sequences are designed for the transcript of interest and then subsequently purified. The intermer region is amplified through a series of thermal cycling reactions, and the intercalator color is Quantification was performed fluorescently using SYBR Green. Primer pairs were engineered against CLT and Assays were performed on RNA extracted from melanoma cell lines or primary patient tissue. Non-melanoma cell lines were used as negative controls. The melanoma cell lines used included COLO 8 29 (ATCC Reference No. CRL-1974), MeWo (ATCC Reference No. HTB-65), SH-4 (ATCC Reference No. CRL-7724) and and control cell lines HepG2 (hepatocellular carcinoma, ATCC reference no. HB-8065), Jurkat (T-cell leukemia, ATCC reference no. Patient-derived melanomas included MCF7 (adenocarcinoma, ATCC reference number HTB-22) and MCF7 (adenocarcinoma, ATCC reference number TIB152). Tissues were derived from six primary lesions and six metastatic sites, all of which were at least stage IIC. RNA was extracted from each sample, reverse transcribed to cDNA, and then analyzed using standard procedures. qRT-PCR analysis with SYBR Green detection according to standard techniques was performed on two regions of each CLT. The relative quantification (RQ) was calculated as follows: It was calculated as: RQ=2[Ct(reference)-Ct(target)] .

[0230] The results of these experiments are shown in Figure 39. Panel A shows the results of two primer sets (76+77 and 78+79). The results of the qRT-PCR assay using 12 melanoma tissue samples and 1 non-melanoma tissue sample were Different regions of CLT (SEQ ID NO: 24) encoding CLT antigen 2 on RNA extracted from laminoma cell lines Panel B shows the results of the PCR using two primer sets (44+45 and 46+47). The results of the qRT-PCR assay were obtained from 12 melanoma tissue samples and 1 non-melanoma tissue sample. Targeting different regions of CLT (SEQ ID NO: 25), which encodes CLT antigen 3 on RNA extracted from cell lines, Panel C shows the results of qRT-PCR using two primer sets (80–81 and 82–83). PCR assay results for 12 melanoma tissue samples and 1 non-melanoma cell line. Targeting different regions of CLT (SEQ ID NO: 28) encoding CLT antigen 6 on RNA extracted from These results demonstrate that melanoma tissue samples are more efficient than non-melanoma cell lines. The specific expression of CLTs was confirmed in RNA extracted from the two or more CLTs analyzed. These tissue samples showed little or no expression, whereas non-melanoma control cell lines showed no expression. was not detected.

[0231] b) RNAScope validation of CLT expression in melanoma cells in situ Transcript expression analysis by in situ hybridization (ISH) is performed using histopathological specimens. This allows visualization of the presence and expression level of a given transcript in a specific context. The assay also uses oligonucleotide probes specific for short strands of the desired RNA sequence. This involves recognizing native RNA molecules in situ, which can be achieved by using antibodies or enzymes. It is visualized by the signal generated by a combination of colorimetric reactions based on RNAScope is a recently developed in s with more advanced probe chemistries. It is a technology based on itu hybridization, ensuring the specificity of the signal generated. and enables highly sensitive single-molecule visualization of target transcripts (Wang et al., 2013). (2012 J Mol Diagn. 14(1):22-29). Positive staining of transcript molecules indicates the presence of Small red dots appear, and multiple dots indicate the presence of multiple transcripts.

[0232] RNAScope probes were designed against CLT and 12 formalin-fixed, paraffin-embedded Sections of cutaneous melanoma tumor cores were assayed. Expression signals were scored as follows: and performed on a representative image from each core as follows: The assessment of % of cells with positive staining for the CLT probe was rounded up to the nearest 10. The estimated per cell level of expression across a given section is: 0 = no staining · 1 = 1-2 dots per cell 2 = 2-6 dots per cell 3 = 6-10 dots per cell · 4 = more than 10 dots per cell.

[0233] Expression of each CLT was detected in tumor cores from multiple different patients, and each independently correlated with tumor-derived R Validate CLT findings from NAseq data and their expression in tumor tissue across a given sample The homogeneity of the CLT was confirmed and the presence of at least one CLT in each patient core analyzed was also confirmed. The results were as follows (Table 5).

[0234] Table 5 - RNAScope scoring within melanoma patient tissue cores [Table 5]

[0235] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprises" " and variations such as "included" and "comprises" include the listed integers, steps, integers, etc. includes any integer, step, group of integers or group of steps, but excludes any other integer, step, group of integers or group of steps. It will be understood to mean that the

[0236] All patents, patent applications and literature references cited in this specification are hereby incorporated by reference. No. 6,299,499, filed on Dec. 1, 2003, which is hereby incorporated in its entirety. The present invention relates to the preferred and more preferred groups and the preferred and more preferred groups listed above. It is intended to include all suitable groups and all combinations of groups of embodiments.

[0237] (Sequence Listing) [ka] TIFF2025124655000008.tif240170TIFF2025124655000009.tif242170TIFF2025124655000010.tif247170TIFF2025124655000011.tif247170TIFF2025124655000012.tif247170TIFF2025124655000013.tif247170TIFF2025124655000014.tif240170TIFF2025124655000015.tif247170TIFF2025124655000016.tif248170TIFF2025124655000017.tif248170TIFF2025124655000018.tif238170TIFF2025124655000019.tif161170

Claims

[Claim 1] The novel products, methods and processes substantially as herein described.

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