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 malignant melanoma, especially cutaneous melanoma and uveal melanoma.

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to antigenic polypeptides and corresponding polynucleotides used for the treatment or prevention of cancer, particularly for the treatment or prevention of melanoma (e.g., cutaneous melanoma or uveal melanoma). The present invention further relates, inter alia, to said nucleic acids and polypeptides, immune cells loaded with and / or stimulated by said polypeptides and polynucleotides, antibodies specific for said polypeptides, and medicaments and immunogenic compositions comprising cells (derived from self or otherwise) genetically engineered with molecules that recognize said polypeptides.

Background Art

[0002] (Background of the Invention) As part of normal immune surveillance against pathogenic microorganisms, all cells break down intracellular proteins to produce peptides that are loaded onto major histocompatibility complex (MHC) class I molecules expressed on the surface of all cells. Most of these peptides derived 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 encoding T cell receptors (TCRs) that tightly bind to MHC I-peptide complexes. This expanded population of T cells can produce effector CD8+ T cells (including cytotoxic T lymphocytes, CTLs) that can eliminate foreign antigen-tagged cells, and can also produce memory CD8+ T cells that can be re-amplified if foreign antigen-tagged cells appear later in the life of the animal.

[0003] The expression of MHC class II molecules is usually restricted to professional antigen-presenting cells (APCs) such as dendritic cells (DCs), and MHC class II molecules are usually loaded with peptides taken up internally from the external environment. In the presence of various factors including T cell adhesion molecules (CD54, CD48) and costimulatory molecules (CD40, CD80, CD86), when the complementary TCR from naive CD4+ T cells binds to the MHCII-peptide complex, the effector cells of CD4+ T cells (e.g., T H 1, T H 2, T H 17, T FH , T reg cells) maturation is induced. These effector CD4+ T cells not only promote the differentiation of B cells into antibody-secreting plasma cells, but also promote the differentiation of antigen-specific CD8+ CTLs, thereby helping to induce an adaptive immune response to foreign antigens, including both short-term effector functions and long-term immune memory. DCs can deliver antigens of exogenous origin (such as peptides or proteins released from pathogens or tumor cells) onto their MHC I molecules, thereby performing the cross-presentation process of peptide antigens and providing an alternative pathway to stimulate the proliferation of naive CD8+ T cells, contributing to the generation of immune memory.

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

[0005] Cancer is the second most common cause of death, accounting for nearly one-sixth of all deaths worldwide. Of the 8.8 million cancer deaths in 2015, the cancers that caused the most deaths 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 in 2010 was estimated to be $1.16 trillion, and the number of new cases is expected to increase by about 70% over the next 20 years (World Health Organization, Cancer facts, 2017).

[0006] Current therapies for cutaneous malignant melanoma are diverse and highly dependent on the location of the tumor and the stage of the disease. The main treatment for non-metastatic malignant melanoma is surgery to remove the tumor and surrounding tissue. Advanced malignant melanoma may require treatment including lymph node dissection, radiation therapy, or chemotherapy. Immune checkpoint inhibition strategies involve the use of antibodies that target negative immune regulators such as PD-1 / PD-L1 and CTLA4, and have recently revolutionized the treatment of various malignancies, including malignant melanoma (Ribas, A. and Wolchok, J. D., (2018) Science, 359:1350-1355). The exceptional value of checkpoint inhibition therapy, and its well-known association with the clinical benefit and the patient's adaptive immune response to their own cancer antigens, particularly T cell-based immune responses, has activated 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 integrated into the germline over generations. 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 mammalian apparent LTR retrotransposons (MaLRs), and thus is collectively known as LTR elements (referred to herein as ERVs collectively to mean all LTR elements). ERVs constitute a significant proportion (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 the prototype retroviral genome structure consisting of the gag, pro, pol, and env genes adjacent to the LTR. Some intact ERV ORFs produce retroviral proteins that share features 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 avoid the selection processes of T-cell and B-cell receptors as well as central and peripheral tolerance (immunotolerance). Immunoreactivity against ERV products may occur spontaneously in infectious diseases and cancers, and ERV products have been implicated as causes of some autoimmune diseases (Kassiotis and Stoye, 2016, Nat. Rev. Immunol. 16:207-219).

[0008] Due to the accumulation of mutation and recombination events in evolution, most ERV-derived sequences have lost some or all of the functional open reading frames of their genes, and as a result, have lost their ability to produce infectious viruses. However, these ERV elements are maintained within the germline DNA like other genes and have the potential to generate proteins from at least some of their genes. In fact, proteins encoded by HERVs have been detected in various human cancers. For example, Rec and Np9, which are splice variants of the HERV-K env gene, are only found in malignant testicular germ cells and not in healthy cells (Ruprecht et al., 2008, Cell Mol Life Sci 65:3366-3382). In cancers such as prostate cancer, an increase in the level of HERV transcripts has also been observed compared to healthy tissues (Wang-Johanning et al., 2003, Cancer 98:187-197; Andersson et al., 1998, Int. J. Oncol, 12:309-313). Furthermore, the 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 mechanism by which HERVs can contribute to cancer progression or pathogenicity remains unclear.

[0009] In addition to deregulating the expression of surrounding adjacent host genes, the activity of ERV regulatory elements and their translocation to new genomic sites may lead to the generation of novel transcripts, some of which may have oncogenic potential (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 an antigenic polypeptide to a subject to enhance the immune response (including B cell and T cell responses) and stimulate immune memory. Alternatively, a polynucleotide may be administered to a subject by a vector such that the immunogenic polypeptide encoded by the polynucleotide is expressed in vivo. The use of viral vectors, such as adenoviral vectors, has been well explored for antigen delivery in both prophylactic vaccination strategies and therapeutic treatment strategies against 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 polynucleotides encoding them can also be used to load patient-derived antigen-presenting cells (APCs), which can then be injected into the subject as a vaccine to elicit a therapeutic or prophylactic immune response. An example of this approach is Provenge, the only currently FDA-approved anti-cancer vaccine.

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

[0012] Antigen-binding biologics are typically composed of multivalent polypeptides engineered to recognize cancer cells modified with an antigen and promote their destruction. The antigen-binding components of these biologics may consist of TCR-based biologics, which include TCRs, high-affinity TCRs, and TCR mimics (including those based on monoclonal antibody technology) generated by various techniques, but are not limited thereto. The cytolytic components of these types of multivalent biologics may consist of cytotoxic chemicals, biological toxins, targeting motifs and / or immunostimulatory 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 removed and stimulated ex vivo with a vaccine antigen preparation (cultured with T cells in the presence or absence of other factors, including cell and cell-free components) (JCI Insight. October 2018, 4;3(19). pii:122467. doi:10.1172 / jci.insight.122467). Alternatively, adoptive cell therapy can be based on cells (including patient-derived or non-patient-derived cells) that have been deliberately engineered to express an antigen-binding polypeptide that recognizes a cancer antigen. These antigen-binding polypeptides are classified into the same classes as those described above for antigen-binding biologics. Thus, lymphocytes (autologous or allogeneic) genetically engineered to express a cancer antigen-binding polypeptide can also be administered to a patient as adoptive cell therapy for the treatment of their cancer.

[0014] The use of ERV-derived antigens to enhance an effective immune response against cancer has shown promising results in murine models of cancer, promoting tumor regression and leading to a more favorable prognosis (Kershaw et al., 2001, Cancer Res. 61:7920-7924; Slansky et al., 2000, Immunity 13:529-538). Thus, despite some limitations in research progress due to the stringent restriction 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 of existing vaccines against infectious pathogens, which commonly enhance cross-reactive immune responses against HERV-K Mel tumor antigens and provide protection against melanoma. WO 00 / 06598 relates to the identification of the HERV-AVL3-B tumor-related gene preferentially expressed in melanoma, and methods and products for diagnosing and treating symptoms characterized by the expression of that gene. WO 2006 / 119527 provides antigenic polypeptides derived from melanoma-associated endogenous retroviruses (MERV), and their use for the detection and diagnosis of melanoma and for the prognosis of this disease. The use of the antigenic polypeptides as anti-cancer vaccines is also disclosed.

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

[0017] The reference by Humer J et al., 2006, Canc. Res., 66:1658-63 identifies melanoma markers derived from endogenous retroviruses associated with melanoma. There is a need to identify additional HERV-related antigen sequences that can be used in the immunotherapy of cancer, particularly melanoma, and more particularly cutaneous melanoma and uveal melanoma. SUMMARY OF THE INVENTION

[0018] (Summary of the Invention) The inventors have surprisingly discovered certain RNA transcripts that are derived from genomic sequences containing or adjacent to LTR elements, are found at high levels in cutaneous malignant melanoma cells, but are undetectable or found at very low levels in normal, healthy tissues (see Example 1). Such transcripts are referred to herein as cancer-specific LTR-element spanning transcripts (CLTs). Furthermore, the inventors have shown that a subset of the potential polypeptide sequences (i.e., open reading frames (ORFs)) encoded by these CLTs are translated within cancer cells, processed by components of the antigen processing apparatus, and further presented on the cell surface as confirmed within tumor tissue, together with class I and class II major histocompatibility complex (MHC class I and MHC class II) and class I and class II human leukocyte antigen (HLA class I, HLA class II) molecules (see Example 2). These findings indicate that these polypeptides (referred to herein as CLT antigens) are in fact antigenic. Thus, presentation of CLT antigens by cancer cells is expected to render these cells susceptible to elimination by T cells having a cognate T cell receptor (TCR) for this CLT antigen, and further, CLT antigen-based vaccination methods / regimens that amplify T cells having these cognate TCRs are expected to elicit an immune response against cancer cells (and tumors containing them), particularly melanoma, and more particularly cutaneous malignant melanoma tumors. Indeed, T cells from melanoma subjects react to CLT antigen-derived peptides disclosed herein, amplifying the T cells and amplifying the T cell receptor sequences (see Example 3). The inventors have confirmed that T cells specific for CLT antigens are not deleted from the T cell repertoire of normal subjects by central tolerance (see Example 4). The presence and killing activity of CLT antigen-specific T cells within in vitro cultures of healthy donor T cells were determined (see Example 5). Finally, qRT-PCR studies confirmed that CLTs were specifically expressed within RNA extracted from malignant melanoma cell lines as compared to non-malignant melanoma cell lines (see Example 7).

[0019] The inventors have also surprisingly discovered that certain CLTs encoding CLT antigens are not only overexpressed in cutaneous malignant melanoma, but also in uveal malignant melanoma. The CLT antigen polypeptide sequences encoded by these CLTs are expected to elicit an immune response against uveal malignant melanoma cells and tumors containing them.

[0020] The CLTs and CLT antigens that are the subject of the present invention are not canonical sequences that can be readily derived from known tumor genomic sequences found in The Cancer Genome Atlas. This CLT is a transcript resulting from complex-transcription and splicing events driven by an ERV-derived transcriptional regulatory sequence. Since the CLT is expressed at high levels and the CLT antigen polypeptide sequence is not a normal human protein sequence, it is likely to elicit a strong and specific immune response (see Examples 3-5, as actually established), and is therefore expected to be suitable for therapeutic use in the context of cancer immunotherapy.

[0021] This CLT antigen, found in highly expressed transcripts that characterize tumor cells, was not previously known to exist in the human body and produce a protein product or stimulate an immune response, and can be used in several forms. First, the CLT antigen polypeptide of the present invention can also 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 have their codons optimized to enhance the expression of the CLT antigen encoded thereby, can be administered directly, and can also be inserted into a vector as a vaccine that produces the encoded protein product in the subject for in vivo delivery, thereby eliciting a therapeutic or prophylactic immune response against tumor cells. 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, thereby eliciting a therapeutic or prophylactic immune response against tumor cells. Fourth, the polynucleotides and / or polypeptides of the present invention can be used to stimulate a subject's T cells ex vivo, prepare a stimulated T cell preparation, and administer it to the subject as a cancer treatment therapy. Fifth, biological molecules such as T cell receptors (TCRs) or TCR mimics can be further modified to recognize the CLT antigen complexed with MHC I molecules and kill (or promote the killing of) cancer cells, and may be administered to a subject as a cancer treatment therapy. Sixth, a chimeric version of a biological molecule that recognizes 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 a subject as a cancer treatment therapy. These and other applications will be described in more detail below.

[0022] Accordingly, the present invention particularly provides an isolated polypeptide comprising a sequence selected from the following: (a) any one of the sequences of SEQ ID NOs: 1 to 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a), (hereinafter referred to as "the polypeptide of the present invention"). The present invention also provides a nucleic acid molecule encoding the polypeptide of the present invention (hereinafter referred to as "the nucleic acid of the present invention").

[0023] The polypeptide of the present invention, the nucleic acid of the present invention, and related aspects of the present invention are expected to be useful in the field of embodiments for the immunotherapy and prevention of cancer, particularly for the immunotherapy and prevention of malignant melanoma, as described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] (Description of the Drawings) For each of FIGS. 1 to 15, the upper panel shows the extracted MS / MS spectrum of the peptide isolated from the patient's tumor sample (along with the assigned fragment ions), and the lower panel shows the rendering of the spectrum, indicating the positions of the linear peptide sequences mapped to the fragment ions.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

[0025]

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

[0026] For each of Figures 29 to 42, the upper panel shows the extracted MS / MS spectrum of the peptide isolated from the patient's tumor sample (along with the assigned fragment ions), and the lower panel shows the rendering of the spectrum, indicating the positions of the linear peptide sequences mapped to the fragment ions.

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

[0027] For each of FIGS. 43 to 50, an alignment is shown that aligns the native MS / MS spectrum (upper) of a peptide isolated from a patient's tumor sample with the native spectrum (lower) of a synthetic peptide corresponding to the same sequence.

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

Figure 50

[0028]

Figure 51

Figure 52

Figure 53

Figure 54

Figure 55

[0029]

Figure 56

Figure 57

Figure 58

Figure 59

[0030] (Description of the Array) SEQ ID NO: 1 is the polypeptide sequence of CLT antigen 1. SEQ ID NO: 2 is the polypeptide sequence of CLT antigen 2. SEQ ID NO: 3 is the polypeptide sequence of CLT antigen 3. SEQ ID NO: 4 is the polypeptide sequence of CLT antigen 4. SEQ ID NO: 5 is the polypeptide sequence of CLT antigen 5. SEQ ID NO: 6 is the polypeptide sequence of CLT antigen 6. SEQ ID NO: 7 is the polypeptide sequence of CLT antigen 7. SEQ ID NO: 8 is the polypeptide sequence of CLT antigen 8. SEQ ID NO: 9 is the polypeptide sequence of CLT antigen 9. SEQ ID NO: 10 is the polypeptide sequence of CLT antigen 10.

[0031] SEQ ID NOs: 11 to 14 are the peptide sequences derived from CLT antigen 1. SEQ ID NOs: 15 and 16 are the peptide sequences derived from CLT antigen 2. SEQ ID NOs: 17 and 18 are the peptide sequences derived from CLT antigen 3. SEQ ID NO: 19 is the peptide sequence derived from CLT antigen 4. SEQ ID NOs: 20 to 22 are the peptide sequences derived from CLT antigen 5. SEQ ID NOs: 23 and 24 are the peptide sequences derived from CLT antigen 6. SEQ ID NO: 25 is the peptide sequence derived from CLT antigen 7. SEQ ID NO: 26 is the peptide sequence derived from CLT antigen 8. SEQ ID NOs: 27 to 29 are the peptide sequences derived from CLT antigen 9. SEQ ID NOs: 30 to 32 are the peptide sequences derived from CLT antigen 10.

[0032] SEQ ID NO: 33 is the cDNA sequence of CLT encoding CLT antigen 1. SEQ ID NO: 34 is the cDNA sequence of CLT encoding CLT antigen 2. SEQ ID NO: 35 is the cDNA sequence of CLT encoding CLT antigens 3 and 4. SEQ ID NO: 36 is the cDNA sequence of CLT encoding CLT antigen 5. SEQ ID NO: 37 is the cDNA sequence of CLT encoding CLT antigen 6. SEQ ID NO: 38 is the cDNA sequence of CLT encoding CLT antigens 7 and 8. SEQ ID NO: 39 is the cDNA sequence of CLT encoding CLT antigen 9. SEQ ID NO: 40 is the cDNA sequence of CLT encoding CLT antigen 10.

[0033] SEQ ID NO: 41 is the cDNA sequence encoding CLT antigen 1. SEQ ID NO: 42 is the cDNA sequence encoding CLT antigen 2. SEQ ID NO: 43 is the cDNA sequence encoding CLT antigen 3. SEQ ID NO: 44 is the cDNA sequence encoding CLT antigen 4. SEQ ID NO: 45 is the cDNA sequence encoding CLT antigen 5. SEQ ID NO: 46 is the cDNA sequence encoding CLT antigen 6. SEQ ID NO: 47 is the cDNA sequence encoding CLT antigen 7. SEQ ID NO: 48 is the cDNA sequence encoding CLT antigen 8. SEQ ID NO: 49 is the cDNA sequence encoding CLT antigen 9. SEQ ID NO: 50 is the cDNA sequence encoding CLT antigen 10.

[0034] SEQ ID NOs: 51 - 52 are peptide sequences derived from CLT antigen 4. SEQ ID NO: 53 is a peptide sequence derived from CLT antigen 3. SEQ ID NO: 54 is a peptide sequence derived from CLT antigen 4. SEQ ID NOs: 55 - 57 are peptide sequences derived from CLT antigen 1. SEQ ID NOs: 58 - 66 are peptide sequences derived from CLT antigen 2. Sequence numbers 67 to 69 are peptide sequences derived from CLT antigen 3. Sequence numbers 70 to 72 are peptide sequences derived from CLT antigen 4. Sequence numbers 73 to 74 are peptide sequences derived from CLT antigen 1. Sequence number 75 is a peptide sequence derived from CLT antigen 2. Sequence numbers 76 to 77 are peptide sequences derived from CLT antigen 4. Sequence number 78 is a peptide sequence derived from CLT antigen 2.

Mode for Carrying Out the Invention

[0035] (Detailed Description of the Invention) (Polypeptide) The terms "protein", "polypeptide" and "peptide" are used interchangeably herein and refer to any peptide-bonded amino acid chain, regardless of length, cotranslation or post-translational modification.

[0036] The term "amino acid" refers to either a naturally occurring amino acid, or an amino acid analogue and amino acid mimetic that function 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 those amino acids that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid and O-phosphoserine. The term "amino acid analogue" refers to a compound having the same basic chemical structure as a naturally occurring amino acid, i.e., a hydrogen, carboxyl group, amino group, and an α-carbon bonded to an R group, but having a modified R group or a modified peptide backbone as compared to a natural amino acid. Examples thereof include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium and norleucine. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to a naturally occurring amino acid. Preferably, the amino acid is a naturally occurring amino acid or an amino acid analogue, particularly a naturally occurring amino acid, and more particularly one of those 20 L-amino acids encoded by the genetic code.

[0037] In this specification, amino acids may be represented by either the generally known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by generally recognized single-letter codes.

[0038] Accordingly, the present invention provides an isolated polypeptide comprising a sequence selected from the following: (a) any one of the sequences of SEQ ID NOs: 1 to 10; and (b) variants of the sequence of (a); and (c) immunogenic fragments of the sequence of (a).

[0039] The present invention also provides an isolated polypeptide comprising a sequence selected from the following: (a) the sequence of any one of SEQ ID NOs: 1 to 10 excluding the first methionine residue; and (b) variants of the sequence of (a); and (c) immunogenic fragments of the sequence of (a).

[0040] Generally, variants of the polypeptide sequences of the present invention include sequences having a high degree of sequence identity thereto. 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.) to the full length of the relevant reference sequence.

[0041] Preferably, the variant is an immunogenic variant. One variant is considered to be one immunogenic variant that elicits a response that is at least 20%, preferably at least 50%, particularly at least 75% (such as at least 90%, etc.) of the activity of the reference sequence (i.e., the sequence to which the variant is one variant). This response can be measured, for example, in an in vitro restimulation assay of PBMC or whole blood using the polypeptide as an antigen (such as restimulation over a period of several hours to up to 1 year, 6 months or less, 1 day to 1 month, or 1 - 2 weeks, etc.), through lymphocyte proliferation (such as T cell proliferation), activation of cells, production of cytokines (such as IFN-γ) in the culture supernatant (measured by ELISA, etc.), or evaluation of the characteristics of the T cell response by intracellular and extracellular staining (using antibodies specific for immune markers such as CD3, CD4, CD8, IL2, TNF-α, IFNg, type I IFN, CD40L, CD69, etc.) followed by analysis with a flow cytometer.

[0042] The variant can be, for example, a conservatively modified variant. A "conservatively modified variant" is one in which the modification results in an amino acid substitution with a functionally similar amino acid, or a substitution / deletion / addition of residues that does not substantially affect the biological function of the variant. Usually, the biological function of such a variant will be to induce an immune response against a cancer antigen of malignant melanoma, such as cutaneous malignant melanoma. Conservative substitution tables providing functionally similar amino acids are well known in the art. The variant can also include homologs of polypeptides found in other species.

[0043] Variants of the polypeptide of the present invention may contain a number of substitutions, for example, conservative substitutions (e.g., 1 to 25, 1 to 10, etc., particularly 1 to 5, and even more particularly 1 amino acid residue may be changed) compared to the reference sequence. The number of substitutions, e.g., the number of conservative substitutions, may be up to 20% of the number of residues in the reference sequence, e.g., up to 10%, e.g., up to 5%, e.g., up to 1%. Generally, conservative substitutions will be included in one of the amino acid groupings identified below, but in some cases other substitutions may also be possible as long as they do not substantially affect the immunogenic properties of the antigen. The following eight groups each contain amino acids that are usually conservative substitutions for each other.

[0044] 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (See, e.g., Creighton, Proteins 1984).

[0045] Preferably, such substitutions do not change the immunological structure of the epitope (e.g., they do not occur within the epitope region mapped to the primary sequence), and thus do not significantly affect the immunogenic properties of the antigen.

[0046] 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 positions 1 or 2, particularly position 1), and the insertions can include, for example, no more than 50 amino acid additions at each position (e.g., no more than 20, particularly no more than 10, and even more particularly no more than 5). Preferably, such insertions do not occur in the epitope region and thus do not significantly affect the immunogenic properties of the antigen. An example of an insertion includes a short stretch of histidine residues (e.g., 2 to 6 residues) to assist in the expression and / or purification of the antigen of interest.

[0047] Polypeptide variants include those in which several amino acids are deleted compared to the reference sequence. For example, such deletions may occur at positions 1 to 10 (e.g., positions 1 to 5, preferably positions 1 or 2, particularly position 1), and the deletions can include, for example, no more than 50 amino acid deletions at each position (e.g., no more than 20, particularly no more than 10, and even more particularly no more than 5). Preferably, such deletions do not occur in the epitope region and thus do not significantly affect the immunogenic properties of the antigen.

[0048] One of ordinary skill in the art will recognize that particular protein variants can include substitutions, deletions, and additions (or any combination thereof). For example, substitutions / deletions / additions may enhance binding to the desired patient's HLA molecules (or have a neutral effect) and may increase immunogenicity (or leave immunogenicity unchanged).

[0049] The immunogenic fragment of the present invention comprises, depending on the length of the CLT antigen, usually at least 9 (e.g., at least 9 or 10), for example at least 12, consecutive amino acids (e.g., at least 15 or at least 20 consecutive amino acids) etc. from the full-length polypeptide sequence, in particular at least 50 consecutive amino acids, for example at least 100 consecutive amino acids (e.g., at least 200 consecutive amino acids) etc. 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% etc. of the length of the full-length polypeptide sequence.

[0050] The immunogenic fragment usually contains at least one epitope. The epitope includes B cell and T cell epitopes. Preferably, the immunogenic fragment contains at least one T cell epitope such as a CD4+ or CD8+ T cell epitope.

[0051] A T cell epitope is a short, consecutive stretch of amino acids that is recognized by T cells (such as CD4+ or CD8+ T cells etc.) when bound to an HLA molecule. Identification of T cell epitopes may be achieved by epitope mapping experiments 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(Suppl. 1):i29 - i37).

[0052] As a result of the decisive involvement of T cell responses in cancer, it is very clear that fragments of the full-length polypeptides of SEQ ID NOs: 1 - 10 containing at least one T cell epitope may be immunogenic and may contribute to immune protection.

[0053] In diverse outbred populations such as humans, it will be understood that different HLA types mean that certain epitopes may not be recognized by all members of the population. As a result, in order to maximize the level of recognition and magnitude of the immune response to a polypeptide, it is generally desirable for an immunogenic fragment to contain multiple epitopes (preferably all epitopes within the CLT antigen) from the full-length sequence.

[0054] Particular fragments of the polypeptides of SEQ ID NOs: 1-10 that may be useful include at least one CD8+ T cell epitope, preferably at least two CD8+ T cell epitopes, more particularly those that include all CD8+ T cell epitopes, especially those associated with multiple HLA alleles, e.g., associated with 2, 3, 4, 5 or more alleles. Particular fragments of the polypeptides of SEQ ID NOs: 1-10 that may be useful include at least one CD4+ T cell epitope, preferably at least two CD4+ T cell epitopes, more particularly those that include all CD4+ T cell epitopes, (especially those associated with multiple HLA alleles, e.g., associated with 2, 3, 4, 5 or more alleles). However, one of ordinary skill in the art of vaccine design will be able to combine exogenous CD4+ T cell epitopes with the CD8+ T cell epitopes of the invention to achieve the desired response to the CD8+ T cell epitopes of the invention.

[0055] When individual fragments of the 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%, particularly at least 75% (e.g., at least 90%) of the activity of the reference sequence (i.e., the sequence of which the fragment is a fragment). This response can be measured, for example, by the activation of cells via lymphocyte proliferation (e.g., T cell proliferation), the production of cytokines (e.g., IFN-γ) in the culture supernatant (measured by ELISA etc.), or the characterization of the T cell response by intracellular and extracellular staining (e.g., using antibodies specific for immune markers such as CD3, CD4, CD8, IL2, TNF-α, IFN-γ, type I IFN, CD40L, CD69 etc.) and subsequent flow cytometer analysis in an in vitro restimulation assay of PBMC or whole blood using the polypeptide as an antigen (e.g., restimulation over a period of several hours to a maximum of 1 year, 6 months or less, 1 day to 1 month, or 1 - 2 weeks etc.).

[0056] In some cases, multiple fragments of the full-length polypeptide (which may or may not overlap and may or may not span the entire full-length sequence) can be used to obtain a biological response equivalent to that against the full-length sequence itself. For example, at least 2 (e.g., 3, 4, or 5 etc.) of the above-mentioned immunogenic fragments are combined to provide at least 50%, preferably at least 75%, more particularly at least 90% of the activity of the reference sequence in an in vitro restimulation assay of PBMC or whole blood (e.g., T cell proliferation and / or IFN-γ production assay).

[0057] Examples of immunogenic fragments of the polypeptides of SEQ ID NOs: 1 to 10, and thus examples of the peptides of the present invention, include polypeptides comprising or consisting of the sequences of SEQ ID NOs: 11 to 32. Further, examples of immunogenic fragments of the polypeptides of SEQ ID NOs: 1 to 4, and thus examples of the peptides of the present invention, include polypeptides comprising or consisting of the sequences of SEQ ID NOs: 51 to 78. The sequences of SEQ ID NOs: 11 to 17, 19 to 28, 30 to 31 and 51 to 54 were confirmed to bind to HLA class I molecules in immunopeptidome analysis (see Examples 2 and 2.1). The sequences of SEQ ID NOs: 18, 29 and 32 were confirmed to bind to HLA class II molecules in immunopeptidome analysis (see Example 2). The sequences of SEQ ID NOs: 55 to 78 were predicted to bind to HLA class I molecules by NetMHC software and were used in immunological validation assays (see Examples 3, 4 and 5).

[0058] (Nucleic acid) The present invention provides an isolated nucleic acid encoding the polypeptide of the present invention (referred to as the nucleic acid of the present invention). For example, the nucleic acid of the present invention comprises or consists of a sequence selected from SEQ ID NOs: 33 to 40 or 41 to 50.

[0059] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to polymeric macromolecules made up of nucleotide monomers, particularly deoxyribonucleotide monomers or ribonucleotide monomers. This term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are of natural and non-natural origin, are intended to have properties similar to a reference nucleic acid, and are intended to be metabolized in a manner similar to a reference nucleotide or to have an extended half-life in vivo. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA). Preferably, the term "nucleic acid" refers to a polymer of deoxyribonucleotide monomers or ribonucleotide monomers of natural origin. Preferably, the nucleic acid molecules of the present invention are recombinant. By recombinant is meant that the nucleic acid molecule is the product of at least one of cloning, restriction treatment or ligation steps, or other manipulations that result in a nucleic acid molecule that is distinct from a nucleic acid molecule found in nature (e.g., in the case of 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 containing non-natural codon usage). In one embodiment, the nucleic acid of the present invention is DNA. Alternatively, the nucleic acid of the present invention is RNA.

[0060] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) refer to nucleic acid molecules having a sugar component backbone that is a deoxyribosyl and ribosyl component, respectively. This sugar component may be linked to bases that are four natural 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 with the thymine (T) of the DNA replaced by uracil (U) in the RNA. The sugar component may also be linked to unnatural bases such as inosine, xanthosine, 7-methylguanosine, dihydrouridine and 5-methylcytidine. The natural phosphodiester bond between the sugar (deoxyribosyl / ribosyl) components may optionally be replaced with a phosphorothioate bond. Preferably, the nucleic acid of the present invention consists of natural bases linked to a deoxyribosyl or ribosyl sugar backbone having a phosphodiester bond between the sugar components.

[0061] In one embodiment, the nucleic acid of the present invention is DNA. For example, this nucleic acid contains, or consists of, a sequence selected from SEQ ID NOs: 33 to 40 or 41 to 50. Also provided is a nucleic acid containing, or consisting of, a variant of a sequence selected from SEQ ID NOs: 33 to 40 or 41 to 50, the variant having the same amino acid sequence but having different nucleic acids based on the degeneracy of the genetic code.

[0062] Accordingly, due to the degeneracy of the genetic code, a very large number of 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 specified by one codon, that codon can be changed to any of its corresponding codons without changing the encoded polypeptide. Such nucleic acid mutations result in "silent" (also sometimes referred to as "degenerate" or "synonymous") variants, which are one type of conservatively modified variant. In all of the nucleic acid sequences disclosed herein that encode a single polypeptide, all silent mutations of that nucleic acid are possible. One of ordinary skill in the art will recognize that each codon within a nucleic acid (except for the AUG codon, which is typically the only codon for methionine, and the UGG codon, which is typically the only codon for tryptophan) can be modified to create a functionally identical molecule. Accordingly, each silent mutation of a nucleic acid encoding a single polypeptide is implicitly included in each of the described sequences and is provided as one aspect of the present invention.

[0063] Degenerate codon substitutions can also be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0064] The nucleic acids of the present invention that comprise or consist of a sequence selected from SEQ ID NOs: 33-40 or 41-50 can also contain a number of silent variants (e.g., 1-50, 1-25, etc., particularly 1-5, more particularly 1 codon may be mutated) when compared to the reference sequence.

[0065] In one embodiment, the nucleic acid of the present invention is RNA. The provided RNA sequences correspond to the DNA sequences herein, have a ribonucleotide backbone instead of a deoxyribonucleotide backbone, and have the side-chain base uracil (U) instead of thymine (T).

[0066] Accordingly, the nucleic acid of the present invention comprises or consists of an RNA equivalent of a cDNA sequence selected from SEQ ID NOs: 33 to 40 or 41 to 50, and may also contain a number of silent variants (e.g., 1 to 50, 1 to 25, etc., particularly 1 to 5, and more particularly 1 codon may be mutated) when compared to the reference sequence. "RNA equivalent" means an RNA sequence that contains the same genetic information as the reference cDNA sequence (i.e., having a ribonucleotide backbone instead of a deoxyribonucleotide backbone and containing the same codons with the side-chain base uracil (U) instead of thymine (T)).

[0067] The present invention also includes sequences complementary to the cDNA and RNA sequences described above. In one embodiment, the nucleic acid of the present invention has codons optimized for expression in human host cells. The nucleic acid of the present invention can be transcribed and translated into the polypeptide of the present invention in the case of DNA nucleic acid, and can also be translated into the polypeptide of the present invention in the case of RNA nucleic acid.

[0068] (Polypeptide and Nucleic Acid) 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 polypeptide or nucleic acid of natural origin is isolated when it is separated from some or all of the substances coexisting in the 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.

[0069] "Of natural origin" when used in relation to a polypeptide or nucleic acid sequence means a sequence found in nature and not synthetically modified. As used with respect to a polypeptide or nucleic acid sequence, "artificial" means a sequence not found in nature, e.g., a synthetic modification of a natural sequence or a sequence that includes non-natural sequences.

[0070] As used with respect to the relationship between one nucleic acid or polypeptide and another nucleic acid or polypeptide, the term "heterologous" indicates that two or more sequences are not found in the same relationship to each other in nature. A "heterologous" sequence can also mean a sequence that is not isolated from, not derived from, or not based on a nucleic acid or polypeptide sequence of natural origin found within a host organism.

[0071] As described above, a polypeptide variant preferably has 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%) relative to the full length of the relevant reference sequence.

[0072] For the purpose of comparing two closely related polypeptide or polynucleotide sequences, the "percent sequence identity" between a first sequence and a second sequence may be calculated. If two polypeptide sequences share 100% sequence identity over their entire length, they can be said to be identical or equal to each other. Residues in a 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 sequences or subsequences that, when compared and aligned to maximize correspondence over a comparison window, are identical or have a specified percentage of amino acid residues that are identical (e.g., 70% identity, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity within a specified region). Preferably, this comparison is performed over a window corresponding to the full length of the reference sequence.

[0073] In array comparison, one array serves as the reference array against which the test array is compared. When using an array comparison algorithm, the test array and the reference array are input into a computer, sub-array coordinates are specified as needed, and further array algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. Then, the array comparison algorithm calculates the percentage of array identity of the test array with respect to the reference array based on the program parameters.

[0074] As used herein, a "comparison window" refers to a segment within which, after optimally aligning one array and a reference array of the same number of consecutive positions, these arrays may be compared. Array alignment methods for comparison are well known in the art. Optimal array alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by 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, Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds., 1995 supplement)).

[0075] An example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignment to create a multiple sequence alignment from a group of related sequences, showing their relationships and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987, J. Mol. Evol. 35:351-360). The method used is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. This program can also align up to 300 sequences if each has a maximum length of 5,000 nucleotides or amino acids. This multiple sequence alignment procedure first performs a pairwise alignment of the two most similar sequences to create a cluster of the two aligned sequences. Next, this cluster is aligned next to the most related cluster of sequences or aligned sequences. The two sequence clusters are aligned by a simple extension of the pairwise alignment of the two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. This program is run by specifying a particular sequence and its amino acid coordinates for the sequence comparison region, and by further specifying program parameters. By using PILEUP, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can also be obtained from the GCG sequence analysis software package, for example, version 7.0 (Devereaux et al., 1984, Nuc. Acids Res. 12:387-395).

[0076] Another example of algorithms suitable for determining percent sequence identity and percent sequence similarity are 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 analyses is publicly available through the National Center for Biotechnology Information (website of www.ncbi.nlm.nih.gov). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that match or satisfy some positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. Word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. The cumulative score for nucleotide sequences is calculated using parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction stops when: the cumulative alignment score drops from its maximum achieved value by an amount X; the cumulative score goes to zero or below for the cumulative of one or more negative-scoring residue alignments; or the end of either sequence is reached.For amino acid sequences, in the BLASTP program, by default, a word length of 3 and an expectation value (E) of 50, as well as a BLOSUM62 scoring matrix of 50 (see Henikoff and Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915) alignment (B), an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands are used.

[0077] The BLAST algorithm also performs a 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 total probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or amino acid sequences occurs by chance.

[0078] A "difference" between sequences refers to the insertion, deletion, or substitution of 1 residue at one position in the second sequence compared to the first sequence. The two sequences can also contain one, two, or more such differences. Otherwise, insertions, deletions, or substitutions in the second sequence that are identical to the first sequence (100% sequence identity) result in a decrease in the % sequence identity. For example, if the identical sequence is 9 residues long, 1 substitution in the second sequence results in 88.9% sequence identity. If the identical sequence is 17 amino acid residues long, 2 substitutions in the second sequence result in 88.2% sequence identity.

[0079] 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 1 residue to the first sequence (including addition to either end of the first sequence). A substitution is the replacement of 1 residue in the first sequence with a different 1 residue. A deletion is the deletion of 1 residue from the first sequence (including deletions at either end of the first sequence).

[0080] (Production of the polypeptide of the present invention) The polypeptide of the present invention can also be obtained and manipulated, for example, using the techniques disclosed in Green and Sambrook, 2012 Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbour Laboratory Press. In particular, artificial gene synthesis techniques can also be used to generate polynucleotides (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 then expressed in a suitable organism to produce the polynucleotide. The gene encoding the polypeptide of the present invention can be synthetically generated, 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, the building blocks are sequentially joined to the growing oligonucleotide chain in the order required by the sequence of the product. When the chain assembly is complete, the product is released from the solid phase into solution, deprotected, and collected. The product can also 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 easily assembled into longer DNA molecules using various gene amplification methods (Methods Mol Biol., 2012;834:93-109), and they are suitable for use in a countless number of recombinant DNA-based expression systems. In the context of the present invention, those skilled in the art will understand that the polynucleotide sequence encoding the polypeptide antigen of the present invention can be readily used in various vaccine production systems, including, for example, viral vectors.

[0081] 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 in the host. Similarly, the polypeptide of the present invention can be produced by transducing a culture of eukaryotic cells (e.g., Chinese hamster ovary cells or Drosophila melanogaster S2 cells) with a nucleic acid of the present invention, which is combined with suitable regulatory and control sequences (including promoters, termination signals, etc.) and sequences for promoting polypeptide secretion suitable for protein production in these cells.

[0082] Improved isolation of the polypeptide of the present invention produced by recombinant techniques can optionally be facilitated by adding a stretch of histidine residues (commonly known as His-tag) towards one end of the polypeptide. The polypeptide may also be prepared synthetically.

[0083] (Vector) In additional embodiments, a gene construct comprising one or more nucleic acids of the present invention is introduced into cells in vivo, and the polypeptide of the present invention is produced in vivo to elicit an immune response. The nucleic acid (e.g., DNA) can be present within any of a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacteria, and some viral expression systems. A number of gene delivery techniques are well known in the art, such as those described by Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, and references cited therein. Some of these approaches are outlined below for illustrative purposes.

[0084] In accordance with the above, vectors (also referred to herein as "DNA expression constructs" or "constructs") comprising the nucleic acid molecules of the present invention are provided. Preferably, this vector contains a nucleic acid encoding regulatory elements (such as suitable promoters and termination signals, etc.) suitable for enabling the transcription of a translationally active RNA molecule in a human host cell. A "translationally active RNA molecule" is an RNA molecule that can be translated into a protein by the translation apparatus of a human cell.

[0085] According to the above, a vector containing the nucleic acid of the present invention (hereinafter referred to as "the vector of the present invention" in this specification) is provided. In particular, this vector may be a viral vector. The viral vector may be an adenovirus, an adeno-associated virus (AAV) (e.g., AAV5 and AAV2), an alphavirus (e.g., Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SIN), Semliki Forest virus (SFV)), a herpesvirus, an arenavirus (e.g., lymphocytic choriomeningitis virus (LCMV)), a measles virus, a poxvirus (such as modified vaccinia Ankara (MVA)), a paramyxovirus, a lentivirus, or a rhabdovirus (such as vesicular stomatitis virus (VSV)) vector, i.e., this vector may be derived from any of the above viruses. Adenovirus is particularly suitable for use as a gene transfer vector due to its medium genome size, ease of manipulation, high titer, broad target cell range, and high infectivity. Both ends of the viral genome contain inverted terminal repeats (ITRs) of 100 to 200 base pairs, which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are split by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins involved in the regulation of transcription of the viral genome and some 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 shutoff (Renan, 1990). The products of the late genes, which include most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript generated by the major late promoter (MLP). The MLP is particularly efficient in the late stage of infection, and all mRNAs transcribed from this promoter carry a tripartite 5'-leader (TPL) sequence, making them favorable mRNAs for translation. Replication-deficient adenoviruses generated from viral genomes with one or more of the early genes deleted are particularly useful because they have restricted replication and a reduced potential for pathogenic spread within vaccinated hosts and for contact with vaccinated hosts.

[0086] (Delivery of Other Polynucleotides) In certain embodiments of the invention, an expression construct comprising one or more polynucleotide sequences may simply consist of a naked recombinant DNA plasmid alone. See Ulmer et al., 1993, Science 259:1745-1749 and Cohen, 1993, Science 259:1691-1692 which reviews it. Introduction of these constructs can also be carried out by any method that physically or chemically permeates the cell membrane. This may be particularly applicable for introduction in vitro, but is equally applicable for in vivo use. It is also envisioned that DNA encoding the gene of interest can be introduced in vivo in a similar manner to express the gene product. Multiple delivery systems have been used to deliver DNA molecules to animal models and humans. Some products based on this technology are approved for use in animals and others are in Phase II and Phase III clinical trials in humans.

[0087] (RNA Delivery) In other embodiments of the invention, the expression construct comprising one or more polynucleotide sequences may consist of RNA molecules derived from naked recombinant DNA (Ulmer et al., 2012, Vaccine 30:4414-4418). For DNA-based expression constructs, various methods can be used to introduce the RNA molecules into cells in vitro or in vivo. RNA-based constructs can be designed to mimic simple messenger RNA (mRNA) molecules such that the introduced biological molecule is directly translated by the translation machinery of the host cell and the polypeptide it encodes is produced within the cell into which it is introduced. Alternatively, the RNA molecules can be designed to self-amplify within the cells into which they are introduced by incorporating them into the structural genes for viral RNA-dependent RNA polymerases. Thus, this type of RNA molecule, known as self-amplifying mRNA (SAM™) molecules (Geall et al., 2012, PNAS, 109:14604-14609), shares properties with some RNA-based viral vectors. Either mRNA-based RNA or SAM™ RNA can be further modified (e.g., by modifying 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 can be formulated (e.g., in emulsions (Brito et al., Molecular Therapy, 2014 22:2118-2129) or in lipid nanoparticles (Kranz et al., 2006, Nature, 534:396-401)) to promote 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 humans, and multiple RNA-based vaccines are being used in ongoing clinical trials.

[0088] (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 all referred to as "the compositions of the present invention"). The compositions of the present invention preferably comprise a polypeptide, nucleic acid or vector of the present invention together with a pharmaceutically acceptable carrier. Accordingly, in one embodiment, there is provided an immunogenic pharmaceutical composition comprising a polypeptide, nucleic acid or vector of the present invention together with a pharmaceutically acceptable carrier.

[0089] In another embodiment, there is provided a vaccine composition comprising a polypeptide, nucleic acid or vector of the present invention together with a pharmaceutically acceptable carrier. The preparation of pharmaceutical compositions is generally described, for example, in Powell and Newman, eds., Vaccine Design (the subunit and adjuvant approach), 1995. The compositions of the present invention may also contain other compounds, which may be biologically active or inactive. Preferably, the compositions of the present invention are sterile compositions suitable for parenteral administration.

[0090] In certain preferred embodiments of the present invention, the pharmaceutical compositions of the present invention are provided as comprising one or more (e.g., one) polypeptides of the present invention in combination with a pharmaceutically acceptable carrier. In certain preferred embodiments of the present invention, the compositions of the present invention are provided as comprising one or more (e.g., one) nucleic acids of the present invention or one or more (e.g., one) vectors of the present invention in combination with a pharmaceutically acceptable carrier.

[0091] In one embodiment, the composition of the invention can also comprise one or more (e.g., one) polynucleotide and one or more (e.g., one) polypeptide components. Alternatively, the composition can also comprise one or more (e.g., one) vector and one or more (e.g., one) polypeptide components. Alternatively, the composition can also comprise one or more (e.g., one) vector and one or more (e.g., one) polynucleotide components. Such compositions may be provided to enhance an immune response.

[0092] (Pharmaceutically acceptable salts) It will be apparent that the compositions of the invention can also comprise pharmaceutically acceptable salts of the nucleic acids or polypeptides provided herein. Such salts can be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., salts of sodium, potassium, lithium, ammonium, calcium and magnesium).

[0093] (Pharmaceutically acceptable carriers) Although many pharmaceutically acceptable carriers known to those skilled in the art can be used in the compositions of the present invention, 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 suitable mode of administration, which includes, for example, administration via parenteral, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular routes, preferably parenteral, such as intramuscular, subcutaneous or intravenous administration. For parenteral administration, the carrier preferably contains water and may also contain a pH-adjusting buffer, stabilizers (such as surfactants and amino acids), and isotonicity regulators (such as salts and saccharides). If the composition is to be provided in lyophilized form to be diluted at the time of use, the formulation may also contain a lyoprotectant, such as a saccharide like trehalose. For oral administration, any of the aforementioned carriers or solid carriers, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate, can be used.

[0094] Accordingly, the compositions of the present invention may also contain a buffer (such as neutral buffered saline or phosphate buffered saline), a carbohydrate (such as glucose, mannose, sucrose or dextran), mannitol, a protein, a polypeptide, or an amino acid such as glycine, an antioxidant, an antibacterial agent, a chelating agent such as EDTA or glutathione, a solute that makes the formulation isotonic, hypotonic or slightly hypertonic with the recipient's blood, a suspending agent, a thickening agent, and / or a preservative. Alternatively, the compositions of the present invention may be formulated as a lyophilized product.

[0095] (Immunostimulant) The composition of the present invention may also contain one or more immunostimulants. The immunostimulant may be any substance that enhances or potentiates the immune response (antibody-mediated and / or cell-mediated) against exogenous antigens. In the context of vaccine formulations, immunostimulants are often referred to as adjuvants, examples of which 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), aminoalkyl glucosaminide 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 interferons. From the above, one or more immunostimulants of the composition of the present invention are preferably selected from aluminum salts, saponins, immunostimulatory oligonucleotides, oil-in-water emulsions, aminoalkyl glucosaminide 4-phosphate, lipopolysaccharides and their derivatives and other TLR4 ligands, TLR7 ligands, TLR8 ligands, and TLR9 ligands. 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.

[0096] In the case of delivery methods with recombinant nucleic acids (e.g., DNA, RNA, viral vectors), a gene encoding a protein-based immunostimulant may be easily delivered together with the gene encoding the polypeptide of the present invention.

[0097] (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 results in a slow / continuous release of the compound following administration.

[0098] (Storage and packaging) The compositions of the present invention may be provided in unit dose or multiple dose containers such as sealed ampoules or vials. Such containers are preferably sealed and capped to maintain the sterility of the formulation until use. The formulation may generally be stored as a suspension, solution or emulsion in an oily or aqueous vehicle. Alternatively, the compositions of the present invention can be stored in a lyophilized state that requires the addition of a sterile liquid carrier (such as water for injection or physiological saline) only immediately prior to use.

[0099] (Dosage) The amount of nucleic acid, polypeptide or vector in each composition of the present invention may be formulated in a manner such that a suitable dosage for therapeutic or prophylactic use is obtained. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf-life, etc., as well as other pharmacological considerations, will be taken into account by those skilled in the art in formulating such compositions, and for this reason, various dosages and treatment regimens will be desirable.

[0100] Typically, compositions containing a therapeutically or prophylactically effective amount deliver from about 0.1 μg to about 1000 μg of the polypeptide of the present invention per administration, more typically from about 2.5 μg to about 100 μg of the polypeptide per administration. When delivered in the form of short synthetic long-chain peptides, the dosage may range from 1 - 200 μg / peptide / dose. With respect to polynucleotide compositions, these typically deliver from about 10 μg to about 20 mg of the nucleic acid of the present invention per administration, more typically from about 0.1 mg to about 10 mg of the nucleic acid of the present invention per administration.

[0101] (Disease to be Treated or Prevented) As described anywhere in the specification, SEQ ID NOs: 1 - 10 are polypeptide sequences corresponding to the CLT antigen overexpressed in cutaneous malignant melanoma. In one embodiment, the present invention provides a polypeptide, nucleic acid, vector or composition of the present invention for use in medicine.

[0102] A further aspect of the invention relates to a method of enhancing an immune response in a human, the method comprising administering to the human a polypeptide, nucleic acid, vector or composition of the invention. The invention also provides a polypeptide, nucleic acid, vector or composition of the invention for use in enhancing an immune response in a human. Use of a polypeptide, nucleic acid, vector or composition of the invention for the manufacture of a medicament for use in enhancing an immune response in a human is also provided.

[0103] Preferably, the immune response is enhanced against a cancerous tumor expressing a corresponding sequence selected from SEQ ID NOs: 1-10 and any variant and immunogenic fragment thereof. "Corresponding" in this context means that if the tumor expresses, for example, SEQ ID NO: A (where A is one of SEQ ID NOs: 1-10) or a variant or immunogenic fragment thereof, the polypeptide, nucleic acid, vector or composition of the invention and medicaments containing them will be based on SEQ ID NO: A or a variant or immunogenic fragment thereof.

[0104] Preferably, the immune response includes CD8+ T cells, CD4+ T cells and / or an antibody response, particularly a CD8+ cytotoxic T cell response and a CD4+ helper T cell response. Preferably, the immune response is elicited against a tumor, particularly one expressing a sequence selected from SEQ ID NOs: 1-10, and variants and immunogenic fragments thereof. In a suitable embodiment, the tumor is a malignant melanoma tumor, for example, a cutaneous malignant melanoma tumor. This tumor can also be a primary tumor or a metastatic tumor.

[0105] A further aspect of the present invention is a method for treating a human cancer patient, wherein the cancer cells thereof express a sequence selected from SEQ ID NOs: 1 to 10, any one of the immunogenic fragments and variants thereof, or a method for preventing a human from suffering from cancer, wherein the cancer will express a sequence selected from SEQ ID NOs: 1 to 10, any one of the immunogenic fragments and variants thereof, and the method comprises administering to the human the corresponding polypeptide, nucleic acid, vector or composition of the present invention.

[0106] The present invention also provides a polypeptide, nucleic acid, vector or composition of the present invention for use in the treatment or prevention of human cancer, wherein the cancer cells thereof express the corresponding sequence selected from SEQ ID NOs: 1 to 10 and any one of the immunogenic fragments thereof.

[0107] The transcripts corresponding to SEQ ID NOs: 33, 35, 36 and 40 were also overexpressed in uveal malignant melanoma. As a result, in another embodiment, the tumor is a uveal malignant melanoma tumor and / or a tumor expressing a sequence selected from SEQ ID NOs: 1, 3, 4, 5 and 10.

[0108] Accordingly, the present invention provides 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) any one of the sequences of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a), and for example, the polypeptide comprises or consists of a sequence selected from any one of SEQ ID NOs: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, 51 to 57, 67 to 74 and 76 to 77, and for example, the nucleic acid comprises or consists of a sequence selected from any one of SEQ ID NOs: 33, 35, 36 or 40, or a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50; and the cancer is uveal malignant melanoma. The terms "prevention" and "prevention" are used interchangeably herein.

[0109] (Treatment and vaccination regimens) A therapeutic regimen can include either (i) a polypeptide, nucleic acid or vector of the invention, (ii) one or more further polypeptides, nucleic acids or vectors of the invention and / or (iii) optionally other components such as other therapeutically useful compounds or molecules such as an antigenic protein to be co-administered with an adjuvant, delivered either simultaneously (co-administration etc.) or sequentially (prime-boost etc.). Examples of co-administration include co-administration to the same outer site and co-administration to opposite sites. Administration "at the same time" preferably relates to all components delivered during the same treatment session. Preferably, all components are co-administered (co-administration of both DNA and protein etc.), but one component may be administered within a few minutes (e.g., during the same consultation appointment or physician visit) or within a few hours.

[0110] In some embodiments, following the “priming” or first administration of the polypeptide, nucleic acid or vector of the invention, one or more “boosts” or additional immunizations of the polypeptide, nucleic acid or vector of the invention are performed (the “prime and boost” method). In one embodiment, the polypeptide, nucleic acid or vector of the invention is used in a single prime-boost vaccination regimen. In one embodiment, both the prime and the boost are the polypeptide of the invention, being in each case the same polypeptide of the invention. In one embodiment, both the prime and the boost are the nucleic acid or vector of the invention, being in each case the same nucleic acid or vector of the invention. Alternatively, the prime may be performed using the nucleic acid or vector of the invention and the boost may be performed using the polypeptide of the invention, or the prime may be performed using the polypeptide of the invention and the boost may be performed using the nucleic acid or vector of the invention. Usually, the first or “priming” administration and the second or “boost” administration are performed after an interval of about 1 to 12 weeks, or up to 4 to 6 months. Subsequent “booster” administrations may be performed at a frequency of every 1 to 6 weeks, or later (up to several years later).

[0111] (Combination of antigens) The polypeptide, nucleic acid or vector 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 the same) that elicit an enhanced immune response against melanoma, such as cutaneous or uveal melanoma. These other antigenic polypeptides may be derived from a variety of sources, and the sources may include melanoma-associated antigens that are well described, such as GPR143, PRAME, MAGE-A3 or pMel (gp100). Alternatively, they can include other species of melanoma antigens, including patient-specific neoantigens (Lauss et al., (2017). Nature Communications, 8(1), 1738. http: / / doi.org / 10.1038 / s41467-017-01460-0), retained intron neoantigens (Smart et al., (2018). Nature Biotechnology. http: / / doi.org / 10.1038 / nbt.4239), splice variant neoantigens (Hoyos et al., Cancer Cell, 34(2), 181-183. http: / / doi.org / 10.1016 / j.ccell.2018.07.008; Kahles et al., (2018), Cancer Cell, 34(2), 211-224.e6. http: / / doi.org / 10.1016 / j.ccell.2018.07.001), melanoma antigens belonging to the category known as antigens encoding T cell epitopes associated with defective peptide processing (TIEPPs; Gigoux, M. and Wolchok, J. (2018), JEM, 215, 2233, Marijt et al., (2018), JEM 215, 2325), or neoantigens (including CLT antigens) that will be discovered.Furthermore, the antigenic peptides derived from these various sources can also be co-administered with (i) non-specific immune activators / adjuvant species and / or (ii) an antigen (delivered as a polypeptide or as a polynucleotide or vector encoding these CD4 antigens) that, for example, contains a universal CD4 helper epitope and is known to elicit strong CD4 helper T cells, to amplify the anti-melanoma specific response elicited by the co-administered antigen.

[0112] 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.

[0113] More generally, when two or more components are used in combination, the components may be, for example: (1) As two or more individual antigenic polypeptide components; (2) As a fusion protein containing both (or further) polypeptide components; (3) As one or more polypeptides and one or more polynucleotide components; (4) As two or more individual polynucleotide components; (5) As a single polynucleotide encoding two or more individual polypeptide components; or (6) As a single polynucleotide encoding a fusion protein containing both (or further) polypeptide components, may be provided.

[0114] For cost reasons, when there are multiple components, it is often desirable for them to be included within a single fusion protein or a polynucleotide encoding a single fusion protein (see below). In one embodiment of the invention, all components are provided as polypeptides (e.g., within a single fusion protein). In another embodiment of the invention, all components are provided as polynucleotides (e.g., a single polynucleotide such as one encoding a single fusion protein).

[0115] (Fusion protein (fusion polypeptide)) As an embodiment of the foregoing discussion regarding antigen combinations, the invention also provides an isolated polypeptide of the invention (hereinafter referred to as the "combination polypeptide of the invention") fused to a second or further polypeptide of the invention by creating a nucleic acid construct that fuses together the sequences encoding the individual antigens. The combination polypeptides of the invention are predicted to have the utilities described herein for the polypeptides of the invention and may have the advantage of enhanced immunogenicity or vaccine activity, or prophylactic or therapeutic effects (including increased breadth and depth of response), and will be particularly useful in outbred populations. The fusion of the polypeptides of the invention will also provide the advantage of enhancing the construction and production efficiency of vaccine antigens and / or vectorized vaccines (including nucleic acid vaccines).

[0116] As described in the "Combination of Antigens" section above, the polypeptides of the invention as well as the combination polypeptides of the invention may also be fused to polypeptide sequences that are not polypeptides of the invention and that include one or more of the following: (a) Other polypeptides that are melanoma-associated antigens and thus may be useful as immunogenic sequences in a vaccine (e.g., GPR143, PRAME, MAGE-A3, and pMel (gp100) mentioned above); and (b) Polypeptide sequences that can enhance the immune response (i.e., immunostimulatory sequences). (c) For example, a polypeptide sequence that contains a universal CD4 helper epitope, provides strong CD4 help, and can increase the CD8+ T cell response against a CLT antigen epitope.

[0117] Exemplary fusion polypeptides include two or more (e.g., 2, 3, or 4) sequences selected from the sequences of SEQ ID NOs: 1, 2, 3, and 4; or, for each such sequence, variants of that sequence, or immunogenic fragments of that sequence.

[0118] One exemplary fusion polypeptide includes the following: (i) A sequence selected from the following: (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) selected, for example, from SEQ ID NOs: 11-14, 55-57, and 73-74; (ii) A sequence selected from the following: (a) The sequence of SEQ ID NO: 2; and (b) Variants of the sequence of (a); and (c) Immunogenic fragments of the sequence of (a) selected, for example, from SEQ ID NOs: 15-16, 58-66, 75, and 78; (iii) A sequence selected from the following: (a) The sequence of SEQ ID NO: 3; and (b) Variants of the sequence of (a); and (c) Immunogenic fragments of the sequence of (a) selected, for example, from SEQ ID NOs: 17-18, 53, and 67-69; and (iv) A sequence selected from the following: (a) The sequence of SEQ ID NO: 4; and (b) Variants of the sequence of (a); and (c) Immunogenic fragments of the sequence of (a) selected, for example, from SEQ ID NOs: 19, 51-52, 54, 70-72, and 76-77. For example, this fusion polypeptide includes the sequences of SEQ ID NOs: 1, 2, 3, and 4.

[0119] Another exemplary fusion polypeptide includes the following: (i) An array selected from the following: (a) The array of SEQ ID NO: 1; and (b) A variant of the array of (a); and (c) An immunogenic fragment of the array of (a) selected from, for example, SEQ ID NOs: 11-14, 55-57, and 73-74; (ii) An array selected from the following: (a) The array of SEQ ID NO: 2; and (b) A variant of the array of (a); and (c) An immunogenic fragment of the array of (a) selected from, for example, SEQ ID NOs: 15-16, 58-66, 75, and 78; and (iii) An array selected from the following: (a) The array of SEQ ID NO: 4; and (b) A variant of the array of (a); and (c) An immunogenic fragment of the array of (a) selected from, for example, SEQ ID NOs: 19, 51-52, 54, 70-72, and 76-77. For example, this fusion polypeptide contains the arrays of SEQ ID NOs: 1, 2, and 4.

[0120] The present invention also provides a nucleic acid encoding the fusion polypeptide with the necessary modifications similar to the polypeptide of the present invention, and other aspects of the present invention (vectors, compositions, cells, etc.).

[0121] (CLT antigen-binding polypeptide) An antigen-binding polypeptide that is immunospecific for a tumor-expressed antigen (the polypeptide of the present invention) could be designed to recruit cytolytic cells to antigen-modified tumor cells and mediate their destruction. One mechanism for the recruitment of cytolytic cells by such an antigen-binding polypeptide is known as antibody-dependent cell-mediated cytotoxicity (ADCC). Accordingly, the present invention provides an antigen-binding polypeptide that is immunospecific for the polypeptide of the present invention. Antigen-binding polypeptides, including monoclonal antibodies and fragments thereof, such as domain antibodies, Fab fragments, Fv fragments, and VHH fragments, may be produced in non-human animal species (e.g., rodents or camels) and humanized, or may be produced in non-human species (e.g., rodents genetically engineered to have a human immune system).

[0122] Antigen-binding polypeptides can be produced by methods well known to those skilled in the art. For example, monoclonal antibodies can be produced by using hybridoma technology to fuse specific antibody-producing B cells with myeloma (B cell cancer) cells selected for their ability to grow in tissue culture and lack of antibody chain synthesis (Kohler and Milstein, 1975, Nature 256(5517):495-497 and Nelson et al., June 2000, Mol Pathol. 53(3):111-7 are hereby incorporated by reference in their entirety).

[0123] Monoclonal antibodies against a desired antigen can be obtained, for example: a) immortalizing lymphocytes obtained from the peripheral blood of an animal (including humans) pre-immunized / exposed with the desired antigen with immortal cells, preferably myeloma cells, to form hybridomas, b) culturing the formed immortalized cells (hybridomas) and recovering the cells that produce antibodies having the desired specificity, can also be obtained by.

[0124] Monoclonal antibodies can also be obtained by a manufacturing process that includes the following steps: a) Cloning steps of DNA or cDNA sequences obtained from lymphocytes of an animal (preferably immunized in advance with a desired antigen), particularly peripheral blood lymphocytes, into a vector, particularly a phage, and more particularly a filamentous bacteriophage b) Transforming prokaryotic cells with the vector under conditions that allow antibody production c) Selecting antibodies by subjecting them to antigen affinity selection d) Recovering antibodies having the desired specificity e) Expressing an antibody-encoding nucleic acid molecule obtained from B cells of a patient exposed to an antigen or an animal subjected to an antigen immunization experiment The selected antibodies may then be produced using conventional recombinant protein production techniques (e.g., from genetically engineered CHO cells).

[0125] The present invention provides an isolated antigen-binding polypeptide that is immunologically specific for 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 conjugated to a cytotoxic component. Examples of cytotoxic components include the Fc domain of an antibody, which would recruit Fc receptor-bearing cells that promote ADCC. Alternatively, the antigen-binding polypeptide may be linked to a biological toxin or a cytotoxic chemical.

[0126] Another important class of antigen-binding polypeptides includes molecules derived from T cell receptors (TCRs) that bind to HLA-displayed fragments of the antigens of the present invention. In this embodiment, a TCR-based biologic (including TCRs directly derived from a patient or specifically engineered high-affinity TCRs) that recognizes a CLT antigen (or a derivative thereof) on the surface of tumor cells may also include targeting components that recognize components on T cells (or another class of immune cells) that attract these immune cells to the tumor and provide a therapeutic effect. In some embodiments, the targeting component may also stimulate beneficial activities (including cytolytic activity) of redirected immune cells.

[0127] Thus, in one embodiment, the antigen-binding polypeptide is immunospecific for an HLA-binding polypeptide that is the polypeptide of the present invention or 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 can also bind to another polypeptide that can bind to cytotoxic cells or other immune components within a subject.

[0128] In one embodiment, the antigen-binding polypeptide is for use in a medicament. In one embodiment, there is provided 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 above.

[0129] The present invention provides a method for treating a human suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NOs: 1 to 10 and any one of its immunogenic fragments and variants, or a method for preventing a human from suffering from cancer, wherein the cancer cells will express a sequence selected from SEQ ID NOs: 1 to 10 and any one of its immunogenic fragments and variants. There is provided a method comprising administering to the human the antigen-binding polypeptide of the present invention or a composition comprising the antigen-binding polypeptide thereof.

[0130] In one embodiment, there is provided the antigen-binding polypeptide of the present invention, which may be bound to a cytotoxic component, or a composition of the present invention comprising the antigen-binding polypeptide thereof, for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NOs: 1 to 10 and any one of its immunogenic fragments. Preferably, in any of the above embodiments, the cancer is malignant melanoma, particularly cutaneous malignant melanoma.

[0131] In one embodiment, an antigen-binding polypeptide or composition for the method of the present invention or for the use of the present invention, wherein the polypeptide comprises a sequence selected from the following: (a) any one of the sequences of SEQ ID NO: 1, 3, 4, 5, and 10; and (b) a variant of the sequence of (a); and, (c) an immunogenic fragment of the sequence of (a), and for example, the polypeptide comprises or consists of a sequence selected from any one of SEQ ID NO: 11-14, 17-18, 19, 20-22, 30-32, 51-57, 67-74, and 76-77, and for example, the nucleic acid comprises or consists of a sequence selected from any one of SEQ ID NO: 33, 35, 36, or 40, or a sequence selected from any one of SEQ ID NO: 41, 43, 44, 45, and 50; and wherein the cancer is uveal malignant melanoma, there is provided an antigen-binding polypeptide or composition for the method or use.

[0132] The antigen-binding polypeptide (such as an antibody or a fragment thereof) may be administered at a dose of, for example, 5-1000 mg, for example, 25-500 mg, for example, 100-300 mg, for example, about 200 mg.

[0133] (Cell therapy to promote antigen presentation in vivo) In order to promote the generation of an antigen-specific immune response, any of a variety of cell delivery vehicles may be used within a pharmaceutical composition. Accordingly, the present invention provides a cell which is an isolated antigen-presenting cell that has been modified by in vitro loading with a polypeptide of the present invention or genetically engineered to express a polypeptide of the present invention (hereinafter referred to as "the APC of the present invention"). Antigen-presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes, and other cells that may be engineered to be efficient APCs. Such cells may be genetically modified, but not necessarily, to enhance antigen-presenting ability, to improve activation and / or maintenance of a T cell response, and / or to be immunologically compatible with a receptor (i.e., having a matching HLA haplotype). APCs can generally be isolated from any of a variety of body fluids and organs and may be autologous, allogeneic, syngeneic, or xenogeneic cells.

[0134] In certain preferred embodiments of the present invention, dendritic cells or their precursors are used as APCs. Thus, in one embodiment, the APCs of the present invention are dendritic cells. Dendritic cells are very powerful APCs (Banchereau and Steinman, 1998, Nature, 392:245-251) and have been shown to be effective as physiological adjuvants for inducing preventive or therapeutic immunity (see Timmerman and Levy, 1999, Ann. Rev. Med. 50:507-529). Generally, dendritic cells can be identified based on their typical shape (star-shaped in situ and having prominent cytoplasmic protrusions (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 that are not normally found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are also contemplated by the present invention. Separately from dendritic cells, secretory vesicles (called exosomes) loaded with antigen may be used in the immunogenic composition (see Zitvogel et al., 1998, Nature Med. 4:594-600). Thus, in one embodiment, exosomes loaded with the polypeptides of the present invention are provided.

[0135] Dendritic cells and precursor cells may be obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, cord blood, or other suitable tissues or body 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 cultures of monocytes obtained from peripheral blood. Alternatively, CD34-positive cells obtained from peripheral blood, cord blood, or bone marrow may be added to a combined culture medium of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and / or other compounds that induce dendritic cell differentiation, maturation, and proliferation to differentiate into dendritic cells.

[0136] Dendritic cells are conveniently categorized into "immature" and "mature" cells and can be easily distinguished between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APCs with high antigen uptake and processing capabilities, which correlates with high expression of Fcγ receptors and mannose receptors. The mature phenotype typically has low expression of these markers but is characterized by high expression of cell surface molecules involved in T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

[0137] The APCs may also be transfected, for example, with a polynucleotide encoding a protein (or a part or other variant thereof) and genetically engineered so that the polypeptide is expressed on the cell surface. Such transfection may occur in vitro, and then a pharmaceutical composition containing the transfected cells may be used as described herein. Alternatively, a gene delivery vehicle targeting dendritic cells or other antigen-presenting cells may be administered to a patient to effect transfection that occurs in vivo. In vivo and in vitro transfection of dendritic cells may be carried out using any method generally known in the art, such as, for example, the methods described in WO97 / 24447 or the gene gun approach described in Mahvi et al., 1997, Immunology and Cell Biology 75:456-460. Antigen loading of dendritic cells may be achieved by incubating the dendritic cells or progenitor cells with a polypeptide, DNA (e.g., plasmid vector) or RNA; or with a recombinant bacterium or virus expressing the antigen (e.g., adenovirus, adeno-associated virus (AAV) (e.g., AAV serotype 5 and 2), alphavirus (e.g., Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SIN), Semliki Forest virus (SFV)), herpes virus, arenavirus (e.g., lymphocytic choriomeningitis virus (LCMV)), measles virus, poxvirus (modified vaccinia Ankara (MVA) or fowlpox, etc.), paramyxovirus, lentivirus, or rhabdovirus (vesicular stomatitis virus (VSV), etc.)). Prior to loading the polypeptide, the polypeptide may be covalently attached to an immunological partner (e.g., a carrier molecule) to provide T cell help. Alternatively, the dendritic cells may be pulsed with a non-conjugated immunological partner, either individually or in the presence of a polypeptide or vector.

[0138] The present invention provides a specifically designed short chemically synthesized epitope-encoding 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 peptide (SLP) and is used to stimulate cells (or load cells) in vitro using the antigenic polypeptides of the present invention (Gornati et al., 2018, Front. Imm, 9:1484), or as a method for introducing a polypeptide antigen into antigen-presenting cells in vivo (Melief and van der Burg et al., 2008, Nat Rev Cancer, 8:351-60).

[0139] In one embodiment, a pharmaceutical composition is provided that comprises an antigen-presenting cell of the present invention, preferably a dendritic cell, 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. In one embodiment, an antigen-presenting cell of the present invention, preferably a dendritic cell, is provided for use in a medicament.

[0140] Similarly, a method for treating a human suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, or a method for preventing a human from suffering from cancer, wherein the cancer cells will express a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, is provided, the method comprising administering to the human an antigen-presenting cell of the present invention, preferably a dendritic cell, or a composition comprising the antigen-presenting cell of the present invention.

[0141] In one embodiment, an antigen-presenting cell of the present invention, preferably a dendritic cell, or a composition comprising the antigen-presenting cell 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 NOs: 1 to 10 and any one immunogenic fragment thereof, is provided.

[0142] In one embodiment, a pharmaceutical composition is provided that includes the exosomes of the present invention together with a pharmaceutically acceptable carrier. Such a composition may be a sterile composition suitable for parenteral administration. For example, see the disclosure of the pharmaceutical composition described above. The composition may optionally also include an immunostimulant. See the disclosure of the immunostimulant described above. In one embodiment, exosomes of the present invention for use in medicine are provided.

[0143] Similarly, a method for treating a human suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, or a method for preventing a human from suffering from cancer, wherein the cancer cells will express a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, a method is provided that includes administering to the human the exosomes of the present invention or a composition comprising the exosomes of the present invention.

[0144] In one embodiment, an exosome of the present invention or a 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 NOs: 1 to 10 and any one immunogenic fragment thereof, is provided. In any one of the above embodiments, preferably, the cancer is malignant melanoma, particularly cutaneous malignant melanoma.

[0145] (Therapy with Stimulated T Cells) In addition to the in vivo or in vitro APC-mediated generation of T cells that are immunospecific for the polypeptide of the present invention, autologous or allogeneic T cells may also be isolated from a subject, for example, from peripheral blood, umbilical cord blood, and / or by apheresis, and stimulated in the presence of tumor-associated antigens loaded on MHC molecules (signal 1) of APC cells to induce the proliferation of T cells having a TCR that is immunospecific for this antigen.

[0146] To successfully activate T cells, the costimulatory surface molecules B7 and CD28 need to bind to antigen-presenting cells and T cells, respectively (signal 2). To achieve optimal T cell activation, both signals 1 and 2 are required. In contrast, antigenic peptide stimulation (signal 1) without costimulation (signal 2) cannot induce complete T cell activation and may result in T cell tolerance. In addition to costimulatory molecules, inhibitory molecules such as CTLA-4 and PD-1 also exist and induce signals to prevent T cell activation.

[0147] Therefore, autologous or non-autologous T cells can be stimulated and proliferated in the presence of the polypeptide of the present invention and then reintroduced into a patient at risk of developing cancer or a cancer patient such that their cancer cells express the corresponding polypeptide of the present invention, provided that their antigen-specific TCR recognizes the antigen presented by the patient's MHC, targets cancer cells expressing the corresponding polypeptide, and will induce the death of those cells.

[0148] In one embodiment, there is provided a polypeptide, nucleic acid, vector or composition of the present invention for use in stimulating and / or amplifying T cells derived from a cancer patient in vitro and then reintroducing the stimulated and / or amplified T cells into the patient for treating the cancer of the patient.

[0149] The present invention provides a method for treating human cancer, wherein the cancer cells express a sequence selected from SEQ ID NOs: 1 to 10 and any immunogenic fragment and variant thereof, the method comprising removing from the human, optionally together with antigen-presenting cells, a leukocyte population comprising at least T 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 into the human at least a part or all of the leukocytes comprising the stimulated and / or amplified T cells. In any one of the above embodiments, preferably, the cancer is malignant melanoma, particularly cutaneous malignant melanoma.

[0150] In one embodiment, there is provided a method for preparing a population of T cells that is cytotoxic to cancer cells expressing a sequence selected from SEQ ID NOs: 1 to 10 and any one of their immunogenic fragments and variants, the method comprising: (a) obtaining T cells and antigen-presenting cells from a cancer patient, and (ii) stimulating and amplifying the population of T cells in vitro with the corresponding polypeptide, nucleic acid, vector or composition of the present invention. In this context, "corresponding" means that when the cancer cells express, for example, SEQ ID NO: A (where A is one of SEQ ID NOs: 1 to 10) or a variant or an immunogenic fragment thereof, the population of T cells is stimulated and amplified in vitro by SEQ ID NO: A or a variant or an immunogenic fragment thereof in the form of a polypeptide, nucleic acid or vector, or a composition comprising one of the foregoing.

[0151] For example, in such a preparation process, culturing and proliferation are carried out in the presence of dendritic cells. The dendritic cells will be transfected with the nucleic acid molecule or vector of the present invention to express the polypeptide of the present invention. The present invention provides a population of T cells (hereinafter referred to as the T cell population of the present invention) that would be obtained by any of the foregoing preparation methods.

[0152] In one embodiment, there is provided a cell that is a T cell stimulated with the polypeptide, nucleic acid, vector or composition of the present invention (hereinafter referred to as the T cell of the present invention). In one embodiment, there is provided a pharmaceutical composition comprising the T cell population or T cell of the present invention together with a pharmaceutically acceptable carrier. Such a composition may be, for example, a sterile composition suitable for parenteral administration. In one embodiment, there is provided the T cell population or T cell of the present invention for use in medicine.

[0153] Similarly, a method for treating a human suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NOs: 1 to 10 and any one of its immunogenic fragments and variants, or a method for preventing a human from suffering from cancer, wherein the cancer cells will express a sequence selected from SEQ ID NOs: 1 to 10 and any one of its immunogenic fragments and variants, a method is provided which comprises administering to the human the T cell population or T cells of the present invention or a composition comprising the T cell population or T cells of the present invention.

[0154] In one embodiment, there is provided a T cell population of the present invention, a T cell of the present invention, 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 NOs: 1 to 10 and any one of its immunogenic fragments. In any one of the above embodiments, preferably, the cancer is melanoma, particularly cutaneous melanoma.

[0155] In one embodiment, there is provided a preparation method, method, or T cell population, T cell, antigen-presenting cell, exosome or composition for use of the present invention, wherein the polypeptide comprises a sequence selected from the following: (a) any one of the sequences of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and, (c) an immunogenic fragment of the sequence of (a), and for example, the polypeptide comprises or consists of a sequence selected from any one of SEQ ID NOs: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, 51 to 57, 67 to 74 and 76 to 77, and for example, the nucleic acid comprises or consists of a sequence selected from any one of SEQ ID NOs: 33, 35, 36 or 40, or a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50; and wherein the cancer is choroidal melanoma, the preparation method, method, or T cell population, T cell, antigen-presenting cell, exosome or composition is provided.

[0156] (Therapy with Genetically Engineered Immune Cells) Derivatives of all of the above types of CLT antigen-binding polypeptides comprise a TCR or TCR mimetic (see Dubrovsky et al., 2016, Oncoimmunology) that recognizes a peptide derived from a CLT antigen that forms a complex with a human HLA molecule, and may be engineered to be expressed on the surface of (self-derived or non-self-derived) T cells, which can then be administered as adoptive T cell therapy for cancer treatment.

[0157] These derivatives fall into the "chimeric antigen receptor (CAR)" category and, as used herein, may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and would further include receptors engineered to confer an artificial specificity to particular immune effector cells. A CAR may be used, for example, for use in adoptive cell therapy to confer the specificity of a monoclonal antibody to T cells, whereby a large number of specific T cells can be generated. A CAR would render the cells specific for a tumor-associated antigen that is a polypeptide of the invention that binds to HLA.

[0158] Another approach for treating a patient's cancer is to genetically modify T cells to target antigens expressed on tumor cells, via the expression of a chimeric antigen receptor (CAR). This technique is reviewed in Wendell and June, 2017, Cell, 168:724-740, which is incorporated herein by reference in its entirety.

[0159] Such CAR T cells can 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 immunospecific for the polypeptide of the present invention that binds to HLA. Such nucleic acids could be integrated into the genome of the cells, and an effective amount of the cells could be administered to the subject to provide a T cell response against the cells expressing the polypeptide of the present invention. For example, a cell sample may be collected from the subject.

[0160] It is understood that the cells used to produce the CAR-expressing T cells may be autologous or allogeneic. The genetically engineered CAR-expressing T cells may exhibit inactivation of the endogenous T cell receptor and / or endogenous HLA expression. For example, the cells may be engineered to eliminate the expression of the endogenous α / β T cell receptor (TCR).

[0161] Methods of cell transfection are well known in the art, but very 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 the cells using a "nucleofection" device.

[0162] The cell population for the CAR-expressing T cells may be enriched after transfection of the cells. For example, the CAR-expressing cells can be selected (e.g., by FACS) from non-expressing cells using an antigen bound by the CAR or a CAR-binding antibody. Another enrichment step may include depleting non-T cells or depleting cells that do not express CAR. For example, CD56+ cells can be depleted from the culture population.

[0163] The population of genetically engineered CAR-expressing cells may be cultured in vitro in a medium that selectively enhances the proliferation of the CAR-expressing T cells. Thus, the CAR-expressing T cells may be expanded in vitro. Samples of CAR cells may be stored (or maintained in culture). For example, the 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.

[0164] In one embodiment, a cytotoxic cell engineered to express any of the antigen-binding polypeptides on its surface is provided. Suitably, the cytotoxic cell is a T cell. In one embodiment, a cytotoxic cell, preferably a T cell, engineered to express any of the antigen-binding polypeptides on its surface for use in medicine is provided. The present invention provides a pharmaceutical composition comprising the cytotoxic cells of the present invention, preferably T cells.

[0165] A method of treating a human cancer patient, wherein the cancer cells thereof express a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, or a method of preventing a human from developing cancer, wherein the cancer would express a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, A method is provided that includes administering to the human the cytotoxic cells of the present invention, preferably T cells.

[0166] In one embodiment, the cytotoxic cells of the present invention, 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 NOs: 1 to 10 and any one immunogenic fragment thereof.

[0167] (Combination therapy) The method for treating cancer of the present invention may be carried out in combination with other therapies, particularly checkpoint inhibitors and interferons. Polypeptides, nucleic acids, vectors, antigen-binding polypeptides, and adoptive cell therapies (based on APCs and T cells) can be used in combination with other components designed to enhance their immunogenicity (e.g., improve the magnitude and / or scope of the induced immune response) or to provide other activities (e.g., activation of other aspects such as innate or adaptive immune responses or destruction of tumor cells).

[0168] Accordingly, the present invention provides kits of several compositions comprising a composition of the invention (i.e., an immunogenic, vaccine or pharmaceutical composition), or a polypeptide, nucleic acid, or vector of the invention, together with a pharmaceutically acceptable carrier, and (i) one or more additional immunogenic or immunostimulatory polypeptides (e.g., interferon, IL-12, checkpoint inhibitory 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 the polypeptide products that are the subject of the present invention.

[0169] Checkpoint inhibitors block normal proteins on cancer cells or proteins on T cells that respond to them, and may be a particularly important class of agents to combine with CLT antigen-based therapies as they attempt to overcome one of the major defenses of cancer against immune system attack.

[0170] Accordingly, one aspect of the invention involves administering the polypeptide, nucleic acid, vector, antigen-binding polypeptide, composition, T cell, T cell population, or antigen-presenting cell of the invention in combination with a checkpoint inhibitor. Examples of checkpoint inhibitors include 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).

[0171] Interferons (such as α, β, and γ) are a family of proteins that are produced in very small amounts by the body. Interferons may delay or stop cancer cell division, reduce the ability of cancer cells to protect themselves from the immune system, and / or enhance multiple aspects of the adaptive immune system. Interferons are typically administered, for example, as a subcutaneous injection in the thigh or abdomen.

[0172] Accordingly, one aspect of the invention involves administering the polypeptide, nucleic acid, vector, antigen-binding polypeptide, or composition of the invention in combination with an interferon, such as interferon α.

[0173] Also, different modes of the invention may be combined. For example, the polypeptide, nucleic acid, and vector of the invention may be combined with the APC, T cell, or T cell population of the invention (as described below). One or more modes of the invention may also be combined with conventional anti-cancer chemotherapy and / or radiation therapy.

[0174] (Diagnosis) In another aspect, the invention provides a method of using one or more polypeptides or nucleic acids of the invention for diagnosing cancer, particularly malignant melanoma, such as cutaneous malignant melanoma, or for diagnosing a human subject suitable for treatment by the polypeptide, nucleic acid, vector, antigen-binding polypeptide, adoptive cell therapy, or composition of the invention.

[0175] Accordingly, the present invention provides a method for diagnosing whether a human is suffering from cancer, comprising: determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NOs: 1 to 10 and any one of its immunogenic fragments or variants (for example, selected from the sequences of SEQ ID NOs: 11 to 32 and 51 to 78); or a nucleic acid encoding the polypeptide sequence (for example, selected from the sequences of SEQ ID NOs: 33 to 40 and SEQ ID NO: 41 to 50); and diagnosing that the human is suffering from cancer when the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells.

[0176] The present invention provides a method for diagnosing a human suffering from cancer which is cutaneous malignant melanoma, comprising: determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NOs: 2, 6, 7, 8 and 9 and any one of its immunogenic fragments or variants, or a nucleic acid encoding the polypeptide sequence; and diagnosing that the human is suffering from cancer which is cutaneous malignant melanoma when 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 that in normal cells.

[0177] The present invention provides a method for diagnosing a human suffering from cancer which is cutaneous malignant melanoma or uveal malignant melanoma, comprising: determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NOs: 1, 3, 4, 5 and 10 and any one of its immunogenic fragments or variants, or a nucleic acid encoding the polypeptide sequence; and diagnosing that the human is suffering from cancer which is cutaneous malignant melanoma or uveal malignant melanoma when the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells.

[0178] Overexpression can be determined by reference to the level of the nucleic acid or polypeptide of the invention within control human subjects known not to have cancer. Thus, overexpression indicates that the nucleic acid or polypeptide of the invention is detected at a significantly higher level (e.g., 30%, 50%, 100% or 500% higher) in the test subject than in the control subject. If the control human subject has the nucleic acid or polypeptide of the invention at a level so low that it cannot be detected, diagnosis is achieved by detecting the nucleic acid or polypeptide of the invention.

[0179] The present invention also provides a method for treating a human suffering from cancer, comprising: (a) determining whether the cancer cells express a polypeptide sequence selected from the polypeptide sequences of SEQ ID NOs: 1 - 10 and any one of its immunogenic fragments or variants (e.g., selected from the sequences of SEQ ID NOs: 11 - 32 and 51 - 78), or a nucleic acid encoding the polypeptide (e.g., selected from the sequences of SEQ ID NOs: 33 - 40 and 41 - 50); and if so, (b) administering to the human a corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cell, antigen - presenting cell, antigen - binding polypeptide or cytotoxic cell of the present invention.

[0180] Similarly, provided is the use of a polypeptide comprising a sequence selected from the following, isolated from a tumor of a human suffering from cancer: (a) any one of the sequences of SEQ ID NOs: 1 - 10; or (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a), or the use of a nucleic acid encoding the polypeptide, as a biomarker for determining whether the human would be suitable for treatment with a vaccine comprising a corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cell, antigen - presenting cell, antigen - binding polypeptide or cytotoxic cell of the present invention. Suitably, the cancer is malignant melanoma, particularly cutaneous malignant melanoma.

[0181] The present invention also relates to a polypeptide comprising a sequence selected from the following: (a) any one of the sequences of SEQ ID NO: 1, 3, 4, 5 and 10; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a), and for example, the polypeptide comprises or consists of a sequence selected from any one of SEQ ID NO: 11-14, 17-18, 19, 20-22, 30-32, 51-57, 67-74 and 76-77, and for example, the nucleic acid comprises or consists of a sequence selected from any one of SEQ ID NO: 33, 35, 36 or 40, or a sequence selected from any one of SEQ ID NO: 41, 43, 44, 45 and 50; and providing a method or use of the present invention, wherein the cancer is choroidal malignant melanoma.

[0182] Preferably, the polypeptide of the present invention has a sequence selected from SEQ ID NO: 1-10, or a fragment such as an immunogenic fragment thereof (for example, selected from the sequences of SEQ ID NO: 11-32 and 51-78). Preferably, the nucleic acid of the present invention has or comprises a sequence selected from any one of SEQ ID NO: 33-40 or 41-50, or a fragment such as an immunogenic fragment thereof.

[0183] 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, enabling the detection and semi-quantification of a specific nucleic acid. Such kits will enable the detection of PCR products through 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 the target DNA) enable the detection and quantification of mRNA, such as the transcript encoding the nucleic acid of the present invention. Assays using certain kits can be set up in a multiplex format to detect multiple nucleic acids simultaneously in one reaction. Kits for detecting active DNA (i.e., DNA having special epigenetic features indicative of 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.

[0184] The nucleic acids of the present invention may also be detected by liquid biopsy using a blood sample from a patient. Such procedures provide a non-invasive biopsy alternative to a surgical biopsy. Plasma from such a blood sample can also be isolated and analyzed for the presence of the nucleic acids of the present invention.

[0185] The polypeptide of the present invention may be detected using an antigen-specific antibody in an ELISA-type assay that detects the polypeptide of the present invention in a preparation obtained by homogenizing a tumor sample of a patient. Alternatively, the polypeptide of the present invention may be detected by immunohistochemical analysis, which involves examining sections of a patient tumor sample stained with an appropriately labeled antibody preparation using an optical 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 tumor sample stained with an appropriately labeled antibody preparation using an optical microscope to identify the presence of the polypeptide antigen.

[0186] The polypeptides of the present invention may also be detected by determining whether they can stimulate T cells to increase their reactivity to the polypeptide. Cells of cancer or tumor, such as malignant melanoma, such as cutaneous malignant melanoma, may be obtained, for example, from a biopsy of cancer, such as malignant melanoma, such as cutaneous malignant melanoma.

[0187] A method for treating human cancer, particularly malignant melanoma, such as cutaneous malignant melanoma, comprises (i) detecting the presence of the nucleic acid or polypeptide of the present invention, and (ii) administering to the subject the nucleic acid, polypeptide, vector, cell, T cell or T cell population or composition of the present invention, (and preferably administering the same nucleic acid or polypeptide or a fragment thereof as that detected).

[0188] A method for treating human cancer, particularly malignant melanoma, such as cutaneous malignant melanoma, also comprises administering to the subject the nucleic acid, polypeptide, vector, cell, T cell or T cell population or composition of the present invention, in which subject the presence of (and preferably the same) nucleic acid or polypeptide of the present invention has been detected.

[0189] In particular, the cancer to be diagnosed and, if possible, treated is malignant melanoma, such as cutaneous malignant melanoma. If a polypeptide of the present invention that is SEQ ID NO: 1, 3, 4, 5, or 10 or a fragment thereof is detected, the cancer is likely to be cutaneous malignant melanoma or uveal malignant melanoma.

[0190] (Specific embodiments) In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 1. Exemplary fragments comprise or consist of any one of SEQ ID NOs: 11-14. Further exemplary fragments comprise two, three, or four of SEQ ID NOs: 11-14. Further exemplary fragments comprise or consist of any one of SEQ ID NOs: 55-57 or 73-74. Further exemplary fragments comprise all of SEQ ID NOs: 11-14, 55-57, and 73-74 (taking into account possible sequence duplications so that there are no multiple occurrences of overlapping sequences). Exemplifications of the nucleic acid encoding the polypeptide sequence comprise or consist of SEQ ID NO: 33 or SEQ ID NO: 41. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. This nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used for the treatment of cancer, particularly malignant melanoma, such as cutaneous malignant melanoma or uveal malignant melanoma. Related diagnostic methods are also provided.

[0191] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 2. Exemplary fragments comprise or consist of SEQ ID NO: 15 or SEQ ID NO: 16. Further exemplary fragments comprise SEQ ID NO: 15 and SEQ ID NO: 16. Further exemplary fragments comprise or consist of any one of SEQ ID NOs: 58-66, 75, and 78. Further exemplary fragments comprise all of SEQ ID NOs: 15-16, 58-66, 75, and 78 (taking into account possible sequence duplications so that no duplicate sequences are present more than once). Exemplification of the nucleic acid encoding the polypeptide sequence comprises or consists of SEQ ID NO: 34 or SEQ ID NO: 42. Provided are the corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as set forth above. This nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used for the treatment of cancer, particularly malignant melanoma, such as cutaneous malignant melanoma. Related diagnostic methods are also provided.

[0192] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 3. Exemplary fragments comprise or consist of SEQ ID NO: 17 or SEQ ID NO: 18. Further exemplary fragments comprise SEQ ID NO: 17 and SEQ ID NO: 18. Further exemplary fragments comprise or consist of SEQ ID NO: 53 and any one of SEQ ID NOs: 67-69. Further exemplary fragments comprise SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 53. Further exemplary fragments comprise all of SEQ ID NOs: 17-18, 53, and 67-69 (taking into account possible sequence duplications so that no duplicate sequences exist more than once). Exemplification of the nucleic acid encoding the polypeptide sequence comprises or consists of SEQ ID NO: 35 or SEQ ID NO: 43. Also provided are the corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as set forth above. This nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used in the treatment of cancer, particularly melanoma, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0193] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 4. Exemplary fragments comprise or consist of SEQ ID NO: 19. Further exemplary fragments comprise or consist of SEQ ID NO: 51 or SEQ ID NO: 52. Further exemplary fragments comprise or consist of SEQ ID NO: 54. Further exemplary fragments comprise or consist of any one of SEQ ID NOs: 70-72 and 76-77. Further exemplary fragments comprise SEQ ID NO: 19 and either SEQ ID NO: 51 or SEQ ID NO: 52. Further exemplary fragments comprise SEQ ID NO: 54 and either SEQ ID NO: 51 or SEQ ID NO: 52. Further exemplary fragments comprise all of SEQ ID NOs: 19, 51-52, 54, 70-72 and 76-77 (taking into account possible sequence duplications so that no duplicate sequences are present more than once). Exemplary nucleic acids encoding the polypeptide sequences comprise or consist of SEQ ID NO: 35 or SEQ ID NO: 44. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes as set forth above are provided. This nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes may be used for the treatment of cancer, particularly malignant melanoma, such as cutaneous malignant melanoma or uveal malignant melanoma. Related diagnostic methods are also provided.

[0194] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 5. Exemplary fragments comprise or consist of any one of SEQ ID NOs: 20-22. Exemplary nucleic acids encoding the polypeptide sequences comprise or consist of SEQ ID NO: 36 or SEQ ID NO: 45. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes as set forth above are provided. This nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes may be used for the treatment of cancer, particularly malignant melanoma, such as cutaneous malignant melanoma or uveal malignant melanoma. Related diagnostic methods are also provided.

[0195] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 6. Exemplary fragments comprise or consist of SEQ ID NO: 23 or SEQ ID NO: 24. Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO: 37 or SEQ ID NO: 46. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. This nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used in the treatment of cancer, particularly malignant melanoma, such as cutaneous malignant melanoma. Related diagnostic methods are also provided.

[0196] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 7. Exemplary fragments comprise or consist of SEQ ID NO: 25. Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO: 38 or SEQ ID NO: 47. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. This nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used in the treatment of cancer, particularly malignant melanoma, such as cutaneous malignant melanoma. Related diagnostic methods are also provided.

[0197] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 8. An exemplary fragment comprises or consists of SEQ ID NO: 26. Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO: 38 or SEQ ID NO: 48. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. These nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used for the treatment of cancer, particularly malignant melanoma, such as cutaneous malignant melanoma. Related diagnostic methods are also provided.

[0198] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 9. An exemplary fragment comprises or consists of any one of SEQ ID NOs: 27-29. Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO: 39 or SEQ ID NO: 49. The corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. These nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used for the treatment of cancer, particularly malignant melanoma, such as cutaneous malignant melanoma. Related diagnostic methods are also provided.

[0199] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO: 10. Exemplary fragments comprise or consist of any one of SEQ ID NOs: 30-32. Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO: 40 or SEQ ID NO: 50. Also provided are the corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above. This nucleic acid (e.g., DNA or RNA), T cells, T cell populations, cytocotic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used for the treatment of cancer, particularly melanoma, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

Example

[0200] (Example) (Example 1 - CLT Identification) The purpose was to identify cancer-specific transcripts that consist entirely or in part of LTR elements. As a first step, a comprehensive cancer-type cross-sectional transcriptome was subjected to de novo assembly analysis. To achieve this, RNA sequencing reads from 768 patient samples, representative of a gender-balanced set of 24 samples (32 cancer types each (31 primary and 1 metastatic malignant melanoma), Table S1), were obtained from the Cancer Genome Atlas (TCGA) consortium and used for genome-guided assembly. These gender-balanced (excluding sex-specific tissues) samples were adapter-trimmed using cutadapt (v1.13) (Marcel M, 2011, EMBnet J., 17:3), quality (Q20) trimmed, length-filtered (both reads of 35 or more nucleotide pairs), and further subjected to kmer normalization (k = 20) with maximum and minimum depths of 200 and 3 respectively using khmer (v2.0) (Crusoe et al., 2015, F1000Res., 4:900). Reads were mapped to GRCh38 using STAR (2.5.2b) with the same settings as those used across TCGA and subjected to genome-guided assembly by disabling the built-in in silico depth normalization in Trinity (v2.2.0) (Trinity, Grabherr, M.G., et al., 2011, Nat. Biotechnol., 29:644-52). Most of the assembly process was completed within 256GB of RAM on 32-core HPC nodes, and failed processes were re-run using a 1.5TB RAM node. The resulting contigs were poly(A)-trimmed (using trimpoly in SeqClean v110222), entropy-filtered (≥0.7), and low-quality and artificial contigs (bbduk within BBMap v36.2) were removed. For each cancer type, the original 24 samples were quasi-mapped to the clean assembly using Salmon (v0.8.2 or v0.9.2) (Patro, R., et al., 2017, Nat. Methods, 14:417-419), and contigs with expression of <0.1 transcripts per million (TPM) were removed.The remaining ones were mapped to GRCh38 using GMAP (v161107) (Wu et al., 2005, Bioinf., 21:1859-1875), and contigs that did not match having 85% or more identity over 85% or more of their length were removed from the assembly. Finally, the assembly combining all cancer types was flattened and merged into the longest continuous transcript using gffread (Cufflinks v2.2.1) (Trapnell et al., 2010, Nat. Biotech., 28:511-515). Since this assembly process was specifically designed to allow the evaluation of repetitive elements, monoexonic transcripts were retained but flagged. The completeness and quality of the transcript assembly were evaluated by comparison with GENCODE v24 basic and MiTranscriptome1 (Iyer et al., 2015, Nat. Genet., 47:199-208). A list of unique splice sites shown in GENCODE was compiled, and it was tested whether those splice sites were present within the transcriptome assembly within a 2-nucleotide tolerance window. This process identified 1,001,931 transcripts, of which 771,006 were spliced and 230,925 were monoexonic.

[0201] Separately from this, the assembled contigs were overlaid with genomic repeat sequence annotation to identify transcripts containing LTR elements. LTR and non-LTR elements were annotated as described above (Attig et al., 2017, Front. In Microbiol., 8:2489). Briefly, a hidden Markov model (HMM) representing known human repeat families (Dfam 2.0 library v150923) was used, and GRCh38 was annotated using RepeatMasker Open-3.0 (Smit, A., R. Hubley and P. Green, http: / / www.repeatmasker.org, 1996 - 2010), which was composed of nhmmer (Wheeler et al., 2013, Bioinform., 29:2487 - 2489). The HMM-based scan improves annotation accuracy compared to the BLAST-based method (Hubley et al., 2016, Nuc. Acid. Res., 44:81 - 89). RepeatMasker annotates LTR and internal regions separately, so 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.

[0202] Salmon was used to estimate transcripts per million (TPM) for all transcripts, and expression within each cancer type was compared to expression in 811 healthy tissue samples (healthy tissue-matched controls for all cancer types obtained from TCGA when available and otherwise from GTEx (The Genotype-Tissue Expression Consortium, 2015, Science, 348:648-60)). Transcripts were considered to be specifically expressed in cancer if detected above 1 TPM in any sample and cancer-specific if the following criteria were met: (i) expressed in 6 or more of 24 samples of each cancer type; (ii) expressed below 10 TPM in 90% or more of all healthy tissue samples; (iii) expressed at least 3-fold higher than the median expression in any control tissue type in the target cancer type; and (iv) expressed at least 3-fold higher than the 90th percentile of each available healthy tissue in the target cancer type. In addition to these expression threshold criteria, transcript selection was based on manual inspection to exclude transcripts with potential misassembled contigs or LTR elements within their 3’ untranslated region (UTR). When the direction of transcription could not be clearly assigned, transcripts corresponding to both strands were considered.

[0203] Next, the list of cancer-specific transcripts was intersected with the list of transcripts containing complete or partial LTR elements to create a list of 5,923 transcripts that met both criteria (referred to as cancer-specific LTR element-spanning transcripts, or CLTs).

[0204] To identify CLTs with the potential to encode proteins, an ORF prediction algorithm was run based on the length and fitness of the dicodon (hexamer) score. The HMM was trained with hexamers from Ensembl CDS sequences and run for ORFs of 300 or more nucleotides, and the sense hexamer score was higher than the antisense score. This filter identified 885 CLTs that have the potential to encode proteins of at least 99 amino acids in length.

[0205] To identify unique protein sequences that may be encoded by the CLTs, the sequences translated from the largest ORFs of the selected CLTs were queried against those translated from all ORFs of 210 or more nucleotides from the entire transcript assembly without soft-masking using tblastn (BLAST+ v2.3.0). CLTs with no hits or with E-values > 10 -5 were retained only if there were hits.

[0206] To further ensure the specificity of cancer-specific antigens encoded by the CLTs, potential cross-reactivity with other proteins that may be expressed in healthy tissues was examined. For this purpose, the translated ORFs having < 85% amino acid sequence identity with any of the other predicted proteins (over the full length of the protein) were retained. For proteins encoded by the CLTs showing > 85% sequence identity with one or more predicted proteins, the expression patterns of the transcripts encoding similar proteins were collated. Also, if these additional transcripts were expressed in a cancer-specific pattern (based on the above criteria), each CLT was left on the selected candidate list. If, on the other hand, the additional transcripts were expressed in healthy tissues, each CLT was deleted. By combining these selection criteria, a final list of 139 CLTs that may encode proteins with sufficiently unique amino acid sequences was created.

[0207] Among these 139 CLTs, 14 were specific to cutaneous malignant melanoma (i.e., it was confirmed that they were specifically upregulated in TGCA cutaneous malignant melanoma samples according to the above method), and 7 were specific to cutaneous malignant melanoma and uveal malignant melanoma (i.e., it was confirmed that they were specifically upregulated in TGCA cutaneous malignant melanoma samples and uveal malignant melanoma samples according to the above method). Four of these CLTs specific to cutaneous malignant melanoma are identified herein as having SEQ ID NOs: 34, 37, 38, and 39. Four of these CLTs specific to cutaneous malignant melanoma and uveal malignant melanoma are identified herein as having SEQ ID NOs: 33, 35, 36, and 40.

[0208] (Example 2 - Immunopeptidome Analysis) Immunopeptidome analysis is a powerful technique that enables the direct detection of specific peptides associated with HLA molecules in cells or tissues. This technique involves affinity purification of HLA molecules from biological samples, followed by elution of the bound peptides from the HLA molecules, and nano ultra-high performance liquid chromatography-mass spectrometry (nUPLC-MS 2) consists of peptide evaluation (Freudenmann et al., 2018, Immunology 154(3):331 - 345). The mass spectrometry (MS) spectra generated by this method can be used to accurately identify short - chain peptides bound to HLA class I and HLA class II molecules. The software used for spectrum interpretation and sequence identification depends on the availability of a defined list of protein sequences for spectrum matching. It is possible to search MS data using a defined list 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), but the interrogation of these extremely large sequence databases leads to a very high false - discovery rate that limits the identification of the presented peptides. Further technical problems (e.g., leucine mass = isoleucine mass) and theoretical problems (e.g., peptide splicing (Liepe et al., 2016, Science 354(6310):354 - 358)) increase the limitations associated with the use of extremely large databases such as those created from known transcriptomes or the entire genome. Therefore, in practice, it is very difficult to perform immunopeptidome analysis to identify novel antigens without referring to a clearly defined set of potential polypeptide sequences.

[0209] Bassani-Sternberg et al. collated MS data collected from HLA-binding peptide samples derived from 25 patients with cutaneous malignant melanoma against polypeptides reported for the entire human proteome (Bassani-Sternberg et al., 2016, Nature Commun., 7:13404). These analyses revealed hundreds of thousands of peptides matching known human proteins. As predicted, these peptides included those found within multiple tumor-associated antigens (TAAs), including PRAME, MAGEA3, and TRPM1 (melastatin). Furthermore, when the MS data of 5 of these patients were collated against a list of polypeptides generated from patient-specific mutant protein sequences detected by genomic analysis of these 5 patients, patient-specific neoantigens presented on the HLA class I and HLA class II molecules of these patients were revealed.

[0210] Many of the predicted polypeptide sequences (ORFs) derived from the 139 CLTs described in Example 1 are not contained within the human proteome. By applying detailed knowledge of immunopeptidome assessment, the inventors collated the RAW data files of Bassani-Sternberg et al. (database link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894) against this series of novel potential CLT antigen sequences.

[0211] To perform this analysis, peptide sequences from all possible ORFs encoded by each CLT were concatenated into a single peptide file for each CLT or not concatenated, and these concatenated files (Analysis A) or single peptide files (Analysis B) were used to collate the raw spectra within the PXD004894 dataset, and the collation was performed against all polypeptides found within the human proteome (UniProt (Analysis A) or UniProt and masDB (Analysis B)), using Peaks™ software (Analysis A) or Mascot software (Analysis B).

[0212] In analysis A, the results of these studies identified 14 peptides associated with HLA class I molecules immunoprecipitated from tumor samples of 25 patients examined by Bassani-Sternberg et al., and these could be attributed to 8 ORFs that were not found in the reported proteome (see Table 1). In analysis B, the results of these studies identified 14 peptides associated with HLA class I or HLA class II molecules immunoprecipitated from tumor samples of 25 patients examined by Bassani-Sternberg et al., and these could be attributed to 7 ORFs that were not found in the reported proteome (see Table 2). Detection of these peptides associated with HLA class I and HLA class II molecules from the cited patients confirmed that the 10 ORFs (Tables 1 and 2, SEQ ID NOs: 1-10) from which they were derived were translated in melanoma tissue and presented to the immune system in complex with HLA class I or HLA class II molecules. Based on this result, the polypeptides encoded by these ORFs were defined as CLT antigens. Tables 1 and 2 show the characteristics of the peptides found in CLT antigens that were not present in the UniProt database. Figures 1-32 show representative mass spectrometry spectra of each of the peptides shown in Tables 1 and 2. These figures show the fragment spectra of the indicated peptide sequences, which were detected in individual SKCM tumor patients by nUPLC-MS 2 (images extracted from the PRIDE dataset by the PEAKS software; Bassani-Sternberg et al.). All detected fragments are shown in the peptide sequences on the spectra, and the most abundant fragment ions are assigned to each spectrum. In Figures 1-15, 29-32 (analysis A), the lower panel of the figure shows the sequence annotation to the predicted spectrum, while similar data are shown in tabular form on the right side of Figures 16-28 (analysis B). Fragment ions are annotated as follows: b: N-terminal fragment ion; y: C-terminal fragment ion; -H2O: water loss; -NH3: ammonia loss; [2+]: doubly charged peptide ion; pre: precursor peptide ion that has not been fragmented.

[0213] From Tables 1 and 2, the number of peptides detected in relation to HLA class I was evaluated to determine the predicted strength of binding to HLA class I supertypes. Specifically, all HLA class I-related peptides of a length of 9 amino acids or more, as cited in Table 3, were collated using the NetMHC 4.0 prediction software (http: / / www.cbs.dtu.dk / services / NetMHC / ), and their binding to HLA class I type A and B supertypes was predicted. The results of these prediction studies indicated that all 11 peptides (or their derived 9-mers) were predicted to bind to at least one of the tested supertypes (see Table 3). Among these, many sequences were predicted to bind with high confidence (low rank score %) to specific types within the tested HLA class I supertypes.

[0214] In summary, the data shown in Tables 1 - 3 and Figures 1 - 32 provide very strong support that this corresponding CLT antigen is being presented in patients with melanoma. In summary, the identification of immunopeptidome peptides derived from the predicted ORFs indicates that these CLTs are translated into polypeptides (SEQ ID NOs: 1 - 10; also referred to as CLT antigens) in tumor tissue. These are then processed by the cell's immune surveillance apparatus, loaded onto HLA class I or HLA class II molecules, and can target the cell for lysis by T cells that recognize the resulting peptide / HLA class I complex or peptide / HLA class II complex. Thus, these CLT antigens and their fragments are predicted to be useful in various therapeutic modalities for the treatment of melanoma in patients whose tumors express these antigens.

[0215] (Table 1: List of peptides identified by immunopeptidome analysis (Analysis A) of SKCM tumor samples, as well as CLT antigen names and cross-referenced SEQ ID NOs)

Table 1

[0216] (Table 2: List of peptides identified by immunopeptidome analysis (Analysis B) of SKCM tumor samples, as well as CLT antigen names and cross-referenced SEQ ID NOs)

Table 2

[0217] (Table 3: Predicted NetMHC 4.0 binding of peptides (length ≥ 9 residues) identified by mass spectrometry to 12 HLA class I supertype alleles (HLA-A0101, HLA-A0201, HLA-A0301, HLA-A2402, HLA-A2601, HLA-B0702, HLA-B0801, HLA-B1501, HLA-B2705, HLA-B3901, HLA-B4001, HLA-B5801), as well as CLT antigen names and cross-referenced SEQ ID numbers)

Table 3

[0218] (Example 2.1 - Additional immunopeptidome analysis) In addition to the analysis described in Example 2, the inventors also identified peptides derived from ORFs predicted through a new immunopeptidome study. This additional study described below further shows that those CLTs are translated into CLT antigen polypeptides within tumor tissue.

[0219] The inventors procured frozen tumor tissues from 10 patients diagnosed with malignant melanoma. Samples of 0.05 - 1 g were homogenized, the lysates were centrifuged at high speed, and the clarified lysates were mixed with protein A (ProA) beads covalently bound to anti - human HLA class I monoclonal antibody (W6 / 32). The mixture was incubated overnight at 4 °C to improve the binding of HLA class I molecules to the antibody (Ternette et al., 2018, Proteomics 18, 1700465). HLA class I - bound peptides were eluted from the antibody using 10% acetic acid, and then the peptides were separated from other high - molecular - weight components using reverse - phase column chromatography (Ternette et al., 2018). The purified eluted peptides were subjected to nUPLC - MS, specific peptides of a given charge - to - mass ratio (m / z) were selected by the mass spectrometer, isolated, fragmented, and subjected to MS / MS to reveal the m / z of the resulting fragment ions (Ternette et al., 2018), and MS / MS datasets corresponding to the immunopeptidomes of each of these tumor samples were generated.

[0220] By applying detailed knowledge of immunopeptidome assessment, the inventors collated the spectra of HLA class I datasets for 10 malignant melanoma tumors created by the inventors using CLT antigen numbers 1, 2, 3, and 4 (Table 4; SEQ ID NOs: 1 - 4), which were searched (for each CLT) against all polypeptide sequences found within the human proteome (UniProt) using PEAKS™ software (v8.5 and vX, Bioinformatics Solutions Inc). Since most of the class I HLA - binding peptides found intracellularly are derived from constitutively expressed proteins, this simultaneous interrogation of these databases with the UniProt proteome helps to confirm that the assignment of the inventors' CLT ORF sequences to the MS / MS spectra is correct.

[0221] The results of these studies identified eight individual peptides (Table 4; SEQ ID NOs: 1-4) that were associated with HLA class I molecules immunoprecipitated from tumor samples from 10 melanoma patient samples procured by the inventors. These peptides corresponded to the amino acid sequences of CLT-derived ORFs and did not correspond to polypeptide sequences present within the known human proteome (UniProt). Of the eight peptides identified from CLT antigen SEQ ID NOs: 1-4 (Table 4) within the inventors' dataset, two were associated with HLA class I molecules immunoprecipitated from tumor samples from patients examined by Bassani-Sternberg et al. (from the same CLT antigen SEQ ID NOs: 1-4 outlined in Example 2 and Tables 1 and 2) and were additional to the 10 individual peptides.

[0222] The detection of these peptides associated with HLA class I molecules confirms that the four ORFs from which they are derived are first translated within melanoma tissue, processed through the HLA class I pathway, and finally presented to the immune system within a complex with HLA class I molecules. Table 4 shows the characteristics of the peptides found within the CLT antigen. Figures 33-42 show representative MS / MS spectra for each of the peptides shown in Table 4. The upper panel of each figure shows the MS / MS peptide fragment profile, along with standard MS / MS annotation (b: N-terminal fragment ion; y: C-terminal fragment ion; -H2O: water loss; -NH3: ammonia loss; [2+]: doubly charged peptide ion; pre: unfragmented precursor peptide ion; a n -n: internal fragment ion), which is the most abundant fragment ion peak within the image extracted from the inventors' dataset by the PEAKS software as shown above. The lower panel of each figure shows a rendering of the spectrum indicating the positions of the linear peptide sequence mapped to the fragment ions. These spectra contain a number of fragments that exactly match the peptide sequences (SEQ ID NOs: 12, 13, 16, 17, 19, 51, 53, and 54) discovered in these analyses, consistent with the high -10lgP scores assigned to the peptides in Table 4.

[0223] All of the peptides detected in relation to HLA class I of Table 4 that are at least 9 amino acids (aa) in length were evaluated using the NetMHCpan 4.0 prediction software (http: / / www.cbs.dtu.dk / services / NetMHCpan / ) to determine the predicted strength of their binding to HLA class I type A and B supertypes. The results of these prediction studies indicated that all of the peptides (or 9-mers contained within each complete sequence) were predicted to bind to at least one of the tested supertypes (see Table 5). Of these, many sequences were predicted to bind with high confidence (low rank score %) to specific types within the tested HLA class I supertypes. The fact that all of the detected peptides were predicted to bind to HLA types expected to be present within the patient population is consistent with their detection results. Furthermore, all of the peptides found in tumor samples from the inventors' dataset were predicted by NetMHCpan 4.0 to bind to one of the HLA types detected in the patient samples.

[0224] To provide further certainty for the assignment of the tumor tissue-derived MS spectra to the peptide sequences found in Example 2.1, peptides having these found sequences were synthesized and subjected to nUPLC-MS using the same conditions as applied to the tumor samples in the inventors' data. 2 The spectral comparison of the selected peptides is shown in FIGS. 43-50. In each figure, the upper spectrum corresponds to the tumor sample (from within the inventors' tumor tissue database - FIGS. 33-42), and the lower spectrum corresponds to a synthetically prepared peptide having the same sequence. The selected m / z values of the detected ion fragments are shown at each of the upper / lower fragment peaks of these MS / MS spectra. These figures clarify the exact alignment of the fragments (the slight differences between the experimentally determined m / z values between the fragment ions from the tumor-derived and synthetic peptides fall within an m / z tolerance of <0.05 Dalton), and confirm the credibility of the assignment of each spectrum from the tumor tissue to the CLT-coded peptides.

[0225] In summary, the peptide data shown in Table 4, Figures 33 - 42 and Figures 43 - 50 provide very strong support for the translation, processing and presentation of the corresponding CLT antigens in melanoma patients.

[0226] To further confirm the cancer - specificity of these CLTs, 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 datasets from these normal tissue samples and searched for all possible peptide sequences derived from the polypeptide sequences of CLT antigen numbers 1, 2, 3 and 4. Peptides derived from CLT antigen numbers 1, 2, 3 and 4 were not detected within the set of normal tissue samples (Table 6), providing additional confirmation that CLTs exhibit cancer - specific expression.

[0227] In summary, this additional immunopeptidome identification, derived from the predicted ORFs, further demonstrates that these CLTs are translated into polypeptides (SEQ ID NOs: 1 - 4; also referred to as CLT antigens) within tumor tissues. Thus, these CLT antigens and their fragments are predicted to be useful in various therapeutic modalities for the treatment of melanoma in patients whose tumors express these antigens.

[0228] (Table 4: List of peptides identified by additional immunopeptidome analysis of melanoma tumor samples, and CLT antigen names and cross - referenced SEQ ID NOs)

Table 4

[0229] (Table 5: Predicted NetMHC 4.0 binding to 12 HLA class I supertype alleles (HLA-A0101, HLA-A0201, HLA-A0301, HLA-A2402, HLA-A2601, HLA-B0702, HLA-B0801, HLA-B1501, HLA-B2705, HLA-B3901, HLA-B4001, HLA-B5801) of peptides (length ≥ 9 residues) identified by mass spectrometry, as well as the CLT antigen name and the cross-referenced sequence number)

Table 5

[0230] (Table 6: Number of peptides derived from CLT antigens 1 - 4 in the normal tissue sample set)

Table 6

[0231] The results shown in Examples 1, 2, and 2.1 of this specification are based in whole or in part on data generated by the Cancer Genome Atlas (TCGA) Research Network (http: / / cancergenome.nih.gov / ) and the Genotype-Tissue Expression (GTEx) project, which is supported by the Common Fund of the Office of the Director of the National Institutes of Health, as well as NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS.

[0232] (Example 3 - HERVFEST) The Functional Expansion of Specific T cells (FEST) technology has been used to identify treatment - relevant tumor - derived epitopes present in the repertoire of “mutation - associated neoantigens” (MANA) found on tumor cells of cancer patients, based on the detection of patient T cells that react to MANA epitopes (Anagnostou et al., Cancer Discovery 2017; Le et al., Science 2017; Forde et al., NEJM 2018; Danilova et al., Cancer Immunol. Res. 2018). The application of the FEST technology to the CLT antigens discovered using the methods described in Examples 1, 2, and 2.1 (Tables 1 - 6, Figures 1 - 50) can be used to identify treatment - relevant T - cell responses to CLT antigens in cancer patients.

[0233] Similar to other assays (e.g., ELISPOT) for identifying epitope-specific T cells within an immunologically exposed subject, the "FEST" technology elicits its specificity by activating / proliferating cognate T cells in an in vitro culture containing antigen-presenting cells and a suitable antigenic peptide. What differentiates this technology from other immunological assays is that it utilizes next-generation sequencing of the T cell receptor (TCR) DNA sequences present within these amplified cultures (specifically, TCRseq targeting the TCR-VβCDR3 region) to detect specific TCRs that have been expanded in cells cultured with individual peptides from a panel of target peptides derived from one antigen (or multiple antigens). Application of TCRseq to tumor tissue from the same patient can likewise be used to show whether the TCR / T cells and peptide-stimulated cultures detected in vitro are also present within tumor-infiltrating lymphocytes found in cancer tissue in situ. Thus, MANAFEST is a powerful technology for identifying MANA epitopes recognized by a patient's T cells, and it has been demonstrated that MANA peptides functionally relevant among the numerous mutant peptides detected by whole-exome sequencing of normal and tumor tissues from cancer patients can be identified (Le et al., Science 2017; Forde et al., NEJM 2018; Danilova et al., Cancer Immunol. Res. 2018; Smith et al., J Immunother Cancer 2019).

[0234] The application of the MANAFEST approach (Danilova et al., Cancer Immunol. Res. 2018) to CLT antigens was performed as follows. This method, referred to herein as HERVFEST, consists of the following steps: Step 1: Peptides predicted to contain epitopes that efficiently bind to the selected HLA class I alleles were identified within the CLT antigens. Step 2: PBMCs from appropriate melanoma patients were matched to the peptide library selected in Step 1 by HLA class I type. Step 3: PBMCs from these patients were separated into T cell and non-T cell fractions. The non-T cells were returned to the patients' T cells and then divided into 20 - 50 wells (containing 250,000 T cells per culture) and grown for 10 days with various T cell growth factors and synthetic peptides derived from individual CLT antigens (selected in Step 1 / 2). Step 4: TCRseq (sequencing of TCR-Vβ CDR3 sequences) was performed on all wells to identify TCR-Vβ CDR3 sequences that were amplified in the presence of individual CLT antigen-derived peptides (but not in the presence of control peptides or in the absence of peptide stimulation). Thus, the presence of amplified TCR-Vβ CDR3 sequences in individual wells of the assay identifies CLT antigen-derived peptides that elicited an immune response in melanoma patients. Step 5: Similarly, TCRseq can be performed on tumor samples to determine whether T cells bearing CLT antigens home to the patient's tumor, providing additional evidence that T cells bearing these TCRs recognize CLT antigen-derived peptides within the patient's tumor.

[0235] The HERVFEST assay was performed with peptides derived from CLT antigens 1-4 (SEQ ID NOs: 1-4). The peptide panel used in these studies (see step 1 above) was based on NetMHC predictions of CLT antigen-derived peptides, and these peptides were predicted to strongly bind to 8 HLA class I types commonly found in patient tumor samples available for the inventors' analysis. Peptides derived from CLT antigens that amplified one or more TCRs in these HERVFEST assays are shown in Table 7. Table 7 also shows the HLA class I types of the CLT antigen peptides tested in cultures derived from each patient's PBMC. Table 8 shows the HLA class I types of the patients whose PBMCs were tested in this study and in which one or more TCRs were amplified in the assay.

[0236] Panel A of FIG. 51 shows publicly available data demonstrating TCR amplification with MANA peptides specific for NSCLC (non-small cell lung cancer) patients (Forde et al., NEJM 2018). The vertical axis shows the prevalence of TCR-Vβ CDR3 sequences, each shown for wells of cells cultured in the presence of the MANA peptides or control peptides listed on the horizontal axis. Amplification within wells containing MANA7 indicates that the patient's T cell repertoire contains T cells that respond to this peptide. Panels B and C of FIG. 51 show representative TCR amplification data from PBMCs derived from two melanoma patients incubated in the presence of the indicated CLT antigen peptides and control peptides. Similar to panel A, the specific amplifications observed in panels B and C indicate that the T cell repertoires of these melanoma patients contain T cells that react with specific CLT antigen-derived peptides. Panel B shows 15 HLA class IA * In all wells stimulated with the 02 peptide panel, the frequency of TCRs detected in wells of PBMCs from melanoma patient 222B stimulated with LMSSFSTLASL is shown. Three TCR sequences were amplified.

Chemical formula

Chemical formula

[0237] The control peptides / conditions used in these experiments are as follows: CEF = a mixture of CMV, EBV, and influenza peptides; SL9, TV9, and QK1 = HIV-1 control peptides; no peptide = cultured in the absence of peptides; baseline = T cells before culture.

[0238] Figure 52 shows a summary of all CLT antigen peptides against CLT antigens 1-4 for which one or more TCRs were amplified during the studies conducted in these patients. Each panel shows the amino acid sequences of CLT antigens 1-4 with the peptides detected by immunopeptidome analysis placed on top (indicated by a dashed underline or bold text. See Examples 2 and 2.1). Below these sequences, the peptides detected by HERVFEST (see Figure 51) are shown together with the numbers of the melanoma patients in which they were detected (Table 8) and the targeted HLA class I types.

[0239] The characteristics of each HERVFEST detection are defined as follows. · Non-decorated text: significant amplification of a single TCR · Bold text: significant amplification of multiple TCRs · Underlined italic text: significant amplification of a single TCR detected in other wells as well · Underlined bold text: significant amplification of multiple TCRs, at least one of which was detected in other wells.

[0240] These results provide strong evidence that CLT antigens 1-4 are present in patients with malignant melanoma, and that peptides derived from these CLT antigens specifically induce T cell responses in these patients with malignant melanoma, confirming the value of these CLT antigens as targets for therapeutic intervention for the treatment of malignant melanoma.

[0241] (Table 7: CLT antigen-derived peptides that amplified one or more TCRs in the HERVFEST assay) [Table 7]

[0242] (Table 8: Characteristics of malignant melanoma patient PBMCs used in the HERVFEST assay) [Table 8]

[0243] (Assay to show that high-affinity T cells specific for CLT antigens were not deleted from the T cell repertoire of normal subjects) 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 thus that they have not been deleted by central tolerance due to the expression of cancer-specific CLT antigens in naive and thymic tissues of these patients. This type of ELISPOT assay involves multiple steps. Step 1: CD8 T cells and CD14 monocytes are isolated from the peripheral blood of normal blood donors, and these cells are HLA typed and matched to the specific CLT antigen to be tested. CD8 T cells can be further differentiated into naive and memory subtypes using a magnetic-labeled antibody 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 expanded CD8 T cells are separated from these cultures and restimulated overnight with fresh monocytes pulsed with peptides. These peptides may include; individual CLT antigen peptides, irrelevant control peptides, or peptides known to elicit a strong response against infectious (e.g., CMV, EBV, influenza, HCV) or self (e.g., Mart-1) antigens. Restimulation is performed on plates coated with anti-interferon gamma (IFNγ) antibody. This antibody captures IFNγ secreted by peptide-stimulated T cells. After overnight activation, the cells are washed off the plates and the IFNγ captured on the plates is detected with additional anti-IFNγ antibody and a standard chromogenic dye. Dark spots remain at the locations where IFNγ-producing cells were initially present on the plates. The data obtained from this assay include the number of spots, the median spot size, and the median spot intensity. These are measurements of the frequency of IFNγ-producing T cells and the amount of IFNγ per cell. And further, a measure of the magnitude of the response to the CLT antigen can be derived from the stimulation index (SI), which is the specific response measured as the number of spots or the median spot size divided by the background response to monocytes without the specific peptide. The criteria for evaluating the stimulation intensity are derived by multiplying the stimulation index of the number of spots by the stimulation index of the spot intensity.Using the comparison of responses to the CLT antigen and responses to a control antigen by this method, it can be shown that naive subjects contain a strong repertoire of CLT antigen-reactive T cells, which can be expanded by vaccination with a CLT antigen-based immunogenic formulation. Table 9 provides a list of CLT antigen-derived peptides that induced significant CD8 T cell responses from HLA-matched normal blood donors. The results are shown in Figures 53-56. The horizontal bars represent the mean values of the data. Statistical significance was measured using one-way analysis of variance of the Kruskal-Wallis test, with Dunnett's correction for repeated measurements. Figure 53 shows HLA-A from CLT antigen 1 (CLT001 in the figure). * shows significant CD8 T cell responses from normal blood donors to the HLA-A*0201-restricted peptide. The example shown in Figure 54 is for a peptide derived from CLT antigen 2 (CLT002 in the figure) that is similarly restricted by HLA-A * *0201, showing the CD8 response from normal donors. Figure 55 shows HLA-A from CLT antigen 4 (CLT004 in the figure). * shows significant CD8 T cell responses from normal blood donors to the HLA-A*0201-restricted peptide. Figure 56 shows the lack of response in memory CD45RO-positive CD8 T cells (panels A and C) to the HLA-B*0702-restricted peptides from CLT antigens 1 and 4 (CLT001 and CLT004 in the figure). * In contrast, naive CD45RO-negative CD8 T cells from the same donors responded significantly to the peptides from both CLT001 and CLT004 (Figure 56, panels B and D).

[0244] (Table 9: CLT antigen-derived peptides that induce significant CD8 T cell responses from HLA-matched normal blood donors)

Table 9

[0245] (Example 5 - Staining of reactive T cells with CLT antigen peptide pentamers) The presence and activity of circulating CD8 T cells specific for CLT antigens in healthy donors and melanoma patients can be measured using HLA class I / peptide pentamer (“pentamer”) staining and / or in vitro killing assays. Accordingly, the application of these techniques to CLT antigens discovered using the methods described in Examples 1, 2, and 2.1 (Tables 1-6, Figures 1-50) can be used to demonstrate the presence of treatment-related T cell responses to CLT antigens in cancer patients.

[0246] For these studies, CD8 T cells isolated from the blood of healthy donors or patients are expanded using various culture methods, such as microbeads coated with anti-CD3 and anti-CD28 and interleukin-2. The expanded cells can then be stained using the CLT peptide pentamer to determine the presence or absence of specific CLT antigen reactivity of their T cell receptors, which consists of pentamers of HLA class I molecules that bind to the relevant CLT antigen peptide within the peptide-binding groove of the HLA molecule. Binding is measured by detection using an antibody fragment conjugated to phycoerythrin or allophycocyanin specific for the coiled-coil multimerization domain of the pentamer structure. In addition to this pentamer staining, surface markers such as the memory marker CD45RO and the lysosome release marker CD107a can also be used for gating. Linking pentamer positivity with specific surface markers can be used to infer both the number and status (memory VS naive / stem) of the pentamer-reactive T cell population.

[0247] Pentaamer-stained cells may also be sorted and purified using a fluorescence-activated cell sorter (FACS). The sorted cells can then be further tested in an in vitro killing assay for their ability to kill target cells. These assays include a CD8 T cell population and a population of fluorescently labeled target cells. In this case, the CD8 population is either specific for the CLT antigen or a pentamer-sorted CD8 T cell specific for a positive control antigen known to induce a strong killing response such as Mart-1. Target cells for these studies include T2 cells pulsed with peptide and expressing HLA-A * 02, T2 cells pulsed with peptide and expressing HLA-A * 02, 03 or B * 07 transfected C1R cells, or malignant melanoma cell lines previously shown to express CLT / CLT antigen, or patient tumor cells may be included. Peptides used to pulse the T2 or C1R cells include the CLT antigen peptide or a positive control peptide. Target cells may be fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE, a cell proliferation dye), and cell death is indicated by the uptake of 7AAD. In this technique, when target cells are killed by CD8 T cell-mediated apoptosis, they acquire red fluorescence and become red / green double positive. Therefore, applying this killing assay to pentamer-sorted CLT antigen-specific CD8 T cells can be used to enumerate the cytotoxic activity of CLT antigen-specific T cells in vitro culture of malignant melanoma patients or healthy donor T cells. Figure 57 shows HLA pentamer staining of healthy donor CD8 T cells with peptides from CLT antigens 1, 2, and 4 (CLT001, CLT002, and CLT004 in the figure). Figure 58 shows that expanded CLT004 pentamer-sorted cells kill C1R-B7 target cells pulsed with CLT004. Significant killing of peptide-pulsed C1R-B7 cells is evident when the effector-to-target cell ratio is 3:1 and 1:1.

[0248] (Example 6 - Study on Mouse Immunogenicity) To demonstrate the immunogenicity of the CLT antigen, mice are inoculated with a replication-deficient adenoviral vector expressing one or more CLT antigens, and T cells obtained from these mice can be tested for the presence of CLT antigen-specific T cells using the IFNγ ELISPOT assay (Mennuni et al., Int. J. Cancer, 2005). Briefly, mice are inoculated with a recombinant adenovirus expressing the CLT antigen, humanely euthanized at an appropriate time point, and a preparation of spleen cells is loaded into wells of a multiwell dish derivatized with a monoclonal antibody against murine IFNγ in the presence (or absence) of overlapping peptides corresponding to the CLT antigen. After an appropriate time period, the immobilized IFNγ is stained with different monoclonal antibodies, the number of cells / spots is counted, and then they are compared to the total number of cells loaded into the well, enabling a quantitative readout of CLT antigen-reactive T cells.

[0249] (Example 7 - Verification Assay of CLT Expression in Malignant Melanoma Cells) a) qRT-PCR Verification of CLT Expression in Malignant Melanoma Cell Lines 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. Specific nucleic acid primer sequences are designed against the target transcript, and then the region between the primers is amplified through a series of thermal cycling reactions and fluorescently quantified using an intercalator dye (SYBR Green). Primer pairs were engineered against CLT and assayed against RNA extracted from malignant melanoma cell lines. Non-malignant melanoma cell lines were utilized as negative controls. Specifically, malignant 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 from 1×10 6 individual snap-frozen cells and reverse transcribed into cDNA. qRT-PCR analysis with SYBR Green detection was performed according to standard techniques using primers designed against two regions of each CLT and a reference gene. Relative quantification values (RQ) were calculated as follows: RQ = 2[Ct(reference) - Ct(target)] .

[0250] These experimental results are shown in Fig. 59. Panel A shows the results of a qRT-PCR assay using two primer sets (1 + 2 and 3 + 4), targeting different regions of CLT (SEQ ID NO: 33) encoding CLT antigen 1 on RNA extracted from three malignant melanoma cell lines and four non-malignant melanoma cell lines. Panel B shows the results of a qRT-PCR assay using two primer sets (5 + 6 and 7 + 8), targeting different regions of CLT (SEQ ID NO: 34) encoding CLT antigen 2 on RNA extracted from three malignant melanoma cell lines and four non-malignant melanoma cell lines. Panel C shows the results of a qRT-PCR assay using two primer sets (9 + 10 and 11 + 12), targeting different regions of CLT (SEQ ID NO: 35) encoding CLT antigen 3 / 4 on RNA extracted from three malignant melanoma cell lines and four non-malignant melanoma cell lines. These results confirmed that CLT was specifically expressed in RNA extracted from malignant melanoma cell lines compared to non-malignant melanoma cells. This CLT was detected in each of the malignant melanoma cell lines tested.

[0251] b) RNAScope verification of CLT expression in in situ malignant melanoma cells The analysis of the expression of transcripts by in situ hybridization (ISH) enables the visualization of the presence and expression levels of a given transcript under the pathological histological context of a specimen. Conventional RNA ISH assays also involve using oligonucleotide probes specific to short strands of the desired RNA sequence to recognize native RNA molecules in situ, which are visualized by signals generated by a combination of colorimetric reactions based on antibodies or enzymes. RNAScope is a recently developed in situ hybridization-based technique with more advanced probe chemistry that ensures the specificity of the generated signals and enables the visualization of single molecules with high sensitivity for the target transcript (Wang et al., 2012 J Mol Diagn. 14(1):22-29). Positive staining of transcript molecules appears as small red dots within a given cell, and multiple dots indicate the presence of multiple transcripts.

[0252] RNAScope probes were designed against CLT and assayed on sections of 12 formalin-fixed paraffin-embedded core biopsies of cutaneous malignant melanoma tumors. Scoring of the expression signals was as follows and was performed on representative images from each core: · Evaluation of the percentage of cells with positive staining for the CLT probe was rounded up to the nearest 10 · The evaluation level per cell of expression across a given section was as follows: · 0 = no staining · 1 = 1 - 2 dots per cell · 2 = 2 - 6 dots per cell · 3 = 6 - 10 dots per cell · 4 = more than 10 dots per cell.

[0253] The expression of each CLT was detected in tumor cores from a number of different patients, and the CLT findings from tumor-derived RNAseq data were verified separately in the cores of each patient analyzed, the uniformity of expression within tumor tissue was confirmed across a defined number of samples, and further, the presence of at least one CLT was highlighted.

[0254] (Table 10 - Scoring of RNAScope in the tissue cores of patients with malignant melanoma)

Table 10

[0255] Throughout this specification and the following claims, unless the context otherwise requires, the term "comprise", and variations such as "comprises" and "comprising", are to be understood to mean that the stated integer, step, group of integers or group of steps includes, but is not limited to, any other integer, step, group of integers or group of steps.

[0256] All patents, patent applications and references cited throughout the specification of the present invention are hereby incorporated by reference in their entirety. The present invention encompasses preferred and more preferred groups, as well as suitable and more suitable groups, and all combinations of the groups of embodiments listed above. The present application provides an invention in the following aspects. (Aspect 1) An isolated polypeptide comprising a sequence selected from the following: (c) Any one of the sequences of SEQ ID NOs: 1 to 10; and (d) A variant of the sequence of (a); and (c) An immunogenic fragment of the sequence of (a). (Aspect 2) The isolated peptide according to Aspect 1, comprising or consisting of a sequence selected from any one of SEQ ID NOs: 11 to 32 and 51 to 78. (Aspect 3) An isolated polypeptide according to embodiment 1 or embodiment 2, which is fused to a second polypeptide, or to a further polypeptide selected from (i) one or more other polypeptides according to embodiment 1 or embodiment 2, (ii) other polypeptides that are melanoma-associated antigens, (iii) polypeptide sequences that can enhance an immune response (i.e., immunostimulatory sequences), and (iv) polypeptide sequences that can provide strong CD4+ help to increase the CD8+ T cell response to an antigen epitope (e.g., including a universal CD4 helper epitope). (Embodiment 4) An isolated nucleic acid encoding a polypeptide according to any one of embodiments 1 to 3. (Embodiment 5) The nucleic acid according to embodiment 4, which is DNA. (Embodiment 6) The nucleic acid according to embodiment 5, which comprises or consists of a sequence selected from any one of SEQ ID NOs: 33 to 40 and 41 to 50. (Embodiment 7) The nucleic acid according to embodiment 6, which has codons optimized for expression in a human host cell. (Embodiment 8) The nucleic acid according to embodiment 4, which is RNA. (Embodiment 9) The nucleic acid according to embodiment 4, 5, 7 or 8, which is an artificial nucleic acid sequence. (Embodiment 10) A vector comprising the nucleic acid according to any one of embodiments 4 to 9. (Embodiment 11) The vector according to embodiment 10, which comprises DNA encoding regulatory elements suitable for enabling transcription of a translationally active RNA molecule in a human host cell. (Embodiment 12) The vector according to embodiment 10 or embodiment 11, which is a viral vector. (Embodiment 13) The vector according to embodiment 12, which is an adenovirus vector, an adeno-associated virus (AAV), an alphavirus, a herpesvirus, an arenavirus, a measles virus, a poxvirus, a paramyxovirus, a lentivirus and a rhabdovirus vector. (Aspect 14) An immunogenic pharmaceutical composition comprising the polypeptide, nucleic acid or vector according to any one of Aspects 1 to 13, together with a pharmaceutically acceptable carrier. (Aspect 15) A vaccine composition comprising the polypeptide, nucleic acid or vector according to any one of Aspects 1 to 13, together with a pharmaceutically acceptable carrier. (Aspect 16) The composition according to Aspect 14 or Aspect 15, comprising one or more immunostimulants. (Aspect 17) The composition according to Aspect 16, wherein the immunostimulant is selected from aluminum salts, saponins, immunostimulatory oligonucleotides, oil-in-water emulsions, aminoalkylglucosaminide 4-phosphate, lipopolysaccharides and derivatives thereof and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and interferons. (Aspect 18) The composition according to any one of Aspects 14 to 17, which is a sterile composition suitable for parenteral administration. (Aspect 19) The polypeptide, nucleic acid, vector or composition according to any one of Aspects 1 to 18 for use in medicine. (Aspect 20) A method for enhancing an immune response in a human, the method comprising administering to the human the polypeptide, nucleic acid, vector or composition according to any one of Aspects 1 to 18. (Aspect 21) The method according to Aspect 20, wherein the immune response is enhanced against a cancerous tumor expressing a sequence selected from SEQ ID NOs: 1 to 10 and variants and immunogenic fragments thereof. (Aspect 22) The polypeptide, nucleic acid, vector or composition according to any one of Aspects 1 to 18 for use in enhancing an immune response in a human. (Aspect 23) The polypeptide, nucleic acid, vector or composition according to embodiment 22, wherein the immune response is enhanced against a cancerous tumor expressing a corresponding sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment or variant thereof. (Embodiment 24) A method for treating a human cancer patient, wherein the cancer cells express a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, or a method for preventing a human from developing cancer, wherein the cancer will express a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, the method comprising administering to the human a corresponding polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18. (Embodiment 25) The polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18 for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment thereof. (Embodiment 26) For use in the stimulation and / or amplification in vitro of T cells derived from a human suffering from cancer, and the stimulated and / or amplified T cells are then reintroduced into the human for treating the cancer of the human, the polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18. (Embodiment 27) A method for treating human cancer, wherein the cancer cells express a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, the method comprising removing from the human a leukocyte population comprising at least T cells, optionally together with antigen-presenting cells, stimulating and / or amplifying the T cells in the presence of a corresponding polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18, and reintroducing into the human a part or all of the leukocytes, comprising at least the stimulated and / or amplified T cells. (Embodiment 28) The method according to any one of aspects 21 and 23 to 27, or the polypeptide, nucleic acid, vector or composition for use, wherein the cancer is malignant melanoma, for example, cutaneous malignant melanoma. (Aspect 29) A method for preparing a population of T cells that is cytotoxic to cancer cells expressing a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, comprising: (a) obtaining T cells from a cancer patient, optionally together with antigen-presenting cells; and (ii) stimulating and amplifying the population of T cells in vitro with the corresponding polypeptide, nucleic acid, vector or composition according to any one of aspects 1 to 18. (Aspect 30) A population of T cells obtainable by the method of aspect 29. (Aspect 31) T cells stimulated with the polypeptide, nucleic acid, vector or composition according to any one of aspects 1 to 18. (Aspect 32) An antigen-presenting cell that has been modified by loading in vitro with the polypeptide, nucleic acid, vector or composition according to any one of aspects 1 to 18, or has been genetically engineered to express the polypeptide according to any one of aspects 1 to 3. (Aspect 33) The antigen-presenting cell of aspect 32, which is a dendritic cell. (Aspect 34) Loaded with the polypeptide, nucleic acid, vector or composition according to any one of aspects 1 to 18 or genetically engineered to express the polypeptide according to any one of Aspects 1 to 3 An exosome loaded with a polypeptide produced from a cell. (Aspect 35) A pharmaceutical composition comprising the population of T cells, T cells, antigen-presenting cell or exosome according to any one of aspects 30 to 34 together with a pharmaceutically acceptable carrier. (Aspect 36) The population of T cells, T cells, antigen-presenting cell or exosome according to any one of aspects 30 to 34 for use in a medicament. (Aspect 37) A method for treating a human suffering from cancer, the method comprising treating a cancer cell expressing a sequence selected from SEQ ID NOs: 1 to 10, any one immunogenic fragment and variant thereof, or a method for preventing a human from suffering from cancer, the method comprising preventing a cancer cell that would express a sequence selected from SEQ ID NOs: 1 to 10, any one immunogenic fragment and variant thereof, the method comprising administering to the human the T cell population, T cell, antigen-presenting cell, exosome or composition according to any one of aspects 30 to 35. (Aspect 38) A T cell population, T cell, antigen-presenting cell, exosome or composition according to any one of aspects 30 to 35 for use in the treatment or prevention of human cancer, wherein the cancer cell expresses a corresponding sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment thereof. (Aspect 39) The cancer is malignant melanoma, such as cutaneous malignant melanoma, the preparation method, method, or T cell population, T cell, antigen-presenting cell, exosome or composition for use according to any one of aspects 29, 37 and 38. (Aspect 40) An isolated antigen-binding polypeptide that is immunospecific for the polypeptide according to any one of aspects 1 to 3. (Aspect 41) The antigen-binding polypeptide according to aspect 40, which is a monoclonal antibody or a fragment thereof. (Aspect 42) The antigen-binding polypeptide according to aspect 40 or aspect 41, which is bound to a cytotoxic component. (Aspect 43) The antigen-binding polypeptide according to any one of aspects 40 to 42 for use in medicine. (Aspect 44) A pharmaceutical composition comprising the antigen-binding polypeptide according to any one of aspects 40 to 42 together with a pharmaceutically acceptable carrier. (Aspect 45) A method for treating a human suffering from cancer, the method comprising treating cancer cells expressing a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, or a method for preventing a human from suffering from cancer, the method comprising preventing cancer cells from expressing a sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment and variant thereof, the method comprising administering to the human the antigen-binding polypeptide or composition according to any one of aspects 40 to 42 and 44. (Aspect 46) An antigen-binding polypeptide or composition according to any one of aspects 40 to 42 and 44 for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NOs: 1 to 10 and any one immunogenic fragment thereof. (Aspect 47) The method, antigen-binding polypeptide or composition according to aspect 45 or aspect 46, wherein the cancer is malignant melanoma, such as cutaneous malignant melanoma. (Aspect 48) An isolated antigen-binding polypeptide that is immunologically specific for an HLA-binding polypeptide that is a polypeptide according to any one of aspects 1 to 3 or a part thereof. (Aspect 49) The antigen-binding polypeptide according to aspect 48, which is a T cell receptor or a fragment thereof. (Aspect 50) The antigen-binding polypeptide according to aspect 48 or aspect 49, which is bound to another polypeptide capable of binding to a cytotoxic cell or other immune component in a subject. (Aspect 51) A cytotoxic cell engineered to express on its surface the antigen-binding polypeptide according to any one of aspects 48 to 50. (Aspect 52) The cytotoxic cell according to aspect 51, which is a T cell. (Aspect 53) The cytotoxic cell according to aspect 51 or aspect 52 for use in medicine. (Aspect 54) A pharmaceutical composition comprising the cell according to aspect 51 or aspect 52. (Aspect 55) A method for treating a human cancer patient, wherein the cancer cells express a sequence selected from SEQ ID NOs: 1 to 10, any one of their 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 NOs: 1 to 10, any one of their immunogenic fragments, and variants, the method comprising administering the cells according to Aspect 51 or Aspect 52 to the human. (Aspect 56) Cytotoxic cells according to Aspect 51 or Aspect 52 for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from SEQ ID NOs: 1 to 10 and any one of their immunogenic fragments. (Aspect 57) A method for diagnosing whether a human has cancer, comprising: determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NOs: 1 to 10, any one of their immunogenic fragments or variants, or a nucleic acid encoding the polypeptide sequence; and diagnosing that the human has cancer when the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells. (Aspect 58) A method for diagnosing a human suffering from cancer that is cutaneous malignant melanoma, comprising: determining whether the cancer cells express a polypeptide sequence of any one of SEQ ID NOs: 2, 6, 7, 8, and 9 and any one of their immunogenic fragments or variants, or a nucleic acid encoding the polypeptide sequence; and diagnosing that the human is suffering from cancer that is cutaneous malignant melanoma when the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells. (Aspect 59) A method for diagnosing a human suffering from cancer, which is cutaneous malignant melanoma or uveal malignant melanoma, comprising the step of determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NO: 1, 3, 4, 5 and 10 and any one of its immunogenic fragments or variants, or a nucleic acid encoding the polypeptide sequence; and the step of diagnosing that the human is suffering from cancer which is cutaneous malignant melanoma or uveal malignant melanoma when the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells. (Aspect 60) A method for treating a human suffering from cancer, comprising: (a) determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NOs: 1 to 10 and any one of its immunogenic fragments or variants, or a nucleic acid encoding the polypeptide (for example, those selected from the sequences of SEQ ID NOs: 33 to 40 and 41 to 50); and if so, (b) administering to the human the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cell, antigen-presenting cell, exosome, antigen-binding polypeptide or cytotoxic cell according to any one of Aspects 1 to 18, 30 to 35, 40 to 42, 44, 50, 51 and 53. The above method. (Aspect 61) Use of a polypeptide isolated from a tumor of a human suffering from cancer and comprising a sequence selected from the following: (a) any one of the sequences of SEQ ID NOs: 1 to 10; or (b) a variant of the sequence of (a); and, (c) an immunogenic fragment of the sequence of (a), or use of a nucleic acid encoding the polypeptide, as a biomarker for determining whether the human would be suitable for treatment with a vaccine comprising the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cell, antigen-presenting cell, exosome, antigen-binding polypeptide or cytotoxic cell according to any one of Aspects 1 to 18, 30 to 35, 40 to 42, 44, 51, 52 and 54. (Aspect 62) The method or use according to embodiment 60 or embodiment 61, wherein the cancer is malignant melanoma, such as cutaneous malignant melanoma. (Embodiment 63) A method according to any one of embodiments 21 and 23 to 27, or a polypeptide, nucleic acid, vector or composition for use, wherein the polypeptide comprises a sequence selected from the following: (a) Any one of the sequences of SEQ ID NO: 1, 3, 4, 5 and 10; and (b) A variant of the sequence of (a); and (c) An immunogenic fragment of the sequence of (a), and for example, the polypeptide comprises or consists of a sequence selected from any one of SEQ ID NO: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, 51 to 57, 67 to 74 and 76 to 77, and for example, the nucleic acid comprises or consists of a sequence selected from any one of SEQ ID NO: 33, 35, 36 or 40, or a sequence selected from any one of SEQ ID NO: 41, 43, 44, 45 and 50; and The cancer is choroidal malignant melanoma, a polypeptide, nucleic acid, vector or composition for the method or use. (Embodiment 64) An antigen-binding polypeptide or composition for the method according to embodiment 45, or the use according to embodiment 46, wherein the polypeptide comprises a sequence selected from the following: (a) Any one of the sequences of SEQ ID NO: 1, 3, 4, 5 and 10; and (b) A variant of the sequence of (a); and, (c) An immunogenic fragment of the sequence of (a), and for example, the polypeptide comprises or consists of a sequence selected from any one of SEQ ID NO: 11 to 14, 17 to 18, 19, 20 to 22, 30 to 32, 51 to 57, 67 to 74 and 76 to 77, and for example, the nucleic acid comprises or consists of a sequence selected from any one of SEQ ID NO: 33, 35, 36 or 40, or a sequence selected from any one of SEQ ID NO: 41, 43, 44, 45 and 50; and An antigen-binding polypeptide or composition for said method or use, wherein the cancer is uveal malignant melanoma. (Aspect 65) A T cell population, T cell, antigen-presenting cell, exosome or composition for the preparation method, method or use according to any one of Aspects 29, 37 and 38, wherein the polypeptide comprises a sequence selected from the following: (a) Any one of the sequences of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) A variant of the sequence of (a); and, (c) An immunogenic fragment of the sequence of (a), and for example, the polypeptide comprises or consists of a sequence selected from any one of SEQ ID NOs: 11-14, 17-18, 19, 20-22, 30-32, 51-57, 67-74 and 76-77, and for example, the nucleic acid comprises or consists of a sequence selected from any one of SEQ ID NOs: 33, 34, 36 or 40, or a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50; and A T cell population, T cell, antigen-presenting cell, exosome or composition for the preparation method, method or use, wherein the cancer is uveal malignant melanoma. (Aspect 66) A method or use according to Aspect 60 or Aspect 61, wherein the polypeptide comprises a sequence selected from the following: (a) Any one of the sequences of SEQ ID NOs: 1, 3, 4, 5 and 10; and (b) A variant of the sequence of (a); and, (c) An immunogenic fragment of the sequence of (a), and for example, the polypeptide comprises or consists of a sequence selected from any one of SEQ ID NOs: 11-14, 17-18, 19, 20-22, 30-32, 51-57, 67-74 and 76-77, and for example, the nucleic acid comprises or consists of a sequence selected from any one of SEQ ID NOs: 33, 35, 36 or 40, or a sequence selected from any one of SEQ ID NOs: 41, 43, 44, 45 and 50; and The method or use, wherein the cancer is uveal malignant melanoma. (Aspect 67) The fusion polypeptide according to embodiment 3, comprising two or more (for example, 2, 3, or 4) sequences selected from the sequences of SEQ ID NOs: 1, 2, 3, and 4; or a variant of the sequence or an immunogenic fragment of the sequence for each of the sequences. (Aspect 68) The fusion polypeptide according to embodiment 67, comprising: (i) A sequence selected from the following: (a) The sequence of SEQ ID NO: 1; and (b) A variant of the sequence of (a); and (c) An immunogenic fragment of the sequence of (a); (ii) A sequence selected from the following: (a) The sequence of SEQ ID NO: 2; and (b) A variant of the sequence of (a); and (c) An immunogenic fragment of the sequence of (a); (iii) A sequence selected from the following: (a) The sequence of SEQ ID NO: 3; and (b) A variant of the sequence of (a); and (c) An immunogenic fragment of the sequence of (a); and (iv) A sequence selected from the following: (a) The sequence of SEQ ID NO: 4; and (b) A variant of the sequence of (a); and (c) An immunogenic fragment of the sequence of (a). (Aspect 69) The fusion polypeptide according to embodiment 68, comprising the sequences of SEQ ID NOs: 1, 2, 3, and 4. (Aspect 70) The fusion polypeptide according to embodiment 68, comprising: (i) A sequence selected from the following: (a) The sequence of SEQ ID NO: 1; and (b) A variant of the sequence of (a); and (c) An immunogenic fragment of the sequence of (a); (ii) A sequence selected from the following: (a) The sequence of SEQ ID NO: 2; and (b) A variant of the sequence of (a); and (c) An immunogenic fragment of the sequence of (a); and (iii) An array selected from the following: (a) The array of SEQ ID NO: 4; and (b) A variant of the array of (a); and (c) An immunogenic fragment of the array of (a). (Aspect 71) The fusion polypeptide according to Aspect 70, comprising the arrays of SEQ ID NOs: 1, 2, and 4. (Aspect 72) An isolated nucleic acid encoding the fusion polypeptide according to any one of Aspects 67 to 71. (Aspect 73) The nucleic acid according to Aspect 72, which is DNA. (Aspect 74) A vector comprising the nucleic acid according to Aspect 73. (Aspect 75) The vector according to Aspect 73, comprising DNA encoding a regulatory element suitable for enabling the transcription of a translationally active RNA molecule in a human host cell. (Aspect 76) The vector according to Aspect 74 or Aspect 75, which is a viral vector.

[0257] (Sequence Listing) SEQ ID NO: 1 (Polypeptide sequence of CLT antigen 1)

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Claims

1. (i) An isolated polypeptide comprising an array selected from the following: (a) The array of SEQ ID NO: 1; and (b) An immunogenic variant of the array of (a) that is at least 90% identical to SEQ ID NO: 1 and elicits a specific immune response against SEQ ID NO: 1; and (c) An immunogenic fragment of the array of (a) comprising at least 9 contiguous amino acids of SEQ ID NO: 1 and eliciting a specific immune response against SEQ ID NO: 1, wherein the at least 9 contiguous amino acids comprise an array selected from any one of SEQ ID NOs: 11-14, 55-57, and 73-74, said immunogenic fragment (ii) (a) (x) A polypeptide having the array of SEQ ID NOs: 2-10, and an immunogenic variant of the polypeptide of (x) that is at least 80% identical thereto, and an immunogenic fragment of the polypeptide of (x) comprising at least 9 contiguous amino acids of the array of the polypeptide, and (y) one or more other polypeptides selected from polypeptides having the arrays of SEQ ID NOs: 15-32, 51-54, 58-72, and 75-78 (b) Other polypeptides that are melanoma-related antigens (c) A polypeptide sequence capable of enhancing an immune response (i.e., an immunostimulatory sequence), and (d) A polypeptide sequence capable of providing strong CD4+ help to increase the CD8+ T cell response to an antigenic epitope A fusion polypeptide comprising the isolated polypeptide described in (i) fused to a second or further polypeptide selected from (iii) An isolated nucleic acid encoding the isolated polypeptide of (i) or the fusion polypeptide of (ii) (iv) A vector comprising an isolated nucleic acid encoding the polypeptide of (i) or the fusion polypeptide of (ii), or comprising the nucleic acid of (iii) (v) A T cell population obtainable by a method comprising stimulating and amplifying a T cell population in vitro using the polypeptide of (i) (vi) T cells stimulated with the polypeptide of (i) (vii) Antigen-presenting cells loaded and modified in vitro with the polypeptide of (i), the fusion polypeptide of (ii), the nucleic acid of (iii), or the vector of (iv), or genetically engineered to express the polypeptide of (i) or the fusion polypeptide of (ii) (viii) Exosomes loaded with the polypeptide of (i) An isolated antigen-binding polypeptide that is immunospecific for the polypeptide of (ix)(i), An isolated antigen-binding polypeptide that is immunospecific for an HLA-binding polypeptide having the sequence of (x)(i) or the sequence of the fusion polypeptide of (ii), or (xi) A cytotoxic cell engineered to express the antigen-binding polypeptide of (x) on its surface An immunogenic pharmaceutical composition for the treatment or prevention of human cancer, comprising: wherein the cancer cells express a corresponding sequence selected from SEQ ID NO: 1, an immunogenic variant of SEQ ID NO: 1 that is at least 90% identical to SEQ ID NO: 1, and an immunogenic fragment of SEQ ID NO: 1 comprising at least 9 contiguous amino acids of SEQ ID NO:

1.

2. The immunogenic pharmaceutical composition according to claim 1, comprising the isolated polypeptide of (i), wherein the isolated polypeptide comprises, or consists of, a sequence selected from any one of SEQ ID NOs: 1, 11-14, 55-57, 73 and 74.

3. The immunogenic pharmaceutical composition according to claim 1, comprising the nucleic acid or vector of (iii) or (iv), wherein the nucleic acid is DNA.

4. The immunogenic pharmaceutical composition according to claim 1, comprising the nucleic acid or vector of (iii) or (iv), wherein the nucleic acid comprises a sequence selected from any one of SEQ ID NOs: 33 and 41.

5. The immunogenic pharmaceutical composition according to any one of claims 1, 3 and 4, comprising the vector of (iv), wherein the vector comprises DNA encoding regulatory elements suitable for enabling transcription of a translationally active RNA molecule in a human host cell.

6. The immunogenic pharmaceutical composition according to any one of claims 1 and 3-5, comprising the vector of (iv), wherein the vector is a viral vector.

7. The immunogenic pharmaceutical composition according to claim 6, wherein the vector is an adenovirus vector, adeno-associated virus (AAV), alphavirus, herpesvirus, arenavirus, measles virus, poxvirus, paramyxovirus, lentivirus or rhabdovirus vector.

8. The immunogenic pharmaceutical composition according to claim 1, wherein the composition further comprises one or more immunostimulants.

9. The immunogenic pharmaceutical composition according to claim 1, comprising the antigen-presenting cell of (vii), wherein the antigen-presenting cell is a dendritic cell.

10. The immunogenic pharmaceutical composition according to claim 1, comprising the antigen-binding polypeptide of (ix), wherein the antigen-binding polypeptide is a monoclonal antibody or an antigen-binding fragment thereof.

11. The immunogenic pharmaceutical composition according to claim 1 or 10, comprising the antigen-binding polypeptide of (ix), wherein the antigen-binding polypeptide is bound to a cytotoxic component.

12. The immunogenic pharmaceutical composition according to claim 1, comprising the antigen-binding polypeptide of (x), wherein the antigen-binding polypeptide is a T cell receptor or a fragment thereof.

13. The immunogenic pharmaceutical composition according to claim 1 or 12, comprising the antigen-binding polypeptide of (x), wherein the antigen-binding polypeptide is bound to a second polypeptide capable of binding to cytotoxic cells or other immune components in a subject.

14. The immunogenic pharmaceutical composition according to claim 1, comprising the cytotoxic cells of (xi), wherein the cytotoxic cells are T cells.

15. Comprising the fusion polypeptide of (ii), wherein the fusion polypeptide (x) a polypeptide comprising a sequence selected from the following: (a) the sequence of SEQ ID NO: 1; and (b) an immunogenic variant of the sequence of (a) that is at least 90% identical to SEQ ID NO: 1; and (c) an immunogenic fragment of the sequence of (a) comprising at least 9 consecutive amino acids of SEQ ID NO: 1, and (y) one or more sequences selected from the sequences of SEQ ID NO: 2, 3, and 4; or, for each of the sequences of SEQ ID NO: 2, 3, and 4, a variant of the sequence that is at least 90% identical to the sequence, or an immunogenic fragment comprising at least 9 consecutive amino acids of the sequence The immunogenic pharmaceutical composition according to claim 1, comprising.

16. The immunogenic pharmaceutical composition according to any one of claims 1 to 15, wherein the cancer is malignant melanoma.

17. The immunogenic pharmaceutical composition according to claim 16, wherein the malignant melanoma is cutaneous malignant melanoma.

Images