New cancer antigens and methods

Abstract

In particular, polypeptides and nucleic acids encoding the same useful for the treatment, prevention and diagnosis of cancer, in particular melanoma, especially cutaneous melanoma and uveal melanoma are disclosed.

Description

Invention Field

[0001] This invention relates to antigenic polypeptides and corresponding polynucleotides for the treatment or prevention of cancer, particularly for the treatment or prevention of melanoma (e.g., cutaneous melanoma or uveal melanoma). The invention further relates, particularly, to pharmaceutical compositions and immunogenic compositions comprising said nucleic acids and polypeptides, immune cells loaded with said polypeptides and polynucleotides and / or stimulated thereon, antibodies specific to said polypeptides, and molecularly engineered (autologous or other) cells for recognizing said polypeptides. Background of the Invention

[0003] As part of normal immune surveillance against pathogenic microorganisms, all cells degrade intracellular proteins to produce peptides that are loaded onto major histocompatibility complex (MHC) class I molecules expressed on the surface of all cells. Most of these peptides, derived from host cells, are considered self and remain invisible to the adaptive immune system. However, foreign (non-self) peptides can stimulate the expansion of naïve CD8+ T cells that encode T cell receptors (TCRs) that tightly bind to the MHC I-peptide complex. This expanded population of T cells can generate effector CD8+ T cells (including cytotoxic T-lymphocytes-CTLs) that can eliminate cells tagged with foreign antigens, as well as memory CD8+ T cells that can re-expand later in the animal's life as tagged cells with foreign antigens reappear.

[0004] MHC class II molecules, whose expression is normally limited to specialized antigen-presenting cells (APCs) such as dendritic cells (DCs), typically load peptides that have already been internalized from the extracellular environment. In the presence of various factors (including T cell adhesion molecules (CD54, CD48) and co-stimulatory molecules (CD40, CD80, CD86)), the binding of complementary TCRs from naive CD4+ T cells to the MHC II-peptide complex induces mature CD4+ T cells into effector cells (e.g., T cells). H 1. T H 2. T H 17. T FH T reg These effector CD4+ T- cells can promote the differentiation of B cells into antibody-secreting plasma cells and the differentiation of antigen-specific CD8+ CTLs, thus contributing to the induction of adaptive immune responses against foreign antigens, including short-term effector function and longer-term immune memory. Dendritic cells (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, facilitating the generation of immune memory by providing a bypass pathway to stimulate the expansion of naïve CD8+ T cells.

[0005] Immune memory (especially antigen-specific B cells / antibodies and antigen-specific CTLs) plays a crucial role in controlling microbial infections, and numerous vaccines have been developed using immune memory to prevent diseases caused by important pathogenic microorganisms. Immune memory is also known to play a key role in controlling tumorigenesis, but effective cancer vaccines are still scarce.

[0006] Cancer is the second leading cause of death worldwide, accounting for one-sixth of all deaths. In 2015, of the 8.8 million deaths caused by cancer, the majority were from lung cancer (1.69 million), liver cancer (788,000), colorectal cancer (774,000), stomach cancer (754,000), and breast cancer (571,000). The economic impact of cancer was estimated at US$1.16 trillion in 2010, and new cases are projected to increase by approximately 70% over the next two decades (World Health Organization Cancer Reality 2017).

[0007] 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 including lymph node dissection, radiation therapy, or chemotherapy. Immune checkpoint blockade strategies, including the use of antibodies targeting negative immunomodulators such as PD-1 / PD-L1 and CTLA4, have recently revolutionized the treatment of various malignancies, including melanoma (Ribas, A. and Wolchok, JD (2018) Science, 359:1350–1355). The extraordinary value of checkpoint blockade therapies and their clinical benefits, linked to patients' full acceptance of their adaptive immune responses to their own cancer antigens (based on specific T cells), have revolutionized research into effective cancer vaccines, vaccine modalities, and cancer vaccine antigens.

[0008] Human endogenous retroviruses (HERVs) are remnants of ancestral germline integrators of exogenous infectious retroviruses. HERVs belong to a group of endogenous retrotransmission elements characterized by the presence of long terminal repeats (LTRs) flanked by the viral genome. This group also includes mammalian epigenetic LTR retrotransposons (MaLRs) and is therefore collectively referred to as LTR elements (here collectively referred to as ERVs to mean all LTR elements). ERVs constitute a significant proportion of the mammalian genome (8%) and can be grouped into approximately 100 families based on sequence homology. Many ERV sequences encode defective proviruses that share a protoviral genome structure consisting of gag, pro, pol, and env genes flanked by LTRs. Some intact ERV ORFs produce retroviral proteins that share characteristics with proteins encoded by exogenous infectious retroviruses such as HIV-1. These proteins can act as antigens that induce strong immune responses (Hurst and Magiokinis, 2015, J. Gen. Virol 96:1207-1218), suggesting that ERV-encoded peptides may evade T-cell and B-cell receptor selection processes and central and peripheral tolerance. Immunoreactivity to ERV products can occur spontaneously in infections or cancers, and ERV products have been implicated as a cause of some autoimmune diseases (Kassiotis and Stoye, 2016, Nat. Rev. Immunol. 16:207-219).

[0009] Due to the accumulation of mutations and recombination events during evolution, most ERVs have lost some or all of their functional reading frames and thus their ability to produce infectious viruses. However, these ERV elements, like other genes, remain in germline DNA and still possess the potential to produce proteins from at least some of their genes. In fact, proteins encoded by HERV have been detected in a variety of human cancers. For example, spliced ​​variants of the HERV-K env gene, namely Rec and Np9, are found only in malignant testicular germ cells and not in healthy cells (Ruprecht et al., 2008, Cell Mol Life Sci 65:3366-3382). Elevated levels of HERV transcripts have also been observed in various cancers, such as prostate cancers, compared to healthy tissue (Wang-Johanning, 2003, Cancer 98:187-197; Andersson et al., 1998, Int. J. Oncol, 12:309-313). Furthermore, overexpression of HERV-E and HERV-H has been shown to have immunosuppressive effects, which may also contribute to carcinogenesis (Mangeney et al., 2001, J. Gen. Virol. 82:2515-2518). However, the exact mechanism by which HERV may contribute to carcinogenesis or pathogenicity remains unknown.

[0010] In addition to deregulating the expression of neighboring host genes, the activity of ERV regulatory elements and transposition to new genomic sites may lead to the production of new transcripts, some of which may have tumorigenic properties (Babaian and Mager, Mob. DNA, 2016; Lock et al., PNAS, 2014, 111:3534-3543).

[0011] A wide range of vaccine modalities are known. One well-described protocol involves the direct delivery of antigenic peptides to subjects to enhance immune responses (including B-cell and T-cell responses) and stimulate immune memory. Alternatively, polynucleotides can be administered to subjects via vectors, thereby expressing the immunogenic peptides encoded by the polynucleotides in vivo. The use of viral vectors (e.g., adenovirus vectors) for antigen delivery in prophylactic vaccination and therapeutic treatment strategies against cancer has been well explored (Wold et al., Current Gene Therapy, 2013, Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy, 13:421–433). Immunogenic peptides, polypeptides, or polynucleotides encoding them can also be used to load patient-derived antigen-presenting cells (APCs), which can then be infused into subjects as vaccines to elicit therapeutic or prophylactic immune responses. An example of this approach is Provenge, which is currently the only FDA-approved anticancer vaccine.

[0012] Various non-vaccine therapeutic modalities can also be generated using cancer antigens, which can be utilized in the treatment and prevention of cancer. These therapeutics are divided into two distinct categories: 1) antigen-binding biologics and 2) adoptive cell therapies.

[0013] Antigen-binding bioproducts generally consist of multivalent engineered peptides that recognize antigen-modified cancer cells and promote their destruction. The antigen-binding component of these bioproducts can be composed of TCR-based bioproducts, including but not limited to TCRs, high-affinity TCRs, and TCR mimics (including those based on monoclonal antibody technology) generated by various technologies. The cytolytic portion of these multivalent bioproducts can consist of cytotoxic chemicals, biotoxins, guiding motifs, and / or immunostimulatory motifs that promote the targeting and activation of immune cells, any of which promote the therapeutic destruction of tumor cells.

[0014] Adoptive cell therapy can be based on a patient’s own T cells, wherein the T cells are extracted and stimulated in vitro with a vaccine antigen preparation (and cultured with the T cells in the presence or absence of other factors, including cellular and non-cellular components) (Yossef et al. 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 engineered to express antigen-binding peptides that recognize cancer antigens. These antigen-binding peptides fall into the same category as described above for antigen-binding biological products. Thus, lymphocytes (autologous or non-autologous) that have been genetically manipulated to express cancer antigen-binding peptides can be administered to a patient as adoptive cell therapy for treating their cancer.

[0015] Elevated immune responses against cancer using ERV-derived antigens have shown promising results in mouse cancer models, promoting tumor regression and improving prognosis (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), although progress has been limited, partly due to the severe limitation of identified tumor-specific ERV antigens.

[0016] WO 2005 / 099750 identified anchored sequences in existing vaccines against infectious pathogens that share the common feature of enhancing cross-reactive immune responses against HERV-K Mel tumor antigens and conferring protection against melanoma.

[0017] WO 00 / 06598 relates to methods and products for identifying the HERV-AVL3-B tumor-associated gene preferentially expressed in melanoma, and for diagnosing and treating conditions characterized by the expression of said gene.

[0018] WO 2006 / 119527 discloses antigenic peptides derived from melanoma-associated endogenous retrovirus (MERV), and their use in the detection and diagnosis of melanoma and in disease prognosis. The use of the antigenic peptides as anticancer vaccines is also disclosed.

[0019] WO 2007 / 137279 discloses methods and compositions for detecting, preventing and treating HERV-K+ cancer, such as using HERV-K+ binding antibodies to prevent or inhibit cancer cell proliferation.

[0020] WO 2006 / 103562 discloses a method for treating or preventing cancer, wherein an immunosuppressive Np9 protein derived from the env gene of HERV-K is expressed. The invention also relates to pharmaceutical compositions comprising nucleic acids or antibodies capable of inhibiting the activity of said protein, or immunogenic or vaccine compositions capable of inducing an immune response against said protein.

[0021] WO 2007 / 109583 provides compositions and methods for preventing or treating neoplastic diseases in mammalian subjects by providing compositions comprising an enriched population of immune cells that are reactive to the HERV-E antigen on tumor cells.

[0022] Humer J et al., 2006, Canc.Res., 66:1658-63 identified melanoma markers derived from melanoma-associated endogenous retroviruses.

[0023] Further identification is needed for HERV-associated antigenic sequences that can be used for immunotherapy of cancers, particularly melanoma, especially cutaneous melanoma and uveal melanoma.

[0024] Invention Summary

[0025] The inventors have surprisingly discovered certain RNA transcripts containing LTR elements, which are present at high levels in cutaneous melanoma cells but undetectable or present at very low levels in normal healthy tissue (see Example 1). These transcripts are referred herein to as cancer-specific transLTR element transcripts (CLTs). Furthermore, the inventors have shown that subsets of potential polypeptide sequences (i.e., open reading frames (ORFs)) encoded by these CLTs are presented when translated in cancer cells, processed by components of an antigen processing apparatus, and presented on the surface of cells present in tumor tissue upon association with human leukocyte antigen class I (HLA class I) molecules (see Example 2). These results, by their very nature, demonstrate that these polypeptides (referred herein to as CLT antigens) are antigenic. Therefore, cancer cell presentation of CLT antigens is expected to make these cells susceptible to clearance by T cells carrying the same family T cell receptor (TCR) of the CLT antigen, and CLT antigen-based inoculation methods / treatment regimens that expand T cells carrying these same family TCRs are expected to elicit an immune response against cancer cells (and tumors containing them), particularly melanoma, especially cutaneous melanoma tumors. T cells from melanoma subjects were indeed reactive to peptides derived from the CLT antigen disclosed herein (see Example 3). The inventors have demonstrated that CLT antigen-specific T cells were not deleted from the T cell bank of normal subjects due to central tolerance (see Example 4). Finally, qRT-PCR studies have confirmed that CLT is specifically expressed in RNA extracted from melanoma tumor tissue, compared to non-melanoma control cell lines (see Example 5).

[0026] The inventors also made the surprising discovery that certain CLT antigens encoded by these CLTs, which are overexpressed in cutaneous melanoma, are also overexpressed in uveal melanoma. These CLT-encoded CLT antigen peptide sequences are expected to elicit an immune response against uveal melanoma cells and tumors containing them.

[0027] The CLT and CLT antigens, which are the subject of this invention, are not typical sequences that can be easily derived from known tumor genome sequences found in cancer genome atlases. CLTs are transcripts produced by complex transcriptional and splicing events driven by ERV-derived transcriptional control sequences. Because CLTs are expressed at high levels and because the CLT antigen polypeptide sequences are not normal human protein sequences, they are expected to be able to elicit strong specific immune responses and are therefore suitable for therapeutic use in cancer immunotherapy settings.

[0028] The CLT antigen, found in highly expressed transcripts characterizing tumor cells, can be used in several modalities, prior to which the presence of said antigen in humans and the production of protein products were unknown. First, the CLT antigen peptide of this invention can be directly delivered to a subject as a vaccine to elicit a therapeutic or prophylactic immune response against tumor cells. Second, the nucleic acid of this invention (which can be codon-optimized to enhance its expression encoding the CLT antigen) can be directly administered or additionally inserted into a vector for in vivo delivery to produce an encoded protein product in a subject as a vaccine to elicit a therapeutic or prophylactic immune response against tumor cells. Third, the polynucleotides and / or peptides of this invention can be used to load patient-derived antigen-presenting cells (APCs), which can then be infused into a subject as a vaccine to elicit a therapeutic or prophylactic immune response against cancer cells. Fourth, the polynucleotides and / or peptides of this invention can be used to stimulate T cells in a subject in vitro, producing a stimulated T cell preparation that can be administered to the subject as a therapeutic agent for treating cancer. Fifth, biomolecules that recognize CLT antigens complexed with MHC I molecules and have been further modified to allow them to kill (or promote the killing of) cancer cells (such as T-cell receptors (TCRs) or TCR mimics) can be administered to subjects as therapeutic agents for cancer treatment. Sixth, chimeric forms of biomolecules that recognize CLT antigens complexed with MHC cells can be introduced into (autologous or non-autologous) T cells, and the resulting cells can be administered to subjects as therapeutic agents for cancer treatment. These and other applications are described in more detail below.

[0029] Therefore, the present invention particularly provides an isolated polypeptide comprising a sequence selected from the following:

[0030] (a) The sequence of any one of SEQ ID NO. 1-8 and

[0031] Variations of the sequences (b)(a); and

[0032] Immunogenic fragments of sequences (c)(a)

[0033] (Hereinafter referred to as "the polypeptide of this invention").

[0034] 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").

[0035] The polypeptides and nucleic acids of the present invention, as well as related aspects of the present invention, are intended to be used in a range of embodiments for cancer immunotherapy and prevention, particularly for melanoma immunotherapy and prevention, as discussed in more detail below. Brief description of the attached diagram

[0037] Figure 1-14The upper inset shows the MS / MS spectrum of the extracted peptide (along with assigned fragment ions), obtained from a patient's tumor sample, and the lower inset shows the spectral presentation indicating the location of the linear peptide sequence, which has been localized to fragment ions.

[0038] Figure 1 Spectroscopy of the peptide SEQ ID NO.9 obtained from a tumor sample from patient Mel-27.

[0039] Figure 2 Spectroscopy of the peptide SEQ ID NO.10 obtained from a tumor sample from patient Mel-21.

[0040] Figure 3 Spectroscopy of the peptide SEQ ID NO.11 obtained from a tumor sample from patient Mel-41.

[0041] Figure 4 Spectroscopy of the peptide SEQ ID NO.12 obtained from a tumor sample from patient Mel-41.

[0042] Figure 5 Spectroscopy of the peptide SEQ ID NO.13 obtained from a tumor sample from patient Mel-41.

[0043] Figure 6 Spectroscopy of the peptide SEQ ID NO.14 obtained from a tumor sample from patient Mel-41.

[0044] Figure 7 Spectroscopy of the peptide SEQ ID NO.15 obtained from a tumor sample from patient Mel-21.

[0045] Figure 8 Spectroscopy of the peptide SEQ ID NO.16 obtained from a tumor sample from patient Mel-21.

[0046] Figure 9 Spectroscopy of the peptide SEQ ID NO.17 obtained from a tumor sample from patient Mel-21.

[0047] Figure 10 Spectroscopy of the peptide SEQ ID NO.18 obtained from a tumor sample from patient Mel-15.

[0048] Figure 11 Spectroscopy of the peptide SEQ ID NO.19 obtained from a tumor sample from patient Mel-27.

[0049] Figure 12 Spectroscopy of the peptide SEQ ID NO.20 obtained from a tumor sample from patient Mel-27.

[0050] Figure 13 Spectroscopy of the peptide SEQ ID NO.21 obtained from a tumor sample from patient Mel-25.

[0051] Figure 14 Spectroscopy of the peptide SEQ ID NO.22 obtained from a tumor sample from patient Mel-25.

[0052] Figure 15-28 Each figure in the image shows a comparison of the native MS / MS spectrum of a peptide obtained from a patient's tumor sample with the native spectrum of a synthetic peptide corresponding to the same sequence.

[0053] Figure 15 Spectroscopy of the peptide SEQ ID NO.9 obtained from a tumor sample from patient Mel-27.

[0054] Figure 16 Spectroscopy of the peptide SEQ ID NO.10 obtained from a tumor sample from patient Mel-20.

[0055] Figure 17 Spectroscopy of the peptide SEQ ID NO.11 obtained from a tumor sample from patient Mel-41.

[0056] Figure 18 Spectroscopy of the peptide SEQ ID NO.12 obtained from a tumor sample from patient Mel-41.

[0057] Figure 19 Spectroscopy of the peptide SEQ ID NO.13 obtained from a tumor sample from patient Mel-41.

[0058] Figure 20 Spectroscopy of the peptide SEQ ID NO.14 obtained from a tumor sample from patient Mel-41.

[0059] Figure 21 Spectroscopy of the peptide SEQ ID NO.15 obtained from a tumor sample from patient Mel-21.

[0060] Figure 22 Spectroscopy of the peptide SEQ ID NO.16 obtained from a tumor sample from patient Mel-21.

[0061] Figure 23 Spectroscopy of the peptide SEQ ID NO.17 obtained from a tumor sample from patient Mel-21.

[0062] Figure 24 Spectroscopy of the peptide SEQ ID NO.18 obtained from a tumor sample from patient Mel-15.

[0063] Figure 25 Spectroscopy of the peptide SEQ ID NO.19 obtained from a tumor sample from patient Mel-27.

[0064] Figure 26 Spectroscopy of the peptide SEQ ID NO.20 obtained from a tumor sample from patient Mel-27.

[0065] Figure 27 Spectroscopy of the peptide SEQ ID NO.21 obtained from a tumor sample from patient Mel-25.

[0066] Figure 28 Spectroscopy of the peptide SEQ ID NO.22 obtained from a tumor sample from patient Mel-25.

[0067] Figure 29 Spectroscopy of the peptide SEQ ID NO.15 obtained from a tumor sample from patient 2MT3.

[0068] Figure 30 Spectroscopy of the peptide SEQ ID NO.20 obtained from a tumor sample from patient 2MT4.

[0069] Figure 31 Spectroscopy of the peptide SEQ ID NO.21 obtained from a tumor sample from patient 2MT4.

[0070] Figure 32 The image shows expanded, pentamer-sorted CD8 T cells killing CaSki cells transfected with the read frame of CLT antigen 6 (SEQ ID NO. 6).

[0071] Figure 33 This shows a CD8 T cell response from a normal blood donor to the HLA-A*02:01 restriction peptide (SEQ ID NO. 9) from CLT antigen 1.

[0072] Figure 34 This shows the CD8 T cell response from a normal blood donor to the HLA-A*03:01 restriction peptide (SEQ ID NO. 10) from CLT antigen 1.

[0073] Figure 35 This shows the CD8 T cell response from a normal blood donor to the HLA-B*07:02 restriction peptide (SEQ ID NO. 13) from CLT antigen 2.

[0074] Figure 36 This shows the CD8 T cell response from a normal blood donor to the HLA-A*03:01 restriction peptide (SEQ ID NO. 15) from CLT antigen 3.

[0075] Figure 37 This shows the CD8 T cell response from a normal blood donor to the HLA-A*03:01 restriction peptide (SEQ ID NO. 18) from CLT antigen 5.

[0076] Figure 38 This shows the CD8 T cell response from a normal blood donor to the HLA-A*02:01 restriction peptide (SEQ ID NO. 39) from CLT antigen 6.

[0077] Figure 39 Figures A through C show the results of qRT-PCR analysis validating the transcription of CLT (SEQ ID NO. 24) encoding CLT antigen 2, CLT (SEQ ID NO. 25) encoding CLT antigen 3, and CLT (SEQ ID NO. 28) encoding CLT antigen 6 in melanoma cancer cell lines.

[0078] Sequence Description

[0079] SEQ ID NO.1 is the polypeptide sequence of CLT antigen 1.

[0080] SEQ ID NO.2 is the polypeptide sequence of CLT antigen 2.

[0081] SEQ ID NO.3 is the polypeptide sequence of CLT antigen 3.

[0082] SEQ ID NO.4 is the polypeptide sequence of CLT antigen 4.

[0083] SEQ ID NO.5 is the polypeptide sequence of CLT antigen 5.

[0084] SEQ ID NO.6 is the polypeptide sequence of CLT antigen 6.

[0085] SEQ ID NO.7 is the polypeptide sequence of CLT antigen 7.

[0086] SEQ ID NO.8 is the polypeptide sequence of CLT antigen 8.

[0087] SEQ ID NOs 9 and 10 are peptide sequences derived from CLT antigen 1; SEQ ID NOs 11-14 are peptide sequences derived from CLT antigen 2; SEQ ID NO 15 is a peptide sequence derived from CLT antigen 3; SEQ ID NOs 16 and 17 are peptide sequences derived from CLT antigen 4; SEQ ID NO 18 is a peptide sequence derived from CLT antigen 5; SEQ ID NOs 19 and 20 are peptide sequences derived from CLT antigen 6; SEQ ID NO 21 is a peptide sequence derived from CLT antigen 7; SEQ ID NO 22 is a peptide sequence derived from CLT antigen 8; SEQ ID NO 23 is a cDNA sequence encoding CLT of CLT antigen 1; SEQ ID NO 24 is a cDNA sequence encoding CLT of CLT antigen 2; SEQ ID NO 25 is a cDNA sequence encoding CLT of CLT antigen 3; SEQ ID NO 26 is a cDNA sequence encoding CLT of CLT antigen 4; and SEQ ID NO 27 is a cDNA sequence encoding CLT of CLT antigen 5. NO.28 is the cDNA sequence encoding CLT antigen 6 (SEQ ID NO.29), the cDNA sequence encoding CLT antigen 7 (SEQ ID NO.30), the cDNA sequence encoding CLT antigen 8 (SEQ ID NO.31), the cDNA sequence encoding CLT antigen 1 (SEQ ID NO.32), the cDNA sequence encoding CLT antigen 2 (SEQ ID NO.33), the cDNA sequence encoding CLT antigen 3 (SEQ ID NO.34), the cDNA sequence encoding CLT antigen 4 (SEQ ID NO.35), the cDNA sequence encoding CLT antigen 5 (SEQ ID NO.36), the cDNA sequence encoding CLT antigen 6 (SEQ ID NO.37), the cDNA sequence encoding CLT antigen 7 (SEQ ID NO.38), and the cDNA sequence encoding CLT antigen 8 (SEQ ID NO.39). SEQ ID NO.39 is a peptide sequence derived from CLT antigen 6. Detailed Implementation Plan

[0088] polypeptide

[0089] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably in this document and refer to any chain of amino acids linked together by a peptide, regardless of its length or whether it is co-translated or post-translational modified.

[0090] The term "amino acid" refers to any naturally occurring amino acid, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are the 20 L-amino acids encoded by the genetic code, and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. The term "amino acid analog" refers to a compound that has the same basic chemical structure as naturally occurring amino acids—an α-carbon bound to a hydrogen, carboxyl, amino, and R group—but has a modified R group or a modified peptide backbone compared to the natural amino acid. Examples include homoserine, ortholeucine, methionine sulfoxide, methionine methylthionium, and ortholeucine. Amino acid mimics refer to chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to that of naturally occurring amino acids. Appropriately, an amino acid is either 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.

[0091] Amino acids can be referred to in this article by their well-known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides can be referred to by their generally accepted single-letter codes.

[0092] Therefore, the present invention provides an isolated polypeptide comprising a sequence selected from the following:

[0093] (a) A sequence of any one of SEQ ID NO. 1-8; and

[0094] Variations of the sequences (b)(a); and

[0095] Immunogenic fragments of sequences (c)(a)

[0096] The present invention also provides an isolated polypeptide comprising a sequence selected from the following:

[0097] (a) Subtracting the sequence of any one of SEQ ID NO. 1-8 from the starting methionine residue; and

[0098] Variations of the sequences (b)(a); and

[0099] Immunogenic fragments of sequences (c)(a)

[0100] Generally speaking, variants of the polypeptide sequence of the present invention comprise sequences having a high degree of sequence identity with it. For example, the variants suitably have at least about 80% identity with the associated reference sequence over its entire length, 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%).

[0101] Appropriately, a variant is an immunogenic variant. A variant is considered immunogenic if, for example, in an in vitro restimulation assay of PBMCs or whole blood using a polypeptide as an antigen (e.g., restimulation lasting from several hours to up to one year (e.g., up to 6 months, 1 day to 1 month, or 1 to 2 weeks), it elicits a reaction that is at least 20%, appropriately at least 50%, and particularly at least 75% (e.g., at least 90%) of the activity of a reference sequence (i.e., the variant is a variant of the reference sequence), wherein the in vitro restimulation assay is performed by means of lymphocyte proliferation (e.g., T cell proliferation), production of cytokines (e.g., IFN-γ) in culture supernatant (measured by ELISA, etc.), or by intracellular and extracellular staining (e.g., using antibodies specific to immunomarkers such as CD3, CD4, CD8, IL2, TNF-α, IFNg, type I IFN, CD40L, CD69, etc.), followed by flow cytometry analysis to characterize the T-cell response and measure cell activation.

[0102] Variants can be, for example, conserved variants. A "conserved variant" is a variant in which the change results in the substitution of a certain amino acid for a functionally similar amino acid or a substitution / deletion / addition of residues that substantially does not affect the biological function of the variant. Generally, this biological function of the variant will induce an immune response against melanoma antigens, such as those found in skin melanoma.

[0103] Conserved substitution tables of functionally similar amino acids are well known in the art. Variants may include polypeptide homologs present in other species.

[0104] When compared to a reference sequence, variants of the polypeptides of the present invention may contain multiple substitutions, for example, conserved 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., conserved substitutions) may be up to 20% of the number of residues in the reference sequence, for example, up to 10%, for example, up to 5%, for example, up to 1%. Generally, conserved substitutions will fall within one of the amino acid groups specified below, however, 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 conserved substitutions for each other:

[0105] 1) Alanine (A), glycine (G);

[0106] 2) Aspartic acid (D), glutamic acid (E);

[0107] 3) Asparagine (N), glutamine (Q);

[0108] 4) Arginine (R), Lysine (K);

[0109] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0110] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0111] 7) Serine (S), threonine (T); and

[0112] 8) Cysteine ​​(C), Methionine (M)

[0113] (See, for example, Creighton, Proteins 1984).

[0114] Appropriately, such substitutions do not alter the immune structure of the epitope (e.g., they do not appear within the epitope region located as in the primary sequence) and therefore do not significantly affect the immunogenic properties of the antigen.

[0115] Peptide variants also include those in which additional amino acids are inserted compared to a reference sequence. For example, such insertions may involve 1-10 positions (e.g., 1-5 positions, suitably 1 or 2 positions, especially 1 position) and may include, for example, the addition of 50 or fewer amino acids at each position (e.g., 20 or fewer, especially 10 or fewer, particularly 5 or fewer). Suitably, such insertions do not appear 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 histidine residue (e.g., 2-6 residues) to aid in the expression and / or purification of the antigen in question.

[0116] Peptide variants include those in which amino acids have been missing compared to a reference sequence. For example, such deletions can occur at 1-10 positions (e.g., 1-5 positions, suitably 1 or 2 positions, especially 1 position), and can involve, for example, the deletion of 50 or fewer amino acids at each position (e.g., 20 or fewer, especially 10 or fewer, particularly 5 or fewer). Suitably, such deletions do not occur within the region of the epitope and therefore do not significantly affect the immunogenic properties of the antigen.

[0117] Those skilled in the art will recognize that specific protein variants can include substitutions, deletions, and additions (or any combination thereof). For example, substitutions / deletions / additions may enhance binding to desired patient HLA molecules (or have a neutral effect on them), thereby potentially increasing immunogenicity (or maintaining immunogenicity unchanged).

[0118] The immunogenic fragment of the present invention will generally comprise 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), particularly 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.

[0119] 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.

[0120] T-cell epitopes are short adjacent amino acid segments that are recognized by T cells (e.g., CD4+ T cells or CD8+ T cells) when they bind to HLA molecules. T-cell epitopes can be identified by epitope localization assays well known to those skilled in the art (see, for example, Paul, Fundamental Immunology, 3rd ed., 243-247 (1993); Beiβbarth et al., 2005, Bioinformatics, 21(Suppl.1): i29-i37).

[0121] As a result of the decisive involvement of T-cell responses in cancer, it is clear that fragments of the full-length polypeptides in SEQ ID NO. 1-8 containing at least one T-cell epitope can be immunogenic and contribute to immune protection.

[0122] It should be understood that in diverse outcrossed populations (such as humans), different HLA types mean that certain epitopes may not be recognized by all members of the population. Therefore, to maximize the level of recognition and the scale of the immune response against the peptide, it is generally desirable for the immunogenic fragment to contain multiple epitopes from the full-length sequence (appropriately all epitopes within the CLT antigen).

[0123] Potentially useful specific fragments of the peptides of SEQ ID NO. 1-8 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, particularly those associated with multiple HLA class I alleles, e.g., those associated with 2, 3, 4, 5, or more alleles. Potentially useful specific fragments of the peptides of SEQ ID NO. 1-8 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 associated with multiple HLA class II alleles, e.g., those associated with 2, 3, 4, 5, or more alleles). However, those skilled in vaccine design may combine exogenous CD4+ T cell epitopes with the CD8+ T cell epitopes of the present invention and achieve desired responses against the CD8+ T cell epitopes of the present invention.

[0124] If an independent fragment of a full-length polypeptide is used, the fragment is considered immunogenic if it elicits a reaction that is at least 20%, suitably at least 50%, and especially at least 75% (e.g., at least 90%) of the activity of a reference sequence (i.e., the activity of the fragment being a fragment of the reference sequence) in an in vitro restimulation assay of PBMCs or whole blood using the polypeptide as an antigen (e.g., for a period of time ranging from several hours to up to one year (e.g., up to 6 months, 1 day to 1 month, or 1 to 2 weeks)). This in vitro restimulation assay is performed by means of lymphocyte proliferation (e.g., T cell proliferation), the production of cytokines (e.g., IFN-γ) in the culture supernatant (measured by ELISA, etc.), or by intracellular and extracellular staining (e.g., using antibodies specific to immunomarkers such as CD3, CD4, CD8, IL2, TNF-α, IFN-γ, type 1 IFN, CD40L, CD69, etc.), followed by flow cytometry analysis to characterize the T-cell response and measure cell activation.

[0125] In some cases, multiple fragments of a full-length polypeptide (which may overlap or not overlap and may or may not cover the entire full-length sequence) can be used to obtain an equivalent biological response against 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 PBMCs or whole blood in vitro restimulation assays (e.g., T cell proliferation and / or IFN-γ production assays).

[0126] Examples of immunogenic fragments of the peptides of SEQ ID NO. 1-8, and therefore examples of peptides of the present invention, include peptides comprising or composed of sequences of SEQ ID NO. 9-22 and 39. The sequences of SEQ ID NO. 9-22 were identified as binding to HLA class I molecules by immunopeptidomics analysis (see Example 2). The sequence of SEQ ID NO. 39 was predicted by NetMHC software to bind to HLA class I molecules and was used in immunological validation analysis (see Example 4).

[0127] Nucleic acid

[0128] This invention provides isolated nucleic acids (referred to as nucleic acids of this invention) encoding polypeptides of this invention. For example, the nucleic acids of this invention comprise or consist of sequences selected from or composed of SEQ ID NO. 23-30 or 31-38.

[0129] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to polymeric macromolecules derived from nucleotide monomers, particularly deoxyribonucleotide monomers or ribonucleotide monomers. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, said nucleic acids being naturally occurring or non-naturally occurring, having properties similar to a reference nucleic acid, and intended to be metabolized in a manner similar to that of a reference nucleotide or intended to have an extended half-life in a system. Examples of such analogs include, but are not limited to, phosphate thioesters, phosphoramide esters, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Suitably, the term “nucleic acid” refers to a naturally occurring polymer 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 a cloning step, a restriction enzyme digestion step, or a ligation step, or the product of other methods that produce nucleic acid molecules distinct from those found in nature (e.g., in the case of cDNA). In one embodiment, the nucleic acid of the present invention is an artificial nucleic acid sequence (e.g., a cDNA sequence or nucleic acid sequence with non-naturally occurring codon selection). In one embodiment, the nucleic acid of the present invention is DNA. Alternatively, the nucleic acid of the present invention is RNA.

[0130] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) refer to nucleic acid molecules with a backbone containing a sugar moiety, namely the deoxyribosyl moiety and the ribosyl moiety, respectively. The sugar moiety can be linked to bases, which are the four 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 RNA that has the same sequence as reference DNA except that thymine (T) in DNA is replaced by uracil (U) in RNA. The sugar moiety can also be linked to non-natural bases such as inosine, xanthoside, 7-methylguanosine, dihydrouridine, and 5-methylcytidine. The natural phosphodiester bonds between the sugar (deoxyribosyl / ribosyl) moieties can optionally be replaced by thiophosphate bonds. Suitable, the nucleic acid of the present invention is composed of natural bases connected to a deoxyribosyl backbone or ribosyl sugar backbone having phosphodiester bonds between sugar moieties.

[0131] In one embodiment, the nucleic acid of the present invention is DNA. For example, the nucleic acid comprises or is composed of a sequence selected from or consisting of SEQ ID NO. 23-30 or 31-38. A nucleic acid is also provided that comprises or is composed of a variant of a sequence selected from or consisting of SEQ ID NO. 23-30 or 31-38, said variant encoding the same amino acid sequence but having different nucleic acids based on genetic code degeneracy.

[0132] Therefore, due to the degeneracy of the genetic code, numerous different but functionally identical nucleic acids can encode any given polypeptide. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified by a certain codon, that codon can be changed to any of the corresponding codons without altering the encoded polypeptide. This variation of nucleic acid results in “silent” (sometimes called “degeneracy” or “synonymous”) variants, which are variations of a kind of conserved modification. Each nucleic acid sequence encoding a polypeptide disclosed herein also enables every possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in the nucleic acid can be modified (with the exceptions of AUG, which is usually the only codon for methionine, and UGG, which is usually the only codon for tryptophan) to produce a functionally identical molecule. Therefore, every silent variation of the nucleic acid encoding a polypeptide is contained within each described sequence and is provided as an aspect of the invention.

[0133] Degenerate codon substitution can also be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0134] When compared with a reference sequence, the nucleic acid of the present invention comprising or composed of sequences selected from SEQ ID NO. 23-30 or 31-38 may contain multiple silent variants (e.g., 1-50, such as 1-25, especially 1-5, and especially 1 codon may be changed).

[0135] The nucleic acids of the present invention may comprise or consist of sequences selected from SEQ ID NO.31-38 that do not have a start codon of methionine (i.e., ATG or AUG), or variants thereof as described above.

[0136] In one embodiment, the nucleic acid of the present invention is RNA. An RNA sequence is provided that corresponds to the DNA sequence provided herein and has a ribonucleotide backbone instead of a deoxyribonucleotide backbone and has a side chain base where uracil (U) replaces thymine (T).

[0137] Therefore, when compared with a reference sequence, the nucleic acid of the present invention comprises or is composed of an RNA equivalent of a cDNA sequence selected from or consisting of cDNA sequences of SEQ ID NO. 23-30 or 31-38, and may contain multiple silent variants (e.g., 1-50, such as 1-25, especially 1-5, and especially 1 codon may be changed). "RNA equivalent" means an RNA sequence containing the same genetic information as the reference cDNA sequence (i.e., containing the same codons, having a ribonucleotide backbone instead of a deoxyribonucleotide backbone, and having a side chain base uracil (U) replacing thymine (T)).

[0138] The present invention also includes sequences complementary to the aforementioned cDNA and RNA sequences.

[0139] In one embodiment, the nucleic acid of the present invention is codon-optimized for expression in human host cells.

[0140] The nucleic acids of the present invention can be transcribed and translated into the polypeptides of the present invention in the case of DNA nucleic acids, and translated into the polypeptides of the present invention in the case of RNA nucleic acids.

[0141] polypeptides and nucleic acids

[0142] Appropriately, the polypeptides and nucleic acids used in this invention are isolated. An "isolated" polypeptide or nucleic acid is one taken from its original environment. For example, it is isolated if a naturally occurring polypeptide or nucleic acid is separated from some or all of the coexisting substances in the natural system. Similarly, a nucleic acid is considered isolated if it is cloned into a vector that is not part of its natural environment.

[0143] When referring to a reference polypeptide or nucleic acid sequence, "naturally occurring" means a sequence that exists in nature and has not been modified in a synthetic manner.

[0144] When referring to a reference polypeptide or nucleic acid sequence, "artificial" means a sequence that does not exist in nature, such as a synthetic modification of a natural sequence or a sequence containing a non-natural sequence.

[0145] When used to refer to the relationship between one nucleic acid or polypeptide and another, the term "heterologous" means that two or more sequences do not exist in the same relationship as each other in nature. A "heterologous" sequence can 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.

[0146] As shown above, the polypeptide variant preferably has at least about 80% identity with the associated reference sequence over its entire length, 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%).

[0147] For the purpose of comparing two closely related polypeptide or polynucleotide sequences, a “sequence identity %” can be calculated between a first and a second sequence. A polypeptide sequence is considered identical or identical to other polypeptide sequences if it shares 100% sequence identity across its entire length. 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 that are identical or have a specified percentage of identical amino acid residues (i.e., 70% identity within a specified region, optionally 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity) when compared and aligned for maximum correspondence within a comparison window. Appropriately, the comparison is made within a window corresponding to the entire length of a reference sequence.

[0148] For sequence comparisons, one sequence serves as a reference sequence to be compared with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and the coordinates of the subsequences are specified if necessary, along with the sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. Based on the program parameters, the sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence.

[0149] As used herein, a “comparison window” refers to a segment in which a sequence can be compared with a reference sequence having the same number of consecutive positions after optimal alignment of the two sequences. Sequence alignment methods for comparison are well known in the art. The best sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444, by the computerized execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds., 1995, ed.)).

[0150] An example of a useful algorithm is PILEUP. Using a progressive pairwise alignment method, PILEUP produces multiple sequence alignment results from a set of related sequences to show relationships and percentages of sequence identity. It also plots phylogenetic trees or dendrograms, which show the clustering relationships used to produce the alignment results. PILEUP uses a simplified form of the Feng and Doolittle progressive alignment method (Feng and Doolittle, 1987, J. Mol. Evol. 35:351-360). The method used is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. The procedure can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins by pairwise aligning the two most similar sequences, producing a cluster of the two aligned sequences. This cluster is then aligned with the next most related sequence or the cluster of aligned sequences. The two sequence clusters are aligned by simply extending the pairwise alignment results of the two independent sequences. The final alignment is achieved through a series of progressive pairwise alignments. The program is run by specifying the specific sequence and its amino acid coordinates for the sequence comparison region and by specifying the program parameters. Using PILEUP, a reference sequence is compared with other test sequences to determine the percentage of sequence identity using the following parameters: default vacancy weight (3.00), default gap length weight (0.10), and weighted terminal vacancy. PILEUP is available from the GCG sequence analysis software package, for example, version 7.0 (Devereaux et al., 1984, Nuc. Acids Res. 12:387-395).

[0151] Another example of an algorithm suitable for determining sequence identity and sequence similarity percentages is the BLAST algorithm and the BLAST 2.0 algorithm, described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information (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, where the short words match or satisfy certain positive threshold scores T when compared to words of the same length in the database sequence. T is called the adjacent word score threshold (Altschul et al., above). These initial adjacent word hits act as seeds, which are used to initiate a search to find longer HSPs containing these seeds. The word hit extends as far as possible along each sequence in both directions, as much as possible to improve the cumulative alignment score. For nucleotide sequences, the cumulative score is calculated using 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. The extension of the word hit in each direction stops when: the cumulative alignment score decreases from its maximum realization value to a value of X; the cumulative score reaches or falls below zero due to the accumulation of one or more negative score 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 expected (E) of 10, along with the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915), alignment result (B) 50, expected (E) 10, M = 5, N = -4, and a comparison of both strands as default.

[0152] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance.

[0153] The "difference" between sequences refers to the insertion, deletion, or substitution of a single residue in the position of a single residue in the second sequence compared to the first sequence. Two sequences can contain one, two, or more of these differences. Otherwise, insertions, deletions, or substitutions in a second sequence that is identical to the first sequence (100% sequence identity) result in a reduced percentage of sequence identity. For example, if the identical sequence is 9 residues long, one substitution in the second sequence produces 88.9% sequence identity. If the identical sequence is 17 amino acid residues long, two substitutions in the second sequence produce 88.2% sequence identity.

[0154] Alternatively, for the purpose of comparing the first reference sequence with the second comparison sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence can be determined. An addition is the addition of a residue to the first sequence (including addition to 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 removal of a residue from the first sequence (including removal from either end of the first sequence).

[0155] The polypeptides of the present invention

[0156] The polypeptides of the present invention can be obtained and manipulated using techniques disclosed, for example, in Green and Sambrook 2012, *Molecular Cloning: A Laboratory Manual*, 4th edition, Cold Spring Harbour Laboratory Press. In particular, artificial gene synthesis 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 suitable organisms to produce polypeptides. Genes encoding the polypeptides of the present invention can be synthesized, for example, by solid-phase DNA synthesis. The complete gene can be synthesized de novo without the need for precursor template DNA. To obtain the desired oligonucleotides, structural units are sequentially coupled to the growing oligonucleotide chain in the order required by the product sequence. Upon completion of chain assembly, the product is released from the solid phase into solution, deprotected, and collected. The product can be separated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity (Verma and Eckstein, 1998, Annu. Rev. Biochem. 67: 99-134). These relatively short segments can be easily assembled into longer DNA molecules suitable for use in numerous recombinant DNA-based expression systems using various gene amplification methods (Methods Mol Biol., 2012; 834: 93-109). In the context of this invention, those skilled in the art will understand that the polynucleotide sequences encoding polypeptide antigens described herein can be readily used in various vaccine production systems, such as viral vectors.

[0157] For the purpose of producing the polypeptides of the present invention in microbial (e.g., bacterial or fungal) hosts, the nucleic acids of the present invention will contain suitable regulatory and control sequences (including promoters, termination signals, etc.) and sequences that promote the secretion of polypeptides suitable for protein production in the host. Similarly, the 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 the 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 the secretion of polypeptides suitable for protein production in these cells.

[0158] Optionally, the separation and improvement of the present invention's polypeptides generated by recombination can be facilitated by adding a histidine residue (commonly referred to as an aHis tag) to one end of the polypeptide.

[0159] It can also synthesize polypeptides.

[0160] carrier

[0161] In an additional embodiment, a genetic construct comprising one or more nucleic acids of the present invention is introduced into cells in vivo, thereby generating the polypeptide of the present invention in vivo and eliciting an immune response. Nucleic acids (e.g., DNA) can be present within any of a variety of delivery systems known to those skilled 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 those described in Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, and those cited in the references therein. Several of these methods are briefly described below for illustrative purposes.

[0162] Therefore, a vector containing the nucleic acid molecule of the present invention is provided (also referred to herein as a “DNA expression construct” or “construct”).

[0163] Appropriately, the vector contains a nucleic acid molecule encoding regulatory elements (such as suitable promoters and termination signals) adapted to allow translationally active RNA molecules to be transcribed in human host cells. "Translationally active RNA molecules" are RNA molecules capable of being translated into proteins by the translational apparatus of human cells.

[0164] Therefore, a vector comprising the nucleic acid of the present invention (hereinafter referred to as "the vector of the present invention") is provided.

[0165] In particular, the vector can be a viral vector. Viral vectors can be adenoviruses, adeno-associated viruses (AAVs) (e.g., AAV types 5 and 2), alphaviruses (e.g., Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SIN), Semliki forest virus (SFV)), herpesviruses, acanthoviruses (e.g., lymphocytic choriomeningitis virus (LCMV)), measles viruses, poxviruses (e.g., modified Ankara vaccinia virus (MVA)), paramyxoviruses, lentiviruses, or rhabdoviruses (e.g., vesicular stomatitis virus (VSV)), meaning the vector can be derived from any of the aforementioned viruses. Adenoviruses are particularly suitable as gene transfer vectors due to their moderate 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 pairs of inverted repeat sequences (ITRs), which are cis elements essential for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units divided according to the initiation of viral DNA replication. The E1 regions (E1A and E1B) encode proteins responsible for regulating the transcription of the viral genome and some cellular genes. Expression of the E2 regions (E2A and E2B) leads to the synthesis of proteins used for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutdown (Renan, 1990). The products of late genes, including most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript released by the major late promoter (MLP). The MLP is particularly efficient during late infection, and all mRNAs transcribed from this promoter possess a 5'-triple leader (TPL) sequence, which makes these mRNAs the preferred mRNAs for translation. Replication-deficient adenoviruses generated from viral genomes lacking one or more early genes are particularly useful because they replicate in a limited manner and are less likely to be pathogenic within the inoculated host and to contacts of the inoculated host.

[0166] Other polynucleotide delivery

[0167] In some embodiments of the invention, the expression construct comprising one or more polynucleotide sequences may consist solely of naked recombinant DNA plasmids. See reviews by Ulm et al., 1993, Science 259:1745-1749 and Cohen, 1993, Science 259:1691-1692. Construct transfer can be performed, for example, by any method that permeates the cell membrane physically or chemically. This is particularly suitable for in vitro transfer, although it can also be used in vivo. DNA conceived to encode a target gene can also be transferred in vivo and express the gene product in a similar manner. Several delivery systems have been used to deliver DNA molecules to animal models and to humans. Several products based on this technology have been licensed for use in animals, and others are in Phase II and Phase III human clinical trials.

[0168] RNA delivery

[0169] In some embodiments of the invention, expression constructs comprising one or more polynucleotide sequences may be composed of naked recombinant DNA-derived plasmids (Ulmer et al., 2012, Vaccine 30:4414–4418). Regarding DNA-based expression constructs, various methods can be used to introduce RNA molecules into cells in vitro or in vivo. RNA-based constructs can be designed to mimic simple messenger RNA (mRNA) molecules, so that the introduced biomolecule is directly translated by the translation apparatus of the host cell to produce its encoded polypeptide in the cell where the biomolecule was introduced. Alternatively, RNA molecules can be designed in such a way that, by incorporating a viral RNA-dependent RNA polymerase gene into the structure of the molecule, they are allowed to self-amplify in the cell where the molecule was introduced. Therefore, these types of RNA molecules are called self-amplifying mRNAs (SAMs). TM The molecule (Geall et al., 2012, PNAS, 109:14604–14609) shares characteristics with some RNA-based viral vectors. It can be further modified into mRNA-based RNA or SAM. TMRNA (e.g., by altering its sequence or by using modified nucleotides) can be formulated 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 humans, and several RNA-based vaccines are in ongoing clinical trials. Pharmaceutical compositions

[0170] The polypeptides, nucleic acids, and carriers of the present invention can be formulated for delivery in pharmaceutical compositions such as immunogenic compositions and vaccine compositions (hereinafter referred to as "compositions of the present invention"). The compositions of the present invention suitably comprise the polypeptides, nucleic acids, or carriers of the present invention together with pharmaceutically acceptable carriers.

[0171] Therefore, in one embodiment, an immunogenic pharmaceutical composition comprising the polypeptide, nucleic acid, or carrier of the present invention together with a pharmaceutically acceptable carrier is provided.

[0172] In another embodiment, a vaccine composition comprising the polypeptide, nucleic acid, or carrier of the present invention together with a pharmaceutically acceptable carrier is provided. The preparation of pharmaceutical compositions is typically described, for example, in Powell and Newman, Vaccine Design (Subunit and Adjuvant Protocols), 1995. The compositions of the present invention may also contain other compounds that may be biologically active or inactive. Suitably, the compositions of the present invention are sterile compositions suitable for parenteral administration.

[0173] In certain preferred embodiments of the invention, a pharmaceutical composition of the invention is provided, the pharmaceutical composition comprising one or more (e.g., one) polypeptides of the invention in combination with a pharmaceutically acceptable carrier.

[0174] In certain preferred embodiments of the invention, a pharmaceutical composition of the invention is provided, the pharmaceutical composition comprising one or more (e.g., one) nucleic acid of the invention or one or more (e.g., one) carrier of the invention in combination with a pharmaceutically acceptable carrier.

[0175] In one embodiment, the compositions of the present invention may comprise one or more (e.g., one) polynucleotides and one or more (e.g., one) polypeptide components. Alternatively, the compositions may comprise one or more (e.g., one) carriers and one or more (e.g., one) polypeptide components. Alternatively, the compositions may comprise one or more (e.g., one) carriers and one or more (e.g., one) polynucleotide components. Such compositions can provide an enhanced immune response.

[0176] Medicinal salt:

[0177] It will be apparent that the compositions of the present invention may contain pharmaceutically acceptable salts of nucleic acids or polypeptides provided herein. Such salts may 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, potassium, lithium, ammonium, calcium, and magnesium salts).

[0178] Pharmaceutical carrier

[0179] Although many pharmaceutically acceptable carriers known to those skilled in the art can be used in the compositions of the present invention, the optimal class of carriers used will vary depending on 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, such as intramuscular, subcutaneous, or intravenous administration. For parenteral administration, the carrier preferably comprises water and may contain a pH-controlled buffer, stabilizers, such as surfactants, and amino acids and tonic agents, such as salts and sugars. If the composition is intended to be provided in lyophilized form for dilution at the point of use, the formulation may contain a lyophilization protectant, such as sugars like trehalose. For oral administration, any of the above-described carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate may be used.

[0180] Therefore, the compositions of the present invention may comprise buffer solutions (e.g., neutral buffered saline or phosphate buffered saline), sugars (e.g., glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, antibacterial agents, chelating agents such as EDTA or glutathione, solutes that make the formulation isotonic, hypotonic, or slightly hypotonic to the recipient's blood, suspending agents, thickeners, and / or preservatives. Alternatively, the compositions of the present invention may be formulated as lyophilized products.

[0181] Immunostimulants

[0182] The compositions of the present invention may also contain one or more immunostimulants. Immunostimulants can be any substance that enhances or stimulates an immune response to exogenous antigens (antibody and / or cell-mediated). 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., where the oil is squalene); aminoalkyl aminoglucosidase 4-phosphate; lipopolysaccharides or derivatives thereof, such as 3-de-O-acylated monophosphate lipid A. And other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and interferon. Therefore, suitably, one or more immunostimulants in the compositions of the present invention are selected from aluminum salts, saponins, immunostimulatory oligonucleotides, oil-in-water emulsions, aminoalkyl aminoglucosidase 4-phosphate, lipopolysaccharides and their derivatives, and other TLR4 ligands, TLR7 ligands, TLR8 ligands, and TLR9 ligands. The immunostimulant may also comprise monoclonal antibodies that specifically interact with other immune components, such as monoclonal antibodies that block interactions with immune checkpoint receptors (including PD-1 and CTLA4).

[0183] In the case of recombinant nucleic acid delivery methods (e.g., DNA, RNA, viral vectors), the gene encoding a protein-based immunostimulant can be easily delivered together with the gene encoding the polypeptide of the present invention.

[0184] Sustained release

[0185] The compositions described herein can be used as part of a sustained-release formulation that achieves slow / sustained release of the compound after administration (i.e., formulations such as capsules, sponges, pastes, or gels (e.g., composed of polysaccharides)).

[0186] Storage and Packaging

[0187] The compositions of the present invention can be contained in single-dose containers or multi-dose containers (such as sealed ampoules or vials). These containers are preferably completely sealed to maintain the sterility of the formulation until use. Typically, the formulation can be stored as a suspension, solution, or emulsion in an oily or aqueous solvent. Alternatively, the compositions of the present invention can be stored in a freeze-dried state, requiring only the addition of a sterile liquid carrier (such as water or saline for injection) just before use.

[0188] dose

[0189] The amounts of nucleic acids, peptides, or carriers in each composition of the present invention can be prepared in such a manner to obtain suitable dosages for therapeutic or preventative use. Those skilled in the art will consider numerous factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and in this regard, multiple dosages and treatment regimens may be desirable.

[0190] Generally, compositions containing a therapeutically effective or preventatively effective amount deliver about 0.1 μg to about 1000 μg of the polypeptide of the present invention per administration, and more generally about 2.5 μg to about 100 μg of the polypeptide per administration. If delivered in the form of a short, synthetic long peptide, the dose may be 1 to 200 μg / peptide / dose. For polynucleotide compositions, these doses generally deliver about 10 μg to about 20 mg of the nucleic acid of the present invention per administration, and more generally about 0.1 mg to about 10 mg of the nucleic acid of the present invention per administration.

[0191] Diseases requiring treatment or prevention

[0192] As shown elsewhere, SEQ ID NO.1-8 are polypeptide sequences corresponding to the CLT antigen overexpressed in cutaneous melanoma.

[0193] In one embodiment, the present invention provides the polypeptide, nucleic acid, carrier, or composition of the present invention for use in a pharmaceutical.

[0194] Other aspects of the invention relate to a method for generating an immune response in humans, the method comprising administering the polypeptide, nucleic acid, carrier, or composition of the invention to the human.

[0195] The present invention also provides polypeptides, nucleic acids, vectors or compositions of the present invention for generating an immune response in humans.

[0196] The invention also provides the use of the polypeptides, nucleic acids, carriers, or compositions thereof for manufacturing pharmaceuticals that generate an immune response in humans.

[0197] Appropriately, an immune response is generated against a cancerous tumor, wherein the cancerous tumor expresses a sequence selected from the corresponding sequences of SEQ ID NO. 1-8 and variants and immunogenic fragments thereof. “Corresponding” here means that if the tumor expresses, for example, SEQ. ID NO. A (A is one of SEQ ID NO. 1-8) or a variant or immunogenic fragment thereof, then the polypeptides, nucleic acids, carriers, or compositions of the present invention and the drugs relating thereto will be based on SEQ ID NO. A or a variant or immunogenic fragment thereof.

[0198] Appropriately, the immune response includes CD8+ T cell, CD4+ T cell and / or antibody responses, especially CD8+ cytolytic T-cell responses and CD4+ helper T-cell responses.

[0199] Appropriately, an immune response is generated against tumors, especially tumors expressing sequences selected from SEQ ID NO. 1-8 and their variants and immunogenic fragments.

[0200] In a preferred embodiment, the tumor is a melanoma tumor, such as a cutaneous melanoma tumor.

[0201] A tumor can be a primary tumor or a metastatic tumor.

[0202] Other aspects of the invention relate to a method of treating a human patient with cancer, wherein the cancer cells express an immunogenic fragment and variant selected from sequences of SEQ ID NO. 1-8 and any of them, or other aspects of the invention relate to a method of preventing cancer in humans, wherein the cancer cells express an immunogenic fragment and variant selected from sequences of SEQ ID NO. 1-8 and any of them, the method comprising administering to the human a corresponding polypeptide, nucleic acid, vector, or composition of the invention.

[0203] The present invention also provides polypeptides, nucleic acids, vectors or compositions thereof for the treatment or prevention of cancer in humans, wherein the cancer cells express sequences selected from the corresponding sequences of SEQ ID NO. 1-8 and immunogenic fragments thereof.

[0204] Transcripts corresponding to SEQ ID NO. 23, 24, 25, 27 and 30 are also overexpressed in uveal melanoma. Therefore, in an alternative embodiment, the tumor is a uveal melanoma tumor and / or a tumor expressing sequences selected from SEQ ID NO. 1, 2, 3, 5 and 8.

[0205] Therefore, the present invention provides a method or polypeptide, nucleic acid, vector, or composition according to the purpose of the invention, wherein the polypeptide comprises a sequence selected from the following:

[0206] (a) The sequence of any one of SEQ ID NO. 1, 2, 3, 5 and 8; and

[0207] Variations of the sequences (b)(a); and

[0208] Immunogenic fragments of sequences (c)(a),

[0209] For example, the polypeptide comprises or is composed of a sequence selected from any of SEQ ID NO. 9-15, 18 and 22, and the nucleic acid comprises or is composed of a sequence selected from any of SEQ ID NO. 23, 24, 25, 27 and 30, or selected from any of SEQ ID NO. 31, 32, 33, 35 and 38;

[0210] And the cancer among them is uveal melanoma.

[0211] The terms “prevent” and “prevent” are used interchangeably in this article.

[0212] Treatment and vaccination program

[0213] Treatment regimens may include the simultaneous (e.g., co-administration) or sequential (e.g., primary-boost) delivery of (i) the polypeptide, nucleic acid, or vector of the present invention together with (ii) one or more further polypeptides, nucleic acids, or vectors of the present invention and / or (iii) other components, such as various other therapeutically useful compounds or molecules, such as antigenic proteins, optionally co-administered with an adjuvant. Examples of co-administration include ipsilateral co-administration and contralateral co-administration. "Simultaneous" administration suitably means that all components are delivered during the same treatment cycle. 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 physician visit) or within hours.

[0214] In some embodiments, a "primary immunization" or first administration of the polypeptide, nucleic acid, or vector of the present invention is followed by one or more "booster" or subsequent administrations of the polypeptide, nucleic acid, or vector of the present invention ("primary and booster" method). In one embodiment, the polypeptide, nucleic acid, or vector of the present invention is used in a primary-booster vaccination regimen. In one embodiment, both the primary immunization and booster use the 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 booster use the 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 booster, 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 booster. Typically, the first or "primary immunization" administration and the second or "booster" administration are given approximately 1-12 weeks later or up to 4-6 months later. Subsequent "booster" administrations can be given at a frequency of 1-6 weeks or can be given considerably later (up to several years later).

[0215] Antigen combination

[0216] The peptides, nucleic acids, or vectors of the present invention can be used in combination with one or more other peptides or nucleic acids, vectors, and / or with other antigenic peptides (or polynucleotides or vectors encoding them) that elicit an immune response against melanoma, such as cutaneous melanoma or uveal melanoma. These other antigenic peptides can be derived from a variety of sources and 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), intron-retaining neoantigens (Smart et al., (2018). Nature Biotechnology. http: / / doi.org / 10.1038 / nbt.4239), and 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 (TIEPPs; Gigoux, M. and Wolchok, J. (2018). JEM, 215, 2233; Marijt et al., (2018). JEM 215, 2325) or undiscovered neoantigens (including CLT antigens) belonging to a class of antigens encoding T-cell epitopes associated with impaired peptide processing. Additionally, antigenic peptides from these diverse sources may be combined with (i) nonspecific immunostimulants / adjuvants and / or (ii) antigens, such as universal CD4 helper epitopes known to strongly elicit CD4 helper T cells (delivered as peptides or as polynucleotides or carriers encoding these CD4 antigens) to amplify anti-melanoma-specific responses elicited by co-administered antigens.

[0217] Different peptides, nucleic acids, or carriers can be formulated in the same formulation or in discrete formulations. Alternatively, the peptide can be provided as a fusion protein in which the peptide of the present invention is fused with a second or other peptide (see below).

[0218] Nucleic acid encoding the aforementioned fusion protein can be provided.

[0219] More generally, when two or more components are used in combination, these components can be present, for example, in the following manner:

[0220] (1) As two or more independent antigenic polypeptide components;

[0221] (2) As a fusion protein containing two (or other) polypeptide components;

[0222] (3) As a component of one or more polypeptides and one or more polynucleotides;

[0223] (4) As two or more independent polynucleotide components;

[0224] (5) As a single polynucleotide encoding two or more independent polypeptide components; or

[0225] (6) As a single polynucleotide encoding a fusion protein, the fusion protein comprises two (or other) polypeptide components.

[0226] For convenience, when multiple components are present, they are often required to 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 peptides (e.g., within a single fusion protein). In an alternative embodiment of the invention, all components are provided as polynucleotides (e.g., single polynucleotides, such as those encoding a single fusion protein).

[0227] Fusion protein

[0228] As an embodiment of the antigen combination discussion above, the present invention also provides isolated polypeptides of the present invention, which are fused with a second or other polypeptide of the present invention (hereinafter referred to as "combined polypeptides of the present invention") by generating a nucleic acid construct that fuses sequences encoding independent antigens together. The combined polypeptides of the present invention are expected to have the uses described herein and may have the advantages of excellent immunogenic or vaccine activity or prophylactic or therapeutic effects (including increased breadth and depth of response), and may be particularly valuable in outcrossed populations. Fusions of the polypeptides of the present invention may also provide the benefit of increased efficiency in constructing and manufacturing vaccine antigens and / or targeted vaccines (including nucleic acid vaccines).

[0229] As described above in the section on antigen combination, the polypeptides and combined polypeptides of the present invention can also be fused to polypeptide sequences that are not polypeptides of the present invention, wherein the polypeptide sequences include one or more of the following:

[0230] (a) Other peptides that are melanoma-associated antigens and therefore may be used as immunogenic sequences in vaccines (e.g., GPR143, PRAME, MAGE-A3, and pMel(gp100, see above); and

[0231] (b) Peptide sequences that can enhance the immune response (i.e., immune stimulation sequences).

[0232] (c) Peptide sequences that can provide strong CD4+ helper to increase CD8+ T cell responses to CLT antigenic epitopes, such as those containing universal CD4 helper epitopes.

[0233] The present invention also provides nucleic acids encoding the aforementioned fusion proteins and other aspects of the invention (vectors, compositions, cells, etc.) with necessary modifications for the polypeptides of the invention.

[0234] CLT antigen-binding peptide

[0235] Antigen-binding peptides with immune specificity to tumor-expressed antigens (the peptides of the present invention) can be designed to recruit cytolytic cells to antigen-modified tumor cells and mediate their destruction. Such a mechanism of recruiting cytolytic cells via antigen-binding peptides is called antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, the present invention provides antigen-binding peptides with immune specificity to the peptides of the present invention. Antigen-binding peptides include antibodies that can be produced and humanized in non-human animal species (e.g., rodents or camels) or that can be produced in non-human species (e.g., genetically modified rodents to have a human immune system), such as monoclonal antibodies and fragments thereof, such as domain antibodies, Fab fragments, Fv fragments, and VHH fragments.

[0236] Antigen-binding peptides can be generated using methods familiar to technicians. For example, hybridoma technology can be used to generate monoclonal antibodies by fusing B cells that produce specific antibodies with myeloma (B-cell carcinoma) cells selected for their ability to grow in tissue culture and the absence of antibody chain synthesis. The references cited in Milstein, 1975, Nature 256(5517):495-497 and Nelson, 2000(Jun), Mol Pathol.53(3):111-7 are incorporated herein by reference in their entirety.

[0237] Monoclonal antibodies against identified antigens can be obtained, for example, in the following ways:

[0238] a) Immortification is achieved by obtaining lymphocytes from the peripheral blood of animals (including humans) that have been previously immunized / exposed to identified antigens, immortalized cells, and preferably myeloma cells, with the aim of forming hybridomas.

[0239] b) Culture the immortalized cells (hybridomas) and recover the cells that produce antibodies with the desired specificity.

[0240] Monoclonal antibodies can be obtained by the following method, which includes the steps of:

[0241] a) Cloning DNA or cDNA sequences obtained from lymphocytes, especially peripheral blood lymphocytes, of animals (appropriately immunized beforehand with identified antigens) into vectors, particularly bacteriophages and more specifically filamentous bacteriophages.

[0242] b) Transform prokaryotic cells using the above-mentioned vector, under conditions that allow antibody production.

[0243] c) Selecting antibodies by subjecting them to antigen-affinity selection.

[0244] d) Recover antibodies with the desired specificity

[0245] e) Expressing a nucleic acid molecule encoding an antibody, said nucleic acid molecule being obtained from B cells of a patient exposed to an antigen or of an animal experimentally immunized with an antigen.

[0246] The selected antibody can then be produced using conventional recombinant protein production techniques (e.g., from genetically engineered CHO cells).

[0247] This invention provides isolated antigen-binding peptides that are immune-specific to the peptides of this invention. Suitably, the antigen-binding peptide is a monoclonal antibody or a fragment thereof.

[0248] In some embodiments, the antigen-binding peptide is coupled to a cytotoxic moiety. An example cytotoxic moiety includes the Fc domain of an antibody that recruits Fc receptor-carrying cells that promote ADCC. Alternatively, the antigen-binding peptide may be linked to a biological toxin or a cytotoxic chemical.

[0249] Another important class of antigen-binding peptides includes T-cell receptor (TCR)-derived molecules that bind to the HLA-display fragment of the antigens of the present invention. In this embodiment, TCR-based bioproducts (including patient-derived TCRs or specifically manipulated high-affinity TCRs) that recognize CLT antigens (or derivatives thereof) on the surface of tumor cells may also include a guiding portion that recognizes components on T cells (or another class of immune cells) that attract these immune cells to the tumor, thereby providing therapeutic benefit. In some embodiments, the guiding portion may also stimulate beneficial activities (including cytolytic activity) of the reguided immune cells.

[0250] Therefore, in one embodiment, the antigen-binding peptide has immune specificity for HLA-binding peptides, said HLA-binding peptides being the peptides of the present invention or portions thereof. For example, the antigen-binding peptide is a T-cell receptor.

[0251] In one embodiment, the antigen-binding polypeptide of the present invention may be coupled to another polypeptide capable of binding to cytotoxic cells or other immune components in a subject.

[0252] In one implementation, the antigen-binding peptide is used in a drug.

[0253] In one embodiment, a pharmaceutical composition comprising the antigen-binding polypeptide of the present invention together with a pharmaceutically acceptable carrier is provided. This composition may be a sterile composition suitable for parenteral administration. See, for example, the pharmaceutical composition disclosure above.

[0254] The present invention provides a method for treating a human with cancer, wherein the cancer cells express an immunogenic fragment and variant selected from sequences selected from SEQ ID NO. 1-8 and any one thereof, or the present invention provides a method for preventing cancer in a human, wherein the cancer cells express an immunogenic fragment and variant selected from sequences selected from SEQ ID NO. 1-8 and 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.

[0255] In one embodiment, an antigen-binding polypeptide of the present invention is provided, which may be coupled to a cytotoxic portion, or a composition comprising the antigen-binding polypeptide of the present invention is provided for the treatment or prevention of cancer in humans, wherein the cancer cells express an immunogenic fragment selected from the corresponding sequences of SEQ ID NO. 1-8 and any one thereof.

[0256] Appropriately, in any of the above embodiments, the cancer is melanoma, particularly cutaneous melanoma.

[0257] 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:

[0258] (a) The sequence of any one of SEQ ID NO. 1, 2, 3, 5 and 8; and

[0259] Variations of the sequences (b)(a); and

[0260] Immunogenic fragments of the sequence (c)(a).

[0261] For example, the polypeptide comprises or is composed of a sequence selected from any of SEQ ID NO. 9-15, 18 and 22, and the nucleic acid comprises or is composed of a sequence selected from any of SEQ ID NO. 23, 24, 25, 27 and 30 or any of SEQ ID NO. 31, 32, 33, 35 and 38;

[0262] And the cancer among them is uveal melanoma.

[0263] Antigen-binding peptides (such as antibodies or fragments thereof) can be administered at doses of, for example, 5-1000 mg, 25-500 mg, 100-300 mg, or about 200 mg.

[0264] Cellular therapy that promotes antigen presentation in vivo

[0265] Any of a variety of cell delivery vehicles can be used within a pharmaceutical composition to promote antigen-specific immune responses. Therefore, the present invention provides a cell, said cell being an isolated antigen-presenting cell, which is loaded with the polypeptide of the present invention through in vitro modification or genetically engineered to express the 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 into highly efficient APCs. These cells can, but do not necessarily need to, be genetically modified to increase antigen-presenting capacity to improve the activation and / or maintenance of T-cell responses and / or immunocompatibility with the recipient (i.e., HLA haplotype matching). APCs can generally be isolated from any of a variety of biological fluids and organs, and can be autologous, allogeneic, syngeneic, or xenogeneic cells.

[0266] Certain preferred embodiments of the present invention use dendritic cells or their progenitor cells as APCs. Thus, in one embodiment, the APC of the present invention is a dendritic cell. 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). Typically, dendritic cells can be identified based on their typical morphology (in situ star-shaped, with prominent cytoplasmic processes visible in vitro), their efficient uptake, processing, and presentation of antigens, and their ability to activate naïve 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 such modified dendritic cells are conceived in the present invention. 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). Therefore, in one embodiment, exosomes loaded with the peptides of the present invention are provided.

[0267] Dendritic cells and progenitor cells can be obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, 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 monocytes harvested from peripheral blood. Alternatively, CD34-positive cells harvested from peripheral blood, 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 dendritic cell differentiation, maturation, and proliferation to the culture medium.

[0268] Dendritic cells are conveniently classified into “immature” and “mature” cells, providing 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, associated with high expression of Fcγ receptors and mannose receptors. Mature phenotypes are generally characterized by lower expression of these markers, but high expression of cell surface molecules responsible for T cell activation, such as class I and II MHC molecules, adhesion molecules (e.g., CD54 and CD11), and co-stimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

[0269] APCs can also be genetically engineered, for example, by transfection with polynucleotides encoding a protein (or a portion thereof or other variants), thereby expressing the polypeptide on the cell surface. This transfection can be performed in vitro, and pharmaceutical compositions containing such transfected cells can then be used as described herein. Alternatively, gene delivery vehicles targeting dendritic cells or other antigen-presenting cells can be administered to a patient to produce in vivo transfection. For example, in vivo and in vitro dendritic cell transfection can be performed using any method known in the art, such as those described in WO 97 / 24447 or the gene gun protocol 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 peptides, DNA (e.g., plasmid vectors), or RNA; or with recombinant bacteria or viruses expressing antigens (e.g., adenovirus, adeno-associated virus (AAV) (e.g., AAV types 5 and 2), alphaviruses (e.g., Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SIN), Semliki forest virus (SFV), herpesviruses, psammoviruses (e.g., lymphocytic choriomeningitis virus (LCMV)), measles virus, poxviruses (e.g., modified Ankara vaccinia virus (MVA) or fowlpox virus), paramyxoviruses, lentiviruses, or rhabdoviruses (e.g., vesicular stomatitis virus (VSV)). Prior to peptide loading, the peptide can be covalently conjugated to an immune partner (e.g., a vector molecule) that provides T cell assistance. Alternatively, dendritic cells can be pulse-labeled with unconjugated immune partners, either independently or in the presence of the peptide or the vector.

[0270] This invention provides specially designed chemically synthesized fragments encoding epitopes for the delivery of polypeptide antigens to antigen-presenting cells. Those skilled in the art will recognize that these types of molecules, also known as synthetic long peptides (SLPs), provide therapeutic platforms for stimulating (or loading) in vitro cells with the antigenic polypeptides of this 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).

[0271] In one embodiment, a pharmaceutical composition comprising antigen-presenting cells (suitably dendritic cells) of the present invention together with a pharmaceutically acceptable carrier is provided. This composition may be a sterile composition suitable for parenteral administration. See, for example, the pharmaceutical composition disclosure above.

[0272] In one embodiment, the antigen-presenting cells (suitably dendritic cells) of the present invention are provided for use in a medicament.

[0273] Also provided is a method for treating a human with cancer, wherein the cancer cells express an immunogenic fragment and variant selected from sequences of SEQ ID NO. 1-8 and any one thereof, or a method for preventing cancer in a human, wherein the cancer cells express an immunogenic fragment and variant selected from sequences of SEQ ID NO. 1-8 and any one thereof, the method comprising administering to the human the antigen-presenting cells of the present invention, suitably dendritic cells, or a composition comprising the antigen-presenting cells of the present invention.

[0274] In one embodiment, the antigen-presenting cells of the present invention are provided, suitably dendritic cells, or compositions comprising the antigen-presenting cells of the present invention, for the treatment or prevention of cancer in humans, wherein the cancer cells express immunogenic fragments selected from the corresponding sequences of SEQ ID NO. 1-8 and any one thereof.

[0275] In one embodiment, a pharmaceutical composition comprising the exosomes of the present invention together with a pharmaceutically acceptable carrier is provided. This composition may be a sterile composition suitable for parenteral administration. See, for example, the pharmaceutical composition disclosure above. The composition may optionally contain an immunostimulant – see the immunostimulant disclosure above.

[0276] In one embodiment, the exosomes of the present invention are provided for use in a pharmaceutical.

[0277] Also provided is a method for treating a human with cancer, wherein the cancer cells express an immunogenic fragment and variant selected from sequences selected from SEQ ID NO. 1-8 and any one thereof, or a method for preventing cancer in humans, wherein the cancer cells will express an immunogenic fragment and variant selected from sequences selected from SEQ ID NO. 1-8 and any one thereof, said method comprising administering to said human exosomes of the present invention or a composition comprising said exosomes of the present invention.

[0278] In one embodiment, the exosomes of the present invention or compositions comprising the exosomes of the present invention are provided for the treatment or prevention of cancer in humans, wherein the cancer cells express immunogenic fragments selected from the corresponding sequences of SEQ ID NO. 1-8 and any one thereof. In any of the above embodiments, the cancer is suitably melanoma, particularly cutaneous melanoma.

[0279] Stimulated T-cell therapy

[0280] In addition to APC-mediated in vivo or in vitro generation of T cells with immune specificity to the peptides of the present invention, autologous or non-autologous T cells can be isolated from the subject, for example, from peripheral blood, umbilical cord blood and / or separated by blood component analysis, and stimulated in the presence of tumor-associated antigens loaded on the MHC molecules (signal 1) of APC cells to induce the proliferation of T cells carrying TCRs with immune specificity to such antigens.

[0281] Successful T cell activation requires the binding of co-stimulatory surface molecules B7 and CD28 (signal 2) on antigen-presenting cells and T cells, respectively. For optimal T cell activation, both signals 1 and 2 are necessary. Conversely, stimulation with antigenic peptides (signal 1) in the absence of co-stimulatory action (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 that induce signals that prevent T cell activation, such as CTLA-4 and PD-1.

[0282] Autologous or non-autologous T cells can therefore be stimulated in the presence of the polypeptides of the present invention and expand and retransmit to patients at risk of or with cancer whose cancer cells express the corresponding polypeptides of the present invention, provided that antigen-specific TCRs will recognize the MHC-presented antigens of the patient, wherein they will target the cancer cells expressing the corresponding polypeptides and induce their killing.

[0283] In one embodiment, the polypeptides, nucleic acids, vectors, or compositions of the present invention are provided for in vitro stimulation and / or expansion of T cells derived from cancer-stricken humans, so as to subsequently reintroduce the stimulated and / or expanded T cells into the humans to treat the cancer in the humans.

[0284] The present invention provides a method for treating cancer in humans, wherein the cancer cells express an immunogenic fragment and variant selected from sequences of SEQ ID NO. 1-8 and any one thereof, comprising obtaining a population of leukocytes comprising at least T cells, optionally together with antigen-presenting cells, from said human, stimulating and / or amplifying said T cells in the presence of a corresponding polypeptide, nucleic acid, vector or composition of the present invention, and further introducing some or all of said leukocytes into the human, comprising at least the stimulated and / or amplified T cells.

[0285] In any of the above embodiments, the cancer is suitably melanoma, particularly cutaneous melanoma.

[0286] In one embodiment, a method is provided for preparing a population of T cells that are cytotoxic to cancer cells expressing sequences selected from SEQ ID NO. 1-8 and any of them, including immunogenic fragments and variants thereof, the method comprising (a) obtaining T cells and antigen-presenting cells from a cancer patient; and (ii) stimulating and expanding the T cell population in vitro with the corresponding polypeptide, nucleic acid, vector, or composition of the present invention.

[0287] "Corresponding" in this context means that if cancer cells express, for example, SEQ ID NO. A (A is one of SEQ ID NO. 1-8) 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 carrier or a composition containing one of the foregoing, in vitro to stimulate and expand the T cell population.

[0288] For example, in such methods, 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.

[0289] The present invention provides a T cell population that can be obtained by any of the foregoing methods (hereinafter referred to as the T cell population of the present invention).

[0290] In one embodiment, a cell is provided, said cell being a T cell (hereinafter referred to as the T cell of the present invention) that has been stimulated with the polypeptide, nucleic acid, carrier or composition of the present invention.

[0291] In one embodiment, a pharmaceutical composition comprising the T cell population or T cells of the present invention together with a pharmaceutically acceptable carrier is provided. Such a composition may, for example, be a sterile composition suitable for parenteral administration.

[0292] In one embodiment, the T cell population or T cells of the present invention are provided for use in a medicament.

[0293] Also provided is a method for treating a human with cancer, wherein the cancer cells express an immunogenic fragment and variant selected from sequences selected from SEQ ID NO. 1-8 and any one thereof, or a method for preventing cancer in a human, wherein the cancer cells express an immunogenic fragment and variant selected from sequences selected from SEQ ID NO. 1-8 and any one thereof, the method comprising administering to the human the T cell population or T cells of the present invention or a composition comprising the T cell population or T cells of the present invention.

[0294] In one embodiment, a population of T cells of the present invention, T cells of the present invention, or a composition comprising the population of T cells or T cells of the present invention are provided for the treatment or prevention of cancer in humans, wherein the cancer cells express sequences selected from the corresponding sequences of SEQ ID NO. 1-8 and immunogenic fragments thereof. In any of the above embodiments, the cancer is suitably melanoma, particularly cutaneous melanoma.

[0295] In one embodiment, a method or population of T cells, T cells, antigen-presenting cells, exosomes, or compositions for use according to the invention are provided, wherein the polypeptide comprises a sequence selected from:

[0296] (a) The sequence of any one of SEQ ID NO. 1, 2, 3, 5 and 8; and

[0297] Variations of the sequences (b)(a); and

[0298] Immunogenic fragments of the sequence (c)(a).

[0299] For example, the polypeptide comprises or is composed of a sequence selected from any of SEQ ID NO. 9-15, 18 and 22, and the nucleic acid comprises or is composed of a sequence selected from any of SEQ ID NO. 23, 24, 25, 27 and 30, or selected from any of SEQ ID NO. 31, 32, 33, 35 and 38;

[0300] And the cancer among them is uveal melanoma.

[0301] Engineered Immunotherapy

[0302] All types of CLT antigen-binding peptide derivatives described above, including TCRs or TCR analogues 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.

[0303] These derivatives fall under the category of "chimeric antigen receptors (CARs)," where, as used herein, a chimeric antigen receptor can refer to, for example, an artificial T-cell receptor, a chimeric T-cell receptor, or a chimeric immunoreceptor, and encompasses engineered receptors that are transplanted with artificial specificity onto specific immune effector cells. CARs can be used to confer specificity of monoclonal antibodies onto T cells, thus allowing for the generation of numerous specific T cells, for example, in adoptive cell therapy. CARs can guide the specificity of cells against tumor-associated antigens (the peptides of this invention), wherein the peptides are HLA-binding.

[0304] Another approach to treating cancer in patients involves genetically modifying T cells by expressing a chimeric antigen receptor (CAR) to target antigens expressed on tumor cells. This technique is reviewed in Wendell and June, 2017, Cell, 168:724-740 (the cited literature is incorporated herein by reference in its entirety).

[0305] These CAR-T cells can be generated by obtaining a sample of cells from a subject (e.g., from peripheral blood, cord blood, and / or via apheresis), wherein the sample contains T cells or T cell progenitor cells, and transfecting the cells with a nucleic acid encoding a chimeric T-cell receptor (CAR) that is immune-specific to the polypeptide of the present invention, wherein the polypeptide is HLA-binding. This nucleic acid will be able to integrate into the cell's genome, and the cells can be administered to the subject in an effective amount to provide a T-cell response against cells expressing the polypeptide of the present invention. For example, a sample of cells from a subject can be collected.

[0306] It is understood that the cells used to generate the CAR-expressing T cells can be autologous or non-autologous.

[0307] Transgenic T cells expressing CAR can have inactivated endogenous T cell receptor and / or endogenous HLA expression. For example, cells can be engineered to eliminate endogenous α / β T cell receptor (TCR) expression.

[0308] Methods for transfecting cells are well known in the art; however, highly efficient transfection methods such as electroporation can be used. For example, a nuclear transfection device can be used to introduce the nucleic acid or vector of the present invention expressing the CAR construct into cells.

[0309] A population of T cells expressing CAR can be enriched after transfection. For example, CAR-expressing cells can be sorted from those that do not express CAR using an antigen bound to CAR or a CAR-binding antibody (e.g., by FACS). Alternatively, the enrichment step may include depletion of non-T cells or depletion of cells lacking CAR expression. For example, CD56+ cells can be depleted from a culture population.

[0310] Transgenic cell populations expressing CAR can be cultured in vitro in media that selectively enhance the proliferation of CAR-expressing T cells. Therefore, CAR-expressing T cells can be expanded in vitro.

[0311] CAR cell samples can be preserved (or maintained in culture). For example, samples can be cryopreserved for later amplification or analysis.

[0312] CAR-expressing T cells can be used in combination with other therapeutic agents, such as checkpoint inhibitors, including PD-L1 antagonists.

[0313] In one embodiment, a cytotoxic cell is provided, which has been engineered to express any of the aforementioned antigen-binding peptides on its surface. Suitably, the cytotoxic cell is a T cell.

[0314] In one embodiment, a cytotoxic cell is provided for use in a drug, said cytotoxic cell being suitably a T cell engineered to express any of the aforementioned antigen-binding peptides on its surface.

[0315] The present invention provides a pharmaceutical composition comprising the cytotoxic cells of the present invention, wherein the cytotoxic cells are suitably T cells.

[0316] A method is provided for treating a human patient with cancer, wherein the cancer cells express an immunogenic fragment and variant selected from sequences of SEQ ID NO. 1-8 and any one thereof, or a method is provided for preventing cancer in a human, wherein the cancer cells express an immunogenic fragment and variant selected from sequences of SEQ ID NO. 1-8 and any one thereof, the method comprising administering the cytotoxic cells of the present invention, suitably T cells, to the human.

[0317] In one embodiment, the cytotoxic cells of the present invention, suitably T cells, are used to treat or prevent cancer in humans, wherein the cancer cells express immunogenic fragments selected from the corresponding sequences of SEQ ID NO. 1-8 and any one thereof.

[0318] combination therapy

[0319] The cancer treatment method of the present invention can be performed in combination with other therapies, especially checkpoint inhibitory proteins and interferons.

[0320] Peptides, nucleic acids, carriers, antigen-binding peptides, and adoptive cell therapies (based on APCs and T cells) can be used in combination with other components to enhance their immunogenicity, for example, to improve the magnitude and / or width of the evoked immune response or to provide other activities (e.g., to activate other aspects of the natural or adaptive immune response or to destroy tumor cells).

[0321] Therefore, the present invention provides kits of the compositions of the present invention (i.e., immunogenic compositions, vaccine compositions, or pharmaceutical compositions) or several such compositions, said compositions comprising the polypeptides, nucleic acids, or carriers of the present invention together with pharmaceutically acceptable carriers, and (i) one or more other immunogenic polypeptides or immunostimulatory polypeptides (e.g., interferon, IL-12, checkpoint blocking molecules, or nucleic acids encoding the former, or carriers comprising such nucleic acids), and (ii) small molecules (e.g., HDAC inhibitors or other drugs regulating the epigenetic characteristics of cancer cells) or biological products that enhance the translation and / or presentation of polypeptide products as the subject of the present invention (delivered as polypeptides, nucleic acids encoding the former, or carriers comprising such nucleic acids).

[0322] Checkpoint inhibitors, which block normal proteins on cancer cells or proteins on T cells that respond to these proteins, may be a particularly important class of drugs to combine with CLT antigen-based therapies, as these inhibitors seek to overcome one of the main defenses that cancer uses against its immune system.

[0323] Therefore, one aspect of the present invention includes administering the polypeptide, nucleic acid, carrier, antigen-binding polypeptide, composition, T cell, T cell population, or 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), bavencio (Bavencio) and durvalumab (Imfinzi), and CTLA-4 inhibitors such as ipilimumab (Yervoy).

[0324] Interferons (e.g., alpha, beta, and gamma) are a family of proteins produced in very small amounts by the body. 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, for example, in the thigh or abdomen.

[0325] Therefore, one aspect of the present invention includes administering the polypeptide, nucleic acid, carrier, antigen-binding polypeptide, or composition of the present invention in combination with an interferon, such as interferon α.

[0326] Different modes of the present invention can also be combined; for example, the polypeptides, nucleic acids and vectors of the present invention can be combined with APCs, T cells or T cell populations of the present invention (especially discussed).

[0327] One or more modes of the present invention can also be combined with conventional anticancer chemotherapy and / or irradiation.

[0328] Diagnostic drugs

[0329] In another aspect, the present invention provides a method for diagnosing cancer, particularly melanoma such as cutaneous melanoma, using one or more of the present invention's polypeptides or nucleic acids, or for diagnosing human subjects suitable for treatment with the present invention's polypeptides, nucleic acids, carriers, antigen-binding polypeptides, adoptive cell therapy, or compositions.

[0330] Therefore, the present invention provides a method for diagnosing a person with cancer, the method comprising the steps of: determining whether the cancerous cells express an immunogenic fragment or variant selected from SEQ ID NO. 1-8 and any of them (e.g., selected from sequences SEQ ID NO. 9-22 and 39), or a nucleic acid encoding the polypeptide sequence (e.g., selected from sequences SEQ ID NO. 23-30 and SEQ ID NO. 31-38), and diagnosing the person with cancer if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells.

[0331] The present invention provides a method for diagnosing a person with cutaneous melanoma, the method comprising the steps of: determining whether the cancerous cells express a polypeptide sequence selected from any of SEQ ID NO.4, 6 and 7 and their immunogenic fragments or variants, or a nucleic acid encoding the polypeptide sequence, and diagnosing the person with cutaneous melanoma if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells.

[0332] As used in this article, “overexpression” in cancer cells means that the expression level in cancer cells is higher than that in normal cells.

[0333] The present invention provides a method for diagnosing a person with cutaneous melanoma or uveal melanoma, the method comprising the steps of: determining whether the cancerous cells express an immunogenic fragment or variant of a polypeptide sequence selected from SEQ ID NO. 1, 2, 3, 5 and 8 or any of the thereof; or a nucleic acid encoding the polypeptide sequence, and diagnosing the person with cutaneous melanoma or uveal melanoma if the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells.

[0334] Overexpression can be determined by referencing the levels of the nucleic acids or peptides of the present invention in known cancer-free control human subjects. Therefore, overexpression indicates that the nucleic acids or peptides of the present invention are detected in experimental subjects at significantly higher levels (e.g., 30%, 50%, 100%, or 500% higher) than in control subjects. If control human subjects have undetectable levels of the nucleic acids or peptides of the present invention, diagnosis can be achieved by detecting the nucleic acids or peptides of the present invention.

[0335] The present invention also provides a method for treating a person suffering from cancer, the method comprising the steps of:

[0336] (a) Determine whether the cancer cells express an immunogenic fragment or variant of SEQ ID NO. 1-8 and any of them (e.g., sequences selected from SEQ ID NO. 9-22 and 39) or a nucleic acid encoding the polypeptide (e.g., sequences selected from SEQ ID NO. 23-30 and 31-38); and if expressed, then

[0337] (b) administering to the person the corresponding peptide, nucleic acid, carrier, composition, T cell population, T cell, antigen-presenting cell, antigen-binding polypeptide or cytotoxic cell of the present invention.

[0338] The use of the polypeptide is also provided, the polypeptide comprising a sequence selected from the following:

[0339] (a) A sequence of any one of SEQ ID NO. 1-8; or

[0340] Variations of the sequences (b)(a); and

[0341] (c)(a) is an immunogenic fragment of the sequence isolated from a tumor in a person with cancer, or is used as a biomarker to determine whether the person is fit to be treated with a vaccine, the vaccine comprising the corresponding polypeptide, nucleic acid, vector, composition, T cell population, T cells, antigen-presenting cells, antigen-binding polypeptide, or cytotoxic cells of the present invention.

[0342] Appropriately, cancer is melanoma, especially cutaneous melanoma.

[0343] The present invention also provides a method or use according to the invention, wherein the polypeptide comprises a sequence selected from the following:

[0344] (a) The sequence of any one of SEQ ID NO. 1, 2, 3, 5 and 8; and

[0345] Variations of the sequences (b)(a); and

[0346] Immunogenic fragments of the sequence (c)(a).

[0347] For example, the polypeptide comprises or is composed of a sequence selected from any of SEQ ID NO. 9-15, 18 and 22, and the nucleic acid comprises or is composed of a sequence selected from any of SEQ ID NO. 23, 24, 25, 27 and 30, or selected from any of SEQ ID NO. 31, 32, 33, 35 and 38;

[0348] And the cancer among them is uveal melanoma.

[0349] Suitable, the polypeptides of the present invention have sequences selected from SEQ ID NO. 1-8 or fragments thereof, such as immunogenic fragments thereof (e.g., selected from sequences SEQ ID NO. 9-22 and 39).

[0350] Suitable, the nucleic acids of the present invention have or contain sequences selected from any one of SEQ ID NO. 23-30 or 31-38, or fragments thereof, such as immunogenic fragments thereof.

[0351] Kits for detecting the presence of nucleic acids are well known. For example, kits comprising at least two oligonucleotides that hybridize with a polynucleotide can be used within a real-time PCR (RT-PCR) reaction to allow the detection and semi-quantification of specific nucleic acids. Such kits can allow detection based on Forster resonance energy transfer (FRET) (e.g., (kit) or when double-stranded DNA binds (e.g., The Green kit generates a fluorescent signal to detect PCR products. Some kits (e.g., those containing exons from transtarget DNA) generate this fluorescent signal. Those probe kits allow for the detection and quantification of mRNA (e.g., transcripts encoding the nucleic acids of this invention). Analysis using certain kits can be established in a multiplex pattern to simultaneously detect multiple nucleic acids within the reaction. Kits can also be used to detect active DNA (i.e., DNA carrying markers indicating specific epigenetic characteristics of expression). Additional components that may be present within such kits include diagnostic reagents or reporter molecules that facilitate the detection of the nucleic acids of this invention.

[0352] The nucleic acids of the present invention can also be detected using a liquid biopsy method, using blood samples from the patient. This method provides a non-invasive alternative to surgical biopsy. Plasma from such blood samples can be separated and the presence of the nucleic acids of the present invention can be analyzed.

[0353] The polypeptides of the present invention can be detected in a homogenized preparation of a patient tumor sample using an antigen-specific antibody in an ELISA-type analysis. Alternatively, the polypeptides of the present invention can be detected using immunohistochemical analysis, which identifies the presence of the polypeptide antigen by examining sections of a patient tumor sample using optical microscopy, wherein the sections have been stained with a suitable labeled antibody preparation. As yet another alternative, the polypeptides of the present invention can be detected using immunohistochemical analysis, which identifies the presence of the polypeptide antigen by examining sections of a patient tumor sample using optical microscopy, wherein the sections have been stained with a suitable labeled antibody preparation.

[0354] The peptides of the present invention can also be detected by determining whether they can stimulate the production of T cells targeting the peptides.

[0355] A method for treating cancer in humans, particularly melanoma, such as cutaneous melanoma, includes (i) detecting the presence of the nucleic acid or polypeptide of the present invention and (ii) administering the nucleic acid, polypeptide, carrier, cell, T cell or T cell population or composition of the present invention to a subject (and preferably administering the same nucleic acid or polypeptide or fragment thereof that has been detected).

[0356] A method of treating cancer in humans, particularly melanoma, such as cutaneous melanoma, further includes administering the nucleic acid, polypeptide, carrier, cell, T cell or T cell population or composition of the present invention to a subject in which the presence of the (and preferably identical) nucleic acid or polypeptide of the present invention has been detected.

[0357] In particular, the cancers awaiting diagnosis and appropriate treatment are melanomas, such as cutaneous melanomas.

[0358] If the polypeptide or fragment thereof of the present invention in SEQ ID NO. 1, 2, 3, 5 or 8 is detected, the cancer may be cutaneous melanoma or uveal melanoma.

[0359] Detailed Implementation Plan

[0360] In one embodiment, the CLT antigen polypeptide comprises or is composed of SEQ ID NO.1. Exemplary fragments comprise or are composed of either SEQ ID NO.9 or 10. Exemplary nucleic acids encoding the polypeptide sequence comprise or are composed of either SEQ ID NO.23 or SEQ ID NO.31. The 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 cancers, particularly melanomas, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0361] In one embodiment, the CLT antigen polypeptide comprises or is composed of SEQ ID NO.2. Exemplary fragments comprise or are composed of any of SEQ ID NOs 11-14. Exemplary nucleic acids encoding the polypeptide sequence comprise or are composed of SEQ ID NO.24 or SEQ ID NO.32. The 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 cancers, particularly melanomas, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0362] In one embodiment, the CLT antigen polypeptide comprises or is composed of SEQ ID NO.3. An exemplary fragment comprises or is composed of SEQ ID NO.15. An exemplary nucleic acid encoding the polypeptide sequence comprises or is composed of SEQ ID NO.25 or SEQ ID NO.33. The corresponding nucleic acid (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 acid (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 cancers, particularly melanomas, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0363] In one embodiment, the CLT antigen polypeptide comprises or is composed of SEQ ID NO. 4. Exemplary fragments comprise or are composed of either SEQ ID NO. 16 or 17. Exemplary nucleic acids encoding the polypeptide sequence comprise or are composed of either SEQ ID NO. 26 or SEQ ID NO. 34. The 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 cancers, particularly melanoma, such as cutaneous melanoma. Related diagnostic methods are also provided.

[0364] In one embodiment, the CLT antigen polypeptide comprises or is composed of SEQ ID NO. 5. An exemplary fragment comprises or is composed of SEQ ID NO. 18. An exemplary nucleic acid encoding the polypeptide sequence comprises or is composed of SEQ ID NO. 27 or SEQ ID NO. 35. The corresponding nucleic acid (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 acid (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 cancers, particularly melanomas, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0365] In one embodiment, the CLT antigen polypeptide comprises or is composed of SEQ ID NO. 6. Exemplary fragments comprise or are composed of either SEQ ID NO. 19 or 20. Further exemplary fragments comprise or are composed of SEQ ID NO. 39. Exemplary nucleic acids encoding the polypeptide sequence comprise or are composed of either SEQ ID NO. 28 or SEQ ID NO. 36. The 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 cancers, particularly melanoma, such as cutaneous melanoma. Related diagnostic methods are also provided.

[0366] In one embodiment, the CLT antigen polypeptide comprises or is composed of SEQ ID NO. 7. An exemplary fragment comprises or is composed of SEQ ID NO. 21. An exemplary nucleic acid encoding the polypeptide sequence comprises or is composed of SEQ ID NO. 29 or SEQ ID NO. 37. The 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 cancers, particularly melanoma, such as cutaneous melanoma. Related diagnostic methods are also provided.

[0367] In one embodiment, the CLT antigen polypeptide comprises or is composed of SEQ ID NO. 8. An exemplary fragment comprises or is composed of SEQ ID NO. 22. An exemplary nucleic acid encoding the polypeptide sequence comprises or is composed of SEQ ID NO. 30 or SEQ ID NO. 38. The 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 cancers, particularly melanomas, such as cutaneous melanoma or uveal melanoma. Related diagnostic methods are also provided.

[0368] Example

[0369] Example 1 – CLT Identification

[0370] The aim is to identify cancer-specific transcripts that are composed entirely or partially of LTR elements.

[0371] As a first step, we assembled a complete pan-cancer transcriptome de novo. To achieve this, RNA sequencing reads from 768 patient samples obtained from the Cancer Genome Atlas (TCGA) consortium and representing a wide range of cancer types (24 sex-balanced samples from each of 32 cancer types (31 primary melanomas and 1 metastatic melanoma); Table S1). Sex-balanced samples (excluding sex-specific tissues) were trimmed for adapters and quality (Q20) and length filtered (≥35 nucleotide pairs for both reads) using cutadapt (v1.13) (Marcel M., 2011, EMBnet J., 17:3), and normalized (k = 20) using khmer (v2.0) (Crusoe et al., 2015, F1000Res., 4:900) for maximum and minimum depths of 200 and 3, respectively. Using STAR (2.5.2b) along with the same settings used across TCGA, 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, with built-in deep normalization disabled on the computer. Most of the assembly process was completed within 256GB of RAM on a 32-core HPC node, with failed processes rerun using a 1.5TB RAM node. The resulting contigs underwent polyadenylation trimming (trimpoly within SeqClean v110222) and entropy filtering (≥0.7) to remove low-quality and artificial contigs (bbduk within BBMap v36.2). Based on cancer type, Salmon (v0.8.2 or v0.9.2) (Patro, R. et al., 2017, Nat. Methods, 14:417-419) was used to quasi-map the original 24 samples to the cleaned assemblies, removing contigs found to be expressed at <0.1 transcripts per million (TPM). GMAP (v161107) (Wu et al., 2005, Bioinf., 21:1859-1875) was used to map the remaining contigs to GRCh38, and contigs not aligned with ≥85% identity within ≥85% of their length were removed from the assemblies. Finally, gffread (Cufflinks v2.2.1) (Trapnell et al., 2010, Nat. Biotech., 28:511-515) was used to flatten all cancer type assemblies together and combine them into the longest continuous transcript.Because this assembly process was specifically designed to enable the assessment of repetitive elements, single-exon transcripts were preserved but tagged. Transcript assembly integrity and quality were assessed by comparison with GENCODEv24basic and MiTranscriptome1 (Iye et al. 2015, Nat. Genet., 47:199-208). We compiled a unique list of splice sites within GENCODE and examined the presence of splice sites within a 2-nucleotide grace window within the transcriptome assembly. This process resulted in the identification of 1,001,931 transcripts, of which 771,006 were spliced ​​and 230,925 were single-exon.

[0372] Individually, the assembled contigs were superimposed with genomic repetitive sequence annotations to identify transcripts containing LTR elements. LTR and non-LTR elements were annotated as previously described (Attig et al., 2017, Front. In Microbiol., 8:2489). Briefly, 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), representing hidden Markov models (HMMs) (Dfam 2.0 library v150923) representing known human repetitive sequence families, was used to annotate GRCh38. Compared to BLAST-based methods, HMM-based scanning improved annotation accuracy (Hubley et al., 2016, Nuc. Acid. Res., 44:81–89). RepeatMasker annotates both the LTR and internal regions separately, thus parsing the tabular output to merge adjacent annotations of the same elements. This process yielded 181,967 transcripts containing one or more, complete or partial, LTR elements.

[0373] Using Salmon, parts-per-million (TPM) of all transcripts were estimated, and expression within each cancer type was compared to expression across 811 healthy tissue samples, with controls matched for all cancer types from TCGA (if available) and separately from GTEx (Genotype-Tissue Expression Consortium, 2015, Science, 348:648-60). Transcripts were considered to be 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. expression in ≥6 of 24 samples for each cancer type; ii. expression at <10 TPM in ≥90% of all healthy tissue samples; iii. expression in the target cancer type ≥3-fold the median expression in any control tissue type; and iv. expression in the target cancer type ≥3-fold the 90th percentile of the corresponding healthy tissue when available.

[0374] The cancer-specific transcript list was then intersected with a list of transcripts containing complete or partial LTR elements to produce a list of 5,923 transcripts that met all the criteria (referred to as the cancer-specific crossover). L TR element transcripts (CLT).

[0375] Forty-three CLTs specifically expressed in melanoma were further managed to exclude those with potential misassembly overlaps and those corresponding to cellular genome assembly. Additional manual evaluation was performed to ensure that the raw RNA sequencing reads from the cancers from which their specific expression was determined supported the splicing pattern. Further CLT classification resulted in the discarding of those with a median expression exceeding 1 TPM in any GTEx normal tissue.

[0376] Of the 403 CLTs in cutaneous melanoma, 97 CLTs passed through these filters.

[0377] Example 2 – Immunopeptidomics Analysis

[0378] Immunopeptidomic analysis based on mass spectrometry (MS) is a powerful technique that allows for the direct detection of specific peptides associated with HLA molecules (HLAp) and presented on the cell surface. This technique involves affinity purification of HLAp from biological samples (such as cells or tissues) via anti-HLA antibody capture. The isolated HLA molecules and bound peptides are then separated from each other, and the eluted peptides are analyzed by nano-ultra-high performance liquid chromatography coupled with mass spectrometry (nUPLC-MS) (Freudenmann et al., 2018, Immunology 154(3):331-345). In the mass spectrometer, specific peptides with a defined mass-charge ratio (m / z) are selected, separated, and fragmented, followed by a second round of mass spectrometry (MS / MS) to reveal the m / z of the resulting fragment ions. The fragment spectrum (MS / MS) can then be queried to precisely identify the amino acid sequence of the selected peptide that produced the detected fragment ions.

[0379] The interpretation of MS / MS spectra and subsequent peptide sequence identification depend on the matching between experimental data and theoretical spectra created from peptide sequences found in reference databases. While MS data can be retrieved using predefined lists corresponding to full open reading frames (ORFs) derived from known transcriptomes or even complete genomes (Nesvizhskii et al., 2014, Nat. Methods 11:1114–1125), querying these very large sequence databases results in extremely high false discovery rates (FDRs), which limit the identification of presented peptides. Other technical issues (e.g., the quality of leucine = the quality of isoleucine) and theoretical issues (e.g., peptide splicing (Liepe et al., 2016, Science 354(6310):354–358)) further increase the limitations associated with using very large databases, such as those derived from known transcriptomes or complete genomes. Therefore, in practice, performing precise immunopeptidomics analysis to identify neoantigens without referencing a well-defined set of potential peptide sequences is exceptionally difficult (Li et al., 2016, BMC Genomics 17(Suppl 13):1031).

[0380] Therefore, we constructed a database of all predicted polypeptide sequences (ORFs) of ≥10 residues from 97 cutaneous melanoma CLTs from Example 1. This yielded 2,269 ORFs ranging in length from 10 to 207 amino acids.

[0381] By comparing peptide sequences reported in the complete human proteome, Bassani-Sternberg et al. (Bassani-Sternberg et al., 2016, Nature Commun., 7:13404; database link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894) queried MS / MS data from HLA-binding peptide samples derived from 25 patients with cutaneous melanoma. These analyses revealed thousands of peptides that match known human proteins. As expected, these peptides included those found within various tumor-associated antigens (TAAs), including PRAME, MAGEA3, and TRPM1 (melastatin).

[0382] The inventors obtained frozen tumor tissue from two patients diagnosed with melanoma. Samples of 0.6–1 g were homogenized, lysed by high-speed centrifugation, and the clarified lysate was mixed with protein A (ProA) beads covalently linked to an 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 HLA class I binding peptide was eluted from the antibody using 10% acetic acid, and then the peptide was separated from other high-molecular-weight components using reversed-phase column chromatography (Ternette et al., 2018). The purified and eluted peptides were subjected to nUPLC-MS, and specific peptides with specific charge-to-mass ratios (m / z) were selected in the mass spectrometer, separated and fragmented, and subjected to a second round of mass spectrometry (MS / MS) to reveal the m / z of the resulting fragment ions (Ternette et al., 2018), generating an MS / MS dataset corresponding to the immune peptide group of each of these tumor samples.

[0383] By applying detailed immunopeptidomics evaluation knowledge, using PEAKS TMThe software (v8.5 and vX, Bioinformatics Solutions Inc.) queried the spectra of the PXD004894 HLA-I class dataset from 25 melanoma patients (Bassani-Sternberg et al., 2016) and the spectra of the HLA-I class dataset from 10 melanoma samples with CLT-derived ORFs greater than 9 AA prepared by the inventors, along with all peptide sequences present in the Human Proteome (UniProt). Since most HLA class I binding peptides present in cells are derived from constitutively expressed proteins, simultaneously querying these databases with the UniProt proteome helps ensure that our CLT ORF sequences are correctly assigned to MS / MS spectra. Similar to other MS / MS query software, the PEAKS software assigns a probability value (-10lgP; see Table 1) to each specified spectrum to quantify the assignment.

[0384] These findings identified over 50 independent peptides associated with HLA class I molecules immunoprecipitated from tumor samples from 25 patients examined by Bassani-Sternberg et al., as well as from samples from two melanoma patients in the inventors' dataset. These peptides correspond to the amino acid sequences of CLT-derived ORFs and do not correspond to polypeptide sequences known within the human proteome (UniProt).

[0385] Further manual analysis of the peptide spectra assigned by the PEAKS software was used to confirm the spectra assigned to the peptides, which were localized to eight CLT-derived ORFs and thus defined as CLT antigens (Table 1; SEQ ID NO. 1-8).

[0386] Detection of these peptides associated with HLA class I molecules confirmed that eight ORFs derived from these peptides are first translated in melanoma tissue, processed via the HLA class I pathway, and ultimately presented to the immune system when complexed with HLA class I molecules. Table 1 shows the characteristics of the peptides found in the CLT antigen. Figure 1-14 Figures 29-31 show representative MS / MS spectra for each of the peptides shown in Table 1. The top small plot of each figure shows the MS / MS peptide fragmentation characteristics, with standard MS / MS annotations (b: N-terminal fragment ion; y: C-terminal fragment ion; -H₂O: dehydration; -NH₃: ammonia loss; [2+]: double-charged peptide ion; pre: undisturbed precursor peptide ion; a...). n -n: internal fragment ions) is shown in the most abundant fragment ion peak (by PEAKS) TMThe software extracts images from the inventors' internal dataset or from the dataset of Bassani-Sternberg et al., stored in the PRIDE database (link: https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894). The lower half of each image indicates the spectral presentation of the location of the linear peptide sequence, which has been localized to fragment ions. Consistent with the high -10lgP scores assigned to the peptides in Table 1, these spectra contain numerous fragments that precisely match the peptide sequences (SEQ ID NO. 9-22) we found in these analyses.

[0387] All peptides longer than 9 AA detected when associating with HLA class I molecules from Table 1 were evaluated using the NetMHCpan 4.0 prediction software. http: / / www.cbs.dtu.dk / services / NetMHCpan / The predictive strength of binding to HLA class A and ultrasound types was determined. The results of these predictive studies indicate that all 14 peptides (or 9-mers contained in each complete sequence) are predicted to bind to at least one tested supertype (see Table 2). Many sequences were predicted to bind to specific types within the examined HLA class I supertypes with high confidence (low-grade score %). The expectation that all detected peptides would bind to HLA types expected to be present in the patient population is consistent with its detection results. Furthermore, while the HLA types reported by Bassani-Sternberg et al. only pertain to a portion (20%) of the queried tumor samples, NetMHCpan 4.0 predicted that every peptide identified in tumor samples from the subtyped patients, or found in tumor samples from the inventors' dataset, would bind to one of the reported patient HLA types.

[0388] To provide further certainty regarding the assignment of tumor tissue-derived MS profiles to the peptide sequences we discovered, peptides with the discovered sequences were synthesized and subjected to nUPLC-MS using the same conditions applicable to tumor samples from the original study (Bassani-Sternberg et al., 2016, Nature Commun., 7:13404; inventors' database). 2 . Figure 15-28 The spectral comparisons of the selected peptides are displayed. In each image, the upper spectrum corresponds to the tumor sample (from the PRIDE database (Bassani-Sternberg et al., 2016, Nature Commun., 7:13404; database link: [link missing]). https: / / www.ebi.ac.uk / pride / archive / projects / PXD004894(or in the inventor's database) and the lower spectra correspond to synthetically produced peptides with the same sequence. Selected m / z values ​​of detected ion fragments are displayed above / below each fragment peak in these MS / MS spectra. These figures reveal precise fragment alignment results (the small differences in experimentally determined m / z values ​​between tumor-derived fragment ions and synthetic peptide-derived fragment ions fall entirely within an m / z tolerance of <0.05 Daltons), confirming the authenticity of the CLT-encoded peptide assignments in the tumor tissue-derived spectra.

[0389] In summary, Tables 1 and 2 Figure 1-14 and 29-31 and Figure 15-28 The peptide data shown provide unusually strong support for the translation, processing, and presentation of the corresponding CLT antigen in melanoma patients.

[0390] 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 spectrally searched the HLA-I dataset from these normal tissue samples, retrieving all possible peptide sequences derived from the polypeptide sequences of CLT antigens 1 through 8. No peptides derived from these CLT antigens were detected in the normal tissue sample set (Table 3), thus further confirming the cancer-specific expression of CLTs.

[0391] In summary, this identification of immunopeptidomic peptides derived from predicted ORFs indicates that these CLTs are translated into polypeptides (SEQ ID NO. 1-8; referred to as CLT antigens) in tumor tissue. These antigens are then processed by the cell's immune monitor, and the component peptides are loaded onto HLA class I molecules, enabling the cells to be targeted by T cells that recognize the resulting peptide / HLA class I complex, leading to cell lysis. Therefore, these CLT antigens and their fragments are expected to be used to treat melanoma in patients whose tumors express these antigens using a variety of therapeutic modalities.

[0392] Table 1: List of peptides identified by immunopeptidomics analysis of melanoma tumor samples, along with CLT antigen names and cross-references to SEQ ID NO.

[0393]

[0394]

[0395] 1 HLA class I peptides identified by mass spectrometry.

[0396] 2Bassani-Sternberg et al., 2016, Nature Comm., 7:13404. Inventors' datasets (2MT3, 2MT4)

[0397] 3 Calculated peptide mass.

[0398] 4 PEAKS TM The procedure displays the 10lgP value of the peptide / patient highest matching degree for the peptide, and obtains more than one spectroscopic detection for the peptide.

[0399] 5 The number of peptides detected was spectral density.

[0400] 6 The deviation between observation quality and computational quality; showing the selected ppm values ​​for peptides for which more than one spectrum was obtained.

[0401] Table 2: Predictions of peptides (≥9 residues in length) identified by mass spectrometry with 18 HLA class I supertype alleles (HLA-A0101, HLA-A0201, HLA-A0301, HLA-A1101, HLA-A2402, HLA-A2501, HLA-A2601, HLA-A6801, HLA-B0702, HLA-B0801, HLA-B1501, HLA-B1801, HLA-B2705, HLA-B3501, HLA-B3503, HLA-B4001, HLA-B4002, HLA-B5101) in NetMHCpan4.0, along with CLT antigen names and cross-references to SEQ ID NO.

[0402]

[0403] 1 The rank score of ≤2.0% was combined with the prediction of the query HLA type I supertype.

[0404] 2 Predict the number of 18HLA class I supertypes (all bound) with a grade score of ≤2.0%.

[0405] 3 Predict the number of 18HLA class I supertypes that bind with a grade score of ≤0.5% (strong binding).

[0406] 4 Bassani-Sternberg et al. 2016, Nature Comm., 7:13404

[0407] Table 3. Number of peptides derived from CLT antigens 1 to 8 in normal tissue sample collections.

[0408] antigen skin lung mammary gland 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 CLT antigen 5 0 / 10 0 / 9 0 / 18 CLT antigen 6 0 / 10 0 / 9 0 / 18 CLT antigen 7 0 / 10 0 / 9 0 / 18 CLT antigen 8 0 / 10 0 / 9 0 / 18

[0409] The results presented here in Examples 1 and 2 are based, in whole or in part, on the Cancer Genome Atlas (TCGA) research network. http: / / cancergenome.nih.gov / Data generated by the Genotype-Tissue Expression (GTEx) project (supported by the Joint Foundation of the Office of the Director of the National Institutes of Health and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS).

[0410] Example 3 – Determination of T-cell specificity against CLT antigen in melanoma patients

[0411] (a) Reactive T cells stained with CLT antigenic peptide pentamer

[0412] The presence and activity of CLT antigen-specific circulating CD8 T cells in melanoma patients can be measured using HLA class I / peptide-pentame (“pentame”) staining and / or in vitro killing assays. Therefore, these methods are applied to CLT antigens (Tables 1-3) identified using the methods elucidated in Examples 1 and 2. Figure 1-31 It can be used to demonstrate the presence of therapeutically significant T-cell responses to CLT antigens in cancer patients.

[0413] For these studies, CD8 T cells isolated from patient blood were expanded using various culture methods, such as anti-CD3 and anti-CD28 coated microbeads plus interleukin-2. Subsequently, the expanded cells were stained reactive to specific CLT antigens for their T cell receptors using CLT peptide pentamers, which consist of HLA class I molecules bound to the peptide-binding groove of HLA molecules. Binding was measured by detecting antibody fragments specific to the coiled-coil polymerized domains of the pentamer structure, conjugated with phycoerythrin or allophycocyanin. In addition to pentamer staining, other surface markers, such as the memory marker CD45RO and the lysosomal release marker CD107a, could be queried. The association between pentamer positivity and specific surface markers could be used to infer the number and status of the pentamer-reactive T cell population (memory cells vs. naïve cells / stem cells).

[0414] Cells stained with pentamers can also be sorted and purified using fluorescence-activated cell sorting (FACS). The ability of sorted cells to kill target cells can then be further tested in in vitro killing assays. These assays include a population of CD8 T cells and a population of fluorescently labeled target cells. In this case, the CD8 population is either CLT antigen-specific to a known potent killing response to a positive control antigen such as Mart-1, or has been sorted by CD8 T cell pentamers and is specific to the aforementioned positive control antigen. Target cells in these studies can include T2 cells pulse-labeled with HLA-A*02, C1R cells pulse-labeled with HLA-A*02,03, or B*07, melanoma cell lines or patient tumor cells previously showing expression of CLT / CLT antigens, or cell lines such as CaSki transfected with CLT read frames. 7AAD uptake indicates target cell death. In this way, as target cells are subjected to CD8 T cell-mediated apoptosis, they acquire red fluorescence. Therefore, the application of this type of kill assay to pentamer-sorted CLT antigen-specific CD8 T cells can be used to count the cytotoxic activity of CLT antigen-specific T cells in ex vivo cultures of T cells from melanoma patients or healthy donors.

[0415] Figure 32 HLA pentamer staining of healthy donor CD8 T cells with a peptide derived from CLT antigen 6 (peptide SLYGHIHNEA (SEQ ID NO. 39)) 14 days after fluorescently activated cell sorting of pentamer-positive cells and amplification with anti-CD3 and anti-CD28-coated zirconia-IL-2. The inset on the right shows that these CD8+ T cells exhibit very weak antigen-specific killing of A2 target cells by the peptide pulse, but potent antigen-specific killing against CaSki cells transfected with the CLT antigen 6 read frame. Negative controls for this in vitro killing assay included unrelated T2 cells without the peptide and untransfected CaSki cells.

[0416] (b) Analysis of T-cell-specific HERVfest in melanoma patients

[0417] Specific T-cell functional expansion (fest) technology has been used to identify specific tumor-derived epitopes present in a library of mutation-associated neoantigens (MANA) in tumor cells of patients who have responded to checkpoint blockade therapy (Anagnostou et al., Cancer Discovery 2017; Le et al., Science 2017). This technology was applied using Examples 1 and 2 (Tables 1 to 3). Figure 1-31 The CLT antigen discovered by the method described in [the paper] can confirm the presence of treatment-associated T-cell responses against the CLT antigen in cancer patients.

[0418] Similar to other analyses that identify epitope-specific T cells in subjects with immune exposure (e.g., ELISPOT), the "fest" technique gains specificity by expanding homologous T cells in in vitro cultures containing antigen-presenting cells and suitable antigenic peptides. This technique differs from other immunological analyses in that it utilizes next-generation sequencing (specifically, TCRseq targeting the TCR-VβCDR3 region) of T cell receptor (TCR) mRNA present in these expanded cultures to detect specific TCRs expanded in cells cultured with the target peptide (pre-selected using a standard HLA binding algorithm to match the patient's HLA type). Applying TCRseq to tumor tissue harvested from the same patient following successful checkpoint blockade therapy can then be used to determine which TCRs / T cells detected in in vitro, peptide-stimulated cultures also reside at immunosuppressive sites in the cancer. In the case of MANAfest, this method is used to identify specific TCRs that recognize MHC-presented neoantigen peptides that evolve in each patient’s tumor and are also detected in the patient’s tumor T cells, thereby allowing the identification of functionally relevant neoantigen peptides among thousands of possible mutant peptides discovered by whole-exome sequencing of normal and tumor tissues from each patient (Le et al., Science 2017).

[0419] The MANAfest (Anagnostou et al., 2017 Cancer Discovery) technology was applied to CLT antigens as described below. Step 1: Identification of peptides in CLT antigens predicted to contain epitopes that effectively bind to selected HLA supertypes. Step 2: Selection of PBMCs from appropriate patients and matching them to the peptide library selected in Step 1 according to HLA type. Step 4: Separation of PBMCs from these patients into T-cell and non-T-cell fractions. Non-T cells were irradiated (to prevent proliferation) and re-added to the patient's T cells, then fractionated into 20–50 samples and cultured for 10–14 days in T-cell growth factors and a separate CLT-specific synthetic peptide (selected in Step 1). Step 5: TCRseq (sequencing of epitope-specific TCR-VβCDR3 sequences) was performed on all wells to identify amplified homologous T cells / TCRs in the presence of the test peptide; the specificity of these TCRs was determined by comparison with TCRs detected in unamplified / proliferating T cells using TCRseq. Data obtained from this step can confirm which peptides elicit an immune response in patients. Step 5: Perform TCRseq on tumor samples to determine which specifically expanded TCRs belong to the tumors of patients who responded to checkpoint blockade therapy, providing evidence that T cells carrying these TCRs may contribute to the effectiveness of checkpoint blockade therapy.

[0420] Example 4 - Demonstrating high affinity for CLT antigens that have not yet been deleted from the normal subject T cell library Analysis of Heli T cells

[0421] ELISPOT analysis can be used to demonstrate the presence of CLT antigen-specific CD8 T cells in a normal T cell pool of healthy individuals, thus not yet due to central tolerance deficiency, because cancer-specific CLT antigens are expressed in the naïve and thymic tissues of these patients. This type of ELISPOT analysis involves multiple steps. Step 1: CD8 T cells and CD14 monocytes can be isolated from peripheral blood of normal blood donors. These cells are HLA-generated to match the specific CLT antigen being tested. CD8 T cells can be further subdivided into naïve and memory subtypes using a magnetically labeled antibody against the memory marker CD45RO. Step 2: CD14 monocytes are pulsed with individual or pooled CLT antigen peptides for three hours, followed by co-culturing 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 may include: individual CLT antigen peptides, irrelevant control peptides, or peptides known to elicit robust responses against infectious antigens (e.g., CMV, EBV, Flu, HCV) or autoantigens (e.g., Mart-1). Restimulation was performed on plates coated with anti-interferon-γ (IFNγ) antibodies. The antibody captured any IFNγ secreted by T cells stimulated by the peptide. After overnight activation, cells were washed off the plates and the captured IFNγ on the plates was detected using other anti-IFNγ antibodies and standard colorimetric dyes. If IFNγ-producing cells were initially on the plate, dark spots were left. Data derived from this type of analysis include spot count, median spot size, and median spot intensity. These data are measures of the frequency of IFNγ-producing T cells and the amount of IFNγ per cell. Additionally, a measure of the magnitude of the CLT antigen response can be derived from the stimulation index (SI), which is a measure of specific response, calculated by dividing the spot count or median spot size by the background response to monocytes without the specific peptide. A measure of stimulation intensity is derived by multiplying the stimulation index of the spot count by the stimulation index of the spot intensity. In this way, comparing the response to the CLT antigen with that to a control antigen can demonstrate that treatment-naïve subjects possess a robust CLT antigen-responsive T cell pool that can be expanded through inoculation with CLT antigen-based immunogenic agents. Table 4 provides a series of CLT antigen-derived peptides that induced significant CD8 T cell responses in HLA-matched healthy blood donors. Results are as follows: Figure 33-38 As shown. The horizontal scale represents the mean of the data. M+t represents the peptide-free, negative control (monocytes and T cells). CEF represents the positive control (a mixture of 23 CMV, EBV, and influenza peptides). Statistical significance was assessed using one-way ANOVA with Kruskall-Walis test, and repeated measures were corrected using Dunns correction. Figure 33This shows a significant CD8 T cell response in normal blood donors to the HLA-A*02:01 restriction peptide from CLT antigen 1 (CLT001 in the figure). Figure 34 This demonstrates a significant CD8 T cell response in normal blood donors to the HLA-A*0301-restricted peptide derived from CLT antigen 1 (CLT001 in the figure). Figure 35 This demonstrates a significant CD8 T cell response in normal blood donors to the HLA-B*0702-restricted peptide derived from CLT antigen 2 (CLT002 ​​in the figure). Figure 36 This demonstrates a significant CD8 T cell response in normal blood donors to the HLA-A*0301-restricted peptide derived from CLT antigen 3 (CLT003 in the figure). Figure 37 This demonstrates a significant CD8 T cell response in normal blood donors to the HLA-A*0301-restricted peptide derived from CLT antigen 5 (CLT005 in the figure). Figure 38 This demonstrates a significant CD8 T cell response in normal blood donors to the HLA-A*0201-restricted peptide derived from CLT antigen 6 (CLT006 in the figure).

[0422] Table 4: CLT antigen-derived peptides that induce significant CD8 T cell responses in HLA-matched healthy blood donors

[0423] Reference CLT antigen SEQ ID NO peptide sequence HLA type of peptide (predicted by NetMHC) 1 SEQ ID.NO.9 GLNSIIWRL HLA-A*02:01 1 SEQ ID.NO.10 ILIQTTGIFK HLA-A*03:01 2 SEQ ID.NO.13 FPFYKDTVLL HLA-B*07:02 3 SEQ ID.NO.15 IVLDAPVTK HLA-A*03:01 5 SEQ ID.NO.18 ALRAVTLTAK HLA-A*03:01 6 SEQ ID.NO.39 SLYGHIHNEA HLA-A*02:01

[0424] Example 5 – Analysis to validate CLT expression in melanoma cells

[0425] a) qRT-PCR validation of CLT expression in melanoma cell lines

[0426] Quantitative real-time polymerase chain reaction (qRT-PCR) is a common technique for determining the quantity of a specific transcript present in RNA extracted from a given biological sample. Specific nucleic acid primer sequences are designed for the target transcript, and the region between the primers is subsequently amplified via a series of thermal cycling reactions, followed by fluorescence quantification using an intercalation dye (SYBR Green). Primer pairs are designed for CLT and analyzed for RNA extracted from melanoma cell lines or primary patient tissues. Non-melanoma cell lines are used as negative controls. The melanoma cell lines used include COLO 829 (ATCC reference number CRL-1974), MeWo (ATCC reference number HTB-65), and SH-4 (ATCC reference number CRL-7724), and the control cell lines HepG2 (hepatocellular carcinoma, ATCC reference number HB-8065), Jurkat (T-cell leukemia, ATCC reference number TIB152), and MCF7 (adenocarcinoma, ATCC reference number HTB-22). Patient-derived melanoma tissues were obtained from 6 primary lesions and 6 metastatic lesions, all from patients with at least stage IIC disease. RNA was extracted from each sample and reverse transcribed into cDNA following standard procedures. qRT-PCR analysis, along with SYBR Green assay, was performed using primers designed for two regions of each CLT and a reference gene, following standard techniques. Relative quantification (RQ) was calculated as follows:

[0427] RQ = 2[Ct(reference) - Ct(target)].

[0428] Figure 39 The results of these experiments are shown in Figure A. Figure A shows the results from qRT-PCR analysis, where two primer sets (76+77 and 78+79) targeted different regions of the CLT encoding CLT antigen 2 (SEQ ID NO. 24) on RNA extracted from 12 melanoma tissue samples and one non-melanoma cell line. Figure B shows the results from qRT-PCR analysis, where two primer sets (44+45 and 46+47) targeted different regions of the CLT encoding CLT antigen 3 (SEQ ID NO. 25) on RNA extracted from 12 melanoma tissue samples and one non-melanoma cell line. Figure C shows the results from qRT-PCR analysis, where two primer sets (80-81 and 82-83) targeted different regions of the CLT encoding CLT antigen 6 (SEQ ID NO. 28) on RNA extracted from 12 melanoma tissue samples and one non-melanoma cell line. These results confirm the specific expression of CLT in RNA extracted from melanoma tissue samples compared to non-melanoma cell lines. Each CLT was detected in two or more tissue samples analyzed, while expression was rarely or not detected in non-melanoma control cell lines.

[0429] b) RNAScope validates CLT expression in orthotopic melanoma cells.

[0430] In situ hybridization (ISH) methods for transcript expression analysis allow visualization of the presence and expression levels of a given transcript within the histopathological context of a specimen. Traditional RNA ISH analysis involves the in situ recognition of native RNA molecules using oligonucleotide probes specific to short segments of the expected RNA sequence, with the recognition visualized by means of a signal generated by a combination of antibody- or enzyme-based colorimetric reactions. RNAScope is a newly developed in situ hybridization-based technique employing more advanced probe chemistry that ensures the specificity of the generated signal and allows for sensitive, single-molecule visualization of the target transcript (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 multiple transcripts present.

[0431] RNAScope probes were designed targeting CLT, and the expression signals of 12 formalin-fixed, paraffin-embedded core sections of cutaneous melanoma tumors were analyzed. The following are representative images from each core section used to assess expression signals:

[0432] • Estimated percentage of CLT probe-positive cells, rounded to the nearest 10%.

[0433] • The estimated expression level per cell across a given slice is:

[0434] 0 = Unstained

[0435] ·1 = 1-2 points per cell

[0436] ·2 = 2-6 points per cell

[0437] ·3 = 6-10 points per cell

[0438] ·4 = >10 points per cell

[0439] The expression of each CLT was detected in tumor cores from multiple different patients, thereby independently validating CLTs discovered from tumor-derived RNAseq data and confirming the homogeneity of expression within tumor tissues across certain samples, as well as highlighting the presence of at least one CLT in the core of each patient analyzed.

[0440] Table 5 – RNAScope Assessment in Core Samples from Melanoma Patients

[0441]

[0442] Throughout this specification and the following claims, unless the content requires otherwise, the word “comprising” and its variations such as “including” and “containing” will be understood to mean including the integer, step, group of integers or group of steps, but will not exclude any other integer, step, group of integers or group of steps.

[0443] All patents, patent applications and references mentioned throughout this invention are incorporated herein by reference in their entirety.

[0444] The present invention includes all combinations of the preferred groups and more preferred groups and suitable groups and more groups and embodiments of the groups mentioned above.

[0445] sequence list

[0446] SEQ ID NO.1 (Polypeptide sequence of CLT antigen 1)

[0447]

[0448] SEQ ID NO.2 (Polypeptide sequence of CLT antigen 2)

[0449]

[0450] SEQ ID NO.3 (Polypeptide sequence of CLT antigen 3)

[0451]

[0452] SEQ ID NO.4 (Polypeptide sequence of CLT antigen 4)

[0453]

[0454] SEQ ID NO.5 (Polypeptide sequence of CLT antigen 5)

[0455]

[0456] SEQ ID NO.6 (Polypeptide sequence of CLT antigen 6)

[0457]

[0458] SEQ ID NO.7 (Polypeptide sequence of CLT antigen 7)

[0459]

[0460] SEQ ID NO.8 (Polypeptide sequence of CLT antigen 8)

[0461]

[0462] SEQ ID NO.9 (peptide sequence derived from CLT antigen 1)

[0463]

[0464] SEQ ID NO.10 (peptide sequence derived from CLT antigen 1)

[0465]

[0466] SEQ ID NO.11 (peptide sequence derived from CLT antigen 2)

[0467]

[0468] SEQ ID NO.12 (peptide sequence derived from CLT antigen 2)

[0469]

[0470] SEQ ID NO.13 (peptide sequence derived from CLT antigen 2)

[0471]

[0472] SEQ ID NO.14 (peptide sequence derived from CLT antigen 2)

[0473]

[0474] SEQ ID NO.15 (peptide sequence derived from CLT antigen 3)

[0475]

[0476] SEQ ID NO.16 (peptide sequence derived from CLT antigen 4)

[0477]

[0478] SEQ ID NO.17 (peptide sequence derived from CLT antigen 4)

[0479]

[0480] SEQ ID NO.18 (peptide sequence derived from CLT antigen 5)

[0481]

[0482] SEQ ID NO.19 (peptide sequence derived from CLT antigen 6)

[0483]

[0484] SEQ ID NO.20 (peptide sequence derived from CLT antigen 6)

[0485]

[0486] SEQ ID NO.21 (peptide sequence derived from CLT antigen 7)

[0487]

[0488] SEQ ID NO.22 (peptide sequence derived from CLT antigen 8)

[0489]

[0490] SEQ ID NO.23 (cDNA sequence encoding CLT antigen 1)

[0491]

[0492]

[0493] SEQ ID NO.24 (cDNA sequence encoding CLT antigen 2)

[0494]

[0495]

[0496]

[0497] SEQ ID NO.25 (cDNA sequence encoding CLT antigen 3)

[0498]

[0499]

[0500] SEQ ID NO 26 (cDNA sequence encoding CLT antigen 4)

[0501]

[0502] SEQ ID NO 27 (cDNA sequence encoding CLT antigen 5)

[0503]

[0504] SEQ ID NO 28 (cDNA sequence encoding CLT antigen 6)

[0505]

[0506]

[0507]

[0508] SEQ ID NO 29 (cDNA sequence encoding CLT antigen 7)

[0509]

[0510]

[0511] SEQ ID NO 30 (cDNA sequence encoding CLT antigen 8)

[0512]

[0513]

[0514] SEQ ID NO.31 (cDNA sequence encoding CLT antigen 1)

[0515]

[0516] SEQ ID NO.32 (cDNA sequence encoding CLT antigen 2)

[0517]

[0518] SEQ ID NO.33 (cDNA sequence encoding CLT antigen 3)

[0519]

[0520] SEQ ID NO.34 (cDNA sequence encoding CLT antigen 4)

[0521]

[0522] SEQ ID NO 35 (cDNA sequence encoding CLT antigen 5)

[0523]

[0524] SEQ ID NO 36 (cDNA sequence encoding CLT antigen 6)

[0525]

[0526] SEQ ID NO 37 (cDNA sequence encoding CLT antigen 7)

[0527]

[0528] SEQ ID NO 38 (cDNA sequence encoding CLT antigen 8)

[0529]

[0530] SEQ ID NO 39 (peptide sequence derived from CLT antigen 6)

[0531]

Claims

1. An isolated polypeptide having an amino acid sequence selected from the following sequences: It consists of the sequence of any one of SEQ ID NO. 2 and 11-14.

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

3. The nucleic acid according to claim 2 is DNA.

4. The nucleic acid according to claim 3, comprising or consisting of a sequence selected from or composed of any one of SEQ ID NO. 24 and 32.

5. The nucleic acid according to claim 4, wherein its expression in human host cells is codon-optimized.

6. The nucleic acid according to claim 2, wherein it is RNA.

7. The nucleic acid according to claim 2, 3, 5 or 6, wherein it is an artificial nucleic acid sequence.

8. A vector comprising the nucleic acid according to any one of claims 2 to 7.

9. The vector of claim 8, comprising DNA encoding a regulatory element adapted to allow translationally active RNA molecules to be transcribed in human host cells.

10. The vector according to claim 8 or claim 9, wherein it is a viral vector.

11. The vector according to claim 10 is an adenovirus vector, adeno-associated virus (AAV), alphavirus vector, herpesvirus vector, arenavirus vector, measles virus vector, poxvirus vector, paramyxovirus vector, lentivirus vector, and rhabdovirus vector.

12. An immunogenic pharmaceutical composition comprising a polypeptide, nucleic acid, or carrier according to any one of claims 1 to 11 together with a pharmaceutically acceptable carrier.

13. A vaccine composition comprising a polypeptide, nucleic acid, or carrier according to any one of claims 1 to 11 together with a pharmaceutically acceptable carrier.

14. The composition according to claim 12 or claim 13, comprising one or more immunostimulants.

15. The composition according to claim 14, wherein the immunostimulant is selected from aluminum salts, saponins, immunostimulatory oligonucleotides, oil-in-water emulsions, aminoalkyl aminoglucosidase 4-phosphate, lipopolysaccharides and their derivatives, and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and interferon.

16. The composition according to any one of claims 13 to 15, wherein it is a sterile composition suitable for parenteral administration.

17. Use of the polypeptide, nucleic acid, carrier, or composition according to any one of claims 1 to 16 in the preparation of a medicament for the treatment of melanoma.

18. Use of the polypeptide, nucleic acid, carrier, or composition according to any one of claims 1 to 16 in the preparation of a medicament for generating an immune response in humans, wherein the immune response is generated against a melanoma tumor.

19. The use according to claim 18, wherein an immune response is generated against a cancerous tumor, said cancerous tumor expressing a sequence selected from SEQ ID NO. 2 and SEQ ID NO. 11-14, wherein said cancerous tumor is melanoma.

20. Use of the polypeptide, nucleic acid, carrier, or composition according to any one of claims 1 to 16 in the preparation of a medicament for treating or preventing cancer in humans, wherein the cancer cell expression is selected from the sequences of SEQ ID NO. 2 and SEQ ID NO. 11-14, wherein the cancer is melanoma.

21. Use of the polypeptide, nucleic acid, carrier, or composition according to any one of claims 1 to 16 in the preparation of a medicament for in vitro stimulation and / or expansion of T cells derived from a human with cancer, so as to subsequently reintroduce the stimulated and / or expanded T cells into the human to treat the cancer in the human, wherein the cancer is melanoma.

22. A method for preparing a population of T cells that are cytotoxic to melanoma cancer cells, said cancer cells expressing sequences selected from SEQ ID NO. 11-14, said method comprising (a) obtaining T cells and antigen-presenting cells from a cancer patient; and (ii) stimulating and expanding the T cell population in vitro with a polypeptide, nucleic acid, vector or composition according to any one of claims 1 to 16.

23. An antigen-presenting cell which is modified or genetically engineered by in vitro loading of any one of the polypeptides, nucleic acids, carriers or compositions according to claims 1 to 16 to express the polypeptide according to claim 1.

24. The antigen-presenting cell according to claim 23, wherein it is a dendritic cell.

25. Exosomes loaded with a polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 16.

26. A pharmaceutical composition comprising an antigen-presenting cell or exosome according to any one of claims 23 to 25 together with a pharmaceutically acceptable carrier.

27. Use of the antigen-presenting cell or exosome according to any one of claims 23 to 25 in the preparation of a medicament for the treatment of melanoma.

28. Use of the antigen-presenting cell, exosome, or composition according to any one of claims 23 to 26 in the preparation of a medicament for treating a human with cancer or for preventing human cancer, wherein the cancer cell expression is selected from the sequences of SEQ ID NO. 2 and SEQ ID NO. 11-14, wherein the cancer is melanoma.

29. The use according to claim 21, wherein the cancer is uveal melanoma.

30. The method of claim 22, wherein the cancer is uveal melanoma.

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