Novel cancer antigens and methods

Abstract

Specifically disclosed are polypeptides and nucleic acids encoding the polypeptides, which are useful in the treatment, prevention and diagnosis of cancer, particularly melanoma, especially cutaneous melanoma and uveal melanoma.

Description

[Technical Field]

[0001] (Field of Invention) The present invention relates to antigenic polypeptides and corresponding polynucleotides for use in the treatment or prevention of cancer, particularly for use in the treatment or prevention of melanoma (e.g., cutaneous melanoma or uveal melanoma). The present invention further relates to pharmaceutical compositions and immunogenic compositions comprising, in particular, nucleic acids and polypeptides, immune cells loaded with and / or stimulated by the polypeptides and polynucleotides, antibodies specific to the polypeptides, and cells (of self or other origin) genetically engineered with molecules that recognize the polypeptides. [Background technology]

[0002] (Background of the invention) As part of normal immune surveillance against pathogenic microorganisms, all cells degrade intracellular proteins to produce peptides, which are loaded onto major histocompatibility complex (MHC) class I molecules, and these molecules are expressed on the surface of all cells. Most of these peptides, originating from host cells, 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 encode T cell receptors (TCRs) that firmly bind the MHC I-peptide complex. This proliferated T cell population can produce effector CD8+ T cells (including cytotoxic T lymphocytes, CTLs) that can eliminate foreign antigen-tagged cells, and further, memory CD8+ T cells that can be re-amplified if foreign antigen-tagged cells reappear later in the animal's lifetime.

[0003] The expression of MHC class II molecules is typically limited to professional antigen-presenting cells (APCs) such as dendritic cells (DCs), and MHC class II molecules are usually loaded along with peptides taken into the cell from the extracellular environment. In the presence of various factors, including T cell adhesion molecules (CD54, CD48) and costimulatory molecules (CD40, CD80, CD86), when a complementary TCR from a naive CD4+ T cell binds to the MHCII-peptide complex, effector cells of the CD4+ T cell (e.g., T cells) H 1. T H 2, T H 17, T FH , T reg Maturation into cells is induced. These effector CD4+ T cells can promote the differentiation of B cells into antibody-secreting plasma cells, as well as the differentiation of antigen-specific CD8+ CTLs, thereby helping to induce an adaptive immune response to foreign antigens, including both short-term effector function and long-term immunological memory. DCs can carry out the cross-presentation process of peptide antigens by delivering exogenous antigens (such as peptides or proteins released from pathogens or tumor cells) onto their MHC1 molecules, contributing to the generation of immunological memory by providing an alternative pathway for stimulating the proliferation of naive CD8+ T cells.

[0004] Immunological memory (specifically antigen-specific B cells / antibodies and antigen-specific CTLs) plays a crucial role in controlling microbial infections, and has been utilized in the development of numerous vaccines to prevent diseases caused by important pathogenic microorganisms. Immunological memory is also known to play an important role in controlling tumorigenesis, yet few effective cancer vaccines have been developed.

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

[0006] Current therapies for cutaneous melanoma are diverse and largely dependent on the tumor's location 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, radiotherapy, or chemotherapy. Immune checkpoint inhibitor strategies, which involve the use of antibodies targeting negative immunomodulatory factors such as PD-1 / PD-L1 and CTLA4, have recently revolutionized the treatment of various malignancies, including melanoma (Ribas, A. and Wolchok, JD, (2018) Science, 359:1350-1355). The exceptional value of checkpoint inhibitor therapy, and the well-known correlation between its clinical benefits and the patient's adaptive immune response to their own cancer antigens (particularly T-cell-based immune responses), have stimulated the search for effective cancer vaccines, vaccine modalities, and cancer vaccine antigens.

[0007] Human endogenous retroviruses (HERVs) are relics of exogenous infectious retroviruses that have been incorporated into the ancestral germline. HERVs belong to a group of endogenous retroelements characterized by the presence of long terminal repeats (LTRs) adjacent to the viral genome. This group also includes apparent LTR retrotransposons (MaLRs) in mammals and is therefore collectively known as LTR elements (hereinafter referred to as ERVs to encompass all LTR elements). ERVs constitute a significant portion (8%) of the mammalian genome and can be grouped into approximately 100 families based on sequence homology. Many ERV sequences encode defective proviruses, which share a prototype retroviral genome structure consisting of gag, pro, pol, and env genes adjacent to the LTR. Some intact ERV ORFs produce retroviral proteins that share characteristics with proteins encoded by exogenous infectious retroviruses such as HIV-1. Such proteins may act as antigens that induce a strong immune response (Hurst and Magiorkinis, 2015, J. Gen. Virol 96:1207-1218), suggesting that polypeptides encoded by ERVs can evade the selection processes of T cell and B cell receptors, as well as central and peripheral tolerance. Immune reactivity to ERV products can occur spontaneously in infections and cancers, and ERV products have been linked to the causes of several autoimmune diseases (Kassiotis and Stoye, 2016, Nat. Rev. Immunol. 16:207-219).

[0008] Due to the accumulation of mutations and recombination events during evolution, most ERVs lose some or all of their functional open reading frames, resulting in the loss of their ability to produce infectious viruses. However, these ERV elements are maintained within germline DNA, just like other genes, and have the potential to produce proteins from at least some of those genes. In fact, proteins encoded by HERVs have been detected in various human cancers. For example, the splice variants Rec and Np9 of the HERV-K env gene 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 also been observed in cancers such as prostate cancer compared to healthy tissue (Wang-Johanning, 2003, Cancer 98:187-197; Andersson et al., 1998, Int. J. Oncol, 12:309-313). Furthermore, overexpression of HERV-E and HERV-H has been shown to be immunosuppressive, and these may also contribute to cancer progression (Mangeney et al., 2001, J. Gen. Virol. 82:2515-2518). However, the actual mechanisms by which HERVs can contribute to cancer progression or pathogenicity remain unclear.

[0009] In addition to deregulating the expression of surrounding, adjacent host genes, the activation and translocation of ERV regulatory elements to new genomic sites may lead to the generation of novel transcripts, some of which may be carcinogenic (Babaian and Mager, Mob. DNA, 2016; Lock et al., PNAS, 2014, 111:3534-3543).

[0010] A wide range of vaccine modalities are known. One well-described approach involves directly delivering antigenic polypeptides to a target, aiming to enhance the immune response (including B-cell and T-cell responses) and stimulate immunological memory. Alternatively, polynucleotides may be administered to the target via a vector such that the immunogenic polypeptide encoded by the polynucleotide is expressed in vivo. The use of viral vectors, such as adenovirus vectors, has been well explored for antigen delivery in both prophylactic vaccination and therapeutic treatment strategies for cancer (Wold et al., Current Gene Therapy, 2013, “Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy” 13:421-433). Immunogenic peptides, polypeptides, or encoded polynucleotides can also be used to load patient-derived antigen-presenting cells (APCs), which can then be injected into the target as vaccines to elicit a therapeutic or prophylactic immune response. An example of this approach is Provenge, currently the only anti-cancer vaccine approved by the FDA.

[0011] Cancer antigens can also be used for the treatment and prevention of cancer, by creating various non-vaccine therapeutic modalities using them. These therapies are classified into two distinct classes: 1) antigen-conjugated biologics and 2) adoptive cell therapies.

[0012] Antigen-binding biologics typically consist of polyvalent polypeptides engineered to recognize antigen-modified cancer cells and promote their destruction. The antigen-binding component of these biologics may consist of TCR-based biologics, including but not limited to TCRs, high-affinity TCRs, and TCR mimics, produced by various technologies (including those based on monoclonal antibody technology). The cytolytic component of these types of polyvalent biologics may consist of cytotoxic chemicals, biotoxins, and targeting and / or immunostimulant motifs that promote the targeting and activation of immune cells, all of which promote the therapeutic destruction of tumor cells.

[0013] Adoptive cell therapy may be based on the patient's own T cells, which are isolated and stimulated in vitro with a vaccine antigen preparation (cultivated with T cells in the presence or absence of other factors, including cellular and acellular components) (Yossef et al., JCI Insight. October 2018, 4;3(19) pii:122467. doi:10.1172 / jci.insight.122467). Alternatively, adoptive cell therapy may be based on cells (including patient-derived or non-patient-derived cells) that have been programmed to express antigen-binding polypeptides that recognize cancer antigens. These antigen-binding polypeptides belong to the same class as those described above for antigen-binding biologics. Therefore, lymphocytes (autologous or non-autologous) genetically engineered to express cancer antigen-binding polypeptides can be administered to the patient as adoptive cell therapy to treat their cancer.

[0014] The use of ERV-derived antigens to enhance an effective immune response against cancer has shown promising results in mouse models of cancer, promoting tumor regression and leading to more favorable prognoses (Kershaw et al., 2001, Cancer Res. 61:7920-7924; Slansky et al., 2000, Immunity 13:529-538). Therefore, despite some limitations in research progress due to the strict limitations of identified tumor-specific ERV antigens, immunotherapy trials centered on HERV antigens are being investigated in humans (Sacha et al., 2012, J.Immunol 189:1467-1479).

[0015] WO 2005 / 099750 identifies anchor sequences for existing vaccines against infectious pathogens that commonly enhance cross-reactive immune responses to the HERV-K Mel tumor antigen, providing protection against melanoma. WO 00 / 06598 relates to the identification of the HERV-AVL3-B tumor-associated gene, which is preferentially expressed in melanoma, and to methods and products for diagnosing and treating conditions characterized by the expression of this gene. WO 2006 / 119527 provides antigenic polypeptides derived from melanoma-associated endogenous retroviruses (MERVs), and their use for the detection and diagnosis of melanoma and for prognostic diagnosis of the disease. The use of antigenic polypeptides as anti-cancer vaccines is also disclosed.

[0016] WO 2007 / 137279 discloses, for example, methods and compositions for detecting, preventing, and treating HERV-K+ cancer using HERV-K+ binding antibodies for preventing or inhibiting cancer cell proliferation. WO 2006 / 103562 discloses a method for treating or preventing cancers in which the immunosuppressive Np9 protein from the env gene of HERV-K is expressed. The invention also relates to a pharmaceutical composition comprising a nucleic acid or antibody capable of inhibiting the activity of the protein, or an immunogen or vaccine composition capable of inducing an immune response to the protein. WO 2007 / 109583 provides compositions and methods for preventing or treating neobiotic diseases in mammals by providing compositions comprising an enriched population of immune cells that react to HERV-E antigens on tumor cells.

[0017] Humer J et al., 2006, Canc. Res., 66:1658-63, identified melanoma markers derived from endogenous retroviruses associated with melanoma. There is a need to identify further HERV-related antigen sequences that can be used for immunotherapy of cancer, particularly melanoma, and even cutaneous and uveal melanoma. [Overview of the Initiative]

[0018] (Summary of the invention) The inventors have surprisingly discovered certain RNA transcripts that contain or are derived from genomic sequences adjacent to LTR elements and are found at high levels in cutaneous melanoma cells but are undetectable or at very low levels in normal, healthy tissue (see Example 1). Such transcripts are referred to herein as cancer-specific LTR-element spanning transcripts (CLTs). Furthermore, the inventors have shown that a specific, potential polypeptide sequence (i.e., an open reading frame (ORF)) encoded by one of these CLTs is translated within cancer cells, processed by components of the antigen processing apparatus, and then presented on the cell surface found in tumor tissue by binding to Class I and Class II major histocompatibility complexes (MHC class I and MHC class II) and Class I and Class II human leukocyte antigens (HLA class I and HLA class II) molecules (see Example 2). These findings indicate that this polypeptide (referred to herein as the CLT antigen) is substantially antigenic. Therefore, it is expected that the presentation of this CLT antigen on cancer cells will make these cells more susceptible to elimination by T cells possessing a congeneral T cell receptor (TCR) for this CLT antigen, and that CLT antigen-based vaccination methods / regimes that amplify these congeneral TCR-possessing T cells will evoke an immune response against cancer cells (and tumors including them), particularly melanoma, and especially cutaneous melanoma. Indeed, T cells from melanoma subjects react to the CLT antigen-derived peptide disclosed herein (see Example 3). We confirmed that CLT antigen-specific T cells are not removed from the normal subject T cell repertoire by central tolerance (see Example 4). Finally, qRT-PCR studies confirmed that CLT is specifically expressed in RNA extracted from melanoma cell lines compared to non-melanoma cell lines (see Example 5).

[0019] The inventors also surprisingly discovered that CLT encoding certain CLT antigens is not only overexpressed in cutaneous melanoma but also in uveal melanoma. The CLT antigen polypeptide sequence encoded by this CLT is expected to elicit an immune response against uveal melanoma cells and tumors containing the same.

[0020] The CLT and CLT antigens of interest in the present invention are not canonical sequences that can be easily derived from known tumor genomic sequences found in the Cancer Genome Atlas. This CLT is a transcript resulting from complex transcriptional and splicing events driven by an ERV-derived transcriptional regulatory sequence. Since this CLT is expressed at high levels and the CLT antigen polypeptide sequence is not a normal human protein sequence, it is expected to be suitable for therapeutic use in the context of cancer immunotherapy as it could elicit a strong and specific immune response.

[0021] This CLT antigen, found in highly expressed transcripts that characterize tumor cells, was not known to exist in the human body and produce a protein product prior to the present invention and can be used in several forms. First, the CLT antigen polypeptide of the present invention can be directly delivered to a subject as a vaccine that elicits a therapeutic or prophylactic immune response against tumor cells. Second, the nucleic acids of the present invention can be codon-optimized to enhance the expression of the CLT antigen they encode and can be directly administered, or alternatively inserted into a vector for in vivo delivery, as a vaccine that elicits a therapeutic or prophylactic immune response against tumor cells to produce the encoded protein product in the subject. Third, the polynucleotides and / or polypeptides of the present invention can be used to load patient-derived antigen-presenting cells (APCs), which can then be injected into the subject as a vaccine that elicits a therapeutic or prophylactic immune response against tumor cells. Fourth, the polynucleotides and / or polypeptides of the present invention can be used to stimulate the subject's T cells ex vivo, generating a stimulated T cell preparation that can be administered to the subject as a cancer treatment. Fifth, biological molecules such as T cell receptors (TCRs) or TCR mimics, which recognize the CLT antigen complexed with MHC I molecules and are further modified to kill (or promote the killing of) cancer cells, may be administered to the subject as a cancer treatment. Sixth, chimeric versions of biological molecules that recognize the CLT antigen complexed with MHC cells may be introduced into (self-derived or non-self-derived) T cells, and the cells after introduction may be administered to the subject as a cancer treatment. These and other applications are described in more detail below.

[0022] Therefore, the present invention particularly provides: (a) the sequence of SEQ ID NO: 1; and (b) variants of the sequence of (a); and (c) immunogenic fragments of the sequence of (a), an isolated polypeptide comprising a sequence selected therefrom (hereinafter referred to as "the polypeptide of the present invention"). The present invention also provides a nucleic acid molecule that encodes the polypeptide of the present invention (hereinafter referred to as "the nucleic acid of the present invention").

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

[0024] (Drawing description) [Figure 1] Figure 1: Spectra of the peptide of SEQ ID NO: 2 obtained from a tumor sample of patient Mel-29. The upper panel shows the extracted MS / MS spectrum of the peptide obtained from the patient's tumor sample (with assigned fragment ions), and the lower panel shows a rendering of the spectrum, indicating the position of the linear peptide sequence mapped to the fragment ions. [Figure 2] Figure 2: Spectrum of the peptide of Sequence ID No. 2 obtained from a tumor sample of patient Mel-29. The figure shows the alignment of the native MS / MS spectrum of the peptide obtained from the patient's tumor sample to the native spectrum of the synthetic peptide corresponding to the same sequence. [Figure 3] Figure 3 shows the response of CD8 T cells from normal blood donors to the HLA-A*03:01-restricted peptide (SEQ ID NO: 6) from CLT antigen 1. [Figure 4] Figure 4 shows the results of a qRT-PCR assay evaluating the transcription of CLT (SEQ ID NO: 3), which encodes CLT antigen 1.

[0025] (Explanation of the array) Sequence ID 1 is the polypeptide sequence of CLT antigen 1. Sequence ID 2 is a peptide sequence derived from CLT antigen 1. Sequence ID 3 is the cDNA sequence of CLT encoding CLT antigen 1. Sequence ID 4 is a cDNA sequence that encodes CLT antigen 1. Sequence ID 5 is a peptide sequence derived from CLT antigen 1. Sequence ID 6 is a peptide sequence derived from CLT antigen 1. [Modes for carrying out the invention]

[0026] (Detailed description of the invention) (polypeptide) The terms "protein," "polypeptide," and "peptide" are used interchangeably herein and refer to any chain of peptide-linked amino acids, regardless of length, translational modification, or post-translational modification.

[0027] The term "amino acid" refers to any naturally occurring amino acid, or any amino acid analog or mimetic that functions in a manner similar to a naturally occurring amino acid. Naturally occurring amino acids are the 20 L-amino acids encoded by the genetic code, and later modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. The term "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a hydrogen atom, a carboxyl group, an amino group, and an α-carbon bonded to an R group, but has a modified R group or a modified peptide skeleton compared to a naturally occurring amino acid. Examples include homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, and norleucine. Amino acid mimetic refers to a chemical compound that has a structure different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid. Preferably, an amino acid is a naturally occurring amino acid or an amino acid analog, in particular a naturally occurring amino acid, and furthermore, one of the 20 L-amino acids encoded by the genetic code.

[0028] In this specification, amino acids may be represented by either a commonly known three-letter symbol or a single-letter symbol, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by a commonly accepted single-letter code.

[0029] Therefore, the present invention: (a) the sequence of sequence number 1; and, (b) Variants of the sequence in (a); and (c) Provides an isolated polypeptide containing a sequence selected from immunogenic fragments of the sequence of (a).

[0030] This invention also: (a) The sequence of Sequence ID No. 1 with the first methionine residue removed; and, (b) Variants of the sequence in (a); and (c) Provides an isolated polypeptide containing a sequence selected from immunogenic fragments of the sequence of (a).

[0031] Generally, variants of the polypeptide sequence of the present invention include sequences that have a high degree of sequence identity with respect to it. For example, the variant preferably has at least about 80% identity, more preferably at least about 85% identity, and most preferably at least about 90% identity (at least about 95%, at least about 98%, or at least about 99%, etc.) with respect to the full length of the reference sequence in question.

[0032] Preferably, the mutant is an immunogenic mutant. A mutant is considered to be an immunogenic mutant that elicits a response of at least 20%, preferably at least 50%, and especially at least 75% (at least 90%, etc.) of the activity of the reference sequence (i.e., the sequence from which the mutant is a mutant). This response is characterized, for example, in an in vitro restorative assay of PBMCs or whole blood using a polypeptide as an antigen (e.g., restorative over a period of 1 to 1 month, or 1 to 2 weeks, ranging from several hours to a maximum of 1 year, up to 6 months, etc.), by cell activation via lymphocyte proliferation (e.g., T cell proliferation), production of cytokines (e.g., IFN-γ) in the culture supernatant (measured by ELISA, etc.), or characterization of the T cell response by intracellular and extracellular staining (e.g., using antibodies specific to immunomarkers such as CD3, CD4, CD8, IL2, TNF-α, IFNg, type 1 IFN, CD40L, CD69, etc.) and subsequent flow cytometry analysis.

[0033] The mutants may be, for example, conservatively modified mutants. A "conservatively modified mutant" is one in which the modification results in amino acid substitutions at functionally similar amino acids, or residue substitutions / deletions / additions that do not substantially affect the biological function of the mutant. Typically, the biological function of such mutants would be to induce an immune response to melanoma, for example, cutaneous melanoma cancer antigens. Conservative substitution tables that provide functionally similar amino acids are well known in this field. Mutants may include homologs of polypeptides found in other species.

[0034] Variants of the polypeptide of the present invention may also contain numerous substitutions, such as conserved substitutions compared to the reference sequence (e.g., 1-25, 1-10, etc., particularly 1-5, and especially 1 amino acid residue may be modified). The number of substitutions, for example, the number of conserved substitutions, may be up to 20% of the number of residues in the reference sequence, for example, up to 10%, for example up to 5%, for example up to 1%. Generally, conserved substitutions will fall into one of the amino acid classifications specified below, but other substitutions may also be present in some cases, as long as they do not substantially affect the immunogenicity of the antigen. The following eight groups each contain amino acids that are usually conserved substitutions of each other.

[0035] 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, for example, Creighton's work, Proteins 1984).

[0036] Preferably, such substitutions do not alter the immunological structure of the epitope (for example, the substitutions do not occur within the epitope region mapped to the primary sequence), and therefore do not significantly affect the immunogenicity of the antigen.

[0037] Polypeptide variants also include those in which additional amino acids are inserted compared to the reference sequence, for example, such insertions may occur at positions 1 to 10 (e.g., positions 1 to 5, preferably at position 1 or 2, particularly at position 1), and the insertions may include, for example, the addition of 50 or fewer amino acids at each position (e.g., 20 or fewer, particularly 10 or fewer, and even 5 or fewer). Preferably, such insertions do not occur in the epitope region and therefore do not significantly affect the immunogenicity of the antigen. An example of an insertion is a short stretch of histidine residues (e.g., 2 to 6 residues) to aid in the expression and / or purification of the antigen of interest.

[0038] Polypeptide variants include those in which amino acids have been deleted compared to a reference sequence. For example, such deletions may occur at positions 1 to 10 (e.g., positions 1 to 5, preferably position 1 or 2, particularly position 1), and the deletions may include, for example, 50 or fewer amino acid deletions at each position (e.g., 20 or fewer, particularly 10 or fewer, and even 5 or fewer). Preferably, such deletions do not occur in epitope regions and therefore do not significantly affect the immunogenicity of the antigen.

[0039] Those skilled in the art will recognize that special protein variants may include substitutions, deletions, and additions (or any combination thereof). For example, substitutions / deletions / additions may enhance (or neutralize) binding to the desired patient's HLA molecule, and may increase (or maintain unchanged) immunogenicity.

[0040] The immunogenic fragment of the present invention will, depending on the length of the CLT antigen, typically contain 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), and particularly at least 50 consecutive amino acids, at least 100 consecutive amino acids (e.g., at least 200 consecutive amino acids), etc., from the full-length polypeptide sequence. Preferably, this immunogenic fragment will be at least 10%, for example at least 20%, for example at least 50%, for example at least 70%, or at least 80% of the length of the full-length polypeptide sequence.

[0041] An immunogenic fragment typically contains at least one epitope. The epitope may include B cell and T cell epitopes, and preferably the immunogenic fragment contains at least one T cell epitope, such as a CD4+ or CD8+ T cell epitope.

[0042] A T cell epitope is a short, continuous stretch of amino acids that is recognized by T cells (e.g., CD4+ or CD8+ T cells) when bound to an HLA molecule. Identification of T cell epitopes may be achieved by epitope mapping experiments, which are well known to those skilled in the art (see, for example, Paul, Fundamental Immunology, 3rd edition, 243-247 (1993); Beiβbarth et al., 2005, Bioinformatics, 21 (Augmented 1): i29-i37).

[0043] As a result of the decisive involvement of T cell responses in cancer, it is quite clear that a full-length polypeptide fragment of SEQ ID NO: 1 containing at least one T cell epitope may be immunogenic and contribute to immune defense.

[0044] In diverse, non-inbred populations such as humans, it will be understood that different HLA types mean that a particular epitope is not recognized by all members of the population. Consequently, to maximize the level of recognition and scale of the immune response to a given polypeptide, it is generally desirable that immunogenic fragments contain multiple epitopes from the full-length sequence (preferably all epitopes within the CLT antigen).

[0045] A specific fragment of the polypeptide of Sequence ID No. 1 that may be useful includes at least one CD8+ T cell epitope, preferably at least two CD8+ T cell epitopes, or even all CD8+ T cell epitopes, particularly those associated with multiple HLA class I alleles (e.g., those associated with 2, 3, 4, 5 or more alleles). A special fragment of the polypeptide of Sequence ID No. 1 that may be useful includes at least one CD4+ T cell epitope, preferably at least two CD4+ T cell epitopes, or even all CD4+ T cell epitopes (particularly those associated with multiple HLA class II alleles, e.g., those associated with 2, 3, 4, 5 or more alleles). However, those skilled in the art of vaccine design will be able to combine exogenous CD4+ T cell epitopes with the CD8+ T cell epitopes of the present invention to achieve a desired response to the CD8+ T cell epitopes of the present invention.

[0046] When individual fragments of a full-length polypeptide are used, such a fragment is considered immunogenic if it elicits a response that is at least 20%, preferably at least 50%, and especially at least 75% (at least 90%, etc.) of the activity of the reference sequence (i.e., the sequence from which the fragment is a single fragment). This response is characterized, for example, by activity in an in vitro restorative assay of PBMCs or whole blood using the polypeptide as an antigen (e.g., restorative stimulation over a period of 1 to 1 month, or 1 to 2 weeks, ranging from several hours to a maximum of 1 year, up to 6 months, etc.), cell activation via lymphocyte proliferation (e.g., T cell proliferation), production of cytokines (e.g., IFN-γ) in the culture supernatant (measured by ELISA, etc.), or by characterization of the T cell response by intracellular and extracellular staining (e.g., using antibodies specific to immunomarkers such as CD3, CD4, CD8, IL2, TNF-α, IFN-γ, type 1 IFN, CD40L, CD69, etc.) and subsequent flow cytometry analysis.

[0047] In some cases, multiple fragments of a full-length polypeptide (which may or may not overlap, and which may or may not span the entire full-length sequence) can be used to obtain a biological response equivalent to that of the full-length sequence itself. For example, a combination of at least two (3, 4, or 5, etc.) immunogenic fragments can provide at least 50%, preferably at least 75%, and even at least 90% of the activity of a reference sequence in an in vitro restimulation assay of PBMCs or whole blood (e.g., a T cell proliferation and / or IFN-γ production assay).

[0048] An example of an immunogenic fragment of the polypeptide of SEQ ID NO: 1, and therefore an example of the peptide of the present invention, is a polypeptide comprising or consisting of the sequence of SEQ ID NO: 2. Another example of the peptide of the present invention is a polypeptide comprising or consisting of the sequence of SEQ ID NO: 5. Another example of the peptide of the present invention is a polypeptide comprising or consisting of the sequence of SEQ ID NO: 6. The sequence of SEQ ID NO: 2 was confirmed to bind to HLA class I molecules by immunopeptidochemical analysis (see Example 2). The sequences of SEQ ID NOs: 5 and 6 were predicted to bind to HLA class I molecules by NetMHC software and were used in immunological validation assays (see Example 4).

[0049] (nucleic acid) The present invention provides isolated nucleic acids (referred to as nucleic acids of the present invention) that encode the polypeptide of the present invention. For example, nucleic acids of the present invention include or consist of sequences selected from SEQ ID NOs: 3 and 4.

[0050] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to polymeric polymers made from nucleotide monomers, particularly deoxyribonucleotide monomers or ribonucleotide monomers. This term encompasses nucleic acids, including known nucleotide analogs or modified skeletal residues or links, which are of natural or non-natural origin, and which have similar properties to the reference nucleic acid, are intended to be metabolized in a similar manner to the reference nucleotide, or are intended to have an extended half-life in system. Examples of such analogs, but not limited to, include phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). Preferably, the term “nucleic acid” refers to polymers of natural origin from deoxyribonucleotide monomers or ribonucleotide monomers. Preferably, the nucleic acid molecules of the present invention are recombinants. Recombinant means that a nucleic acid molecule is the product of at least one of the following: cloning, restriction or ligation steps, or other operations that produce a nucleic acid molecule distinct from naturally occurring nucleic acid molecules (e.g., cDNA). In one embodiment, the nucleic acid of the present invention is an artificial nucleic acid sequence (e.g., a cDNA sequence or nucleic acid sequence that utilizes non-natural codons). In one embodiment, the nucleic acid of the present invention is DNA. Alternatively, the nucleic acid of the present invention is RNA.

[0051] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) refer to nucleic acid molecules having a sugar component backbone that is a deoxyribosyl component and a ribosyl component, respectively. This sugar component may be bound to bases that are four native bases (adenine (A), guanine (G), cytosine (C), thymine (T) in DNA, and adenine (A), guanine (G), cytosine (C), and uracil (U) in RNA). As used herein, "corresponding RNA" is RNA having the same sequence as the reference DNA, but where thymine (T) in DNA is replaced with uracil (U) in RNA. The sugar component may also be bound to non-native bases such as inosine, xanthosine, 7-methylguanosine, dihydrouridine, and 5-methylcytidine. The native phosphate diester bond between the sugar (deoxyribosyl / ribosyl) components may optionally be replaced with a phosphorothioate bond. Preferably, the nucleic acids of the present invention consist of a native base bonded to a deoxyribosyl or ribosyl sugar skeleton having a phosphate diester bond between sugar components.

[0052] In one embodiment, the nucleic acid of the present invention is DNA. For example, this nucleic acid contains or consists of sequences selected from SEQ ID NOs: 3 and 4. Similarly provided is a nucleic acid containing or consisting of variants of sequences selected from SEQ ID NOs: 3 and 4, the variants encoding the same amino acid sequence but having different nucleic acids based on genetic coding degeneracy.

[0053] Therefore, due to the degeneracy of the gene code, a great many different but functionally identical nucleic acids can encode any given polypeptide. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at all positions where alanine is identified by codon 1, that codon can be modified to any of the corresponding codons without altering the encoded polypeptide. Such nucleic acid mutations result in “silent” (sometimes referred to as “degenerate” or “synonymous”) variants, which are a type of conservatively modified variant. All nucleic acid sequences disclosed herein encoding polypeptide 1 also allow for all possible silent mutations in that nucleic acid. Those skilled in the art will recognize that each codon in the nucleic acid (except AUG, which is usually the sole codon for methionine, and UGG, which is usually the sole codon for tryptophan) can be modified to produce functionally identical molecules. Accordingly, each silent mutation of the nucleic acid encoding polypeptide 1 is implicit in each described sequence and is provided as one aspect of the present invention.

[0054] Degenerate codon substitution can also be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0055] The nucleic acids of the present invention, which include or consist of sequences selected from sequence numbers 3 and 4, may contain many silent variants (e.g., 1-50, 1-25, and especially 1-5, or even one codon may be modified) compared to the reference sequence.

[0056] The nucleic acid of the present invention may include, or consist of, a sequence selected from Sequence ID No. 4 or its variants as described above, excluding the first codon for methionine (i.e., ATG or AUG).

[0057] In one embodiment of the present invention, the nucleic acid is RNA. The provided RNA sequence corresponds to the DNA sequence provided herein, having a ribonucleotide backbone instead of a deoxyribonucleotide backbone, and having uracil (U) instead of thymine (T) as the side chain base.

[0058] Accordingly, the nucleic acids of the present invention include or consist of RNA equivalents of cDNA sequences selected from Sequence IDs 3 and 4, and may contain many silent variants compared to the reference sequence (e.g., 1-50, 1-25, etc., especially 1-5, or even one codon may be modified). "RNA equivalent" means an RNA sequence that contains the same genetic information as the reference cDNA sequence (i.e., it has a ribonucleotide backbone instead of a deoxyribonucleotide backbone and contains the same codons with uracil (U) instead of thymine (T) in the side chain base).

[0059] The present invention also includes sequences complementary to the cDNA and RNA sequences. In one embodiment of the present invention, the nucleic acid is a codon optimized for expression in a human host cell. The nucleic acids of the present invention can be transcribed and translated into the polypeptides of the present invention if they are DNA nucleic acids, and can be translated into the polypeptides of the present invention if they are RNA nucleic acids.

[0060] (Polypeptides and nucleic acids) Preferably, the polypeptides and nucleic acids used in the present invention are isolated. An "isolated" polypeptide or nucleic acid is one that has been removed from its original environment. For example, a naturally occurring polypeptide or nucleic acid is isolated when it is separated from some or all of the substances that coexist with it in its natural system. A nucleic acid is considered isolated, for example, when it is cloned into a vector that is not part of its natural environment.

[0061] When used in reference to polypeptides or nucleic acid sequences, "of natural origin" means sequences that are found in nature and have not been synthetically modified. When used in reference to polypeptides or nucleic acid sequences, "artificial" means, for example, a synthetic modification of a natural sequence, or a sequence that does not exist in nature, including non-natural sequences.

[0062] The term “heterogeneous” is used in reference to the relationship between one nucleic acid or polypeptide and another, indicating that two or more sequences are not found in nature in the same relationship to one another. “Heterogeneous” sequences can also mean sequences that are not isolated from, derived from, or based on naturally occurring nucleic acid or polypeptide sequences found within a host organism.

[0063] As described above, polypeptide variants preferably have at least about 80% identity, more preferably at least about 85% identity, and most preferably at least about 90% identity (e.g., at least about 95%, at least about 98%, or at least about 99%) with respect to the full length of the reference sequence in question.

[0064] For the purpose of comparing two closely related polypeptide or polynucleotide sequences, a "sequence identity percentage" may be calculated between the first and second sequences. If a polypeptide sequence has 100% sequence identity over its entire length, it can be said to be identical or equal to the other polypeptide sequence. The residues in the sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus of the polypeptide. The terms "identical" or "percent identity" in the context of two or more polypeptide sequences refer to two or more sequences or subsequences that, when compared and aligned to correspond maximally on a comparison window, are identical or have a specific percentage of identical amino acid residues (i.e., 70% identity, and optionally 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity within a specified region). Preferably, this comparison is performed on a window corresponding to the full length of the reference sequence.

[0065] In sequence comparison, sequence 1 acts as the reference sequence for which the test sequence is compared. When using the sequence comparison algorithm, the test sequence and reference sequence are entered into the computer, sub-sequence coordinates are specified as needed, and sequence algorithm program parameters are also specified. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence with respect to the reference sequence based on the program parameters.

[0066] As used herein, a "comparison window" refers to a segment within which one sequence and the same number of consecutive reference sequences can be compared, and this comparison is performed after the two sequences have been optimally aligned. Sequence alignment methods for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482; the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; the similarity search method of Pearson and Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); or by manual alignment and visual inspection (see, for example, "Current Protocols in Molecular Biology," edited by Ausubel et al., 1995 augmented edition).

[0067] One example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignment to create multiple sequence alignments from related sequences, showing their relationships and sequence identity percentages. It also plots a tree or dendrogram showing the clustering relationships used to create the alignments. PILEUP uses a simplified version of Feng and Doolittle's progressive alignment method (1987, J. Mol. Evol. 35:351-360). The method used is similar to that described in the literature by Higgins and Sharp, 1989, CABIOS 5:151-153. This program can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. This multiple sequence alignment procedure first performs pairwise alignment on two of the most similar sequences to create a cluster of two aligned sequences. This cluster is then aligned to the most related or aligned sequence cluster. Two sequence clusters are aligned by a simple extension of pairwise alignments of the two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. This program is executed by specifying specific sequences and their amino acid coordinates for the sequence comparison region, and by specifying program parameters. Using PILEUP, a reference sequence is compared to the other test sequence, and the percentage of sequence identity relationship is determined using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gap. PILEUP is available from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., 1984, Nuc. Acids Res. 12:387-395).

[0068] Another example of a suitable algorithm for determining the percentage of sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov / ). This algorithm involves initially identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that, when aligned to words of the same length in the database sequence, match or satisfy a certain positive threshold score T, where T refers to the neighbor word score threshold (Altschul et al., cited above). These initial neighbor word hits act as seeds to initiate a search for longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Word hit extension in each direction stops when: the cumulative alignment score falls by X from its maximum achieved value; the cumulative score becomes 0 or less due to a negative scoring residue alignment accumulation of 1 or more; or the end of either sequence is reached. For amino acid sequences, the BLASTP program uses default settings: word length of 3 and expected value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff's literature, 1989, Proc. Natl. Acad. Sci. USA 89:10915) alignment (B) of 50, expected value (E) of 10, M=5, N=-4, and comparison of both strands is used.

[0069] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide sequences or two amino acid sequences occurs by chance.

[0070] A "difference" between sequences refers to the insertion, deletion, or substitution of one residue at a single position in the second sequence compared to the first sequence. Two sequences can contain one, two, or more such differences. An insertion, deletion, or substitution in the second sequence that is otherwise identical to the first sequence (100% sequence identity) results in a decrease in sequence identity percentage. For example, if the identical sequence is 9 residues long, one substitution in the second sequence results in 88.9% sequence identity. If the identical sequence is 17 amino acid residues long, two substitutions in the second sequence result in 88.2% sequence identity.

[0071] Alternatively, for the purpose of comparing the first reference sequence with the second comparison sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence may be determined. An addition is the addition of one residue to the first sequence (including additions at either end of the first sequence). A substitution is the replacement of one residue in the first sequence with one different residue. A deletion is the deletion of one residue from the first sequence (including deletions at either end of the first sequence).

[0072] (Generation of polypeptides according to the present invention) The polypeptides of the present invention can be obtained and manipulated using techniques disclosed, for example, in Green and Sambrook's 2012 book, "Molecular Cloning: A Laboratory Manual," 4th edition, Cold Spring Harbour Laboratory Press. In particular, polynucleotides can also 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 Grundstrom et al., 1985, Nucl. Acids Res., 13:3305-3316), and subsequently expressed in a suitable organism to produce polypeptides. The gene encoding the polypeptide of the present invention can be synthesized, for example, by solid-phase DNA synthesis. The entire gene may be synthesized de novo without the need for a precursor template DNA. To obtain the desired oligonucleotide, building blocks are sequentially attached to the growing oligonucleotide chain in the order required by the product sequence. Once the chain assembly is complete, the product is released from the solid phase into solution, deprotected, and recovered. The product can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotide in high purity (Verma and Eckstein, 1998, Annu. Rev. Biochem. 67:99-134). These relatively short segments can be readily assembled into longer DNA molecules using various gene amplification methods (Methods Mol Biol., 2012;834:93-109), and they are suitable for use in countless recombinant DNA-based expression systems. In the context of the present invention, those skilled in the art will understand that the polynucleotide sequences encoding polypeptide antigens described in the present invention can be readily used in various vaccine production systems, for example, including viral vectors.

[0073] For the purpose of producing the polypeptide of the present invention in a microbial host (e.g., bacterial or fungal), the nucleic acid of the present invention will include suitable regulatory and control sequences (including promoters, termination signals, etc.), as well as sequences for promoting polypeptide secretion suitable for protein production within the host. Similarly, the polypeptide of the present invention can be produced by transduction of a culture of eukaryotic cells (e.g., Chinese hamster ovary cells or Drosophila S2 cells) with the nucleic acid of the present invention, which is combined with suitable regulatory and control sequences (including promoters, termination signals, etc.), as well as sequences for promoting polypeptide secretion suitable for protein production within these cells.

[0074] The isolation of polypeptides of the present invention generated by recombinant methods can optionally be facilitated by adding a histidine residue stretch (commonly known as a His tag) to one end of the polypeptide. Polypeptides may also be prepared synthetically.

[0075] (vector) In additional embodiments, a gene construct comprising one or more nucleic acids of the present invention is introduced into cells in vivo to produce polypeptides of the present invention in vivo and induce an immune response. These nucleic acids (e.g., DNA) may reside in any variety of delivery systems known to those skilled in the art, including nucleic acid expression systems, bacteria, and some viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described in Rolland's work, 1998, Crit. Rev. Therap. "Drug Carrier Systems" 15:143-198, and the references cited therein. Some of these approaches are outlined below for illustrative purposes.

[0076] Based on the above, a vector containing the nucleic acid molecule of the present invention (also referred to herein as a "DNA expression construct" or "construct") is provided. Preferably, the vector contains nucleic acids encoding regulatory elements (such as suitable promoters and termination signals) suitable for enabling the transcription of translationally active RNA molecules in human host cells. A "translationally active RNA molecule" is an RNA molecule that can be translated into a protein by the translation machinery of a human cell. Based on the above, a vector containing the nucleic acid of the present invention (hereinafter referred to as "the vector of the present invention") is provided.

[0077] In particular, this vector may be a viral vector. Viral vectors may include adenovirus, adeno-associated virus (AAV) (e.g., AAV types 5 and 2), alphavirus (e.g., Venezuelan encephalitis virus (VEEV), Sindbisvirus (SIN), Semryqui forest virus (SFV)), herpesvirus, arenavirus (e.g., lymphocytic choriomeningitis virus (LCMV)), measles virus, poxvirus (modified vaccinia ankala (MVA), etc.), paramyxovirus, lentivirus, or rhabdovirus (e.g., vesicular stomatitis virus (VSV)), meaning that this vector can be derived from any of the aforementioned viruses. Adenoviruses are particularly suitable for use as gene transfer vectors due to their medium genome size, ease of handling, high titer, broad target cell range, and high infectivity. The viral genome contains 100-200 base pair reverse repeats (ITRs) at both ends, which are cis-elements necessary for viral DNA replication and packaging. The genome contains different transcription units in its early (E) and late (L) regions, which are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins involved in regulating the transcription of the viral genome and several cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell blockade (Renan, 1990). The products of late genes, including most of the viral capsid proteins, are expressed only after critical processing of a single primary transcript generated by the major late promoter (MLP). The MLP is particularly efficient in the late stages of infection, and all mRNA transcribed from this promoter possesses a 5'-tripartite leader (TPL) sequence, making it favorable for translation. Replication-deleted adenoviruses, which are produced from viral genomes in which one or more initial genes are deleted, are particularly useful because their replication is limited within vaccinated hosts, reducing the likelihood of pathogenic spread and minimizing the possibility of pathogenic contact in vaccinated hosts.

[0078] (Other polynucleotide delivery) In certain embodiments of the present invention, the expression construct containing one or more polynucleotide sequences may consist solely of a naked recombinant DNA plasmid. See Ulmer et al., 1993, Science 259:1745-1749 and Cohen, 1993, Science 259:1691-1692, which reviews it. The introduction of this construct can be carried out, for example, by making the cell membrane permeable either physically or chemically. This is particularly applicable to in vitro introduction, but is equally applicable to in vivo use. It is also assumed that the DNA encoding the gene of interest can be introduced in vivo in a similar manner to express the gene product. Numerous delivery systems have been used to deliver DNA molecules to animal models and humans. Several products based on this technology are approved for use in animals, and others are in Phase II and Phase III clinical trials in humans.

[0079] (RNA delivery) In other embodiments of the present invention, the expression construct comprising one or more polynucleotide sequences may consist of naked recombinant DNA-derived RNA molecules (Ulmer et al., 2012, Vaccine 30:4414-4418). For DNA-based expression constructs, various methods can be used to introduce RNA molecules into cells in vitro or in vivo. RNA-based constructs can be designed to mimic simple messenger RNA (mRNA) molecules so that the introduced biological molecule is directly translated by the host cell's translation mechanism, producing the polypeptide it encodes within the introduced cell. Alternatively, RNA molecules may be designed in such a way that they can self-amplify within the introduced cell by incorporating their structural gene for viral RNA-dependent RNA polymerase. Thus, this type of RNA molecule, known as self-amplifying mRNA (SAM™) molecules (Geall et al., 2012, PNAS, 109:14604-14609), shares properties with several RNA-based viral vectors. Either mRNA-based RNA or SAM(trademark) RNA can be further modified (e.g., by altering their sequences or by using modified nucleotides) to enhance stability and translation (Schlake et al., RNA Biology, 9:1319-1330), and both types of RNA may be formulated (e.g., into emulsions (Brito et al., Molecular Therapy, 2014 22:2118-2129) or into lipid nanoparticles (Kranz et al., 2006, Nature, 534:396-401)) to enhance stability and / or entry into cells in vitro or in vivo. Myriad formulations of modified (and unmodified) RNA have been tested as vaccines in animal models and in humans, and numerous RNA-based vaccines are in ongoing clinical trials.

[0080] (Pharmaceutical composition) The polypeptides, nucleic acids, and vectors of the present invention may be formulated for delivery within pharmaceutical compositions such as immunogenic compositions and vaccine compositions (hereinafter referred to as "compositions of the present invention"). Preferably, the compositions of the present invention contain the polypeptides, nucleic acids, or vectors of the present invention together with a pharmaceutically acceptable carrier. Accordingly, one embodiment provides an immunogenic pharmaceutical composition comprising the polypeptide, nucleic acid, or vector of the present invention together with a pharmaceutically acceptable carrier.

[0081] In another embodiment, a vaccine composition is provided comprising the polypeptide, nucleic acid, or vector of the present invention together with a pharmaceutically acceptable carrier. The preparation of the pharmaceutical composition is generally described, for example, in Powell and Newman, eds., "Vaccine Design (the subunit and adjuvant approach)," 1995. The composition of the present invention may also contain other compounds that may be biologically active or inactive. Preferably, the composition of the present invention is a sterile composition suitable for parenteral administration.

[0082] In certain preferred embodiments of the present invention, the pharmaceutical composition of the present invention is provided as comprising one or more (e.g., one) polypeptides of the present invention combined with a pharmaceutically acceptable carrier. In certain preferred embodiments of the present invention, the composition of the present invention is provided comprising one or more nucleic acids of the present invention or one or more vectors of the present invention, combined with a pharmaceutically acceptable carrier.

[0083] In one embodiment, the composition of the present invention may include one or more (e.g., one) polynucleotides and one or more (e.g., one) polypeptide components. Alternatively, the composition may include one or more (e.g., one) vectors and one or more (e.g., one) polypeptide components. Alternatively, the composition may include one or more (e.g., one) vectors and one or more (e.g., one) polynucleotide components. Such compositions may be provided to enhance an immune response.

[0084] (Salt that is acceptable as a medicine) It will be apparent that the compositions of the present invention may include pharmaceutically acceptable salts of nucleic acids or polypeptides provided herein. Such salts may be prepared from pharmaceutically acceptable non-toxic bases, which include organic bases (e.g., salts of primary, secondary, and tertiary amines and basic amino acids) and salts from inorganic bases (e.g., salts of sodium, potassium, lithium, ammonium, calcium, and magnesium).

[0085] (A career path acceptable in the medical field) Many pharmaceutically acceptable carriers known to those skilled in the art may be used in the compositions of the present invention, but the optimal type of carrier used will vary depending on the mode of administration. The compositions of the present invention may be formulated for any preferred mode of administration, which may include, for example, parenteral, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration, preferably parenteral, for example, intramuscular, subcutaneous, or intravenous administration. For parenteral administration, the carrier preferably contains water and may include a pH adjusting buffer, stabilizers (e.g., surfactants and amino acids), and isotonic modifiers (e.g., salts and sugars). If the composition is to be provided in a lyophilized form for dilution at use, the formulation may include a lyophilization protective agent, such as sugars such as trehalose. For oral administration, either the carriers or solid carriers, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be used.

[0086] Accordingly, the composition of the present invention may contain a buffer (e.g., neutral buffered saline or phosphate-buffered saline), a carbohydrate (e.g., glucose, mannose, sucrose, or dextran), mannitol, a protein, a polypeptide, or an amino acid such as glycine, an antioxidant, a bacteriostatic agent, a chelating agent such as EDTA or glutathione, a solute to make the formulation isotonic, hypotonic, or weakly hypertonic with the recipient's blood, a suspending agent, a thickener, and / or a preservative. Alternatively, the composition of the present invention may be formulated as a lyophilized product.

[0087] (Immunostimulant) The compositions of the present invention may also contain one or more immunostimulants. The immunostimulants may be any substance that enhances or strengthens the immune response (antibody-mediated and / or cell-mediated) to an exogenous antigen. Immunostimulants are often referred to as adjuvants in the context of vaccine formulations, and examples include aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate, saponins such as QS21, immunostimulatory oligonucleotides such as CPG, oil-in-water emulsions (e.g., when the oil is squalene), aminoalkylglucosaminide 4-phosphate, lipopolysaccharides or derivatives thereof (e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPL®) and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and interopropyl alcohols). - Feron is included. From the above, one or more immunostimulants of the composition of the present invention are preferably selected from aluminum salts, saponins, immunostimulant oligonucleotides, oil-in-water emulsions, aminoalkylglucosaminide 4-phosphate, lipopolysaccharides and their derivatives, and other TLR4 ligands, TLR7 ligands, TLR8 ligands and TLR9 ligands. The immunostimulants may also include monoclonal antibodies that specifically interact with other immune components, such as monoclonal antibodies that block the interaction of immune checkpoint receptors including PD-1 and CTLA4.

[0088] In the case of recombinant nucleic acid delivery methods (e.g., DNA, RNA, viral vectors), the gene encoding a protein-based immunostimulant may be conveniently delivered together with the gene encoding the polypeptide of the present invention.

[0089] (Sustained release) The compositions described herein may be administered as part of a sustained-release formulation (i.e., a formulation such as a capsule, sponge, patch, or gel (e.g., consisting of a polysaccharide) that produces a sustained / continuous release of the compound after administration).

[0090] (Storage and Packaging) The compositions of the present invention may be provided in unit-dose or multi-dose containers such as sealed ampoules or vials. Such containers are preferably sealed tightly to maintain the sterility of the formulation until use. The formulations may generally be stored as suspensions, solutions, or emulsions in oily or aqueous vehicles. Alternatively, the compositions of the present invention may be stored in a lyophilized state, requiring only the addition of a sterile liquid carrier (such as water or saline for injection) immediately before use.

[0091] (Dosage of medication) The amounts of nucleic acids, polypeptides, or vectors in each composition of the present invention may be formulated in such a manner that suitable dosages for therapeutic or prophylactic use are obtained. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art who prepare such compositions, and for this purpose, a variety of dosages and therapeutic regimens will be desirable.

[0092] Typically, compositions containing therapeutic or prophylactic effective doses deliver about 0.1 ug to about 1000 ug of the polypeptide of the present invention per dose, more typically about 2.5 ug to about 100 ug of polypeptide per dose. When delivered in the form of short synthetic long-chain peptides, the dosage may range from 1 to 200 ug / peptide / dose. With respect to polynucleotide compositions, these typically deliver about 10 ug to about 20 mg of the nucleic acid of the present invention per dose, more typically about 0.1 mg to about 10 mg of the nucleic acid of the present invention per dose.

[0093] (Diseases that are treated or prevented) As stated throughout the specification, Sequence ID No. 1 is a polypeptide sequence corresponding to the CLT antigen overexpressed in cutaneous melanoma. In one embodiment, the present invention provides a polypeptide, nucleic acid, vector, or composition for use in pharmaceuticals.

[0094] A further aspect of the present invention relates to a method for increasing the immune response in a human, comprising administering a polypeptide, nucleic acid, vector, or composition of the present invention to the human. The present invention also provides polypeptides, nucleic acids, vectors, or compositions for use in enhancing the immune response in humans. The use of polypeptides, nucleic acids, vectors, or compositions of the present invention for the manufacture of pharmaceuticals used to enhance the immune response in humans is also provided.

[0095] Preferably, this immune response is elevated against cancerous tumors expressing a corresponding sequence selected from SEQ ID NO: 1 and its variants and its immunogenic fragments. In this context, "corresponding" means that if the tumor expresses, for example, SEQ ID NO: 1 or its variants or its immunogenic fragments, then the polypeptides, nucleic acids, vectors or compositions of the present invention and pharmaceuticals containing them will be based on SEQ ID NO: 1 or its variants or its immunogenic fragments.

[0096] Preferably, the immune response includes a CD8+ T cell response, a CD4+ T cell response, and / or an antibody response, particularly a CD8+ cytolytic T cell response and a CD4+ helper T cell response. Preferably, the immune response is triggered against tumors, particularly those expressing sequences selected from Sequence ID No. 1 and its variants, as well as its immunogenic fragments.

[0097] In a preferred embodiment, the tumor is a melanoma, such as a cutaneous melanoma. This tumor may be a primary tumor or a metastatic tumor.

[0098] Further aspects of the present invention relate to a method for treating a human patient with cancer, wherein the cancer cells express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, or a method for preventing a human from developing cancer, wherein the cancer will express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, the method comprising administering a corresponding polypeptide, nucleic acid, vector, or composition of the present invention to the human.

[0099] The present invention also provides a polypeptide, nucleic acid, vector, or composition for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NO: 1 and its immunogenic fragment.

[0100] The transcript corresponding to Sequence ID No. 3 was also overexpressed in uveal melanoma. Therefore, in another embodiment, the tumor is a uveal melanoma tumor and / or a tumor expressing the sequence of Sequence ID No. 1.

[0101] Accordingly, the present invention relates to a method of the present invention, or a polypeptide, nucleic acid, vector, or composition for use, wherein the polypeptide comprises a sequence selected from the following: (a) the sequence of sequence number 1; and (b) Variants of the sequence in (a); and (c) Immunogenic fragment of the sequence in (a), For example, the polypeptide contains or consists of any one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6, and for example, the nucleic acid contains or consists of a sequence selected from any one of SEQ ID NOs: 3 and 4; and, The cancer is uveal melanoma, and the present invention provides polypeptides, nucleic acids, vectors, or compositions for the aforementioned method or use. The terms "prevention" and "prevention" are used interchangeably in this specification.

[0102] (Treatment and vaccination regimens) A therapeutic regimen may include the delivery of (i) a polypeptide, nucleic acid, or vector of the present invention, and (ii) one or more further polypeptides, nucleic acids, or vectors of the present invention and / or (iii) various other therapeutically useful compounds or molecules such as antigenic proteins or other further components, optionally in co-administration with an adjuvant, either at the same time (e.g., co-administration) or sequentially (e.g., prime boost). Examples of co-administration include co-administration to the same lateral site and co-administration to opposing contralateral sites. "Co-administration" preferably means that all components are delivered during the same therapeutic session. Preferably, all components are administered simultaneously (e.g., co-administration of both DNA and protein), but one component may be administered within minutes (e.g., during the same appointment or doctor's visit) or within hours.

[0103] In some embodiments, one or more "boosts" or additional doses of the polypeptide, nucleic acid, or vector of the present invention are administered following a "priming" or first dose of the polypeptide, nucleic acid, or vector of the present invention ("prime and boost" method). In one embodiment, the polypeptide, nucleic acid, or vector of the present invention is used in one prime-boost vaccination regimen. In one embodiment, both the prime and boost are polypeptides of the present invention, and in each case, they are the same polypeptide of the present invention. In one embodiment, both the prime and boost are nucleic acids or vectors of the present invention, and in each case, they are the same nucleic acid or vector of the present invention. Alternatively, the prime may be performed using the nucleic acid or vector of the present invention and the boost using the polypeptide of the present invention, or the prime may be performed using the polypeptide of the present invention and the boost using the nucleic acid or vector of the present invention. Typically, the first or "priming" dose and the second or "boost" dose are administered about 1 to 12 weeks, or up to 4 to 6 months apart. The next "booster" dose may be administered every 1 to 6 weeks, or even later (up to several years later).

[0104] (combination of antigens) The polypeptides, nucleic acids, or vectors of the present invention can be used in combination with one or more other polypeptides, nucleic acids, or vectors of the present invention, and / or in combination with other antigenic polypeptides (or polynucleotides or vectors encoding them) that elicit an immune response against melanoma, such as cutaneous melanoma or uveal melanoma. These other antigenic polypeptides may be derived from a variety of sources, which may include melanoma-related antigens described in detail, such as GPR143, PRAME, MAGE-A3, or pMel(gp100). Alternatively, they may contain melanoma antigens from other species, including patient-specific novel antigens (Lauss et al., (2017) Nature Communications, 8(1), 1738. http: / / doi.org / 10.1038 / s41467-017-01460-0), retained intron novel antigens (Smart et al., (2018) Nature Biotechnology. http: / / doi.org / 10.1038 / nbt.4239), and splice variant novel antigens (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). This includes melanoma antigens (TIEPPs; Gigoux, M. and Wolchok, J. (2018), JEM, 215, 2233; Marijt et al., (2018), JEM 215, 2325) belonging to the category of antigens that encode T cell epitopes associated with damaged peptide processing, or novel antigens that may be discovered (including CLT antigens).Furthermore, antigenic peptides derived from these various sources may also be combined with (i) nonspecific immunostimulants / adjuvants and / or (ii) antigens (delivered as polypeptides or as polynucleotides or vectors encoding these CD4 antigens) that are known to induce potent CD4 helper T cells, for example, including universal CD4 helper epitopes, to amplify the anti-melanoma-specific response induced by the concurrently administered antigens.

[0105] Different polypeptides, nucleic acids, or vectors may be formulated as the same formulation or as separate formulations. Alternatively, the polypeptide may be provided as a fusion protein in which the polypeptide of the present invention is fused to a second or further polypeptide (see below). The nucleic acid may be provided as encoding the fusion protein.

[0106] Furthermore, more generally, when using two or more components in combination, those components are, for example, (1) Two or more individual antigenic polypeptide components, (2) As a fusion protein containing both (or further) polypeptide components, (3) One or more polypeptides and one or more polynucleotide components, (4) Two or more individual polynucleotide components, (5) As a single polynucleotide encoding two or more individual polypeptide components, or (6) As a single polynucleotide encoding a fusion protein containing both (or further) polypeptide components, It may be possible to exist.

[0107] For convenience, when many components are present, it is often desirable that they be contained within a single fusion protein or a polynucleotide encoding a single fusion protein (see below). In one embodiment of the present invention, all components are provided as polypeptides (e.g., within a single fusion protein). In another embodiment of the present invention, all components are provided as polynucleotides (e.g., a single polynucleotide such as one encoding a single fusion protein).

[0108] (Fusion protein) As an embodiment of the foregoing discussion concerning antigen combinations, the present invention also provides isolated polypeptides of the present invention fused to a second or further polypeptide of the present invention (hereinafter referred to as "combined polypeptides of the present invention") by creating nucleic acid constructs that fuse sequences encoding individual antigens together. The combined polypeptides of the present invention are expected to have the utility described herein for the polypeptides of the present invention and may have advantages of superior immunogenicity or vaccine activity, or prophylactic or therapeutic effects (including increased range and depth of response), and will be of particular value in non-inbred populations. The fusion of polypeptides of the present invention will also provide the advantage of increasing the efficiency of constructing and manufacturing vaccine antigens and / or vectorized vaccines (including nucleic acid vaccines).

[0109] As described in the "Antigen Combinations" section above, the polypeptide of the present invention and the combined polypeptide of the present invention may also be fused to a polypeptide sequence that is not the polypeptide of the present invention and contains one or more of the following: (a) Other polypeptides that are melanoma-related antigens and therefore potentially useful as immunogenic sequences in vaccines (e.g., GPR143, PRAME, MAGE-A3, and pMel(gp100) mentioned above); and (b) A polypeptide sequence capable of enhancing the immune response (i.e., an immunostimulatory sequence). (c) For example, a polypeptide sequence that contains a universal CD4 helper epitope, can provide potent CD4+ support, and can enhance the CD8+ T cell response to a CLT antigen epitope.

[0110] The present invention also provides nucleic acids encoding the fusion protein, and other embodiments of the present invention (vectors, compositions, cells, etc.) with necessary modifications to the polypeptide of the present invention.

[0111] (CLT antigen-binding polypeptide) Antigen-binding polypeptides that are immune-specific to tumor-expressing antigens (polypeptides of the present invention) may be designed to recruit cytolytic cells to antigen-modified tumor cells and mediate their destruction. One known mechanism of cytolytic cell recruitment by such antigen-binding polypeptides is antibody-dependent cell-mediated cytotoxicity (ADCC). Accordingly, the present invention provides antigen-binding polypeptides that are immune-specific to the polypeptides of the present invention. Antigen-binding polypeptides containing antibodies, such as monoclonal antibodies and their fragments, e.g., domain antibodies, Fab fragments, Fv fragments, and VHH fragments, may be produced and humanized in non-human animal species (e.g., rodents or camels), or in non-human species (e.g., rodents genetically engineered to have a human immune system).

[0112] Antigen-binding polypeptides can be prepared by methods well known to those skilled in the art. For example, monoclonal antibodies can be produced by fusing specific antibody-producing B cells with myeloma (B-cell carcinoma) cells selected for their ability to proliferate in tissue culture and for their lack of antibody chain synthesis, using hybridoma technology (Kohler and Milstein, 1975, Nature 256(5517):495-497 and Nelson et al., June 2000, Mol Pathol. 53(3):111-7 are incorporated herein by reference in their entirety).

[0113] A monoclonal antibody against a desired antigen is, for example: a) Lymphocytes obtained from the peripheral blood of animals (including humans) that have been pre-immunized / exposed with a desired antigen are immortalized with immortal cells, preferably myeloma cells, to form hybridomas. b) Culture the formed immortalized cells (hybridomas) and collect cells that produce antibodies with the desired specificity. It can be obtained by [method].

[0114] Monoclonal antibodies can be obtained by a process that includes the following steps: a) Cloning step of DNA or cDNA sequences obtained from animal lymphocytes, particularly peripheral blood lymphocytes, (preferably pre-immunized with a desired antigen) into a vector, especially a phage, and more specifically, a filamentous bacteriophage. b) A step of transforming prokaryotic cells with the above vector under conditions that enable antibody production, c) A step of selecting antibodies by subjecting them to antigen affinity selection, d) A step of recovering an antibody having the desired specificity, e) A step of expressing an antibody-encoding nucleic acid molecule obtained from B cells of a patient exposed to the antigen or from an animal immunized with the antigen in a laboratory. The selected antibodies may then be produced using conventional recombinant protein production techniques (for example, from genetically engineered CHO cells).

[0115] The present invention provides an isolated antigen-binding polypeptide that is immunospecific to the polypeptide of the present invention. Preferably, the antigen-binding polypeptide is a monoclonal antibody or a fragment thereof. In certain embodiments, the antigen-binding polypeptide is bound to a cytotoxic component. An example of this cytotoxic component is the Fc domain of an antibody, which would mobilize Fc receptor-possessing cells that promote ADCC. Alternatively, the antigen-binding polypeptide may be bound to a biotoxin or a cytotoxic chemical.

[0116] Another important type of antigen-binding polypeptide includes T cell receptor (TCR)-derived molecules that bind to the HLA-presenting fragment of the antigen of the present invention. In this embodiment, a TCR-based biologic that recognizes the CLT antigen (or its derivatives) on the surface of tumor cells (including TCRs directly derived from the patient or specifically engineered high-affinity TCRs) may also include a targeted component that recognizes a component present on T cells (or other types of immune cells) that attracts these immune cells to the tumor, thereby providing a therapeutic effect. In some embodiments, this targeted component may also stimulate beneficial activity (including cytolytic activity) of the redirected immune cells.

[0117] Therefore, in one embodiment, the antigen-binding polypeptide is immunospecific to the polypeptide of the present invention or to an HLA-binding polypeptide that is a part thereof. For example, the antigen-binding polypeptide is a T cell receptor. In one embodiment, the antigen-binding polypeptide of the present invention may be bound to another polypeptide capable of binding to cytotoxic cells or other immune components within the subject.

[0118] In one embodiment, the antigen-binding polypeptide is intended for use in pharmaceuticals. One embodiment provides a pharmaceutical composition comprising the antigen-binding polypeptide of the present invention together with a pharmaceutically acceptable carrier. Such a composition may be a sterile composition suitable for parenteral administration. See, for example, the disclosure of the pharmaceutical composition described above.

[0119] The present invention provides a method for treating a person suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, or a method for preventing a person from developing cancer, wherein the cancer cells will express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, the method comprising administering to the person an antigen-binding polypeptide of the present invention or a composition containing the antigen-binding polypeptide.

[0120] One embodiment provides an antigen-binding polypeptide of the present invention, which may be bound to a cytotoxic component, or a composition comprising the antigen-binding polypeptide of the present invention, for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NO: 1 and its immunogenic fragment.

[0121] Preferably, in any of the above embodiments, the cancer is melanoma, in particular cutaneous melanoma.

[0122] In one embodiment of the present invention, an antigen-binding polypeptide or composition for use, wherein the polypeptide comprises a sequence selected from the following: (a) the sequence of sequence number 1; and (b) Variants of the sequence of (a); and, (c) Immunogenic fragment of the sequence in (a), For example, the polypeptide contains or consists of any one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6, and for example, the nucleic acid contains or consists of a sequence selected from any one of SEQ ID NOs: 3 and 4; and, The cancer is uveal melanoma, and the method or antigen-binding polypeptide or composition for use is provided.

[0123] Antigen-binding polypeptides (antibodies or fragments thereof, etc.) may be administered in doses such as 5-1000 mg, 25-500 mg, 100-300 mg, or approximately 200 mg.

[0124] (Cell therapy that promotes antigen presentation in vivo) Various cell delivery vehicles may be used in the pharmaceutical composition to promote the development of an antigen-specific immune response. Accordingly, the present invention provides isolated antigen-presenting cells (hereinafter referred to as "APCs of the present invention") that have been modified by loading the polypeptide of the present invention in vitro or that have been genetically engineered to express the polypeptide of the present invention. Antigen-presenting cells (APCs) include, for example, dendritic cells, macrophages, B cells, monocytes, and other cells that may be engineered to be efficient APCs. Such cells may, though not necessarily, be genetically modified to enhance antigen-presenting ability, improve the activation and / or persistence of the T cell response, and / or be immunocompatible with receptors (i.e., HLA haplotype matching). APCs can generally be isolated from any of various bodily fluids and organs and may be autologous, allogeneic, syngeneic, or heterogeneic cells.

[0125] In certain preferred embodiments of the present invention, dendritic cells or their precursors are used as APCs. Thus, in one embodiment, the APC of the present invention is a dendritic cell. Dendritic cells are very potent APCs (see Banchereau and Steinman, 1998, Nature, 392:245-251) and have been shown to be effective as physiological adjuvants for inducing prophylactic or therapeutic immunity (see Timmerman and Levy, 1999, Ann. Rev. Med. 50:507-529). Generally, dendritic cells may be identified based on their typical morphology (star-shaped in situ and possessing prominent cytoplasmic projections (dendrites) visible in vitro), their ability to efficiently take up, process, and present antigens, and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell surface receptors or ligands not normally found on dendritic cells in vivo or in vitro, and such modified dendritic cells are also intended by the present invention. Instead of dendritic cells, antigen-loaded secretory vesicles (referred to as exosomes) may be used in the immunogenic composition (see Zitvogel et al., 1998, Nature Med. 4:594-600). Thus, one embodiment provides exosomes loaded with polypeptides of the present invention.

[0126] Dendritic cells and progenitor cells may be obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood, or other suitable tissues or bodily fluids. For example, dendritic cells may be differentiated in vitro by adding a combination of cytokines such as GM-CSF, IL-4, IL-13, and / or TNFα to a culture of monocytes collected from peripheral blood. Alternatively, CD34-positive cells collected from peripheral blood, umbilical cord blood, or bone marrow may be differentiated into dendritic cells by adding a combination culture medium of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and / or other compounds that induce differentiation, maturation, and proliferation of dendritic cells.

[0127] Dendritic cells are conveniently categorized into "immature" and "mature" cells, which allows for easy distinction between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages in differentiation. Immature dendritic cells are characterized as APCs with high antigen uptake and processing capabilities, which correlate with high expression of Fcγ receptors and mannose receptors. Mature phenotypes typically have low expression of these markers but are characterized by high expression of cell surface molecules involved in T cell activation, such as class I and class II MHCs, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

[0128] Alternatively, APCs may be genetically engineered and transfected, for example, with a polynucleotide encoding a protein (or a part thereof or other variant thereof) so that the polypeptide is expressed on the cell surface. Such transfection may be performed in vitro, and the pharmaceutical composition containing the transfected cells can then be used as described herein. Alternatively, transfection may be induced in vivo by administering a gene transduction vehicle targeting dendritic cells or other antigen-presenting cells to a patient. In vivo and in vitro transfection of dendritic cells may be carried out using any method generally known in the art, such as the method described in WO97 / 24447, or the gene gun approach described in Mahvi et al., 1997, Immunology and Cell Biology 75:456-460. Antigen loading into dendritic cells involves loading dendritic cells or progenitor cells with polypeptides, DNA (e.g., plasmid vectors), or RNA; or by using recombinant bacteria or viruses expressing antigens (e.g., adenoviruses, adeno-associated viruses (AAVs) (e.g., AAV types 5 and 2), alphaviruses (e.g., Venezuelan encephalitis virus (VEEV), Sindbisvirus (SIN), Semryqui forest virus (SFV), herpesviruses, arenaviruses (e.g., lymphocytic choriomeningitis virus (LCMV)), measles virus). This may be achieved by incubation with a poxvirus (such as modified vaccinia ancara (MVA) or fowlpox), paramyxovirus, lentivirus, or rhabdovirus (such as vesicular stomatitis virus (VSV)). Before loading the polypeptide, it may be covalently bound to an immunological partner (e.g., a carrier molecule) that provides T cell support. Alternatively, dendritic cells may be pulsed with a non-conjugate immunological partner, either individually or in the presence of the polypeptide or vector.

[0129] The present invention provides a short, chemically synthesized, specifically designed epitope-coding fragment of a polypeptide antigen for delivery to antigen-presenting cells. Those skilled in the art will understand that this type of molecule is known as a synthetic long-chain peptide (SLP) and provides a therapeutic platform for stimulating (or loading into) cells in vitro using the antigenic polypeptide of the present invention (Gornati et al., 2018, Front. Imm, 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).

[0130] One embodiment provides a pharmaceutical composition comprising antigen-presenting cells, preferably dendritic cells, of the present invention together with a pharmaceutically acceptable carrier. Such a composition may be a sterile composition suitable for parenteral administration. See, for example, the disclosure of the pharmaceutical composition described above. In one embodiment, antigen-presenting cells, preferably dendritic cells, of the present invention are provided for use in pharmaceuticals.

[0131] Similarly, there is a method for treating a person suffering from cancer, wherein the cancer cells express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, or a method for preventing a person from developing cancer, wherein the cancer cells will express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, the method comprising administering to the person an antigen-presenting cell of the present invention, preferably a dendritic cell, or a composition containing such antigen-presenting cell of the present invention.

[0132] One embodiment provides antigen-presenting cells, preferably dendritic cells, or a composition comprising such antigen-presenting cells for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NO: 1 and its immunogenic fragment.

[0133] One embodiment provides a pharmaceutical composition comprising the exosomes of the present invention together with a pharmaceutically acceptable carrier. Such a composition may be a sterile composition suitable for parenteral administration. See, for example, the disclosure of the pharmaceutical composition above. The composition may optionally contain an immunostimulant. See, for example, the disclosure of the immunostimulant above. One embodiment provides the exosomes of the present invention for use in pharmaceuticals.

[0134] Similarly, there is a method for treating a person suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, or a method for preventing a person from developing cancer, wherein the cancer cells will express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, the method comprising administering to the person an exosome of the present invention or a composition containing an exosome of the present invention.

[0135] One embodiment provides an exosome or composition of the present invention comprising the exosome for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NO: 1 and its immunogenic fragment. In any one of the embodiments described above, the cancer is preferably melanoma, in particular cutaneous melanoma.

[0136] (Therapy using stimulated T cells) In addition to the in vivo or in vitro APC-mediated generation of immune-specific T cells to the polypeptide of the present invention, autologous or non-autologous T cells may be isolated from a subject, for example, from peripheral blood, umbilical cord blood, and / or by apheresis, and stimulated in the presence of a tumor-associated antigen loaded onto the MHC molecule (signal 1) of the APC cells to induce proliferation of T cells having an immune-specific TCR to this antigen.

[0137] For successful T cell activation, the co-stimulatory surface molecules B7 and CD28 must bind to antigen-presenting cells and T cells, respectively (signal 2). Both signals 1 and 2 are necessary to achieve optimal T cell activation. In contrast, antigenic peptide stimulation (signal 1) without co-stimulation (signal 2) may fail to induce complete T cell activation, potentially leading to T cell tolerance. Besides co-stimulatory molecules, inhibitory molecules such as CTLA-4 and PD-1 also exist, inducing signals that inhibit T cell activation.

[0138] Therefore, autologous or non-autologous T cells may be stimulated and proliferated in the presence of the polypeptide of the present invention and reintroduced into patients at risk of developing or already suffering from cancer in which the cancer cells express the corresponding polypeptide of the present invention, provided that the antigen-specific TCR recognizes the antigen presented by the patient's MHC, targets cancer cells expressing the corresponding polypeptide, and induces cell death.

[0139] One embodiment of the present invention provides a polypeptide, nucleic acid, vector, or composition for use in vitro to stimulate and / or amplify T cells derived from a human with cancer, and then to reintroduce the stimulated and / or amplified T cells into that human to treat the cancer of that human.

[0140] The present invention provides a method for treating human cancer, wherein the cancer cells express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, and the method comprises: taking a population of leukocytes, including at least T cells, from the human, optionally together with antigen-presenting cells; stimulating and / or amplifying the T cells in the presence of the corresponding polypeptide, nucleic acid, vector, or composition of the present invention; and reintroducing some or all of the leukocytes, including at least the stimulated and / or amplified T cells, into the human.

[0141] In any one of the embodiments described above, the cancer is preferably melanoma, in particular cutaneous melanoma.

[0142] One embodiment provides a process for preparing a population of T cells that are cytotoxic to cancer cells expressing sequences selected from Sequence ID No. 1 and its immunogenic fragments and variants, the process comprising (a) obtaining T cells and antigen-presenting cells from a cancer patient, and (ii) stimulating and amplifying the T cell population in vitro with the corresponding polypeptide, nucleic acid, vector or composition of the present invention.

[0143] In this context, "corresponding" means that when cancer cells express SEQ ID NO: 1 or its variants or its immunogenic fragments, a T cell population is stimulated and amplified in vitro by SEQ ID NO: 1 or its variants or its immunogenic fragments in the form of a polypeptide, nucleic acid, vector, or a composition containing one of the above.

[0144] For example, in such a process, culture and proliferation are carried out in the presence of dendritic cells. These dendritic cells will be transfected with the nucleic acid molecule or vector of the present invention to express the polypeptide of the present invention.

[0145] The present invention provides a T cell population (hereinafter referred to as the T cell population of the present invention) that can be obtained by any of the above processes. One embodiment provides a cell (hereinafter referred to as the T cell of the present invention) that is a T cell stimulated with the polypeptide, nucleic acid, vector, or composition of the present invention.

[0146] One embodiment provides a pharmaceutical composition comprising a T cell population or T cells of the present invention together with a pharmaceutically acceptable carrier. Such a composition may be, for example, a sterile composition suitable for parenteral administration. One embodiment provides a T cell population or T cells of the present invention for use in pharmaceuticals.

[0147] Similarly, there is a method for treating a person suffering from cancer, wherein the cancer cells express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, or a method for preventing a person from developing cancer, wherein the cancer cells will express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, the method comprising administering to the person a T cell population or T cells of the present invention or a composition containing such T cell population or T cells of the present invention.

[0148] One embodiment provides a T cell population, T cells, or a composition comprising the T cell population or T cells of the present invention for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NO: 1 and its immunogenic fragment. In any one of the above embodiments, the cancer is preferably melanoma, in particular cutaneous melanoma.

[0149] One embodiment of the present invention provides a process, method, or T cell population, T cells, antigen-presenting cells, exosomes, or composition, the polypeptide comprising a sequence selected from the following: (a) the sequence of sequence number 1; and (b) Variants of the sequence of (a); and, (c) Immunogenic fragment of the sequence in (a), For example, the polypeptide contains or consists of any one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6, and for example, the nucleic acid contains or consists of a sequence selected from any one of SEQ ID NOs: 3 and 4; and The cancer is uveal melanoma, and the above-mentioned preparation method, method, or T cell population, T cells, antigen-presenting cells, exosomes, or composition is provided.

[0150] (Therapy using genetically modified immune cells) Derivatives of all types of CLT antigen-binding polypeptides described above may include a TCR or TCR mimetic (see Dubrovsky et al., 2016, Oncoimmunology) that recognizes a CLT antigen-derived peptide in complex with a human HLA molecule, and may be genetically engineered to be expressed on the surface of (autologous or non-autologous) T cells, which may then be administered as adoptive T cell therapy for cancer treatment.

[0151] These derivatives are classified under the “chimeric antigen receptor (CAR)” category, which, as used herein, may also refer to, for example, artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors, and may include receptors engineered to confer artificial specificity to specific immune effector cells. CARs may also be used to confer monoclonal antibody specificity to T cells, thereby enabling the generation of a large number of specific T cells for use, for example, in adoptive cell therapy. CARs will direct the cell specificity to tumor-associated antigens, which are polypeptides of the present invention bound to HLA.

[0152] Another approach to treating cancer in patients involves genetically modifying T cells to target antigens expressed on tumor cells, mediated by the expression of chimeric antigen receptors (CARs). This technique is reviewed in the literature Wendell and June, 2017, Cell, 168:724-740 (the entire work is incorporated herein by reference).

[0153] Such CAR T cells may be produced by obtaining a cell sample containing T cells or T cell precursors from a subject, for example, from peripheral blood, umbilical cord blood, and / or by apheresis, and transfecting the cells with a nucleic acid encoding a chimeric T cell receptor (CAR) that is immune-specific to the polypeptide of the present invention that is bound to HLA. Such nucleic acids may be incorporated into the genome of the cells, and an effective amount of the cells may be administered to the subject to provide a T cell response to cells expressing the polypeptide of the present invention. For example, a cell sample may be taken from the subject.

[0154] It is understood that the cells used to produce the aforementioned CAR-expressing T cells may be of autologous or non-autologous origin. Transgenic CAR-expressing T cells may also exhibit inactivation of endogenous T cell receptors and / or endogenous HLA. For example, cells may be manipulated to eliminate the expression of endogenous α / β T cell receptors (TCRs).

[0155] While cell transfection methods are well known in this field, highly efficient transfection methods such as electroporation can also be used. For example, the nucleic acid or vector of the present invention expressing the CAR construct may be introduced into cells using a "nucleofection" apparatus.

[0156] A cell population for CAR-expressing T cells may be enriched after cell transfection. For example, CAR-expressing cells can be separated from non-expressing cells (e.g., by FACS) using CAR-bound antigens or CAR-binding antibodies. Another enrichment step may involve depleting non-T cells or non-CAR-expressing cells. For example, CD56+ cells can be depleted from the culture population.

[0157] A population of gene-transformed CAR-expressing cells may be cultured in vitro in a medium that selectively enhances the proliferation of CAR-expressing T cells. Therefore, CAR-expressing T cells can be proliferated in vitro.

[0158] CAR cell samples may be stored (or maintained in culture). For example, samples may be cryopreserved for later growth or analysis. CAR-expressing T cells may be used in combination with other therapies, such as checkpoint inhibitors including PD-L1 antagonists.

[0159] One embodiment provides a cytotoxic cell that has been engineered to express one of the antigen-binding polypeptides on its surface. Preferably, the cytotoxic cell is a T cell.

[0160] One embodiment provides cytotoxic cells, preferably T cells, that have been manipulated to express one of the antigen-binding polypeptides on their surface for use in pharmaceuticals. The present invention provides a pharmaceutical composition comprising cytotoxic cells, preferably T cells.

[0161] A method for treating a human patient suffering from cancer is provided, wherein the cancer cells express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, or a method for preventing a human from developing cancer is provided, wherein the cancer will express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, the method comprising administering the cytotoxic cells of the present invention, preferably T cells, to the human.

[0162] In one embodiment of the present invention, the cytotoxic cells, preferably T cells, are for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NO: 1 and its immunogenic fragment.

[0163] (Combination therapy) The cancer treatment method of the present invention may be used in combination with other therapies, particularly checkpoint inhibitors and interferon. Polypeptides, nucleic acids, vectors, antigen-binding polypeptides, and adoptive cell therapies (APC and T cell-based) can be used in combination with other components that have been engineered to enhance their immunogenicity (e.g., improve the magnitude and / or scope of the immune response they induce) or to provide other activities (e.g., activation of other aspects of innate or adaptive immune responses, or destruction of tumor cells).

[0164] Accordingly, the present invention provides compositions of the present invention (i.e., immunogenic, vaccine or pharmaceutical compositions), or kits of some such compositions, comprising polypeptides, nucleic acids, or vectors of the present invention together with a pharmaceutically acceptable carrier, and (i) one or more further immunogenic or immunostimulatory polypeptides (e.g., interferon, IL-12, checkpoint inhibitor molecules or nucleic acids encoding them, or vectors containing such nucleic acids), (ii) small molecules (e.g., HDAC inhibitors or other agents that modify the epigenetic profile of cancer cells), or biologics (delivered as polypeptides or nucleic acids encoding them, or vectors containing such nucleic acids) that enhance the translation and / or presentation of polypeptide products that are the subject of the present invention.

[0165] Checkpoint inhibitors block normal proteins on cancer cells, or proteins on T cells that respond to them, and these inhibitors can be a particularly important type of drug to combine with CLT antigen-based therapies in an attempt to overcome one of cancer's main defenses against immune system attacks.

[0166] Accordingly, one aspect of the present invention includes administering the polypeptide, nucleic acid, vector, antigen-binding polypeptide, composition, T cells, T cell population, or antigen-presenting cells of the present invention in combination with a checkpoint inhibitor. Examples of checkpoint inhibitors are selected from PD-1 inhibitors such as pembrolizumab (Keytruda) and nivolumab (Opdivo), PD-L1 inhibitors such as atezolizumab (Tecentriq), avelumab (Bavencio) and durvalumab (Imfinzi), and CTLA-4 inhibitors such as ipilimumab (Yervoy).

[0167] Interferons (α, β, and γ, etc.) are a family of proteins produced in very small amounts by the body. Interferons can delay or halt cancer cell division, reduce the ability of cancer cells to defend themselves from the immune system, and / or enhance several aspects of the adaptive immune system. Interferons are usually administered by subcutaneous injection, for example, into the thigh or abdomen.

[0168] Accordingly, one aspect of the present invention includes administration of the polypeptide, nucleic acid, vector, antigen-binding polypeptide, or composition of the present invention in combination with interferon, such as interferon-α.

[0169] Furthermore, different forms of the present invention may be combined; for example, the polypeptide, nucleic acid, and vector of the present invention may be combined with the APC, T cell, or T cell population of the present invention (as described below). One or more of the present invention may also be combined with conventional anti-cancer chemotherapy and / or radiotherapy.

[0170] (diagnosis) In another aspect, the present invention provides a method of using one or more polypeptides or nucleic acids of the present invention for diagnosing cancer, particularly melanoma, such as cutaneous melanoma, or for diagnosing a human subject suitable for treatment with the polypeptides, nucleic acids, vectors, antigen-binding polypeptides, adoptive cell therapy, or compositions of the present invention.

[0171] Accordingly, the present invention provides a method for diagnosing that a person has cancer, comprising the steps of determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NO: 1 and its immunogenic fragment or variant (for example, any one sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 6), or a nucleic acid encoding that polypeptide sequence (for example, selected from the sequences of SEQ ID NO: 3 and 4), and diagnosing that the person has cancer if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells. As used herein, "overexpressed" in cancer cells means that the expression level in cancer cells is higher than in normal cells.

[0172] The present invention provides a method for diagnosing whether a person has cancer that is cutaneous melanoma or uveal melanoma, comprising the steps of: determining whether the cancer cells express a polypeptide sequence selected from Sequence ID No. 1 and its immunogenic fragment or variant thereof, or a nucleic acid encoding the polypeptide sequence; and diagnosing that the person has cancer that is cutaneous melanoma or uveal melanoma if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells.

[0173] Overexpression can be determined by referencing the levels of the nucleic acid or polypeptide of the present invention in a control human subject known to be cancer-free. Therefore, overexpression indicates that the nucleic acid or polypeptide of the present invention is detected at significantly higher levels (e.g., 30%, 50%, 100%, or 500% higher) in the test subject than in the control subject. If the control human subject has undetectable levels of the nucleic acid or polypeptide of the present invention, a diagnosis can be made at the time of detection of the nucleic acid or polypeptide of the present invention.

[0174] The present invention also relates to a method for treating a person suffering from cancer: (a) A step of determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NO: 1 and its immunogenic fragment or variant thereof (e.g., any sequence of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6), or a nucleic acid encoding the polypeptide (e.g., selected from sequences of SEQ ID NO: 3 and 4); and if they do express, (b) The method described above, comprising the step of administering to a human being the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cells, antigen-presenting cells, antigen-binding polypeptide, or cytotoxic cells of the present invention.

[0175] Similarly, isolated from tumors of humans suffering from cancer, (a) The sequence of sequence number 1; or (b) Variants of the sequence of (a); and, (c) Immunogenic fragment of the sequence in (a), The use of a polypeptide comprising a sequence selected from, or the use of a nucleic acid encoding such polypeptide, is provided as a biomarker for determining whether a person would be suitable for treatment with a vaccine comprising the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cells, antigen-presenting cells, antigen-binding polypeptide, or cytotoxic cells of the present invention. More accurately, the cancer is melanoma, specifically cutaneous melanoma.

[0176] The present invention also includes a polypeptide sequence selected from the following: (a) the sequence of sequence number 1; and (b) Variants of the sequence in (a); and (c) Immunogenic fragment of the sequence in (a), For example, the polypeptide contains or consists of any one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6, and for example, the nucleic acid contains or consists of a sequence selected from any one of SEQ ID NOs: 3 and 4; and The present invention provides a method or use for the cancer, which is uveal melanoma.

[0177] Preferably, the polypeptide of the present invention has a sequence selected from SEQ ID NO: 1, or a fragment such as an immunogenic fragment thereof (for example, any one sequence of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6). Preferably, the nucleic acid of the present invention has or contains a sequence selected from either SEQ ID NOs: 3 or 4.

[0178] Kits for detecting the presence of nucleic acids are well known. For example, a kit containing at least two oligonucleotides that hybridize to a single polynucleotide may be used in a real-time PCR (RT-PCR) reaction to enable the detection and semi-quantification of specific nucleic acids. Such kits may enable the detection of PCR products by the generation of a fluorescent signal as a result of Förster resonance energy transfer (FRET) (e.g., TaqMan® kits) or by binding to double-stranded DNA (e.g., SYBR® Green kits). Some kits (e.g., those containing TaqMan® probes that span multiple exons of target DNA) enable the detection and quantification of mRNA, e.g., the transcript-encoding nucleic acids of the present invention. Assays using certain kits may be set up in a multiplex format to detect multiple nucleic acids simultaneously in one reaction. Kits for detecting active DNA (i.e., DNA with specific epigenetic features that indicate expression) may also be used. Additional components that may be present in such kits include diagnostic reagents or reporters that facilitate the detection of the nucleic acids of the present invention.

[0179] The nucleic acids of the present invention may also be detected by liquid biopsy using a blood sample from a patient. Such a procedure provides a non-invasive biopsy as an alternative to surgical biopsy. Plasma can be isolated from such a blood sample and analyzed for the presence of the nucleic acids of the present invention.

[0180] The polypeptide of the present invention may be detected using an antigen-specific antibody in an ELISA-type assay, which involves detecting the polypeptide in a homogenized preparation of a patient's tumor sample. Alternatively, the polypeptide of the present invention may be detected by immunohistochemical analysis, which involves examining sections of a patient's tumor sample stained with a suitably labeled antibody preparation using a light microscope to identify the presence of the polypeptide antigen. As a further alternative, the polypeptide of the present invention may be detected by immunohistochemical analysis, which involves examining sections of a patient's tumor sample stained with a suitably labeled antibody preparation using a light microscope to identify the presence of the polypeptide antigen.

[0181] The polypeptides of the present invention may also be detected by determining whether they can stimulate T cells that have increased reactivity to the polypeptide.

[0182] A method for treating human cancer, particularly melanoma, such as cutaneous melanoma, comprises (i) detecting the presence of the nucleic acid or polypeptide of the present invention, and (ii) administering the nucleic acid, polypeptide, vector, cells, T cells or T cell population or composition of the present invention to the subject (preferably administering the same nucleic acid or polypeptide or fragment thereof that was detected).

[0183] A method for treating human cancer, particularly melanoma, such as cutaneous melanoma, also comprises administering the nucleic acid, polypeptide, vector, cells, T cells or T cell population or composition of the present invention to a subject in which the presence of the nucleic acid or polypeptide of the present invention has been detected (preferably the same as the present invention).

[0184] In particular, cancers that are diagnosed and, if possible, treated are melanomas, such as cutaneous melanomas. If the polypeptide of the present invention, which is Sequence ID No. 1 or a fragment thereof, is detected, the cancer may be cutaneous melanoma or uveal melanoma.

[0185] (Special Embodiment) In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 1. An example of a fragment comprises or consists of any one of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6. An example of a nucleic acid encoding the polypeptide sequence comprises or consists of SEQ ID NO: 3 or 4. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes are provided. These nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used in the treatment of cancer, particularly melanoma, e.g., cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided. [Examples]

[0186] (Examples) (Example 1 - CLT specific) The objective was to identify cancer-specific transcripts consisting entirely or partially of LTR elements. As a first step, a comprehensive, cross-cancer-type transcriptome was assembled and analyzed de novo. To achieve this, RNA sequencing reads from 768 patient samples, representative of a wide variety of cancer types (24 sex-balanced samples, each derived from 32 cancer types (31 primary and 1 metastatic melanoma); Table S1), obtained from the Cancer Genome Atlas (TCGA) Consortium, were used as genome guide assemblies. These sex-balanced (excluding sex-specific tissues) samples were quality-trimmed (Q20) and length-filtered using cutadapt(v1.13) (Marcel M., 2011, EMBnet J., 17:3) as an adapter (both reads were 35 or more nucleotide pairs), and then KMER normalization (k=20) was performed using khmer(v2.0) (Crusoe et al., 2015, F1000Res., 4:900) with maximum and minimum depths of 200 and 3, respectively. Reads were mapped to GRCh38 using STAR(2.5.2b) with the same settings as used throughout TCGA, and genome-guided assembly was performed using Trinity(v2.2.0) (Trinity, Grabherr, MG et al., 2011, Nat. Biotechnol., 29:644-52) with the built-in in silico depth normalization disabled. Most of the assembly process was completed within 256GB RAM on a 32-core HPC node, and failed processes were rerun using a 1.5TB RAM node. The resulting contigs were poly(A) trimmed (using trimpoly in SeqClean v110222) and entropy filtered (≧0.7) to remove low-quality, artificial contigs (bbduk in BBMap v36.2). For each cancer type, the original 24 samples were quasi-mapped to a cleaned assembly using Salmon (v0.8.2 or v0.9.2) (Patro, R., et al., 2017, Nat. Methods, 14:417-419), and contigs showing only transcript expression (TPM) < 0.1 per million were removed.The remaining components were mapped to GRCh38 using GMAP (v161107) (Wu et al., 2005, Bioinf., 21:1859-1875), and contigs that did not align with more than 85% identity over more than 85% of their length were removed from the assembly. Finally, the assembly of all cancer types together was flattened and merged using gffread (Cufflinks v2.2.1) (Trapnell et al., 2010, Nat. Biotech., 28:511-515) to obtain the longest continuous transcript. This assembly process was specifically designed to allow evaluation of repeating elements, so monoexonal transcripts were retained but flagged. The completeness and quality of the transcript assembly were evaluated by comparison with GENCODE v24basic and MiTranscriptome1 (Iyer et al., 2015, Nat. Genet., 47:199-208). The list of unique splice sites indicated by GENCODE was compiled, and it was checked whether these splice sites existed within the transcriptome assembly within a 2-nucleotide grace window. This process identified 1,001,931 transcripts, of which 771,006 were spliced ​​and 230,925 were monoexonal.

[0187] Separately, the assembled contigs were overlaid with genome repeat sequence annotation to identify transcripts containing LTR elements. LTR and non-LTR elements were annotated as previously described (Attig et al., 2017, Front. In Microbiol., 8:2489). Briefly, a hidden Markov model (HMM) representing a known human repeat family was used (Dfam 2.0 library v150923), GRCh38 was annotated using RepeatMasker Open-3.0 (Smit, A.,R. Hubley and P. Green, http: / / www.repeatmasker.org, 1996-2010), and the resulting dataset was constructed using nhmmer (Wheeler et al., 2013, Bioinform., 29:2487-2489). HMM-based scanning improves annotation accuracy compared to BLAST-based methods (Hubley et al., 2016, Nuc. Acid. Res., 44:81-89). Since RepeatMasker annotates LTRs and internal regions separately, the tabular output was analyzed to merge adjacent annotations for the same element. This process yielded 181,967 transcripts containing one or more complete or partial LTR elements.

[0188] The total product per million units (TPM) of all transcripts was estimated using Salmon, and the expression within each cancer type was compared to the expression in 811 healthy tissue samples (from TCGA where available, or from GTEx (The Genotype-Tissue Expression Consortium, 2015, Science, 348:648-60), tissue-matched healthy controls for all cancer types). A transcript was considered expressed within a cancer if it was detected at more than 1 TPM in any sample, and was considered cancer-specific if the following criteria were met: (i) expressed in 6 or more of the 24 samples for each cancer type; (ii) expressed at less than 10 TPM in 90% or more of all healthy tissue samples; (iii) expressed at more than 3 times the median expression in any control tissue type in the target cancer type; and (iv) expressed at more than 3 times the 90th percentile in each available healthy tissue in the target cancer type.

[0189] Next, we cross-referenced a list of cancer-specific transcripts with a list of transcripts containing complete or partial LTR elements to create a list of 5,923 transcripts that met all the criteria (referred to as CLT for cancer-specific LTR element-spanning transcripts).

[0190] Further curation was performed on 403 CLTs specifically expressed in melanoma, excluding those corresponding to potentially misassembled contigs and cytogene assemblies. Additional manual evaluation was performed to confirm that the splicing patterns were supported by the original RNA sequencing reads. CLTs were further triaged, and those with a median expression level exceeding 1 TPM in any GTEx-normal tissue were discarded. Of the 403 CLTs related to cutaneous melanoma, 97 passed through these filters.

[0191] (Example 2 - Immunopeptide analysis) Mass spectrometry (MS)-based immunopeptideome analysis is a powerful technique that enables the direct detection of specific peptides associated with HLA molecules (HLAp) presented on the cell surface. This technique involves affinity purification of HLAp from biological samples such as cells or tissues by anti-HLA antibody capture. The isolated HLA molecules and bound peptides are then separated from each other, and the eluted peptides are analyzed by nano-ultrafast liquid chromatography-mass spectrometry (nUPLC-MS) (Freudenmann et al., 2018, Immunology 154(3):331-345). In mass spectrometry, specific peptides with a defined charge-to-mass ratio (m / z) are selected, isolated, fragmented, and then subjected to a second mass spectrometry (MS / MS) to obtain the m / z of the detected fragment ions. By then comparing these fragmentation spectra (MS / MS), the amino acid sequence of the selected peptide that generated the detected fragment ions can be precisely identified.

[0192] MS / MS spectral interpretation and subsequent peptide sequence identification depend on the agreement between experimental data and theoretical spectra constructed from peptide sequences contained in reference databases. While it is possible to search MS data using predefined lists corresponding to all open reading frames (ORFs) derived from known transcriptomes, or even the entire genome (Nesvizhskii et al., 2014, Nat. Methods 11:1114-1125), matching these enormous sequence databases leads to a very high false-detection rate (FDR), limiting the identification of presented peptides. Furthermore, technical issues (e.g., leucine mass = isoleucine mass) and theoretical issues (e.g., peptide splicing (Liepe et al., 2016, Science 354(6310):354-358)) increase the limitations associated with the use of enormous databases, such as databases constructed from known transcriptomes or the entire genome. Therefore, in practice, it is extremely difficult to perform accurate immunopeptidomic analysis to identify novel antigens without referring to a clearly defined set of possible polypeptide sequences (Li et al., 2016, BMC Genomics 17 (Augmented 13):1031).

[0193] Accordingly, the inventors constructed a database of all predicted polypeptide sequences (ORFs) of 10 residues or more derived from 97 cutaneous melanoma CLTs in Example 1. This yielded 2,269 ORFs ranging in length from 10 to 207 amino acids.

[0194] Bassani-Sternberg et al. compared MS / MS data collected from HLA-binding peptide samples derived from 25 cutaneous melanoma patients against polypeptides reported for the entire human proteome (Bassani-Sternberg et al., 2016, Nature Commun., 7:13404; 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 predicted, these peptides included those found within several tumor-associated antigens (TAAs), including PRAME, MAGEA3, and TRPM1 (melastatin).

[0195] By applying detailed knowledge of immunopeptidomic assessment, the inventors used PEAKS® software (v8.5 and vX, Bioinformatics Solutions Inc.) to match spectra from the PXD004894 HLA class I dataset alongside all polypeptide sequences found within the human proteome (UniProt). Since the majority of class I HLA-binding peptides found in cells originate from constitutively expressed proteins, simultaneous matching of these databases with the UniProt proteome helps confirm the correctness of the inventors' assignment of CLT ORF sequences to MS / MS spectra. Like other MS / MS matching software, PEAKS software quantifies each spectral assignment by assigning a significance probability (p-value) (-10 lgP; see Table 1) to that assignment.

[0196] The results of these studies identified more than 50 individual peptides, which were associated with HLA class I molecules immunoprecipitated from tumor samples from 25 patients examined by Bassani-Sternberg et al. These corresponded to amino acid sequences of CLT-derived ORFs and not to polypeptide sequences present in the known human proteome (UniProt).

[0197] The peptide spectra assigned by the PEAKS software were further manually evaluated, and this evaluation was used to confirm the spectral assignment to peptides mapped to CLT-derived ORFs, which were then defined as CLT antigens. Peptide 1 was repeatedly observed in Bassani-Sternberg MS / MS datasets mapped to 45-codon ORFs and was defined as CLT antigen 1 (Table 1; SEQ ID NO: 1).

[0198] The repeated detection of these peptides associated with HLA class I molecules indicates that CLT antigen 1 (SEQ ID NO: 1) is translated within melanoma tissue, processed via the HLA class I pathway, and finally presented to the immune system as a complex with HLA class I molecules. Table 1 shows the characteristics of peptides detected in three patients and mapped to this CLT antigen. Figure 1 shows representative MS / MS spectra obtained from one patient sample containing the peptides shown in Table 1. The top panel of Figure 1 shows MS / MS peptide fragment profiles with standard MS / MS annotations (b: N-terminal fragment ion; y: C-terminal fragment ion; -H2O: water loss; -NH3: ammonia loss; [2+]: divalently charged peptide ion; pre: unfragmented precursor peptide ion; a n -n: internal fragment ion). The upper panel of Figure 1 shows the extraction of the most abundant fragment ion peaks, which were assigned by PEAKS software and obtained from the PRIDE database (Bassani-Sternberg et al., 2016, Nature Commun., 7: 13404; database link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894). The lower panel of Figure 1 shows a rendering of the spectrum showing the positions of the linear peptide sequence mapped to the fragment ions. This spectrum contains numerous fragments that precisely match the peptide sequence (SEQ ID NO: 2) found in these analyses, as it is consistent with the high -10 lgP score assigned to the peptide in Table 1.

[0199] The peptides detected in relation to HLA class I in Table 1 were evaluated using NetMHCpan 4.0 prediction software (http: / / www.cbs.dtu.dk / services / NetMHCpan / ) to determine the predicted binding strength to HLA class I type A and type B molecules in patients. The results of these prediction studies showed that this peptide strongly binds to the HLA class I B07:02 allele found in both types of patients (see Table 2). The fact that this CLT antigen-derived peptide was expected to bind to one of the HLA alleles found in both types of patients is consistent with these detection results.

[0200] To provide further certainty in the assignment of tumor tissue-derived MS spectra to the peptide sequence discovered by the inventors, a peptide having this discovered sequence was synthesized and nUPLC-MS was performed using the same conditions as those applied to the tumor samples in the original study. 2 (Bassani-Sternberg et al., 2016, Nature Commun., 7: 13404). A spectral comparison of this synthetic peptide and the selected endogenous peptide is shown in Figure 2. In this figure, the upper spectrum corresponds to the tumor sample (from the PRIDE database (Bassani-Sternberg et al., 2016, Nature Commun., 7: 13404; database link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894)), and the lower spectrum corresponds to a synthetically prepared peptide of the same sequence. The selected m / z values ​​of the detected ion fragments are shown in the upper / lower fragment peaks of these MS / MS spectra. This figure clarifies the precise alignment of the fragments (the slight difference between experimentally determined m / z values ​​between the tumor-derived and synthetic peptide-derived fragment ions is within the fragment tolerance range of <0.05 Daltons) and confirms the reliability of the assignment of the tumor tissue-derived spectrum to the CLT-encoded peptide spectrum.

[0201] In summary, the data shown in Tables 1 and 2, and Figures 1 - 2 provide very strong support for the translation, processing and presentation of the corresponding CLT antigen within melanoma patients.

[0202] To further confirm the cancer specificity of this CLT, the inventors processed 37 normal tissue samples (10 normal skins, 9 normal lungs and 18 normal breast tissues) and prepared them for immunopeptidome analysis. The inventors collated the spectra of the HLA class I dataset from these normal tissue samples and searched for all possible peptide sequences derived from the polypeptide sequence of CLT antigen 1. Peptides derived from CLT antigen 1 were not detected within the set of normal tissue samples (Table 3), additionally confirming that this CLT exhibits cancer - specific expression.

[0203] In summary, the iterative identification of peptides of the immunopeptidome derived from the predicted ORF indicates that this CLT (SEQ ID NO: 3) is translated into a polypeptide (SEQ ID NO: 1; referred to as CLT antigen 1) within tumor tissue. Thus, this antigen is processed by the cell's immune surveillance apparatus, the peptide component (e.g., SEQ ID NO: 2) is loaded onto HLA class I molecules, enabling the resulting peptide / class I complex to be recognized by T cells, which can target that cell for lysis. Therefore, CLT antigen 1 and its fragments are predicted to be useful in various therapeutic modalities for the treatment of melanoma in patients whose tumors express these antigens.

[0204] (Table 1: List of peptides identified by immunopeptidome analysis of SKCM tumor samples, and cross - reference with CLT antigen names and SEQ ID NOs) [Table 1] 1 : HLA class I peptides identified by mass spectrometry. 2 : Bassani - Sternberg et al., 2016, Nature Comm., 7: 13404. 3 :Calculated peptide mass. 4 The PEAKS(trademark) program-101gP value indicates the peptide that best matched the patient / peptide for which a detection spectrum greater than 1 was obtained. 5 : The number of spectra in which the peptide was detected. 6 : The deviation between the observed mass and the calculated mass; for peptides from which spectra greater than 1 were obtained, the selected ppm value is shown.

[0205] (Table 2: Predicted NetMHCpan4.0 binding of peptides identified by mass spectrometry to patient HLA types) [Table 2] 1 :Bassani-Sternberg et al., 2016, Nature Comm., 7: 13404; NA = Not applicable 2 Predicted rank score (%) from NetMHCpan 4.0; a score less than 0.5% predicts strong coupling.

[0206] (Table 3: Number of peptides derived from CLT antigens 1-8 in a normal tissue sample set) [Table 3]

[0207] The results shown in whole or in part in Examples 1 and 2 of this specification are based on data generated by the Cancer Genome Atlas (TCGA) Research Network (http: / / cancergenome.nih.gov / ) and the Genotype-Tissue Expression (GTEx) Project (supported by the Common Fund of the Office of the Director of the National Institutes of Health (NIH), as well as by the NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS).

[0208] (Example 3 - Assay to demonstrate T cell specificity to CLT antigen in melanoma patients) (a) Staining of reactive T cells with CLT antigen peptide pentamer) The presence and activity of CLT antigen-specific circulating CD8 T cells in melanoma patients can be measured using HLA class I / peptide pentamer ("pentamer") staining and / or in vitro toxicization assays. Therefore, the application of these techniques to CLT antigens discovered using the methods described in Examples 1 and 2 (Tables 1-3, Figures 1-2) can be used to demonstrate the presence of a treatment-related T cell response to CLT antigens in cancer patients.

[0209] For these studies, CD8 T cells isolated from patient blood are grown using various culture methods, such as anti-CD3 and anti-CD28 coated microbeads and interleukin-2. The grown cells can then be stained with a CLT peptide pentamer for the specific CLT antigen reactivity of their T cell receptor, which consists of a pentamer of an HLA class I molecule that binds to the relevant CLT antigen peptide within the peptide binding groove of the HLA molecule. Binding is measured by detection using antibody fragments conjugated with phycoerythrin or allophycocyanin, which are specific to the coiled-coil multimerization domain of the pentamer structure. In addition to this pentamer staining, further matching can be performed with surface markers such as the memory marker CD45RO and the lysosomal release marker CD107a. The positive binding affinity between the pentamer and specific surface markers can be used to infer both the number and state (memory vs. naive / stem) of the pentamer-reactive T cell population.

[0210] Pentamer-stained cells may also be sorted and purified using a fluorescence-activated cell sorter (FACS). The sorted cells can then be further tested in in vitro killing assays for their ability to kill target cells. These assays include a CD8 T cell population and a fluorescently labeled target cell population. In this case, the CD8 population consists of CLT antigen-specific cells or CD8 T cells that are pentamer-sorted and specific to positive control antigens known to induce a potent killing response, such as Mart-1. The target cells in these studies are peptide-pulsed HLA-A cells. * T2 cells expressing O2 are subjected to peptide pulses, and HLA-A * 02, 03, or B * The test can include C1R cells transfected with 07, or melanoma cell lines already shown to express CLT / CLT antigen, or tumor cells from a patient. The peptide used to pulse the T2 or C1R cells may include a CLT antigen peptide or a positive control peptide. Target cells may be double-labeled with a vital dye such as a red nuclear stain (nuclight rapid red) that is taken up by the nucleus of healthy cells. Additional evidence of attack on target cells by specific T cells can be shown by a green caspase 3 / 7 activity indicator indicating caspase 3 / 7-mediated apoptosis. Thus, when target cells are killed by CD8 T cell-mediated apoptosis, the caspase 3 / 7 activity inherent to apoptosis causes the target cells to lose their red fluorescence and emit green fluorescence. Therefore, the application of such a killing assay to pentamer-selected CLT antigen-specific CD8 T cells can be used to demonstrate the cytotoxic activity of CLT antigen-specific T cells in in vitro culture of melanoma patient T cells.

[0211] (b) HERVfest analysis of T cell specificity in melanoma patients Specific T cell enhancement (fest) techniques have been used to identify specific tumor-derived epitopes present in the “mutation-associated novel antigen” (MANA) repertoire found in tumor cells of patients responding to checkpoint inhibitor therapy (Anagnostou et al., Cancer Discovery 2017; Le et al., Science 2017). Applying this technique to CLT antigens discovered using the methods described in Examples 1 and 2 (Tables 1 and 2, Figures 1-2) allows for confirmation of the presence of a treatment-related T cell response to CLT antigens in cancer patients.

[0212] Similar to other assays for identifying epitope-specific T cells within immunohistochemically exposed subjects (e.g., ELISPOT), the "fest" technology derives its specificity by growing homologous T cells in an in vitro culture containing antigen-presenting cells and suitable antigenic peptides. What distinguishes this technology from other immunological assays is its use of next-generation sequencing (specifically, TCRseq targeting the TCR-VβCDR3 region) of T cell receptor (TCR) mRNA present in these amplified cultures to detect specific TCRs grown in cells cultured with the target peptide (pre-selected to match the patient's HLA type using a standard HLA-binding algorithm). Application of TCRseq to tumor tissue from the same patient, collected after successful checkpoint inhibitor therapy, can then be used to determine which TCRs / T cells detected in the peptide-stimulated culture in vitro are similarly present in the immunosuppressive sites of the cancer. In the case of MANAfest, this method is used to identify specific TCRs that recognize novel MHC-presented antigen peptides produced in each patient's tumor and detected within T cells in the patient's tumor, enabling the identification of functionally relevant novel antigen peptides from thousands of possible mutant peptides discovered by whole exome sequencing of normal tissue and tumor tissue from each patient (Le et al., Science 2017).

[0213] The application of the MANAfest technology (Anagnostou et al., 2017, Cancer Discovery) to CLT antigens is carried out as follows: Step 1: Identify peptides within the CLT antigen that are predicted to contain epitopes that efficiently bind to the selected HLA supertype. Step 2: Select PBMCs from appropriate patients and match them to the peptide library selected in Step 1 by HLA type. Step 4: Separate these patient PBMCs into T cell fractions and non-T cell fractions. Non-T cells are radioactively treated (to prevent proliferation), returned to patient T cells, then divided into 20-50 samples and grown for 10-14 days with T cell growth factor and individual CLT-specific synthetic peptides (selected in Step 1). Step 4: Perform TCRseq (sequencing of epitope-specific TCR-Vβ CDR3 sequences) on all wells to identify amplified homologous T cells / TCRs in the presence of test peptides. The specificity of these TCRs is determined by comparing the TCRs detected in unamplified T cells / amplified T cells using TCRseq. The data obtained from this step can be used to identify which peptides triggered the patient's immune response. Step 5: TCRseq is performed on tumor samples to determine which of the specifically amplified TCRs homed to the tumors of patients who responded to checkpoint inhibitor therapy, providing evidence that T cells with these TCRs may contribute to the effectiveness of checkpoint inhibitor therapy.

[0214] (Example 4 - Assay to demonstrate that CLT antigen-specific high-affinity T cells have not been removed from the normal target T cell repertoire) The ELISPOT assay may be used to show that CLT antigen-specific CD8 T cells are present within the normal T cell repertoire of healthy individuals, and therefore can also be used to show that they were not removed by central tolerance due to the expression of cancer-specific CLT antigen in the naive and thymic tissues of these patients. This type of ELISPOT assay involves several steps. Step 1: CD8 T cells and CD14 monocytes are isolated from the peripheral blood of normal donors, and these cells can be HLA-typed to match the specific CLT antigen to be tested. CD8 T cells can be further subdivided into naive and memory subtypes using magnetically labeled antibodies against the memory marker CD45RO. Step 2: CD14 monocytes are pulsed with individual or pooled CLT antigen peptides for 3 hours and then co-cultured with CD8 T cells for 14 days. Step 3: The proliferated CD8 T cells are isolated from these cultures and re-stimulated overnight with fresh monocytes pulsed with the peptide. These peptides may include individual CLT antigen peptides, unrelated control peptides, or peptides known to elicit a potent response to infectious (e.g., CMV, EBV, influenza, HCV) or auto (e.g., MART-1) antigens. Restimulation is performed on a plate coated with an anti-interferon-gamma (IFNγ) antibody. This antibody captures any IFNγ secreted by peptide-stimulated T cells. After overnight activation, the cells are washed off the plate, and the captured IFNγ is detected with further anti-IFNγ antibodies and standard colorimetric dyes. Dark spots remain where IFNγ-producing cells were initially present on the plate. Data obtained from this assay include the number of spots, median spot size, and median spot intensity. These represent the frequency of IFNγ-producing T cells and the amount of IFNγ per cell. Furthermore, the magnitude of the response to the CLT antigen can be measured using the stimulation index (SI), which is the specific response, measured as the number of spots or median spot size, divided by the background response to monocytes that do not contain the specific peptide.The criteria for evaluating stimulation intensity are derived by multiplying the stimulation index of the number of spots by the stimulation index of the spot intensity. Using this method, by comparing the response to the CLT antigen and the response to the control antigen, it is possible to show that naive subjects contain a robust repertoire of CLT antigen-reactive T cells, which is amplified by vaccination with CLT antigen-based immunogenic agents. Table 4 provides a list of CLT antigen-derived peptides that induced a significant CD8 T cell response from HLA-matched normal blood donors. Representative results are shown in Figure 3. The horizontal bar represents the mean of the data. M+t indicates a negative control (monocytes and T cells) without peptides. CEF indicates a positive control (a mixture of 23 CMV, EBV, and influenza peptides). Statistical significance was measured using one-way ANOVA with the Kruskal-Wallis test, and repeated measures corrected with Dunn correction. Figure 3 provides an example of a significant CD8 T cell response from normal blood donors to the HLA-A*03:01-restricted peptide from CLT antigen 1 (SEQ ID NO: 6; RPDLILLQL CLT001 in Figure 3).

[0215] (Table 4: CLT antigen-derived peptides that induce a significant CD8 T cell response from HLA-matched normal blood donors) [Table 4]

[0216] (Example 5 - Assay for verifying CLT expression in melanoma cells) a) qRT-PCR validation of CLT expression in melanoma cells Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for determining the amount of a specific transcript present in RNA extracted from a given biological sample. A specific nucleic acid primer sequence is designed for the target transcript, and then (subeqeuntly) the interprimer region is amplified through a series of thermal cycling reactions and quantified fluorescently using an intercalator dye (SYBR Green). The primer pairs were manipulated against CLT and assayed against RNA extracted from melanoma cell lines. Non-melanoma cell lines were used as negative controls. Specifically, melanoma cell lines COLO 829 (ATCC reference number CRL-1974), MeWo (ATCC reference number HTB-65), SH-4 (ATCC reference number CRL-7724) and control cell lines HepG2 (hepatocellular carcinoma, ATCC reference number HB-8065), Jurkat (T-cell leukemia, ATCC reference number TIB152), and MCF7 (adenocarcinoma, ATCC reference number HTB-22) were grown in vitro, and RNA was extracted at a rate of 1 × 10⁶. 6 Extracts were obtained from individual flash-frozen cells and reverse-transcribed into cDNA. Following standard techniques, qRT-PCR analysis with SYBR Green detection was performed on two regions of each CLT and using primers designed for the reference gene. Relative quantification (RQ) values ​​were calculated as follows: RQ=2[Ct(reference)-Ct(target)]

[0217] These experimental results are shown in Figure 4. Figure 4 shows the results of a qRT-PCR assay using the primer set (88+89), targeting CLT (SEQ ID NO: 3), which encodes CLT antigen 1 on RNA extracted from three melanoma cell lines and three non-melanoma cell lines. These results confirmed that CLT was specifically expressed in RNA extracted from melanoma cell lines compared to non-melanoma cells. This CLT was detected in two-thirds of the melanoma cell lines tested.

[0218] b) RNAScope validation of CLT expression in in situ melanoma cells In situ hybridization (ISH) allows for the visualization of the presence and expression level of a given transcript under histopathological conditions. Conventional RNA ISH assays also involve the in situ recognition of native RNA molecules using oligonucleotide probes specific to short chains of desired RNA sequences, which are visualized by signals generated by a combination of antibody- or enzyme-based colorimetric reactions. RNAScope is a recently developed in situ hybridization-based technique with more advanced probe chemistry, ensuring the specificity of the generated signals and enabling highly sensitive, single-molecule visualization of target transcripts (Wang et al., 2012 J Mol Diagn. 14(1):22-29). When transcript molecules are positively stained, small red dots appear within a given cell, and multiple dots indicate the presence of multiple transcripts.

[0219] An RNAScope probe was designed for CLT, and sections of 12 formalin-fixed, paraffin-embedded cutaneous melanoma tumor cores were assayed. Expression signal scoring was performed on representative images from each core, as follows: • The percentage of cells that show positive staining with the CLT probe should be rounded up to the nearest ten. The evaluation level of expression per cell across a given section is as follows: •0 = No dyeing • 1 = 1-2 dots per cell ·2 = 2-6 dots per cell • 3 = 6-10 dots per cell • 4 = More than 10 dots per cell.

[0220] Expression of each CLT was detected in tumor cores from numerous different patients, and the findings were independently validated from tumor-derived RNA-seq data. The relative uniformity of expression within tumor tissue across the given samples was confirmed, and its presence across multiple patients was highlighted (Table 5).

[0221] (Table 5 - Scoring of RNAScope within the tissue cores of melanoma patients)

Table 5

[0222] Throughout this specification and the following claims, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising", are to be understood to imply the inclusion of the stated integer, step, group of integers or group of steps but not the exclusion of any other integer, step, group of integers or group of steps.

[0223] All patents, patent applications and references cited in the specification of the present invention are hereby incorporated by reference in their entirety. The present invention encompasses all combinations of the groups of preferred and more preferred, suitable and more suitable, and embodiments listed above.

[0224] (Sequence Listing) (Chemical Formula 1) TIFF0007887250000006.tif40170TIFF0007887250000007.tif249170TIFF0007887250000008.tif141170 This application provides the invention in the following embodiments. (Aspect 1) (a) the sequence of sequence number 1; and, (b) Variants of the sequence in (a); and (c) Immunogenic fragment of the sequence in (a), An isolated polypeptide containing a sequence selected from the above. (Aspect 2) The isolated peptide according to embodiment 1, comprising or consisting of any one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6. (Aspect 3) The isolated polypeptide according to Embodiment 1 or Embodiment 2, wherein the isolated polypeptide is fused to a second polypeptide or further polypeptide selected from (i) one or more other polypeptides according to Embodiment 1 or Embodiment 2, (ii) another polypeptide that is a melanoma-associated antigen, (iii) a polypeptide sequence capable of enhancing the immune response (i.e., an immunostimulatory sequence), and (iv) a polypeptide sequence that includes, for example, a universal CD4 helper epitope and can provide potent CD4+ support that increases the CD8+ T cell response to the antigen epitope. (Aspect 4) An isolated nucleic acid encoding a polypeptide as described in any one of embodiments 1 to 3. (Appendix 5) The nucleic acid described in embodiment 4, which is DNA. (Aspect 6) The nucleic acid according to embodiment 5, comprising a sequence selected from either sequence number 3 or 4, or comprising said sequence. (Aspect 7) The nucleic acid according to embodiment 6, which is a codon optimized for expression in human host cells. (Pattern 8) The nucleic acid described in embodiment 4, which is RNA. (Aspect 9) The nucleic acid according to embodiment 4, 5, 7, or 8, which is an artificial nucleic acid sequence. (Aspect 10) A vector comprising the nucleic acid described in any one of embodiments 4 to 9. (Aspect 11) The vector according to embodiment 10, comprising DNA encoding a regulatory element suitable for enabling the transcription of translationally active RNA molecules in human host cells. (Aspect 12) A viral vector, as described in embodiment 10 or embodiment 11. (Aspect 13) The vector according to embodiment 12, which is an adenovirus vector, adeno-associated virus (AAV), alphavirus, herpesvirus, arenavirus, measles virus, poxvirus, paramyxovirus, lentivirus, and rhabdovirus vector. (Aspect 14) An immunogenic pharmaceutical composition comprising a polypeptide, nucleic acid, or vector according to any one of embodiments 1 to 13, together with a pharmaceutically acceptable carrier. (Aspect 15) A vaccine composition comprising a polypeptide, nucleic acid, or vector according to any one of embodiments 1 to 13, together with a pharmaceutically acceptable carrier. (Aspect 16) A composition according to embodiment 14 or embodiment 15, comprising one or more immunostimulants. (Aspect 17) The composition according to embodiment 16, wherein the immunostimulant is selected from aluminum salts, saponins, immunostimulant oligonucleotides, oil-in-water emulsions, aminoalkylglucosaminide 4-phosphate, lipopolysaccharides and their derivatives, and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and interferon. (Aspect 18) A sterile composition suitable for parenteral administration, as described in any one of embodiments 14 to 17. (Aspect 19) A polypeptide, nucleic acid, vector, or composition according to any one of embodiments 1 to 18, for use in pharmaceuticals. (Aspect 20) A method for increasing the immune response in a human, comprising administering to the human a polypeptide, nucleic acid, vector, or composition described in any one of embodiments 1 to 18. (Aspect 21) The method according to embodiment 20, wherein the immune response is elevated against a cancerous tumor expressing sequence selected from Sequence ID No. 1, its variants, and immunogenic fragments. (Aspect 22) A polypeptide, nucleic acid, vector, or composition according to any one of embodiments 1 to 18, for use in enhancing the immune response in humans. (Aspect 23) The polypeptide, nucleic acid, vector, or composition according to embodiment 22, wherein the immune response is elevated against a cancerous tumor expressing a corresponding sequence selected from SEQ ID NO: 1 and its immunogenic fragment or variant. (Aspect 24) A method for treating a human patient having cancer, wherein the cancer cells express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, or a method for preventing a human from developing cancer, wherein the cancer will express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, the method comprising administering to the human a corresponding polypeptide, nucleic acid, vector, or composition according to any one of embodiments 1 to 18. (Aspect 25) A polypeptide, nucleic acid, vector, or composition according to any one of embodiments 1 to 18, wherein the cancer cells express a corresponding sequence selected from Sequence ID No. 1 and its immunogenic fragment, for use in the treatment or prevention of human cancer. (Aspect 26) A polypeptide, nucleic acid, vector, or composition according to any one of embodiments 1 to 18 for use in vitro to stimulate and / or amplify T cells derived from a human with cancer, wherein the polypeptide, nucleic acid, vector, or composition is used to reintroduce the stimulated and / or amplified T cells into the human for the treatment of the human cancer. (Aspect 27) A method for treating human cancer, wherein cancer cells express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, the method comprising: taking a population of leukocytes, including at least T cells, from the human, optionally together with antigen-presenting cells; stimulating and / or amplifying the T cells in the presence of the corresponding polypeptide, nucleic acid, vector, or composition described in any one of embodiments 1 to 18; and reintroducing at least the stimulated and / or amplified T cells, which are some or all of the leukocytes, into the human. (Aspect 28) The cancer is melanoma, for example, cutaneous melanoma, according to any one of embodiments 21 and 23-27, or a polypeptide, nucleic acid, vector or composition for use. (Aspect 29) A process for preparing a population of T cells that are cytotoxic to cancer cells expressing sequences selected from Sequence ID No. 1 and its immunogenic fragments and variants, comprising: (a) obtaining T cells, optionally together with antigen-presenting cells, from a cancer patient; and (ii) stimulating and amplifying the T cell population in vitro with the corresponding polypeptide, nucleic acid, vector, or composition described in any one of embodiments 1 to 18. (Aspect 30) A T cell population that can be obtained from the process described in embodiment 29. (Aspect 31) T cells stimulated with a polypeptide, nucleic acid, vector, or composition according to any one of embodiments 1 to 18. (Aspect 32) Antigen-presenting cells modified by loading a polypeptide, nucleic acid, vector, or composition described in any one of embodiments 1 to 18 in vitro, or genetically engineered to express a polypeptide described in any one of embodiments 1 to 3. (Aspect 33) A dendritic cell, which is an antigen-presenting cell according to embodiment 32. (Aspect 34) Exosomes loaded with polypeptides produced from cells loaded with the polypeptide, nucleic acid, vector, or composition described in any one of embodiments 1 to 18, or genetically engineered to express the polypeptide described in any one of embodiments 1 to 3. (Aspect 35) A pharmaceutical composition comprising a T cell population, T cells, antigen-presenting cells, or exosomes described in any one of embodiments 30 to 34, together with a pharmaceutically acceptable carrier. (Aspect 36) A T cell population, T cell, antigen-presenting cell, or exosome according to any one of embodiments 30 to 34, for use in pharmaceuticals. (Aspect 37) A method for treating a person having cancer, wherein cancer cells express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, or a method for preventing a person from developing cancer, wherein cancer cells will express a sequence selected from SEQ ID NO: 1 and its immunogenic fragments and variants, the method comprising administering to the person a T cell population, T cells, antigen-presenting cells, exosomes, or composition according to any one of embodiments 30 to 35. (Aspect 38) A T cell population, T cells, antigen-presenting cells, exosomes, or composition according to any one of embodiments 30 to 35 for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NO: 1 and its immunogenic fragment. (Aspect 39) The cancer is melanoma, for example, cutaneous melanoma, and the process, method, or T cell population, T cells, antigen-presenting cells, exosomes, or composition for use according to any one of embodiments 29, 37, and 38. (Approach 40) An isolated antigen-binding polypeptide that is immune-specific to the polypeptide described in any one of the embodiments 1 to 3. (Aspect 41) The antigen-binding polypeptide according to embodiment 40, which is a monoclonal antibody or a fragment thereof. (Aspect 42) An antigen-binding polypeptide according to embodiment 40 or embodiment 41, which is bound to a cytotoxic component. (Aspect 43) An antigen-binding polypeptide according to any one of embodiments 40 to 42, for use in pharmaceuticals. (Aspect 44) A pharmaceutical composition comprising an antigen-binding polypeptide according to any one of embodiments 40 to 42, together with a pharmaceutically acceptable carrier. (Aspect 45) A method for treating a person suffering from cancer, wherein the cancer cells express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, or a method for preventing a person from developing cancer, wherein the cancer cells will express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, the method comprising administering to the person an antigen-binding polypeptide or composition according to any one of embodiments 40-42 and 44. (Aspect 46) An antigen-binding polypeptide or composition according to any one of embodiments 40 to 42 and 44, wherein the cancer cells express a corresponding sequence selected from SEQ ID NO: 1 and its immunogenic fragment, for use in the treatment or prevention of human cancer. (Aspect 47) The cancer is melanoma, for example, cutaneous melanoma, according to the method of embodiment 45 or embodiment 46, antigen-binding polypeptide or composition. (Aspect 48) An isolated antigen-binding polypeptide that is immune-specific to the polypeptide described in any one of the embodiments 1 to 3, or to an HLA-binding polypeptide which is a part thereof. (Aspect 49) The antigen-binding polypeptide according to embodiment 48, which is a T cell receptor or a fragment thereof. (Appearance 50) The antigen-binding polypeptide according to embodiment 48 or embodiment 49, which is bound to another polypeptide capable of binding to cytotoxic cells or other immune components within the target. (Aspect 51) Cytotoxic cells are modified to express an antigen-binding polypeptide described in any one of embodiments 48 to 50 on their surface. (Appearance 52) A cytotoxic cell, which is a T cell, as described in embodiment 51. (Aspect 53) Cytotoxic cells according to embodiment 51 or embodiment 52 for use in pharmaceuticals. (Aspect 54) A pharmaceutical composition comprising the cells described in embodiment 51 or embodiment 52. (Aspect 55) A method for treating a human patient having cancer, wherein the cancer cells express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, or a method for preventing a human from developing cancer, wherein the cancer will express a sequence selected from Sequence ID No. 1 and its immunogenic fragments and variants, the method comprising administering the cells described in aspect 51 or aspect 52 to the human. (Aspect 56) Cytotoxic cells according to embodiment 51 or embodiment 52 for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from Sequence ID No. 1 and its immunogenic fragment. (Aspect 57) A method for diagnosing whether a person has cancer: The steps include determining whether the cancer cells express a polypeptide sequence selected from Sequence ID No. 1 and its immunogenic fragment or variant, or a nucleic acid encoding the polypeptide sequence, and The method comprising the step of diagnosing a person having cancer if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells. (Pattern 58) A method for diagnosing whether a person has cancer that is cutaneous melanoma or uveal melanoma: The steps include determining whether the cancer cells express a polypeptide sequence selected from Sequence ID No. 1 and its immunogenic fragment or variant, or a nucleic acid encoding the polypeptide sequence, and The method comprising the step of diagnosing that a person has cancer, which is cutaneous melanoma or uveal melanoma, if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells. (Aspect 59) A method of treating people who have cancer: (a) A step of determining whether the cancer cells express a polypeptide sequence selected from Sequence ID No. 1 and its immunogenic fragment or variant, or a nucleic acid encoding the polypeptide (for example, selected from the sequences of Sequence ID Nos. 3 and 4); and if they do express, (b) The step of administering to the human being the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cells, antigen-presenting cells, exosomes, antigen-binding polypeptide, or cytotoxic cells as described in any one of embodiments 1 to 18, 30 to 35, 40 to 42, 44, 50, 51, and 53. The method, including the method described above. (Appendix 60) Isolated from tumors of humans suffering from cancer, (a) The sequence of sequence number 1; or (b) Variants of the sequence of (a); and, (c) Immunogenic fragment of the sequence in (a), The use of a polypeptide comprising a sequence selected from, or the use of a nucleic acid encoding said polypeptide, as a biomarker for determining whether said human would be suitable for treatment with a vaccine comprising the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cells, antigen-presenting cells, exosomes, antigen-binding polypeptide, or cytotoxic cells as described in any one of embodiments 1-18, 30-35, 40-42, 44, 51, 52, and 54. (Aspect 61) The method or use according to aspect 59 or aspect 60, wherein the cancer is melanoma, for example, cutaneous melanoma. (Aspect 62) The aforementioned polypeptide is (a) the sequence of sequence number 1; and, (b) Variants of the sequence in (a); and (c) A polypeptide comprising a sequence selected from immunogenic fragments of the sequence of (a), for example, comprising or consisting of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6, for example, the nucleic acid comprising or consisting of a sequence selected from one of SEQ ID NOs: 3 and 4; and The cancer is uveal melanoma, the method according to any one of embodiments 21 and 23-27, or a polypeptide, nucleic acid, vector or composition for use. (Aspect 63) The aforementioned polypeptide is (a) the sequence of sequence number 1; and, (b) Variants of the sequence in (a); and (c) A polypeptide comprising a sequence selected from immunogenic fragments of the sequence of (a), for example, comprising or consisting of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6, for example, the nucleic acid comprising or consisting of a sequence selected from one of SEQ ID NOs: 3 and 4; and The cancer is uveal melanoma, according to the method of embodiment 45, or the antigen-binding polypeptide or composition for use according to embodiment 46. (Personal aspect 64) The aforementioned polypeptide is (a) the sequence of sequence number 1; and, (b) Variants of the sequence in (a); and (c) A polypeptide comprising a sequence selected from immunogenic fragments of the sequence of (a), for example, comprising or consisting of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6, for example, the nucleic acid comprising or consisting of a sequence selected from one of SEQ ID NOs: 3 and 4; and The cancer is uveal melanoma, and the process, method, or T cell population, T cells, antigen-presenting cells, exosomes, or composition for use according to any one of embodiments 29, 37, and 38. (Patent 65) The aforementioned polypeptide is (a) the sequence of sequence number 1; and, (b) Variants of the sequence in (a); and (c) A polypeptide comprising a sequence selected from immunogenic fragments of the sequence of (a), for example, comprising or consisting of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6, for example, the nucleic acid comprising or consisting of a sequence selected from one of SEQ ID NOs: 3 and 4; and The method or use according to aspect 59 or aspect 60, wherein the cancer is uveal melanoma.

Claims

1. Isolated polypeptide, (A)(a) Sequence of sequence number 1; and (b) A sequence selected from immunogenic variants of the sequence of Sequence ID No. 1, which are at least 90% identical to Sequence ID No. 1 and include one or more sequences of Sequence ID No. 2, 5, and 6; or (B) The isolated polypeptide comprising any one of the sequences of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO:

6.

2. The isolated polypeptide according to claim 1, comprising the sequence of sequence number 1.

3. An isolated polypeptide according to claim 1 or claim 2, (i) (a) the sequence of SEQ ID NO: 1; (b) an immunogenic variant of the sequence of SEQ ID NO: 1 that is at least 90% identical to SEQ ID NO: 1 and includes one or more sequences of SEQ ID NO: 2, 5, and 6; and (c) an immunogenic fragment of the sequence of SEQ ID NO: 1 that includes at least nine consecutive amino acids of SEQ ID NO: 1 and includes one amino acid sequence of any one of SEQ ID NO: 2, 5, and 6; one or more other polypeptides comprising a sequence selected from these; (ii) Other polypeptides that are melanoma-associated antigens, (iii) polypeptide sequences capable of enhancing the immune response, and (iv) A polypeptide sequence containing a universal CD4 helper epitope that can provide potent CD4+ support to increase the CD8+ T cell response to an antigen epitope, The isolated polypeptide is fused to a further polypeptide selected from the above.

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

5. A vector comprising the nucleic acid described in claim 4.

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

7. The composition according to claim 6, for use in enhancing the immune response in humans.

8. The composition for use according to claim 7, wherein the immune response is elevated against a cancerous tumor expressing SEQ ID NO: 1 and a corresponding sequence selected from immunogenic variants of the SEQ ID NO: 1 sequence, which is at least 90% identical to SEQ ID NO: 1 sequence and includes one or more sequences of SEQ ID NO: 2, 5, and 6.

9. The composition according to claim 6, for use in vitro to stimulate and / or amplify T cells derived from a person with cancer, and for then reintroducing the stimulated and / or amplified T cells into the person in order to treat the person's cancer.

10. A process for preparing a population of T cells that are cytotoxic to cancer cells expressing Sequence ID No. 1 and a sequence selected from immunogenic variants of Sequence ID No. 1, which is at least 90% identical to Sequence ID No. 1 and includes one or more sequences of Sequence ID No. 2, 5, and 6, the process comprising stimulating and amplifying T cells obtained from a cancer patient in vitro with a polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 6.

11. T cells expressing an immune-specific TCR to a polypeptide selected from sequences of sequence number 1, an immunogenic variant of sequence number 1 that is at least 90% identical to sequence number 1 and includes one or more sequences of sequence numbers 2, 5, and 6, and sequence number 2, 5, and 6.

12. Antigen-presenting cells expressing the polypeptide described in any one of claims 1 to 3.

13. The antigen-presenting cell according to claim 12, wherein the antigen-presenting cell is a dendritic cell.

14. An exosome comprising a polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 6, or genetically engineered to express a polypeptide according to any one of claims 1 to 3.

15. An isolated antigen-binding polypeptide that is immune-specific to the polypeptide described in any one of claims 1 to 3.

16. A pharmaceutical composition comprising a T cell, antigen-presenting cell, exosome, or antigen-binding polypeptide according to any one of claims 11 to 15.

17. An isolated antigen-binding polypeptide that is immune-specific to an HLA-binding polypeptide, which is a polypeptide according to any one of claims 1 to 3.

18. The antigen-binding polypeptide according to claim 17, which is a T cell receptor or a fragment thereof.

19. A cytotoxic cell, wherein the cytotoxic cell is a T cell, which is a cytotoxic cell that has been manipulated to express the antigen-binding polypeptide described in claim 17 or 18 on its surface.

20. The composition according to claim 6 or 16 for use in pharmaceuticals.

21. A pharmaceutical composition comprising the cells described in claim 19.

22. A composition according to any one of claims 6, 16, and 21, for use in the treatment or prevention of human cancer, The composition wherein the cancer cells express Sequence ID No. 1 and a corresponding sequence selected from immunogenic variants of Sequence ID No. 1, which are at least 90% identical to Sequence ID No. 1 and include one or more sequences of Sequence ID No. 2, 5, and 6.

23. Isolated from tumors of humans suffering from cancer, (A)(a) Sequence of sequence number 1; and (b) A sequence selected from immunogenic variants of the sequence of Sequence ID No. 1 that are at least 90% identical to Sequence ID No. 1 and include one or more sequences of Sequence ID No. 2, 5, and 6; or (B) Consists of one of the sequences of sequence number 2, sequence number 5, and sequence number 6, The use of a polypeptide, or the use of a nucleic acid that encodes said polypeptide, The use as a biomarker for obtaining data to determine whether the human would be suitable for treatment with a vaccine comprising the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cells, antigen-presenting cells, exosome, antigen-binding polypeptide, or cytotoxic cells as described in any one of claims 1 to 6, 11 to 16, 19, and 21.

24. The process or use according to claim 10 or 23, wherein the cancer is melanoma.

25. The aforementioned polypeptide is (A)(a) Sequence of sequence number 1; and (b) A sequence selected from immunogenic variants of the sequence of Sequence ID No. 1, which are at least 90% identical to Sequence ID No. 1 and include one or more sequences of Sequence ID No. 2, 5, and 6; or (B) Consists of any one sequence of sequence number 2, sequence number 5, and sequence number 6, or The nucleic acid consists of a sequence selected from either SEQ ID NOs: 3 or 4; and The aforementioned cancer is uveal melanoma. The process or use described in claim 10 or 23.

26. The composition for use according to any one of claims 8, 9, and 22, wherein the cancer or malignant tumor is melanoma.

27. The aforementioned polypeptide is (A)(a) Sequence of sequence number 1; and (b) A sequence selected from immunogenic variants of the sequence of Sequence ID No. 1, which are at least 90% identical to Sequence ID No. 1 and include one or more sequences of Sequence ID No. 2, 5, and 6; or (B) Consists of any one sequence of sequence number 2, sequence number 5, and sequence number 6, or The nucleic acid consists of a sequence selected from either SEQ ID NOs: 3 or 4; and The aforementioned cancer or malignant tumor is uveal melanoma. A composition for use according to any one of claims 8, 9, and 22.

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