Novel cancer antigens and methods

Abstract

Disclosed, inter alia, are polypeptides and nucleic acids encoding the polypeptides useful for the treatment, prevention and diagnosis of cancer, in particular melanoma, in particular skin melanoma and uveal melanoma.

Description

[0001] This application is a divisional application of Chinese application No. 201980068630.3, whose application date is October 18, 2019 and whose invention name is “New Cancer Antigens and Methods”. Field of the Invention

[0002] The present invention relates to antigenic polypeptides and corresponding polynucleotides for treating or preventing cancer, especially for treating or preventing melanoma (e.g., skin melanoma or uveal melanoma). The present invention further relates to pharmaceutical compositions and immunogenic compositions comprising the nucleic acids and polypeptides, immune cells loaded with the polypeptides and polynucleotides and / or stimulated therewith, antibodies specific to the polypeptides, and (autologous or other) cells genetically engineered with molecules that recognize the polypeptides. Background of the Invention

[0004] As part of normal immune surveillance for pathogenic microorganisms, all cells degrade intracellular proteins to produce peptides, which 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 regarded as self and remain invisible to the adaptive immune system. However, foreign (non-self) peptides can stimulate initial CD8+T cell expansion, and the cell encoding is tightly bound to the T cell receptor (TCR) of MHC I-peptide complex. This expanded T cell colony can produce effector CD8+T cells (including cytotoxic T-lymphocytes-CTLs), which can eliminate cells with foreign antigen labels, and memory CD8+T cells, which can be expanded again when the cells with foreign antigen labels appear later in the life of animals.

[0005] MHC class II molecules, whose expression is normally restricted to professional antigen presenting cells (APCs) such as dendritic cells (DCs), are usually loaded with peptides that have been internalized from the foreign environment. In the presence of various factors, including T cell adhesion molecules (CD54, CD48) and co-stimulatory molecules (CD40, CD80, CD86), the binding of the complementary TCR from naive CD4+ T cells to the MHC II-peptide complex induces mature CD4+ T cells into effector cells (e.g., T H 1. T H 2. T H 17. T FH、 T regCells). These effector CD4+T-cells can promote the differentiation of B cells into plasma cells that secrete antibodies and promote the differentiation of antigen-specific CD8+CTLs, thereby helping to induce adaptive immune responses to foreign antigens, including short-term effector functions and longer-term immune memory. DCs can perform cross-presentation of peptide antigens by delivering exogenously derived antigens (such as peptides or proteins released from pathogens or tumor cells) to their MHC I molecules, helping to generate immune memory by providing a bypass pathway that stimulates the expansion of initial CD8+T cells.

[0006] Immune memory (especially antigen-specific B cells / antibodies and antigen-specific CTLs) is a key role in controlling microbial infections, and many vaccines have been developed to prevent diseases caused by important pathogenic microorganisms using immune memory. It is also known that immune memory plays a key role in controlling tumor formation, but effective cancer vaccines have hardly been developed.

[0007] Cancer is the second leading cause of disease, accounting for 1 in 6 deaths worldwide. In 2015, of the 8.8 million deaths caused by cancer, most of the lives were lost to lung cancer (1.69 million), liver cancer (788,000), colorectal cancer (774,000), stomach cancer (754,000) and breast cancer (571,000). The economic impact of cancer was estimated at $1.16 trillion in 2010, and the number of new cases is expected to rise by approximately 70% over the next two decades (World Health Organization Cancer Reality 2017).

[0008] Current treatments for cutaneous melanoma vary and are highly dependent on tumor location and disease stage. The primary treatment for non-metastatic melanoma is surgical removal of the tumor and surrounding tissue. Advanced melanoma may require treatment that includes lymph node dissection, radiation therapy, or chemotherapy. Immune checkpoint blockade strategies, including the use of antibodies targeting negative immune regulators such as PD-1 / PD-L1 and CTLA4, have recently revolutionized the treatment of a variety of malignancies, including melanoma (Ribas, A. and Wolchok, JD (2018) Science, 359:1350–1355). The extraordinary value of checkpoint blockade therapy and its well-recognized link to the patient's adaptive immune response to their own cancer antigens (specific T cell-based immune responses) have revolutionized the search for effective cancer vaccines, vaccine modalities, and cancer vaccine antigens.

[0009] Human endogenous retroviruses (HERVs) are remnants of ancestral germline integrations of exogenous infectious retroviruses. HERVs belong to a group of endogenous retroviral elements characterized by the presence of long terminal repeats (LTRs) flanking the viral genome. This group also includes mammalian epi-LTR retrotransposons (MaLRs) and are therefore collectively referred to as LTR elements (herein collectively referred to as ERVs to mean all LTR elements). ERVs constitute a significant proportion of mammalian genomes (8%) and can be grouped into approximately 100 families based on sequence homology. Many ERV sequences encode defective proviruses that share a prototypical retroviral genomic structure consisting of gag, pro, pol, and env genes flanked by LTRs. Some intact ERV ORFs produce retroviral proteins that share features with proteins encoded by exogenous infectious retroviruses such as HIV-1. Such proteins can act as antigens that induce strong immune responses (Hurst and Magiorkinis, 2015, J. Gen. Virol 96: 1207-1218), showing that ERV-encoded polypeptides may escape T cell receptor selection processes and B cell receptor selection processes and central tolerance and peripheral tolerance. Immune reactivity against ERV products can occur spontaneously in infection or cancer, and ERV products have been implicated as the cause of some autoimmune diseases (Kassiotis and Stoye, 2016, Nat. Rev. Immunol. 16: 207-219).

[0010] Due to the accumulation of mutations and recombination events during evolution, most of the sequences derived from ERV have lost some or all of their genes' functional open reading frames and therefore lost their ability to produce infectious viruses. However, these ERV elements are maintained in germline DNA like other genes and still have the potential to produce proteins from at least some of their genes. In fact, HERV-encoded proteins have been detected in a variety of human cancers. For example, HERV-K env genes, i.e., splice variants of Rec and Np9, are only present in malignant testicular germ cells and not in healthy cells (Ruprecht et al., 2008, Cell Mol Life Sci 65:3366-3382). As compared with healthy tissues, elevated levels of HERV transcripts have also been observed in a variety of cancers such as those of prostate cancer (Wang-Johanning, 2003, Cancer 98:187-197; Andersson et al., 1998, Int. J. Oncol, 12:309-313). Additionally, overexpression of HERV-E and HERV-H has been shown to be immunosuppressive, which may also contribute to carcinogenesis (Mangeney et al., 2001, J. Gen. Virol. 82:2515-2518). However, the exact mechanism by which HERVs may contribute to carcinogenesis or pathogenicity remains unknown.

[0011] In addition to deregulating the expression of surrounding neighboring host genes, the activity and transposition of ERV regulatory elements to new genomic locations may lead to the generation of new transcripts, some of which may have tumorigenic properties (Babaian and Mager, Mob. DNA, 2016; Lock et al., PNAS, 2014, 111:3534-3543).

[0012] Vaccine patterns of a wide range of types are known. A well-described scheme includes direct delivery of antigenic polypeptides to a subject to increase immune responses (including B cell responses and T cell responses) and stimulate immune memory. Alternatively, polynucleotides can be administered to a subject by means of a carrier, so that the immunogenic polypeptides encoded by the polynucleotides are expressed in vivo. It has been fully explored to use viral vectors (such as adenovirus vectors) to deliver antigens in preventive vaccinations and therapeutic treatment strategies for cancer (Wold et al. Current Gene Therapy, 2013, Adenovirus Vectors for Gene Therapy (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 infused into a subject as a vaccine that stimulates a therapeutic or preventive immune response. An example of this method is Provenge, which is currently the only anticancer vaccine approved by the FDA.

[0013] Cancer antigens can also be used to generate a variety of non-vaccine therapeutic modalities that can be exploited in the treatment and prevention of cancer. These therapeutic agents fall into two distinct categories: 1) antigen-binding biologics, and 2) adoptive cell therapy agents.

[0014] Antigen-binding biological products are generally composed of multivalent engineered polypeptides that recognize antigen-modified cancer cells and promote the destruction of cancer cells. The antigen-binding elements of these biological products can be composed of biological products based on TCR, including but not limited to TCR, high-affinity TCR and TCR mimics (including those based on monoclonal antibody technology) produced by various technologies. The cytolytic part of this type of multivalent biological product can be composed of cytotoxic chemicals, biotoxins, guidance motifs and / or immunostimulatory motifs that promote targeting and activation of immune cells, and any of the aforementioned promotes therapeutic destruction of tumor cells.

[0015] Adoptive cell therapy can be based on the patient's own T cells, wherein the T cells are taken out and stimulated in vitro with a vaccine antigen preparation (cultured with T cells in the presence or absence of other factors (including cellular components and non-cellular components)) (JCI Insight. 2018 Oct 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 artificially engineered to express antigen-binding polypeptides that recognize cancer antigens. These antigen-binding polypeptides belong to the same category as described above for antigen-binding biological products. Therefore, lymphocytes (autologous or non-autologous) that have been genetically manipulated to express cancer antigen-binding polypeptides can be administered to patients as adoptive cell therapy for treating their cancer.

[0016] The use of ERV-derived antigens to elevate effective immune responses against cancer has shown promising results in promoting tumor regression and more favorable prognosis in murine cancer models (Kershaw et al., 2001, Cancer Res. 61: 7920-7924; Slansky et al., 2000, Immunity 13: 529-538). Therefore, HERV antigen-centric immunotherapy trials have been conceived in humans (Sacha et al., 2012, J. Immunol 189: 1467-1479), but progress has been limited, in part due to the severe limitation of tumor-specific ERV antigens that have been identified.

[0017] WO 2005 / 099750 identifies anchored sequences in existing vaccines against infectious pathogens that are common in raising cross-reactive immune responses against HERV-K Mel tumor antigens and conferring protection against melanoma.

[0018] WO 00 / 06598 relates to the identification of a HERV-AVL3-B tumor-associated gene that is preferentially expressed in melanoma, and methods and products for diagnosing and treating conditions characterized by the expression of said gene.

[0019] WO 2006 / 119527 provides antigenic polypeptides derived from melanoma-associated endogenous retroviruses (MERVs), and uses thereof in detecting and diagnosing melanoma and in disease prognosis, and also discloses the use of the antigenic polypeptides as anti-cancer vaccines.

[0020] WO 2007 / 137279 discloses methods and compositions for detecting, preventing and treating HERV-K+ cancers, such as using HERV-K+ binding antibodies to prevent or inhibit cancer cell proliferation.

[0021] WO 2006 / 103562 discloses a method for treating or preventing cancer, wherein an immunosuppressive Np9 protein from the env gene of HERV-K is expressed. The invention also relates to a pharmaceutical composition comprising a nucleic acid or antibody capable of inhibiting the activity of the protein, or an immunogen or vaccine composition capable of inducing an immune response against the protein.

[0022] WO 2007 / 109583 provides compositions and methods for preventing or treating a neoplastic disease in a mammalian subject by providing a composition comprising an enriched population of immune cells reactive with HERV-E antigens on tumor cells.

[0023] Humer J et al., 2006, Canc. Res., 66: 1658-63 identified melanoma markers derived from melanoma-associated endogenous retroviruses.

[0024] There is a need to further identify HERV-associated antigenic sequences that can be used for immunotherapy of cancer, particularly melanoma, especially cutaneous melanoma and uveal melanoma. Brief description of the invention

[0026] The inventors have surprisingly discovered that certain RNA transcripts, which contain LTR elements or are derived from genomic sequences adjacent to LTR elements, are present at high levels in skin melanoma cells, but are undetectable or present at very low levels in normal healthy tissues (see Example 1). Such transcripts are referred to herein as cancer-specific trans-LTR element transcripts (CLTs). Further, the inventors have shown that a subset of potential polypeptide sequences encoded by these CLTs (i.e., open reading frames (ORFs)) are translated in cancer cells, processed by components of the antigen processing apparatus, and presented on the surface of cells present in tumor tissue when associated with class I and class II major histocompatibility complexes (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). By themselves, these results show that these polypeptides (referred to herein as CLT antigens) are antigenic. Therefore, cancer cell presentation of the CLT antigen is expected to render these cells susceptible to T cell clearance by cognate T cell receptors (TCRs) bearing the CLT antigen, and CLT antigen-based vaccination methods / therapeutic regimens that expand T cells carrying these cognate TCRs are expected to stimulate immune responses against cancer cells (and tumors containing them), particularly melanomas, especially skin melanoma tumors. T cells from melanoma subjects are indeed reactive with peptides derived from the CLT antigen disclosed herein and expand T cells and amplify T cell receptor sequences (see Example 3). The inventors have demonstrated that T cells specific for the CLT antigen are not deleted from the T cell pool of normal subjects due to central tolerance (see Example 4). The presence and killing activity of CLT antigen-specific T cells in ex vivo cultures of healthy donor T cells have been determined (see Example 5). Finally, qRT-PCR studies have confirmed that CLT is specifically expressed in RNA extracted from melanoma cell lines as compared to non-melanoma cell lines (see Example 7).

[0027] The inventors have also surprisingly found that certain CLT antigens encoding CLT and overexpressed in skin melanoma are also overexpressed in uveal melanoma. These CLT-encoding CLT antigen polypeptide sequences are expected to stimulate an immune response against uveal melanoma cells and tumors containing them.

[0028] CLT and CLT antigens, which are the subject of the present invention, are not typical sequences that can be easily derived from known tumor genome sequences present in the Cancer Genome Atlas. CLT is a transcript produced as a result of complex transcription and splicing events driven by ERV-derived transcriptional control sequences. Since CLT is expressed at high levels and since CLT antigen polypeptide sequences are not normal human protein sequences, it is expected that they will be able to stimulate a strong specific immune response (as indeed has been established - see Examples 3-5) and are therefore suitable for therapeutic use in the context of cancer immunotherapy.

[0029] The CLT antigen found in highly expressed transcripts characterizing tumor cells, which prior to the present invention was not known to exist in humans and did not produce a protein product and was not known to stimulate an immune response, can be used in several modes. First, the CLT antigen polypeptide of the present invention can be delivered directly to a subject as a vaccine to stimulate a therapeutic or prophylactic immune response against tumor cells. Second, the nucleic acid of the present invention (which can be codon-optimized to enhance its expression of the encoded CLT antigen) can be directly administered or otherwise inserted into a vector for in vivo delivery to produce the encoded protein product in a subject as a vaccine to stimulate 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 infused into a subject as a vaccine to stimulate a therapeutic or prophylactic immune response against cancer cells. Fourth, the polynucleotides and / or polypeptides of the present invention can be used to stimulate T cells of a subject ex vivo to produce a stimulated T cell preparation, which can be administered to a subject as a therapeutic drug for treating cancer. Fifth, biomolecules (such as T cell receptors (TCRs) or TCR mimetics) that recognize CLT antigens complexed with MHC I molecules and have been further modified to allow them to kill (or promote killing) cancer cells can be administered to a subject as a therapeutic for treating cancer. Sixth, chimeric forms of biomolecules that recognize CLT antigens complexed with MHC I molecules can be introduced into (autologous or non-autologous) T cells, and the resulting cells can be administered to a subject as a therapeutic for treating cancer. These and other applications are described in more detail below.

[0030] Therefore, the present invention particularly provides an isolated polypeptide comprising a sequence selected from the group consisting of:

[0031] (a) any one of SEQ ID NOs. 1-10 and

[0032] (b) a variant of the sequence of (a); and

[0033] (c) Immunogenic fragments of the sequence of (a)

[0034] (hereinafter referred to as "the polypeptide of the present invention").

[0035] The present invention also provides nucleic acid molecules encoding the polypeptides of the present invention (hereinafter referred to as "nucleic acids of the present invention").

[0036] The polypeptides of the invention and the nucleic acids of the invention, as well as related aspects of the invention, are contemplated to be useful in cancer immunotherapy and prevention, particularly melanoma immunotherapy and prevention, in a range of embodiments, as discussed in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] for Figure 1-Figure 15 In each of the , the upper panel shows the extracted MS / MS spectrum of the peptide (along with the assigned fragment ions) isolated from a patient's tumor sample, and the lower panel indicates the spectral presentation of the position of the linear peptide sequence that has been mapped to the fragment ions.

[0039] Figure 1 . Spectrum of the peptide of SEQ ID NO. 11 isolated from a tumor sample of patient Mel-3.

[0040] Figure 2 . Spectrum of the peptide of SEQ ID NO. 12 isolated from a tumor sample of patient Mel-3.

[0041] Figure 3 . Spectrum of the peptide of SEQ ID NO. 13 isolated from a tumor sample of patient Mel-5.

[0042] Figure 4 . Spectrum of the peptide of SEQ ID NO. 13 isolated from a tumor sample of patient Mel-16.

[0043] Figure 5 . Spectrum of the peptide of SEQ ID NO. 15 isolated from a tumor sample of patient Mel-26.

[0044] Figure 6 . Spectrum of the peptide of SEQ ID NO. 16 isolated from a tumor sample of patient Mel-20.

[0045] Figure 7 . Spectrum of the peptide of SEQ ID NO. 17 isolated from a tumor sample of patient Mel-35.

[0046] Figure 8 . Spectrum of the peptide of SEQ ID NO. 19 isolated from a tumor sample of patient Mel-3.

[0047] Fig. 9 . Spectrum of the peptide of SEQ ID NO. 21 isolated from a tumor sample of patient Mel-27.

[0048] Fig.10 . Spectrum of the peptide of SEQ ID NO. 20 isolated from a tumor sample of patient Mel-27.

[0049] Fig.11 . Spectrum of the peptide of SEQ ID NO. 22 isolated from a tumor sample of patient Mel-27.

[0050] Fig.12. Spectrum of the peptide of SEQ ID NO. 23 isolated from a tumor sample of patient Mel-27.

[0051] Fig.13 . Spectrum of the peptide of SEQ ID NO. 24 isolated from a tumor sample of patient Mel-27.

[0052] Fig.14 . Spectrum of the peptide of SEQ ID NO. 25 isolated from a tumor sample of patient Mel-16.

[0053] Fig.15 . Spectrum of the peptide of SEQ ID NO. 26 isolated from a tumor sample of patient Mel-41.

[0054] Fig.16 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 27 from the immunopeptidomic analysis of patient Mel-15.

[0055] Fig.17 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 29 from the immunopeptidomic analysis of patient Mel-10.

[0056] Fig.18 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 14 from the immunopeptidomic analysis of patient Mel-5.

[0057] Fig.19 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 21 from the immunopeptidomic analysis of patient Mel-4.

[0058] Fig. 20 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 31 from the immunopeptidomic analysis of patient Mel-18.

[0059] Fig.21 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 13 from the immunopeptidomic analysis of patient Mel-16.

[0060] Fig. 22 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 19 from the immunopeptidomic analysis of patient Mel-3.

[0061] Fig.23 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 28 from the immunopeptidomic analysis of patient Mel-6.

[0062] Fig.24Shown is the mass spectrometry spectrum of peptide fragments attributed to SEQ ID NO. 18 from the immunopeptidomic analysis of patient Mel-18.

[0063] Fig.25 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 30 from the immunopeptidomic analysis of patient Mel-4.

[0064] Fig.26 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 20 from the immunopeptidomic analysis of patient Mel-4.

[0065] Fig. 27 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 16 from the immunopeptidomic analysis of patient Mel-20.

[0066] Fig.28 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 12 from the immunopeptidomic analysis of patient Mel-3.

[0067] for Figure 29-Figure 42 In each of the , the upper panel shows the extracted MS / MS spectrum of the peptide (along with the assigned fragment ions) isolated from a patient's tumor sample, and the lower panel indicates the spectral presentation of the position of the linear peptide sequence that has been mapped to the fragment ions.

[0068] Fig.29 . Spectrum of the peptide of SEQ ID NO. 51 isolated from a tumor sample of patient Mel-40.

[0069] Fig.30 . Spectrum of the peptide of SEQ ID NO. 51 isolated from a tumor sample of patient Mel-41.

[0070] Fig.31 . Spectrum of the peptide of SEQ ID NO. 52 isolated from a tumor sample of patient Mel-27.

[0071] Fig.32 . Spectrum of the peptide of SEQ ID NO. 52 isolated from a tumor sample of patient Mel-39.

[0072] Fig.33 . Spectrum of the peptide of SEQ ID NO. 13 isolated from a tumor sample of patient 2MT3.

[0073] Fig.34 . Spectrum of the peptide of SEQ ID NO. 13 isolated from a tumor sample of patient 2MT10.

[0074] Fig.35. Spectrum of the peptide of SEQ ID NO. 12 isolated from a tumor sample of patient 2MT3.

[0075] Fig.36 . Spectrum of the peptide of SEQ ID NO. 16 isolated from a tumor sample of patient 2MT4.

[0076] Fig.37 . Spectrum of the peptide of SEQ ID NO. 17 isolated from a tumor sample of patient 2MT3.

[0077] Fig.38 . Spectrum of the peptide of SEQ ID NO. 53 isolated from a tumor sample of patient 1MT1.

[0078] Fig.39 . Spectrum of the peptide of SEQ ID NO. 51 isolated from a tumor sample of patient 2MT3.

[0079] Fig.40 . Spectrum of the peptide of SEQ ID NO. 19 isolated from a tumor sample of patient 2MT3.

[0080] Fig.41 . Spectrum of the peptide of SEQ ID NO. 19 isolated from a tumor sample of patient 2MT1.

[0081] Fig.42 . Spectrum of the peptide of SEQ ID NO. 54 isolated from a tumor sample of patient 2MT12.

[0082] Figure 43-Figure 50 Each shows an alignment of a native MS / MS spectrum of a peptide isolated from a patient tumor sample (upper) with a native spectrum of a synthetic peptide corresponding to the same sequence (lower).

[0083] Fig.43 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 13 from the immunopeptidomic analysis of patient 2MT3.

[0084] Fig.44 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 12 from the immunopeptidomic analysis of patient 2MT3.

[0085] Fig.45 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 16 from the immunopeptidomic analysis of patient 2MT4.

[0086] Fig.46 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 17 from the immunopeptidomic analysis of patient 2MT3.

[0087] Fig.47 Shown is the mass spectrometry spectrum of the peptide fragment attributed to SEQ ID NO. 53 from the immunopeptidomic analysis of patient 1MT1.

[0088] Fig.48 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 51 from the immunopeptidomic analysis of patient 2MT3.

[0089] Fig.49 Shown is the mass spectrometry spectrum of the peptide fragments attributed to SEQ ID NO. 19 from the immunopeptidomic analysis of patient 2MT3.

[0090] Fig.50 Shown is the mass spectrometry spectrum of the peptide fragment attributed to SEQ ID NO. 54 from the immunopeptidomic analysis of patient 2MT12.

[0091] Fig.51 Panels A to C show the expansion of tumor antigen-specific T cells from patient PBMC cultures in response to incubation with specific tumor antigen-derived peptides.

[0092] Fig.52 Panels A to D provide a summary of CLT antigen-derived peptides (SEQ ID NO. 55 to SEQ ID NO. 72) that are able to expand TCR-bearing specific T cells from melanoma patient PBMCs.

[0093] Fig.53 Shown are CD8 T cell responses from normal blood donors against an HLA-A*0201 restricted peptide from CLT antigen 1 (SEQ ID NO. 73).

[0094] Fig.54 CD8 T cell responses from normal blood donors against an HLA-A*0201 restricted peptide from CLT antigen 2 (SEQ ID NO. 75) are shown.

[0095] Fig.55 CD8 T cell responses from normal blood donors against an HLA-A*0201 restricted peptide from CLT antigen 4 (SEQ ID NO. 76) are shown.

[0096] Fig.56 Panels A to D show the reactivity against HLA-B*0702 restricted peptides (SEQ ID NOs. 74 and 77) from CLT antigen 1 and CLT antigen 4, respectively, in memory CD45RO positive CD8 T cells compared to naive CD45RO negative CD8 T cells from the same donor.

[0097] Fig.57HLA pentamer staining of normal CD8 T cells specific for a peptide derived from CLT antigen 1 (SEQ ID NO. 73), a peptide derived from CLT antigen 2 (SEQ ID NO. 78), and a peptide derived from CLT antigen 4 (SEQ ID NO. 77) is shown.

[0098] Fig.58 Expanded, pentamer-sorted CD8 T cells pulsed with a peptide derived from CLT antigen 4 (SEQ ID NO. 77) are shown to kill C1RB7 target cells.

[0099] Fig.59 Panels A to C show the results of qRT-PCR analysis validating the transcription of CLT encoding CLT antigen 1 (SEQ ID NO. 33), CLT encoding CLT antigen 2 (SEQ ID NO. 34); and CLT encoding CLT antigens 3 and 4 (SEQ ID NO. 35) in melanoma cancer cell lines.

[0100] Sequence Description

[0101] SEQ ID NO.1 is the polypeptide sequence of CLT antigen 1

[0102] SEQ ID NO.2 is the polypeptide sequence of CLT antigen 2

[0103] SEQ ID NO.3 is the polypeptide sequence of CLT antigen 3

[0104] SEQ ID NO.4 is the polypeptide sequence of CLT antigen 4

[0105] SEQ ID NO.5 is the polypeptide sequence of CLT antigen 5

[0106] SEQ ID NO.6 is the polypeptide sequence of CLT antigen 6

[0107] SEQ ID NO.7 is the polypeptide sequence of CLT antigen 7

[0108] SEQ ID NO.8 is the polypeptide sequence of CLT antigen 8

[0109] SEQ ID NO.9 is the polypeptide sequence of CLT antigen 9

[0110] SEQ ID NO.10 is the polypeptide sequence of CLT antigen 10

[0111] SEQ ID NO. 11-14 are peptide sequences derived from CLT antigen 1

[0112] SEQ ID NO. 15 and 16 are peptide sequences derived from CLT antigen 2

[0113] SEQ ID NOs. 17 and 18 are peptide sequences derived from CLT antigen 3

[0114] SEQ ID NO.19 is a peptide sequence derived from CLT antigen 4

[0115] SEQ ID NO. 20-22 are peptide sequences derived from CLT antigen 5

[0116] SEQ ID NOs. 23 and 24 are peptide sequences derived from CLT antigen 6

[0117] SEQ ID NO.25 is a peptide sequence derived from CLT antigen 7

[0118] SEQ ID NO.26 is a peptide sequence derived from CLT antigen 8

[0119] SEQ ID NO. 27-29 are peptide sequences derived from CLT antigen 9

[0120] SEQ ID NO. 30-32 are peptide sequences derived from CLT antigen 10

[0121] SEQ ID NO.33 is the cDNA sequence of CLT encoding CLT antigen 1

[0122] SEQ ID NO.34 is the cDNA sequence of CLT encoding CLT antigen 2

[0123] SEQ ID NO.35 is the cDNA sequence of CLT encoding CLT antigens 3 and 4

[0124] SEQ ID NO.36 is the cDNA sequence of CLT encoding CLT antigen 5

[0125] SEQ ID NO.37 is the cDNA sequence of CLT encoding CLT antigen 6

[0126] SEQ ID NO.38 is the cDNA sequence of CLT encoding CLT antigens 7 and 8

[0127] SEQ ID NO.39 is the cDNA sequence of CLT encoding CLT antigen 9

[0128] SEQ ID NO.40 is the cDNA sequence of CLT encoding CLT antigen 10

[0129] SEQ ID NO.41 is the cDNA sequence encoding CLT antigen 1

[0130] SEQ ID NO.42 is the cDNA sequence encoding CLT antigen 2

[0131] SEQ ID NO.43 is the cDNA sequence encoding CLT antigen 3

[0132] SEQ ID NO.44 is the cDNA sequence encoding CLT antigen 4

[0133] SEQ ID NO.45 is the cDNA sequence encoding CLT antigen 5

[0134] SEQ ID NO.46 is the cDNA sequence encoding CLT antigen 6

[0135] SEQ ID NO.47 is the cDNA sequence encoding CLT antigen 7

[0136] SEQ ID NO.48 is the cDNA sequence encoding CLT antigen 8

[0137] SEQ ID NO.49 is the cDNA sequence encoding CLT antigen 9

[0138] SEQ ID NO.50 is the cDNA sequence encoding CLT antigen 10

[0139] SEQ ID NO. 51-52 are peptide sequences derived from CLT antigen 4

[0140] SEQ ID NO.53 is a peptide sequence derived from CLT antigen 3

[0141] SEQ ID NO.54 is a peptide sequence derived from CLT antigen 4

[0142] SEQ ID NO. 55-57 are peptide sequences derived from CLT antigen 1

[0143] SEQ ID NO. 58-66 are peptide sequences derived from CLT antigen 2

[0144] SEQ ID NO. 67-69 are peptide sequences derived from CLT antigen 3

[0145] SEQ ID NO. 70-72 are peptide sequences derived from CLT antigen 4

[0146] SEQ ID NO. 73-74 are peptide sequences derived from CLT antigen 1

[0147] SEQ ID NO.75 is a peptide sequence derived from CLT antigen 2

[0148] SEQ ID NO. 76-77 are peptide sequences derived from CLT antigen 4

[0149] SEQ ID NO.78 is a peptide sequence derived from CLT antigen 2 Specific implementation plan

[0150] Peptides

[0151] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length, co-translationally or post-translationally modified.

[0152] The term "amino acid" refers to any naturally occurring amino acid, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those 20 L-amino acids encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, γ-carboxyglutamate and O-phosphoserine. The term "amino acid analog" refers to a compound having the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bound to a hydrogen, a carboxyl group, an amino group and an R group, and an R group, but having a modified R group or a modified peptide backbone compared to a natural amino acid. Examples include homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium and norleucine. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid. Suitably, an amino acid is a naturally occurring amino acid or an amino acid analog, especially a naturally occurring amino acid and particularly one of the 20 L-amino acids encoded by the genetic code.

[0153] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0154] Therefore, the present invention provides an isolated polypeptide comprising a sequence selected from the group consisting of:

[0155] (a) the sequence of any one of SEQ ID NOs. 1-10; and

[0156] (b) a variant of the sequence of (a); and

[0157] (c) Immunogenic fragments of the sequence of (a)

[0158] The present invention also provides an isolated polypeptide comprising a sequence selected from the group consisting of:

[0159] (a) the sequence of any one of SEQ ID NOs. 1-10 minus the initial methionine residue; and

[0160] (b) a variant of the sequence of (a); and

[0161] (c) Immunogenic fragments of the sequence of (a)

[0162] In general, variants of polypeptide sequences of the present invention include sequences with a high degree of sequence identity therewith. For example, variants are suitably at least about 80% identical, more preferably at least about 85% identical, and most preferably at least about 90% identical (e.g., at least about 95%, at least about 98%, or at least about 99%) to the relevant reference sequence over its entire length.

[0163] Suitably, the variant is an immunogenic variant. A variant is considered to be an immunogenic variant if it stimulates a response that is at least 20%, suitably at least 50% and in particular at least 75% (e.g. at least 90%) of the activity of a reference sequence (i.e., the variant is a variant of said reference sequence), for example in an in vitro re-stimulation assay of PBMC or whole blood using the polypeptide as an antigen (e.g., the re-stimulation lasts for a period of between a few hours and up to 1 year (e.g., up to 6 months, 1 day to 1 month or 1 to 2 weeks), wherein the in vitro re-stimulation assay measures the activation of cells by means of lymphocyte proliferation (e.g., T cell proliferation), cytokine (e.g., IFN-γ) production in the culture supernatant (measured by ELISA, etc.) or by intracellular and extracellular staining (e.g., using antibodies specific for immune markers such as CD3, CD4, CD8, IL2, TNF-α, IFNg, type 1 IFN, CD40L, CD69, etc.), followed by characterization of T-cell responses by flow cytometric analysis.

[0164] Variants can be, for example, conservatively modified variants. "Conservatively modified variants" are variants in which the changes result in substitution of an amino acid with a functionally similar amino acid or substitution / deletion / addition of residues that do not substantially affect the biological function of the variant. Typically, such biological functions of the variant will induce an immune response against melanoma, such as skin melanoma cancer antigens.

[0165] Conservative substitution tables providing functionally similar amino acids are well known in the art. Variants may include polypeptide homologs existing in other species.

[0166] When compared to a reference sequence, a variant of a polypeptide of the invention may contain multiple substitutions, e.g., conservative substitutions (e.g., 1-25, such as 1-10, especially 1-5 and especially 1 amino acid residue may be changed). The number of substitutions (e.g., conservative substitutions) may be up to 20%, e.g., up to 10%, e.g., up to 5%, e.g., up to 1%, of the number of residues in the reference sequence. Typically, conservative substitutions will fall within the scope of one of the amino acid groupings specified below, although in some cases, other substitutions may be possible without significantly affecting the immunogenic properties of the antigen. The following eight groups each contain amino acids that are generally conservative substitutions of each other:

[0167] 1) Alanine (A), glycine (G);

[0168] 2) Aspartic acid (D), glutamic acid (E);

[0169] 3) Asparagine (N), glutamine (Q);

[0170] 4) Arginine (R), Lysine (K);

[0171] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0172] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0173] 7) serine (S), threonine (T); and

[0174] 8) Cysteine ​​(C), Methionine (M)

[0175] (See, e.g., Creighton, Proteins 1984).

[0176] Suitably, such substitutions do not alter the immunological structure of the epitope (eg, they do not occur within the region of the epitope as mapped in the primary sequence), and therefore do not significantly affect the immunogenic properties of the antigen.

[0177] Polypeptide variants also include those variants in which additional amino acids are inserted compared to the reference sequence, for example, such insertions may involve 1-10 positions (such as 1-5 positions, suitably 1 or 2 positions, especially 1 position), and may, for example, include adding 50 or less (such as 20 or less, especially 10 or less, especially 5 or less) amino acids at each position. Suitably, such insertions do not occur within the region of the epitope, and therefore do not significantly affect the immunogenic properties of the antigen. An example of an insertion includes a short stretch of histidine residues (e.g., 2-6 residues) that assist in the expression and / or purification of the antigen in question.

[0178] Polypeptide variants include those variants in which amino acids have been deleted compared to the reference sequence, for example, such deletions may occur at 1-10 positions (such as 1-5 positions, suitably 1 or 2 positions, especially 1 position), and may, for example, involve deleting 50 or less (such as 20 or less, especially 10 or less, particularly 5 or less) amino acids at each position. Suitably, such deletions do not occur in the region of the epitope, and therefore do not significantly affect the immunogenic properties of the antigen.

[0179] Those of ordinary skill in the art will recognize that a particular protein variant may comprise substitutions, deletions, and additions (or any combination thereof). For example, a substitution / deletion / addition may enhance binding to (or have a neutral effect on) a desired patient HLA molecule, thereby potentially increasing immunogenicity (or maintaining immunogenicity unchanged).

[0180] The immunogenic fragments of the present invention will generally contain at least 9 (e.g., at least 9 or 10) consecutive amino acids from the full-length polypeptide sequence, such as at least 12 consecutive amino acids (e.g., at least 15 or at least 20 consecutive amino acids), especially at least 50 consecutive amino acids, such as at least 100 consecutive amino acids (e.g., at least 200 consecutive amino acids), depending on the length of the CLT antigen. Suitably, the immunogenic fragment will be at least 10%, such as at least 20%, such as at least 50%, such as at least 70% or at least 80% of the length of the full-length polypeptide sequence.

[0181] Immunogenic fragments generally contain at least one epitope. Epitopes include B cell epitopes and T cell epitopes, and suitably, immunogenic fragments contain at least one T cell epitope such as a CD4+ T cell epitope or a CD8+ T cell epitope.

[0182] T cell epitopes are short stretches of contiguous amino acids that are recognized by T cells (e.g., CD4+T cells or CD8+T cells) when bound to HLA molecules. The identification of T cell epitopes can be achieved by epitope mapping experiments that are well known to those skilled in the art (see, e.g., Paul, Fundamental Immunology, 3rd edition, 243-247 (1993); Beiβbarth et al., 2005, Bioinformatics, 21 (Suppl. 1): i29-i37).

[0183] As a result of the crucial involvement of T-cell responses in cancer, it is evident 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 immunoprotection.

[0184] It will be appreciated that in a diverse outbred population (such as humans), different HLA types mean that a particular epitope may not be recognized by all members of the population. Therefore, in order to maximize the level of recognition and the size of the immune response to the polypeptide, it is generally desirable that the immunogenic fragment contains multiple epitopes from the full-length sequence (suitably all epitopes within the CLT antigen).

[0185] Potentially useful specific fragments of the polypeptides of SEQ ID NO.1-10 include those containing at least one CD8+T cell epitope, suitably at least two CD8+T cell epitopes and especially all CD8+T cell epitopes, especially those fragments associated with multiple HLA alleles, for example, those fragments associated with 2, 3, 4, 5 or more alleles. Potentially useful specific fragments of the polypeptides of SEQ ID NO.1-10 include those containing at least one CD4+T cell epitope, suitably at least two CD4+T cell epitopes and especially all CD4+T cell epitopes (especially those fragments associated with multiple HLA alleles, for example, those fragments associated with 2, 3, 4, 5 or more alleles). However, a technician designing a vaccine may combine an exogenous CD4+T cell epitope with a CD8+T cell epitope of the present invention and achieve a desired response to a CD8+T cell epitope of the present invention.

[0186] If an isolated fragment of a full-length polypeptide is used, this fragment is considered to be immunogenic if it stimulates a response that is at least 20%, suitably at least 50% and in particular at least 75% (e.g. at least 90%) of the activity of the reference sequence (i.e. of which the fragment is a fragment) (e.g. in an in vitro re-stimulation assay of PBMC or whole blood using the polypeptide as antigen (e.g., re-stimulation lasting from a few hours to up to 1 year (e.g., up to 6 months, 1 day to 1 month or 1 to 2 weeks)), wherein the in vitro re-stimulation assay measures the activation of cells by means of lymphocyte proliferation (e.g., T cell proliferation), cytokine (e.g., IFN-γ) production in the culture supernatant (measured by ELISA, etc.) or by intracellular and extracellular staining (e.g., using antibodies specific for immune markers such as CD3, CD4, CD8, IL2, TNF-α, IFN-γ, type 1 IFN, CD40L, CD69, etc.), followed by characterization of the T-cell response by flow cytometric analysis.

[0187] In some cases, multiple fragments of a full-length polypeptide (which may or may not overlap and may or may not cover the entirety of the full-length sequence) may be used to obtain an equivalent biological response to the full-length sequence itself. For example, at least two immunogenic fragments (such as three, four or five) as described above, when combined, provide at least 50%, suitably at least 75% and especially at least 90% of the activity of the reference sequence in a PBMC or whole blood in vitro re-stimulation assay (e.g., T cell proliferation and / or IFN-γ production assay).

[0188] Exemplary immunogenic fragments of the polypeptides of SEQ ID NO. 1-10, and therefore exemplary peptides of the present invention, include polypeptides comprising or consisting of sequences of SEQ ID NO. 11-32. Other exemplary immunogenic fragments of the polypeptides of SEQ ID NO. 1-4, and therefore exemplary peptides of the present invention, include polypeptides comprising or consisting of sequences of SEQ ID NO. 51-78. Sequences of SEQ ID NO. 11-17, 19-28, 30-31 and 51-54 were identified from immunopeptidomic analysis as binding to HLA class I molecules (see Examples 2 and 2.1). Sequences of SEQ ID NO. 18, 29 and 32 were identified from immunopeptidomic analysis as binding to HLA class II molecules (see Example 2). Sequences of SEQ ID NO 55-78 were predicted by NetMHC software to bind to HLA class I molecules and were used in immunological validation analysis (see Examples 3, 4 and 5).

[0189] Nucleic Acids

[0190] The present invention provides an isolated nucleic acid encoding a 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 NO. 33-40 or 41-50.

[0191] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to polymeric macromolecules produced from nucleotide monomers, especially deoxyribonucleotide monomers or ribonucleotide monomers. The term encompasses nucleic acids containing known nucleotide analogs or modified main chain residues or bonds, which are naturally occurring and non-naturally occurring, have properties similar to reference nucleic acids, and are intended to be metabolized in a manner similar to reference nucleotides or intended to have an extended half-life in the system. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, peptide nucleic acids (PNAs). Suitably, the term "nucleic acid" refers to naturally occurring polymers of deoxyribonucleotide monomers or ribonucleotide monomers. Suitably, the nucleic acid molecules of the present invention are recombinant. Recombinant means that the nucleic acid molecule is the product of at least one of the cloning step, restriction step or ligation step or the product of other methods that produce nucleic acid molecules (e.g., in the case of cDNA) that are very different from nucleic acid molecules present in nature. In one embodiment, the nucleic acid of the invention is an artificial nucleic acid sequence (e.g., a cDNA sequence or nucleic acid sequence with non-natural codon selection). In one embodiment, the nucleic acid of the invention is DNA. Alternatively, the nucleic acid of the invention is RNA.

[0192] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) refer to nucleic acid molecules with a main chain of sugar moieties, which are deoxyribosyl and ribosyl, respectively. Sugar moieties can be linked to bases, which are 4 natural bases (adenine (A), guanine (G), cytosine (C) and thymine (T) in DNA and adenine (A), guanine (G), cytosine (C) and uracil (U) in RNA). As used herein, "corresponding RNA" is an RNA with the same sequence as the reference DNA except that thymine (T) in DNA is replaced with uracil (U) in RNA. Sugar moieties can also be linked to non-natural bases such as inosine, xanthosine, 7-methylguanosine, dihydrouridine and 5-methylcytidine. The natural phosphodiester bond between the sugar (deoxyribosyl / ribosyl) moieties can be optionally replaced with a phosphorothioate bond. Suitably, the nucleic acids of the invention consist of natural bases linked to a deoxyribosyl or ribosyl sugar backbone with phosphodiester bonds between the sugar moieties.

[0193] In one embodiment, the nucleic acid of the present invention is DNA. For example, the nucleic acid comprises or consists of a sequence selected from SEQ ID NO.33-40 or 41-50. A nucleic acid is also provided, which comprises or consists of a variant of a sequence selected from SEQ ID NO.33-40 or 41-50, wherein the variant encodes the same amino acid sequence but has a different nucleic acid based on the degeneracy of the genetic code.

[0194] Therefore, due to the degeneracy of the genetic code, many different but functionally identical nucleic acids can encode any given polypeptide. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified by a certain codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variants result in "silent" (sometimes referred to as "degenerate" or "synonymous") variants, which are conservatively modified variations of a type. Each nucleic acid sequence encoding a polypeptide disclosed herein also enables each possible silent variation of the nucleic acid. The skilled person will recognize that each codon in the nucleic acid can be modified (exceptions are AUG, which is usually the only codon for methionine, and UGG, which is usually the only codon for tryptophan) to produce functionally identical molecules. Therefore, each silent variation of a nucleic acid encoding a polypeptide is contained in each described sequence and is provided as an aspect of the present invention.

[0195] Degenerate codon substitutions can also be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0196] When compared to a reference sequence, a nucleic acid of the invention comprising or consisting of a sequence selected from SEQ ID NO. 33-40 or 41-50 may contain multiple silent variations (e.g., 1-50, such as 1-25, especially 1-5 and especially 1 codon may be changed).

[0197] In one embodiment, the nucleic acid of the invention is RNA. RNA sequences are provided that correspond to the DNA sequences provided herein and have a ribonucleotide backbone rather than a deoxyribonucleotide backbone and have side chain bases uracil (U) instead of thymine (T).

[0198] Therefore, when compared to the reference sequence, the nucleic acid of the present invention comprises or consists of an RNA equivalent of a cDNA sequence selected from SEQ ID NO. 33-40 or 41-50, and may contain multiple silent variations (e.g., 1-50, such as 1-25, especially 1-5 and especially 1 codon may be changed). "RNA equivalent" means an RNA sequence that contains the same genetic information as the reference cDNA sequence (i.e., contains the same codons, has a ribonucleotide backbone instead of a deoxyribonucleotide backbone, and has a side chain base uracil (U) instead of thymine (T)).

[0199] The present invention also includes sequences complementary to the aforementioned cDNA sequences and RNA sequences.

[0200] In one embodiment, the nucleic acids of the invention are codon-optimized for expression in human host cells.

[0201] The nucleic acids of the invention can be transcribed and translated into the polypeptides of the invention in the case of DNA nucleic acids, and translated into the polypeptides of the invention in the case of RNA nucleic acids.

[0202] Peptides and nucleic acids

[0203] Suitably, the polypeptides and nucleic acids used in the present invention are isolated. An "isolated" polypeptide or nucleic acid is one that is removed from its original environment. For example, a naturally occurring polypeptide or nucleic acid is isolated if it is separated from some or all of the coexisting materials in the natural system. For example, a nucleic acid is considered isolated if it is cloned into a vector that is not part of its natural environment.

[0204] "Naturally occurring" when used in reference to a polypeptide or nucleic acid sequence means a sequence that occurs in nature and has not been synthetically modified.

[0205] "Artificial" when used in reference to a polypeptide or nucleic acid sequence refers to a sequence that does not occur in nature, where, for example, the sequence is a synthetic modification of a native sequence or contains non-natural sequences.

[0206] The term "heterologous," when used in reference to the relationship of one nucleic acid or polypeptide to another nucleic acid or polypeptide, means that the two or more sequences do not exist in the same relationship to each other as in nature. A "heterologous" sequence may also mean a sequence that is not isolated from, derived from, or based on a naturally occurring nucleic acid or polypeptide sequence present in a host organism.

[0207] As indicated above, polypeptide variants preferably have at least about 80% identity, more preferably at least about 85% identity, and most preferably at least about 90% identity (e.g., at least about 95%, at least about 98%, or at least about 99%) to the relevant reference sequence over its entire length.

[0208] For the purpose of comparing two closely related polypeptide sequences or polynucleotide sequences, the "sequence identity %" between the first sequence and the second sequence can be calculated. If a polypeptide sequence has 100% sequence identity over its entire length, the polypeptide sequence is said to be identical or identical to the other polypeptide sequence. The residues in the sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus of the polypeptide. In the case of two or more polypeptide sequences, the term "identical" or "identity" percentage refers to two or more sequences or subsequences of the same amino acid residues (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity) that are identical or have a specified percentage when compared and aligned for maximum correspondence within a comparison window. Suitably, the comparison is performed within a window corresponding to the entire length of the reference sequence.

[0209] For sequence comparison, a sequence serves as a reference sequence compared to a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Based on the program parameters, the sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence.

[0210] As used herein, "comparison window" refers to a segment in which a sequence can be compared to a reference sequence having the same number of consecutive positions after optimal alignment of the two sequences. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences 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 search for similarity method of Pearson and Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds., 1995 suppl.)).

[0211] An example of a useful algorithm is PILEUP. Using a progressive pairwise alignment method, PILEUP generates multiple sequence alignment results from a group of related sequences to display relationships and percentages of sequence identity. It also draws an evolutionary tree or dendrogram, which displays the clustering relationships used to generate the alignment results. PILEUP uses a simplified form of Feng and Doolittle's progressive alignment method (Feng and Doolittle, 1987, J.Mol.Evol.35:351-360). The method used is similar to the method described by Higgins and Sharp, 1989, CABIOS 5:151-153. The program can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. The multiple alignment program begins with a pairwise alignment of the two most similar sequences, generating a cluster of two aligned sequences. This cluster is then aligned with the next most related sequence or cluster of aligned sequences. Align the two sequence clusters by simply extending the pairwise alignment results of two independent sequences. The final alignment is achieved by a series of progressive pairwise alignments. By specifying specific sequence and amino acid coordinates thereof for sequence comparison region and by specifying program parameters, run the program.Use PILEUP, reference sequence and other test sequences are compared to use the following parameters to determine the sequence identity percentage relationship: default gap weight (3.00), default gap length weight (0.10) and weighted end gap.PILEUP can be obtained from GCG sequence analysis software package, for example, version 7.0 (Devereaux et al., 1984, Nuc.Acids Res.12:387-395).

[0212] Another example of an algorithm suitable for determining percentages of sequence identity and sequence similarity is the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al., 1977, Nuc. Acids Res. 25: 3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215: 403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (website address: www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by determining short words of length W in the query sequence, wherein when compared with words of the same length in the database sequence, the short words match or meet certain positive threshold scores T. T is referred to as the adjacent word score threshold (Altschul et al., supra). These initial adjacent word hits serve as seeds, which are used to initiate searches to find longer HSPs containing these seeds. The word hits are extended as far as possible along each sequence in both directions as long as the cumulative alignment score can be improved. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hits in each direction is stopped when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score reaches or falls below zero due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. For amino acid sequences, the BLASTP program uses a word length of 3 and an expectation (E) of 10, as well as the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89: 10915), alignments (B) 50, expectations (E) 10, M = 5, N = -4, and comparison of both chains as defaults.

[0213] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance.

[0214] A "difference" between sequences refers to an insertion, deletion or substitution of a single residue in a position of the second sequence compared to the first sequence. Two sequences may contain one, two or more such differences. An insertion, deletion or substitution in a second sequence that is otherwise identical to the first sequence (100% sequence identity) results in a decrease in the % sequence identity. For example, if the identical sequence is 9 residues long, one substitution in the second sequence results in 88.9% sequence identity. If the identical sequence is 17 amino acid residues long, two substitutions in the second sequence result in 88.2% sequence identity.

[0215] Alternatively, for the purpose of comparing a first reference sequence to a second comparison sequence, the number of additions, substitutions and / or deletions made to the first sequence to generate the second sequence can be determined. An addition is the addition of a residue to the first sequence (including additions at either end of the first sequence). A substitution is the replacement of a residue in the first sequence with a different residue. A deletion is the deletion of a residue from the first sequence (including deletions at either end of the first sequence).

[0216] Producing the polypeptides of the invention

[0217] The polypeptides of the present invention can be obtained and manipulated using techniques disclosed in, for example, Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual 4th edition Cold Spring Harbour Laboratory Press. In particular, artificial gene synthesis methods can be used to produce 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), which are then expressed in a suitable organism to produce the polypeptides. Genes encoding the polypeptides of the present invention can be synthetically produced, for example, by solid phase DNA synthesis. Complete genes can be synthesized de novo without the need for precursor template DNA. In order to obtain the desired oligonucleotide, the structural units are sequentially coupled to the growing oligonucleotide chain in the order required by the product sequence. When the chain assembly is completed, the product is released from the solid phase to the solution, deprotected, and collected. The product can be separated by high performance liquid chromatography (HPLC) to obtain the desired oligonucleotide (Verma and Eckstein, 1998, Annu. Rev. Biochem. 67: 99-134) of high purity. By using a variety of gene amplification methods (Methods Mol Biol., 2012; 834: 93-109), these relatively short segments are easily assembled into longer DNA molecules suitable for countless recombinant DNA-based expression systems. In the context of the present invention, those skilled in the art will understand that the polynucleotide sequences encoding polypeptide antigens described in the present invention can be easily used in a variety of vaccine production systems, such as viral vectors.

[0218] For the purpose of producing polypeptides of the present invention in a microbial (e.g., bacterial or fungal) host, nucleic acids of the present invention will comprise suitable regulatory and control sequences (including promoters, termination signals, etc.) and sequences that promote secretion of polypeptides suitable for producing proteins in the host. Similarly, polypeptides of the present invention can be produced by transducing cultures of eukaryotic cells (e.g., Chinese hamster ovary cells or Drosophila S2 cells) with nucleic acids of the present invention, wherein the nucleic acids have been combined with suitable regulatory and control sequences (including promoters, termination signals, etc.) and sequences that promote secretion of polypeptides suitable for producing proteins in these cells.

[0219] Improved isolation of polypeptides of the invention produced by recombinant means may optionally be facilitated by the addition of a stretch of histidine residues, commonly known as an aHis tag, to one terminus of the polypeptide.

[0220] Polypeptides can also be produced synthetically.

[0221] Carrier

[0222] In additional embodiments, a genetic construct comprising one or more nucleic acids of the present invention is introduced into a cell in vivo to produce a polypeptide of the present invention in vivo, stimulating an immune response. Nucleic acid (e.g., DNA) may be present in any one of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial expression systems, and some viral expression systems. Numerous gene delivery techniques are well known in the art, such as Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198 and those described in references cited therein. Several of these schemes are briefly described below for illustrative purposes.

[0223] Thus, provided are vectors (also referred to herein as "DNA expression constructs" or "constructs") comprising the nucleic acid molecules of the invention.

[0224] Suitably, the vector comprises a nucleic acid molecule encoding regulatory elements (such as a suitable promoter and termination signal) suitable for allowing transcription of the translationally active RNA molecule in a human host cell."Translationally active RNA molecule" is an RNA molecule that can be translated into protein by the translation machinery of a human cell.

[0225] Therefore, a vector comprising the nucleic acid of the present invention (hereinafter referred to as "the vector of the present invention") is provided.

[0226] In particular, the vector may be a viral vector. The viral vector may be an adenovirus, adeno-associated virus (AAV) (e.g., AAV type 5 and type 2), an alphavirus (e.g., Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SIN), Semliki Forest virus (SFV)), a herpes virus, an arenavirus (e.g., lymphocytic choriomeningitis virus (LCMV)), a measles virus, a poxvirus (e.g., modified vaccinia Ankara virus (MVA)), a paramyxovirus, a lentivirus, or a rhabdovirus (e.g., vesicular stomatitis virus (VSV)) vector, i.e., the vector may be derived from any of the aforementioned viruses. Adenovirus is particularly suitable for use as a gene transfer vector because of its medium genome size, ease of manipulation, high titer, wide target cell range, and high infectivity. The two ends of the viral genome contain 100-200 base pair inverted repeat sequences (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) region and the late (L) region of the genome contain different transcription units divided according to the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for regulating the 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 shutdown (Renan, 1990). The products of the late genes, including most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript released from the major late promoter (MLP). The MLP is particularly efficient during the late stages of infection, and all mRNAs transcribed from this promoter possess a 5'-triplet leader (TPL) sequence that makes these mRNAs preferred for translation. Replication-defective adenoviruses generated from viral genomes lacking one or more early genes are particularly useful because they have limited replication and are less likely to spread pathogenicity within and to contacts of inoculated hosts.

[0227] Other polynucleotide delivery

[0228] In certain embodiments of the present invention, the expression construct comprising one or more polynucleotide sequences can be composed of naked recombinant DNA plasmids alone. See Ulm et al., 1993, Science 259:1745-1749 and Cohen, 1993, Science 259:1691-1692 for review. Construct transfer can be performed, for example, by any method of permeabilizing the cell membrane physically or chemically. This is particularly suitable for in vitro transfer, but it can also be used in vivo. It is contemplated that the DNA encoding the target gene can also be transferred in vivo in a similar manner and express the gene product. Multiple delivery systems have been used to deliver DNA molecules to animal models and to humans. Some products based on this technology have been licensed for use in animals, and others are in Phase II and Phase III human clinical trials.

[0229] RNA delivery

[0230] In certain embodiments of the invention, an expression construct comprising one or more polynucleotide sequences may consist of a naked recombinant DNA-derived plasmid (Ulmer et al., 2012, Vaccine 30:4414–4418). With respect to DNA-based expression constructs, a variety of methods can be used to introduce RNA molecules into cells in vitro or in vivo. RNA-based constructs can be designed to mimic simple messenger RNA (mRNA) molecules, whereby the introduced biomolecules are directly translated by the translation apparatus of the host cell to produce the polypeptides encoded by them in the cells into which the biomolecules are introduced. Alternatively, RNA molecules can be designed in such a way that they allow them to self-amplify in the cells into which the molecules are introduced by incorporating viral RNA-dependent RNA polymerase genes into the structure of the molecules. Therefore, these types of RNA molecules, referred to as self-amplifying mRNAs (SAMs), are known as self-amplifying mRNAs. TM ) molecules (Geall et al., 2012, PNAS, 109:14604–14609), which share properties with some RNA-based viral vectors. mRNA-based RNA or SAM can be further modified TMRNA (e.g., by changing its sequence 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 lipid nanoparticles (Kranz et al., 2006, Nature, 534: 396-401)) to promote stability and / or entry into cells in vitro or in vivo. Numerous formulations of modified (and unmodified) RNA have been tested as vaccines in models and in humans, and multiple RNA-based vaccines are being used in ongoing clinical trials.

[0231] Pharmaceutical composition

[0232] The polypeptides, nucleic acids and vectors of the invention can be formulated for delivery in pharmaceutical compositions such as immunogenic compositions and vaccine compositions (hereinafter referred to as "compositions of the invention"). The compositions of the invention suitably comprise the polypeptides, nucleic acids or vectors of the invention together with a pharmaceutically acceptable carrier.

[0233] Thus, in one embodiment, an immunogenic pharmaceutical composition comprising a polypeptide, nucleic acid or vector of the invention together with a pharmaceutically acceptable carrier is provided.

[0234] In another embodiment, a vaccine composition comprising a polypeptide, nucleic acid or vector of the invention together with a pharmaceutically acceptable carrier is provided. The preparation of pharmaceutical compositions is generally described, for example, in Powell and Newman, Vaccine Design (Subunit and Adjuvant Program), 1995. The composition of the invention may also contain other compounds that may be biologically active or inactive. Suitably, the composition of the invention is a sterile composition suitable for parenteral administration.

[0235] In certain preferred embodiments of the present invention, pharmaceutical compositions of the present invention are provided, comprising one or more (eg, one) polypeptides of the present invention in combination with a pharmaceutically acceptable carrier.

[0236] In certain preferred embodiments of the present invention, pharmaceutical compositions of the present invention are provided, comprising one or more (eg, one) nucleic acids of the present invention or one or more (eg, one) vectors of the present invention in combination with a pharmaceutically acceptable carrier.

[0237] In one embodiment, the composition of the present invention can include one or more (e.g., one) polynucleotides and one or more (e.g., one) polypeptide components. Alternatively, the composition can include one or more (e.g., one) carriers and one or more (e.g., one) polypeptide components. Alternatively, the composition can include one or more (e.g., one) carriers and one or more (e.g., one) polynucleotide components. Such compositions can provide an enhanced immune response.

[0238] Pharmaceutically acceptable salts:

[0239] It will be apparent that the compositions of the invention can contain 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., sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts and magnesium salts).

[0240] Pharmaceutically acceptable carrier

[0241] Although many pharmaceutically acceptable carriers known to those of ordinary skill in the art can be used in the compositions of the present invention, the best class of the carrier used will vary according to the mode of administration. The compositions of the present invention can be formulated for any suitable mode of administration, including, for example, parenteral, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration, preferably parenteral administration, for example, intramuscular, subcutaneous or intravenous administration. For parenteral administration, the carrier preferably comprises water and can contain a pH control buffer, a stabilizer, for example, a surfactant and an amino acid and a tension regulator, for example, salt and sugar. If the composition is intended to be provided in a lyophilized form for dilution at the point of use, the preparation can contain a lyophilization protectant, for example, a sugar such as trehalose. For oral administration, any of the above-mentioned carriers or solid carriers, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose and magnesium carbonate can be used.

[0242] Thus, the compositions of the invention may include a buffer (e.g., neutral buffered saline or phosphate buffered saline), a sugar (e.g., glucose, mannose, sucrose or dextran), mannitol, a protein, a polypeptide or an amino acid such as glycine, an antioxidant, an antibacterial agent, a complexing agent such as EDTA or glutathione, a solute that makes the formulation isotonic, hypotonic or slightly hypotonic to the blood of the recipient, a suspending agent, a thickening agent and / or a preservative. Alternatively, the compositions of the invention may be formulated as a lyophilized product.

[0243] Immunostimulants

[0244] The compositions of the present invention may also include one or more immunostimulants. An immunostimulant may be any substance that enhances or excites an immune response (antibody and / or cell-mediated) to an exogenous antigen. Examples of immunostimulants often referred to as adjuvants in the context of vaccine formulations include aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; saponins (including QS21), immunostimulatory oligonucleotides such as CPG, oil-in-water emulsions (e.g., wherein the oil is squalene), aminoalkylaminoglucosides 4-phosphate, lipopolysaccharides or their derivatives such as 3-de-O-acylated monophosphate lipid A (3D-MPL ) and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12 and interferon. Therefore, suitably, one or more immunostimulants of the composition of the present invention are selected from aluminum salts, saponins, immunostimulatory oligonucleotides, oil-in-water emulsions, aminoalkyl aminoglucosides 4-phosphates, lipopolysaccharides and derivatives thereof, and other TLR4 ligands, TLR7 ligands, TLR8 ligands and TLR9 ligands. Immunostimulants can 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).

[0245] In the case of recombinant nucleic acid delivery (eg, DNA, RNA, viral vectors), a gene encoding a protein-based immunostimulatory agent can be easily delivered along with a gene encoding a polypeptide of the present invention.

[0246] Sustained Release

[0247] The compositions described herein may be administered as part of a sustained release formulation (ie, a formulation such as a capsule, sponge, paste or gel (eg, composed of a polysaccharide)) that effects slow / sustained release of the compound following administration.

[0248] Storage and packaging

[0249] The compositions of the present invention may be present in unit dose containers or multidose containers (such as sealed ampoules or vials). Such containers are preferably fully sealed to maintain the sterility of the preparation until use. Typically, the preparation may be stored as a suspension, solution or emulsion in an oily or aqueous solvent. Alternatively, the compositions of the present invention may be stored in a freeze-dried state, requiring only the addition of a sterile liquid carrier (such as water or saline for injection) prior to use.

[0250] dose

[0251] The amount of nucleic acid, polypeptide or vector in each composition of the present invention can be prepared in such a way that a suitable dose for therapeutic or prophylactic use will be obtained. Those skilled in the art of preparing such compositions will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life and other pharmacological considerations, and in this regard, a variety of doses and treatment regimens may be desirable.

[0252] Typically, compositions comprising a therapeutically effective amount or a prophylactically effective amount deliver about 0.1 ug to about 1000 ug of a polypeptide of the invention per administration, more typically about 2.5 ug to about 100 ug of a polypeptide of the invention per administration. If delivered in the form of a short, synthetic long peptide, the dose may be 1 to 200 ug / peptide / dose. For polynucleotide compositions, these doses typically deliver about 10 ug to about 20 mg of a nucleic acid of the invention per administration, more typically about 0.1 mg to about 10 mg of a nucleic acid of the invention per administration.

[0253] Disease to be treated or prevented

[0254] As shown elsewhere, SEQ ID NOs. 1-10 are polypeptide sequences corresponding to the CLT antigen overexpressed in cutaneous melanoma.

[0255] In one embodiment, the invention provides a polypeptide, a nucleic acid, a vector or a composition of the invention for use in medicine.

[0256] Other aspects of the invention relate 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.

[0257] The present invention also provides the polypeptide, nucleic acid, vector or composition of the present invention for use in enhancing an immune response in a human.

[0258] Also provided is the use of a polypeptide, nucleic acid, vector or composition of the invention for the manufacture of a medicament for enhancing an immune response in a human.

[0259] Suitably, an immune response is generated against a cancerous tumor that expresses a corresponding sequence selected from SEQ ID NO. 1-10 and variants and immunogenic fragments of any of them. "Corresponding" in this case means that if the tumor expresses SEQ. ID NO. A (A is one of SEQ ID NO. 1-10) or a variant or immunogenic fragment thereof, the polypeptide, nucleic acid, vector or composition of the invention and the medicaments related thereto will be based on SEQ ID NO. A or a variant or immunogenic fragment thereof.

[0260] Suitably, the immune response comprises a CD8+ T cell, a CD4+ T cell and / or an antibody response, in particular a CD8+ cytolytic T-cell response and a CD4+ helper T cell response.

[0261] Suitably, an immune response is generated against a tumour, in particular a tumour expressing a sequence selected from SEQ ID NO. 1-10 and variants thereof and immunogenic fragments thereof.

[0262] In a preferred embodiment, the tumor is a melanoma tumor, such as a cutaneous melanoma tumor.

[0263] The tumor can be a primary tumor or a metastatic tumor.

[0264] Other aspects of the present invention relate to a method for treating a human patient suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO.1-10 and immunogenic fragments and variants of any one thereof, or other aspects of the present invention relate to a method for preventing a human from suffering from cancer, wherein the cancer expresses a sequence selected from SEQ ID NO.1-10 and immunogenic fragments and variants of any one thereof, the method comprising administering to the human a corresponding polypeptide, nucleic acid, vector or composition of the present invention.

[0265] The present invention also provides a polypeptide, nucleic acid, vector or composition of the present invention for treating or preventing cancer in humans, wherein the cancer cells express the corresponding sequence selected from SEQ ID NO. 1-10 and an immunogenic fragment of any one thereof.

[0266] Transcripts corresponding to SEQ ID NOs. 33, 35, 36 and 40 are also overexpressed in uveal melanoma. Thus, in alternative embodiments, the tumor is a uveal melanoma tumor and / or a tumor expressing a sequence selected from SEQ ID NOs.

[0267] Therefore, the present invention provides a method or a polypeptide, a nucleic acid, a vector or a composition for use according to the present invention, wherein the polypeptide comprises a sequence selected from the group consisting of:

[0268] (a) the sequence of any one of SEQ ID NOs. 1, 3, 4, 5 and 10; and

[0269] (b) a variant of the sequence of (a); and

[0270] (c) an immunogenic fragment of the sequence of (a),

[0271] 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 selected from any one of SEQ ID NOs. 41, 43, 44, 45 and 50;

[0272] And wherein the cancer is uveal melanoma.

[0273] The words "prevent" and "preventing" are used interchangeably herein.

[0274] Treatment and vaccination regimen

[0275] The treatment regimen may include simultaneous (e.g., co-administration) or sequential (e.g., priming-boosting) delivery of (i) a polypeptide, nucleic acid or vector of the invention together with (ii) one or more further polypeptides, nucleic acids or vectors of the invention and / or (iii) other components, such as a variety of other therapeutically useful compounds or molecules, such as antigenic proteins, optionally administered simultaneously with an adjuvant. Examples of co-administration include ipsilateral co-administration and contralateral co-administration. "Simultaneous" administration suitably refers to delivery of all components during the same treatment round. Suitably, all components are administered at the same time (e.g., simultaneous administration of DNA and protein), however, a component may be administered within minutes (e.g., at the same medical appointment or doctor's visit) or within hours.

[0276] In some embodiments, the "primary immunization" or first administration of a polypeptide, nucleic acid or vector of the present invention is followed by one or more "boosting" or subsequent administration of a polypeptide, nucleic acid or vector of the present invention ("primary immunization and boosting" method). In one embodiment, the polypeptide, nucleic acid or vector of the present invention is used in a primary immunization-boosting vaccination scheme. In one embodiment, both the primary immunization and the boosting use a polypeptide of the present invention, and the same polypeptide of the present invention is used in each case. In one embodiment, both the primary immunization and the boosting use a nucleic acid or vector of the present invention, and the same nucleic acid or vector of the present invention is used in each case. Alternatively, the nucleic acid or vector of the present invention can be used for primary immunization and the polypeptide of the present invention can be used for boosting, or the polypeptide of the present invention can be used for primary immunization and the nucleic acid or vector of the present invention can be used for boosting. Usually the first or "primary immunization" administration and the second or "boosting" administration are given about 1-12 weeks later or at most 4-6 months later. Subsequent "boosting" administration can be given as frequently as every 1-6 weeks or can be given quite late (up to several years later).

[0277] Antigen combination

[0278] The polypeptides, nucleic acids or vectors of the present invention can be used in combination with one or more other polypeptides or nucleic acids, vectors of the present invention and / or with other antigenic polypeptides (or polynucleotides or vectors encoding them) that induce an immune response to melanoma, such as skin melanoma or uveal melanoma. These other antigenic polypeptides can be derived from a variety of sources, and they may include well-described melanoma-associated antigens, such as GPR143, PRAME, MAGE-A3 or pMel (gp100). Alternatively, they may include other types 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), neoantigens that retain introns (Smart et al., (2018). Nature Biotechnology. http: / / doi.org / 10.1038 / nbt.4239), spliced ​​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 a class called antigens encoding T cell epitopes associated with impaired peptide processing (TIEPPs; Gigoux, M. and Wolchok, J. (2018). JEM, 215, 2233; Marijt et al., (2018). JEM 215, 2325) or new antigens to be discovered (including CLT antigens). In addition, antigenic peptides from these diverse sources may also be combined with (i) non-specific immunostimulatory agents / adjuvants and / or (ii) antigens, such as universal CD4 helper epitopes (delivered as polypeptides or as polynucleotides or vectors encoding these CD4 antigens) that are known to stimulate strong CD4 helper T cells to amplify the anti-melanoma specific response stimulated by the co-administered antigens.

[0279] The different polypeptides, nucleic acids or vectors may be formulated in the same preparation or in separate preparations. Alternatively, the polypeptide may be provided as a fusion protein in which a polypeptide of the invention is fused to a second or further polypeptide (see below).

[0280] Nucleic acids encoding the aforementioned fusion proteins may be provided.

[0281] More generally, when two or more components are used in combination, these components may be present, for example, as follows:

[0282] (1) as two or more independent antigenic polypeptide components;

[0283] (2) as a fusion protein comprising two (or other) polypeptide components;

[0284] (3) as one or more polypeptide and one or more polynucleotide components;

[0285] (4) as two or more independent polynucleotide components;

[0286] (5) as a single polynucleotide encoding two or more independent polypeptide components; or

[0287] (6) As a single polynucleotide encoding a fusion protein comprising two (or other) polypeptide components.

[0288] For convenience, when multiple components are present, it is often desirable that they be contained within a single fusion protein or a polynucleotide encoding a single fusion protein (see below). In one embodiment of the invention, all components are provided as polypeptides (e.g., within a single fusion protein). In an alternative embodiment of the invention, all components are provided as polynucleotides (e.g., a single polynucleotide, such as that encoding a single fusion protein).

[0289] Fusion protein (fusion peptide)

[0290] As an embodiment of the above antigen combination discussion, the present invention also provides isolated polypeptides of the present invention, which are fused to the second or other polypeptides of the present invention (hereinafter referred to as "combination polypeptides of the present invention") by producing a nucleic acid construct that fuses the sequences encoding independent antigens together. The combination polypeptides of the present invention are expected to have the uses described herein for the polypeptides of the present invention, and may have the advantages of excellent immunogenic activity or vaccine activity or preventive or therapeutic effects (including increasing the breadth and depth of the response), and may be particularly valuable in outbred populations. The fusions of the polypeptides of the present invention can also provide benefits: increasing the efficiency of constructing and manufacturing vaccine antigens and / or guided vaccines (including nucleic acid vaccines).

[0291] As described above in the antigen combination section, the polypeptides of the present invention and the combination polypeptides of the present invention may also be fused to a polypeptide sequence that is not a polypeptide of the present invention, said polypeptide sequence comprising one or more of the following:

[0292] (a) other polypeptides that are melanoma-associated antigens and therefore potentially useful as immunogenic sequences in vaccines (e.g., GPR143, PRAME, MAGE-A3 and pMel (gp100), see above); and

[0293] (b) polypeptide sequences capable of enhancing the immune response (i.e., immunostimulatory sequences).

[0294] (c) A polypeptide sequence capable of providing strong CD4+ help to increase the CD8+ T cell response to CLT antigen epitopes, for example comprising a universal CD4 helper epitope.

[0295] Exemplary fusion polypeptides comprise two or more (eg, two, three or four) sequences selected from SEQ ID NO. 1, 2, 3 and 4; or variants of the sequences or immunogenic fragments of the sequences relative to each of the sequences.

[0296] Exemplary fusion polypeptides include:

[0297] (i) a sequence selected from the group consisting of:

[0298] (a) the sequence of SEQ ID NO. 1; and

[0299] (b) a variant of the sequence of (a); and

[0300] (c) an immunogenic fragment of the sequence of (a), for example selected from SEQ ID NOs. 11-14, 55-57 and 73-74; and

[0301] (ii) a sequence selected from the group consisting of:

[0302] (a) the sequence of SEQ ID NO. 2; and

[0303] (b) a variant of the sequence of (a); and

[0304] (c) an immunogenic fragment of the sequence of (a), for example selected from SEQ ID NOs. 15-16, 58-66, 75 and 78; and

[0305] (iii) a sequence selected from the group consisting of:

[0306] (a) the sequence of SEQ ID NO. 3; and

[0307] (b) a variant of the sequence of (a); and

[0308] (c) an immunogenic fragment of the sequence of (a), for example selected from SEQ ID NOs. 17-18, 53 and 67-69; and

[0309] (iv) a sequence selected from the group consisting of:

[0310] (a) the sequence of SEQ ID NO. 4; and

[0311] (b) a variant of the sequence of (a); and

[0312] (c) An immunogenic fragment of the sequence of (a), for example, selected from SEQ ID NOs. 19, 51-52, 54, 70-72 and 76-77.

[0313] For example, the fusion polypeptide comprises the sequences of SEQ ID NOs. 1, 2, 3 and 4.

[0314] Another exemplary fusion polypeptide includes:

[0315] (i) a sequence selected from the group consisting of:

[0316] (a) the sequence of SEQ ID NO. 1; and

[0317] (b) a variant of the sequence of (a); and

[0318] (c) an immunogenic fragment of the sequence of (a), for example selected from SEQ ID NOs. 11-14, 55-57 and 73-74; and

[0319] (ii) a sequence selected from the group consisting of:

[0320] (a) the sequence of SEQ ID NO. 2; and

[0321] (b) a variant of the sequence of (a); and

[0322] (c) an immunogenic fragment of the sequence of (a), for example selected from SEQ ID NOs. 15-16, 58-66, 75 and 78; and

[0323] (iii) a sequence selected from the group consisting of:

[0324] (a) the sequence of SEQ ID NO. 4; and

[0325] (b) a variant of the sequence of (a); and

[0326] (c) An immunogenic fragment of the sequence of (a), for example, selected from SEQ ID NOs. 19, 51-52, 54, 70-72 and 76-77.

[0327] For example, the fusion polypeptide comprises the sequences of SEQ ID NOs. 1, 2 and 4.

[0328] The present invention also provides nucleic acids encoding the aforementioned fusion polypeptides and other aspects of the present invention (vectors, compositions, cells, etc.) mutatis mutandis with respect to the polypeptides of the present invention.

[0329] CLT antigen binding polypeptide

[0330] Antigen-binding polypeptides with immunological specificity for tumor-expressed antigens (polypeptides of the present invention) can be designed to recruit cytolytic cells to antigen-modified tumor cells to mediate the destruction of the latter. Such a mechanism of recruiting cytolytic cells by antigen-binding polypeptides is called antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, the present invention provides antigen-binding polypeptides with immunological specificity for polypeptides of the present invention. Antigen-binding polypeptides include antibodies that can be produced in non-human animal species (e.g., rodents or camelids) and humanized or can be produced in non-human species (e.g., rodents genetically modified to have a human immune system), such as monoclonal antibodies and fragments thereof, such as domain antibodies, Fab fragments, Fv fragments, and VHH fragments.

[0331] Antigen-binding polypeptides can be produced by methods well known to the skilled artisan. For example, monoclonal antibodies can be produced using hybridoma technology by fusing B cells that produce specific antibodies with myeloma (B cell cancer) cells that are selected for their ability to grow in tissue culture and the absence of antibody chain synthesis. and Milstein, 1975, Nature 256(5517):495-497 and Nelson and, 2000 (Jun), Mol Pathol. 53(3):111-7, which are incorporated herein by reference in their entireties).

[0332] Monoclonal antibodies directed against a defined antigen can be obtained, for example, by:

[0333] a) immortalizing lymphocytes obtained from the peripheral blood of animals (including humans) previously immunized / exposed to defined antigens, immortalized cells and preferably immortalized cells with myeloma cells, with the aim of forming hybridomas,

[0334] b) Culturing the resulting immortalized cells (hybridomas) and recovering cells producing antibodies with the desired specificity.

[0335] The monoclonal antibody can be obtained by the following method, which comprises the steps of:

[0336] a) cloning into a vector, in particular a bacteriophage and more particularly a filamentous phage, a DNA or cDNA sequence obtained from lymphocytes, in particular peripheral blood lymphocytes, of an animal (suitably previously immunized with a defined antigen),

[0337] b) transforming prokaryotic cells with the above-mentioned vector under conditions that allow the production of antibodies,

[0338] c) selecting the antibody by subjecting the antibody to antigen-affinity selection,

[0339] d) Recovering antibodies with desired specificity

[0340] e) expressing a nucleic acid molecule encoding the antibody obtained from B cells of a patient exposed to the antigen or of an animal experimentally immunized with the antigen.

[0341] The selected antibodies can then be produced using conventional recombinant protein production techniques (eg, from genetically engineered CHO cells).

[0342] The present invention provides an isolated antigen-binding polypeptide immunospecific for a polypeptide of the present invention. Suitably, the antigen-binding polypeptide is a monoclonal antibody or a fragment thereof.

[0343] In certain embodiments, the antigen binding polypeptide is coupled to a cytotoxic moiety. Example cytotoxic moieties include the Fc domain of an antibody, which will recruit cells carrying Fc receptors that promote ADCC. Alternatively, the antigen binding polypeptide can be linked to a biological toxin or a cytotoxic chemical.

[0344] Another important category of antigen-binding polypeptides includes T cell receptor (TCR) derived molecules that bind to HLA display fragments of the antigens of the present invention. In this embodiment, TCR-based biologics (including TCRs derived directly from patients, or high-affinity TCRs operated specifically) that recognize CLT antigens (or derivatives thereof) on the surface of tumor cells can also include a guide portion that recognizes components on T cells (or another category of immune cells) that attract these immune cells to the tumor, thereby providing a therapeutic benefit. In some embodiments, the guide portion can also stimulate the beneficial activities (including cytolytic activity) of the re-directed immune cells.

[0345] Thus, in one embodiment, the antigen binding polypeptide is immunospecific for an HLA-bound polypeptide that is a polypeptide of the invention or a portion thereof. For example, the antigen binding polypeptide is a T cell receptor.

[0346] In one embodiment, the antigen binding polypeptides of the invention may be coupled to another polypeptide capable of binding to cytotoxic cells or other immune components in a subject.

[0347] In one embodiment, the antigen binding polypeptide is for use in medicine.

[0348] In one embodiment, a pharmaceutical composition comprising an antigen-binding polypeptide of the invention together with a pharmaceutically acceptable carrier is provided. Such a composition may be a sterile composition suitable for parenteral administration. See, for example, the disclosure of pharmaceutical compositions above.

[0349] The present invention provides a method for treating a human suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO.1-10 and immunogenic fragments and variants of any one thereof, or the present invention provides a method for preventing a human from suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO.1-10 and immunogenic fragments and variants of any one thereof, the method comprising administering an antigen-binding polypeptide or a composition comprising the antigen-binding polypeptide of the present invention to the human.

[0350] In one embodiment, an antigen-binding polypeptide of the present invention is provided, which can be coupled to a cytotoxic portion, or a composition comprising the antigen-binding polypeptide of the present invention is provided for use in treating or preventing cancer in humans, wherein the cancer cells express the corresponding sequences selected from SEQ ID NO.1-10 and immunogenic fragments of any one of them.

[0351] Suitably, in any of the above embodiments, the cancer is melanoma, particularly cutaneous melanoma.

[0352] In one embodiment, a method or antigen-binding polypeptide or composition for use according to the invention is provided, wherein the polypeptide comprises a sequence selected from the group consisting of:

[0353] (a) the sequence of any one of SEQ ID NOs. 1, 3, 4, 5 and 10; and

[0354] (b) a variant of the sequence of (a); and

[0355] (c) Immunogenic fragments of the sequence of (a).

[0356] 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 selected from any one of SEQ ID NOs. 41, 43, 44, 45 and 50;

[0357] And wherein the cancer is uveal melanoma.

[0358] The antigen binding polypeptide (such as an antibody or fragment thereof) may be administered at a dose of, for example, 5-1000 mg, such as 25-500 mg, such as 100-300 mg, such as about 200 mg.

[0359] Cell therapy to enhance antigen presentation in vivo

[0360] Any of a variety of cell delivery vehicles can be used inside a pharmaceutical composition to promote the production of an antigen-specific immune response. Therefore, the present invention provides a cell, which is a separated antigen-presenting cell, which is loaded with a polypeptide of the present invention by ex vivo modification or genetically engineered to express a polypeptide of the present invention (hereinafter referred to as "APC of the present invention"). Antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells can be engineered to become efficient APCs. Such cells can, but not necessarily, be genetically modified to increase antigen presenting capacity to improve the activation and / or maintenance of T-cell responses and / or be immunologically compatible with the recipient (i.e., HLA haplotype matching). APCs can be generally isolated from any of a variety of biological fluids and organs, and can be autologous, allogeneic, isogenic or xenogeneic cells.

[0361] Certain preferred embodiments of the present invention use dendritic cells or their progenitor cells as APCs. Thus, in one embodiment, the APCs of the present invention are dendritic cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, 1998, Nature, 392: 245-251) and have been shown to be effective as physiological adjuvants for stimulating prophylactic or therapeutic immunity (see Timmerman and Levy, 1999, Ann. Rev. Med. 50: 507-529). In general, dendritic cells can be identified based on their typical shape (star-shaped in situ, with obvious cytoplasmic processes visible in vitro), their ability to efficiently take up, process and present antigens, and their ability to activate initial T-cell responses. Of course, dendritic cells can be engineered to express specific cell surface receptors or ligands that are not normally present on dendritic cells in vivo or in vitro, and the present invention contemplates such modified dendritic cells. As an alternative to dendritic cells, antigen-loaded secretory vesicles (called exosomes) can be used within immunogenic compositions (see Zitvogel et al., 1998, Nature Med. 4:594-600). Thus, in one embodiment, exosomes loaded with a polypeptide of the invention are provided.

[0362] Dendritic cells and progenitor cells can be obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood, or any other suitable tissue or fluid. For example, dendritic cells can be differentiated in vitro by adding a combination of cytokines (such as GM-CSF, IL-4, IL-13, and / or TNFα) to a culture of mononuclear cells harvested from peripheral blood. Alternatively, CD34-positive cells harvested from peripheral blood, umbilical cord blood, or bone marrow can be differentiated into dendritic cells by adding a combination of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and / or other compounds that induce differentiation, maturation, and proliferation of dendritic cells to the culture medium.

[0363] Dendritic cells are conveniently classified into "immature" cells and "mature" cells, which brings a simple way to distinguish 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 are associated with high expression of Fcγ receptors and mannose receptors. The mature phenotype is generally characterized by low expression of these markers, but high expression of cell surface molecules responsible for T cell activation, such as class I and class II MHC molecules, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

[0364] APCs can also be genetically engineered, for example, by transfection with a polynucleotide encoding a protein (or a portion or other variant thereof) such that the polypeptide is expressed on the cell surface. Such transfection can be performed ex vivo, and a pharmaceutical composition comprising such transfected cells can then be used as described herein. Alternatively, a gene delivery vehicle targeting dendritic cells or other antigen presenting cells can be administered to a patient, resulting in transfection occurring in vivo. For example, dendritic cells can be transfected in vivo and ex vivo using any method known in the art, such as those described in WO 97 / 24447 or the gene gun approach described in Mahvi et al., 1997, Immunology and Cell Biology 75: 456-460. Antigen loading of dendritic cells can be achieved by incubating dendritic cells or progenitor cells with polypeptides, DNA (e.g., plasmid vectors) or RNA; or with recombinant bacteria or viruses expressing the antigen (e.g., adenovirus, adeno-associated virus (AAV) (e.g., AAV types 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 (e.g., modified vaccinia Ankara virus (MVA) or fowlpox virus), paramyxovirus, lentivirus, or rhabdovirus (e.g., vesicular stomatitis virus (VSV)). Prior to polypeptide loading, the polypeptide can be covalently conjugated to an immunizing partner (e.g., a carrier molecule) that provides T cell help. Alternatively, dendritic cells can be pulse-labeled with an unconjugated immunizing partner, either independently or in the presence of the polypeptide or carrier.

[0365] The present invention provides a fragment of a specially designed chemically synthesized encoding epitope for delivering polypeptide antigens to antigen presenting cells. It will be appreciated by those skilled in the art that these types of molecules, also known as synthetic long peptides (SLPs), provide therapeutic platforms to stimulate (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 polypeptide antigens into antigen presenting cells in vivo (Melief and van der Burg, 2008, Nat Rev Cancer, 8: 351-60).

[0366] In one embodiment, a pharmaceutical composition comprising an antigen presenting cell of the invention (which is suitably a dendritic cell) together with a pharmaceutically acceptable carrier is provided. This composition may be a sterile composition suitable for parenteral administration. See, for example, the disclosure of pharmaceutical compositions above.

[0367] In one embodiment, an antigen presenting cell of the invention, suitably a dendritic cell, is provided for use in medicine.

[0368] Also provided is a method for treating a human suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, or a method for preventing a human from suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, the method comprising administering to the human the antigen-presenting cell of the present invention, which is suitably a dendritic cell, or a composition comprising the antigen-presenting cell of the present invention.

[0369] In one embodiment, an antigen-presenting cell of the invention, suitably a dendritic cell, or a composition comprising the antigen-presenting cell of the invention is provided for use in treating or preventing cancer in humans, wherein the cancer cells express the corresponding sequences selected from SEQ ID NO. 1-10 and immunogenic fragments of any one thereof.

[0370] In one embodiment, a pharmaceutical composition comprising the exosomes of the invention together with a pharmaceutically acceptable carrier is provided. Such a composition may be a sterile composition suitable for parenteral administration. See, e.g., the disclosure of pharmaceutical compositions above. The composition may optionally comprise an immunostimulant - see the disclosure of immunostimulants above.

[0371] In one embodiment, the exosomes of the invention are provided for use in medicine.

[0372] Also provided is a method for treating a human suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, or a method for preventing a human from suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, the method comprising administering to the human an exosome of the present invention or a composition comprising the exosome of the present invention.

[0373] In one embodiment, the exosomes of the invention or compositions comprising the exosomes of the invention are provided for use in treating or preventing cancer in humans, wherein the cells of the cancer express the corresponding sequences selected from SEQ ID NO. 1-10 and immunogenic fragments of any of them. In any of the above embodiments, the cancer is suitably melanoma, in particular skin melanoma.

[0374] Stimulated T cell therapy

[0375] In addition to APC-mediated in vivo or ex vivo generation of T cells immunospecific for a polypeptide of the invention, autologous or non-autologous T cells can be isolated from a subject, e.g., from peripheral blood, umbilical cord blood, and / or by apheresis, and stimulated in the presence of a tumor-associated antigen loaded on an APC cell MHC molecule (signal 1) to induce proliferation of T cells carrying a TCR immunospecific for such an antigen.

[0376] Successful T cell activation requires binding to the co-stimulatory surface molecules B7 and CD28 (signal 2) on antigen presenting cells and T cells, respectively. Signals 1 and 2 are required to achieve optimal T cell activation. In turn, antigenic peptide stimulation (signal 1) in the absence of co-stimulation (signal 2) cannot induce complete T cell activation and may lead to T cell tolerance. In addition to co-stimulatory molecules, there are also inhibitory molecules such as CTLA-4 and PD-1 that induce signals that prevent T cell activation.

[0377] Autologous or non-autologous T cells can therefore be stimulated in the presence of the polypeptides of the present invention, and expanded and transferred to a patient at risk of or suffering from cancer whose cancer cells express the corresponding polypeptides of the present invention, provided that the antigen-specific TCR will recognize the antigen presented by the patient's MHC, where they will target the cancer cells expressing the corresponding polypeptide and induce killing thereof.

[0378] In one embodiment, a polypeptide, nucleic acid, vector or composition of the invention is provided for use in ex vivo stimulation and / or expansion of T cells derived from a human suffering from cancer, so as to subsequently reintroduce the stimulated and / or expanded T cells into the human to treat the cancer in the human.

[0379] The present invention provides a method for treating cancer in humans, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one of them, which comprises obtaining a population of leukocytes comprising at least T cells, optionally together with antigen presenting cells, from the human, stimulating and / or expanding the T cells in the presence of the corresponding polypeptide, nucleic acid, vector or composition of the present invention, and reintroducing some or all of the leukocytes into the human, which comprises at least the stimulated and / or expanded T cells.

[0380] In any of the above embodiments, the cancer is suitably a melanoma, in particular a cutaneous melanoma.

[0381] In one embodiment, a method is provided for preparing a T cell population that is cytotoxic to cancer cells, wherein the cancer cells express a sequence selected from SEQ ID NO.1-10 and immunogenic fragments and variants of any one of them, the method comprising (a) obtaining T cells and antigen presenting cells from a cancer patient; and (ii) stimulating and expanding the T cell population ex vivo with the corresponding polypeptide, nucleic acid, vector or composition of the present invention.

[0382] "Corresponding" in this case means that if the cancer cells express SEQ ID NO. A (A is one of SEQ ID NO. 1-10) or a variant or immunogenic fragment thereof, then SEQ ID NO. A or a variant or immunogenic fragment thereof in the form of a polypeptide, nucleic acid or vector or a composition containing one of the foregoing stimulates and expands the T cell population in vitro.

[0383] For example, in such methods, the culture and expansion are carried out in the presence of dendritic cells. Dendritic cells can be transfected with the nucleic acid molecules or vectors of the present invention and express the polypeptides of the present invention.

[0384] The present invention provides a T cell population obtainable by any of the aforementioned methods (hereinafter referred to as the T cell population of the present invention).

[0385] In one embodiment, a cell is provided, which is a T cell that has been stimulated with the polypeptide, nucleic acid, vector or composition of the present invention (hereinafter referred to as the T cell of the present invention).

[0386] In one embodiment, a pharmaceutical composition comprising a T cell population or T cells of the invention together with a pharmaceutically acceptable carrier is provided. Such a composition may, for example, be a sterile composition suitable for parenteral administration.

[0387] In one embodiment, a T cell population or T cell of the invention is provided for use in medicine.

[0388] Also provided is a method for treating a human suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO.1-10 and immunogenic fragments and variants of any one of them, or a method for preventing a human from suffering from cancer, wherein the cancer cells express a sequence selected from SEQ ID NO.1-10 and immunogenic fragments and variants of any one of them, the method comprising administering to the human the T cell population or T cells of the present invention or a composition of the T cell population or T cells of the present invention.

[0389] In one embodiment, a T cell population of the invention, a T cell of the invention or a composition comprising the T cell population or T cell of the invention is provided for use in treating or preventing cancer in humans, wherein the cells of the cancer express an immunogenic fragment of a corresponding sequence selected from SEQ ID NO. 1-10 and any of them. In any of the above embodiments, the cancer is suitably a melanoma, in particular a skin melanoma.

[0390] In one embodiment, a method or a T cell population, a T cell, an antigen presenting cell, an exosome or a composition for use according to the invention is provided, wherein the polypeptide comprises a sequence selected from the group consisting of:

[0391] (a) the sequence of any one of SEQ ID NOs. 1, 3, 4, 5 and 10; and

[0392] (b) a variant of the sequence of (a); and

[0393] (c) Immunogenic fragments of the sequence of (a).

[0394] 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 selected from any one of SEQ ID NOs. 41, 43, 44, 45 and 50;

[0395] And wherein the cancer is uveal melanoma.

[0396] Engineered immune cell therapy

[0397] Derivatives of all types of CLT antigen-binding polypeptides described above, including TCRs or TCR mimetics that recognize CLT antigen-derived peptides complexed with human HLA molecules (see Dubrovsky et al., 2016, Oncoimmunology), can be engineered to be expressed on the surface of (autologous or non-autologous) T cells, which can then be administered as adoptive T cell therapy for the treatment of cancer.

[0398] These derivatives belong to the "chimeric antigen receptor (CAR)" category, wherein as used herein, the chimeric antigen receptor may refer, for example, to an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor and encompasses an engineered receptor that transplants artificial specificity to a specific immune effector cell. CAR can be used to confer the specificity of monoclonal antibodies to T cells, thereby allowing numerous specific T cells to be generated, for example, for adoptive cell therapy. CAR can direct the specificity of cells for tumor-associated antigens (polypeptides of the present invention), wherein the polypeptide is HLA-bound.

[0399] Another approach to treating cancer in patients is to genetically modify T cells to target antigens expressed on tumor cells by expressing chimeric antigen receptors (CARs). This technology is reviewed in Wendell and June, 2017, Cell, 168: 724-740 (incorporated by reference in its entirety).

[0400] Such CART cells can be produced by the following method: a sample of cells is obtained from a subject (e.g., from peripheral blood, cord blood and / or by apheresis), wherein the sample comprises T cells or T cell progenitor cells, and the cells are transfected with nucleic acids encoding a chimeric T cell receptor (CAR) having immunospecificity for the polypeptide of the present invention, wherein the polypeptide is HLA-bound. This nucleic acid will be able to be integrated into the genome of the cell, and the cell can be administered to the subject in an effective amount to provide a T cell response for cells expressing the polypeptide of the present invention. For example, a sample of cells from a subject can be collected.

[0401] It will be appreciated that the cells used to generate the CAR-expressing T cells may be autologous or non-autologous.

[0402] The transgenic T cells expressing the CAR can have inactivated endogenous T cell receptor and / or endogenous HLA expression. For example, the cells can be engineered to eliminate endogenous α / β T cell receptor (TCR) expression.

[0403] Methods for transfecting cells are well known in the art, but highly efficient transfection methods such as electroporation can be used. For example, a nuclear transfection device can be used to introduce nucleic acids or vectors of the invention expressing CAR constructs into cells.

[0404] The cell population of T cells expressing CAR can be enriched after transfection of cells. For example, cells expressing CAR can be separated from those cells that do not express (e.g., by FACS) by using an antigen bound by CAR or CAR binding antibody. Alternatively, the enrichment step includes depleting non-T cells or depleting cells lacking CAR expression. For example, CD56+ cells can be depleted from the culture population.

[0405] The population of transgenic cells expressing CAR can be cultured ex vivo in a medium that selectively enhances the proliferation of CAR-expressing T cells. Thus, CAR-expressing T cells can be expanded ex vivo.

[0406] Samples of CAR cells can be stored (or maintained in culture). For example, samples can be stored frozen for later expansion or analysis.

[0407] CAR-expressing T cells can be used in combination with other therapeutic agents, such as checkpoint inhibitors, including PD-L1 antagonists.

[0408] In one embodiment, there is provided a cytotoxic cell which has been engineered to express any of the aforementioned antigen binding polypeptides on its surface. Suitably, the cytotoxic cell is a T cell.

[0409] In one embodiment, there is provided a cytotoxic cell for use in medicine, suitably a T cell, engineered to express any of the aforementioned antigen binding polypeptides on its surface.

[0410] The present invention provides a pharmaceutical composition comprising a cytotoxic cell of the present invention, suitably a T cell.

[0411] A method of treating a human patient suffering from cancer, wherein the cells of the cancer express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one of them, or a method of preventing a human from suffering from cancer, wherein the cancer expresses a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one of them, is provided, the method comprising administering to the human a cytotoxic cell of the invention, which is suitably a T cell.

[0412] In one embodiment, the cytotoxic cells of the invention, suitably T cells, are used to treat or prevent cancer in humans, wherein the cancer cells express the corresponding sequences selected from SEQ ID NO. 1-10 and immunogenic fragments of any of them.

[0413] Combination therapy

[0414] The cancer treatment methods of the present invention can be performed in combination with other therapies, particularly checkpoint inhibitors and interferons.

[0415] Peptides, nucleic acids, vectors, antigen-binding polypeptides and adoptive cell therapies (APC- and T-cell-based) can be used in combination with other components to enhance their immunogenicity, e.g., to improve the magnitude and / or breadth of the immune response elicited or to provide other activities (e.g., to activate other aspects of the innate or adaptive immune response or to destroy tumor cells).

[0416] Thus, the present invention provides a composition of the invention (i.e., an immunogenic composition, a vaccine composition, or a pharmaceutical composition) or a kit of several such compositions, comprising a polypeptide, a nucleic acid, or a vector of the invention together with a pharmaceutically acceptable carrier, and (i) one or more other immunogenic polypeptides or immunostimulatory polypeptides (e.g., interferon, IL-12, a checkpoint blocking molecule, or a nucleic acid encoding the former, or a vector comprising such a nucleic acid), (ii) a small molecule (e.g., an HDAC inhibitor or other drug that modulates the epigenetic characteristics of cancer cells) or a biologic (delivered as a polypeptide or a nucleic acid encoding the former, or a vector comprising such a nucleic acid) that enhances the translation and / or presentation of a polypeptide product that is the subject of the invention.

[0417] Checkpoint inhibitor proteins, which block normal proteins on cancer cells or on T cells that respond to those proteins, may be a particularly important class of drugs to combine with CLT antigen-based therapies because these inhibitors seek to overcome one of cancer's main defenses against immune system attack.

[0418] Therefore, one aspect of the present invention includes administering a polypeptide of the present invention, a nucleic acid, a vector, an antigen-binding polypeptide, a composition, a T cell, a T cell colony, or an antigen presenting cell in combination with a checkpoint inhibitor protein. Examples of checkpoint inhibitor proteins are selected from PD-1 inhibitors such as pembrolizumab (Keytruda) and nivolumab (Opdivo), PD-L1 inhibitors such as atezolizumab (Tecentriq), avelumab (Bavencio) and durvalumab (Imfinzi) and CTLA-4 inhibitors such as ipilimumab (Yervoy).

[0419] Interferons (e.g., α, β, and γ) are a family of proteins produced by the body in very small amounts. Interferons can slow 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 generally administered as subcutaneous injections, such as in the thigh or abdomen.

[0420] Thus, one aspect of the invention includes administering a polypeptide, nucleic acid, vector, antigen-binding polypeptide or composition of the invention in combination with an interferon, such as interferon alpha.

[0421] The different modes of the invention may also be combined, for example the polypeptides, nucleic acids and vectors of the invention may be combined with the APCs, T cells or T cell populations of the invention (particularly discussed).

[0422] One or more modalities of the present invention may also be combined with conventional anti-cancer chemotherapy and / or irradiation.

[0423] Diagnostic drugs

[0424] In another aspect, the invention provides methods of using one or more polypeptides or nucleic acids of the invention to diagnose cancer, particularly melanoma, e.g., cutaneous melanoma, or to diagnose a human subject suitable for treatment with a polypeptide, nucleic acid, vector, antigen-binding polypeptide, adoptive cell therapy or composition of the invention.

[0425] Therefore, the present invention provides a method for diagnosing a person as having cancer, the method comprising the steps of determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NO.1-10 and an immunogenic fragment or variant of any one thereof (for example, selected from sequences SEQ ID NO.11-32 and 51-78), or a nucleic acid encoding the polypeptide sequence (for example, selected from sequences SEQ ID NO.33-40 and SEQ ID NO.41-50), and if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, diagnosing the person as having cancer.

[0426] The present invention provides a method for diagnosing a person as having cancer as skin melanoma, the method comprising the steps of determining whether cells of the cancer express a polypeptide sequence selected from SEQ ID NO. 2, 6, 7, 8 and 9 and immunogenic fragments or variants thereof; or a nucleic acid encoding the polypeptide sequence, and if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, diagnosing the person as having cancer as skin melanoma.

[0427] As used herein, "overexpression" in cancer cells means that the expression level in cancer cells is higher than that in normal cells.

[0428] The present invention provides a method for diagnosing a person as having cancer as skin melanoma or uveal melanoma, the method comprising the steps of determining whether cells of the cancer express a polypeptide sequence selected from any one of SEQ ID NO. 1, 3, 4, 5 and 10 and an immunogenic fragment or variant of any one of them; or a nucleic acid encoding the polypeptide sequence, and if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, diagnosing the person as having cancer as skin melanoma or uveal melanoma.

[0429] Overexpression can be determined by reference to the levels of nucleic acids or polypeptides of the present invention in control human subjects known not to suffer from cancer. Thus, overexpression means that the nucleic acids or polypeptides of the present invention are detected in the test subject at significantly higher levels (e.g., 30%, 50%, 100% or 500% higher levels) than in the control subjects. If the control human subjects have undetectable levels of nucleic acids or polypeptides of the present invention, diagnosis can be achieved by detecting the nucleic acids or polypeptides of the present invention.

[0430] The present invention also provides a method of treating a human suffering from cancer, the method comprising the steps of:

[0431] (a) determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NO.1-10 and an immunogenic fragment or variant thereof (e.g., selected from SEQ ID NO.11-32 and 51-78) or a nucleic acid encoding the polypeptide sequence (e.g., selected from SEQ ID NO.33-40 and 41-50); and if expressed,

[0432] (b) administering to the human a corresponding peptide, nucleic acid, vector, composition, T cell population, T cell, antigen presenting cell, antigen binding polypeptide or cytotoxic cell of the present invention.

[0433] Also provided is the use of a polypeptide comprising a sequence selected from the group consisting of:

[0434] (a) the sequence of any one of SEQ ID NOs. 1-10; or

[0435] (b) a variant of the sequence of (a); and

[0436] (c) Use of an immunogenic fragment of the sequence of (a), isolated from a tumor of a human suffering from cancer, or a nucleic acid encoding the polypeptide, as a biomarker for determining whether the human will be suitable for treatment with a vaccine comprising the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cell, antigen presenting cell, antigen binding polypeptide or cytotoxic cell of the present invention.

[0437] Suitably, the cancer is melanoma, in particular cutaneous melanoma.

[0438] The present invention also provides a method or use according to the present invention, wherein the polypeptide comprises a sequence selected from the group consisting of:

[0439] (a) the sequence of any one of SEQ ID NOs. 1, 3, 4, 5 and 10; and

[0440] (b) a variant of the sequence of (a); and

[0441] (c) Immunogenic fragments of the sequence of (a).

[0442] 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 selected from any one of SEQ ID NOs. 41, 43, 44, 45 and 50;

[0443] And wherein the cancer is uveal melanoma.

[0444] Suitably, the polypeptide of the invention has a sequence selected from SEQ ID NO. 1-10 or a fragment thereof, such as an immunogenic fragment thereof (eg, a sequence selected from SEQ ID NO. 11-32 and 51-78).

[0445] Suitably, 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 thereof (such as an immunogenic fragment thereof).

[0446] Kits for detecting the presence of nucleic acids are well known. For example, a kit comprising at least two oligonucleotides that hybridize to a polynucleotide can be used within a real-time PCR (RT-PCR) reaction to allow for the detection and semi-quantification of specific nucleic acids. Such kits can allow for the detection of nucleic acids based on Forster resonance energy transfer (FRET) (e.g., TaqMan Kit) or when double-stranded DNA binds (e.g., SYBR Green kit) to detect PCR products. Some kits (e.g., TaqMan Kits that contain probes (such as those that contain probes) allow for the detection and quantification of mRNA (e.g., transcripts encoding nucleic acids of the invention). Assays using certain kits can be set up in a multiplex format for simultaneous detection of multiple nucleic acids within a reaction. Kits that detect active DNA (i.e., DNA that carries specific epigenetic signatures that indicate expression) can also be used. Additional components that may be present within such kits include diagnostic agents or reporter molecules that facilitate detection of nucleic acids of the invention.

[0447] The nucleic acids of the present invention can also be detected by liquid biopsy using a blood sample from a patient. This method provides a non-invasive alternative to surgical biopsy. The plasma from such a blood sample can be separated and analyzed for the presence of the nucleic acids of the present invention.

[0448] The polypeptides of the present invention can be detected in an ELISA-type assay that detects the polypeptides of the present invention in homogenized preparations of patient tumor samples with the aid of antigen-specific antibodies. Alternatively, the polypeptides of the present invention can be detected with the aid of an immunohistochemical assay that identifies the presence of the polypeptide antigen by examining sections of patient tumor samples using optical microscopy, wherein the sections have been stained with an antibody preparation using a suitable label. As yet another alternative, the polypeptides of the present invention can be detected with the aid of an immunohistochemical assay that identifies the presence of the polypeptide antigen by examining sections of patient tumor samples using optical microscopy, wherein the sections have been stained with an antibody preparation using a suitable label.

[0449] The polypeptides of the invention may also be tested by determining whether they are able to stimulate the production of T cells directed against the polypeptide.

[0450] Cells of a cancer or tumor (eg, a melanoma, such as a skin melanoma) can be obtained, for example, from a biopsy of a cancer (eg, a melanoma, such as a skin melanoma).

[0451] A method of treating cancer, particularly melanoma, e.g., skin melanoma, in a human comprises (i) detecting the presence of a nucleic acid or polypeptide of the invention and (ii) administering to the subject a nucleic acid, polypeptide, vector, cell, T cell or T cell population or composition of the invention (and preferably administering the same nucleic acid or polypeptide or fragment thereof that has been detected).

[0452] A method of treating cancer in humans, in particular melanoma, e.g., cutaneous melanoma, further comprises administering a nucleic acid, polypeptide, vector, cell, T cell or T cell population or composition of the invention to a subject in which the presence of a nucleic acid or polypeptide of the invention (and preferably the same) has been detected.

[0453] In particular, the cancer to be diagnosed and, if appropriate, treated is melanoma, such as cutaneous melanoma.

[0454] If the polypeptide of the present invention of SEQ ID NO. 1, 3, 4, 5 or 10 or a fragment thereof is detected, the cancer may be cutaneous melanoma or uveal melanoma.

[0455] Specific implementation plan

[0456] 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 NO.11-14. Other exemplary fragments comprise two, three or four of SEQ ID NO.11-14. Other exemplary fragments comprise or consist of any one of SEQ ID NO.55-57 or 73-74. Other exemplary fragments comprise or consist of all of SEQ ID NO.11-14, 55-57 and 73-74 (allowing for possible sequence overlap, so that any overlapping sequence does not need to exist more than once). Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO 33 or SEQ ID NO.41. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes as described above are provided. The nucleic acids (eg, DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen binding polypeptides, antigen presenting cells and exosomes can be used to treat cancer, particularly melanoma, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0457] 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. Other exemplary fragments comprise SEQ ID NO.15 and SEQ ID NO.16. Other exemplary fragments comprise or consist of any one of SEQ ID NO.58-66, 75 and 78. Other exemplary fragments comprise all of SEQ ID NO.15-16, 58-66, 75 and 78 (allowing for possible sequence overlap, so that any overlapping sequence does not need to exist more than once). Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO.34 or SEQ ID NO.42. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes as described above are provided. The nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes can be used to treat cancer, particularly melanoma, such as skin melanoma. Related diagnostic methods are also provided.

[0458] 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. Other exemplary fragments comprise SEQ ID NO.17 and SEQ ID NO.18. Other exemplary fragments comprise or consist of any one of SEQ ID NO.53 and 67-69. Other exemplary fragments comprise SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.53. Other exemplary fragments comprise all of SEQ ID NO.17-18, 53 and 67-69 (allowing for possible sequence overlap, so that any overlapping sequence does not need to exist more than once). Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO.35 or SEQ ID NO.43. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes as described above are provided. The nucleic acids (eg, DNA or RNA), T cells, T cell populations, cytotoxic cells, antigen binding polypeptides, antigen presenting cells and exosomes can be used to treat cancer, particularly melanoma, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0459] 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. Other exemplary fragments comprise or consist of SEQ ID NO.51 or SEQ ID NO.52. Other exemplary fragments comprise or consist of SEQ ID NO.54. Other exemplary fragments comprise or consist of any one of SEQ ID NOs.70-72 and 76-77. Other exemplary fragments comprise SEQ ID NO.19 and SEQ ID NO.51 or SEQ ID NO.52. Other exemplary fragments comprise SEQ ID NO.54 and SEQ ID NO.51 or SEQ ID NO.52. Other exemplary fragments comprise all of SEQ ID NOs.19, 51-52, 54, 70-72 and 76-77 (allowing for possible sequence overlap, so that any overlapping sequence need not exist more than once). Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO.35 or SEQ ID NO.44. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and allochthons as described above are provided. The nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and allochthons can be used to treat cancer, especially melanoma, such as skin melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0460] 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 NO.20-22. Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO.36 or SEQ ID NO.45. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. The nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes can be used to treat cancer, particularly melanoma, such as skin melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0461] 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. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. The nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes can be used to treat cancer, particularly melanoma, such as skin melanoma. Related diagnostic methods are also provided.

[0462] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO.7. An exemplary fragment comprises or consists of SEQ ID NO.25. An exemplary nucleic acid encoding the polypeptide sequence comprises or consists of SEQ ID NO.38 or SEQ ID NO.47. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. The nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes can be used to treat cancer, particularly melanoma, such as skin melanoma. Related diagnostic methods are also provided.

[0463] 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. An exemplary nucleic acid encoding the polypeptide sequence comprises or consists of SEQ ID NO.38 or SEQ ID NO.48. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. The nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes can be used to treat cancer, particularly melanoma, such as skin melanoma. Related diagnostic methods are also provided.

[0464] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO.9. Exemplary fragments comprise or consist of any one of SEQ ID NO.27-29. Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO.39 or SEQ ID NO.49. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. The nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes can be used to treat cancer, particularly melanoma, such as skin melanoma. Related diagnostic methods are also provided.

[0465] 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 NO.30-32. Exemplary nucleic acids encoding the polypeptide sequence comprise or consist of SEQ ID NO.40 or SEQ ID NO.50. Corresponding nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes as described above are provided. The nucleic acids (e.g., DNA or RNA), T cells, T cell colonies, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes can be used to treat cancer, particularly melanoma, such as skin melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0466] Specifically,

[0467] 1. An isolated polypeptide comprising a sequence selected from the group consisting of:

[0468] (a) the sequence of any one of SEQ ID NOs. 1-10; and

[0469] (b) a variant of the sequence of (a); and

[0470] (c) Immunogenic fragments of the sequence of (a).

[0471] 2. The isolated peptide according to embodiment 1, comprising or consisting of a sequence selected from any one of SEQ ID NOs. 11-32 and 51-78.

[0472] 3. An isolated polypeptide according to embodiment 1 or embodiment 2, fused to a second or other polypeptide, wherein the second or other polypeptide is selected from (i) one or more other polypeptides according to embodiment 1 or embodiment 2; (ii) other polypeptides of melanoma-associated antigens; (iii) polypeptide sequences that can enhance immune responses (i.e., immunostimulatory sequences) and (iv) polypeptide sequences that can provide strong CD4+ help to increase CD8+ T cell responses against antigenic epitopes, such as comprising universal CD4 helper epitopes.

[0473] 4. An isolated nucleic acid encoding a polypeptide according to any one of embodiments 1 to 3.

[0474] 5. The nucleic acid according to embodiment 4, which is DNA.

[0475] 6. The nucleic acid according to embodiment 5, comprising or consisting of a sequence selected from any one of SEQ ID NOs. 33-40 and 41-50.

[0476] 7. The nucleic acid according to embodiment 6, which is codon-optimized for expression in human host cells.

[0477] 8. The nucleic acid according to embodiment 4, which is RNA.

[0478] 9. The nucleic acid according to embodiment 4, 5, 7 or 8, which is an artificial nucleic acid sequence.

[0479] 10. A vector comprising the nucleic acid according to any one of embodiments 4 to 9.

[0480] 11. The vector according to embodiment 10, comprising DNA encoding regulatory elements suitable for allowing transcription of the translationally active RNA molecule in a human host cell.

[0481] 12. The vector according to embodiment 10 or embodiment 11, which is a viral vector.

[0482] 13. The vector according to embodiment 12, which is an adenovirus vector, an adeno-associated virus (AAV), an alphavirus vector, a herpesvirus vector, an arenavirus vector, a measlesvirus vector, a poxvirus vector, a paramyxovirus vector, a lentivirus vector, and a rhabdovirus vector.

[0483] 14. An immunogenic pharmaceutical composition comprising the polypeptide, nucleic acid or vector according to any one of embodiments 1 to 13 together with a pharmaceutically acceptable carrier.

[0484] 15. A vaccine composition comprising the polypeptide, nucleic acid or vector according to any one of embodiments 1 to 13 together with a pharmaceutically acceptable carrier.

[0485] 16. The composition of embodiment 14 or embodiment 15, comprising one or more immunostimulants.

[0486] 17. A composition according to embodiment 16, wherein the immunostimulatory agent is selected from aluminum salts, saponins, immunostimulatory oligonucleotides, oil-in-water emulsions, aminoalkylaminoglucosides 4-phosphates, lipopolysaccharides and their derivatives and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12 and interferons.

[0487] 18. The composition according to any one of embodiments 14 to 17, which is a sterile composition suitable for parenteral administration.

[0488] 19. A polypeptide, a nucleic acid, a vector or a composition according to any one of embodiments 1 to 18 for use in medicine.

[0489] 20. A method of increasing an immune response in a human, comprising administering to said human a polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18.

[0490] 21. A method according to embodiment 20, wherein an immune response is generated against a cancerous tumor that expresses a sequence selected from SEQ ID NO. 1-10 and variants and immunogenic fragments of any one thereof.

[0491] 22. A polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18 for use in raising an immune response in a human.

[0492] 23. A polypeptide, nucleic acid, vector or composition according to embodiment 22, wherein an immune response is generated against a cancerous tumor, which expresses a corresponding sequence selected from SEQ ID NO.1-10 and an immunogenic fragment or variant of any one thereof.

[0493] 24. A method for treating a human patient suffering from cancer, wherein the cancer cells express a gene selected from the group consisting of SEQ ID NO.

[0494] 1-10 and immunogenic fragments and variants of any one of them, or a method for preventing a human from developing cancer, the cancer expressing a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one of them, the method comprising administering to the human a corresponding polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18.

[0495] 25. A polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18 for use in treating or preventing cancer in humans, wherein the cells of the cancer express the corresponding sequence selected from SEQ ID NO. 1-10 and an immunogenic fragment of any one thereof.

[0496] 26. A polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18, for use in ex vivo stimulation and / or expansion of T cells derived from a human suffering from cancer, so as to subsequently reintroduce the stimulated and / or expanded T cells into the human to treat the cancer in the human.

[0497] 27. A method for treating cancer in a human, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, comprising obtaining a population of leukocytes comprising at least T cells, optionally together with antigen presenting cells, from the human, stimulating and / or expanding 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 some or all of the leukocytes, at least the stimulated and / or expanded T cells, into the human.

[0498] 28. The method or polypeptide, nucleic acid, vector or composition for use according to any one of embodiments 21 and 23 to 27, wherein the cancer is melanoma, such as cutaneous melanoma.

[0499] 29. A method for preparing a T cell population that is cytotoxic to cancer cells, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, the method comprising (a) obtaining T cells, optionally together with antigen presenting cells, from a cancer patient; and (ii) stimulating and expanding the T cell population ex vivo with a corresponding polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18.

[0500] 30. A T cell population obtainable by the method according to embodiment 29.

[0501] 31. A T cell which has been stimulated with a polypeptide, a nucleic acid, a vector or a composition according to any one of embodiments 1 to 18.

[0502] 32. An antigen presenting cell modified by ex vivo loading with a polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18 or genetically engineered to express a polypeptide according to any one of embodiments 1 to 3.

[0503] 33. The antigen presenting cell according to embodiment 32, which is a dendritic cell.

[0504] 34. Exosomes loaded with a polypeptide prepared from a cell loaded with a polypeptide, nucleic acid, vector or composition according to any one of embodiments 1 to 18 or genetically engineered to express a polypeptide according to any one of embodiments 1 to 3.

[0505] 35. A pharmaceutical composition comprising a T cell population, a T cell, an antigen presenting cell or an exosome according to any one of embodiments 30 to 34 together with a pharmaceutically acceptable carrier.

[0506] 36. A T cell population, a T cell, an antigen presenting cell or an exosome according to any one of embodiments 30 to 34 for use in medicine.

[0507] 37. A method for treating a human having cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, or a method for preventing a human from developing cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, the method comprising administering to the human a T cell population, T cell, antigen presenting cell, exosome or composition according to any one of embodiments 30 to 35.

[0508] 38. A T cell population, T cell, antigen presenting cell, exosome or composition according to any one of embodiments 30 to 35 for treating or preventing cancer in humans, wherein the cells of the cancer express a corresponding sequence selected from SEQ ID NO.1-10 and an immunogenic fragment of any one thereof.

[0509] 39. The method for use or the T cell population, T cell, antigen presenting cell, exosome or composition according to any one of embodiments 29, 37 and 38, wherein the cancer is melanoma, such as cutaneous melanoma.

[0510] 40. An isolated antigen-binding polypeptide immunospecific for the polypeptide of any one of embodiments 1 to 3.

[0511] 41. The antigen-binding polypeptide of embodiment 40, which is a monoclonal antibody or a fragment thereof.

[0512] 42. An antigen binding polypeptide according to embodiment 40 or embodiment 41, which is conjugated to a cytotoxic moiety.

[0513] 43. An antigen binding polypeptide according to any one of embodiments 40 to 42, for use in medicine.

[0514] 44. A pharmaceutical composition comprising an antigen-binding polypeptide according to any one of embodiments 40 to 42 together with a pharmaceutically acceptable carrier.

[0515] 45. A method for treating a human suffering from cancer, wherein the cells of the cancer express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, or a method for preventing a human from suffering from cancer, wherein the cells of the cancer express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, the method comprising administering to the human an antigen-binding polypeptide or composition according to any one of embodiments 40 to 42 and 44.

[0516] 46. ​​An antigen-binding polypeptide or composition according to any one of embodiments 40 to 42 and 44 for use in treating or preventing cancer in humans, wherein the cells of the cancer express the corresponding sequences selected from SEQ ID NO. 1-10 and immunogenic fragments of any one thereof.

[0517] 47. The method, antigen-binding polypeptide or composition of embodiment 45 or embodiment 46, wherein the cancer is melanoma, eg, cutaneous melanoma.

[0518] 48. An isolated antigen-binding polypeptide immunospecific for an HLA-binding polypeptide that is a polypeptide or a portion thereof according to any one of embodiments 1 to 3.

[0519] 49. An antigen-binding polypeptide according to embodiment 48, which is a T cell receptor or a fragment thereof.

[0520] 50. The antigen-binding polypeptide of embodiment 48 or embodiment 49, coupled to another polypeptide capable of binding to a cytotoxic cell or other immune component in a subject.

[0521] 51. A cytotoxic cell which has been engineered to express on its surface the antigen binding polypeptide of any one of embodiments 48 to 50.

[0522] 52. The cytotoxic cell of embodiment 51, which is a T cell.

[0523] 53. A cytotoxic cell according to embodiment 51 or embodiment 52, for use in medicine.

[0524] 54. A pharmaceutical composition comprising the cell according to embodiment 51 or embodiment 52.

[0525] 55. A method for treating a human patient suffering from cancer, wherein the cancer cells express a gene selected from the group consisting of SEQ ID NO.

[0526] 1-10 and immunogenic fragments and variants of any one of them, or a method for preventing a human from developing cancer, the cancer expressing a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one of them, the method comprising administering to the human a cell according to embodiment 51 or embodiment 52.

[0527] 56. The cytotoxic cell according to embodiment 51 or embodiment 52, for use in treating or preventing cancer in humans, wherein the cancer cells express the corresponding sequences selected from SEQ ID NO. 1-10 and immunogenic fragments of any one thereof.

[0528] 57. A method of diagnosing a person as having cancer, comprising the steps of:

[0529] Determine whether the cancer cells express a polypeptide sequence selected from SEQ ID NO. 1-10 and an immunogenic fragment or variant of any one of them, or a nucleic acid encoding the polypeptide sequence, and diagnose the human as having cancer if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells.

[0530] 58. A method for diagnosing a person with skin melanoma, comprising the steps of: determining whether cells of the cancer express a polypeptide sequence of any one of SEQ ID NO. 2, 6, 7, 8 and 9 and an immunogenic fragment or variant thereof; or a nucleic acid encoding the polypeptide sequence, and if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, diagnosing the person with skin melanoma.

[0531] 59. A method for diagnosing a person with cutaneous melanoma or uveal melanoma, comprising the steps of: determining whether cells of the cancer express a polypeptide sequence selected from any one of SEQ ID NO. 1, 3, 4, 5 and 10 and an immunogenic fragment or variant of any one of them; or a nucleic acid encoding the polypeptide sequence, and if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, diagnosing the person with cutaneous melanoma or uveal melanoma.

[0532] 60. A method of treating a human suffering from cancer, comprising the steps of:

[0533] (a) determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NO. 1-10 and an immunogenic fragment or variant of any one of them or a nucleic acid encoding the polypeptide sequence (e.g., a sequence selected from SEQ ID NO. 33-40 and 41-50); and if expressed,

[0534] (b) the human administers 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 embodiments 1 to 18, 30 to 35, 40 to 42, 44, 50, 51 and 53.

[0535] 61. Use of a polypeptide comprising a sequence selected from the group consisting of:

[0536] (a) the sequence of any one of SEQ ID NOs. 1-10; or

[0537] (b) a variant of the sequence of (a); and

[0538] (c) Use of an immunogenic fragment of the sequence of (a), isolated from a tumor of a human suffering from cancer, or a nucleic acid encoding the polypeptide, as a biomarker for determining whether the human will be suitable for treatment with a vaccine, the 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 embodiments 1 to 18, 30 to 35, 40 to 42, 44, 51, 52 and 54.

[0539] 62. The method or use of embodiment 60 or embodiment 61, wherein the cancer is melanoma, such as cutaneous melanoma.

[0540] 63. The method or polypeptide, nucleic acid, vector or composition for use according to any one of embodiments 21 and 23 to 27, wherein the polypeptide comprises a sequence selected from the group consisting of:

[0541] (a) the sequence of any one of SEQ ID NOs. 1, 3, 4, 5 and 10; and

[0542] (b) a variant of the sequence of (a); and

[0543] (c) an immunogenic fragment of the sequence of (a),

[0544] 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 selected from any one of SEQ ID NOs. 41, 43, 44, 45 and 50;

[0545] And wherein the cancer is uveal melanoma.

[0546] 64. An antigen-binding polypeptide or composition for use according to embodiment 45, wherein the polypeptide comprises a sequence selected from the group consisting of:

[0547] (a) the sequence of any one of SEQ ID NOs. 1, 3, 4, 5 and 10; and

[0548] (b) a variant of the sequence of (a); and

[0549] (c) an immunogenic fragment of the sequence of (a),

[0550] 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 selected from any one of SEQ ID NOs. 41, 43, 44, 45 and 50;

[0551] And wherein the cancer is uveal melanoma.

[0552] 65. The method for use or the T cell population, T cell, antigen presenting cell, exosome or composition according to any one of embodiments 29, 37 and 38, wherein the polypeptide comprises a sequence selected from the group consisting of:

[0553] (a) the sequence of any one of SEQ ID NOs. 1, 3, 4, 5 and 10; and

[0554] (b) a variant of the sequence of (a); and

[0555] (c) an immunogenic fragment of the sequence of (a),

[0556] 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 selected from any one of SEQ ID NOs. 41, 43, 44, 45 and 50;

[0557] And wherein the cancer is uveal melanoma.

[0558] 66. The method or use according to embodiment 60 or embodiment 61, wherein the polypeptide comprises a sequence selected from the group consisting of:

[0559] (a) the sequence of any one of SEQ ID NOs. 1, 3, 4, 5 and 10; and

[0560] (b) a variant of the sequence of (a); and

[0561] (c) Immunogenic fragments of the sequence of (a).

[0562] 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 any one of SEQ ID NOs. 33, 35, 36 or 40 or selected from SEQ ID NOs. 41, 43,

[0563] 44, 45 and 50; and wherein the cancer is uveal melanoma. 67. The fusion polypeptide according to embodiment 3, comprising two or more (e.g., two, three or four) sequences selected from the sequence of SEQ ID NO. 1, 2, 3 and 4; or a variant of the sequence or an immunogenic fragment of the sequence relative to each of the sequences.

[0564] 68. The fusion polypeptide of embodiment 67, comprising:

[0565] (i) a sequence selected from the group consisting of:

[0566] (a) the sequence of SEQ ID NO. 1; and

[0567] (b) a variant of the sequence of (a); and

[0568] (c) an immunogenic fragment of the sequence of (a); and

[0569] (ii) a sequence selected from the group consisting of:

[0570] (a) the sequence of SEQ ID NO. 2; and

[0571] (b) a variant of the sequence of (a); and

[0572] (c) an immunogenic fragment of the sequence of (a); and

[0573] (iii) a sequence selected from the group consisting of:

[0574] (a) the sequence of SEQ ID NO. 3; and

[0575] (b) a variant of the sequence of (a); and

[0576] (c) an immunogenic fragment of the sequence of (a); and

[0577] (iv) a sequence selected from the group consisting of:

[0578] (a) the sequence of SEQ ID NO. 4; and

[0579] (b) a variant of the sequence of (a); and

[0580] (c) Immunogenic fragments of the sequence of (a).

[0581] 69. A fusion polypeptide according to embodiment 68, which comprises the sequences of SEQ ID NO.1, 2, 3 and 4.

[0582] 70. The fusion polypeptide of embodiment 68, comprising:

[0583] (i) a sequence selected from the group consisting of:

[0584] (a) the sequence of SEQ ID NO. 1; and

[0585] (b) a variant of the sequence of (a); and

[0586] (c) an immunogenic fragment of the sequence of (a); and

[0587] (ii) a sequence selected from the group consisting of:

[0588] (a) the sequence of SEQ ID NO. 2; and

[0589] (b) a variant of the sequence of (a); and

[0590] (c) an immunogenic fragment of the sequence of (a); and

[0591] (iii) a sequence selected from the group consisting of:

[0592] (a) the sequence of SEQ ID NO. 4; and

[0593] (b) a variant of the sequence of (a); and

[0594] (c) Immunogenic fragments of the sequence of (a).

[0595] 71. A fusion polypeptide according to embodiment 70, which comprises the sequences of SEQ ID NO.1, 2 and 4.

[0596] 72. An isolated nucleic acid encoding a fusion polypeptide according to any one of embodiments 67-71.

[0597] 73. The nucleic acid of embodiment 72, which is DNA.

[0598] 74. A vector comprising the nucleic acid according to embodiment 73.

[0599] 75. A vector according to embodiment 73, comprising DNA encoding regulatory elements suitable for allowing transcription of a translationally active RNA molecule in a human host cell.

[0600] 76. The vector of embodiment 74 or embodiment 75, which is a viral vector.

[0601] Example

[0602] Example 1 - CLT Identification

[0603] The goal was to identify cancer-specific transcripts that consist entirely or partially of LTR elements.

[0604] As a first step, we assembled a complete pan-cancer transcriptome from scratch. To achieve this goal, RNA sequencing reads from 768 patient samples were used for genome-guided assembly, where the patient samples were obtained from the Cancer Genome Atlas TCGA consortium and represented a wide variety of cancer types (24 gender-balanced samples from each of 32 cancer types (31 primary melanomas and 1 metastatic melanoma); Table S1). Cutadapt (v1.13) (Marcel M., 2011, EMBnet J., 17: 3) was used to trim the adapter and quality (Q20) and length filter (two reads were ≥ 35 nucleotide pairs) for gender-balanced samples (excluding sex-specific tissues), and khmer (v2.0) (Crusoe et al., 2015, F1000 Res., 4: 900) was used for kmer normalization (k = 20) for the maximum and minimum depths of 200 and 3, respectively. Reads were mapped to GRCh38 and transferred to Trinity (v2.2.0) (Trinity, Grabherr, MG et al., 2011, Nat. Biotechnol., 29:644-52) for genome-guided assembly using STAR (2.5.2b) with the same settings as those used across TCGA, with built-in deep normalization disabled on the computer. Most of the assembly process was completed within 256GB RAM on a 32-core HPC node, with failed processes repeated using a 1.5TB RAM node. The resulting contigs were subjected to poly-A trimming (trimpoly inside SeqClean v110222) and entropy filtering (≥0.7) to remove low-quality and artificial contigs (bbduk inside BBMap v36.2). Depending on the cancer type, the original 24 samples were mapped to the cleaned assembly using Salmon (v0.8.2 or v0.9.2) (Patro, R. et al., 2017, Nat. Methods, 14: 417-419), removing contigs found to be expressed at <0.1 transcripts per million (TPM). The remaining contigs were mapped to GRCh38 using GMAP (v161107) (Wu et al., 2005, Bioinf., 21: 1859-1875), and contigs that were not aligned with ≥85% identity within their ≥85% length were removed from the assembly. Finally, gffread (Cufflinks v2.2.1) (Trapnell et al., 2010, Nat. Biotech., 28: 511-515) was used to flatten the assemblies of all cancer types together and composite them into the longest continuous transcript.Since this assembly process is specially designed to make it possible to evaluate repetitive elements, single exon transcripts are retained, but marked. By comparing with GENCODEv24basic and MiTranscriptome1, transcript assembly integrity and quality (Iye et al. 2015, Nat.Genet., 47: 199-208) are assessed. We compiled a unique splice site list inside GENCODE and checked whether the splice site exists in a 2-nucleotide grace window (grace window) inside the transcriptome assembly. This process leads to the identification of 1,001,931 transcripts, of which 771,006 are spliced ​​and 230,925 are single exons.

[0605] Separately, the assembled contigs were superimposed with genomic repeat annotations to identify transcripts containing LTR elements. LTR elements and non-LTR elements were annotated as previously described (Attig et al., 2017, Front. In Microbiol., 8: 2489). In short, using RepeatMasker Open-3.0 (Smit, A., R. Hubley and P. Green, http: / / www.repeatmasker.org, 1996-2010) configured with nhmmer (Wheeler et al., 2013, Bioinform., 29: 2487-2489), hidden Markov models (HMMs) (Dfam 2.0 library v150923) representing known human repeat families were used to annotate GRCh38. Compared with BLAST-based methods, HMM-based scanning improves annotation accuracy (Hubley et al., 2016, Nuc. Acid. Res., 44: 81-89). RepeatMasker annotates LTRs and internal regions separately, so the tabular output was parsed to merge adjacent annotations of identical elements. This process yielded 181,967 transcripts containing one or more, complete or partial, LTR elements.

[0606] Using Salmon, transcripts per million (TPM) were estimated for all transcripts, and expression within each cancer type was compared with expression across 811 healthy tissue samples, where healthy tissue matched controls for all cancer types were from TCGA (if available) and separately from GTEx (Genotype-Tissue Expression Consortium, 2015, Science, 348: 648-60). Transcripts were considered to be specifically expressed in cancer if detected at more than 1 TPM in any sample, and were considered cancer-specific if the following criteria were met: i. expressed in ≥6 of the 24 samples of each cancer type; ii. expressed at <10 TPM in ≥90% of all healthy tissue samples; iii. expression in the cancer type of interest ≥3 times the median expression of any control tissue type; and iv. expression in the cancer type of interest ≥3 times the 90th percentile of the corresponding healthy tissue, when available. In addition to these expression thresholds, transcript selection was based on manual inspection, excluding possible misassembled contigs or transcripts with LTR elements in the 3' untranslated region (UTR). If the transcription direction could not be assigned unambiguously, transcripts corresponding to both strands were considered.

[0607] The list of cancer-specific transcripts was then intersected with the list of transcripts containing complete or partial LTR elements to generate a list of 5,923 transcripts that met both criteria (referred to as the cancer-specific cross-transcript list). L TR element transcript, CLT).

[0608] To identify CLTs with protein coding potential, we ran an ORF prediction algorithm based on length and dicodon (hexamer) score suitability. The HMM was trained on hexamers derived from Ensembl CDS sequences and ORFs of ≥300 nucleotides were treated where their hexamer sense score exceeded the antisense score. This filtering procedure identified 885 CLTs that potentially encode proteins with a length of at least 99 amino acids.

[0609] To identify unique protein sequences that CLTs may encode, sequences translated from the largest ORF of the selected CLTs were interrogated against those translated from all ORFs of ≥210 nucleotides derived from the complete transcript assembly using tblastn (BLAST+ v2.3.0) without soft-masking. Only hits with no hits or with an E-value >10 were retained. -5 The hit CLT.

[0610] To further ensure the specificity of the cancer-specific antigens encoded by CLTs, we examined their potential cross-reactivity with other proteins that may be expressed in healthy tissues. For this purpose, the translated ORFs that have <85% amino acid sequence identity with any other predicted protein (over the entire length of the protein) are retained. For those CLT-encoded proteins that show >85% sequence identity with one or more predicted proteins, we queried the expression pattern of transcripts encoding similar proteins. If these additional transcripts are also expressed in a cancer-specific manner (based on the criteria listed above), it is clear that the corresponding CLT remains in the selected candidate list. If the additional transcripts are also expressed in healthy tissues, the corresponding CLT is discarded. The combination of these selection criteria produces a final list of 139 CLTs that may encode proteins with sufficiently unique amino acid sequences.

[0611] Of these 139 CLTs, 14 were specific for skin melanoma (i.e., they were found to be specifically upregulated in skin melanoma samples of TGCA according to the above methodology) and 7 were specific for skin melanoma and uveal melanoma (i.e., they were found to be specifically upregulated in skin melanoma samples and uveal melanoma samples of TGCA according to the above methodology). Four of these skin melanoma-specific CLTs are identified herein as having SEQ ID NOs. 34, 37, 38, and 39. Four of these skin melanoma and uveal melanoma-specific CLTs are identified herein as having SEQ ID NOs. 33, 35, 36, and 40.

[0612] Example 2 - Immunopeptidomic Analysis

[0613] Immunopeptidomic analysis is a powerful technique that allows direct detection of specific peptides associated with HLA molecules in cells or tissues. The technique consists of affinity purification of HLA molecules from biological samples and subsequent elution of bound peptides from HLA molecules and analysis by nano-ultra-performance liquid chromatography mass spectrometry (nUPLC-MS 2) (Freudenmann et al., 2018, Immunology 154(3):331-345) to evaluate peptides. Mass spectrometry (MS) spectra generated by this method can be used to accurately identify short peptides that bind to HLA class I and HLA class II molecules. Software for spectrum interpretation and sequence identification relies on the availability of a predefined list of protein sequences for spectrum matching. Although MS data can be retrieved by using a predefined list corresponding to all open reading frames (ORFs) derived from a known transcriptome or even a complete genome (Nesvizhskii et al., 2014, Nat. Methods 11:1114–1125), querying these very large sequence databases results in extremely high false discovery rates, which limit the identification of the presented peptides. Other technical issues (e.g., mass of leucine = mass of isoleucine) and theoretical issues (e.g., peptide splicing (Liepe et al., 2016, Science 354(6310):354–358)) add to the limitations associated with using very large databases such as those generated from known transcriptomes or complete genomes. Therefore, in practice, it is extremely difficult to perform immunopeptidomic analysis to identify new antigens without reference to a well-defined set of potential polypeptide sequences.

[0614] Bassani-Sternberg et al. (Bassani-Sternberg et al., 2016, Nature Commun., 7: 13404) queried MS data collected from HLA-bound peptide samples derived from 25 patients with cutaneous melanoma in comparison to the peptides reported for the complete human proteome. These analyses revealed thousands of peptides that matched known human proteins. As expected, these peptides included peptides found within a variety of tumor-associated antigens (TAAs), including PRAME, MAGEA3, and TRPM1 (melastatin). In addition, the MS data from these five patients were queried with a list of peptides created from patient-specific mutant protein sequences detected by genomic analysis of five of these patients, revealing patient-specific neoantigens presented on HLA class I and HLA class II molecules in these patients.

[0615] Many of the predicted polypeptide sequences (ORFs) derived from the 139 CLTs mentioned in Example 1 are not contained in the human proteome. By applying the knowledge of detailed immunopeptidomics evaluation, the inventors used this new set of potential CLT antigen sequences to query the RAW data file of Bassani-Sternberg et al. (database link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894).

[0616] 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 and analyzed using Peaks TM Software (Analysis A) or Mascot software (Analysis B), these concatenated files (Analysis A) or individual peptide files (Analysis B) were used to query the raw spectra in the PXD004894 dataset, together with all peptides found in the human proteome (UniProt (Analysis A) or UniProt and masDB (Analysis B)).

[0617] 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., which peptides could be assigned to 8 ORFs that were not present in the reported proteome (see Table 1). In analysis B, the results of these studies identified 14 peptides associated with HLA class I molecules or HLA class II molecules immunoprecipitated from tumor samples of 25 patients examined by Bassani-Sternberg et al., which peptides could be assigned to 7 ORFs that were not present in the reported proteome (see Table 2). The detection of these peptides associated with HLA class I and HLA class II molecules from the cited patients confirmed that the 10 ORFs from which these peptides were derived (Tables 1 and 2, SEQ ID NOs. 1-10) were translated in melanoma tissue and presented to the immune system in complex with HLA class I or HLA class II molecules. On this basis, the polypeptides encoded by these ORFs were defined as CLT antigens. Tables 1 and 2 show the properties of peptides found within the CLT antigen that are not part of the UniProt database. Figure 1-Figure 32 Representative mass spectrometry spectra are shown from each of the peptides shown in Tables 1 and 2. These figures show the mass spectra of each patient's SKCM tumor as detected by nUPLC-MS 2 Detected fragmentation spectra for the indicated peptide sequences (from Bassani-Sternberg et al.; images extracted from the PRIDE dataset using PEAKS software). All fragments that have been detected are indicated in the peptide sequence above the spectra and the most abundant fragment ion is assigned in each spectrum. Figure 1-Figure 15 , Figure 29-Figure 32 In (Analysis A), the lower panel of the figure shows the sequence annotation of the predicted spectra, while similar data are shown in tabular form in Figure 16-Figure 28 on the right (Analysis B). The fragment ions are annotated as follows: b: N-terminal fragment ion; y: C-terminal fragment ion; -H2O: loss of water; -NH3: loss of ammonia; [2+]: doubly charged peptide ion; pre: unfragmented precursor peptide ion.

[0618] A number of peptides detected in association with HLA class I from Tables 1 and 2 were evaluated to determine the predicted strength of binding to HLA class I supertypes. Specifically, the predicted strength of binding to HLA class I supertypes was determined by using NetMHC 4.0 prediction software ( http: / / www.cbs.dtu.dk / services / NetMHC / ) All HLA class I associated peptides of 9 amino acids or longer mentioned in Table 3 were queried for prediction of their binding to HLA class I supertypes A and B. The results of these prediction studies showed that all 11 peptides (or 9-mers derived from them) were predicted to bind to at least one of the supertypes tested (see Table 3). Among these sequences, many were predicted to bind with high confidence (low % rank score) to specific types within the HLA class I supertypes examined.

[0619] In summary, Tables 1-3 and Figure 1-Figure 32 The data presented in provide unusually strong support for the presence of the corresponding CLT antigen in melanoma patients.

[0620] Summary: Identification of immunopeptidomic peptides derived from predicted ORFs indicated that these CLTs are translated into polypeptides (SEQ ID NOs. 1-10; referred to as CLT antigens) in tumor tissues. These polypeptides are subsequently processed by the immune surveillance machinery of the cell and loaded onto HLA class I or HLA class II molecules, making it possible to target the cells for lysis by T cells that recognize the resulting peptide / HLA class I complex or peptide / HLA class II complex. Therefore, these CLT antigens and fragments thereof are expected to be useful in a variety of therapeutic modalities for the treatment of melanoma in patients whose tumors express these antigens.

[0621] Table 1: List of peptides identified by immunopeptidomic analysis of SKCM tumor samples (Analysis A), together with CLT antigen names and cross references to SEQ ID NOs.

[0622]

[0623]

[0624] 1 Peptides identified by mass spectrometry. All peptides are HLA class I peptides.

[0625] 2 Bassani-Sternberg et al., 2016, Nature Comm., 7: 13404

[0626] 3 Calculated peptide masses.

[0627] 4 Peaks from mass spectra TMProgram Area; displays selected area values ​​for peptides for which more than one spectrum was obtained.

[0628] 5 Number of spectra in which the peptide was detected.

[0629] 6 Deviation between observed and calculated masses; selected ppm values ​​are shown for peptides for which more than one spectrum was obtained.

[0630] Table 2: List of peptides identified by immunopeptidomic analysis of SKCM tumor samples (Analysis B), together with CLT antigen names and cross references to SEQ ID NOs.

[0631]

[0632]

[0633] 1 Peptides identified by mass spectrometry. All peptides are HLA class I peptides unless indicated by * (which are HLA class II)

[0634] 2 Bassani-Sternberg et al., 2016, Nature Comm., 7: 13404

[0635] 3 Calculated peptide masses.

[0636] 4 Number of spectra in which the peptide was detected.

[0637] 5 Difference between observed and calculated masses; selected mass delta values ​​are shown for peptides for which more than one spectrum was obtained.

[0638] *Indicates HLA class II peptide

[0639] Table 3: Peptides identified by mass spectrometry (length > 9 residues) 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), along with the CLT antigen name and SEQ ID NO cross-reference.

[0640]

[0641] 1 Binding to the query HLA class I supertype was predicted at any grade score.

[0642] 2 Proportion of HLA class I supertypes predicted to bind with a grade score <5.1% (weak binding).

[0643] 3 Proportion of HLA class I supertypes predicted to bind with a grade score <2.1% (stronger binding).

[0644] 4 Bassani-Sternberg et al., 2016, Nature Comm., 7: 13404

[0645] Example 2.1 - Additional immunopeptidomic analyses

[0646] In addition to the analysis described in Example 2, the inventors have also identified peptides derived from the predicted ORFs by novel immunopeptidomic studies. This additional study described below further demonstrated that these CLTs are translated into CLT antigen polypeptides in tumor tissues.

[0647] The inventors obtained frozen tumor tissue from 10 patients diagnosed with melanoma. The samples between 0.05-1g were homogenized, the lysate was centrifuged at high speed and the clarified lysate was mixed with protein A (ProA) beads covalently linked 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). The peptides bound to HLA class I were eluted from the antibody using 10% acetic acid, and then reverse phase column chromatography was used to separate the peptides from other high molecular weight components (Ternette et al., 2018). The eluted purified peptides were subjected to nUPLC-MS and specific peptides with defined charge / mass ratios (m / z) were selected inside the mass spectrometer, separated, fragmented and subjected to MS / MS to reveal the m / z of the resulting fragment ions (Ternette et al., 2018), generating MS / MS data sets corresponding to the respective immunopeptidomes of these tumor samples.

[0648] By applying detailed knowledge of immunopeptidomics evaluation, the inventors queried the spectrum of the HLA-I class dataset of 10 melanoma tumors prepared by the inventors with CLT antigen numbers 1, 2, 3 and 4 (Table 4; SEQ ID NO.1-4), using PEAKS TMThe spectra were searched together with all polypeptide sequences present in the human proteome (UniProt) using the software (v8.5 and vX, Bioinformatics Solutions Inc) (for each CLT). Since the majority of HLA class I binding peptides present in cells are derived from constitutively expressed proteins, simultaneous querying of these databases with the UniProt proteome helped ensure that our CLTORF sequences were correctly assigned to the MS / MS spectra.

[0649] As a result of these studies, eight independent peptides were identified that were associated with HLA class I molecules immunoprecipitated from tumor samples obtained by the inventors from ten melanoma patients (Table 4; SEQ ID NOs. 1-4). These peptides correspond to the amino acid sequence of the CLT-derived ORF and do not correspond to polypeptide sequences present within the known human proteome (UniProt). Two of the eight peptides identified from CLT antigen SEQ ID NOs. 1-4 (Table 4) in the inventors' data set were additional to the ten independent peptides associated with HLA class I molecules immunoprecipitated from tumor samples of 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).

[0650] Detection of these HLA class I-associated peptides demonstrates that the four ORFs from which these peptides are derived are first translated in melanoma tissue, processed via the HLA class I pathway and ultimately presented to the immune system in complex with HLA class I molecules. Table 4 shows the identity of the peptides present within the CLT antigen. Figure 33-Figure 42 Representative MS / MS spectra are shown from each of the peptides shown in Table 4. The top panel of each of these figures shows the MS / MS peptide fragmentation characteristics, standard MS / MS annotations (b: N-terminal fragment ion; y: C-terminal fragment ion; -H2O: loss of water; -NH3: loss of ammonia; [2+]: doubly charged peptide ion; pre: unfragmented precursor peptide ion; a n -n: internal fragment ion) is shown above the most abundant fragment ion peak in the image extracted by PEAKS software from the inventors' data set. The lower panel of each figure indicates the spectral presentation of the position of the linear peptide sequence, which has been mapped to the fragment ion. Consistent with the high -101gP scores assigned to the peptides in Table 4, these mass spectra contain numerous fragments that exactly match the peptide sequences (SEQ ID NO. 12, 13, 16, 17, 19, 51, 53 and 54) we found in these analyses.

[0651] All peptides of at least 9 AA in length detected in association with HLA class I from Table 4 were evaluated for the presence of http: / / www.cbs.dtu.dk / services / NetMHCpan / ), and their predicted strength of binding to HLA class I supertypes A and B was determined. The results of these prediction studies showed that all peptides (or 9-mers contained within each full sequence) were predicted to bind to at least one of the supertypes tested (see Table 5). Among these sequences, many were predicted to bind with high confidence (low % rank score) to specific types within the HLA class I supertypes examined. The fact that all detected peptides are expected to bind to HLA types expected to be present in the patient population is consistent with the detection of these peptides. In addition, every peptide found in tumor samples from the inventors' dataset was predicted by NetMHCpan 4.0 to bind to one of the HLA types we detected in patient samples.

[0652] To provide further certainty in the assignment of tumor tissue-derived MS spectra to the peptide sequences we discovered in Example 2.1, peptides with these discovered sequences were synthesized and subjected to nUPLC-MS using the same conditions applied to the tumor samples in our data. 2 . Figure 43-Figure 50 Comparison of spectra of selected peptides is shown in Figure 2. In each figure, the upper spectrum corresponds to a tumor sample (from the inventors’ tumor tissue database – Figure 33-Figure 42 ) and the lower spectrum corresponds to the synthetically produced peptides of the same sequence. Selected m / z values ​​of detected ion fragments are shown above / below each fragmentation peak in these MS / MS spectra. These figures reveal accurate fragmentation alignments (the small differences in experimentally determined m / z values ​​between tumor-derived fragment ions and synthetic peptide-derived fragment ions fall well within the m / z tolerance of <0.05 Daltons), confirming the authenticity of the assignment of each tumor tissue-derived mass spectrum to CLT-encoded peptides.

[0653] In summary, Table 4 Figure 33-Figure 42 and Figure 43-Figure 50 The peptide data shown in provide unusually strong support for translation, processing and presentation of the corresponding CLT antigen in melanoma patients.

[0654] To further confirm the cancer specificity of these CLTs, the inventors processed 37 normal tissue samples (10 normal skin samples, 9 normal lung samples, and 18 normal breast tissue samples) and prepared them for immunopeptidomics analysis. The inventors queried the spectra of the HLA-I class data set from these normal tissue samples, retrieving all possible peptide sequences derived from the polypeptide sequences of CLT antigens No. 1, No. 2, No. 3, and No. 4. Peptides derived from CLT antigens No. 1, No. 2, No. 3, and No. 4 were not detected in the normal tissue sample collection (Table 6), thereby additionally confirming that CLT has cancer-specific expression.

[0655] Summary: This additional identification of immunopeptidomic peptides derived from the predicted ORFs further indicated that these CLTs are translated into polypeptides (SEQ ID NOs. 1-4; referred to as CLT antigens) in tumor tissues. Therefore, these CLT antigens and fragments thereof are expected to be useful in a variety of therapeutic modalities for treating melanoma in patients whose tumors express these antigens.

[0656] Table 4: List of peptides identified by additional immunopeptidomic analysis of melanoma tumor samples, together with CLT antigen names and cross-references to SEQ ID NOs.

[0657]

[0658]

[0659] ND – Not Determined

[0660] 1 HLA class I peptides identified by mass spectrometry.

[0661] 2 Inventors' datasets (1MT1, 1MT2, 1MT3, 2MT1, 2MT2, 2MT3, 2MT4, 2MT9, 2MT10, 2MT12).

[0662] 3 Calculated peptide masses.

[0663] 4 Peaks from mass spectra TM Program Area; displays selected area values ​​for peptides for which more than one spectrum was obtained.

[0664] 5 Number of spectra in which the peptide was detected.

[0665] 6 Deviation between observed and calculated masses; selected ppm values ​​are shown for peptides for which more than one spectrum was obtained.

[0666] Table 5: Peptides identified by mass spectrometry (length > 9 residues) 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), along with the CLT antigen name and SEQ ID NO cross-reference.

[0667]

[0668]

[0669] 1 Binding to the query HLA class I supertype was predicted at any grade score.

[0670] 2 Proportion of HLA class I supertypes predicted to bind with a grade score <5.1% (weak binding).

[0671] 3 Proportion of HLA class I supertypes predicted to bind with a grade score <2.1% (stronger binding).

[0672] 4 Inventors' datasets (1MT1, 1MT2, 1MT3, 2MT1, 2MT2, 2MT3, 2MT4, 2MT9, 2MT10, 2MT12).

[0673] Table 6 Number of peptides derived from CLT antigens 1 to 4 in a pool of normal tissue samples.

[0674] antigen skin lung Breast CLT antigen 1 0 / 10 0 / 9 0 / 18 CLT antigen 2 0 / 10 0 / 9 0 / 18 CLT antigen 3 0 / 10 0 / 9 0 / 18 CLT antigen 4 0 / 10 0 / 9 0 / 18

[0675] The results presented herein in Examples 1, 2 and 2.1 are based in whole or in part on the Cancer Genome Atlas (TCGA) research network ( http: / / cancergenome.nih.gov / ) and the Genotype-Tissue Expression (GTEx) Project (supported by the NIH Office of the Director Common Fund and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS).

[0676] Example 3 HERVFEST

[0677] The functional expansion of specific T cells (FEST) technology has been used to identify therapeutically relevant tumor-derived epitopes present in the "mutation-associated neoantigen" (MANA) library, where the mutation-associated neoantigens are found in 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 FEST technology was applied to the CLT antigens discovered using the methods described in Examples 1, 2, and 2.1 (Tables 1-6, Figure 1-Figure 50 ) can be used to identify therapeutically useful T cell responses against CLT antigens in cancer patients.

[0678] Like other assays for identifying epitope-specific T cells in subjects that have undergone immune exposure (e.g., ELISPOT), the "FEST" technique derives its specificity by activating / expanding cognate T cells in ex vivo cultures containing antigen presenting cells and appropriate antigenic peptides. This technique differs from other immunological assays in that it utilizes next generation sequencing of T cell receptor (TCR) DNA sequences present in these expanded cultures (particularly: TCRseq targeting the TCR-VβCDR3 region) to detect specific TCRs expanded in cells cultured with respective peptides from a set of target peptides derived from an antigen (or antigens). The application of TCRseq to tumor tissue from the same patient can also be used to show whether TCRs / T cells detected in peptide-stimulated ex vivo cultures are also present within tumor-infiltrating lymphocytes found in carcinoma in situ tissue. Thus, MANAFEST has been shown to be a powerful technique for identifying MANA epitopes recognized by patient T cells, allowing the identification of mutant peptides among a variety of mutant peptides found with the aid of whole exome sequencing of normal and tumor tissues from cancer patients. MANA peptides with functional significance (Le et al., Science 2017; Forde et al., NEJM 2018; Danilova et al., Cancer Immunol. Res. 2018; Smith et al., J Immunother Cancer 2019).

[0679] The MANAFEST method system (Danilova et al., Cancer Immunol. Res. 2018) is applied to the CLT antigen as follows. This method, which we will call HERVFEST, consists of the following steps: Step 1: Identify peptides in the CLT antigen that are predicted to contain epitopes that efficiently bind to selected HLA class I alleles. Step 2: PBMCs from suitable melanoma patients are matched to the peptide library selected in step 1 according to the HLA class I type. Step 3: PBMCs derived from these patients are separated into T cell parts and non-T cell parts. The non-T cells are added back to the patient's T cells and then divided into 20-50 wells (each culture contains 250,000 T cells) and proliferated for 10 days with a variety of T cell growth factors and independent CLT antigen-derived synthetic peptides (selected in step 1 / 2). Step 4: TCRseq (TCR-VβCDR3 sequence sequencing) is performed on all wells, and TCR-VβCDR3 sequences that are amplified in the presence of independent CLT antigen-derived peptides (but not in the presence of control peptides or in the absence of peptide stimulation) are identified. The presence of amplified TCR-VβCDR3 sequences in each well of this analysis is therefore identified as CLT antigen-derived peptides that stimulate immune responses in melanoma patients. Step 5: TCRseq can also be performed on tumor samples to determine whether T cells carrying CLT antigen-amplified TCRs home to patient tumors, providing additional evidence that T cells carrying these TCRs recognize CLT antigen-derived peptides within patient tumors.

[0680] HERVFEST analysis was performed with peptides derived from CLT antigens 1-4 (SEQ ID NO. 1-4). The set of peptides used for these studies (see step 1 above) was based on NetMHC predictions of CLT antigen-derived peptides, which were predicted to strongly bind to 8 HLA class I types commonly present in patient tumor samples available for our analysis. CLT antigen-derived peptides that amplified one or more TCRs in the HERVFEST analysis are provided in Table 7. Table 7 also indicates the HLA class I types of the CLT antigen peptides tested with each patient's PBMC-derived culture. The HLA class I types of patients whose PBMCs were tested in the study and amplified one or more TCRs in the analysis are shown in Table 8.

[0681] Fig.51 Panel A shows published data demonstrating TCR expansion with NSCLC patient-specific MANA peptides (Forde et al., NEJM 2018). The vertical axis shows the prevalence of each of the indicated TCR-Vβ CDR3 sequences in wells of cells cultured in the presence of the MANA or control peptides listed on the horizontal axis. Expansion in wells containing MANA7 indicates that the patient's T cell repertoire contains T cells reactive to this peptide. Fig.51 Panel B and panel C show representative TCR amplification data from PBMCs of 2 melanoma patients, wherein the PBMCs were incubated in the presence of the CLT antigen peptides and control peptides shown. As with panel A, the specific amplification observed in panels B and C shows that the T cell pools of these melanoma patients contain T cells reactive with specific CLT antigen-derived peptides. Panel B shows the frequency of TCR detected in the PBMC wells stimulated by LMSSFSTLASL from melanoma patient 222B in all wells stimulated with 15 HLA class I A*02 peptide combinations from CLT antigens 1, 2, and 4. All three TCR sequences were amplified. LMSSFSTLASL (SEQ ID NO.61) is an A*02 binding peptide derived from CLT antigen 2. Panel C shows the frequency of TCR detected in wells of PBMC stimulated with MVACRIKTFR from melanoma patient 224B in all wells stimulated with a combination of 15 HLA class I A*02 peptides from CLT antigens 1, 2 and 4 and 24 HLA class I A*03 peptides from CLT antigens 1, 2, 3 and 4. One TCR sequence was amplified. MVACRIKTFR (SEQ ID NO. 64) is an A*03 binding peptide derived from CLT antigen 2.

[0682] The control peptides / conditions used in these experiments were as follows: CEF = a mixture of CMV, EBV, and influenza peptides; SL9, TV9, and QK1 = HIV-1 control peptides; No peptide = culture in the absence of peptide; Baseline = T cells prior to culture.

[0683] Fig.52 A summary of all CLT antigen peptides for CLT antigens 1-4 that amplified one or more TCRs in studies performed with these patients is shown. Each panel shows the amino acid sequence of CLT antigens 1-4 superimposed with peptides detected by immunopeptidomics analysis (by 2 and 2.1). Below these sequences, the peptides detected by HERVFEST are shown (see Fig.51 ) together with the number of melanoma patients in which these peptides were detected (Table 8) and the targeted HLA class I type.

[0684] The characteristics of each HERVFEST assay are defined as follows:

[0685] Plain text: Significant amplification of a single TCR

[0686] Bold text: Significant amplification of multiple TCRs

[0687] Underlined italic text: Significant amplification of a single TCR detected in other wells

[0688] · Underline bold text : Significantly amplify multiple TCRs of which at least one was detected in other wells

[0689] These results provide strong evidence that CLT antigens 1-4 are present in melanoma patients and that peptides derived from these CLT antigens have elicited specific T-cell responses in melanoma patients, confirming the value of these CLT antigens as targets for therapeutic intervention to treat melanoma.

[0690] Table 7: CLT antigen-derived peptides that amplify one or more TCRs in the HERVFEST assay

[0691]

[0692]

[0693] Table 8: Characteristics of melanoma patient PBMCs used in the HERVFEST analysis

[0694]

[0695] Example 4 - Demonstration that high affinity T cells specific for CLT antigen have not been deleted from the T cell pool of normal subjects Analysis

[0696] ELISPOT assays can be used to show that CLT antigen-specific CD8 T cells are present in the normal T cell pool of healthy individuals and therefore have not been lost due to central tolerance because the cancer-specific CLT antigen is expressed in naive and thymic tissues in these patients. This type of ELISPOT assay involves multiple steps. Step 1: CD8 T cells and CD14 monocytes can be isolated from the peripheral blood of normal donors and these cells are HLA typed to match the specific CLT antigen being tested. Using magnetically labeled antibodies against the memory marker CD45RO, CD8 T cells can be further subdivided into naive and memory subtypes. Step 2: CD14 monocytes are pulsed with independent or pooled CLT antigen peptides for three hours and then co-cultured with CD8 T cells for 14 days. Step 3: Expanded CD8 T cells are isolated from these cultures and restimulated overnight with fresh monocytes pulsed with peptides. These peptides can include; individual CLT antigen peptides, unrelated control peptides, or peptides known to stimulate robust responses against infectious antigens (e.g., CMV, EBV, Flu, HCV) or self-antigens (e.g., Mart-1). Re-stimulation is performed on a plate coated with anti-interferon gamma (IFNγ) antibodies. The antibody captures any IFNγ secreted by peptide-stimulated T cells. After overnight activation, the cells are washed off the plate and the IFNγ captured on the plate is detected with other anti-IFNγ antibodies and standard colorimetric dyes. If the cells producing IFNγ are initially on the plate, dark spots are left. Data derived from this type of analysis include point counts, point median size, and point median intensity. These data measure the frequency of T cells producing IFNγ and the amount of IFNγ per cell. In addition, the measurement of the CLT antigen response amplitude can be derived from the stimulation index (SI), which is a specific response, measured in point counts or point median size, divided by the background response to monocytes in the absence of a specific peptide. The measurement of the stimulation intensity is derived by multiplying the stimulation index of the number of points by the stimulation index of the point intensity. In this way, comparison of responses to CLT antigens with responses to control antigens can be used to demonstrate that treatment-naive subjects contain a robust CLT antigen-reactive T cell repertoire that can be expanded by vaccination with an immunogenic formulation based on the CLT antigen. Table 9 provides a series of CLT antigen-derived peptides that induced significant CD8 T cell responses from HLA-matched normal blood donors. Figure 53-Figure 56 Results are shown in Figure 2. Horizontal scales represent the mean of the data. Statistical significance was measured using one-way ANOVA with Kruskall-Wallis test and Dunns correction for repeated measures. Fig.53 Significant CD8 T cell responses from normal blood donors are shown against HLA-A*0201 restricted peptides from CLT antigen 1 (CLT001 in this figure). Fig.54The example shown in shows a CD8 response from a normal donor to a peptide derived from CLT antigen 2 (CLT002 ​​in this figure), which is also restricted by HLA-A*0201. Fig.55 Significant CD8 T cell responses from normal blood donors are shown against HLA-A*0201 restricted peptides from CLT antigen 4 (CLT004 in this figure). Fig.56 The lack of responses to HLA-B*0702 restricted peptides from CLT antigens 1 and 4 (CLT001 and CLT004 in this figure) in memory CD45RO positive CD8 T cells is shown (Panels A and C). In contrast, naive CD45RO negative CD8 T cells from the same donor responded significantly to peptides from CLT001 and CLT004 ( Fig.56 , Panel B and Panel D).

[0697] Table 9: A list of CLT antigen-derived peptides that induced significant CD8 T cell responses from HLA-matched normal blood donors.

[0698]

[0699]

[0700] Example 5 - Staining of reactive T cells containing CLT antigen peptide pentamer

[0701] The presence and activity of CLT antigen-specific circulating CD8 T cells in healthy donors and melanoma patients can be measured by using HLA class I / peptide-pentamer ("pentamer") staining and / or in vitro killing assays. Thus, these methods are applied to the CLT antigens discovered using the methods set forth in Examples 1, 2, and 2.1 (Tables 1-6, Figure 1-Figure 50 ) can be used to demonstrate the presence of therapeutically meaningful T cell responses against CLT antigens in cancer patients.

[0702] For these studies, CD8 T cells isolated from the blood of healthy donors or patients are expanded using various culture methods, such as anti-CD3 and anti-CD28 coated microbeads plus interleukin-2. The expanded cells can then be stained for specific CLT antigen reactivity of their T cell receptors using CLT peptide pentamers, which consist of pentamers of HLA class I molecules bound to the relevant CLT antigen peptide in the peptide-binding groove of the HLA molecule. Binding is measured by detection with phycoerythrin-conjugated or allophycocyanin-conjugated antibody fragments specific for the coiled-coil multimerization domain of the pentamer structure. In addition to pentamer staining, other surface markers can be interrogated, such as the memory marker CD45RO and the lysosomal release marker CD107a. The association of pentamer positivity with specific surface markers can be used to infer the number and status of the pentamer-reactive T cell population (memory cells vs. naive cells / stem cells)

[0703] The pentamer-stained cells may also be sorted and purified using fluorescence activated cell sorting (FACS). The sorted cells may then be further tested for their ability to kill target cells in in vitro killing assays. These assays involve a population of CD8 T cells and a population of fluorescently labeled target cells. In this case, the CD8 population is specific for the CLT antigen or has been sorted with CD8 T cells pentamer specific for a positive control antigen known to induce a strong killing response such as Mart-1. Target cells for these studies may include T2 cells pulsed with peptides expressing HLA-A*02, C1R cells pulsed with peptides transfected with HLA-A*02, 03, or B*07, or melanoma cell lines or patient tumor cells previously shown to express CLT / CLT antigen. Peptides used to pulse label T2 cells or C1R cells include CLT antigen peptides or positive control peptides. Target cells may be fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE, a cell proliferation dye) and death is indicated by uptake of 7AAD. In this way, as target cells undergo CD8 T cell-mediated apoptotic killing, they acquire red fluorescence and become red / green double positive. Therefore, the application of this type of 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 ex vivo cultures of T cells from melanoma patients or healthy donors. Fig.57 Shown are HLA pentamer staining of healthy donor CD8 T cells with peptides derived from CLT antigens 1, 2 and 4 (CLT001, CLT002 ​​and CLT004 in this figure). Fig.58 Expanded CLT004 pentamer sorted cells are shown to kill CLT004 pulsed C1R-B7 target cells. Significant killing of peptide pulsed C1R-B7 cells was evident at both 3:1 and 1:1 effector cell to target cell ratios.

[0704] Example 6 - Immunogenicity studies in mice

[0705] To demonstrate the immunogenicity of CLT antigens, mice can be inoculated with replication-defective adenoviral vectors 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 IFN ELISPOT assay (Mennuni et al., Int. J. Cancer, 2005). Briefly, saplings are inoculated with recombinant adenovirus expressing CLT antigens, humanely euthanized at appropriate time points, and splenocyte preparations are injected into wells of multiwell culture dishes derivatized with monoclonal antibodies against murine IFNs in the presence (or absence) of overlapping peptides corresponding to CLT antigens. After an appropriate time, the fixed IFNs are stained with different monoclonal antibodies, allowing Count cells / spots , which are then compared to the total cells loaded into the well to generate a quantitative readout of CLT antigen-reactive T cells.

[0706] Example 7 - Analysis to validate CLT expression in melanoma cells

[0707] a) qRT-PCR validation of CLT expression in melanoma cell lines

[0708] Quantitative real-time polymerase chain reaction (qRT-PCR) is a universal technique for determining the number of specific transcripts present in RNA extracted from a given biological sample. Specific nucleic acid primer sequences are designed for the transcript of interest, and the region between the primers is subsequently amplified by a series of thermal cycling reactions and quantified fluorescently by using an intercalating dye (SYBR Green). Primer pairs were designed for CLT and analyzed for RNA extracted from melanoma cell lines. Non-melanoma cell lines were used as negative controls. Specifically, melanoma cell lines COLO 829 (ATCC reference CRL-1974), MeWo (ATCC reference HTB-65), SH-4 (ATCC reference CRL-7724) and control cell lines HepG2 (hepatocellular carcinoma, ATCC reference HB-8065), Jurkat (T cell leukemia, ATCC reference TIB152) and MCF7 (adenocarcinoma, ATCC reference HTB-22) were expanded in vitro and 1x10 6 RNA was extracted from frozen cells and reverse transcribed into cDNA. Following standard techniques, qRT-PCR analysis was performed with primers designed for two regions of each CLT and reference genes together with SYBR Green detection. Relative quantification (RQ) was calculated as:

[0709] RQ = 2[Ct(reference) - Ct(target)].

[0710] Fig.59 The results of these experiments are shown in Figure 2. Panel A shows the results of RNA extracted from three melanoma cell lines and four non-melanoma cell lines by qRT-PCR analysis, and the analysis adopts two primer sets (1+2 and 3+4) of different regions of CLT (SEQ ID33) encoding CLT antigen 1. Panel B shows the results of RNA extracted from three melanoma cell lines and four non-melanoma cell lines by qRT-PCR analysis, and the analysis adopts two primer sets (5+6 and 7+8) of different regions of CLT (SEQ ID 34) encoding CLT antigen 2. Panel C shows the results of RNA extracted from three melanoma cell lines and four non-melanoma cell lines by qRT-PCR analysis, and the analysis adopts two primer sets (9+10 and 11+12) of different regions of CLT (SEQ ID 35) encoding CLT antigen 3 / 4. These results confirm that CLT is specifically expressed in RNA extracted from melanoma cell lines compared with non-melanoma cells. CLT is detected in each melanoma cell line tested.

[0711] b) RNAScope validates CLT expression in melanoma cells in situ

[0712] The in situ hybridization (ISH) method of transcript expression analysis allows visualization of the presence and expression level of a given transcript within the histopathological background of a specimen. Traditional RNA ISH analysis includes in situ recognition of natural RNA molecules with oligonucleotide probes specific for short segments of expected RNA sequences, and the recognition is visualized by signals generated by a combination of antibody- or enzyme-based colorimetric reactions. RNAScope is a recently developed technique based on in situ hybridization that uses more advanced probe chemistry, which ensures the specificity of the signals generated and allows sensitive, single-molecule visualization of target transcripts (Wang et al. 2012 J Mol Diagn. 14 (1): 22-29). Positive staining of transcript molecules appears as small red dots in a given cell, with multiple dots representing the presence of multiple transcripts.

[0713] RNAScope probes were designed for CLT and sections of 12 formalin-fixed, paraffin-embedded skin melanoma tumor cores were analyzed. Representative images from each core were scored for expression signal as follows:

[0714] Estimated % of cells positively stained with CLT probe, rounded to the nearest 10

[0715] The estimated per-cell level across a given slice is expressed as:

[0716] 0 = No staining

[0717] 1 = 1-2 spots per cell

[0718] 2 = 2-6 spots per cell

[0719] 3 = 6-10 spots per cell

[0720] 4 = >10 spots per cell

[0721] Expression of each CLT was detected across multiple different patient tumor cores, independently validating the discovery of CLTs from tumor-derived RNAseq data and confirming the uniformity of expression within tumor tissue across certain samples, and also highlighting the presence of at least one CLT in each patient tumor core analyzed.

[0722] Table 10 - RNAScope assessment in melanoma patient tissue cores

[0723]

[0724] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprising" and "including" will be understood to mean the inclusion of stated integers, steps, groups of integers or groups of steps, but will not exclude any other integers, steps, groups of integers or groups of steps.

[0725] All patents, patent applications, and references mentioned throughout the present specification are incorporated herein by reference in their entirety.

[0726] The present invention includes all combinations of the preferred groups and more preferred groups and suitable groups and further groups and embodiments of the groups mentioned above.

[0727] Sequence Listing

[0728] SEQ ID NO.1 (polypeptide sequence of CLT antigen 1)

[0729] MWNFFRRELTSNGFPENFSLDVPANTYNALKSRLCDPNADHTSCPSPCSLHAAGALPGTGRQRWRVELAHLADRKLSLRDVSRLRQGGERRSGIAVKVVRGGAGFAARLQGSVTLVQQGWFFPRLGGCQAWWRMGAVVWCGELLTCTS

[0730] SEQ ID NO.2 (polypeptide sequence of CLT antigen 2)

[0731] MTGVLIRRGDLVTDMVACRIKTFRGHTEKAAICKTRKESSSAETSPADSLILDFQPLQLMSSFSTLASLDK

[0732] SEQ ID NO.3 (polypeptide sequence of CLT antigen 3)

[0733] MNTPNIVSLRAHQPEVGIIPSVLLMRPLRIKGVFHHIHSPLHGENQGFTLCLQGAPPSSSV

[0734] SEQ ID NO.4 (polypeptide sequence of CLT antigen 4)

[0735] MAKTKGSLSVFRELHPAAAFDRAVHFLFLELWLPEPMLSSSPPSSTAPLLGSEPLRHWEASLSR

[0736] SEQ ID NO.5 (polypeptide sequence of CLT antigen 5)

[0737] MKRKANRWRLSLRNGLLPSTPRATQQIPMEFLNSRVADIPIKPW

[0738] SEQ ID NO.6 (polypeptide sequence of CLT antigen 6)

[0739] MRGFLWRVETRGVEGSMRGPQKVLGNRLPGAGRNARSRDNFAVW

[0740] SEQ ID NO.7 (polypeptide sequence of CLT antigen 7)

[0741] MVYYGNPESSPGISLVFGLLRLDRMQPGFSVSQEGDPVGITDHLGC

[0742] SEQ ID NO.8 (polypeptide sequence of CLT antigen 8)

[0743] MPAQLKFTLQVNPATKMRVTLLSQPMETYEGDVLGVQTPYSSDSTILVL

[0744] SEQ ID NO.9 (polypeptide sequence of CLT antigen 9)

[0745] MGSSRVGERMMEEESRTGQKVNPGNTGKLFVGVGISRIAKVKYGECGQGFSDKSDVITHQRTHTGGKPYVCRECGRALAGSQTSSVTRGHTQGRSLMSAESVSGALAGSQSSSFTRGHTGETPQSAGRMSKSLVIKPYLNSHKKTNVITTHLHTPALRWLQRKSANPLHSPRV

[0746] SEQ ID NO.10 (polypeptide sequence of CLT antigen 10)

[0747] MHSLQIFSLCLFTLLIVSFIVQKPFNLIRSNLSIFLLVEIAFEDLVMNYLPKLTSRRVFSRFSYMTFYSFRSYIKVFVSSQIDFFSLVKGRGPVQAHFSMWFCYSG

[0748] SEQ ID NO.11 (peptide sequence derived from CLT antigen 1)

[0749] VQQGWFFPR

[0750] SEQ ID NO.12 (peptide sequence derived from CLT antigen 1)

[0751] VVRGGAGFAAR

[0752] SEQ ID NO.13 (peptide sequence derived from CLT antigen 1)

[0753] HLADRKLSL

[0754] SEQ ID NO 14 (peptide sequence derived from CLT antigen 1)

[0755] QGSVTL

[0756] SEQ ID NO.15 (peptide sequence derived from CLT antigen 2)

[0757] ADSLILDF

[0758] SEQ ID NO.16 (peptide sequence derived from CLT antigen 2)

[0759] SSFSTLASLDK

[0760] SEQ ID NO.17 (peptide sequence derived from CLT antigen 3)

[0761] NTPNIVSLR

[0762] SEQ ID NO.18 (peptide sequence derived from CLT antigen 3)

[0763] QPEVGIIPSVLLMRP

[0764] SEQ ID NO.19 (peptide sequence derived from CLT antigen 4)

[0765] KTKGSLSVFR

[0766] SEQ ID NO.20 (peptide sequence derived from CLT antigen 5)

[0767] RVADIPIKPW

[0768] SEQ ID NO.21 (peptide sequence derived from CLT antigen 5)

[0769] DIPIKPW

[0770] SEQ ID NO.22 (peptide sequence derived from CLT antigen 5)

[0771] RVADIPIKP

[0772] SEQ ID NO.23 (peptide sequence derived from CLT antigen 6)

[0773] RSRDNFAVW

[0774] SEQ ID NO.24 (peptide sequence derived from CLT antigen 6)

[0775] RSRDNFA

[0776] SEQ ID NO.25 (peptide sequence derived from CLT antigen 7)

[0777] SPGISLVF

[0778] SEQ ID NO.26 (peptide sequence derived from CLT antigen 8)

[0779] SSDSTILVL

[0780] SEQ ID NO.27 (peptide sequence derived from CLT antigen 9)

[0781] AGRMSKSLVIK

[0782] SEQ ID NO.28 (peptide sequence derived from CLT antigen 9)

[0783] PQSAGRM

[0784] SEQ ID NO.29 (peptide sequence derived from CLT antigen 9)

[0785] AGSQSSSFTRGHTGETPQ

[0786] SEQ ID NO.30 (peptide sequence derived from CLT antigen 10)

[0787] RSNLSIFL

[0788] SEQ ID NO.31 (peptide sequence derived from CLT antigen 10)

[0789] FSLCLFTL

[0790] SEQ ID NO.32 (peptide sequence derived from CLT antigen 10)

[0791] SRRVFSRFSYMTFYSFRSYIKVFV

[0792] SEQ ID NO.33 (cDNA sequence of CLT encoding CLT antigen 1)

[0793]

[0794] SEQ ID NO.34 (cDNA sequence of CLT encoding CLT antigen 2)

[0795]

[0796] SEQ ID NO.35 (cDNA sequence of CLT encoding CLT antigens 3 and 4)

[0797]

[0798] SEQ ID NO 36 (cDNA sequence of CLT encoding CLT antigen 5)

[0799]

[0800] SEQ ID NO 37 (cDNA sequence of CLT encoding CLT antigen 6)

[0801]

[0802] SEQ ID NO 38 (cDNA sequence of CLT encoding CLT antigens 7 and 8)

[0803]

[0804] SEQ ID NO 39 (cDNA sequence of CLT encoding CLT antigen 9)

[0805]

[0806] SEQ ID NO 40 (cDNA sequence of CLT encoding CLT antigen 10)

[0807]

[0808] SEQ ID NO.41 (cDNA sequence encoding CLT antigen 1)

[0809] ATGTGGAACTTCTTCAGGAGAGAATTAACATCCAATGGATTCCCAGAAAACTTTTCCCTCGATGTACCAGCAAACACCTACAATGCCCTGAAAAGCCGCCTCTGCGACCCCAATGCAGATCACACGTCCTGTCCCAGCCCCTGCAGCCTCCACGCGGCGGGTGCACTGCCAGGCACGGGAAGGCAGCGCTGGCGAGTAGAACTGGCCCATCTCGCAGATAGGAAGCTGAGCCTCAGGGACGTTTCACGCCTTCGTCAAGGTGGTGAGAGGAGGAGCGGGATTGCCGTGAAGGTGGTGAGAGGAGGAGCGGGGTTTGCTGCCCGACTTCAGGGATCTGTCACCCTCGTCCAGCAGGGTTGGTTCTTCCCGAGGCTGGGAGGATGCCAAGCCTGGTGGAGGATGGGGGCGGTGGTGTGGTGTGGGGAGCTTCTGACTTGCACATCC

[0810] SEQ ID NO.42 (cDNA sequence encoding CLT antigen 2)

[0811] ATGACTGGTGTTCTTATAAGAAGAGGAGATTTGGTCACAGACATGGTTGCATGCAGAATAAAGACTTTTCGAGGACACACTGAGAAGGCAGCCATCTGCAAAACAAGGAAAGAGTCCTCAGCAGAAACCAGTCCTGCAGACTCCTTGATCTTGGACTTCCAGCCACTGCAATTGATGTCAAGCTTCAGCACCCTTGCATCTCTGGATAAA

[0812] SEQ ID NO.43 (cDNA sequence encoding CLT antigen 3)

[0813] ATGAATACTCCTAACATTGTCTCTTTAAGAGCTCACCAGCCTGAGGTAGGAATCATTCCATCTGTGTTACTAATGAGACCGCTGAGGATCAAAGGGGTTTTCCACCACATCCACTCACCTCTACATGGCGAAAACCAAGGGTTCACTCTCTGTCTTCAGGGAGCTCCACCCAGCAGCAGCGTT

[0814] SEQ ID NO.44 (cDNA sequence encoding CLT antigen 4)

[0815] ATGGCGAAAACCAAGGGTTCACTCTCTGTCTTCAGGGAGCTCCACCCAGCAGCAGCGTTTGACAGAGCTGTTCACTTCCTCTTCCTGGAGCTGTGGCTTCCAGAGCCCATGCTCAGCAGTTCCCCTCCTTCTTCGACTGCTCCTCTCTTAGGCTCAGAGCCACTCAGACATTGGGAAGCAAGTTTGTCAAGA

[0816] SEQ ID NO.45 (cDNA sequence encoding CLT antigen 5)

[0817] ATGAAGAGGAAAGCAAACAGGTGGAGACTCAGCCTGAGAAATGGTCTGTTGCCAAGCACACCCAGAGCTACCCAACAGATTCCTATGGAGTTCTTGAATTCCAGGGTGGCGGATATTCCAATAAAGCCATGG

[0818] SEQ ID NO 46 (cDNA sequence encoding CLT antigen 6)

[0819] ATGCGTGGATTTCTGTGGAGAGTGGAAACACGGGGAGTCGAGGGGAGCATGCGCGGGCCTCAGAAAGTTCTGGGAAACCGACTCCCGGGAGCAGGGAGGAACGCGCGCTCCAGAGACAACTTCGCGGTGTGG

[0820] SEQ ID NO 47 (cDNA sequence encoding CLT antigen 7)

[0821] ATGGTTTATTATGGGAATCCTGAATCCAGCCCAGGGATTTCTCTTGTCTTTGGCCTTCTACGGCTGGACAGGATGCAGCCTGGGTTTTCAGTCTCCCAGGAAGGAGATCCAGTGGGAATCACTGACCACCTCGGCTGC

[0822] SEQ ID NO 48 (cDNA sequence encoding CLT antigen 8)

[0823] ATGCCAGCACAATTGAAATTCACACTGCAAGTGAATCCTGCAACAAAAATGAGGGTGACCCTGCTCTCCCAACCCATGGAGACCTATGAAGGGGATGTGCTGGGGGTCCAGACCCCATATTCCTCAGACTCAACAATTCTTGTTCTT

[0824] SEQ ID NO 49 (cDNA sequence encoding CLT antigen 9)

[0825] ATGGGGAGCTCTAGAGTGGGAGAAAGAATGATGGAAGAAGAGTCCAGAACAGGCCAGAAAGTGAATCCAGGGAACACAGGCAAATTATTTGTGGGGGTAGGAATCTCAAGAATTGCGAAAGTCAAATATGGAGAGTGTGGGCAAGGTTTCAGTGATAAGTCAGATGTTATTACACACCAAAGGACACACACAGGGGGGAAGCCCTACGTCTGCAGAGAGTGTGGGAGGGCTTTAGCCGGAAGTCAGACCTCCTCAGTCACCAGAGGACACACACAGGGGAGAAGCCTTATGTCTGCAGAGAGTGTGAGCGGGGCTTTAGCCGGAAGTCAGTCCTCCTCATTCACCAGAGGACACACAGGGGAGACGCCCCAGTCTGCAGGAAGGATGAGTAAGTCATTAGTAATAAAACCTTATCTCAATAGCCACAAGAAGACAAACGTGATCACCACACACTTGCACACCCCAGCTCTGAGGTGGCTTCAGCGAAAGTCTGCTAACCCCTTACATTCCCCGAGAGTG

[0826] SEQ ID NO 50 (cDNA sequence encoding CLT antigen 10)

[0827] ATGCATAGTTTGCAAATATTTTCTCTCTGTCTGTTTACCCTGTTGATAGTTTCTTTTATTGTGCAGAAACCCTTTAATTTAATCAGGTCCAACTTATCCATTTTTCTTTTGGTTGAAATTGCTTTTGAGGACTTAGTTATGAATTATTTGCCAAAGCTG ACGTCAAGAAGGGGTATTCTCTAGGTTTTCTTATATGACATTTTATAGTTTTAGGTCTTATATTAAAGTCTTTGTTTCATCTCAAATTGACTTTTTTTCTCTGGTAAAAGGTAGGGGTCCAGTTCAAGCTCATTTTTCCATGTGGTTTTGTTACAGTGGG

[0828] SEQ ID NO.51 (peptide sequence derived from CLT antigen 4)

[0829] AAFDRAVHF

[0830] SEQ ID NO.52 (peptide sequence derived from CLT antigen 4)

[0831] AFDRAVHF

[0832] SEQ ID NO.53 (peptide sequence derived from CLT antigen 3)

[0833] RPLRIKGVF

[0834] SEQ ID NO.54 (peptide sequence derived from CLT antigen 4)

[0835] KTKGSLSVF

[0836] SEQ ID NO.55 (peptide sequence derived from CLT antigen 1)

[0837] VPANTYNALK

[0838] SEQ ID NO.56 (peptide sequence derived from CLT antigen 1)

[0839] RLGGCQAWWR

[0840] SEQ ID NO.57 (peptide sequence derived from CLT antigen 1)

[0841] ANTYNALKSR

[0842] SEQ ID NO.58 (peptide sequence derived from CLT antigen 2)

[0843] LVTDMVACRI

[0844] SEQ ID NO.59 (peptide sequence derived from CLT antigen 2)

[0845] LILDFQPLQL

[0846] SEQ ID NO.60 (peptide sequence derived from CLT antigen 2)

[0847] MSSFSTLASL

[0848] SEQ ID NO.61 (peptide sequence derived from CLT antigen 2)

[0849] LMSSFSTLASL

[0850] SEQ ID NO.62 (peptide sequence derived from CLT antigen 2)

[0851] LMSSFSTLA

[0852] SEQ ID NO.63 (peptide sequence derived from CLT antigen 2)

[0853] QLMSSFSTLA

[0854] SEQ ID NO.64 (peptide sequence derived from CLT antigen 2)

[0855] MVACRIKTFR

[0856] SEQ ID NO.65 (peptide sequence derived from CLT antigen 2)

[0857] VTDMVACRIK

[0858] SEQ ID NO.66 (peptide sequence derived from CLT antigen 2)

[0859] SPADSLIL

[0860] SEQ ID NO.67 (peptide sequence derived from CLT antigen 3)

[0861] NTPNIVSLRA

[0862] SEQ ID NO.68 (peptide sequence derived from CLT antigen 3)

[0863] VLLMRPLRIK

[0864] SEQ ID NO.69 (peptide sequence derived from CLT antigen 3)

[0865] MRPLRIKGVF

[0866] SEQ ID NO.70 (peptide sequence derived from CLT antigen 4)

[0867] FLFLELWL

[0868] SEQ ID NO.71 (peptide sequence derived from CLT antigen 4)

[0869] SVFRELHPA

[0870] SEQ ID NO.72 (peptide sequence derived from CLT antigen 4)

[0871] SPPSSTAPL

[0872] SEQ ID NO.73 (peptide sequence derived from CLT antigen 1)

[0873] RLQGSVTLV

[0874] SEQ ID NO.74 (peptide sequence derived from CLT antigen 1)

[0875] VPANTYNAL

[0876] SEQ ID NO.75 (peptide sequence derived from CLT antigen 2)

[0877] QLMSSFSTL

[0878] SEQ ID NO.76 (peptide sequence derived from CLT antigen 4)

[0879] FLELWLPEPML

[0880] SEQ ID NO.77 (peptide sequence derived from CLT antigen 4)

[0881] APLLGSEPL

[0882] SEQ ID NO.78 (peptide sequence derived from CLT antigen 2)

[0883] SLILDFQPL

Claims

1. An isolated polypeptide comprising a sequence selected from the group consisting of: (a) the sequence of any one of SEQ ID NOs. 1-10; and (b) a variant of the sequence of (a); and (c) Immunogenic fragments of the sequence of (a).

2. An isolated nucleic acid encoding the polypeptide according to claim 1.

3. A vector comprising the nucleic acid according to claim 2.

4. An immunogenic pharmaceutical composition comprising the polypeptide, nucleic acid or vector according to any one of claims 1 to 3 together with a pharmaceutically acceptable carrier.

5. A vaccine composition comprising the polypeptide, nucleic acid or vector according to any one of claims 1 to 3 together with a pharmaceutically acceptable carrier.

6. A method of increasing an immune response in a human, comprising administering to the human a polypeptide, a nucleic acid, a vector or a composition according to any one of claims 1 to 5.

7. A method for treating a human patient suffering from cancer, wherein the cancer cells express a gene selected from the group consisting of SEQ ID NO. 1-10 and immunogenic fragments and variants of any one of them, or a method for preventing a human from developing cancer, the cancer expressing a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one of them, the method comprising administering to the human a corresponding polypeptide, nucleic acid, vector or composition according to any one of claims 1 to 5.

8. A method for treating cancer in a human, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, comprising obtaining a population of leukocytes comprising at least T cells, optionally together with antigen presenting cells, from the human, stimulating and / or expanding the T cells in the presence of a corresponding polypeptide, nucleic acid, vector or composition according to any one of claims 1 to 5, and reintroducing some or all of the leukocytes, at least the stimulated and / or expanded T cells, into the human.

9. A method for preparing a T cell population that is cytotoxic to cancer cells, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, the method comprising (a) obtaining T cells, optionally together with antigen presenting cells, from a cancer patient; and (ii) stimulating and expanding the T cell population ex vivo with a corresponding polypeptide, nucleic acid, vector or composition according to any one of claims 1 to 5.

10. A T cell population obtainable by the method according to claim 9.

11. T cells which have been stimulated with the polypeptide, nucleic acid, vector or composition according to any one of claims 1 to 5.

12. An antigen presenting cell which has been modified or genetically engineered to express the polypeptide according to claim 1 by ex vivo loading with the polypeptide, nucleic acid, vector or composition according to any one of claims 1 to 5.

13. Exosomes loaded with a polypeptide prepared from a cell loaded with a polypeptide, nucleic acid, vector or composition according to any one of claims 1 to 5 or genetically engineered to express a polypeptide according to claim 1.

14. A pharmaceutical composition comprising the T cell population, T cell, antigen presenting cell or exosome according to any one of claims 10 to 13 together with a pharmaceutically acceptable carrier.

15. A method for treating a human with cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, or a method for preventing a human from developing cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, the method comprising administering to the human a T cell population, T cell, antigen presenting cell, exosome or composition according to any one of claims 10 to 14.

16. An isolated antigen-binding polypeptide immunospecific for the polypeptide of claim 1.

17. A pharmaceutical composition comprising the antigen-binding polypeptide according to claim 16 together with a pharmaceutically acceptable carrier.

18. A method for treating a human with cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, or a method for preventing a human from developing cancer, wherein the cancer cells express a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one thereof, the method comprising administering to the human an antigen-binding polypeptide or composition according to any one of claims 16 and 17.

19. An isolated antigen-binding polypeptide immunospecific for an HLA-binding polypeptide which is a polypeptide according to claim 1 or a portion thereof.

20. A cytotoxic cell which has been engineered to express on its surface the antigen-binding polypeptide of any one of claim 19.

21. A pharmaceutical composition comprising the cell according to claim 20.

22. A method for treating a human patient suffering from cancer, wherein the cancer cells express a gene selected from the group consisting of SEQ ID NO. 1-10 and immunogenic fragments and variants of any one of them, or a method for preventing a human from developing cancer, the cancer expressing a sequence selected from SEQ ID NO. 1-10 and immunogenic fragments and variants of any one of them, the method comprising administering a cell according to claim 20 to the human.

23. A method of diagnosing a person as having cancer, comprising the steps of: Determine whether the cancer cells express a polypeptide sequence selected from SEQ ID NO. 1-10 and an immunogenic fragment or variant of any one of them, or a nucleic acid encoding the polypeptide sequence, and diagnose the human as having cancer if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells.

24. A method for diagnosing a person with skin melanoma, comprising the steps of: determining whether cells of the cancer express a polypeptide sequence of any one of SEQ ID NO. 2, 6, 7, 8 and 9 and an immunogenic fragment or variant thereof; or a nucleic acid encoding the polypeptide sequence, and if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, diagnosing the person with skin melanoma.

25. A method for diagnosing a person with cutaneous melanoma or uveal melanoma, comprising the steps of: determining whether cells of the cancer express a polypeptide sequence selected from any one of SEQ ID NO. 1, 3, 4, 5 and 10 and an immunogenic fragment or variant of any one of them; or a nucleic acid encoding the polypeptide sequence, and if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, diagnosing the person with cutaneous melanoma or uveal melanoma.

26. A method of treating a human suffering from cancer comprising the steps of: (a) determining whether the cancer cells express a polypeptide sequence selected from SEQ ID NO. 1-10 and an immunogenic fragment or variant of any one of them or a nucleic acid encoding the polypeptide sequence (e.g., a sequence selected from SEQ ID NO. 33-40 and 41-50); and if expressed, (b) administering 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 claims 1 to 25 to the human.

27. Use of a polypeptide comprising a sequence selected from the group consisting of: (a) the sequence of any one of SEQ ID NOs. 1-10; or (b) a variant of the sequence of (a); and (c) Use of an immunogenic fragment of the sequence of (a), isolated from a tumor of a human suffering from cancer, or a nucleic acid encoding the polypeptide, as a biomarker for determining whether the human will be suitable for treatment with a vaccine, the 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 claims 1 to 25.

28. An isolated nucleic acid encoding the fusion polypeptide of claim 27.

29. A vector comprising the nucleic acid according to claim 28.

Images