Novel cancer antigen and method

Abstract

A polypeptide useful for the treatment, prevention, and diagnosis of cancer, particularly ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC, and a nucleic acid encoding said polypeptide are disclosed.

Description

Technology Field

[0001] The present invention relates to an antigenic polypeptide and a corresponding polynucleotide for use in the treatment or prevention of cancer, particularly cancer, e.g., ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, colon cancer, breast cancer, skin cancer, melanoma, lung cancer, osteosarcoma, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or for use in the treatment or prevention of EAC or ESSC. The present invention further relates to a pharmaceutical and immunogenic composition, particularly comprising said nucleic acid and polypeptide, said polypeptide and polynucleotide loaded and / or stimulated by said polypeptide and polynucleotide, said polypeptide genetically modified by a molecule that recognizes said polypeptide, and an antibody specific to the cell (autologous or other). Background Technology

[0002] As part of normal immune surveillance against pathogenic microorganisms, all cells degrade intracellular proteins to produce peptides that load onto the Major Histocompatibility Complex (MHC), which is expressed on the surface of all cells. Most of these peptides originate from host cells, which are recognized as self-contained and are not detected by the adaptive immune system. However, non-self peptides can stimulate the expansion of naive CD8+ T cells encoding T cell receptors (TCRs) that bind tightly to the MHC I-peptide complex. This expanded T cell population can produce effector CD8+ T cells (including cytotoxic T-lymphocytes - CTLs) capable of clearing foreign antigen-tagged cells, as well as memory CD8+ T cells that can be re-amplified if foreign antigen-tagged cells appear later in the animal's life.

[0003] MHC class II molecules are typically restricted to professional antigen-presenting cells (APCs), such as dendritic cells (DCs), and are usually loaded with peptides internalized from the external environment. The binding of complementary TCRs from naive CD4+ T cells to MHC II-peptide complexes in the presence of various factors, including T-cell adhesion molecules (CD54, CD48) and co-stimulatory molecules (CD40, CD80, CD86), involves CD4+ T-cells acting as effector cells (e.g., T H 1, T H 2, T H 17, T FH, T reg These effector CD4+ T cells are induced to mature into antibody-secreting plasma cells. These effector CD4+ T cells not only promote B-cell differentiation into antibody-secreting plasma cells but also promote the differentiation of antigen-specific CD8+ CTLs, thereby helping to induce an adaptive immune response to foreign antigens that includes both short-term effector function and long-term immune memory. DCs can perform the process of cross-presentation of peptide antigens by delivering exogenously derived antigens (e.g., peptides or proteins released from pathogens or tumor cells) to their MHC I molecules, which contributes to the generation of immune memory by providing an alternative pathway that stimulates the expansion of naive CD8+ T-cells.

[0004] Immune memory (specifically antigen-specific B cells / antigens and antigen-specific CTLs) plays a crucial role in eliminating microbial infections, and it has been utilized in the development of numerous vaccines to prevent diseases caused by significant pathogenic microorganisms. Although immune memory is known to play an important role in controlling tumor formation, effective cancer vaccines have rarely been developed.

[0005] Cancer is the second leading cause of morbidity worldwide and accounts for nearly one-sixth of all deaths globally. Of the 8.8 million deaths caused by cancer in 2015, the most life-threatening cancers were lung (1.69 million), liver (788,000), colorectal (774,000), gastric (754,000), esophageal (439,025), and breast (571,000) cancers. The economic loss due to cancer was estimated at $1.16 trillion in 2010, and the number of new cancer cases is expected to increase by approximately 70% over the next 20 years (WHO Cancer Statistics 2017).

[0006] Current therapies for cancer are highly diverse and vary significantly depending on the stage of the disease. One treatment for cancer is surgery to remove the tumor and surrounding tissues. Late-stage cancer may require treatment including lymph node dissection, radiation therapy, or chemotherapy. Immune checkpoint blocking strategies, including the use of antibody-targeted negative immunomodulators such as PD-1 / PD-L1 and CTLA4, have recently revolutionized the treatment of various malignancies (Ribas, A., & Wolchok, JD (2018) Science , 359: 1350-1355.). Significant recognition that the excellent value of checkpoint block therapies and their clinical benefits are related to the patient's own adaptive immune response to cancer antigens (particularly T cell-based immune response) has revitalized research on effective cancer vaccines, vaccine modalities, and cancer vaccine antigens.

[0007] Historically, it has been recognized that only a portion of the inventors' genome is transcribed into mRNA to produce proteins, and that the majority of the human genome consists of non-coding RNA that is not translated into proteins. In fact, less than approximately 1.5% of the human genome encodes proteins. The human genome is typically divided into the 'coding' genome, which generates approximately 20,000 annotated human protein-coding genes, and the 'dark' genome, which does not encode proteins. The dark genome likely occupies approximately 98.5% of the genomic space where repetitive elements, enhancers, regulatory sequences, and non-coding RNA exist. The most important gene regions are protein-coding sequences, and the remaining genome was considered to remain in an unclear state. More recently, it has been determined that these RNAs play important roles in various cellular and physiological processes, including gene regulation, chromatin packaging, cell differentiation, and development. Consequently, a significant portion of the human transcriptome remains biologically unknown and uncharacterized, continuing to contain the 'dark material' of the genome. Recent advancements in cancer vaccines and immunotherapy have led to the development of various treatments that utilize T-cell immunity to recognize and eliminate cancer cells, providing benefits to cancer patients with unmet needs. However, targeted immunotherapy has traditionally relied on tumor-associated antigens, which lack cancer specificity and are broadly expressed across the entire patient population. Genomic dark matter (genomic regions previously considered non-coding) is now recognized as offering opportunities to identify and characterize 'dark antigens,' which are novel, cancer-specific antigens uniquely presented on the surface of cancer cells and primary tumors by MHC receptors. Probing genomic dark matter requires a complex process of generating pan-cancer transcriptome assemblies using RNA sequence reads obtained from assembled cancer genome databases for specific tumors.Transcriptome sequences subject to differential expression analysis can be selected from sequences that exhibit abundant expression in tumors compared to a composite panel of healthy tissues. Subsequently, dark antigens encoded by tumor-specific transcriptomes are identified by translating all possible open reading frames (ORFs) and comparing them with mass spectrometry-based immunopeptidemics data obtained from selected cancer samples. This allows for the identification of peptides mapping to putative antigen-related ORF sequences, followed by immunopeptidemics validation regarding transcriptome expression frequency and cancer specificity. The immunogenicity of the identified dark antigens can be further evaluated by characterizing antigen-specific T cell responses from healthy donors. These dark antigens, as cancer antigens, can form the basis of therapeutic cancer vaccines.

[0008] A wide variety of vaccine modalities are known. One well-described approach involves directly delivering antigenic polypeptides to a subject for the purpose of enhancing immune responses (including B- and T-cell responses) and stimulating immune memory. Alternatively, polynucleotides can be administered to a subject via a vector so that polynucleotide-encoded immunogenic polypeptides are expressed in vivo. The use of viral vectors, such as adenovirus vectors, has been well studied for antigen delivery in both vaccination and cancer treatment strategies (Wold et al. Current Gene Therapy, 2013, Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy, 13:421-433). Immunogenic peptides, polypeptides, or polynucleotides encoding them can also be used to load patient-derived antigen-presenting cells (APCs), which can then be injected into a subject as a vaccine to induce a therapeutic or prophylactic immune response. An example of this approach is Provenge™ (cipurucel-T), currently the only FDA-approved anticancer vaccine.

[0009] Cancer antigens can also be studied in the treatment and prevention of cancer by using them to generate various non-vaccine therapeutic modalities. These therapies are divided into two distinct classes: 1) antigen-conjugated bioagents, and 2) adoptive cell therapies.

[0010] Antigen-binding bioagents may typically comprise a binding domain thereof capable of binding to a TCR or antibody or MHC-presented antigen peptide or epitope (pMHC), or may consist of a polyvalently engineered polypeptide that recognizes antigen-decorated cancer cells and promotes their destruction. The antigen-binding component of such polyvalently engineered polypeptide may consist of a TCR, a high-affinity TCR or its pMHC binding domain, and a TCR mimic produced by various technologies (including those based on monoclonal antibody technology) (e.g., an antibody-derived binding domain capable of recognizing and binding to pMHC), but is not limited to these, and may consist of a TCR-based bioagent. An antigen-binding bioagent that is a polyvalently engineered polypeptide may combine a TCR and an antibody domain in the same molecule, or combine a TCR or antibody domain with a cell lysis moiety. The cytolytic moiety of this type of multivalent bioagent may consist of cytotoxic chemicals, biological toxins, targeting motifs, and / or immunostimulating motifs that promote the targeting and activation of immune cells, and these promote the therapeutic destruction of tumor cells.

[0011] Adoptive cell therapy may be based on the patient's own T cells that are removed and stimulated in vitro with vaccine antigen preparations (cultured with T cells in the presence or absence of other factors including cellular and non-cellular components) (JCI Insight. 2018 Oct 4;3(19). pii: 122467. doi: 10.1172 / jci.insight.122467). Alternatively, adoptive cell therapy may be based on cells (including patient- or non-patient-derived cells) that have been intentionally engineered to express antigen-binding polypeptides that recognize cancer antigens. These antigen-binding polypeptides belong to the same class as those described above for antigen-binding bioagents. Thus, lymphocytes (autologous or non-autologous) genetically engineered to express cancer antigen-binding polypeptides can be administered to patients as adoptive cell therapy to treat their cancer.

[0012] The use of HERV (human endogenous retrovirus)-associated DAC antigens to enhance an effective immune response against cancer has shown promising results in promoting tumor regression and a more favorable prognosis in murine models of cancer (Kershaw et al., 2001, Cancer Res. 61:7920-7924; Slansky et al., 2000, Immunity 13:529-538). Additional HERV-associated DAC antigens were identified in WO2020 / 260898 (The Francis Crick Institute Ltd. and Enara Bio Ltd.), which discloses ovarian cancer antigens identified by a method involving the step of identifying cancer-specific transcripts consisting wholly or partially of LTR elements. Although HERV-related dark antigen-centered immunotherapy trials have been considered in humans (Sacha et al., 2012, J. Immunol 189:1467-1479), progress has been partially limited due to several limitations of identified tumor-specific ERV antigens. It is desirable to identify additional cancer-specific peptide-HLA antigens derived from abnormal epigenetic activity in the genomic 'dark matter,' which is a previously estimated non-coding genomic region of tumor cells, and which are distinct from ERV-related antigens. These novel cancer-related antigen sequences can be used in the immunotherapy of cancers, e.g., ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, colon cancer, breast cancer, skin cancer, melanoma, osteosarcoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer or EAC or ESSC.

[0013] The inventors have surprisingly discovered a specific RNA transcript that is found at high levels in ovarian cancer cells, pancreatic cancer cells, gastric cancer cells, stromal cancer cells, non-small cell lung cancer (NSCLC) cells, osteosarcoma cells, colon cancer cells, breast cancer cells, skin cancer cells, melanoma cells, head and neck cancer cells, sarcoma cells, rectal cancer cells, lung cancer (e.g., LUSC, LUAD, NSCLC) cells, gastric cancer cells, cervical cancer cells, uterine cancer cells, and particularly in esophageal cancer cells, e.g., esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), but is undetectable or found at very low levels in normal, healthy tissues (see Example 4). Furthermore, the inventors have shown that a subset of potential polypeptide sequences (i.e., open reading frames (ORFs)) is translated in cancer cells, processed by components of an antigen-processing unit, and presented on the surface of cells found in tumor tissue in relation to class I and class II major histocompatibility complexes (MHC class I and MHC class II) and class I and class II human leukocyte antigens (HLA class I, HLA class II) molecules (see Example 2). The inventors discovered that these peptides were mapped to ORFs encoded by a single cancer-specific transcript. These transcripts are referred to herein as cancer-specific transcripts [CSTs]. These results demonstrate that these polypeptides (referred to herein as CST antigens) are also antigenic.Therefore, the presentation of CST antigens to cancer cells is expected to provide these cells, which are likely to be eliminated by T cells possessing a cognate T cell receptor (TCR) for the CST antigen, and a CST antigen-based vaccination method / therapy that amplifies T cells possessing such cognate TCRs is expected to induce an immune response against cancer cells (and tumors containing them), e.g., ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, and in particular esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC). The CST and CST antigens subject to the present invention are not standard sequences that can be easily derived from known tumor genome sequences found in the cancer genome atlas. CST is a transcript resulting from transcription from a non-standard ORF. Because CSTs are expressed at high levels and the CST antigen polypeptide sequence is not that of a normal human protein, they can induce a strong, specific immune response and are therefore expected to be suitable for therapeutic use in the field of cancer immunotherapy.

[0014] CST antigens have been found to be usable in various forms in highly expressed transcripts for characterizing tumor cells that were previously unknown to exist and were not known to produce protein products in the human body prior to the present invention. First, the CST antigen polypeptide of the present invention can be delivered directly to a subject as a vaccine to induce a therapeutic or prophylactic immune response against tumor cells. Second, the nucleic acids of the present invention, which can be codon-optimized to enhance the expression of their encoded CST antigens, can be administered directly or otherwise inserted into a vector for in vivo delivery to produce the encoded protein product in the subject as a vaccine to induce a therapeutic or prophylactic immune response against tumor cells. Third, the polynucleotides and / or polypeptides of the present invention can be used to load patient-derived antigen-presenting cells (APCs), which can then be injected into the subject as a vaccine to induce a therapeutic or prophylactic immune response against tumor cells. Fourth, the polynucleotides and / or polypeptides of the present invention can be used for ex vivo stimulation of a subject's T cells to produce stimulated T cell preparations that can be administered to the subject as a therapy for treating cancer. Fifth, biological molecules such as T cell receptors (TCRs) or TCR mimics (i.e., antibody-derived molecules, e.g., antibody binding domains as described herein) that recognize CST antigens complexed to MHC I molecules (e.g., pMHC or peptide MHC) can be further modified to selectively kill (or promote) cancer cells, which can be administered to the subject as a therapy for treating cancer.These biological molecules also include multivalent bioagents that may contain targeting motifs and / or immune-stimulating motifs (e.g., antibodies or antibody-derived molecules capable of targeting / binding to immune response cells), which promote the targeting and activation of immune cells in combination with antigen-binding components that may consist of TCRs, high-affinity TCRs, and TCR mimics produced by various technologies (e.g., mimics comprising or composed of antibodies or antibody-derived molecules such as MHC-presented antigens and / or peptides, e.g., binding domains of antibodies capable of recognizing and binding to pMHC), any of which may promote the therapeutic destruction of tumor cells. Sixth, biological molecules in the form of chimeric molecules or biological molecules containing heterogeneous sequences that recognize CST antigens complexed with MHC cells may be introduced into T cells (autologous or non-autologous), and the resulting cells may be administered to subjects as a therapy for treating cancer. These and other applications are described in more detail below.

[0015] Therefore, the present invention particularly

[0016] (a) any one of SEQ ID NOs 1 to 17 and 57, and

[0017] (b) Variants of the sequence of (a); and

[0018] (c) Fragments of the sequences of (a) and (b), e.g., immunogenic fragments

[0019] Provides an isolated polypeptide comprising a sequence selected from (hereinafter referred to herein as 'polypeptide of the present invention').

[0020] The present invention also provides a nucleic acid molecule encoding the polypeptide of the present invention (hereinafter referred to herein as the 'nucleic acid of the present invention').

[0021] The polypeptide and nucleic acid of the present invention, as well as related aspects of the present invention, are expected to be useful in cancer immunotherapy and prevention, particularly in any of cancers, particularly ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly in esophageal cancer, or in various embodiments of immunotherapy and prevention of EAC or ESSC.

[0022] Explanation of the hierarchy

[0023] MKRYNRIMHDELT, SEQ ID NO. 1, polypeptide sequence of CST antigen 1 (EVA001).

[0024] VVLPGTLCFMKLLKLNMGSLAPRSALTLETPAKYVR, SEQ ID NO. 2, polypeptide sequence of CST antigen 2 (EVA002).

[0025] LAANSVSVTSVPLWCLYSISLASSLMHPKTDAALLSPVPSQQTRGCFYPCLDF, SEQ NO. 3, polypeptide sequence of CST antigen 3 (EVA003).

[0026] LARETLLPASSAFPSSPFRTVPHLLLAQCCLKMQLLFANNRNTS, SEQ ID NO. 4, polypeptide sequence of CST antigen 4 (EVA004).

[0027] LSGMSLSGVMIKLYSPLFMTRYNSPSPLKSQLLVSLSWTSVIGRLLMSIFNLC, SEQ ID NO. 5, polypeptide sequence of CST antigen 5 (EVA005).

[0028] MLKTNNKKQTTKKKLKKKTEIEL, SEQ ID NO. 6, polypeptide sequence of CST antigen 6 (EVA006).

[0029] MLIAVSLTILAVLIFWIN, SEQ No. 7, polypeptide sequence of CST antigen 7 (EVA007).

[0030] MQKVPDVLWNRKLRKKKRSFKRSSPVNSVLSKHWTRSASR, SEQ ID NO. 8, polypeptide sequence of CST antigen 8 (EVA008).

[0031] VEMPAIKWHPDGLIHITLL, SEQ ID NO. 9, polypeptide sequence of CST antigen 9 (EVA009).

[0032] MSQGRASPFTTSRPRPGKGSLWRRRFVREEGEAPTHLGLTRSFAKRLGGVPGATKLPPS, SEQ ID NO. 10, polypeptide sequence of CST antigen 10 (EVA010).

[0033] MDSPIPYHLSPTLAPEILNSTM, SEQ ID NO. 11, polypeptide sequence of CST antigen 11 (EVA011).

[0034] TFLELKLFSTEEDRADSRDHGVSLGPSPQMVHPLAEAPAHSLTSNLLEPAHHCQAHY, SEQ No. 12, polypeptide sequence of CST antigen 12 (EVA012).

[0035] VQNSVDSSLSLLWRSSIPYLEMTLFMCLSLVMML, SEQ NO. 13, polypeptide sequence of CST antigen 13 (EVA013).

[0036] MASRKKMIGFVK, SEQ ID NO. 14, polypeptide sequence of CST antigen 14 (EVA014).

[0037] LFKMPQVKHQRRNLRDPLPNRSLIKQRPPRKWQSPTLASFQLGSRLLTTPPCPTRK polypeptide sequence of SEQ ID No. 15, CST antigen 15 (EVA015).

[0038] MPGRPPPTVGPLISPVAPRLPFTFGPSKLKSTFGAEIRGPFRFTSTPGPFSSPETCGPLALTCKSDPGVEMPNWGSFILGSLMLPSDASKLRSGAPTDTLGHLMSPETARSPCRLGPLSVTSGAPTLSFVVASIPGTFTPSPPTCILPPTPLRLSPGACTFMLGTDASRSPFKLGPFKFRSRSGPETFGIERFTEGKSTPGPFRETSGPSSLTSGPLKSPSREGSWAWTSAPPPSIFTPGMPILGME, SEQ ID No. 16, polypeptide sequence of CST antigen 16 (EVA016).

[0039] VYNIKMKGFVLHCEVQAEVGIRF, SEQ No. 17, polypeptide sequence of CST antigen 17 (EVA017).

[0040] KRYNRIMHDEL, SEQ No. 18, peptide sequence derived from CST antigen 1 (EVA001).

[0041] KLLKLNMGSL, SEQ No. 19, peptide sequence derived from CST antigen 2 (EVA002).

[0042] HPKTDAALL, SEQ No. 20, peptide sequence derived from CST antigen 3 (EVA003).

[0043] SSLMHPKTDAAL, SEQ ID NO. 21, peptide sequence derived from CST antigen 3 (EVA003).

[0044] FPSSPFRTV, SEQ No. 22, peptide sequence derived from CST antigen 4 (EVA004).

[0045] FRTVPHLLL, SEQ ID NO. 23, peptide sequence derived from CST antigen 4 (EVA004).

[0046] SPSPLKSQL, SEQ ID NO. 24, peptide sequence derived from CST antigen 5 (EVA005).

[0047] LKKKTEIEL, SEQ No. 25, peptide sequence derived from CST antigen 6 (EVA006).

[0048] AVSLTILAV, SEQ No. 26, peptide sequence derived from CST antigen 7 (EVA007).

[0049] KRSSPVNSV, SEQ No. 27, peptide sequence derived from CST antigen 8 (EVA008).

[0050] KRSSPVNSVLSK, SEQ ID NO. 28, peptide sequence derived from CST antigen 8 (EVA008).

[0051] GLIHITLL, SEQ No. 29, peptide sequence derived from CST antigen 9 (EVA009).

[0052] HPDGLIHITL, SEQ No. 30, peptide sequence derived from CST antigen 9 (EVA009).

[0053] RPRPGKGSL, SEQ No. 31, peptide sequence derived from CST antigen 10 (EVA010).

[0054] SPTLAPEIL, SEQ No. 32, peptide sequence derived from CST antigen 11 (EVA011).

[0055] SRDHGVSL, SEQ ID NO. 33, peptide sequence derived from CST antigen 12 (EVA012).

[0056] VSLGPSPQMV, SEQ No. 34, peptide sequence derived from CST antigen 12 (EVA012).

[0057] VDSSLSLL, SEQ No. 35, peptide sequence derived from CST antigen 13 (EVA013).

[0058] MASRKKMI, SEQ No. 36, peptide sequence derived from CST antigen 14 (EVA014).

[0059] SLIKQRPPRK, SEQ No. 37, a peptide sequence derived from CST antigen 15 (EVA015).

[0060] RSLIKQRPPR, SEQ No. 38, peptide sequence derived from CST antigen 15 (EVA015).

[0061] SLIKQRPPR, SEQ No. 39, a peptide sequence derived from CST antigen 15 (EVA015).

[0062] DPLPNRSLI, SEQ No. 40, peptide sequence derived from CST antigen 15 (EVA015).

[0063] RSLIKQRPPRK, SEQ No. 41, peptide sequence derived from CST antigen 15 (EVA015).

[0064] ILPPTPLRL, SEQ ID NO. 42, a peptide sequence derived from CST antigen 16 (EVA016).

[0065] GAPTLSFVVA, SEQ ID NO. 43, peptide sequence derived from CST antigen 16 (EVA016).

[0066] GSFILGSLM, SEQ ID NO. 44, peptide sequence derived from CST antigen 16 (EVA016).

[0067] LMLPSDASKLR, SEQ No. 45, peptide sequence derived from CST antigen 16 (EVA016).

[0068] LRSGAPTDTL, SEQ ID NO. 46, a peptide sequence derived from CST antigen 16 (EVA016).

[0069] RFTEGKSTPGPFR, SEQ ID NO. 47, a peptide sequence derived from CST antigen 16 (EVA016).

[0070] ILPPTPLRL, SEQ ID NO. 48, a peptide sequence derived from CST antigen 16 (EVA016).

[0071] KLKSTFGAEI, SEQ ID NO. 49, peptide sequence derived from CST antigen 16 (EVA016).

[0072] SFILGSLML, SEQ ID NO. 50, peptide sequence derived from CST antigen 16 (EVA016).

[0073] GAPTLSFVVA, SEQ ID NO. 51, peptide sequence derived from CST antigen 16 (EVA016).

[0074] GSFILGSLM, SEQ ID NO. 52, peptide sequence derived from CST antigen 16 (EVA016).

[0075] TSGPLKSPSR, SEQ ID NO. 53, peptide sequence derived from CST antigen 16 (EVA016).

[0076] VQAEVGIRF, SEQ No. 54, peptide sequence derived from CST antigen 17 (EVA017).

[0077] DPLPNRSLI, SEQ No. 55, peptide sequence derived from CST antigen 15 (EVA015).

[0078] KRSSPVNSVLSK, SEQ ID NO. 56, peptide sequence derived from CST antigen 8 (EVA008).

[0079] MDPPGHQGLQVQEATLESLVFQGPLGLQAHQVKQSCLRVL, SEQ ID NO. 57, peptide sequence derived from CST antigen 18 (EVA018).

[0080] GPLGLQAHQV, SEQ No. 58, peptide sequence derived from CST antigen 18 (EVA018).

[0081] AKFVAAWTLKAAA, sequence number 59, PADRE epitope sequence,

[0082] PKYVKQNTLKLAT, sequence number 60, HA307-319 epitope sequence,

[0083] QYIKANSKFIGITE, sequence number 61 TT830-843 epitope sequence,

[0084] FNNFTVSFWLRVPKVSASHLE, sequence number 62, VP2 epitope sequence,

[0085] ISQAVHAAHAEINEAGR, sequence number 63, ovalbumin epitope sequence, Brief explanation of the drawing

[0086] List of abbreviations for cancer types and cells used in drawings ACC - Adrenal Cortical Carcinoma BLCA - Bladder urothelial carcinoma BRCA - Invasive breast carcinoma CESC - Uterine squamous cell carcinoma and endocervical adenocarcinoma CHOL- Bile duct cancer (bile duct) COAD - Colon Adenocarcinoma DLBC - Lymphoid neoplasm, diffuse large B-cell lymphoma ESCA- Esophageal Carcinoma GBM - Polymorphic glioblastoma H&N- Head and Neck HNSC - Head and Neck Squamous Cell Carcinoma LAML - Acute Myeloid Leukemia KICH - Kidney Chromophobe KIRC- Clear renal cell carcinoma KIRP - Renal papillary carcinoma LAML - Acute Myeloid Leukemia LGG - Low-grade glioma LIHC - Hepatocellular Carcinoma LUAD - Lung Adenocarcinoma LUSC - Lung squamous cell carcinoma MESO- Mesothelioma OV- Ovarian serous cystic carcinoma PAAD - Pancreatic Adenocarcinoma PCPG - Paraganglioma and collateral ganglion PRAD - Prostate Adenocarcinoma READ- Rectal adenocarcinoma SARC - Breeding SKCM - Skin Melanoma STAD- Gastric adenocarcinoma TGCT - Testicular germ cell tumor THCA - Thyroid carcinoma THYM - Thymoma UCS / UCSC - Uterine carcinosarcoma UVM - Uveal Melanoma SkMus = Skin Muscle Liv = liver Pro = Prostate Lun = lung Spl = spleen Kid = height Bra = Brain Hea = Head and neck Flo-1 = Human esophageal adenocarcinoma OE19 = Human esophageal adenocarcinoma OE33 = Human esophageal adenocarcinoma OACM5.1C = Human esophageal adenocarcinoma SK-GT4 = Human distal esophageal carcinoma ESO51 = Human esophageal adenocarcinoma ESO62 = Adenocarcinoma of the gastroesophageal junction KYAE-1 = Esophageal adenocarcinoma EBC-1 = Lung squamous cell carcinoma cell line SKL-U1 = Adenocarcinoma HCT116 = Colon carcinoma DMS53 = Colon carcinoma CL-40 = RPMI7951 = Melanoma HARA = Epstein-Barr virus (EBV) transformed by lymphoblast cell lines FIGS. 1 to 17 and FIG. 26. Identification of cancer-specific transcripts specific to squamous lung carcinoma types using de novo assembly - Transcripts per million [TPM] were estimated for all transcripts, and expression in healthy tissue samples from GTEx (Fig. (A)) was compared with expression in various cancer types from TGCA (Fig. (B)). Fig. 1:EVA001 Sequence No. 1. Cancer specificity: ESCA, HNSC, UCSC. Expression (normal): Low expression in tibial nerve and colon (1 to 2 TPM). Fig. 2. EVA002 Sequence No. 2. Cancer specificity: ESCA, DLBC, PRAAD, COAD. Expression (normal): Low expression in various organs (TPM <1). Fig. 3. EVA003 Sequence No. 3. Cancer specificity: ESCA. Expression (normal): Low ovarian (2 to 3 TPM). Fig. 4. EVA004 Sequence No. 4. Cancer specificity: ESCA. Expression (normal): Esophagus_muc(2 TPM), Colon(1 TPM), Small intestine(1 TPM). Fig. 5. EVA005 Sequence No. 5. Cancer specificity: H&N, ESCA, LALM, STAD. Expression (normal): Esophageal_mun (TPM <1), Testis (approx. 1 TPM). Fig. 6. EVA006 Sequence No. 6. Cancer specificity: BRCA, CHOL, ESCA, LUAD, LUSC, OV, PAAD, STAD. Expression (normal): msg, arterial_coronary and pulmonary (approx. 1 TPM). Fig. 7. EVA007 Sequence No. 7. Cancer specificity: ESCA, HNSC, LUSC. Expression (normal): Esophagus_muc, Colon (2 to 3 TPM). Fig. 8. EVA008 Sequence No. 8. Cancer specificity: ESCA, HNSC, LUSC. Expression (normal): Esophagus_muc, Colon (2 to 3 TPM). Fig. 9. EVA009 Sequence No. 9. Cancer specificity: LUAD, KIRC, ESCA. Expression (normal): Clear in normal tissues, with no significant level of expression. Fig. 10. EVA010 Sequence No. 10. HNSC, LUAD, LUSC. Expression (Normal): Lungs, fat, intestines, spleen (approx. 1 TPM). Fig. 11. EVA011 Sequence No. 11. Cancer specificity: STAD, MESO, LUSC, and ESCA. Expression (normal): Nerve_Tibial (3 TPM), Stomach (2 to 3 TPM). Fig. 12.EVA012 Sequence No. 12. Cancer specificity: COAD, LUAD, STAD. Expression (normal): Clear in normal tissues, with no significant level of expression. Fig. 13. EVA013 Sequence No. 13. Cancer specificity: H&N, LUSC, ESCA. Expression (normal): esophagus_muc(approx. 1 TPM). Fig. 14. EVA014 Sequence No. 14. Cancer specificity: H&N, LUSC, ESCA. Expression (normal): esophagus_muc(approx. 1 TPM). Fig. 15. EVA015 Sequence No. 15. Cancer specificity: LCA, CESC, COAD, ESCA, HNSC, LUSC, OV, READ, SARC, STAD, TGCT. Expression (normal): Esophagus, testis, transverse colon (3 to 4 TPM). Fig. 16. EVA016 Sequence No. 16. Cancer specificity: ESCA. Expression (normal): Clear in normal tissues, with no significant level of expression. Fig. 17. EVA017 Sequence No. 17. HNSC, CESC, LUSC. Expression (Normal): Esophagus_muc, Spleen, Thyroid (approx. 1 TPM). Fig. 18 : EVA001 transcript expression by RT-qPCR in EAC esophageal adenocarcinoma (indicated as ESOAD in the figure) tissues and EAC cell lines. Data show low expression levels in most LUSC tissues (8 / 11) and intermediate expression levels in most NSCLC cell lines (11 / 13) compared to reference genes TBP and PGK1, which are expressed at consistent levels across EAC tissues. The following cell lines are shown: Flo-1 esophageal adenocarcinoma (EAC) cells, OE19 (EAC) cells, OE33 (EAC) cells, OACM5.1C (EAC) cells, SKGT4 distal esophageal EAC, ESO26 distal esophageal EAC, ESO51 distal esophageal EAC, and KYAE-1 distal esophageal EAC. Positive control cell lines are known to express the EVA001 transcript. These are NCI-H1299 lung carcinoma cells. The negative control cell line This is the MCF7 breast cancer cell line known not to express EVA001. EVA001 has a high prevalence in EAC, with expression observed in 11 / 14 tumor tissue samples and 7 / 7 tumor cell lines. In most cases, when present, EVA001 is expressed at high levels compared to the TBP and PGK1 reference genes. (NAT = Normal adjacent tissue) Fig. 19: A representative example of an RNAscope score level assigned by microscopic observation. Fig. 20: EVA001 transcript expression detected by the RNAscope probe EVA001-20zz-RNAscope in control tissues known by pre-sequencing analysis to express or not express one of the transcript sequences. EVA001-20zz-RNAscope binds within the region of SEQ ID NO. 17 containing the sequence encoding the peptide of SEQ ID NO. 18. Fig. 21: EVA001 transcript expression in control EAC tissue detected by the RNAscope probe EVA001-20zz-RNAscope. Expression (score ≥ 1) was observed in 7 / 16 EAC tissues (44%), and positivity was observed across almost all tumor stages tested. Fig. 22: EVA001 transcript expression in normal tissues by RNAscope probe EVA001-20zz-RNAscope. EVA001 expression (score <1) was generally observed across normal tissues: values ​​>1 were seen in normal samples from testes alone (3 / 3 tissues). Fig. 23: EVA001 transcript expression in selected tumor tissues by RNAscope probe EVA001-20zz-RNAscope. EVA001 expression was observed in 7 / 11 generalized tumor tissue types. Fig. 24: IFN-gamma CD8 T-cell response to HLA-restricted CST antigen peptide SEQ ID NO. 18 (EVA001) from normal blood donors Fig. 25:IFN-gamma CD8 T-cell response to HLA-restricted CST antigen peptide sequence number 26 (EVA007) from a normal blood donor. Fig. 26. EVA018 Sequence No. 57. Cancer specificity: BRCA, ESCA, LUAD, PAAD, STAD. Expression (normal): Thyroid (approx. 1 TPM). Fig. 27. Transcript expression of EVA001, EVA006, and EVA007 in normal tissue by RNAscope probes: EVA001-20zz-RNAscope, EVA006-20zz-RNAscope, EVA007-20zz-RNAscope. Fig. 28. Transcript expression of EVA001, EVA006, and EVA007 in esophageal adenocarcinoma (EAC) tissue by RNAscope probes: EVA001-20zz-RNAscope, EVA006-20zz-RNAscope, EVA007-20zz-RNAscope. Fig. 29. Transcript expression of EVA001, EVA006, and EVA007 in additional tumor tissues by RNAscope probes: EVA001-20zz-RNAscope, EVA006-20zz-RNAscope, EVA007-20zz-RNAscope. Fig. 30: IFN-gamma CD8 T-cell responses from normal blood donors to HLA-restricted CST antigen peptides, SEQ NO. 18 (EVA001), SEQ NO. 26 (EVA007) and SEQ NO. 25 (EVA006). Fig. 31: Bar chart report of IFN gamma CD8 T-cell responses from normal blood donors to HLA-restricted CST antigen peptides, SEQ NO 18 (EVA001), SEQ NO 26 (EVA007) and SEQ NO 25 (EVA006). Fig. 32: Transcript expression of EVA015 (SEQ No. 15) by RT-qPCR screening in esophageal adenocarcinoma tissue. Fig. 33: EVA015 transcript expression in normal tissue by RNAscope probe: EVA015-20zz-RNAscope. Fig. 34:EVA015 transcript expression in tumor tissue by RNAscope probe: EVA015-20zz-RNAscope. Fig. 35: Transcript expression of EVA004 (SEQ No. 4) by RT-qPCR screening in esophageal adenocarcinoma tissue. Fig. 36: EVA004 transcript expression in normal tissue by RNAscope probe: EVA004-20zz-RNAscope. Fig. 37: EVA004 Transcript Expression in Tumor Tissue by RNAscope Probe: EVA004-20zz-RNAscope. Fig. 38: Transcript expression of EVA007 (SEQ No. 7) by RT-qPCR screening in esophageal adenocarcinoma tissue. Fig. 39: Transcript expression of EVA008 (SEQ No. 8) by RT-qPCR screening in esophageal adenocarcinoma tissue. Fig. 40: EVA007 / EVA008 transcript expression in normal tissue by RNAscope probe: EVA007-20zz-RNAscope. Fig. 41: EVA007 / EVA007 transcript expression in tumor tissue by RNAscope probe: EVA007008-20zz-RNAscope. Fig. 42: Transcript expression of EVA005 (SEQ No. 5) by RT-qPCR screening in esophageal adenocarcinoma tissue. Fig. 43: EVA005 transcript expression in normal tissue by RNAscope probe: EVA005-20zz-RNAscope. Fig. 44: EVA005 transcript expression in tumor tissue by RNAscope probe: EVA005-20zz-RNAscope. Fig. 45: Transcript expression of EVA006 (SEQ No. 6) by RT-qPCR screening in esophageal adenocarcinoma tissue. Fig. 46: EVA005 transcript expression in normal tissue by RNAscope probe: EVA006-20zz-RNAscope. Fig. 47: EVA005 transcript expression in tumor tissue by RNAscope probe: EVA006-20zz-RNAscope. Fig. 48: Transcript expression of EVA013 (SEQ No. 13) by RT-qPCR screening in esophageal adenocarcinoma tissue. Fig. 49: EVA013 transcript expression in normal tissue by RNAscope probe: EVA013-20zz-RNAscope. Fig. 50: EVA013 Transcript Expression in Tumor Tissue by RNAscope Probe: EVA013-20zz-RNAscope. Fig. 51: Transcript expression of EVA011 (SEQ No. 11) by RT-qPCR screening in esophageal adenocarcinoma tissue. Fig. 52: EVA011 transcript expression in normal tissue by RNAscope probe: EVA011-20zz-RNAscope. Fig. 53: EVA011 Transcript Expression in Tumor Tissue by RNAscope Probe: EVA011-20zz-RNAscope. Fig. 54: Report of IFN-gamma CD8 T-cell responses and bar charts of IFN-gamma CD8 T-cell responses to HLA-restricted CST antigen peptides from normal blood donors, SEQ ID NO. 28 (EVA008), SEQ ID NO. 37 (EVA015), SEQ ID NO. 38 (EVA015) and SEQ ID NO. 39 (EVA015). Fig. 55: Report of IFN-gamma CD8 T-cell responses and bar charts of IFN-gamma CD8 T-cell responses to HLA-restricted CST antigen peptides from normal blood donors, SEQ ID NO. 25 (EVA006), SEQ ID NO. 24 (EVA005), SEQ ID NO. 28 (EVA008), SEQ ID NO. 37 (EVA015), SEQ ID NO. 38 (EVA015) and SEQ ID NO. 39 (EVA015). Fig. 56: Report of IFN-gamma CD8 T-cell responses and bar charts of IFN-gamma CD8 T-cell responses to HLA-restricted CST antigen peptides from normal blood donors, SEQ ID NO. 25 (EVA006), SEQ ID NO. 22 (EVA004), SEQ ID NO. 24 (EVA005), SEQ ID NO. 27 (EVA008), SEQ ID NO. 37 (EVA015) and SEQ ID NO. 35 (EVA013). Fig. 57: Report of IFN-gamma CD8 T-cell responses and bar charts of IFN-gamma CD8 T-cell responses to HLA-restricted CST antigen peptides from normal blood donors, SEQ ID NO. 25 (EVA006), SEQ ID NO. 22 (EVA004), SEQ ID NO. 24 (EVA005), SEQ ID NO. 27 (EVA008), SEQ ID NO. 37 (EVA015) and SEQ ID NO. 35 (EVA013). Fig. 58: Report of IFN-gamma CD8 T-cell responses and bar charts of IFN-gamma CD8 T-cell responses to HLA-restricted CST antigen peptides from normal blood donors, SEQ ID NO. 18 (EVA001), SEQ ID NO. 22 (EVA004), SEQ ID NO. 19 (EVA002), SEQ ID NO. 24 (EVA005), SEQ ID NO. 26 (EVA007) and SEQ ID NO. 27 (EVA008). Specific details for implementing the invention

[0087] polypeptide

[0088] The terms ‘protein’, ‘polypeptide’, and ‘peptide’ are used interchangeably herein and refer to any peptide chain of amino acids regardless of length, co-translation, or post-translational modification.

[0089] The term 'amino acid' refers to naturally occurring amino acids as well as any one of amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Natural amino acids are the 20 L-amino acids encoded by the genetic code, as well as subsequently modified amino acids, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphorine. The term 'amino acid analog' refers to a compound having the same chemical structure as a naturally occurring amino acid, namely an α-carbon bonded to a hydrogen, carboxyl, amino, and R group, but having a modified R group or a modified peptide backbone compared to the natural amino acid. Examples include homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, and norleucine. Amino acid mimics refer to chemical compounds that have a structure different from the general chemical structure of an amino acid but function in a manner similar to naturally occurring amino acids. Suitablely, the amino acid is a naturally occurring amino acid or an amino acid analog, in particular a naturally occurring amino acid and in particular one of the 20 L-amino acids encoded by the genetic code.

[0090] Amino acids may be referred to here by the commonly known three-letter symbol or by the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by the commonly accepted single-letter code.

[0091] Therefore, the present invention

[0092] (a) any one of SEQ ID NOs 1 to 17 and 57, and

[0093] (b) Variants of the sequence of (a); and

[0094] (c) A fragment of the sequence of (a) and / or (b), e.g., an immunogenic fragment

[0095] Provides an isolated polypeptide comprising a sequence selected from.

[0096] The present invention also

[0097] (a) any one of SEQ ID NOs 1 to 17 and 57 with the initial methionine residue removed; and

[0098] (b) Variants of the sequence of (a); and

[0099] (c) A fragment of the sequence of (a) and / or (b), e.g., an immunogenic fragment

[0100] Provides an isolated polypeptide comprising a sequence selected from.

[0101] Generally, variants of the polypeptide sequence of the present invention comprise sequences having a high degree of sequence identity with respect to it. For example, the variants suitably have at least about 75% or 80% identity with respect to the relevant reference sequence over the entire length, or any of at least about 81, 82, 83, 84% identity, more preferably at least about 85% identity or any of at least about 86, 87, 88, 89% identity, and most preferably at least about 90% identity (e.g., any of at least about 91, 92, 93, 94, 96, or 97%, or at least about 98% or at least about 99%).

[0102] Suitablely, the variant (which may be a fragment) is an immunogenic variant. The variant is considered to be an immunogenic variant, wherein, for example, induces a response of at least 20%, suitably at least 50%, and particularly at least 75% (e.g., at least 90%) of the activity of the reference sequence (i.e., the sequence to which the variant belongs) in an in vitro restimulation assay of PBMC or whole blood, or optionally T-cells derived therefrom, using the polypeptide as an antigen (e.g., restimulation for a period of several hours to up to 1 year, e.g., up to 6 months, 1 day to 1 month, or 1 to 2 weeks, or, e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 hours, or any of 12 to 24 hours, e.g., any of 14, 16, 18, 20, or 22 hours), and this assay induces a response of at least 20%, suitably at least 50%, and particularly at least 75% (e.g., at least 90%) of the activity of the reference sequence (i.e., the sequence to which the variant belongs), and this assay induces a response of cells, e.g., through lymphocyte proliferation (e.g., T-cell proliferation). After measuring the activation of T-cells, the production of cytokines (e.g., IFN-gamma) in the supernatant of the culture (measured by ELISA, etc.), or the characterization of T-cell responses by intracellular and extracellular staining (e.g., using antibodies specific to immune markers such as CD3, CD4, CD8, IL2, TNF-alpha, IFNg, type 1 IFN, CD40L, CD69, etc.), analysis by a flow cytometer follows. For example, variants may be presented by MHC on antigen-presenting cells such as dendritic cells or on MHC 'multimers' (e.g., dextramers, pentamers, on solid substrates such as polymer beads or plates).

[0103] The polypeptide of the present invention, comprising fragments and variants, is also considered 'immunogenic,' wherein they bind to at least one MHC molecule and / or induce an immune response, and, for example, as a complex with said MHC, induce immune response cells such as, for example, T cells, for example, T-cells that cross-react with said polypeptide. For example, 'cross-reacting' immune response cells or T-cells can be characterized by their ability to bind to both the polypeptide and polypeptide variants (or polypeptide fragments and polypeptide fragment variants) when presented, for example, on MHC, for example, as pMHC (in MHC multimers or on antigen-presenting cells). For example, an 'immune response' induced in immunoreactive cells (e.g., T-cells or NK cells) that bind to polypeptides or pMHCs may be the induction of T-cell proliferation and / or the induction of production of cytokines, e.g., IFN-gamma, IL-2, TNF-alpha, or, e.g., the granzyme of tumor cell death or apoptosis and / or the induction of immunoreactive cells, and the immune response may be measured in cell proliferation or immunological assays known in the art.The polypeptide of the present invention, comprising fragments and variants, preferably has at least one MHC molecule, for example, an MHC I or MHC II molecule, preferably has at least one MHC allele, for example, at least one of HLA A, B, or C alleles, preferably HLA-A*01:01, HLA-A*02, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, HLA-A*30:01, HLA-A*31:01, HLA-B*07, HLA-B*07:02, HLA-B*08:01, HLA-B*15:01, HLA-B*35:02, HLA-B*37:01, HLA-B*39:01, HLA-B*44:02, HLA-B*51:01, It binds to at least one HLA allele selected from the group consisting of HLA-C*2:02, HLA-C*03:03, HLA-C*03:04, HLA-C*04:01, HLA-C*05:01, HLA-C*06:02, HLA-C*07, and HLA-C*07:02. The variant sequence of the polypeptide preferably induces T cells that maintain the ability to bind to MHC molecules or molecules as listed herein, more specifically, to the same MHC molecules or molecules, or cross-react with said variant.

[0104] According to the present invention, the variant may be, for example, a conservatively modified variant. A 'conservatively modified variant' is one in which the modification(s) result in an amino acid substitution to a functionally similar amino acid or a substitution / deletion / addition of a residue that does not substantially affect the biological function or immunogenicity of the variant, and may be any of, for example, 70 to 75% or 75% to 80% or 80 to 90% or 90 to 95%, preferably 96, 97, 98, 99% or more, when tested by binding and / or immunological analysis. Typically, this immunogenicity or biological function of the variant will bind to, for example, polypeptide (or non-variant / non-modified polypeptide) or its pMHC and / or preferably to ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly to esophageal cancer or EAC or ESSC or to one or more of its tumor cells, for example, and / or induce an immune response in these cells. Immunogenicity or biological function may be tested by the analyses presented herein, for example, the ELISPOT cytokine assay or the (tumor) cell death assay known in the art, and by the binding affinity assay known in the art.

[0105] Conservative substitution tables providing functionally similar amino acids are known in the art. Variants may include homologs of polypeptides found in other species.

[0106] Variants of the polypeptide of the present invention may include a number of substitutions, for example, conservative substitutions, compared to the reference sequence (e.g., 1 to 25, e.g., 1 to 10, particularly any of 1, 2, 3, 4, or 5, and particularly one amino acid residue(s) may be changed). The number of substitutions, for example, conservative substitutions, may be up to 20%, e.g., up to 10%, e.g., up to 5%, e.g., up to 1% of the number of residues of the reference sequence. Generally, conservative substitutions will belong to one of the amino acid groups specified below, but in some cases, other substitutions may be possible without substantially affecting the immunogenic properties of the polypeptide. The following eight groups each include amino acids that are typically conservative substitutions relative to one another:

[0107] 1) Alanine (A), Glycine (G);

[0108] 2) Aspartic acid (D), glutamic acid (E);

[0109] 3) Asparagine (N), Glutamine (Q);

[0110] 4) Arginine(R), Lysine(K);

[0111] 5) Isoleucine (I), leucine (L), methionine (M), valine (V);

[0112] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0113] 7) Serine (S), Threonine (T); and

[0114] 8) Cysteine ​​(C), Methionine (M)

[0115] (For example, literature [Creighton, Proteins [Refer to 1984]).

[0116] Suitablely, these substitutions do not alter the immunological structure and / or features of the epitope (e.g., they do not occur within the epitope region as mapped in the primary sequence), and therefore do not have a significant effect on the tertiary structure or immunogenic properties of the polypeptide.

[0117] Polypeptide variants also include additional amino acids inserted relative to the reference sequence, for example, such insertions may occur at 1 to 10 positions (e.g., any of 1, 2, 3, 4, or 5 positions; suitably, 1 or 2 positions, particularly 1 position), and may involve the addition of 50 or fewer amino acids (e.g., 20 or fewer, particularly 10 or fewer, particularly 5 or fewer, e.g., 4, 3, 2, or 1) at each position. Suitably, such insertions do not occur within or within the region of the epitope and thus do not have a significant or substantial effect on the immunogenic properties of the polypeptide. One example of an insertion is, for example, a short extension of histidine residues (e.g., 2 to 6 residues) at the C or N terminus to aid in the expression and / or purification of the antigen.

[0118] Polypeptide variants comprise deletions of amino acids relative to the reference sequence, for example, such deletions may occur at 1 to 10 positions (e.g., any of 1, 2, 3, 4, or 5 positions; suitably, 1 or 2 positions, particularly 1 position), for example, and may involve deletions of 50 or fewer amino acids (e.g., 20 or fewer, particularly 10 or fewer, particularly 5 or fewer, e.g., 4, 3, 2, or 1) at each position. Suitably, such deletions do not occur within the region of the epitope or within the epitope and thus do not have a significant or substantial effect on the immunogenic properties of the polypeptide.

[0119] Those skilled in the art will recognize that certain protein variants may include substitutions, deletions, and additions (or any combination thereof). For example, substitutions / deletions / additions may enhance (or neutralize) binding to the target patient HLA molecule, potentially increasing (or maintaining) immunogenicity without altering it.

[0120] A fragment according to the present invention, e.g., an immunogenic fragment or a variant thereof, typically comprises, depending on the length of the CST antigen, at least 8 or 9 adjacent amino acids (e.g., at least 7 or 8 to 11, or 9 or 10), e.g., at least 12 adjacent amino acids (e.g., at least 13 or 14 or 15 or at least 18, e.g., 16 or 17 or at least 19 or 20 adjacent amino acids), (e.g., any of up to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 adjacent amino acids) or 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to It will include any of 18, 8 to 19, or 8 to 20 adjacent amino acids), alternatively any of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 adjacent amino acids, in particular at least 50 adjacent amino acids, e.g., at least 100 adjacent amino acids (e.g., at least 200 adjacent amino acids). Suitably, the fragment will be at least 10%, e.g., at least 20%, e.g., at least 50%, e.g., at least 70%, or at least 80% of the full polypeptide sequence length.

[0121] Immunogenic fragments, such as fragments or variants thereof, typically contain at least one epitope. The epitopes include B cell and T cell epitopes, and suitably, the fragment contains at least one T-cell epitope, e.g., a CD4+ or CD8+ T-cell epitope.

[0122] T cell epitopes are short, adjacent elongations of amino acids that are recognized by T cells (e.g., CD4+ or CD8+ T cells) when selectively bound to HLA molecules. Identification of T cell epitopes can be achieved through epitope mapping experiments well known to those skilled in the art (e.g., the literature [Paul, Fundamental Immunology , 3rd ed., 243-247 (1993); Beiβbarth et al., 2005; Bioinformatics ,21(Suppl. 1):i29-i37] see reference).

[0123] It is evident that a fragment of the full-length polypeptide of SEQ ID NOs. 1 to 17 and 57, or a variant thereof, comprising at least one T cell epitope as a result of decisive involvement in the T cell response in cancer, may be immunogenic and may contribute to biological function and / or immunogenicity or immunoprotection (i.e., the ability to induce an immune response in immunoreactive cells (e.g., T-cells or NK cells) against the cancer or tumor cells disclosed herein, e.g., causing apoptosis or cell death).

[0124] In diverse crossbreed populations such as humans, it will be understood that different HLA types mean that certain epitopes may not be recognized by all members of the population. Consequently, to maximize the level of recognition and the scale of the immune response to polypeptides, it is generally desirable for a fragment, such as an immunogenic fragment, to contain multiple epitopes from the full-length sequence (suitably all epitopes within the CST antigen).

[0125] A specific fragment of a polypeptide of any one of sequences 1 to 17 and 57 that can be used according to the present invention; or a variant thereof comprises at least one CD8+ T-cell epitope, suitably at least two CD8+ T-cell epitopes and in particular all CD8+ T-cell epitopes, in particular associated with one or more HLA class I alleles, e.g., associated with two, three, four, five or more alleles (e.g., as described above in this specification). A specific fragment of the polypeptide of SEQ ID NOs. 1 to 17 and 57, or a variant thereof, which can be used according to the present invention, comprises at least one CD4+ T-cell epitope, suitably at least two CD4+ T-cell epitopes, and in particular all CD4+ T-cell epitopes (in particular those associated with one or more HLA class II alleles, e.g., those associated with two, three, four, five or more alleles). However, a person skilled in the art of vaccine design could combine the exogenous CD4+ T-cell epitope with the CD8+ T-cell epitope of the present invention and achieve the desired response to the CD8+ T-cell epitope of the present invention.

[0126] Where individual fragments of a full-length polypeptide are used, such fragments are considered immunogenic if they induce a response of at least 20%, suitably at least 50%, and particularly at least 75%, 80%, or 85% (e.g., at least 90% or 95%) of the activity of a reference sequence (i.e., the sequence to which the fragment belongs), for example, in an in vitro restimulation assay of PBMC or whole blood using the polypeptide as an antigen (e.g., several hours, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 hours or 10 to 24 hours, or 12 to 14 hours or 16 to 18 hours or 20 to 22 hours to a maximum of one year, e.g., a maximum of six months, or restimulation for a period of one day to one month or one to two weeks), which is the activation of cells through lymphocyte proliferation (e.g., NK cell, B-cell, or T-cell proliferation), After measuring the production of cytokines (e.g., IFN-gamma, IL-2, TNF-alpha) in the supernatant of the culture (measured by ELISA, etc.) or the characterization of immune cell or T cell responses by intracellular and extracellular staining (e.g., using antibodies specific to immune markers such as CD3, CD4, CD8, IL2, TNF-alpha, IFN-gamma, type 1 IFN, CD40L, CD69, etc.), analysis by a flow cytometer follows.

[0127] In some situations, a biological response equivalent to the full-length sequence itself can be obtained by using multiple fragments of a full-length polypeptide (which may or may not overlap and may or may not encompass the entire full-length sequence). For example, at least two fragments, for example, immunogenic fragments combined as described above (e.g., three, four, or five fragments), provide at least 50%, suitably at least 75%, 80%, 85%, and particularly at least 90% to 95% activity of the reference sequence in an in vitro assay of PBMC or whole blood (e.g., T cell proliferation and / or IFN-gamma production assay).

[0128] An exemplary fragment of the polypeptide of SEQ ID NOs. 1 to 17 and 57, e.g., an immunogenic fragment thereof, and an exemplary peptide of the present invention according thereto comprises a sequence selected from any one of SEQ ID NOs. 18 to 56 and 58, e.g., a polypeptide comprising or composed of one of SEQ ID NOs. 18 to 56 and 58, or a variant or immunogenic variant of SEQ ID NOs. 18 to 56 and 58 having 1, 2, 3, or 4 amino acid variations selected from addition, substitution, and deletion in relation thereto. The sequences of SEQ ID NOs. 18 to 56 and 58 were confirmed to bind to HLA class I molecules from immunopeptidemix analysis (see Example 2).

[0129] The present invention further relates to a polypeptide according to the present invention, wherein the polypeptide comprises non-peptide bonds. Additionally, the polypeptide, variant, or fragment may be further modified to improve stability and / or binding to MHC molecules to induce a stronger immune response. Methods for such optimization of peptide sequences are well known in the art and include, for example, the introduction of reverse peptide bonds or non-peptide bonds. In reverse peptide bonds, amino acid residues are not bound by peptide (-CO-NH-) linkages, but the peptide bond is inverted. Peptides comprising the bonds described above may be synthesized by additional chemical groups present at their amino and / or carboxyl terminals to improve the stability, bioavailability, and / or affinity of the peptides. For example, hydrophobic groups, such as carbobenzoxyl, monocrystalline, or t-butyloxycarbonyl groups, may be added to the amino terminals of the peptides. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be located at the amino terminus of the peptide. Additionally, a hydrophobic group, a t-butyloxycarbonyl group, or an amido group may be added to the carboxy terminus of the peptide. The present invention further relates to a polypeptide according to the present invention, wherein the polypeptide comprises a modified stereochemical configuration. For example, one or more D-isomers of the amino acid residues of the peptide may be used instead of the usual L-isomer. For example, at least one amino acid residue of the polypeptide or peptide of the present invention may be substituted with one of the well-known non-naturally occurring amino acid residues. Such modifications may serve to enhance the stability, bioavailability, and / or binding activity of the polypeptide of the present invention. Similarly, the polypeptide or fragment or variant of the present invention may be chemically modified by the reaction of specific amino acids before or after the synthesis of the peptide.Chemical modifications of amino acids include, but are not limited to, acylation, amideation, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups by 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide modification of carboxyl groups and sulfhydryl modification by perforic acid oxidation of cysteine, formation of mercury derivatives, formation of mixed disulfides with other thiol compounds, reaction with maleimide, carboxymethylation by iodoacetic acid or iodoacetamide, and modification by carbamoylation using cyanate at alkaline pH. The present invention further relates to non-naturally occurring polypeptides, preferably in each case, their immunogenicity, said polypeptides consisting of or essentially of amino acid sequences or fragments or variants according to SEQ ID NOs 1 to 58, and produced by synthesis as pharmaceutically acceptable salts.

[0130] nucleic acids

[0131] The present invention provides an isolated nucleic acid encoding the polypeptide of the present invention (referred to as the nucleic acid of the present invention). For example, the nucleic acid of the present invention comprises or consists of a sequence or a fragment thereof that encodes any of SEQ ID NOs 1 to 58, preferably a sequence selected from an immunogenic fragment. Preferably, the nucleic acid is an encoding nucleic acid sequence comprising or consisting of a mammalian or human nucleic acid sequence or a sequence according to mammals, preferably human codon usage frequency and / or codon optimization. The sequence may include one or more synonymous codons for each of the encoded amino acids of the polypeptide. The sequence may include one or more axially weighted codons, and one or more or each codon may encode all possible synonymous codons for a specific amino acid, or a preferred codon using only the most frequent synonymous codon, or a codon selected by probability based on the frequency distribution observed for a highly expressed human polypeptide sequence.

[0132] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to polymeric macromolecules prepared from nucleotide monomers, in particular deoxyribonucleotide or ribonucleotide monomers. The terms encompass nucleic acids that are naturally occurring and non-natural, possess properties similar to reference nucleic acids, are intended to be metabolized in a manner similar to reference nucleotides, or are intended to have an extended half-life within the system, or contain known nucleotide analogs or modified backbone residues or linkages. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotide, and peptide-nucleic acids (PNA). Suitably, the term “nucleic acid” refers to naturally occurring polymers of deoxyribonucleotide or ribonucleotide monomers. Suitably, the nucleic acid molecules of the present invention are recombinants. A recombinant means that the nucleic acid molecule is the product of at least one of a cloning, restriction or ligation step, or other procedure that produces a nucleic acid molecule distinct from a nucleic acid molecule found in nature (e.g., in the case of cDNA). In embodiments, the nucleic acid of the present invention is an artificial nucleic acid sequence (e.g., a cDNA sequence or a nucleic acid sequence having non-natural codon usage frequencies). In one embodiment, the nucleic acid of the present invention is DNA. Alternatively, the nucleic acid of the present invention is RNA.

[0133] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) refer to nucleic acid molecules having a backbone of sugar moiety, which are deoxyribosyl and ribosyl moiety, respectively. The sugar moiety may be linked to 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). 'Corresponding RNA' as used herein refers to RNA having the same sequence as reference DNA, but in which thymine (T) in DNA is substituted with uracil (U) in RNA. The sugar moiety may also be linked to non-natural bases, e.g., inosine, xanthosine, 7-methylguanosine, dihydrouridine, and 5-methylcytidine. Natural phosphodiester linkages between sugar (deoxyribosyl / ribosyl) moietyes can optionally be replaced with phosphothioate linkages. Suitably, the nucleic acid of the present invention consists of natural bases attached to a deoxyribosyl or ribosyl sugar backbone having phosphodiester linkages between sugar moietyes.

[0134] In one embodiment, the nucleic acid of the present invention is DNA. For example, the nucleic acid comprises or consists of a sequence encoding a sequence selected from any of SEQ ID NOs 1 to 58. Additionally, a nucleic acid is provided that encodes a sequence comprising or consisting of a variant of a sequence selected from any of SEQ ID NOs 1 to 58, and such variants encode the same amino acid sequence but have different nucleic acids based on the axis of the genetic code.

[0135] Therefore, due to the axing of the genetic code, multiple 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 any position where alanine is specified by a codon, the codon can be changed to any of the corresponding disclosed codons without altering the encoded polypeptide. Such nucleic acid variations give rise to 'silence' (sometimes referred to as 'axing' or 'synonym') variants, which are a type of conservedly modified variant. All nucleic acid sequences disclosed herein that encode polypeptides also enable all possible silence variants of the nucleic acid. A person skilled in the art will recognize that each codon of the nucleic acid (except AUG, which is typically the only codon for methionine, and UGG, which is the only codon for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, each silencing variant of the nucleic acid encoding the polypeptide is potential in each described sequence and is provided as an aspect of the present invention.

[0136] Accumulation codon substitution can also be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted 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).

[0137] The nucleic acid of the present invention, comprising or composed of a sequence or a fragment or variant thereof selected from any of SEQ ID NOs 1 to 58, preferably a sequence encoding an immunogenic fragment, may include a number of silencing variants (e.g., 1 to 50, e.g., 1 to 25, particularly 1 to 5, e.g., 1, 2, 3, 4 or 5, and particularly 1 codon(s) may be changed) compared to a reference encoding sequence.

[0138] The nucleic acid of the present invention may comprise or consist of a sequence selected from any of SEQ ID NOs 1 to 58, or a fragment or variant thereof, preferably an immunogenic fragment, without an initial codon for methionine (i.e., ATG or AUG), or a variant thereof as described above.

[0139] In one embodiment, the nucleic acid of the present invention is RNA. The present invention provides an RNA sequence corresponding to the DNA sequence provided and mentioned herein, having a ribonucleotide backbone instead of a deoxyribonucleotide backbone and side chain bases that are uracil (U) instead of thymine (T).

[0140] Accordingly, the nucleic acid of the present invention comprises or consists of RNA equivalents of an RNA sequence of a DNA or cDNA sequence, wherein the nucleic acid comprises or consists of a sequence or fragment or variant selected from SEQ ID NOs 1 to 58, preferably a sequence encoding an immunogenic fragment, and the RNA sequence may comprise a number of silencing variants compared to the reference sequence (e.g., 1 to 50, e.g., 1 to 25, particularly, 1 to 5, e.g., 1, 2, 3, 4, or 5, and particularly one codon(s) may be changed). 'RNA equivalent' means an RNA sequence containing the same genetic information as the reference cDNA or DNA sequence (i.e., having a ribonucleotide backbone instead of a deoxyribonucleotide backbone and having the same codon having the side chain base uracil (U) instead of thymine (T)).

[0141] The present invention also includes sequences complementary to the aforementioned cDNA, DNA, and RNA sequences.

[0142] In an embodiment, the nucleic acid of the present invention is codon-optimized for expression in a host cell such as a human host cell.

[0143] The nucleic acid of the present invention can be transcribed and translated into the polypeptide of the present invention in the case of DNA nucleic acid, and translated into the polypeptide of the present invention in the case of RNA nucleic acid. Accordingly, the present invention provides an isolated nucleic acid as described herein encoding a polypeptide according to the present invention, for example, (a) a sequence of any of SEQ ID NOs 1 to 17 and 57; and (b) a variant of the sequence of (a); and (c) a sequence selected from any one of the sequences of (a) and (b), for example, SEQ ID NOs 18 to 56 and 58, or a fragment or variant thereof, preferably a fragment such as an immunogenic fragment of an immunogen.

[0144] polypeptides and nucleic acids

[0145] Suitably, polypeptides and nucleic acids used in the present invention are isolated. An 'isolated' polypeptide or nucleic acid is one that has been removed from its original environment. For example, a naturally occurring polypeptide or nucleic acid is isolated if it is separated from some or all of the material coexisting in nature. A nucleic acid is considered isolated if, for example, it is cloned into a vector that is not part of the natural environment.

[0146] When used in relation to polypeptide or nucleic acid sequences, 'naturally occurring' refers to a sequence found in nature that is not modified by synthesis.

[0147] When used in relation to polypeptide or nucleic acid sequences, 'artificial' refers to, for example, a synthetic modification of a natural sequence or a sequence not found in nature that includes non-natural sequences.

[0148] The term 'heterogeneity,' when used to describe the relationship between one nucleic acid or polypeptide and another, indicates that two or more sequences are not found in nature in an identical relationship. 'Heterogeneity' sequences may also refer to sequences that are not isolated from, derived from, or based on naturally occurring nucleic acid or polypeptide sequences found in a host organism.

[0149] The term 'chimera', when used for biological molecules such as polypeptides or nucleic acids, refers to a molecule having more than one origin; that is, its sequence contains heterogeneous sequences. The second or subsequent origin may occur within the same organism, or in a different organism or artificial source, even if it has an unnatural association with the first origin.

[0150] As mentioned above, the polypeptide variant preferably has at least about 65% identity with the related reference sequence over its entire length, e.g., at least about 66, 67, 68, or 69% identity, preferably at least about 74% identity, e.g., at least about 70, 71, 72, or 73% identity, preferably at least about 75% identity, e.g., at least about 76, 77, 78, or 79% identity, preferably at least about 80% identity, more preferably at least about 85% identity, e.g., at least about 86, 87, 88, or 89% identity, most preferably at least about 90% identity (e.g., at least about 95%, at least about 98%, or at least about 99%).

[0151] For the purpose of comparing two closely related polypeptide or polynucleotide sequences, a 'sequence identity %' between the first sequence and the second sequence may be calculated. Polypeptide sequences are referred to as identical or the same as another polypeptide sequence if they share 100% sequence identity over their entire length. Residues within the sequence are numbered from left to right, that is, from the N- to the C-terminus in the case of polypeptides. With respect to two or more polypeptide sequences, the term 'identical' or 'identity' percentage refers to two or more sequences or subsequences having amino acid residues that are identical or of a specified percentage (i.e., 65% identity for a specified region, optionally at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity) when compared and aligned to the maximum correspondence across a comparison window. Suitably, the composition is performed over a window corresponding to the entire length of the reference sequence.

[0152] For sequence comparison, one sequence serves as a reference sequence to be compared with the test sequence. When using the sequence comparison algorithm, the test and reference sequences are input into a computer, sequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters may be used, or other parameters may be specified. Subsequently, the sequence comparison algorithm calculates the sequence identity percentage for the test sequence relative to the standard sequence based on the program parameters.

[0153] As used herein, the term 'comparison window' refers to a segment in which a sequence can be compared with an equal number of reference sequences at adjacent positions after two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment for comparison is, for example, described in the literature [Smith & Waterman, 1981, Adv . Appl . Math By the local homology algorithm of . 2:482], literature [Needleman & Wunsch, 1970, J. Mol. Biol. By the homology sorting algorithm of [48:443], the literature [Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. USA By searching for similarity methods of [85:2444], by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA of the Genetics Computer Group, Wisconsin Genetics Software Package, 575 Madison Science Drive, Wisconsin), or by manual alignment and visual inspection (e.g., literature[ Current Protocols in Molecular Biology (Ausubel et al [Refer to ., eds. 1995 supplement)] It can be performed.

[0154] An example of a useful algorithm is PILEUP. PILEUP generates multiple sequence alignments from groups of related sequences using progressive, pairwise alignment to show relationships and percentages of sequence identity. It also plots a tree or phylogenetic tree showing the clustering relationships used to generate the alignments. PILEUP [Feng & Doolittle, 1987, J. Mol. Evol. It uses a simplified progressive alignment method from [35:351-360]. The method of use is [Higgins & Sharp, 1989, CABIOS It is similar to the method described in [5:151-153]. The program can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with pairwise alignment of the two most similar sequences to produce a cluster of the two aligned sequences. The cluster is then aligned to the next cluster of aligned sequences or the next most related sequence. The two clusters of sequences are aligned by a simple expansion of the pairwise alignment of the two individual sequences. Final alignment is achieved by a series of progressive, pairwise alignments. The program is executed by specifying specific sequences and their amino acid coordinates for the region of sequence comparison, and by specifying program parameters. Using PILEUP, a reference sequence is compared with another test sequence to determine the sequence identity percentage relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gap. PILEUP is a GCG sequence analysis software package, for example, version 7.0 (Devereaux et al., 1984 , Nuc. Acids Res. It can be obtained from 12:387-395).

[0155] Other examples of algorithms suitable for determining sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are respectively [Altschul et al., 1977 , Nuc. Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol. It is described in [215:403-410]. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (website www.ncbi.nlm.nih.gov / ). The algorithm involves the first identification of a highly scored sequence pair (HSP) by identifying a short word of length W in the query sequence that matches or satisfies some positive-evaluated threshold score T when aligned with words of the same length within the database sequence. T is referred to as the neighbor word score threshold (Altschul et al., (See above). These initial neighbor word hits serve as seeds to initiate a search to find longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (compensation score for a pair of mismatched residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction is stopped when the cumulative alignment score corresponds to a quantity X from its maximum achieved value; when the cumulative score is 0 or less due to the accumulation of one or more negative scoring residue alignments; or when the end of either sequence is reached. For amino acid sequences, the BLASTP program defaults to a word length of 3 and an estimate (E) of 10 and a BLOSUM62 scoring matrix (reference [Henikoff & Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915]) Use alignment value (B) 50, (E) estimate 10, M=5, N=4 and comparison of both strands.

[0156] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., literature [Karlin & Altschul, 1993, Proc. Nat'l. Acad. Sci. USA [Refer to 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 occurs by chance.

[0157] 'Differences' between sequences refer to the insertion, deletion, or substitution of a single residue at the position of the second sequence relative to the first sequence. The two sequences may contain one or more of these differences. An insertion, deletion, or substitution of the second sequence that is identical to the first sequence in other respects (100% sequence identity) results in a decrease in the sequence identity percentage. For example, if the identical sequence is 9 residues long, one substitution in the second sequence results in 88.9% sequence identity. If the identical sequence is 17 amino acid residues long, two substitutions in the second sequence result in 88.2% sequence identity.

[0158] Alternatively, for the purpose of comparing the first reference sequence and the second comparison sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence may be identified. An addition is the addition of a residue into the first sequence (including an addition at one of the ends of the first sequence). A substitution is the substitution of one residue in the first sequence with another residue. A deletion is the deletion of a residue from the first sequence (including a deletion at one of the ends of the first sequence).

[0159] With respect to the binding of A to B, terms such as 'bind,' 'specific binding,' 'specifically bind,' or 'specifically binding' as used herein mean that the affinity of A binding to B is greater or much greater than the affinity of A binding to other molecules not targeted by A, for example, at or within each specific binding site, domain, or pocket, with an affinity generally associated with the binding of a ligand to a receptor or with an affinity generally associated with immune system molecules such as antibodies and T-cell receptors, or optionally at the micromolar or nanomolar level. Binding is selective, and binding to an antigen can be distinguished from unwanted or non-specific interactions. The ability to bind to a specific epitope can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, for example, surface plasmon resonance techniques and traditional binding assays. Preferably, when measured by an appropriate technique, the degree of binding to an unrelated protein is less than about 10% of the degree of binding to the antigen, and preferably, the dissociation constant (K D ) is less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13It is M). The term 'specifically bound' should be interpreted in a similar sense. 'Affinity' refers to the magnitude of the total sum of non-covalent interactions between a single binding site of a binding molecule (e.g., MHC, TCR, antibody) and its binding counterpart (e.g., ligand, antigen, epitope, polypeptide, or peptide); thus, 'binding affinity' refers to the intrinsic binding affinity resulting from the 1:1 interaction between the members of such binding pairs. Affinity can be measured by methods known in the art, for example, surface plasmon resonance (SPR).

[0160] Production of polypeptide of the present invention

[0161] The polypeptide of the present invention can be obtained and manipulated, for example, using techniques disclosed in the literature [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) Subsequently, polypeptides can be produced by expressing them in a suitable organism. The nucleic acid or DNA sequence or gene encoding the polypeptide of the present invention can be produced by synthesis, for example, by solid-phase DNA synthesis. The entire encoding nucleic acid or DNA or gene sequence can be synthesized de novo without the need for a precursor template DNA. To obtain the desired oligonucleotide, building blocks are sequentially coupled to an oligonucleotide chain growing in the order required by the product sequence. Upon completion of chain assembly, the product is released from the solid phase into a solution, deprotected, and collected. The product can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotide in high purity (Verma and Eckstein, 1998, Annu. Rev. Biochem. 67:99-134). These relatively short segments are easily assembled into longer DNA molecules suitable for use in numerous recombinant DNA-based expression systems by using various gene amplification methods (Methods Mol Biol., 2012; 834:93-109). In relation to the present invention, those skilled in the art will understand that the polynucleotide sequence encoding the polypeptide antigen described in the present invention could be easily used in various vector production systems, for example, including viral vectors.

[0162] For the purpose of producing the polypeptide of the present invention in a microbial host (e.g., bacteria or fungi), the nucleic acid or vector of the present invention will comprise suitable regulatory and control sequences (including promoters, termination signals, etc.) and sequences that promote the secretion of the polypeptide suitable for protein production in the host. Similarly, the polypeptide of the present invention may be produced by transducing a culture of the nucleic acid of the present invention combined with suitable regulatory and control sequences (including promoters, termination signals, etc.) and sequences that promote the secretion of the polypeptide suitable for protein production in eukaryotic cells, mammalian cells, e.g., human cells (e.g., Chinese hamster ovary cells, HEK-293 cells, or Drosophila S2 cells) into eukaryotic cells, mammalian cells, e.g., human cells (e.g., Chinese hamster ovary cells, HEK-293 cells, or Drosophila S2 cells). Thus, the present invention provides the nucleic acid of the present invention, or one or more nucleic acids of the present invention, which further comprise regulatory and / or control sequences suitable for the production of the polypeptide encoded by the present invention.

[0163] Improved isolation of the polypeptide of the present invention produced by the recombinant can be selectively promoted by the addition of an extension of a histidine residue (typically a His-tag) toward one end of the polypeptide. The polypeptide can also be produced by synthesis.

[0164] vector

[0165] In additional embodiments, a gene construct comprising one or more of the nucleic acids of the present invention is introduced into a cell in vivo to produce a polypeptide of the present invention, or one or more thereof, selectively fused into a protein, or is expressed separately and / or simultaneously in vivo to induce an in vivo immune response. The nucleic acid (e.g., DNA) may be present in any various delivery system known to those skilled in the art, including nucleic acid expression systems, bacterial and some viral expression systems. Numerous gene delivery techniques, e.g., the literature [Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems[15:143-198] and the references cited therein are well known in the art. For the purpose of explanation, some of these approaches are outlined below.

[0166] Accordingly, a vector comprising a nucleic acid molecule of the present invention (also referred to herein as a 'DNA expression construct' or 'construct') is provided.

[0167] Suitablely, the vector contains regulatory elements (e.g., a suitable promoter and termination signal) suitable for allowing the transcription of a translation-active RNA molecule in a host cell, such as a human host cell. A 'translation-active RNA molecule' is an RNA molecule that can be translated into a protein by the translation machinery of a human cell.

[0168] Accordingly, a vector comprising the nucleic acid of the present invention (hereinafter referred to herein as 'vector of the present invention') is provided.

[0169] In particular, the vector may be a viral vector. The viral vector may be an adenovirus, an adeno-associated virus (AAV) (e.g., AAV types 5 and 2), an alphavirus (e.g., Venezuelan equine encephalitis virus (VEEV), Sindvis virus (SIN), forest semliki virus (SFV)), a herpes virus, an arenavirus (e.g., lymphocytic choriomeningitis virus (LCMV)), a measles virus, a poxvirus (e.g., modified vaccinia ankara (MVA)), a paramyxovirus, a lentivirus, or a rhabdovirus (e.g., vesicular stomatitis virus (VSV)), that is, the vector may be derived from any of the aforementioned viruses. Adenoviruses are particularly suitable for use as gene transfer vectors due to their medium-sized genome, ease of manipulation, high titer, broad target cell range, and high infectivity. Both ends of the viral genome contain 100 to 200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcriptional units separated by the initiation of viral DNA replication. The E1 regions (E1A and E1B) encode proteins responsible for the transcriptional regulation of the viral genome and minority cellular genes. Expression of the E2 regions (E2A and E2B) results in the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutdown (Renan, 1990). The products of late genes, including most viral capsid proteins, are expressed only after significant processing of a single primary transcript generated from a major late promoter (MLP). MLPs are particularly efficient in the late stages of infection, and all mRNA transcribed from this promoter has a 5'-tripartite leader (TPL) sequence, making them preferred mRNAs for translation.Replication-defective adenoviruses generated from viral genomes in which one or more of the initial genes are deleted are particularly useful because their replication is limited and the potential for pathogen spread within the vaccinated host and to the vaccinated host's contacts is low.

[0170] Other polynucleotide delivery

[0171] In a specific embodiment of the present invention, an expression construct comprising one or more polynucleotide sequences may simply consist of a naked recombinant DNA plasmid. Accordingly, the present invention provides a DNA plasmid comprising the nucleic acid of the present invention or one or more nucleic acids of the present invention, which further comprises a regulatory and / or control sequence suitable for the expression or production of the polypeptide encoded by the present invention or polypeptides. Literature [Ulmer et al. , 1993, Science 259:1745-1749] and literature[Cohen, 1993, Science [259:1691-1692] Refer to the review. Delivery of the product can be performed, for example, by any method that physically or chemically penetrates the cell membrane. This applies particularly to in vitro delivery, but also to in vivo use. DNA encoding the gene of interest is also believed to be capable of being delivered in vivo in a similar manner and expressing the gene product. Various delivery systems have been used to deliver DNA molecules to animal models and the human body. Some products based on this technology have been approved for animal studies, while others are in Phase 2 and Phase 3 clinical trials in humans.

[0172] RNA delivery

[0173] In another embodiment of the present invention, an expression construct comprising one or more polynucleotide sequences may consist of a naked, recombinant DNA-derived RNA molecule (Ulmer et al., 2012, Vaccine 30:4414-4418). Accordingly, the present invention provides a DNA-derived RNA molecule or plasmid comprising the nucleic acid of the present invention or one or more nucleic acids of the present invention, which optionally further comprises a regulatory and / or control sequence suitable for the expression or production of the polypeptide encoded by the present invention. For DNA-based expression constructs, RNA molecules may be introduced into cells in vitro or in vivo using various methods. Since RNA-based constructs may be designed to mimic simple messenger RNA (mRNA) molecules, the introduced biological molecules are directly translated by the translation mechanism of the host cell to produce the polypeptide encoded within the cell into which they are introduced. Alternatively, RNA molecules may be designed in such a way that they enable self-amplification within the cell into which they are introduced by incorporating their structural genes for viral RNA-dependent RNA polymerases. Therefore, this type of RNA molecule, also known as self-amplifying mRNA (SAM™) molecules (Geall et al. 2012, PNAS, 109:14604-14609), shares characteristics with some RNA-based viral vectors. Either the mRNA-based or SAM™ RNA can be further modified (e.g., by altering their sequences or by using modified nucleotides) to improve stability and translation (Schlake et al., RNA Biology, 9: 1319-1330), and both RNA types are (e.g., emulsions (Brito et al., Molecular Therapy, 2014 22:2118-2129) or lipid nanoparticles (Kranz et al., 2006, Nature,It can be formulated to facilitate in vitro or in vivo stability and / or entry into cells. Numerous formulations of modified (and non-modified) RNA have been tested as vaccines in animal models and humans, and many RNA-based vaccines are being used in ongoing clinical trials.

[0174] Pharmaceutical composition

[0175] The polypeptides, nucleic acids, and vectors of the present invention may be formulated for delivery in pharmaceutical compositions, such as immunogenic compositions and vaccine compositions (all referred to herein as 'compositions of the present invention'). The compositions of the present invention suitably comprise the polypeptides, nucleic acids, or vectors of the present invention together with a pharmaceutically acceptable carrier.

[0176] Accordingly, in an embodiment, a pharmaceutical composition such as an immunogenic pharmaceutical composition comprising a polypeptide, nucleic acid, or vector of the present invention together with a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt is provided.

[0177] In another embodiment, a vaccine composition is provided comprising the polypeptide, nucleic acid, or vector of the present invention together with a pharmaceutically acceptable carrier and / or adjuvant. The preparation of the pharmaceutical composition is generally, for example, described in the literature [Powell & Newman, eds., Vaccine Design It is described in [the subunit and adjuvant approach, 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. It will be understood that vaccine compositions include not only therapeutic vaccine compositions but also preventive vaccine compositions.

[0178] In a specific preferred embodiment of the present invention, a pharmaceutical composition of the present invention is provided comprising one or more (e.g., one) polypeptides of the present invention in combination with a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt.

[0179] In a specific preferred embodiment of the present invention, a pharmaceutical composition of the present invention is provided comprising one or more (e.g., one) nucleic acids of the present invention or one or more (e.g., one) vectors of the present invention in combination with a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt.

[0180] In an embodiment, the composition of the present invention may comprise one or more (e.g., one) polynucleotides of the present invention and / or one or more (e.g., one) polypeptides of the present invention as components. Alternatively, the composition may comprise one or more (e.g., one) vectors and / or one or more (e.g., one) polypeptide components. Alternatively, the composition may comprise one or more (e.g., one) vectors and / or one or more (e.g., one) polynucleotides as components. Such compositions may provide an enhanced immune response.

[0181] Pharmaceutically acceptable salts

[0182] It will be apparent that the composition of the present invention may contain a pharmaceutically acceptable salt of the nucleic acid, vector, or polypeptide provided herein. Such salts may be prepared from pharmaceutically acceptable non-toxic bases comprising organic bases (e.g., salts of primary, secondary, and tertiary amines and basic amino acids) and inorganic bases (e.g., salts of sodium, potassium, lithium, ammonium, calcium, and magnesium). For example, the pharmaceutically acceptable salt may comprise an anion selected from any of chlorides, sulfates, maleates, chlorides, and acetates, and / or a cation selected from sodium, potassium, magnesium, and calcium.

[0183] Pharmaceutically acceptable carrier

[0184] Although a number of pharmaceutically acceptable carriers known to those skilled in the art may be used in the compositions of the present invention, the optimal type of carrier used will vary depending on the mode of administration. The compositions of the present invention may be formulated for any suitable mode of administration, including, for example, parenteral, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration, preferably parenteral, for example, intramuscular, subcutaneous, or intravenous administration. For parenteral administration, the carrier may preferably contain water and may contain a buffer for pH control, a stabilizer, for example, a surfactant and an amino acid, and an isotonic modifier, for example, a salt and a sugar. If the composition is intended to be provided in a lyophilized form for dilution at the time of use, the formulation may contain a cryoprotectant, for example, a sugar such as trehalose. For oral administration, any of the above carriers or solid carriers, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate may be used.

[0185] Accordingly, the composition of the present invention may comprise a buffer (e.g., neutral buffered saline or phosphate buffered saline), a carbohydrate (e.g., glucose, mannose, sucrose, or dextran), mannitol, a protein, a polypeptide, or an amino acid, e.g., glycine, an antioxidant, a bacteriostatic agent, a chelating agent, e.g., EDTA or glutathione, a solute to form a formulation that is isotonic, hypoosmolar, or weakly hyperosmolar with the recipient's blood, a suspending agent, a thickening agent, and / or a preservative. Alternatively, the composition of the present invention may be formulated as a lyophilized material.

[0186] Immunostimulant

[0187] The composition of the present invention may include one or more immunostimulators. Immunostimulators may be any substance that enhances or amplifies the immune response (antibody and / or cell-mediated) to exogenous antigens. Examples of immunostimulators commonly referred to as adjuvants in relation to vaccine formulations include aluminum salts, e.g., aluminum hydroxide gel (alum) or aluminum phosphate; saponins including QS21; immunostimulating oligonucleotides such as CPG; oil-in-water emulsions (e.g., where the oil is squalene); aminoalkyl glucosamini 4-phosphate; lipopolysaccharides or derivatives thereof, e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPL®); and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and interferon. Further suitable examples include the following: incomplete Freund adjuvant (IFA); aluminum-based adjuvants, e.g., aluminum hydroxide (Alhydrogel™). Aluminum phosphate (Adjut-phos™), oil-in-water emulsions, e.g., MF59, AS03; adjuvants, e.g., TLR agonist-based adjuvants, e.g., TLR2 agonists, e.g., bacterial lipopeptides (e.g., MALP-2, Pam2Cys, Pam3Cys); TLR3 agonists, e.g., palmitic acid-modified TLR7 / 8 agonists (C16-R848), polyinosine, polycythidyl acid (poly I:C), poly ICLC (Hiltonol), poly I:C12U; TLR4 agonists, e.g., monophosphoryl lipid A (MPLA); TLR7 / 8 agonists, e.g., imiquimod (Aldara cream), resiquimod; TLR9 agonists, e.g., CpG oligonucleotides (ODN), e.g., CpG A, CpG B, CpG C, CpG ODN 7909, IC31; synthetic double-stranded RNA (dsRNA), e.g., poly-I:C, poly-ICLC; glucopyranosyl lipid A (GLA), e.g., GLA-SE, GLA-AF, GLA-LS, GLA-alum;Imidazocinolines, e.g., imiquimod (R837), resiquimod (R848), 3M-052; cyclic dinucleotides (CDN), e.g., 2',3'-cGAMP, 3',3'-cGAMP, c-di-GMP, c-di-AMP; manganese-based adjuvants, e.g., Mn2+ solution, manganese jelly (MnJ), NanoMn; metabolic adjuvants, e.g., bisphosphonates, statins; nanoparticle-based adjuvants, e.g., lipid nanoparticles (LNP), poly(lactide-co-glycolide) (PLGA) particles, chitosan; water-in-oil nanoemulsions, e.g., Montanide ISA 51, Montanide ISA 720; Micro or nanoparticle adjuvants, e.g., poly(lact-co-glycolic acid) (PLGA) particles, poly(lactic acid) (PLA), polystyrene particles, liposomes (e.g., coated with polyethylene glycol (PEG) or modified into immunoliposomes), poly(DL-lactide-co-glycolide) microspheres, poly(propylene sulfide) nanoparticles, cGAS-STING / STING agents, e.g., cyclic GMP-AMP (cGAMP) and analogs thereof, STING agents such as CpG ODN 1018, AS04 (containing monophosphoryl lipid A), CF501, and ADU-S100; Cytokines, e.g., interleukin-2 (IL-2), e.g., IL-2 mutain, IL-2 pre-conjugated with antibodies, IL-2 conjugated with polyethylene glycol, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferons, e.g., IFN-α, IFN-β, IFN-γ; self-assembling peptides, e.g., nanofiber-forming peptides for adjuvant-free vaccines, virus-like particles (VLPs), e.g., Qβ, Eastern mosaic virus (CPMV); inorganic nanoparticles, e.g., gold nanoparticles (AuNP), silica nanoparticles, mesoporous silica rods (MSR), calcium phosphate nanoparticles, iron oxide nanoparticles, manganese-based nanoparticles, nanoMn, MnJ (manganese jelly), carbon-based nanomaterials, graphene oxide, and carbon nanotubes;Cage protein nanoparticles, e.g., ferritin nanoparticles, virus-like particles (VLPs), self-assembling protein nanoparticles (SAPNs), heat shock protein nanoparticles (HSPs). Additional suitable examples particularly useful in nucleic acid antigen vaccines and compositions include the following: nucleoside-unmodified mRNA, e.g., poly-uracil (U) sequences, double-stranded RNA (dsRNA), RNA molecules without nucleoside modification, RNA oligonucleotides having phosphothioate-nucleotide linkages, short double-stranded RNA (dsRNA) having 5'-triphosphate ends; PRR ligands (pattern recognition receptor ligands), e.g., IRF, interferon regulatory factors, e.g., NF-κB, nuclear factor kappa light chain; reactive oxygen species (ROS), e.g., NLRP3, NOD, LRR, and pyrin domain-containing protein-like 3; 5'-ppp, 5'-triphosphate; mRNA-coding functional proteins, for example, a combination of STING, CD40L, CD70, and constitutively active TLR4; Ingredient lipid-based adjuvants, e.g., ionizable cationic lipids in LNP, e.g., DLinDMA, C12-TLRa, lipid components (DOTMA and DOPE), A2-Iso5-2DC18 (A2-ionizable cationic lipid-like substance), SAL12 (C12-TLRaLipidoidA2 (C1-cationic lipid-like substance)), ionizable amino lipids, LCP lipid-coated calcium phosphate, DOPA dioleoylphosphatidic acid, DLinDMA 1,2-dilinoleyloxy-n,n-dimethyl-3-aminopropane, DDA dimethyldioctadecylammonium, DOTMA trimethyl[2,3-(dioleyloxy)propyl]ammonium chloride, DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;Peptide and glycolipid immunostimulators, e.g., lipid ALC-0315, ionizable lipid SM-102, protamine, DP7 (cationic peptide), α-GC glycolipid, arginine-rich protamine peptide, cholesterol-modified cationic peptide DP7, liposomes containing DP7, palmitic acid-modified TLR7 / 8 agonist R848 (C16-R848). Accordingly, suitably, one or more immunostimulators of the composition of the present invention are aluminum salts, saponins, immunostimulating oligonucleotides, oil-in-water emulsions, aminoalkyl glucosamine 4-phosphate, lipopolysaccharides and derivatives thereof and other TLR4 ligands, TLR7 ligands, TLR8 ligands and TLR9 ligands, IL12, interferon, incomplete Freund adjuvant (IFA), aluminum-based adjuvants, TLR agonists, synthetic double-stranded RNA (dsRNA), glucopyranosyl lipid α (GLA), imidazoquinoline, CPG oligodeoxynucleotides (ODN), cyclic dinucleotides (CDN), manganese-based adjuvants; The adjuvants are selected from metabolic adjuvants, nanoparticle-based adjuvants, water-in-oil nanoemulsions, micro or nanoparticle adjuvants, CGAS-STING / STING agonists, cytokines, self-assembling peptides, virus-like particles (VLPs), inorganic nanoparticles, cage protein nanoparticles, nucleoside-unmodified mRNA, PRR ligands (pattern recognition receptor ligands), mRNA-coding functional proteins, component lipid-based adjuvants, peptide and glycolipid immunostimulators, and TLR agonists. Additionally or alternatively, one or more immunostimulators of the compositions of the present invention may also be selected from monoclonal antibodies that specifically interact with other immune components, for example, monoclonal antibodies that block the interaction of immune checkpoint receptors including PD-1 and CTLA4.

[0188] In the case of a recombinant nucleic acid delivery method (e.g., DNA, RNA, viral vector), a gene encoding a protein-based immunostimulator can be easily delivered together with a gene encoding the polypeptide of the present invention.

[0189] Continuous release

[0190] The compositions described in this specification may be administered as part of a sustained-release formulation (i.e., a formulation such as a capsule, sponge, patch, or gel (e.g., composed of a polysaccharide)) that results in sustained / sustained release of the compound after administration.

[0191] Storage and Packaging

[0192] The composition of the present invention may be provided in a single-dose or multi-dose container, such as a sealed ampoule or vial. Such containers are preferably sealed to preserve the sterility of the formulation until use. Generally, the formulation may be stored as a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the composition of the present invention may be stored under freeze-drying conditions and may require only the addition of a sterile liquid carrier (e.g., water for injection or saline solution) immediately before use.

[0193] Dosage

[0194] In each composition of the present invention, the amount of nucleic acid, polypeptide, or vector may be prepared in a manner that obtains a suitable dosage for therapeutic or prophylactic use. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmaceutical considerations, will be considered by those skilled in the art who manufacture these compositions, and as such, various dosages and therapeutic regimens may be desirable.

[0195] Typically, a composition containing a therapeutically or prophylactically effective amount delivers about 0.1 µg to about 1000 µg of the polypeptide of the present invention per dose, more typically about 2.5 µg to about 100 µg of the polypeptide per dose. When delivered in the form of short synthetic long peptides, the dose could be in the range of 1 to 200 µg / peptide / dose. For polynucleotide compositions, they typically deliver about 10 µg to about 20 mg of the nucleic acid of the present invention per dose, more typically about 0.1 mg to about 10 mg of the nucleic acid of the present invention per dose.

[0196] Diseases to be treated or prevented

[0197] As mentioned elsewhere, SEQ ID NOs 1 to 17 and 57 and SEQ ID NOs 18 to 56 and 58 are polypeptide sequences corresponding to CST antigens overexpressed in ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC (see Table 1).

[0198]

[0199] In one embodiment, the present invention provides a polypeptide, nucleic acid, vector, or composition of the present invention for use in medicine.

[0200] A further aspect of the present invention relates to a method for inducing an immune response in a human, comprising the step of administering a polypeptide, nucleic acid, vector, or composition of the present invention to said human.

[0201] The present invention also provides a polypeptide, nucleic acid, vector, or composition of the present invention for use in enhancing an immune response in humans.

[0202] Uses of the polypeptide, nucleic acid, vector, or composition of the present invention for the manufacture of medicines to be used to enhance an immune response in humans are provided.

[0203] Suitably, an immune response occurs against a cancer tumor expressing a sequence comprising or composed of a corresponding sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and variants and fragments thereof, e.g., an immunogenic fragment, e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and variant sequences thereof. In this regard, 'corresponding' means that when expressing any of SEQ ID NOs 1 to 17 and 57 as disclosed in this specification, or a variant or fragment thereof, e.g., an immunogenic fragment (SEQ ID NOs 18 to 56 and 58 or a variant thereof), the polypeptide, nucleic acid, vector, or composition of the present invention and the medicine comprising the same may comprise a sequence selected from any one of SEQ ID NOs 1 to 17 and 57 or a variant or fragment thereof, e.g., an immunogenic fragment, e.g., SEQ ID NOs 18 to 56 and 58 and a variant thereof, or may be based on a sequence composed of these.

[0204] Suitably, the immune response comprises an immune response of immunoreactive cells, e.g., CD8+ T-cells, or CD4+ T-cells and / or antibody responses, in particular CD8+ cell lysis T-cell responses and / or CD4+ helper T-cell responses. The immune response as disclosed may include any of cell proliferation, cytokine or granzyme production, and induction of tumor cell death or apoptosis.

[0205] Suitably, an immune response occurs upon a tumor or tumor cell expressing a sequence selected from any of SEQ ID NOs. 1 to 17 and 57 as disclosed herein, variants and fragments thereof, e.g., immunogenic fragments thereof (SEQ ID NOs. 18 to 56 and 58 or variants thereof). In a preferred embodiment, the tumor may be any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC, e.g.,

[0206] Ovarian cancer may include any of ovarian carcinomas, serous types, e.g., ovarian serous cystadenocarcinomas;

[0207] Colorectal cancer may include mesenchymal or mesenchymal chondrosarcoma, gastrointestinal stromal tumor (GIST), lymphoma, carcinoid-Turcott syndrome, Peutz-Jeghers syndrome (PJS), familial colorectal cancer (FCC), and any adenocarcinoma that may be pediatric colorectal polyposis;

[0208] Esophageal cancer may include squamous cell carcinoma, which often originates in the squamous cells covering the esophagus and usually occurs in the upper and middle parts of the esophagus; adenocarcinoma, which often originates in the glandular tissue of the lower esophagus where the esophagus meets the stomach; small cell carcinoma; and / or any of esophageal adenocarcinoma (EAC) or esophageal squamous cell carcinoma (ESSC);

[0209] Breast cancer may include any carcinoma, lobular carcinoma in situ (LCIS), angiosarcoma of the breast, Paget's disease, inflammatory breast cancer, invasive breast cancer, triple-negative breast cancer, invasive lobular breast cancer, metastatic breast cancer, medullary breast cancer, mucinous (mucinous or colloidal) breast cancer, tubular breast cancer, adenocystic carcinoma of the breast, metaplastic breast cancer, lymphoma of the breast, basal-type breast cancer, phyllodes or cystosarcoma phyllodes, and papillary breast cancer;

[0210] Skin cancer may include any of basal cell carcinoma, squamous cell carcinoma, and melanoma; for example, melanoma is cutaneous melanoma or uveal melanoma, particularly cutaneous melanoma.

[0211] The gastrointestinal tract, or GI tract, includes the mouth, pharynx, esophagus, stomach, small intestine, duodenum, jejunum and ileum, the large intestine (including the colon starting from the cecum and ending at the rectum and anus), and accessory digestive organs of the gastrointestinal tract, such as the tongue, salivary glands, pancreas, liver, and gallbladder. Thus, gastrointestinal (GI) cancer includes cancer of any of the aforementioned structures, most specifically one of colorectal cancer / tumor and esophageal cancer / tumor, e.g., colorectal adenocarcinoma and esophageal adenocarcinoma. Gastrointestinal cancer may also include gastrointestinal stromal tumors (GISTs) that commonly occur in the small intestine or stomach, which may include cancerous forms of the soft mesenchymal tissue of the gastrointestinal tract and interstitial cells of Cajal (ICC).

[0212] Stromal cancer includes any stroma-rich cancer type, for example, any gastrointestinal stromal tumor (GIST), pancreatic cancer, prostate cancer, and breast cancer.

[0213] Prostate cancer includes prostate adenocarcinoma, transitional cell carcinoma of the prostate, squamous cell carcinoma of the prostate, and small cell prostate cancer.

[0214] Pancreatic cancer may be an exocrine, endocrine, neuroendocrine, or pancreatic islet tumor; for example, pancreatic cancer may be an adenocarcinoma or an exocrine adenocarcinoma, and pancreatic cancer may additionally include mucocystic neoplasms, intraductal papillary mucinous tumors (IPMNs), and acinar cell carcinomas. Types of pancreatic endocrine cancer include any of gastrinomas, glucagonomas, insulinomas, somatostatinomas, VIPomas (VIP = vascular intestinal peptide), non-functional pancreatic islet cell tumors, or cancers.

[0215] Head and neck cancer includes cancer of the hypopharynx, nasopharynx, oropharynx, sinuses and nasal cavity, salivary glands, oral cavity, and larynx; head and neck melanoma; oral and oropharyngeal cancer; head and neck basal cell carcinoma; and head and neck squamous cell carcinoma, and these cancers may be squamous cell carcinoma (SCC) or sarcoma. Sarcoma may include any of Kaposi sarcoma, liposarcoma, osteosarcoma, Ewing sarcoma, and soft tissue sarcoma.

[0216] Rectal cancer may include any of the following: adenocarcinoma of the rectum, signet ring cell adenocarcinoma, mucinous adenocarcinoma, adenosquamous cell carcinoma (ASC), neuroendocrine carcinoma, small cell carcinoma, melanoma, squamous cell carcinoma, carcinoid tumor, gastrointestinal stromal tumor, lymphoma, and hereditary rectal cancer.

[0217] Lung cancer may include any of the following: pulmonary squamous cell carcinoma (LUSC), pulmonary adenocarcinoma (LUAD), large-cell carcinoma, small cell lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenosquamous cell carcinoma, sarcomatoid carcinoma, pulmonary sarcoma, and pulmonary lymphoma.

[0218] Gastric cancer may include any of gastric adenocarcinoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, lymphoma, neuroendocrine tumor, carcinoid tumor, squamous cell carcinoma, gastric lymphoma, and leiomyosarcoma.

[0219] Cervical cancer may include either cervical squamous cell carcinoma or adenocarcinoma.

[0220] Uterine cancer may include endometrial cancer, and any of uterine sarcoma, clear cell adenocarcinoma, serous adenocarcinoma, mucinous neoplasm, small cell carcinoma, endometrial stromal sarcoma, carcinosarcoma, endometrioid tumor, uterine carcinosarcoma, and squamous cell carcinoma.

[0221] Tumors or cancer can be primary or metastatic.

[0222] Further aspects of the present invention relate to a method for treating cancer in a human patient with cancer in which cancer cells express a sequence selected from SEQ ID NOs 1 to 17 and 57 and variants and fragments thereof, e.g., immunogenic fragments, or a method for preventing cancer in a human patient with cancer in which a sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and variants and fragments thereof, e.g., immunogenic fragments, e.g., SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, or any sequence selected from the sequences comprising these, wherein the method comprises the step of administering the corresponding polypeptide, nucleic acid, vector, or composition of the present invention to said human.

[0223] The present invention also provides a polypeptide, nucleic acid, vector, or composition of the present invention for use in treating or preventing human cancer, wherein the cancer cells express a corresponding sequence selected from any of SEQ ID NOs. 1 to 17 and 57 and variants and fragments thereof, e.g., immunogenic fragments as described herein. The cancer or tumor may be any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC.

[0224] In this description of various aspects and embodiments of the present invention and the following description, particularly in the therapeutic embodiments and aspects thereof, a fragment of a polypeptide of any of SEQ ID NOs 1 to 17 and 57, e.g., an immunogenic fragment thereof, and an exemplary peptide of the present invention according thereto comprises a polypeptide comprising or composed of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof, e.g., having one, two, or three amino acid variations selected from addition, substitution, and deletion in relation thereto, e.g., SEQ ID NOs 18 to 56 and 58 or any variant of SEQ ID NOs 18 to 56 and 58 or an immunogenic variant thereof.

[0225] The words 'prevention' and 'prevention' are used interchangeably in this specification.

[0226] Treatment and vaccination therapy

[0227] The therapeutic regimen may involve one of the separate, simultaneous (e.g., co-administration), or sequential (e.g., prime-boost) delivery of (i) the polypeptide, nucleic acid, or vector of the present invention and (ii) one or more additional polypeptides, nucleic acid, or vectors of the present invention and / or (iii) additional components, e.g., various other therapeutically useful compounds or molecules, e.g., antigenic proteins administered concurrently with optional adjuvants. Examples of co-administration include ipsilateral co-administration and contralateral co-administration. 'Simultaneous administration' appropriately refers to all components being delivered during the same course of treatment. Suitably, all components are administered simultaneously (e.g., simultaneous administration of both DNA and protein), but one component could be administered within minutes (e.g., at the same appointment or doctor's visit) or within hours.

[0228] In some embodiments, the 'priming' or first administration of the polypeptide, nucleic acid, or vector of the present invention is followed by one or more 'boosting' or subsequent administrations of the polypeptide, nucleic acid, or vector of the present invention ('prime and boost' method). In one embodiment, the polypeptide, nucleic acid, or vector of the present invention is a prime-boost vaccination regimen. In an embodiment, both the prime and the boost are polypeptides of the present invention, and in each case, the same polypeptide of the present invention. In an embodiment, both the prime and the boost are nucleic acids of the present invention, and in each case, the same nucleic acid or vector of the present invention. Alternatively, the prime may be performed using the nucleic acid or vector of the present invention and the boost may be performed using the polypeptide of the present invention, or the prime may be performed using the polypeptide of the present invention and the boost may be performed using the nucleic acid or vector of the present invention. Typically, the first or 'priming' administration and the second or 'boosting' administration are provided after about 1 to 12 weeks or up to 4 to 6 months. Subsequent 'booster' administration may be provided frequently every 1 to 6 weeks or much later (up to several years later).

[0229] Antigen combination

[0230] According to the present invention, a polypeptide, nucleic acid, or vector of the present invention may be used in combination with one or more other polypeptides, nucleic acids, or vectors of the present invention (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) and / or one or more other antigenic polypeptides, polynucleotides, or vectors encoding these (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), which may be used for ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cells. It induces an immune response against any of the carcinomas (ESSC), particularly esophageal cancer, or EAC or ESSC. These other antigenic polypeptides could be derived from various sources and could include well-described cancer-associated antigens or cancer-tumor-associated antigens (TAAs) for the aforementioned cancer or tumor types, e.g., MUC16, CRABP1 / 2, FOLR1 and KLK10, CEA, MAGE-A1, MAGE-A3, MAGE-A4, PRAME, hTERT, HER2, MUC1, Survivin, STEAP1, SOX2, NY-ESO-1, LAGE-1, OIP5, TTK, PLU1, DKKL1, FBXO39, AFP, SSX, EGFR, hTERT, ALDH, IDO, PSMA, VEGF, TEM-1, or antigenic peptides derived therefrom.

[0231] Specific cancer-associated antigens or tumor-associated antigens (TAAs) are known to be associated with specific types of cancer; for example, esophageal cancer TAAS include TP53, CDKN2A, PIK3CA, NOTCH1, MUC16, EPHA2, and CTNNB1 and peptides derived therefrom. Head and neck cancer TAAS include TP53, NOTCH1, HRAS, CASP8, FAT1, HPV E6 and E7 polypeptides and peptides derived therefrom. Lung cancer TAAS include ALK fusion-derived peptides, EGFR, KRAS, ATM, BRCA1 / 2, RB1, TP53, NOTCH1, PIK3CA, BRAF, ALK, EML4-ALK fusion, MET, STK11, KEAP1 and peptides derived therefrom. Colorectal cancer TAAS includes APC, KRAS, TP53, BRAF, PIK3CA, TGFBR2, SMAD4, MLH1, MSH2, MSH6, PMS2, and peptides derived therefrom. Pancreatic cancer TAAS includes TMOD3, TPX2, WNT7A, CA19-9, MUC16CD, ADAM9, EFNB2, and peptides derived therefrom. Breast cancer TAAS includes TP53, RB1, and PTEN, TP53, PIK3CA, GATA3, BRCA1 / BRCA2, ERBB2(HER2), AKT1, ESR1, and peptides derived therefrom. Bladder cancer TAAS includes TP53, FGFR3, PIK3CA, HRAS, KRAS, RB1, ERBB2(HER2), TSC1, ARID1A, KDM6A, STAG2, and peptides derived therefrom. Kidney cancer TAAS includes VHL, PBRM1, SETD2, BAP1, MTOR, PIK3CA, PTEN, TP53, KDM5C, and peptides derived therefrom. Ovarian cancer TAAS includes TP53, BRCA1 / BRCA2, PIK3CA, KRAS, CTNNB1, ARID1A, NF1, PTEN, RB1, NOTCH3, and peptides derived therefrom.Melanoma cancer TAAS includes BRAF, NRAS, KIT, TP53, CDKN2A, PTEN, NF1, RAC1, TERT, and peptides derived therefrom, e.g., antigenic peptides. Any one or more of these aforementioned TAAs and / or TAAs known to be associated with a specific cancer type may be combined with, for example, a combination or fusion protein, in an embodiment of a fusion protein or an embodiment of a combination of the present invention (e.g., for co-administration), for example, in particular, said TAA and said polypeptide of the present invention may be combined with a polypeptide of the present invention disclosed herein as being associated with the same cancer type (see Table 1 of the present specification), e.g., a combination or fusion protein, thereby expressing a sequence of a polypeptide of the present invention suitable for the treatment or prevention of said cancer type or for inducing an immune response against a cancerous tumor of said cancer type.

[0232] Additionally, the combination or fusion protein of the present invention and / or the antigenic peptide from such source (cancer-related antigen) could also be combined with (i) non-specific immunostimulator / adjuvant species and / or (ii) antigens known to induce strong CD4 helper T cells (e.g., including universal CD4 helper epitopes) (as polypeptides, or as polynucleotides or vectors encoding such CD4 antigens) to amplify the anticancer-specific response induced by the co-administered antigen. Examples of universal CD4+ helper epitopes include PADRE (universal-DR epitope, sequence AKFVAAWTLKAAA, SEQ NO. 59), HA307-319 (erythropoietin epitope from influenza A, sequence: PKYVKQNTLKLAT, SEQ NO. 60), TT830-843 (tetanus toxin epitope, sequence: QYIKANSKFIGITE, SEQ NO. 61), VP2 (epitope from VP2 capsid protein, sequence: FNNFTVSFWLRVPKVSASHLE, SEQ NO. 62) and ovalbumin-derived (OVA323-339, sequence: ISQAVHAAHAEINEAGR, SEQ NO. 63).

[0233] Different polypeptides, nucleic acids, or vectors may be formulated in the same formulation or in separate formulations for use, for example, as a combination. Alternatively, the polypeptide may be provided as a fusion protein in which the polypeptide of the present invention is fused with a second or additional polypeptide (e.g., any of the aforementioned TAA and / or one or more additional different polypeptides of the present invention). For example, additional polypeptides are cancer-related antigens, e.g., ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly associated with one or more of esophageal cancers, e.g., proteins or polypeptides or antigens, or EAC or ESSC-related proteins or polypeptides or antigens, e.g., MUC16, CRABP1 / 2, FOLR1 and KLK10, CEA, MAGE-A1, MAGE-A3, MAGE-A4, PRAME, hTERT, HER2, MUC1, Servibin, STEAP1, SOX2, NY-ESO-1, LAGE-1, OIP5, It is any one or more of TTK, PLU1, DKKL1, FBXO39, AFP, SSX, EGFR, hTERT, ALDH, IDO, PSMA, VEGF, TEM-1 or antigenic peptides derived therefrom and / or any one or more of the aforementioned disclosed TAAs or TAAs or antigenic peptides derived therefrom known to be associated with a specific type of cancer.

[0234] The nucleic acid may be provided as a nucleic acid encoding the aforementioned fusion protein.

[0235] More generally, when two or more components are used in combination, the components are, for example,

[0236] (1) as two or more individual antigenic polypeptide components;

[0237] (2) A fusion protein comprising both polypeptide components (or additionally, i.e., two or more);

[0238] (3) as one or more polypeptide and one or more polynucleotide components;

[0239] (4) as two or more individual polynucleotide components;

[0240] (5) as a single polynucleotide encoding two or more individual polypeptide components; or

[0241] (6) As a single polynucleotide encoding a fusion protein containing both (or additionally, i.e., two or more) polypeptide components

[0242] It could be presented.

[0243] For convenience, when multiple components are present, it is often desirable to include them within a single fusion protein or a polynucleotide encoding a single fusion protein. In one embodiment of the present invention, all components are provided as polypeptides (e.g., within a single fusion protein). In an alternative embodiment of the present invention, all components are provided as polynucleotides (e.g., single polynucleotides, e.g., encoding a single fusion protein).

[0244] fusion protein

[0245] As an embodiment of the above discussion of antigen combinations, the present invention also provides an isolated polypeptide according to the present invention fused to a second or additional polypeptide of the present invention (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12), (hereinafter referred to herein as 'fusion protein' or 'combination polypeptide of the present invention'), wherein optionally each polypeptide is different. Such combination polypeptides of the present invention may be produced by generating a nucleic acid construct that fuses with a sequence encoding an individual antigen. The combination polypeptide of the present invention is expected to have the utility described herein for the polypeptide of the present invention and may have the advantage of excellent immunogenicity or vaccine activity or prophylactic or therapeutic effects (e.g., increasing the range and depth of in vitro reactions in cancer cells or in vivo, e.g., in subjects suffering from cancer), and may be particularly valuable in crossbreed populations. The fusion of polypeptides of the present invention can also provide the advantage of increasing the efficiency of the formulation and manufacturing of vaccine antigens and / or vector vaccines (including nucleic acid vaccines).

[0246] As described in the antigen combination section above, the polypeptide of the present invention and the combination polypeptide of the present invention may also be fused to a polypeptide sequence other than the polypeptide of the present invention, which comprises one or more of the polypeptides selected from the following (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12):

[0247] (a) another polypeptide that is a cancer-associated antigen (as defined herein) and is therefore potentially useful as an immunogenic sequence in a vaccine (e.g., selected from MUC16, CRABP1 / 2, FOLR1, KLK10, CEA, MAGE-A1, MAGE-A3, MAGE-A4, PRAME, hTERT, HER2, MUC1, Servibin, STEAP1, SOX2, NY-ESO-1, LAGE-1, OIP5, TTK, PLU1, DKKL1, FBXO39, AFP, SSX, EGFR, hTERT, ALDH, IDO, PSMA, VEGF, TEM-1, or any one or more antigenic peptides derived from the above) and / or is a cancer-associated antigen known to be associated with any one or more cancer-associated antigens (TAAs) or a specific cancer type as disclosed herein or an antigenic peptide derived therefrom; and

[0248] (b) a polypeptide sequence capable of enhancing an immune response (i.e., an immunostimulator sequence), and

[0249] (c) For example, a polypeptide sequence containing a universal CD4 helper epitope and capable of providing strong CD4+ assistance to increase the CD8+ T cell response to a CST antigen epitope.

[0250] Examples of immunostimulator sequences include thymosin alpha-1 (Tα1), muramyl dipeptide (MDP) and the muramyl dipeptide derivative FK-565, poly-L-arginine, beta-defensin, BAMBI-derived peptide, P40 peptide, and epitalone. The present invention also provides nucleic acids encoding the aforementioned fusion proteins of the present invention and other embodiments (vectors, compositions, cells, etc.) applicable to the polypeptides of the present invention.

[0251] CST antigen-binding polypeptide

[0252] An antigen-binding polypeptide that is immune-specific to a tumor-expressed antigen (the polypeptide of the present invention) can be designed to replenish lysed cells to antigen-decorated tumor cells, thereby mediating their destruction. One such mechanism of replenishment of lysed cells by an antigen-binding polypeptide is known as antibody-dependent cell-mediated cytotoxicity (ADCC). Accordingly, the present invention provides an antigen-binding polypeptide that is immune-specific to the polypeptide of the present invention, which is optionally complexed (i.e., bound) with HLA as described herein. The antigen-binding polypeptide comprises antibody-derived molecules, such as monoclonal antibodies, antibodies and fragments, particularly their antigen-binding fragments, such as domain antibodies, Fab fragments, Fv fragments, such as scFv and VHH fragments, which may be produced in non-human animal species (e.g., rodents or camelid species) and humanized, or may be produced in non-human species (e.g., rodents genetically modified to have a human immune system).

[0253] Antigen-binding polypeptides, which may be antibody-derived molecules, can be produced by methods well known to those skilled in the art. For example, monoclonal antibodies can be produced using hybridoma technology by fusing specific antibody-producing B cells with myeloma (B-cell cancer) cells selected for their ability to grow in tissue cultures and for lacking antibody chain synthesis (the literature in which the full text is referenced herein [ and Milstein, 1975, Nature 256(5517): 495-497 and Nelson et al., 2000 (Jun), Mol Pathol. 53(3):111-7]).

[0254] A monoclonal antibody directed toward an antigen (e.g., the CST antigen of the present invention) is, for example,

[0255] a) a step of forming a hybridoma by immortalizing lymphocytes obtained from the peripheral blood of an animal (including humans) previously immune / exposed to a determined antigen into immortal cells, preferably myeloma cells,

[0256] b) A step of culturing the formed immortalized cells (hybridomas) and recovering the cells that produce antibodies with the desired specificity.

[0257] It can be obtained by.

[0258] Monoclonal antibodies

[0259] a) a step of cloning a DNA or cDNA sequence obtained from animal lymphocytes, particularly animal peripheral blood lymphocytes (suitably previously immunized with a determined antigen) into a vector, particularly phages and more specifically filamentous bacteriophages,

[0260] b) a step of transforming prokaryotic cells using the above vector under conditions where antibody production is possible,

[0261] c) A step of selecting antibodies by performing antigen-affinity selection on these,

[0262] d) A step of recovering antibodies having the desired specificity

[0263] e) a step of expressing an antibody-coding nucleic acid molecule obtained from B cells of a patient exposed to the antigen, or of an animal experimentally immunized with the antigen, or a nucleic acid encoding its antigen-binding fragment.

[0264] It can be obtained by a process including.

[0265] Subsequently, the selected antibody, or its antigen-binding fragment, can be produced using conventional recombinant protein production technology (e.g., from genetically engineered cells, e.g., HEK 298 cells or CHO cells).

[0266] The present invention provides an isolated antigen-binding polypeptide that is immunospecific to the polypeptide of the present invention. Suitably, the antigen-binding polypeptide is any of a monoclonal antibody or a fragment thereof, for example, an antigen-binding fragment thereof, for example, Fab, Fab', diabody, tribody, minibody, scFv, scFv-Fc, F(ab')2, VHH, V-NAR, dab, dab-Fc, free-LC, half antibody, preferably for example, scFv. The term "immunospecific" refers to the ability of an antigen-binding polypeptide, e.g., an antibody-derived molecule, e.g., an antibody or its antigen-binding fragment, or a T cell receptor-derived molecule, e.g., a T cell receptor, or its antigen-binding fragment, to bind to or specifically bind to a specific antigen or epitope according to the present invention, e.g., an MHC-presented peptide antigen or polypeptide, not to bind to unrelated or peptide molecules, and to selectively induce an immune response in each B-cell or T-cell / NK-cell that produces / presents said antigen-binding polypeptide. The same definition applies to each B-cell or T-cell that produces said antigen-binding polypeptide.

[0267] In certain embodiments, the antigen-binding polypeptide is coupled to a cytotoxic moiety. An exemplary cytotoxic moiety comprises the Fc domain of an antibody, which will promote ADCC by supplementing Fc receptor-bearing cells. Alternatively, the antigen-binding polypeptide may be linked to a biological toxin or cytotoxic chemical, e.g., calicemycin, doxorubicin, or monomethylauristatin E.

[0268] Another important class of antigen-binding polypeptides includes T-cell receptor (TCR)-derived molecules, fragments or variants of the present invention, for example, HLA-display fragments of the antigen of the present invention that bind to the HLA-display polypeptide. In the present embodiment, the TCR-based bioagent or T-cell receptor (TCR)-derived molecule (including TCRs, e.g., TCRs directly derived from or specifically engineered from a subject or patient, high-affinity TCRs, e.g., recombinant TCRs, or T-cell receptor (TCR)-derived molecules) recognizes the CST antigen (or derivatives or variants thereof) on the surface of a tumor cell, e.g., the HLA-display polypeptide, fragment, or variant of the present invention.

[0269] Accordingly, the antigen-binding polypeptide according to the present invention may be, for example, a T cell receptor or TCR comprising an α chain and a β chain, for example, wherein the extracellular region of each chain comprises three CDRs (CDR1, CDR2, CDR3) and four framework regions that are sides of the CDRs, and a constant region. Each chain has a linking peptide region that connects a transmembrane region and an intracellular region to the extracellular domain at the C-terminus. The TCR may also be a soluble form of the TCR, for example, consisting of an extracellular domain (also called an antigen-binding domain). The soluble TCR may lack a transmembrane domain and, ideally, also an intracellular domain, for example, may consist of α chain and β chain variable domains that optionally include each constant domain or a part thereof, or alternatively lack a constant domain, for example, wherein the α chain and β chain variable domains are linked to form a single-chain variable domain called scTv.

[0270] The antigen-binding polypeptide of the present invention, which is a TCR-derived molecule, can be produced by a method well known to those skilled in the art. For example, (a) isolating T-cells from a sample of peripheral blood; (ii) contacting the T-cells with the polypeptide of the present invention as presented on, for example, antigen-presenting cells, e.g., dendritic cells, or on a multimeric MHC reagent, e.g., monomeric, dextramer, or pentamer reagent; (iii) identifying and isolating T-cells expressing a TCR that specifically binds and / or whose binding induces an immune response in the T-cells (e.g., induction of T-cell proliferation and / or production of cytokines, e.g., IFN-gamma, IL-2, TNF-alpha, or, e.g., granzymes of tumor cell death or apoptosis and / or induction of immunoreactive cells); (iv) isolating a nucleic acid encoding the TCR and / or its antigen-binding fragment; and optionally (v) transfecting cells, e.g., CHO cells, KED 293 cells, T-cells, or Jurkat cells with the nucleic acid to form a TCR Alternatively, a step of expressing the antigen-binding fragment thereof, selectively expressing it on the cell surface or secreting it as a soluble TCR-derived molecule, and additionally, selectively purifying the soluble molecule.

[0271] Accordingly, in the embodiments, the antigen-binding polypeptide is immunospecific to and / or binds to an HLA-binding polypeptide that is a polypeptide of the present invention or a part thereof, i.e., a polypeptide of the present invention, a variant thereof, or an immunogenic fragment thereof. For example, the antigen-binding polypeptide is a recombinant T-cell receptor or a chimeric antigen receptor (CAR) or an antigen-binding fragment thereof.

[0272] In an embodiment, the antigen-binding polypeptide of the present invention (which may include an antibody-derived molecule or a TCR-derived molecule) may be coupled to an immune cell activating component or ligand, for example, another polypeptide (e.g., an antibody or a binding domain of an antibody, e.g., Fab or a Fab fragment, Fv or a Fv fragment, e.g., scFv), which can bind to an immune cell reactive cell of a target, e.g., a T-cell or a cytotoxic cell or a cytotoxic T-cell or other immune component, e.g., a cell surface receptor of said cell, e.g., CD3, CD4, CD8, CD70, CD137, CD28, preferably CD28 or CD3, more preferably CD3, and preferably the immune cell activating component or ligand is an antibody-derived polypeptide that binds to an immune component, preferably CD3.

[0273] Therefore, the antigen-binding polypeptide is a T-cell receptor (TCR)-derived molecule; For example, it may be a TCR-based bioagent comprising a TCR or an antigen-binding fragment thereof comprising a TCR, e.g., a TCR derived directly from a subject or patient, or a specifically engineered, high-affinity TCR, e.g., a recombinant TCR or a T-cell receptor (TCR)-derived molecule or an antibody mimic thereof (i.e., an antibody-derived molecule, antibody or antibody fragment or binding domain as described herein that is immunospecific to and / or binds to an HLA-binding polypeptide that is or is part of the polypeptide of the present invention), which may comprise or be coupled thereto a targeting moiety or immune cell-activating component or ligand that recognizes a component on a T-cell or T-cell (or other class of immune cells) that recognizes a CST antigen (or a derivative thereof) on the surface of a tumor cell and also attracts these immune cells to the tumor to provide therapeutic benefit, and for example, the targeting moiety may be provided as an antibody or antibody fragment or binding domain, in particular, e.g., an Fv or scFv that binds to CD3. In some embodiments, the targeting moiety can also stimulate the beneficial activity (including cytolytic activity) of the redirected immune cells.

[0274] For example, according to the present embodiment, a bispecific molecule or bispecific construct is provided comprising an antigen-binding polypeptide comprising, for example, a TCR-derived molecule, a TCR or cancer-specific binding fragment (e.g., an antigen-binding fragment), such as a soluble TCR, e.g., scTv, and, for example, a TCR variable alpha and beta domain having an optional constant alpha and / or beta domain or a cleavage thereof, or, for example, a TCR V-alpha-V-beta domain or scTv or an antibody mimic of any of these (i.e., an antibody-derived molecule, antibody or antibody fragment or binding domain that binds to the HLA-presenting antigen or peptide of the present invention), which is coupled to an immune cell-activating component or ligand (e.g., an antibody protein or a binding domain of an antibody, e.g., scFv, Fv domain or Fab) that binds to and activates immune cells, e.g., T-cells, e.g., CD4+ or CD8+ T-cells. For example, an immune cell activating component or ligand activates immune cells through binding to CD3. For example, one specific bispecific molecule or construct may comprise a polypeptide sequence of the TCR or cancer-specific binding fragment / antigen binding fragment of the present invention, e.g., a soluble TCR or scTv (or an antibody mimic thereof), and an antibody protein that binds to CD3 (in particular, an agonist antibody protein), e.g., scFv, an Fv domain, or Fab.In an embodiment, a bispecific construct / molecule is provided that may comprise, for example, a TCR or a cancer-specific binding fragment / antigen-binding fragment of a TCR, e.g., a soluble TCR, e.g., a TCR V-alpha-V-beta domain or scTv or an antibody mimic thereof (i.e., an antibody-derived molecule, antibody or antibody fragment or binding domain that binds to the HLA-presented antigen or polypeptide / peptide of the present invention), and an antigen-binding polypeptide that may be an immune cell-activating component or ligand that binds to and activates immune cells, e.g., T-cells, e.g., CD8+ T-cells (e.g., an antibody-derived molecule, antibody protein, particularly an Fv domain, Fab domain, or scFv domain). For example, the immune cell-activating component or ligand activates the immune cell through binding to CD3. For example, one specific bispecific construct / molecule may comprise the TCR or cancer-specific binding fragment / antigen-binding fragment of the present invention, e.g., the soluble TCR of the present invention, e.g., the TCR V-alpha-V-beta domain or scTv, and the polypeptide sequence of an immune cell activating component or ligand, e.g., an antibody protein binding to CD3, particularly an Fv domain or scFv domain or Fab (particularly an agonist antibody protein). The antibody protein containing the immune cell activating component is suitably an antigen-binding domain of an antibody such as VH (variable heavy chain domain from a four-chain antibody) or VHH (variable heavy chain domain from a two-chain (heavy chain only) antibody such as a camelid antibody), or scFv (i.e., a fusion protein comprising variable regions of the light and heavy chains of an antibody linked by an optionally short (e.g., 10 to 25 amino acids) linker). A number of anti-CD3 agonist antibodies are available in the prior art. For example, blinatumomab, an approved bispecific T-cell agent (BiTE) product, consists of a CD19-targeting antibody (heavy chain scFv) linked to a CD3-targeting agonist antibody (light chain scFv).The bispecific molecule or construct according to the present invention may further comprise an antibody Fc domain or a part thereof.

[0275] A linker may be provided to link a chain of a TCR or cancer-specific binding fragment / its antigen-binding fragment to another part of a bispecific construct / molecule, e.g., an immune cell activating component. More generally, a fusion protein is provided comprising any of the following: a TCR-derived molecule of the present invention or a TCR or cancer-specific / its antigen-binding fragment (or an antibody mimic thereof, i.e., an antibody-derived molecule as described herein) and a heterogeneous protein providing immune cell stimulating and / or activating activity, e.g., an antibody which may be a monoclonal antibody, or an antibody fragment or binding domain, e.g., Fab, Fab', diabody, triabody, minibody, scFv, scFv-Fc, F(ab')2, VHH, V-NAR, dab, dab-Fc, free-LC, anti-antibody, which is optionally coupled by a linker. Additionally, a polynucleotide encoding the bispecific, bispecific construct / molecule, or fusion protein is provided. The linker may include a short polypeptide of 5 to 20 amino acids containing glycine and / or serine.

[0276] In an embodiment, the antigen-binding polypeptide, which may be a bispecific composition or molecule of the present invention, is intended for use in medicine.

[0277] In an embodiment, a pharmaceutical composition comprising an antigen-binding polypeptide of the present invention is provided, which may have a pharmaceutically acceptable carrier together with a bispecific construct or molecule of the present invention. Such a composition may be a sterile composition suitable for parenteral administration. For example, refer to the disclosure of said pharmaceutical composition.

[0278] The present invention provides a method for treating a human with cancer in which cancer cells express a sequence selected from SEQ ID NOs 1 to 17 and 57 and a variant thereof and a fragment thereof, e.g., an immunogenic fragment (e.g., a sequence comprising or composed of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant thereof) or a method for preventing cancer in a human with cancer in which cancer cells express a sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and a variant thereof and a fragment thereof, e.g., an immunogenic fragment, e.g., a sequence comprising or composed of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant thereof, the method comprising the step of administering the antigen-binding polypeptide of the present invention or a composition comprising said antigen-binding polypeptide to said human.

[0279] In an embodiment, an antigen-binding polypeptide of the present invention that can be coupled to a cytotoxic moiety of the present invention for use in the treatment or prevention of human cancer, or a composition comprising said antigen-binding polypeptide of the present invention is provided, wherein the cancer cells express a corresponding sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and a fragment of any one of these as described herein (e.g., a sequence including or composed of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof).

[0280] Suitably, in any of the above embodiments, the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or EAC or ESSC.

[0281] The antigen-binding polypeptide (e.g., an antibody or a fragment thereof) may be administered in doses of, for example, 5 to 1000 mg, for example, 25 to 500 mg, for example, 100 to 300 mg, for example, about 200 mg.

[0282] Cell therapy that facilitates in vivo antigen presentation

[0283] Any various cell delivery vehicles may be used in pharmaceutical compositions to facilitate the production of antigen-specific immune responses. Accordingly, the present invention provides isolated antigen-presenting cells that are modified by loading the polypeptide of the present invention in vitro (e.g., by pulsing the polypeptide of the present invention into cells) or genetically engineered to express the polypeptide of the present invention, e.g., by transduction or transfection by the nucleic acid or vector of the present invention, so that the encoded polypeptide is expressed and presented by the cells, e.g., by complexation with MHC (hereinafter referred to herein as 'APC of the present invention'). Antigen-presenting cells (APCs), e.g., dendritic cells, macrophages, B cells, monocytes, and other cells, may be engineered to become efficient APCs. These cells may be genetically modified to increase the ability to present antigens and / or to improve the activation and / or maintenance of T cell responses and / or to be immunologically compatible with the recipient (i.e., HLA haplotype matching), but are not necessarily required to be so. APCs can generally be isolated from any various biological fluids and organs and may be autologous, homologous, common gene, or xenogeneic cells.

[0284] A specific preferred embodiment of the present invention uses dendritic cells or their progenitor cells as an APC. Accordingly, in the embodiment, the APC of the present invention is a dendritic cell. Dendritic cells are a significantly potent APC (Banchereau & Steinman, 1998, Nature,392:245-251) It has been shown to be effective as a physiological adjuvant to induce preventive or therapeutic immunity (Literature [Timmerman & Levy, 1999, Ann. Rev. Med. [Refer to 50:507-529]). Generally, dendritic cells can be identified based on their typical morphology (star-shaped in their natural position, and showing prominent cytoplasmic projections (dendritic projections) in vitro), their ability to absorb, process, and present antigens with high efficiency, and their ability to activate naive T cell responses. Dendritic cells can be engineered to express specific cell-surface receptors or ligands not typically found in dendritic cells, either in vivo or in vitro, and such modified dendritic cells are conceived by the present invention. As an alternative to dendritic cells, antigen-loading secretory vesicles (called exosomes) may be used in immunogenic compositions (refer to [Zitvogel]). et al. , 1998 Nature Med. [See 4:594-600]). Accordingly, in an embodiment, an exosome loaded with a polypeptide of the present invention is provided. An antigen-loaded secretory vesicle (exosome) can be produced by mixing an isolated exosome with a polypeptide or nucleic acid or vector of the present invention, and passing the mixture through a lipid extruder with a membrane of 100 to 400 nm or by transfecting the exosome with a vector.

[0285] Dendritic cells and progenitor cells may be obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood, or any other suitable tissue or fluid. For example, dendritic cells may be differentiated in vitro by adding a combination of cytokines, such as GM-CSF, IL-4, IL-13, and / or TNFα, to a culture of monocytes collected from peripheral blood. Alternatively, CD34-positive cells collected from peripheral blood, umbilical cord blood, or bone marrow may be differentiated into dendritic cells by adding a combination of a culture medium containing GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and / or other compound(s) that induce differentiation, maturation, and proliferation of dendritic cells.

[0286] Dendritic cells can be conveniently classified into 'immature' and 'mature' cells, which allows for a simple distinction between two well-known phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized by antigen-presenting cells (APCs) with excellent antigen uptake and processing capabilities, which is associated with high expression of Fcγ receptors and mannose receptors. The mature phenotype is typically characterized by low expression of these markers but high expression of cell surface molecules involved in T cell activation, such as class I and class II MHCs, adhesion molecules (e.g., CD54 and CD11), and co-stimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

[0287] According to the present invention, APCs may also be genetically engineered and transfected, for example, with a protein according to the present invention (or a part thereof or other variants thereof), for example, a polynucleotide encoding a polypeptide according to the present invention, for example, a polynucleotide or a vector, so that the polypeptide is complexed with, for example, MHC and expressed on the cell surface. Such transfection may occur in vitro, and subsequently, a pharmaceutical composition comprising such transfected cells may be used as described herein. Alternatively, a gene delivery vehicle targeting dendritic cells or other antigen-presenting cells may be administered to a patient to induce transfection in vivo. In vivo and in vitro transfection of dendritic cells may be, for example, by any method known in the art, e.g., that described in WO 97 / 24447, or in the literature [Mahvi et al. , 1997, Immunology and Cell BiologyAntigen loading of dendritic cells can generally be performed using the total gene approach described by [75:456-460]. Dendritic cell antigen loading involves dendritic cells or progenitor cells with polypeptides, DNA (e.g., plasmid vectors), or RNA; Alternatively, this can be achieved by incubating with antigen-expressing recombinant bacteria or viruses (e.g., adenovirus, adeno-associated virus (AAV) (e.g., AAV types 5 and 2), alphavirus (e.g., Venezuelan equine encephalitis virus (VEEV), Sindvis virus (SIN), Semliki forest virus (SFV)), herpes virus, arenavirus (e.g., lymphocytic choriomeningitis virus (LCMV)), measles virus, poxvirus (e.g., modified vaccinia ankara (MVA) or fowlpox), paramyxovirus, lentivirus, or rhabdovirus (e.g., vesicular stomatitis virus (VSV)). Prior to polypeptide loading, the polypeptide may be covalently conjugated to an immunological counterpart (e.g., a carrier molecule) that provides T cell assistance. Alternatively, dendritic cells may be pulsed to a non-conjugated immunological counterpart, either separately from or in the presence of the polypeptide or vector.

[0288] The present invention provides for delivering a specially designed short, chemically synthesized epitope-encoded polypeptide antigen fragment, for example, the polypeptide of the present invention, to an antigen-presenting cell, for example, a polypeptide according to the present invention, for example, a sequence selected from any of SEQ ID NOs 1 to 17 and 57 and variants thereof and fragments, for example, immunogenic fragments, for example, a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof, or a sequence comprising or consisting of these. Those skilled in the art will know that this type of molecule, also known as synthetic long peptide (SLP), provides a therapeutic platform as a method for stimulating (or loading) cells in vitro using the antigenic polypeptide of the present invention (Gornati et al., 2018, Front. Imm, 9:1484) or introducing polypeptide antigens into antigen-presenting cells in vivo (Melief & van der Burg, 2008, Nat Rev Cancer, 8:351-60).

[0289] In an embodiment, a pharmaceutical composition is provided comprising the antigen-presenting cells of the present invention, suitably dendritic cells, together with a pharmaceutically acceptable carrier. Such a composition may be a sterile composition suitable for parenteral administration. For example, refer to the disclosure of said pharmaceutical composition.

[0290] In an embodiment, an antigen-presenting cell of the present invention for use in medicine, suitably a dendritic cell, is provided.

[0291] Additionally, a method for treating a human with cancer in which cancer cells express a sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and a variant thereof and a fragment thereof, e.g., an immunogenic fragment, e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant thereof, or a sequence consisting thereof is provided, or a method for preventing cancer in a human with cancer in which cancer cells express a sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and a variant thereof and a fragment thereof, e.g., an immunogenic fragment (e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant thereof) or a sequence consisting thereof is provided, the method comprising the step of administering to the human the antigen-presenting cell of the present invention, suitably a dendritic cell, or a composition comprising the antigen-presenting cell of the present invention. In an embodiment, an antigen-presenting cell of the present invention, suitably a dendritic cell, or a composition comprising said antigen-presenting cell of the present invention is provided for use in treating or preventing human cancer, wherein the cancer cell expresses a corresponding sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and variants and fragments thereof, e.g., an immunogenic fragment, e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, or a sequence composed of these.

[0292] In an embodiment, a pharmaceutical composition is provided comprising the exosomes of the present invention together with a pharmaceutically acceptable carrier. Such a composition may be a sterile composition suitable for parenteral administration. For example, refer to the disclosure of said pharmaceutical composition. The composition may optionally comprise an immunostimulator—refer to the disclosure of said immunostimulator.

[0293] In an embodiment, an exosome comprising a polypeptide, nucleic acid, or vector according to the present invention is provided.

[0294] In an embodiment, an exosome of the present invention for use in medicine is provided.

[0295] Additionally, a method for treating a human with cancer in which cancer cells express a sequence selected from any one of SEQ ID NOs 1 to 17 and 57, a variant thereof and a fragment thereof, for example, an immunogenic fragment as described in this specification (for example, a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant thereof) or a sequence consisting thereof is provided, or a method for preventing cancer in a human with cancer in which cancer cells express a sequence selected from any one of SEQ ID NOs 1 to 17 and 57, a variant thereof and a fragment thereof, for example, an immunogenic fragment as described in this specification (for example, a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant thereof) or a sequence consisting thereof is provided, and the method comprises the step of administering the exosome of the present invention or a composition comprising the exosome of the present invention to the human.

[0296] In an embodiment, an exosome of the present invention or a composition comprising said exosome of the present invention is provided for use in the treatment or prevention of human cancer, wherein the cancer cells express a corresponding sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and a fragment of any one of these (e.g., a sequence including or composed of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof).

[0297] In any of the above embodiments, suitably the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or EAC or ESSC.

[0298] Stimulated T-cell therapy

[0299] In addition to the in vivo or in vitro APC-mediated production of immune-specific T-cells to the polypeptide of the present invention, autologous or non-autologous T-cells may be isolated from a subject, for example, from peripheral blood, umbilical cord blood, and / or by apheresis, and stimulated in the presence of a tumor-associated antigen loaded on the MHC molecule of the APC cells (signal 1), for example as described herein, to induce proliferation of T-cells having a TCR immune-specific to this antigen (optionally providing a co-stimulation signal in the presence of an anti-CD3 antibody and an anti-CD28 antibody).

[0300] Successful T-cell activation requires the co-stimulatory surface molecules B7 and CD28 on antigen-presenting cells and T cells, respectively (Signal 2). Optimal T-cell activation requires both Signal 1 and Signal 2. Conversely, antigenic peptide stimulation (Signal 1) cannot induce full T-cell activation in the absence of co-stimulation (Signal 2) and may result in T-cell tolerance. In addition to co-stimulatory molecules, inhibitory molecules such as CTLA-4 and PD-1, which induce signals that prevent T-cell activation, are also present.

[0301] Accordingly, autologous or non-autologous T-cells can be stimulated in the presence of the polypeptide or nucleic acid of the present invention, and this is extended and delivered to a patient at risk of developing cancer or who is suffering from cancer, wherein the cancer cells express the corresponding polypeptide of the present invention, provided that the antigen-specific TCR recognizes the antigen presented by the patient's MHC, targets the cancer cells expressing the corresponding polypeptide, and induces apoptosis.

[0302] In an embodiment, a polypeptide, nucleic acid, vector, or composition of the present invention is provided for stimulating and / or amplifying T-cells derived from a human having cancer in vitro, and subsequently reintroducing said stimulated and / or amplified T-cells into said human to use in treating said cancer in said human.

[0303] The present invention provides a method for treating cancer in humans, wherein the cancer cells express a sequence selected from any one of SEQ ID NOs. 1 to 17 and 57 and variants and fragments thereof, e.g., an immunogenic fragment, e.g., a sequence selected from any one of SEQ ID NOs. 18 to 56 and 58 and variant sequences thereof, and the method comprises the steps of: collecting a population of leukocytes from said human that selectively includes at least T-cells together with antigen-presenting cells; stimulating and / or amplifying said T-cells in the presence of a corresponding polypeptide, nucleic acid, vector, or composition of the present invention; and reintroducing some or all of said leukocytes, including at least the stimulated and / or amplified T-cells, into said human.

[0304] In any of the above embodiments, suitably the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or EAC or ESSC.

[0305] In an embodiment, a method for producing a T-cell population cytotoxic to cancer cells expressing a sequence selected from any one of SEQ ID NOs. 1 to 17 and 57 and a variant thereof and a fragment thereof, e.g., an immunogenic fragment, e.g., a sequence selected from any one of SEQ ID NOs. 18 to 56 and 58 and a variant thereof, or a sequence consisting thereof is provided, the method comprising the steps of (a) obtaining T-cells and antigen-presenting cells from a cancer patient and (ii) stimulating and amplifying the T-cell population with a corresponding polypeptide, nucleic acid, vector, or composition of the present invention.

[0306] In this regard, 'corresponding' means that when cancer cells express, for example, any of SEQ ID NOs 1 to 17 and 57 or variants or fragments thereof, for example, immunogenic variants or fragments thereof as described herein (for example, sequences comprising or consisting of sequences selected from any one of SEQ ID NOs 18 to 56 and 58 and variant sequences thereof), a T-cell population is stimulated and amplified in vitro by any of SEQ ID NOs 1 to 17 and 57 or variants or fragments thereof (optionally identical) as described herein (for example, sequences comprising or consisting of sequences selected from any one of SEQ ID NOs 18 to 56 and 58 and variant sequences thereof) in the form of a polypeptide, nucleic acid or vector, or a composition comprising one of the aforementioned.

[0307] For example, in this process, the steps of culture and expansion or stimulation and amplification may be performed in the presence of dendritic cells, e.g., APCs of the present invention. Dendritic cells may be transfected with nucleic acid molecules or vectors of the present invention and may express polypeptides of the present invention or be pulsed with polypeptides as described herein.

[0308] The present invention provides a T-cell population (hereinafter referred to herein as the T-cell population of the present invention) that can be obtained by any of the aforementioned processes.

[0309] In an embodiment, a cell is provided that is a T-cell (hereinafter referred to herein as the T-cell of the present invention) stimulated by the polypeptide, nucleic acid, vector, or composition of the present invention. Preferably, the T-cell is immunoreactive to the polypeptide, nucleic acid, vector, or composition of the present invention, preferably to the polypeptide of the present invention, and, for example, the T-cell may bind to exhibit an 'immune response,' e.g., induction of T-cell proliferation and / or production of cytokines, e.g., IFN-gamma, IL-2, TNF-alpha and / or production of, e.g., granzyme and / or induction of tumor cell death or apoptosis.

[0310] In an embodiment, a pharmaceutical composition comprising a population of T-cells of the present invention or T-cells together with a pharmaceutically acceptable carrier is provided. Such a composition may be, for example, a sterile composition suitable for parenteral administration.

[0311] In an embodiment, a T-cell population or T-cells of the present invention for use in medicine are provided.

[0312] Additionally, a method for treating a human with cancer in which cancer cells include a sequence selected from any one of SEQ ID NOs 1 to 17 and 57, a variant thereof and a fragment thereof, for example, an immunogenic fragment as described in this specification (for example, a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant thereof) or express a sequence consisting thereof, or a method for preventing cancer in a human with cancer in which cancer cells include a sequence selected from any one of SEQ ID NOs 1 to 17 and 57, a variant thereof and a fragment thereof, for example, an immunogenic fragment as described in this specification (for example, a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant thereof) or express a sequence consisting thereof is provided, and the method comprises the step of administering to the human the T-cell population or T-cell of the present invention, or a composition comprising the T-cell population or T-cell of the present invention.

[0313] In an embodiment, a T-cell population of the present invention, a T-cell of the present invention, or a composition comprising said T-cell population or T-cell of the present invention is provided for use in treating or preventing human cancer, wherein the cancer cells express a corresponding sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and variants and fragments thereof, e.g., an immunogenic fragment as described in this specification (e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof) or a sequence composed of these.

[0314] In any of the above embodiments, suitably the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or EAC or ESSC.

[0315] The present invention further provides a method for producing immunoreactive cells or a population of immunoreactive cells that bind to or induce an immune response from a polypeptide sequence according to the present invention, for example, a polypeptide sequence selected from any one of SEQ ID NOs. 1 to 17 and 57 and variants and fragments thereof, for example, immunogenic fragments thereof, for example, a sequence selected from any one of SEQ ID NOs. 18 to 56 and 58 or a variant thereof or an immunogenic variant thereof, or a polypeptide sequence composed thereof, wherein the method

[0316] (a) A step of obtaining immunoreactive cells or a population of immunoreactive cells,

[0317] (b) polypeptide sequence and In the contact stage A step of selecting immunoreactive cells or a population of immunoreactive cells,

[0318] (c) a step of identifying an immunoreactive cell or a population of immunoreactive cells that bind to a polypeptide sequence, and optionally

[0319] (d) a step of isolating identified combined immunoreactive cells or a population of immunoreactive cells

[0320] Includes

[0321] According to one embodiment, a variant or immunogenic variant of any of SEQ ID NOs 1 to 17 and 57 or SEQ ID NOs 18 to 56 and 58 has 1, 2 or 3 amino acids selected from additions, substitutions, and deletions related thereto.

[0322] Accordingly, the present invention provides isolated immunoreactive cells or a population of immunoreactive cells produced according to the aforementioned process of the present invention, which bind to a polypeptide sequence according to the present invention. Additionally, optionally, the immunoreactive cells or a population of immunoreactive cells that bind to the polypeptide sequence of the present invention may be T-cells or NKT cells, and / or the process may also include the step of extracting or isolating nucleic acids encoding a T-cell receptor that binds to the polypeptide, and additionally optionally involve the step of transducing additional immunoreactive cells or a population of immunoreactive cells to express a T-cell receptor. According to the present embodiment, the present invention provides modified immunoreactive cells or a population of immunoreactive cells that express a T-cell receptor that binds to a polypeptide sequence according to the present invention. Alternatively, optionally, the immunoreactive cell or population of immunoreactive cells binding to the polypeptide sequence of the present invention may be a T-cell or NKT cell, and / or the process may also include the step of extracting or isolating a nucleic acid encoding a T-cell receptor or an antigen-binding fragment thereof binding to the polypeptide of the present invention, and additionally optionally include the step of transducing a host cell to express said TCR or its fragment, wherein preferably, said TCR or fragment is secreted as a soluble TCR as described herein.

[0323] The present invention further provides identified combined immunoreactive cells or a population of immunoreactive cells or modified immunoreactive cells or a population of immunoreactive cells for use in treating cancer. In a preferred embodiment, the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or EAC or ESSC.

[0324] Immunoreactive cells or cells may be cells of the lymphatic system, e.g., peripheral blood mononuclear cells (PBMCs), leukocytes, or lymphocytes, and include B, T, or natural killer (NK) cells. Immunoreactive cells may be cells of the lymphatic system, including T cells, natural killer T (NKT) cells, embryonic stem cells, and their precursors, including pluripotent stem cells (e.g., cells that can differentiate from lymphocytes). T cells may include, but are not limited to, helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated constant T cells, and gamma-delta T cells. Preferably, immunooreactive cells are T cells, optionally CD4 + T cells or CD8 + It is a T cell. Therefore, the immunoreactive cell may be a T-cell, selectively a CD4+ T cell or a CD8+ T cell, or the immunoreactive cell may be a T-cell, selectively a population of CD4+ T cells; or a CD8+ T cell, or a mixed population of CD4+ T cells and CD8+ T cells.

[0325] Binding can be determined by known methods such as equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE™ assay). Binding to the polypeptide of the present invention may be binding to the polypeptide presented as a complex with an antigen-presenting molecule, such as HLA, presented by an antigen-presenting cell, for example, binding to the polypeptide complexed with an MHC tetramer or dextramer, for example, chip or plate-bound tetramer or dextramer, or optionally binding to a free-labeled polypeptide.

[0326] Engineered Immunotherapy

[0327] Derivatives of all types of CST antigen-binding polypeptides described above, including antibody-derived molecules, antibodies, TCR-derived molecules, TCRs, or TCR mimics that recognize CST antigen-derived peptides complexed with human HLA molecules (Reference [Dubrovsky] et al [See ., 2016, Oncoimmunology]) can be engineered to be expressed on the surface of immunoreactive cells, e.g., T cells (autologous or non-autologous), and then administered as adoptive cell / T cell therapy to treat cancer.

[0328] These derivatives include, for example, 'Chimeric Antigen Receptors (CARs)' as used herein, and may refer to artificial T-cell receptors, chimeric T-cell receptors, or chimeric immune receptors, and encompass, for example, engineered receptors that conjugate artificial specificity to specific immune effector cells. CARs may be used to generate a number of specific T cells for use, for example in adoptive cell therapy, by conferring specificity of a monoclonal antibody onto T cells. CARs may induce cell specificity to tumor-associated antigens, specifically the polypeptide of the present invention, wherein, preferably, the polypeptide is HLA-conjugated.

[0329] Another approach to treating cancer in patients is to genetically modify T-cells to target antigens expressed on tumor cells through the expression of chimeric antigen receptors (CARs). This technique is reviewed in the literature [Wendell & June, 2017, Cell, 168: 724-740 (the full text is cited by reference)].

[0330] Another approach to treating cancer in patients is to genetically modify T cells to target antigens expressed on tumor cells through the expression of a recombinant T-cell receptor (TCR) that targets HLA-binding target antigens expressed on tumor cells, for example, the polypeptide of the present invention, wherein the polypeptide is HLA-binding.

[0331] These T-cells (including CAR T-cells) may be produced by a method of obtaining a sample of cells from a subject, for example, from peripheral blood, umbilical cord blood and / or by apheresis (said that the sample comprises T-cells or T-cell progenitor cells) and by transfecting said cells with a nucleic acid encoding an immune-specific T-cell receptor (e.g., recombinant TCR or CAR) to the polypeptide of the present invention (the polypeptide is HLA-bound). Such nucleic acid may be incorporated into the genome of the cells, and the cells may be administered to the subject in an effective amount to provide a T-cell response to the cells expressing the polypeptide of the present invention. For example, a sample from the subject may be collected, for example, in a separate step.

[0332] It is understood that the cells used to produce the above-mentioned recombinant TCR- or CAR-expressing T-cells may be autologous or non-autologous.

[0333] Transplanted CAR-expressing or TCR-expressing T cells may have the expression of endogenous T-cell receptors and / or endogenous HLA inactivated. For example, cells may be processed to remove the expression of endogenous alpha / beta T-cell receptors (TCRs).

[0334] Although methods for transfecting cells are widely known in the art, highly efficient transfection methods such as electroporation can be used. For example, the nucleic acid or vector of the present invention expressing a TCR or CAR construct can be introduced into a cell using a nucleofection device.

[0335] A cell population for recombinant TCR- or CAR-expressing T-cells can be enriched after cell transfection. For example, cells expressing recombinant TCR or CAR can be distinguished from non-expressing cells (e.g., via FACS) by the use of antigens bound to the recombinant TCR or CAR or by the use of TCR or CAR-binding antibodies. Alternatively, the enrichment step involves the depletion of non-T-cells or cells lacking recombinant TCR or CAR expression. For example, CD56+ cells can be depleted from the culture population.

[0336] A population of genetically modified, recombinant TCR- or CAR-expressing cells can be cultured in vitro in a medium that selectively enhances the proliferation of CAR-expressing T-cells. Thus, recombinant TCR- or CAR-expressing T-cells can be expanded in vitro in the presence, for example, of CD3 and / or CD28 agonists, for example, agonist antibodies.

[0337] Samples of recombinant TCR- or CAR-expressing T-cells can be preserved (or maintained in a culture). For example, samples can be cryopreserved for subsequent enlargement or analysis.

[0338] Recombinant TCR- or CAR-expressing T cells can be used in combination with other therapeutic agents, for example, checkpoint inhibitors including PD-L1 antagonists, for example, anti-PD1 antibodies or anti-PDL1 antibodies.

[0339] In an embodiment, a T-cell or a population of T-cells engineered to express any of the above antigen-binding polypeptides (e.g., recombinant TCR or CAR) on its surface is provided. Suitably, the T-cell is a cytotoxic T-cell.

[0340] In an embodiment, a T-cell or a group of T-cells engineered to express any of the above antigen-binding polypeptides (e.g., recombinant TCR or CAR) for use in medicine is provided.

[0341] The present invention provides a pharmaceutical composition comprising a T-cell or a population of T-cells of the present invention (e.g., any of the antigen-binding polypeptides above, engineered to express a recombinant TCR or CAR on the surface).

[0342] T cells can be, for example, helper (CD4+) or cytotoxic (CD8+) T cells. The T cell population can be a mixed population of CD4+ and CD8+ T cells. Suitablely, the T cells are cytotoxic T cells.

[0343] Additionally, a method for treating a human patient with cancer in which cancer cells include a sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and variants and fragments thereof, e.g., an immunogenic fragment (e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof) or express a sequence consisting thereof is provided, or a method for preventing cancer in a human patient with cancer in which cancer includes a sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and variants and fragments thereof, e.g., an immunogenic fragment as described in this specification (e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof) or expresses a sequence consisting thereof is provided, and the method comprises the step of administering the T-cells or a population of T-cells of the present invention to said human.

[0344] In an embodiment, the T-cell or population of T-cells of the present invention is intended for use in treating or preventing human cancer, wherein the cancer cells express a sequence comprising a corresponding sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and a variant and fragment thereof, e.g., an immunogenic fragment, e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof, or a sequence composed of these.

[0345] Combination therapy

[0346] The cancer treatment method according to the present invention can be performed in combination with other therapies, in particular, with a checkpoint inhibitor and interferon.

[0347] According to the present invention, polypeptides, nucleic acids, vectors, exosomes, antigen-binding polypeptides, and adoptive cell therapies (APC and T cell-based) may be used in combination with other components designed to enhance their immunogenicity, for example, to improve the scale and / or scope of the induced immune response or to provide other activities (e.g., activation of other modes of innate or adaptive immune response, or destruction of tumor cells).

[0348] Accordingly, the present invention provides a kit of several such compositions comprising a composition of the present invention (i.e., an immunogenic, vaccine, or pharmaceutical composition) or a polypeptide, nucleic acid, or vector of the present invention together with a pharmaceutically acceptable carrier; and (i) one or more additional immunogenic or immunostimulatory polypeptides (e.g., interferon, IL-12, a checkpoint blocking molecule or a nucleic acid encoding the same, or a vector comprising such nucleic acid) or (ii) a small molecule (e.g., an HDAC inhibitor or another drug that modifies the epigenetic profile of cancer cells) or a bioagent (delivered as a polypeptide or a nucleic acid encoding the same, or a vector comprising such nucleic acid) that enhances the translation and / or presentation of the polypeptide product of the present invention.

[0349] Checkpoint inhibitors that block normal proteins on cancer cells or proteins on T cells that respond to them can be a particularly important class of drugs for combination with CST-antigen-based therapies, because these inhibitors attempt to overcome one of the cancer's major defenses against immune system attacks.

[0350] Accordingly, an aspect of the present invention comprises the step of administering the polypeptide, nucleic acid, vector, exosome, antigen-binding polypeptide, composition, immunoreactive cell, immunoreactive cell population, T-cell, T-cell population, or antigen-presenting cell of the present invention in combination with a checkpoint inhibitor. Exemplary checkpoint inhibitors are selected from PD-1 inhibitors, e.g., pembrolizumab (Keytruda) and nivolumab (Opdivo), PD-L1 inhibitors, e.g., atezolizumab (Tecentriq), avelumab (Bavencio) and durvalumab (Imfinzi), and CTLA-4 inhibitors, e.g., ipilimumab (Yervoy).

[0351] Interferons (e.g., alpha, beta, and gamma) are a family of proteins that the body produces in very small amounts. Interferons can slow or stop cancer cell division and / or reduce the ability of cancer cells to protect themselves from the immune system and / or enhance various aspects of the adaptive immune system. Interferons are typically administered by subcutaneous injection, for example, in the thigh or abdomen.

[0352] Accordingly, an aspect of the present invention comprises the step of administering a polypeptide, nucleic acid, vector, antigen-binding polypeptide vector, exosome, antigen-binding polypeptide, composition, immunoreactive cell, immunoreactive cell population, T-cell, T-cell population, or antigen-presenting cell, or a composition of the present invention in combination with an interferon selected from, for example, one or more of interferon alpha, gamma, beta, or lambda and / or interleukin (for example, selected from one or more of IL1, 2, 4, 6, 7, 15, 17, and 23).

[0353] Different modes of the present invention may also be combined, for example, polypeptides, nucleic acids, vectors, and antigen-binding polypeptides of the present invention may be combined with APCs, immunoreactive cells, immunoreactive cell populations, T-cells, or T-cell populations of the present invention (discussed below).

[0354] One or more modes of the present invention may also be used in combination with conventional anticancer chemotherapy and radiation. The present invention further provides the polypeptide, nucleic acid, vector, exosome, antigen-binding polypeptide, composition, immunoreactive cell, immunoreactive cell population, T-cell, T-cell population, or antigen-presenting cell of the present invention in combination with one or more additional therapeutic agents. Preferably, additional therapeutic agents are designed to enhance their immunogenicity or improve the scale and / or extent of the induced immune response, and are selected from one or more of, for example, anticancer antibodies, small molecule anticancer therapeutic agents, checkpoint inhibitors, interleukins, or interferons; alternatively, additional therapeutic agents may be selected from one or more polypeptides, fusion proteins, nucleic acids, vectors, exosomes, antigen-binding polypeptides, compositions, immunoreactive cells, immunoreactive cell populations, T-cells, T-cell populations, or antigen-presenting cells and / or other antigenic polypeptides (or polynucleotides or vectors encoding the same), preferably, cancer, preferably ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cells It causes an immune response to any of the carcinomas (ESSC), particularly esophageal cancer, or EAC or ESSC.

[0355] Accordingly, the present invention provides a polypeptide, nucleic acid, vector, exosome, antigen-binding polypeptide, composition, immunoreactive cell, immunoreactive cell population, T-cell, T-cell population, or antigen-presenting cell of the present invention for use in causing an immune response against cancer or tumor or for the treatment of cancer or tumor, wherein the polypeptide, nucleic acid, vector, exosome, antigen-binding polypeptide, composition, immunoreactive cell, immunoreactive cell population, T-cell, T-cell population, or antigen-presenting cell is intended for use in combination with one or more additional therapeutic agents, or is intended for use or administration, or is optionally intended for separate, sequential, or simultaneous administration. According to the present invention, the cancer or tumor may be any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or EAC or ESSC.

[0356] diagnosis

[0357] In another aspect, the present invention provides a method for diagnosing cancer using one or more of the polypeptides or nucleic acids of the present invention, or diagnosing a human subject suitable for treatment by the polypeptide, nucleic acid, vector, antigen-binding polypeptide, adoptive cell therapy, exosome, or composition of the present invention.

[0358] Accordingly, the present invention provides a method for diagnosing that a human has cancer, comprising the steps of determining whether the cancer cells express a polypeptide sequence and variant and fragment selected from any one of SEQ ID NOs 1 to 17 and 57, e.g., an immunogenic fragment thereof, e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof, or a polypeptide sequence selected from a sequence composed thereof; or a nucleic acid encoding said polypeptide sequence; and diagnosing that the human has cancer if said polypeptide or the corresponding nucleic acid or the nucleic acid encoding said polypeptide is overexpressed in said cancer cells.

[0359] The present invention provides a method for diagnosing whether a human has any of the following cancers, particularly esophageal cancer, or cancers that are EAC or ESSC: for example, ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC); wherein the cells of said cancer comprise a polypeptide sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and variants and fragments thereof, for example, immunogenic fragments, for example, a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, or a sequence composed thereof; or a step of determining whether to express a nucleic acid encoding the polypeptide sequence, and a step of diagnosing the human having any of, for example, ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or cancer that is EAC or ESSC.

[0360] As used herein, 'overexpressed' in cancer cells means that the expression level in cancer cells is higher than in normal cells. Overexpression can be determined by reference to the levels of the nucleic acid or polypeptide of the present invention in control human subjects known not to have cancer. Thus, overexpression indicates that the nucleic acid or polypeptide of the present invention is detected at a significantly higher level in the test subject than in the control subject (e.g., any of 5 to 500%, 10 to 250%, 20 to 100%, 30 to 50%, 30%, 50%, 100%, or 500% or more). If the control human subject has an undetectable level of the nucleic acid or polypeptide of the present invention, diagnosis can be achieved by detecting the nucleic acid or polypeptide of the present invention.

[0361] The present invention also provides a method for treating a human being suffering from cancer, which is

[0362] (a) determining whether the cells of the cancer selectively express a polypeptide sequence selected from any of SEQ ID NOs 1 to 17 and 57 and variants and fragments thereof, such as an immunogenic fragment (e.g., a sequence comprising or consisting of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof) or a nucleic acid encoding said polypeptide in a biological sample obtained from said human; if so, (b) administering the corresponding polypeptide, nucleic acid, vector, composition, immunoreactive cell, immunoreactive cell population, T-cell population, T-cell, antigen-presenting cell, or antigen-binding polypeptide or combination of said human to said human.

[0363] also,

[0364] (a) any sequence of SEQ ID NOs 1 to 17 and 57;

[0365] (b) Variants of the sequence of (a); and

[0366] The use of a polypeptide comprising sequences (a) and (b) isolated from a tumor of a human suffering from cancer, a fragment of (e.g., a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof or a sequence composed thereof), such as a sequence selected from an immunogenic fragment, or the use of a nucleic acid encoding said polypeptide as a biomarker to determine whether said human is suitable for treatment by the corresponding polypeptide, nucleic acid, vector, composition, immunoreactive cell, immunoreactive cell population, T-cell population, T-cell, antigen-presenting cell, or vaccine comprising an antigen-binding polypeptide or combination of said polypeptide is provided.

[0367] Suitably, the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or EAC or ESSC.

[0368] Suitably, the polypeptide of the present invention has a sequence selected from any one of SEQ ID NOs 1 to 17 and 57 or a fragment thereof, e.g., an immunogenic fragment (e.g., a sequence including or composed of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof).

[0369] Suitably, the nucleic acid of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs 1 to 17 and 57 and variants thereof and fragments thereof, for example, a sequence encoding an immunogenic fragment, for example, a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, or has a sequence composed of these or comprises the same.

[0370] Kits for detecting the presence of nucleic acids are well known. For example, a kit containing at least two oligonucleotides that hybridize to a polynucleotide can be used in a real-time PCR (RT-PCR) reaction to enable the detection and semi-quantification of specific nucleic acids. Such kits enable the detection of PCR products by the generation of a fluorescent signal as a result of Forster Resonance Energy Transfer (FRET) (e.g., TaqMan® kit) or upon binding of double-stranded DNA (e.g., SYBR® Green kit). Some kits (e.g., those containing a TaqMan® probe spanning the exons of target DNA) enable the detection and quantification of mRNA, e.g., transcripts encoding the nucleic acids of the present invention. The analysis using a specific kit can be configured in a multiplex format to detect multiple nucleic acids simultaneously within a single reaction. Kits for detecting active DNA (i.e., DNA possessing specific epigenetic features indicating expression) may also be used. Additional components that may be included in such kits include diagnostic reagents or reporters that facilitate nucleic acid detection of the present invention.

[0371] The nucleic acid of the present invention can also be detected through a liquid biopsy using a patient's blood sample. This procedure provides a non-invasive alternative to surgical biopsy. Plasma from such blood samples can be isolated and analyzed for the presence of the nucleic acid of the present invention.

[0372] The polypeptide of the present invention can be detected by an antigen-specific antibody in an ELISA type, thereby detecting the polypeptide of the present invention in a homogenized preparation of a patient tumor sample. Alternatively, the polypeptide of the present invention can be detected by immunohistochemical analysis, which identifies the presence of the polypeptide antigen by using an optical microscope to examine sections of a patient tumor sample stained with an appropriately labeled antibody preparation. As a further alternative, the polypeptide of the present invention can be detected by immunohistochemical analysis, which identifies the presence of the polypeptide antigen by using an optical microscope to examine sections of a patient tumor sample stained with an appropriately labeled antibody preparation.

[0373] The polypeptides of the present invention can also be detected by determining whether they can stimulate T-cells generated for said polypeptide.

[0374] The present invention provides a method for treating any of cancers, e.g., ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC, in humans, comprising: (i) selectively detecting the presence of a nucleic acid or polypeptide according to the present invention in a biological sample obtained from said human; and (ii) administering a nucleic acid, polypeptide, vector, cell, antigen-binding polypeptide, immunoreactive cell, immunoreactive cell population, T-cell or T-cell population or composition or combination according to the present invention to a subject (preferably administering the same nucleic acid or polypeptide or a fragment thereof detected).

[0375] The present invention provides a method for treating any of cancers, e.g., ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC, wherein the method comprises the step of administering a nucleic acid, polypeptide, vector, antigen-binding polypeptide, cell, T-cell or T-cell population or composition or combination according to the present invention to a subject, wherein the presence of the nucleic acid or polypeptide according to the present invention (preferably the same) is selectively detected in a biological sample obtained from said human.

[0376] In particular, any of the cancers that are diagnosed and appropriately treated are ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC).

[0377] When a polypeptide of the present invention or a fragment thereof of any of SEQ ID NOs 1 to 17 and 57, for example, a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof, or a sequence composed thereof is detected, the cancer may be any of cancer, for example, ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or EAC or ESSC.

[0378] Specific embodiments

[0379] In an embodiment, the CST antigen polypeptide comprises or consists of any of SEQ ID NOs 1 to 17 and 57, variants thereof, and fragments thereof, such as immunogenic fragments. An exemplary fragment comprises or consists of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, or a sequence composed thereof. An exemplary nucleic acid comprises encoding a sequence selected from any one of SEQ ID NOs 1 to 17 and 57, variants thereof, and fragments thereof, such as immunogenic fragments, such as a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, or a sequence composed thereof. Corresponding nucleic acids (e.g., DNA or RNA) as described above, T-cells, T-cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells, and exosomes are provided. The above nucleic acids (e.g., DNA or RNA), T-cells, T-cell populations, antigen-binding polypeptides, antigen-presenting cells, and exosomes may be used in the treatment of any of cancers, particularly ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC. Related diagnostic methods are also provided.

[0380] Further embodiments of the present invention are defined by the following provisions:

[0381] 1. As an isolated polypeptide,

[0382] (a) any one of SEQ ID NOs 1 to 17 and 57, and

[0383] (b) Variants of the sequence of (a); and

[0384] (c) A fragment of the sequence of (a) or (b)

[0385] An isolated polypeptide comprising a sequence selected from any one of the following, wherein optionally the fragment and / or variant is an immunogenic fragment and / or immunogenic variant.

[0386] 2. An isolated polypeptide according to Clause 1, comprising or composed of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof, wherein, when optionally bound to MHC, the variant sequence optionally is immunogenic and / or retains the ability to bind to MHC molecule(s) and / or induces T cells that cross-react with said polypeptide and / or variant.

[0387] 3. In Clause 1 or 2, the polypeptide has the ability to bind to one of an MHC molecule, preferably an MHC class 1 or class 2 molecule, and / or the polypeptide is an isolated polypeptide that, when bound to the MHC, is immunogenic and / or can be recognized by CD4 and / or CD8 T cells (binding to them).

[0388] 4. In any one of provisions 1 to 3, the polypeptide or fragment or variant is an isolated polypeptide having a total length of 7 to 30 amino acids, optionally any of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, or 8 to 20 adjacent amino acids.

[0389] 5. In any one of clauses 1 to 4, the polypeptide is an isolated polypeptide comprising non-peptide bonds.

[0390] 6. In any one of Clauses 1 to 5, the polypeptide is an isolated polypeptide that is part of a fusion protein.

[0391] 7. In Clause 6, the polypeptide is an isolated polypeptide fused to a second or additional polypeptide selected from (i) one or more other polypeptides according to Clauses 1 to 5, (ii) one or more other polypeptides that are cancer-associated antigens, optionally esophageal cancer-associated antigens, (iii) one or more polypeptide sequences capable of enhancing an immune response (i.e., an immunostimulator sequence), and (iv) one or more polypeptide sequences comprising, for example, a universal CD4 helper epitope capable of providing strong CD4+ assistance to increase a CD8+ T-cell response to an antigen epitope.

[0392] 8. An isolated polypeptide comprising any of two or more sequences selected from sequences selected from polypeptides according to Clauses 1 to 5, preferably 2, 3, 4 or 5, 6, 7, 8, 9, 10, 11 or 12, wherein, optionally, all polypeptide sequences are different.

[0393] 9. Isolated nucleic acid encoding a polypeptide according to any one of Clauses 1 through 8.

[0394] 10. In Clause 9, the nucleic acid, which is DNA.

[0395] 11. In Clause 9 or 10, a codon-optimized nucleic acid for expression in human host cells.

[0396] 12. In Clause 9, nucleic acid, which is RNA.

[0397] 13. In any one of Clauses 9 to 12, a nucleic acid that is an artificial nucleic acid sequence.

[0398] 14. A vector comprising a nucleic acid according to any one of clauses 9 to 13.

[0399] 15. A vector comprising a DNA-coding regulatory element suitable for allowing transcription of a translationally active RNA molecule in a human host cell, in accordance with Clause 14.

[0400] 16. A vector that is a virus vector in Clause 14 or Clause 15.

[0401] 17. In Clause 16, vectors that are adenovirus vectors, adeno-associated virus (AAV), alphavirus, herpes virus, arenavirus, measles virus, poxvirus, paramyxovirus, lentivirus and rhabdovirus vectors.

[0402] 18. A pharmaceutical composition comprising a polypeptide, nucleic acid, or vector according to any one of provisions 1 to 17 together with a pharmaceutically acceptable carrier, wherein, preferably, the pharmaceutical composition is an immunogenic pharmaceutical composition.

[0403] 19. A vaccine composition comprising a polypeptide, nucleic acid, or vector according to any one of provisions 1 to 17 together with a pharmaceutically acceptable carrier.

[0404] 20. A composition comprising one or more immunostimulators according to Clause 18 or Clause 19.

[0405] 21. In Clause 20, the above one or more immunostimulators are aluminum salts, saponins, immunostimulating oligonucleotides, oil-in-water emulsions, aminoalkyl glucosamine 4-phosphate, lipopolysaccharides and derivatives thereof and other TLR4 ligands, TLR7 ligands, TLR8 ligands and TLR9 ligands, IL12, interferon, incomplete Freund adjuvant (IFA), aluminum-based adjuvants, TLR agonists, synthetic double-stranded RNA (dsRNA), glucopyranosyl lipid α (GLA), imidazoquinoline, CPG oligodeoxynucleotides (ODN), cyclic dinucleotides (CDN), manganese-based adjuvants; A composition selected from metabolic adjuvants, nanoparticle-based adjuvants, water-in-oil nanoemulsions, micro or nanoparticle adjuvants, CGAS-STING / STING agonists, cytokines, self-assembling peptides, virus-like particles (VLPs), inorganic nanoparticles, cage protein nanoparticles, nucleoside-unmodified mRNA, PRR ligands (pattern recognition receptor ligands), mRNA-coding functional proteins, component lipid-based adjuvants, peptides and glycolipid immunostimulators, and TLR agonists.

[0406] 22. A composition that is a sterile composition suitable for parenteral administration, in any one of clauses 18 to 21.

[0407] 23. A polypeptide, nucleic acid, vector, or composition according to any one of provisions 1 to 22 for use in medicine.

[0408] 24. A method for inducing an immune response in a human, comprising the step of administering a polypeptide, nucleic acid, vector, or composition according to any one of Clauses 1 to 22 to the human.

[0409] 25. The method of Clause 24, wherein the immune response is induced in a cancerous tumor expressing or presenting on the surface thereof a sequence selected from SEQ ID NOs. 1 to 17 and 57 and any one of the variants thereof and an immunogenic fragment, or (b) SEQ ID NOs. 18 to 56 and 58; and the variant sequences thereof, wherein, when selectively bound to MHC, the variant sequence selectively induces T cells that are immunogenic and / or retain the ability to bind to MHC molecule(s) and / or cross-react with said polypeptide and / or variant.

[0410] 26. A polypeptide, nucleic acid, vector, or composition according to any one of provisions 1 to 22 for use in inducing a human immune response.

[0411] 27. A polypeptide, nucleic acid, vector, or composition according to Clause 26, wherein the immune response is induced in a cancerous tumor expressing (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments or variants thereof, or (b) a corresponding sequence selected from SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, wherein, when selectively bound to MHC, the variant sequence selectively induces T cells that are immunogenic and / or retain the ability to bind to MHC molecule(s) and / or cross-react with said polypeptide and / or variant.

[0412] 28. A method for treating a human patient with cancer, wherein cancer cells express a sequence selected from (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and optionally, said variant sequences retain the ability to bind to MHC molecule(s) and / or induce T cells that cross-react with said variants, or a method for preventing cancer in a human patient with cancer, wherein cancer expresses (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and optionally, when bound to MHC, said variant sequences are immunogenic and / or retain the ability to bind to MHC molecule(s) and / or induce T cells that cross-react with said polypeptide and / or variants, wherein the method comprises provisions A method comprising the step of administering a corresponding polypeptide, nucleic acid, vector, or composition according to any one of 1 to 22 to the human.

[0413] 29. A polypeptide, nucleic acid, vector, or composition according to any one of provisions 1 to 22 for use in treating or preventing human cancer, wherein cancer cells express (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) a corresponding sequence selected from SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, selectively induce T cells in which said variant sequence is immunogenic and / or retains the ability to bind to MHC molecule(s) and / or cross-reacts with said polypeptide and / or variant.

[0414] 30. A polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 22, for use in in vitro stimulation and / or amplification of T-cells derived from a human having cancer, and subsequently for reintroducing said stimulated and / or amplified T-cells into said human for the treatment of said cancer.

[0415] 31. A method for treating cancer in humans, comprising the steps of: (a) inducing T cells selected from SEQ ID NOs. 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs. 18 to 56 and 58 and variant sequences thereof, wherein, when selectively bound to MHC, the variant sequence selectively induces T cells that are immunogenic and / or retain the ability to bind to MHC molecule(s) and / or cross-react with said polypeptide and / or variant, and wherein the T cells are selectively obtained from said human, the population of leukocytes including at least T cells together with antigen-presenting cells; stimulating and / or amplifying said T cells in the presence of a corresponding polypeptide, nucleic acid, vector, or composition according to any one of Clauses 1 to 22; and reintroducing at least some or all of said leukocytes of the stimulated and / or amplified T cells into said human.

[0416] 32. In any one of provisions 23 to 31, the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or selected from EAC or ESSC, a method or polypeptide, nucleic acid, vector or composition.

[0417] 33. A process for producing a T-cell population that is cytotoxic to cancer cells expressing (a) any one of SEQ ID NOs 1 to 17 and 57 and an immunogenic fragment and variant thereof, or (b) a sequence selected from SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, wherein, when selectively bound to MHC, the variant sequence selectively induces T cells that are immunogenic and / or maintain the ability to bind to MHC molecule(s) and / or cross-react with said polypeptide and / or variant; wherein the process comprises (i) obtaining T-cells using selectively antigen-presenting cells from a cancer patient and (ii) stimulating and amplifying an in vitro T-cell population with a corresponding polypeptide, nucleic acid, vector, or composition according to any one of Clauses 1 to 22.

[0418] 34. A T-cell population obtainable by the process of Clause 33, wherein the T-cells are preferably immunoreactive to the polypeptide, nucleic acid, vector, or composition.

[0419] 35. T-cells stimulated by a polypeptide, nucleic acid, vector, or composition according to any one of Clauses 1 to 22, wherein, preferably, the T-cells are T-cells and are immunoreactive to the polypeptide, nucleic acid, vector, or composition.

[0420] 36. Antigen-presenting cells modified by in vitro loading of a polypeptide, nucleic acid, vector, or composition according to any one of Clauses 1 to 22, or genetically engineered to express a polypeptide according to any one of Clauses 1 to 8.

[0421] 37. In Clause 36, an antigen-presenting cell that is a dendritic cell.

[0422] 38. Exosomes loaded with a polypeptide prepared from a cell loaded with a polypeptide, nucleic acid, vector, or composition according to any one of Clauses 1 to 22, or genetically engineered to express a polypeptide according to any one of Clauses 1 to 8.

[0423] 39. A pharmaceutical composition comprising a T-cell population, T-cells, antigen-presenting cells, or exosomes according to any one of provisions 34 to 38 together with a pharmaceutically acceptable carrier.

[0424] 40. A T-cell population, T-cell, antigen-presenting cell, exosome, or composition according to any one of provisions 34 to 39 for use in medicine.

[0425] 41. A treatment method for a human with cancer, wherein cancer cells express a sequence selected from (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, selectively induce T cells in which said variant sequence is immunogenic and / or retains the ability to bind to MHC molecule(s) and / or cross-reacts with said polypeptide and / or variant; or cancer cells express a sequence selected from (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, said variant sequence is selectively immunogenic and / or retains the ability to bind to MHC molecule(s) and / or cross-reacts with said polypeptide and / or variant A method for preventing cancer in a human with cancer, which induces responsive T cells, comprising the step of administering a T-cell population, T-cells, antigen-presenting cells, and exosomes according to any one of provisions 34 to 39 to said human.

[0426] 42. A T-cell population, T-cells, antigen-presenting cells, exosomes, or composition according to any one of provisions 34 to 39 for use in treating or preventing human cancer, wherein cancer cells express (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) a corresponding sequence selected from SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, selectively induce T cells in which said variant sequence is immunogenic and / or retains the ability to bind to MHC molecule(s) and / or cross-reacts with said polypeptide and / or variant.

[0427] 43. In any one of Clauses 33, 41 and 42, the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or selected from EAC or ESSC, a process, method or T-cell population, T-cells, antigen-presenting cells, exosomes or compositions.

[0428] 44. An isolated antigen-binding polypeptide that is immunospecific to another polypeptide in any one of provisions 1 to 8, when the polypeptide is optionally bound to an MHC molecule.

[0429] 45. An antigen-binding polypeptide in accordance with Clause 44, wherein the antibody is a monoclonal antibody or a fragment thereof or a T cell receptor or a fragment thereof, and optionally, the fragment is an antigen-binding fragment.

[0430] 46. ​​An antigen-binding polypeptide coupled to a cytotoxic moiety in Clause 44 or Clause 45.

[0431] 47. A pharmaceutical composition comprising an antigen-binding polypeptide according to any one of claims 44 to 46 together with a pharmaceutically acceptable carrier.

[0432] 48. An antigen-binding polypeptide according to any one of provisions 44 to 46 or a pharmaceutical composition according to provision 47 for use in medicine.

[0433] 49. A treatment method for a human with cancer, wherein cancer cells express a sequence selected from (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, selectively induce T cells in which said variant sequence is immunogenic and / or retains the ability to bind to MHC molecule(s) and / or cross-reacts with said polypeptide and / or variant; or cancer cells express a sequence selected from (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, said variant sequence is selectively immunogenic and / or retains the ability to bind to MHC molecule(s) and / or cross-reacts with said polypeptide and / or variant A method for preventing cancer in a human with cancer, which induces responding T cells, comprising the step of administering an antigen-binding polypeptide or composition according to any one of provisions 44 to 46 and 47 to said human.

[0434] 50. An antigen-binding polypeptide or composition according to any one of provisions 44 to 46 and 47 for use in treating or preventing human cancer, wherein cancer cells express (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) a corresponding sequence selected from SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, selectively say variant sequences are immunogenic and / or retain the ability to bind to MHC molecule(s) and / or induce T cells that cross-react with said polypeptide and / or variant.

[0435] 51. In Clause 49 or Clause 50, the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or EAC or ESSC, a method, antigen-binding polypeptide or composition.

[0436] 52. An isolated antigen-binding polypeptide that is immunospecific to and / or binds to an HLA-binding polypeptide that is a polypeptide or part thereof according to any one of Clauses 1 to 8.

[0437] 53. An antigen-binding polypeptide in Clause 52, wherein the molecule derived from a T-cell receptor or a T-cell receptor or a fragment thereof, preferably an antigen-binding fragment thereof, or an antibody mimic of any of these.

[0438] 54. An antigen-binding polypeptide that, in accordance with Clause 52 or Clause 53, is preferably coupled to an immunoreactive cell of a target capable of binding to a T-cell, or to a T-cell or cytotoxic cell or other immune component, wherein, optionally, the polypeptide is an antibody-derived molecule.

[0439] 55. A cell engineered to express any one of the antigen-binding polypeptides of provisions 52 to 54 on its surface, and alternatively to secrete said antigen-binding polypeptide as a soluble polypeptide or molecule.

[0440] 56. In Clause 55, cells that are immune cells (e.g., immunoreactive cells) and / or cytotoxic cells and / or T-cells.

[0441] 57. A pharmaceutical composition comprising an antigen-binding fragment of any of provisions 52 to 54 or a cell according to provision 55 or provision 56.

[0442] 58. Cells according to Article 55 or Article 56 for use in medicine, antigen-binding proteins of any of Articles 52 to 54, or pharmaceutical compositions according to Article 57.

[0443] 59. A method for treating a human patient with cancer, wherein cancer cells express a sequence selected from (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, selectively induce T cells in which said variant sequence is immunogenic and / or retains the ability to bind to MHC molecule(s) and / or cross-reacts with said polypeptide and / or variant; or cancer expresses a sequence selected from (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, said variant sequence is immunogenic and / or retains the ability to bind to MHC molecule(s) and / or cross-reacts with said polypeptide and / or variant A method for preventing cancer in a human with cancer, which induces responsive T cells, comprising the step of administering an antigen-binding polypeptide according to any one of provisions 52 to 54, a cell according to provision 55 or 56, or a pharmaceutical composition according to provision 57 to the cell, wherein the cell is optionally a human cell.

[0444] 60. An antigen-binding polypeptide according to any of provisions 52 to 54 or cells according to provision 55 or 56 or a pharmaceutical composition according to provision 57 for use in treating or preventing human cancer, wherein cancer cells express (a) SEQ ID NOs 1 to 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) a corresponding sequence selected from SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, and when selectively bound to MHC, selectively say variant sequences are immunogenic and / or retain the ability to bind to MHC molecule(s) and / or induce T cells that cross-react with said polypeptide and / or variant.

[0445] 61. A method for diagnosing a human having cancer, comprising the step of determining whether the cancer cells express (a) an immunogenic fragment and variant thereof of SEQ ID NOs 1 to 17 and 57 and any one thereof, or (b) a polypeptide sequence selected from SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, wherein, when selectively bound to MHC or a nucleic acid encoding said polypeptide sequence, said variant sequence is immunogenic and / or retains the ability to bind to MHC molecule(s) and / or induces T cells that cross-react with said polypeptide and / or variant; and the step of diagnosing said human having cancer if said polypeptide or encoding nucleic acid is overexpressed in said cancer cells.

[0446] 62. A method for diagnosing that a human has any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC, wherein the cancer cells express a polypeptide sequence selected from any of SEQ ID NOs 1 to 17 and 57 or 18 to 56 and 58, and a fragment or immunogenic fragment and / or variant or immunogenic variant thereof, wherein, when selectively bound to MHC or a nucleic acid encoding said polypeptide sequence, said variant sequence is selectively immunogenic and / or has the ability to bind to MHC molecule(s). A method comprising the step of determining to induce T cells that maintain and / or cross-react with said polypeptide and / or variant, and the step of diagnosing that the human has any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC cancer.

[0447] 63. A method for diagnosing that a human has any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC, wherein the cancer cells express a polypeptide sequence selected from any of SEQ ID NOs 1 to 17 and 57 or 18 to 56 and 58, and a fragment or immunogenic fragment and / or variant or immunogenic variant thereof, wherein, when selectively bound to MHC or a nucleic acid encoding said polypeptide sequence, said variant sequence is selectively immunogenic and / or has the ability to bind to MHC molecule(s). A method comprising the step of determining, wherein the corresponding nucleic acid is overexpressed in the cancer cells, the step of inducing T cells that maintain or cross-react with the polypeptide and / or variant, and the step of diagnosing the human having any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or EAC or ESSC.

[0448] 64. As a treatment method for humans suffering from cancer,

[0449] (a) a step of determining whether the cancer cells express a polypeptide sequence selected from any of SEQ ID NOs 1 to 17 and 57 or 18 to 56 and 58, and a fragment or immunogenic fragment and / or variant or immunogenic variant thereof, wherein, when selectively bound to MHC or a nucleic acid encoding said polypeptide sequence, said variant sequence is immunogenic and / or retains the ability to bind to MHC molecule(s) and / or induces T cells that cross-react with said polypeptide and / or variant, if so,

[0450] (b) a step of administering to the human a corresponding polypeptide, nucleic acid, vector, composition, T-cell population, T-cell, antigen-presenting cell, exosome, antigen-binding polypeptide, or cell according to any one of provisions 1 to 22, 34 to 39, 44 to 47, and 52 to 57.

[0451] A method including

[0452] 65. For use as a polypeptide or nucleic acid, isolated from a tumor of a human with cancer,

[0453] (a) any one of the sequences SEQ ID NOs 1 to 17 and 57, or

[0454] (b) Variants of the sequence of (a) or immunogenic variants; and

[0455] (c) Use of a polypeptide comprising a fragment of a sequence selected from any of SEQ ID NOs. 18 to 56 and 58 and variant sequences thereof, or a sequence selected from an immunogenic fragment, wherein, when optionally bound to MHC, the variant sequence is optionally immunogenic and / or retains the ability to bind to MHC molecule(s) and / or induces T cells that cross-react with said polypeptide and / or variant, or use of a nucleic acid encoding said polypeptide as a biomarker for determining whether said human is suitable for treatment by a polypeptide, nucleic acid, vector, composition, T-cell population, T-cell, antigen-presenting cell, exosome, antigen-binding polypeptide or cell according to any one of provisions 1 to 22, 34 to 39, 44 to 47, and 52 to 57.

[0456] 66. A method or use in any one of Clause 59, Clause 60, Clause 61, Clause 64 or Clause 65, wherein the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, particularly esophageal adenocarcinoma (EAC) or esophageal squamous cell carcinoma (ESSC).

[0457] 67. In any one of Clauses 25 and 27 to 31, the polypeptide is

[0458] (a) any one of SEQ ID NOs 1 to 17 and 57, and

[0459] (b) Variants of the sequence of (a) or immunogenic variants; and

[0460] (c) A fragment of the sequence of (a) or (b) or an immunogenic fragment

[0461] A method or polypeptide, nucleic acid, vector or composition comprising a sequence selected from, wherein optionally the polypeptide comprises a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 or is composed of these, or optionally the nucleic acid comprises a sequence encoding any one of SEQ ID NOs 1 to 58 or is composed of these; and wherein the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or selected from EAC or ESSC.

[0462] 68. A method or antigen-binding polypeptide according to Clause 49 or 59, a cell or composition for use according to Clause 50 or 60, wherein the polypeptide is

[0463] (a) any one of SEQ ID NOs 1 to 17 and 57, and

[0464] (b) Variants of the sequence of (a) or immunogenic variants; and

[0465] (c) A fragment of the sequence of (a) or (b) or an immunogenic fragment

[0466] A method, cell, or composition comprising a sequence selected from, wherein optionally the polypeptide comprises a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 or is composed of these, or optionally the nucleic acid comprises a sequence encoding any one of SEQ ID NOs 1 to 58 or is composed of these; and wherein the cancer is any one of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or selected from EAC or ESSC.

[0467] 69. A process, method, or T-cell population, T-cell, antigen-presenting cell, exosome, or composition for use in accordance with any one of Clauses 33, 41, and 42, wherein the polypeptide

[0468] (a) any one of SEQ ID NOs 1 to 17 and 57, and

[0469] (b) Variants of the sequence of (a) or immunogenic variants; and

[0470] (c) A fragment of the sequence of (a) or (b) or an immunogenic fragment

[0471] A process, method, or T-cell population, T-cells, antigen-presenting cells, exosomes, or compositions comprising a sequence selected from, wherein optionally, the polypeptide comprises a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 or is composed of these, or optionally, the nucleic acid comprises a sequence encoding any one of SEQ ID NOs 1 to 58 or is composed of these; and the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or selected from EAC or ESSC.

[0472] 70. In Clause 64 or Clause 65, the polypeptide is

[0473] (a) any one of SEQ ID NOs 1 to 17 and 57, and

[0474] (b) Variants of the sequence of (a) or immunogenic variants; and

[0475] (c) A fragment of the sequence of (a) or (b) or an immunogenic fragment

[0476] A method or use comprising a sequence selected from, wherein optionally the polypeptide comprises a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 or is composed of these, or optionally the nucleic acid comprises a sequence encoding any one of SEQ ID NOs 1 to 58 or is composed of these; and the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), in particular esophageal cancer, or selected from EAC or ESSC.

[0477] Examples

[0478] Example 1 - Cancer-specific transcript identification

[0479] The objective was to identify a novel transcript specific to human esophageal adenocarcinoma (EAC).

[0480] High-quality EAC RNAseq data were obtained from The Cancer Genome Atlas Consortium (TCGA) and used to generate transcriptome assemblies via a reference-guided approach. Potentially cancer-specific transcripts were identified through differential expression analysis, and their expression distribution (measured by TPM) was investigated across 33 TCGA-defined indications and 48 anatomical regions for which RNAseq data were available (GTEx, The Genotype-Tissue Expression Consortium, 2015, Science, 348:648-60) (Figs. 1–17 and Fig. 26). Estimated ORFs were inferred from these transcripts in all three read frames: starting with a conventional start codon (ATG), encoding eight or more amino acids, and terminating at a stop codon or reaching the end of the transcript.

[0481] Example 2 - Immunopeptide Mix Analysis

[0482] Mass spectrometry (MS)-based immunopeptidemix analysis is a powerful technique capable of directly detecting specific peptides present on the cell surface associated with HLA molecules (HLAp). This technique involves purifying the affinity of HLAp in biological samples, such as cells or tissues, by capturing anti-HLA antibodies. Subsequently, the isolated HLA molecules and bound peptides are separated from each other, and the eluted peptides are analyzed by nano-ultra-high performance liquid chromatography-mass spectrometry (nUPLC-MS) (Freudenmann et al., 2018, Immunology 154(3):331-345). In the mass spectrometer, specific peptides with a specific charge-to-mass ratio (m / z) were selected, isolated, and fragmented, and then a second round of mass spectrometry (MS / MS) was performed to determine the m / z values ​​of the generated fragment ions. Subsequently, by analyzing the fragmentation spectrum (MS / MS), the amino acid sequence of the selected peptide that generated the detected fragment ions can be accurately identified.

[0483] MS / MS spectrum interpretation and subsequent peptide sequence identification depend on the agreement between experimental data and theoretical spectra generated from peptide sequences found in reference databases. While it is possible to search MS data using a predefined list corresponding to all Open Reading Frames (ORFs) derived from known transcripts or the whole genome (Nesvizhskii et al., 2014, Nat. Methods 11: 1114-1125), examining such very large sequence databases results in a very high false discovery rate (FDR), which limits the identification of presented peptides. Additional technical issues (e.g., mass of leucine = mass of isoleucine) and theoretical issues (e.g., peptide splicing (Liepe et al., 2016, Science 354 (6310): 354-358)) increases the limitations associated with the use of very large databases, such as those generated from known transcripts or the whole genome. Therefore, it is actually very difficult to perform accurate immunopeptidemix analysis to identify novel antigens without referring to a well-defined set of potential polypeptide sequences (Li, et al., 2016, BMC Genomics 17 (Suppl 13):1031).

[0484] The inventors obtained frozen tumor tissues from 60 patients with ovarian carcinoma (OV), 40 with breast cancer, 5 with renal cancer, 33 with skin cancer, 11 with esophageal adenocarcinoma (EAC), 5 with esophageal squamous cell carcinoma (ESSC), and 66 with colorectal adenocarcinoma (COAD). 0.2 to 1 g of the sample was homogenized, the lysate was centrifuged at high speed, and the supernatant was mixed with Protein A (ProA) beads covalently bound to anti-human HLA class I monoclonal antibodies (BB7 and / or W6 / 32). For W6 / 32 monoclonal antibody cultures, the mixture was incubated overnight at 4 °C to improve antibody binding to HLA class I molecules (Ternette et al., 2018). Proteomics 18, 1700465). HLA class I-binding peptides were eluted from antibodies using 10% acetic acid and subsequently separated from other high molecular weight components using reverse-phase column chromatography (see Ternette et al., 2018 above). The purified and eluted peptides were subjected to nUPLC-MS, and specific peptides with specific charge-to-mass ratios (m / z) within the mass spectrometer were selected, separated, and fragmented to show the m / z of the fragment ions generated through secondary mass spectrometry (MS / MS) (see Ternette et al., 2018 above), thereby generating MS / MS datasets corresponding to immunopeptides for each tumor sample.

[0485] By applying detailed knowledge of immunopeptidemix evaluation, the inventors investigated the spectra of an HLA-class I dataset prepared by the inventors for 60 patients with ovarian carcinoma (OV), 40 patients with breast cancer, 5 patients with renal cancer, 33 patients with skin cancer, 11 patients with esophageal adenocarcinoma (EAC), 5 patients with esophageal squamous cell carcinoma (ESSC), and 66 patients with colorectal adenocarcinoma (COAD), using the cancer-specific transcript-derived ORF of Example 1. Since most HLA-class I binding peptides found in cells originate from always-expressed proteins, searching this database in conjunction with the UniProt proteome allows for verification of the accuracy of the assignment of ORF sequences to MS / MS spectra. Two methods, data-dependent acquisition (DDA) and data-independent acquisition (DIA), were used to obtain MS / MS spectra from mass spectrometry. DDA MS / MS spectra were analyzed using PEAKS software, which quantifies assignments by assigning a probability value (-10lgP) to each spectral assignment. DIA MS / MS spectra were investigated using DIA-NN (Data Independent Acquisition by Neural Network) (Nature Methods. 2020 Jan; 17(1): 41-44) against a database of peptide sequences within putative cancer-specific ORF sequences predicted to bind to common HLA alleles by NetMHCpan 4.0. Only peptide identification results with a total Q value of less than 1% were accepted, and the identified peptides and their associated Q values ​​are shown in Table 2 below.

[0486] The results of this study identified individual peptides associated with immunoprecipitated HLA class I molecules in 60 ovarian carcinomas (OV), 40 breast cancers, 5 kidney cancers, 33 skin cancers, 11 esophageal adenocarcinomas (EAC), 5 esophageal squamous cell carcinomas (ESSC), and 66 colorectal adenocarcinomas (COAD) tumor samples included in the dataset held by the inventors, which corresponded to amino acid sequences of ORFs derived from cancer-specific transcripts and did not correspond to polypeptide sequences present in the known human proteome (UniProt and / or masDB).

[0487] Further manual review of the peptide spectra assigned to these ORF sequences in PEAKS and DIA-NN software confirmed that the spectra were assigned to peptides mapped to 18 ORFs (EVA001 to EVA018, SEQ ID NOs 1 to 17 and 57) encoded by a single cancer-specific transcript. Thus, the ORFs were defined as CST antigens (SEQ ID NOs 1 to 17 and 57) as specified in Table 3.

[0488] The detection of peptides associated with HLA class I molecules confirms that the 18 ORFs from which they originated are first translated in different tumor types, processed through the HLA class I pathway, and finally form complexes with HLA class I molecules to be presented to the immune system. Table 3 shows the characteristics of peptides (SEQNs 18 to 56 and 58) found in CST antigens (SEQNs 1 to 17 and 57) and their presentation in patient-derived tumor tissues. Figures 1 to 17 and 26 show the identification of cancer-specific transcripts specific to the esophageal adenocarcinoma (EAC) cancer type using de novo assembly, the number of transcripts per million (TPM) for all transcripts were estimated, and the expression of TGCA in various cancer types (Figures 1 to 17 and 26, Part B) was compared with the expression of GTEx in healthy tissue samples (Figures 1 to 17 and 26, Part A).

[0489] Representative MS / MS spectra for peptide sequence numbers 18 to 56 and 58 were analyzed using PEAKS software to obtain MS / MS peptide fragment profiles from the tumor MS / MS dataset, and through this, spectra showing the positions of linear peptide sequences mapped to the peptides presented in Table 3 were generated using PEAKS and DIA-NN, and these spectra contained numerous fragments that exactly matched the sequences of the peptides (sequence numbers 18 to 56 and 58) found in the analysis.

[0490] The peptides detected in association with HLA class I molecules in Table 3 were further evaluated using NetMHCpan 4.0 prediction software (http: / / www.cbs.dtu.dk / services / NetMHCpan / ) to predict binding strengths for HLA class IA, B, and C supertypes. The results of this prediction study show that the peptides are predicted to bind to at least one haplotype expressed in patient-derived tissues (see Table 3). The fact that the detected peptides are predicted to bind to a standard set of HLA types provides further validation for the detection of the peptides. Additionally, the peptides found in tumor samples from the dataset held by the inventors were predicted by NetMHCpan 4.0 to bind to one of the HLA types detected in patient samples.

[0491] Taken together, the peptide data shown in Tables 2 and 3, and Figures 1 to 17 and 26 provide strong evidence for the translation, processing, and presentation of corresponding CST antigens in patients with ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC), particularly in patients with esophageal cancer, or EAC or ESSC.

[0492] To further confirm the cancer specificity of these CST antigens, the inventors processed normal tissue samples (including normal lung, kidney, liver, spleen, head / neck tissue, and breast tissue) and prepared them for immunopeptidemix analysis. Using Peaks™ software (X), the inventors investigated the spectra of the HLA-Class I dataset from these normal tissue samples by searching for all possible peptide sequences derived from the polypeptide sequences of CST antigens 1 through 18, along with all polypeptides found in the human proteome (UniProt). The absence of peptides derived from CST antigens EVA001 through EVA018 (SEQ ID NOs 1 through 17 and 57) provided additional evidence that CST exhibits cancer-specific expression. To accurately link the MS spectra derived from tumor tissue to the peptide sequences discovered by the inventors, peptides containing the corresponding sequences were synthesized and nUPLC-MS was performed under the same conditions applied to tumor samples in previous studies. 2 MS / MS was performed. The spectra of the synthetic peptide and the endogenous (i.e., tumor) peptide were compared, and the m / z (mass-to-charge ratio) values ​​were determined for each fragment peak detected in these MS / MS spectra. This analysis confirmed the accurate alignment of the fragments (the minute difference in experimentally measured m / z values ​​between the tumor-derived fragment ions and the synthetic peptide-derived fragment ions was within an m / z tolerance range of less than 0.05 daltons), indicating that each tumor tissue-derived spectrum was assigned to a CST-encoding peptide (i.e., peptides derived from EVA001 to EVA018).

[0493] In summary, the identification of immunopeptide mix peptides derived from the predicted ORF demonstrates that the CST transcript is translated into polypeptides (SEQ Nos. 1 to 17 and 57; referred to as CST antigens EVA001 to EVA018) in tumor tissue. These are subsequently processed by the cellular immune surveillance apparatus, and the component peptides bind to HLA class I molecules, allowing the cells to become targets for cell lysis by T cells that recognize the generated peptide / HLA class I complex. Therefore, the identified CST antigens and fragments derived from these CST antigens are expected to be useful for various therapeutic approaches for the treatment of esophageal cancer patients whose tumors express these antigens.

[0494]

[0495]

[0496] Example 3 - Transcript expression by RT-qPCR in tumor versus normal tissue

[0497] Transcriptional expression of antigen-encoding transcripts EVA001 to EVA018 (i.e., SEQ ID NOs 1 to 17 and 57) was evaluated by RT-qPCR on a panel of esophageal adenocarcinoma (EAC) tissue samples and cell lines.

[0498] Approximately 1,000,000 cells were obtained from tumor samples (derived from human esophageal adenocarcinoma (EAC) tissue) for RNA extraction. RNA extraction was performed using the Maxwell® RSC simplyRNA cell kit (Promega) according to the manufacturer's instructions. The RNA extracts were treated with DNAse enzymes to remove any contaminated genomic DNA. RNA was quantified from RNA samples with an RNA Integrity Index (RIN) of 6 or higher and sufficient yield, and cDNA was synthesized using the iScript™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions.

[0499] For each candidate sequence, real-time quantitative PCR (RT-qPCR) analysis was performed using dye-labeled sequence-specific oligonucleotide probe (TaqMan hydrolysis probe) chemistry or SYBR Green dsDNA binding dye chemistry. Amplification was performed over a 75 to 200 bp region of each DA (DA = DAC antigen, or CST antigen) candidate to avoid regions with long single nucleotide repeats and to select regions with a GC content in the range of 50 to 60%.

[0500] Forward and reverse oligonucleotide primers were synthesized to be used for amplifying candidate DA (cancer antigen) and to include the peptide sequences determined for the ORF coding region and SEQ ID NOs 18 through 56 and 58, respectively, and the nucleotide length for analysis in each case was approximately 130 bp (data not shown). Using the probes, the associated transcript ORFs from EVA001 to EVA018 can be detected using TaqMan chemistry.

[0501] The RT-qPCR analysis procedure was carried out in two steps: (1) running a temperature gradient to optimize the primer annealing temperature (TA) using positive and negative cDNA samples for DA expression designed based on the Cancer Cell Line Encyclopaedia (CCLE) RNASeq dataset; (2) running a standard curve using serial dilutions of plasmid DNA containing DA sequences with known copy numbers. The PCR products from the heat gradient RT-qPCR runs of (1) were stored at -20°C until the RT-qPCR assay with the best performance in (2) was selected, i.e., the most sensitive, specific, and efficient assay. After selection, the PCR products were purified, cloned into a backbone, and then the sequences of the amplified products were verified through SANGER sequencing.

[0502] The expression levels of candidate DAs were calculated based on the reference gene expression levels of PGK1 (phosphoglycerate kinase 1) and TBP (TATA binding protein), which are expressed at consistent levels throughout the EAC tissue, and these were used as reference gene controls for analysis. The expression levels of each EAC DA were calculated using the ΔCT method with two reference genes (TBP and PGK1) as described below.

[0503] First, calculate the average CT (Cq, or quantification cycle) of the reference gene:

[0504]

[0505] Second, calculate the ΔCT method using the mean of the reference gene:

[0506]

[0507] The obtained result reflects the relative expression of DA with respect to the reference gene.

[0508] Example 4 - Transcript expression by RNAscope™ in tumor versus normal tissue

[0509] Transcriptome expression of DA EVA001 to EVA018 (SEQ NOs 1 to 17 and 57) was further experimentally verified in tissue microarrays (TMAs) of esophageal adenocarcinoma (EAC), normal tissue, and multiple tumor tissue using RNA ISH (RNAscope™) with a BOND RX (Leica Biosystems) automated staining instrument. RNAscope™ enables the detection of short RNA target sequences (mRNA or ncRNA) of greater than 300 nt. The methodology involved hybridizing to regions of candidate dark antigen transcripts encoding the EVA001 to EVA018 series (SEQ NOs 1 to 17 and 57) using probes designed for each CST sequence, and in each case, the probes were designed to hybridize to regions containing sequences encoding peptide sequences (i.e., SEQ NOs 18 to 56 and 58 in each case), and performing TMA quality control to verify the overall quality and integrity of the RNA. 20ZZ RNAscope probes were designed for each relevant EVA sequence, for example, EVA001 transcript expression was detected by the RNAscope probe EVA001-20zz-RNAscope, which aligns to regions 1342 to 2769 of EVA001. EVA007 transcript expression was detected by the RNAscope probe EVA007-20zz-RNAscope, which aligns to regions 1623 to 2998 of EVA007. EVA006 transcript expression was detected by the RNAscope probe EVA006-20zz-RNAscope, which aligns to regions 774 to 1760 of EVA006. Probes for other EVA sequences were designed and manufactured accordingly. Subsequently, DAC (DA) staining was performed on tissues, and the expression of DA content for esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC) candidates was evaluated and reported for tissues that passed quality control (QC).

[0510] For the quality control step and RNA quality evaluation of TMA, the processed slides of each TMA were tested with a negative control probe (Bacillus subtilis ( Bacillus subtilis ) was stained with dihydrodipicolinate reductase (DapB) and a positive control probe (peptidylpropyl isolease B, PPIB). Some TMAs were additionally stained with a second positive control probe (ubiquitin C, UBC) for additional quality control regarding RNA quality.

[0511] Once TMA was stained, the overall histological integrity and sample RNA quality were evaluated. Tissue integrity was assessed by confirming the presence of a core, a sufficient tissue region of interest, and appropriate nuclear morphology based on hematoxylin counterstaining, while RNA quality was assessed through semi-quantitative scoring of the tissue core. A semi-quantitative scoring method was used for the evaluation of QC and DA expression. Detailed information on the RNAscope scoring criteria can be found in Table 4 below, and representative tissue scoring results are presented in Figure 19.

[0512] For all TMAs, if the PPIB score was 2 or higher (relatively uniform across the entire tissue core) and the DapB score was less than 1 (no non-specific signal in DapB), the core was considered to have passed QC.

[0513]

[0514] To screen for normal and tumor tissues, RNAscope probes were hybridized to each TMA of the study subjects, and full slide scan images (40x magnification) were used for evaluation.

[0515] For example, Figure 20 shows the testing and detection reliability of the RNAscope tissue probe for tissue cores known to express or not express the EVA001 transcript sequence through prior sequencing (based on the CCLE dataset).

[0516] Results and Discussion of Transcriptome Expression by RT-qPCR and RNAScope in Examples 3 and 4

[0517] EVA001 (Sequence No. 1):

[0518] EVA001 (Sequence No. 1) was confirmed to be expressed in several esophageal adenocarcinoma (ESOAD) tissue samples (11 / 14) and tumor cell lines (8 / 8) via RT-qPCR. Very low or almost no expression was observed in all normal esophageal tissues (NAT) adjacent to the tumors. Very low or non-existent expression of EVA01 was observed in various normal tissues (Fig. 18). In most cases, when present, EVA001 is expressed at high levels compared to the PGK1 and TBP reference genes. The analysis included lung carcinoma (NCI-H1299, indicated by '+'), a positive control cell line known to express the EVA001 transcript, and breast adenocarcinoma (MCF7, indicated by '-'), a negative control cell line known not to express the EVA001 transcript.

[0519] EVA001 transcript expression was evaluated in normal tissues using RNAScope probe staining specific to the ORF region (probe EVA001-20zz) (Fig. 22). High-risk tissues ( Brain, heart, kidneys, liver, lungs No positive signal (score ≥ 1) was observed in ) or other normal tissues, except for the testis.

[0520] RNAScope probe staining confirmed positive signals for EVA001 in 60 different tissues across 9 different cancer indications (positive expression of EVA001 was observed in 1 / 9 COAD cases, 4 / 9 H&N cases, 3 / 12 melanoma cases, 2 / 9 NSCLC cases, 1 / 10 ovarian ADC cases, 1 / 1 osteosarcoma case, and 2 / 9 bladder urothelial carcinoma cases (Fig. 23)). Among 16 EAC tissues, 7 were measured at stage 1 or higher, highlighting the high prevalence of EVA001 in EAC tissues, specifically esophageal cancer (esophageal adenocarcinoma) (Fig. 21). Additionally, EVA001 also exhibited moderate to high expression in tumor tissues of head and neck cancer (head and neck squamous cell carcinoma), melanoma, ovarian cancer (ovarian adenocarcinoma), lung cancer (non-small cell lung cancer, NSCLC), bladder cancer (bladder cholangiocarcinoma), and osteosarcoma.

[0521] EVA015 (Sequence No. 15):

[0522] EVA015 (sequence number 15) was confirmed to be expressed in all esophageal adenocarcinoma (ESOAD) tissue samples (14 / 14) and tumor cell lines (8 / 8) by RT-qPCR. Low expression was observed in all normal esophageal tissues (NAT) adjacent to the tumor. Low or no expression of EVA015 was observed in various normal tissues (Fig. 32). Depending on the cell line, low expression (1,000 to 5,000 copy numbers / 10 ng cDNA), intermediate expression (5,000 to 10,000 copy numbers / 10 ng cDNA), and high expression (>10,000 copy numbers / 10 ng cDNA). Positive controls are indicated by '+' in the figure.

[0523] EVA015 transcript expression was evaluated in normal tissues using RNAScope probe staining specific to the ORF region (probe EVA015-20zz) (Fig. 33). High-risk tissues ( Brain, heart, kidneys, liver, lungsNo positive signal (score ≥ 1) was observed in 4 / 5 normal esophagus, as well as 3 / 3 testes, 3 / 3 lymph nodes, 3 / 3 thymus, 3 / 3 small intestine, 2 / 2 larynx, 1 / 3 bladder, 1 / 3 colon, 1 / 3 stomach, and 1 / 3 uterus. RNAScope probe staining confirmed positive signals for EVA015 in 60 different tissues and 9 different cancer indications (12 / 12 COAD cases, 10 / 11 H&N cases, 10 / 11 melanoma cases, 8 / 10 NSCLC cases, 6 / 8 ovarian ADC cases, 6 / 8 pancreatic ADC cases, 4 / 8 breast carcinoma cases, and 1 / 9 RCCC cases showed positive expression of EVA015 (Fig. 34)). In addition to esophageal cancer (esophageal adenocarcinoma) in which 100% of the tissue showed intermediate to high expression, EVA015 also showed intermediate to high expression in tumor tissues of head and neck cancer (head and neck squamous cell carcinoma), melanoma, ovarian cancer (ovarian adenocarcinoma), lung cancer (non-small cell lung cancer, NSCLC), and colorectal cancer (colorectal adenocarcinoma COAD).

[0524] EVA004, Antigen 4, Sequence No. 4:

[0525] EVA004 (Sequence No. 4) was confirmed to be expressed at high to moderate levels in all esophageal adenocarcinoma tissue samples (10 / 10) and tumor cell lines (8 / 8) via RT-qPCR. Moderate expression was observed in all esophageal normal tissues (NAT) adjacent to the tumor. Across various normal tissues (Fig. 35), for example, the spleen (1 / 1), lung (2 / 2), and brain (2 / 2), low or no expression of EVA004 was observed. Positive controls are indicated by '+' in the figure.

[0526] EVA004 transcript expression was evaluated in normal tissues using RNAScope probe staining specific to the ORF region (probe EVA004-20zz) (Fig. 36). High-risk tissues ( Brain, heart, kidneys, liver, lungsNo positive signal (score ≥ 1) was observed in ). Low positive signal (score ≥ 1) was observed in 2 / 3 normal tissue and 1 / 3 uterus.

[0527] RNAScope probe staining confirmed low expression levels in 31.25% of the tissues of esophageal cancer (esophageal adenocarcinoma), and EVA004 also showed low expression in tumor tissues of head and neck cancer (head and neck squamous cell carcinoma), melanoma, pancreatic cancer (pancreatic adenocarcinoma), lung cancer (non-small cell lung cancer, NSCLC), and colorectal cancer (colorectal adenocarcinoma COAD) (Fig. 37).

[0528] EVA007 (Sequence No. 7) and EVA008 (Sequence No. 8):

[0529] EVA007 (Sequence No. 7) and EVA008 (Sequence No. 8) were confirmed to be expressed at high to moderate levels in all esophageal adenocarcinoma tissue samples (14 / 14) and tumor cell lines (8 / 8) via RT-qPCR. Four esophageal NAT tissues showed moderate expression, normal tissues showed very low expression (Figs. 38 / 39), and the spleen (1 / 1) showed moderate expression in both cases. Positive controls are indicated by '+' in the figure.

[0530] Since EVA007 / EVA008 are transcribed from the same RNA transcript, they can be detected using the same probe in RNAScope analysis. EVA007 / EVA008 transcript expression was evaluated in normal tissues using RNAScope probe staining specific to the ORF region (Probe EVA007008-20zz) (Fig. 40). High-risk tissues ( Brain, heart, kidneys, liver, lungs No positive signal (score ≥ 1) was observed in the esophagus (4 / 5), thymus (3 / 3), and testis (3 / 3). Expression was observed in the small intestine (3 / 3), lymph nodes (2 / 2), and larynx (2 / 2), and expression was lower in the fallopian tubes, ovaries (2 / 3), tonsils (1 / 3), and cervix (1 / 2).

[0531] RNAScope probe staining (Fig. 41) confirmed intermediate to high expression levels in 100% of the tissues of esophageal cancer (esophageal adenocarcinoma), and EVA007 also showed low expression in tumor tissues of head and neck cancer (head and neck squamous cell carcinoma), melanoma, pancreatic cancer (pancreatic adenocarcinoma), lung cancer (non-small cell lung cancer, NSCLC), colorectal cancer (colorectal adenocarcinoma COAD), breast cancer (breast duct carcinoma), and bladder cancer (cholangiocarcinoma).

[0532] EVA005 (Sequence No. 5):

[0533] EVA005 (sequence number 5) was confirmed to be expressed at high to moderate levels in all esophageal adenocarcinoma tissue samples (13 / 14) and tumor cell lines (8 / 8) via RT-qPCR. One esophageal NAT tissue showed low expression, and normal tissue showed very little expression (Fig. 42). Positive controls are indicated by '+' in the figure.

[0534] EVA005 transcript expression was evaluated in normal tissues using RNAScope probe staining specific to the ORF region (probe EVA005-20zz) (Fig. 43). High-risk tissues ( Brain, heart, kidneys, liver, lungs No positive signal (score ≥ 1) was observed in ). Expression was low in the esophagus (2 / 5), colon (2 / 5), thymus (2 / 3), lymph nodes (1 / 2), larynx (1 / 2) and uterus (1 / 3), and high in the testes (3 / 3).

[0535] RNAScope probe staining (Fig. 44) confirmed intermediate to high expression levels in 50% of the tissues of esophageal cancer (esophageal adenocarcinoma), and EVA005 also showed intermediate expression in tumor tissues of head and neck cancer (head and neck squamous cell carcinoma), melanoma, and lung cancer (non-small cell lung cancer, NSCLC), and appeared to a lower degree in colorectal cancer (colorectal adenocarcinoma COAD), pancreatic cancer (pancreatic adenocarcinoma), breast cancer (breast duct carcinoma), bladder cancer (cholangiocarcinoma), kidney cancer (renal clear cell carcinoma), and ovarian cancer (ovarian adenocarcinoma).

[0536] EVA006 (Sequence No. 6):

[0537] EVA006 (sequence number 6) was expressed in some esophageal adenocarcinoma tissue samples (6 / 14) by RT-qPCR, and was strongest in late (III and IV) tumor tissues. No expression was observed in esophageal NAT tissue or normal tissue. No expression was observed in esophageal tumor cell lines (Fig. 45). Positive controls are indicated by '+' in the figure.

[0538] EVA006 transcript expression was evaluated in normal tissues using RNAScope probe staining specific to the ORF region (probe EVA006-20zz) (Fig. 46). High-risk tissues ( Brain, heart, kidneys, liver, lungs No positive signal (score ≥ 1) was observed in ) or other normal tissues.

[0539] RNAScope probe staining (Fig. 47) confirmed high expression levels in 100% of the tissues in esophageal cancer (esophageal adenocarcinoma), and EVA006 also showed high expression in tumor tissues of breast cancer (breast duct carcinoma), bladder cancer (cholangiocarcinoma), colorectal cancer (colorectal adenocarcinoma COAD), lung cancer (non-small cell lung cancer, NSCLC), and pancreatic cancer (pancreatic adenocarcinoma), and showed intermediate expression in head and neck cancer (head and neck squamous cell carcinoma) and ovarian cancer (ovarian adenocarcinoma).

[0540] It was noted that RNAScope probe staining signals from tumor tissue sections were detected only in the stromal region of the tumor, which may suggest that expression correlates with more advanced / aggressive disease states, particularly in stromal cancers such as breast cancer / carcinoma, pancreatic cancer / adenocarcinoma (and GIST carcinomas including colorectal and esophageal cancer). Additionally, it was confirmed that the majority of expression was observed in tumor tissue samples where the cancer had metastasized to the lymph nodes.

[0541] EVA013 (Sequence No. 13):

[0542] EVA013 (sequence number 13) was confirmed to be expressed at high to moderate levels in all esophageal adenocarcinoma tissue samples (13 / 13) and tumor cell lines (8 / 8) via RT-qPCR. All esophageal NAT tissues showed partial expression of EVA013 at moderate to high levels. Normal tissues generally showed low expression, and some showed moderate expression in the spleen (Fig. 48). Positive controls are indicated by '+' in the figure.

[0543] EVA013 transcript expression was evaluated in normal tissues using RNAScope probe staining specific to the ORF region (probe EVA014-20zz) (Fig. 49). High-risk tissues ( Brain, heart, kidneys, liver, lungs No positive signal (score ≥ 1) was observed in ). Low expression was observed in the esophagus (1 / 5), colon (1 / 3), thymus (1 / 3), and lymph nodes (1 / 2), intermediate expression was observed in the larynx (1 / 1) and uterus (1 / 4), and intermediate to high expression was observed in the testes (5 / 5).

[0544] RNAScope probe staining (Fig. 50) confirmed low levels of expression in 45% of the tissues of esophageal cancer (esophageal adenocarcinoma). Positive signals (score ≥ 1) were observed from 11 donors across five different cancer diseases. EVA014 showed low expression in tumor tissues of head and neck cancer (head and neck squamous cell carcinoma), melanoma, and lung cancer (non-small cell lung cancer, NSCLC); [expressed to a lower degree in breast cancer (breast duct carcinoma) and bladder cancer (cholangiocarcinoma)].

[0545] EVA011 (Sequence No. 11):

[0546] EVA011 (Sequence No. 11) was confirmed to be expressed at high to moderate levels in all esophageal adenocarcinoma tissue samples (13 / 13) and tumor cell lines (8 / 8) via RT-qPCR. All esophageal NAT tissues showed partial expression of EVA011 at moderate to high levels. Normal tissues generally showed low expression, and some showed moderate expression in the spleen (Fig. 51). Positive controls are indicated by '+' in the figure.

[0547] EVA011 transcript expression was evaluated in normal tissues using RNAScope probe staining specific to the ORF region (probe EVA014-20zz) (Fig. 52). High-risk tissues ( Brain, heart, kidneys, liver, lungs No positive signal (score ≥ 1) was observed in ). Low to moderate expression was observed in the esophagus (5 / 5), and low expression was observed in the colon (1 / 3), cervix (1 / 3), small intestine (2 / 3), larynx (1 / 2), skin (1 / 3) and stomach (1 / 3).

[0548] RNAScope probe staining (Fig. 53) confirmed intermediate to low levels of expression in 50% of the tissues of esophageal cancer (esophageal adenocarcinoma). Positive signals (score ≥ 1) were observed from 17 donors across 10 different cancer diseases. EVA011 showed high expression in 5 / 6 of the pancreatic cancer (pancreatic adenocarcinoma) tissues, low to intermediate expression in 5 / 11 of the head and neck cancer (head and neck squamous cell carcinoma) tumor tissues, and low expression in 3 / 10 of the lung cancer (non-small cell lung cancer, NSCLC) and 3 / 10 of the colorectal cancer (colorectal adenocarcinoma) tissues [lower levels in bladder cancer (cholangiocarcinoma)].

[0549] Observations on EVA001, EVA006, and EVA007:

[0550] Transcriptome expression for each of EVA001, EVA006, and EVA007 was evaluated by RNAscope™ across major healthy tissues, GI (gastrointestinal) tumor tissues (esophageal adenocarcinoma, EAC tissue samples), and a number of common tumor tissue types including melanoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, and breast cancer. The data presented in Figure 27 confirm that there is no transcriptome expression for any of EVA001, EVA006, or EVA007 across major healthy tissues. Transcriptome staining in Figure 28 can be noted as showing that both EVA001 (scores of 3 to 4) and EVA007 (scores of 1 to 2) were significantly expressed in GI tumor tissues, and that transcriptome staining for EVA006 (scores of 1 to 2) was observed to be much more intense in stromal cells compared to tumor cells, confirming that transcriptome targeting is much more oriented toward the tumor stromal compartment. Figure 29 confirms that EVA001 was strongly detected at high levels in melanoma and ovarian tumor tissues, EVA007 in breast cancer and non-small cell lung cancer tissues, and especially EVA006 in stroma-rich cancers or tumors such as breast cancer (69% of tumor cells) and pancreatic cancer / adenocarcinoma (81% of tumor cells).

[0551] conclusion: Transcriptome expression data determined by RT-qPCR and RNAScope show high levels of EVA001, EVA004, EVA005, EVA006, EVA007, EVA008, EVA011, EVA013, EVA015, and DAC antigen transcriptome expression in EAC tissues, as indicated by RNA inoculatory hybridization (RNA ISH). Expression in normal tissues was detected at a negligible level (no positive signal (score ≥ 1) was observed in high-risk tissues such as the brain, heart, kidney, liver, and lungs, and no positive signal (score ≥ 1) was observed in normal tissues), and these antigens are cancer-specific and indicate that they are validated therapeutic targets for their indicated cancer therapies in, for example, ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, osteosarcoma, esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC), particularly esophageal cancer, or in EAC or ESSC (see also Table 1 in this specification). EVA006 is known to be a strong therapeutic target for the treatment of stroma-rich cancers such as breast cancer, pancreatic cancer, prostate cancer, gastric cancer, etc.

[0552] Example 5 - Immunogenicity of peptides derived from CST antigens. CST antigens identified by a preceding immunopeptide mix analysis and subsequently detected in esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESSC) were further tested for immunogenicity against donor PBMCs. The purpose of this study is to investigate the ability to identify the presence of potentially therapeutically significant immune responses to CST antigens in subjects or cancer patients.

[0553] Cytokine release assays can indicate the presence of CST antigen-specific CD8 T cells in the normal T cell repertoire of healthy individuals. Due to the expression of CST antigens in naive and thymic tissues, T cells were not cleared by central tolerance. This type of analysis involves several steps performed over 7 weeks. Step 1: PBMCs, CD14 monocytes, CD19 + B cells and naive CD8 T-cells were isolated from the peripheral blood of normal blood donors (HLA type); these cells were HLA class I type, matching the specific binding precipitate of the CST antigen under test. In this case, analysis was performed on several HLA donors matching the relevant CST sequences (Table 3). CD14 cells were cultured for 9 days with a specific cytokine cocktail to mature into dendritic cells, and then B cells were expanded over 2 weeks in an hCD40L-transfected NIH / 3T3 cell line. Step 2: Mature dendritic cells were pulsed with individual CST antigen peptides for 4 hours and then co-cultured for 9 days with isolated naive CD8 T-cells. Step 3: CD8 T-cells were restimulated three times at 7-day intervals using peptide-pulsed expanded B cells. Step 4: Read ELISpot assay is performed to detect IFNγ released by activated CD8 T cells. CD8 T cells are cultured and activated with peptides on Elispot plates coated with an IFN-gamma capture antibody. After activation overnight, the cells are washed from the plate, and the captured IFN-gamma on the plate is detected by a detection antibody and a streptavidin-substrate reaction, which forms a purple precipitate in which IFN-gamma is captured.

[0554] The data obtained from this analysis include the number of spots, the median size of the spots, and the median intensity of the spots. These are indicators representing the frequency of IFN-gamma-producing T cells and the amount of IFN-gamma per cell. A comparison of responses to CST antigens and control antigens reveals that inexperienced subjects possess a strong repertoire of CST antigen-reactive T-cells, demonstrating that this can be expanded through vaccination with CST antigen-based immunogenic formulations.

[0555] Results of immunogenicity analysis:

[0556] Figures 24 and 25 show significant CD8 T-cell responses to HLA-restricted peptides of CST antigen peptides from normal blood donors (peptide derived from EVA001, SEQ NO. 18 and peptide derived from EVA007, SEQ NO. 26). The data in Figures 30 and 31 indicate that for the peptide derived from EVA006, SEQ NO. 25, the incidence of reactive T-cell immunogenicity in T-cells derived from a single normal donor sample tested is low but observable.

[0557] FIGS. 54 and 55 show CD8 T-cell responses to HLA-restricted peptides of CST antigen peptides from normal blood donors: EVA015-derived peptide, SEQ NOs. 37, 38, 39, EVA006-derived peptide SEQ NO. 25, EVA005-derived peptide SEQ NO. 24, EVA008-derived peptide SEQ NO. 28.

[0558] Figure 56 shows the CD8 T-cell response to HLA-restricted peptides of CST antigen peptides from normal blood donors: peptide derived from EVA006 SEQ No. 25, peptide derived from EVA004 SEQ No. 22.

[0559] FIG. 57 shows the CD8 T-cell response to HLA-restricted peptides of CST antigen peptides from normal blood donors: EVA005-derived peptide SEQ NO. 24, EVA008-derived peptide SEQ NO. 27 and EVA015-derived peptide SEQ NO. 37,

[0560] FIG. 58 shows the CD8 T-cell response to HLA-restricted peptides of CST antigen peptides from normal blood donors: peptide derived from EVA002, SEQ NO. 19; peptide derived from EVA005, SEQ NO. 24; peptide derived from EVA007, SEQ NO. 26; and peptide derived from EVA008, SEQ NO. 27.

[0561] In conclusion, evidence of immunogenicity was observed for the following antigenic peptides: EVA001 peptide, SEQ NO. 18; EVA002 peptide, SEQ NO. 19; EVA004-derived peptide, SEQ NO. 22; EVA005-derived peptide, SEQ NO. 24; EVA006-derived peptide, SEQ NO. 25; EVA007 peptide, SEQ NO. 26; EVA008-derived peptides, SEQ NO. 27 and SEQ NO. 28; Strong evidence of immunogenicity was observed in 1 / 25 individual C*07:02 donors for KRYNRIMHDEL (SEQ No. 18, peptide sequence derived from CST antigen 1 from EVA001) and in 1 / 16 individual A*02:01 donors for AVSLTILAV (SEQ No. 26, peptide sequence derived from CST antigen 7 from EVA007).

[0562] Example 6: Antigen-specific TCR isolation

[0563] PBMCs and CD8 T-cells were isolated from the peripheral blood of HLA-matched healthy blood donors. Subsequently, the isolated CD8 populations were stained with DNA barcoding MHC dextramer reagents, and peripheral CD8 T-cells recognizing pHLA of interest (specific target peptide-HLA complex) were identified via flow cytometry. The classified cells were used for single-cell sequencing and immune profiling using the 10x Chromium platform (10X Genomics). TCR sequences were subsequently identified using Cell Ranger software (10X Genomics), which performs sample demultiplexing, barcode processing, single-cell 3' and 5' gene counting, V(D)J transcriptome sequence assembly and annotation, and barcode analysis from single-cell data. The collected data underwent further in-house computer analysis. This analysis was performed using peripheral blood from 12 HLA-matched healthy blood donors for antigen peptides derived from EVA015 (SEQ No. 15), specifically peptide antigen SEQ No. 37 SLIKQPPRK, and a total of 17 T cell clones were identified, and antigen-specific TCRs were isolated, cloned, and sequenced from each clone (Table 5).

[0564] Using the same procedure, T-cell clones were identified, and antigen-specific TCRs were isolated, cloned, and sequenced for (i) antigen peptides derived from EVA001 (SEQ No. 1), particularly peptide antigen SEQ No. 18 KRYNRIMHDEL, and (ii) antigen peptides derived from EVA007 (SEQ No. 7), particularly peptide antigen SEQ No. 26 AVSLTILAV (see Table 5).

[0565]

[0566] conclusionSpecifically, immunoreactive T-cells were isolated from antigenic peptides derived from EVA001, EVA007, and EVA015, particularly antigenic peptides SEQ NO. 18, SEQ NO. 26, and SEQ NO. 37. From each of the T-cell clones, a specifically immunoreactive TCR recognizing each antigenic peptide was obtained, cloned and isolated, and further sequencing of the TCR chain was performed.

Claims

Claim 1 An isolated polypeptide comprising: (a) any one of sequence numbers 15, 1 to 14, 16, 17 and 57; and (b) a variant of the sequence of (a); and (c) a sequence selected from any one of the fragments of the sequence of (a) or (b). Claim 2 In claim 1, an isolated polypeptide comprising or composed of a sequence selected from any one of SEQ ID NOs 18 to 56 and 58 and a variant sequence thereof, Claim 3 In claim 1 or 2, the polypeptide is an isolated polypeptide having the ability to bind to an MHC molecule. Claim 4 In any one of claims 1 to 3, the polypeptide is an isolated polypeptide having a total length of 8 to 30 amino acids. Claim 5 In any one of claims 1 to 4, the polypeptide is an isolated polypeptide comprising non-peptide bonds. Claim 6 In any one of claims 1 to 5, the polypeptide is an isolated polypeptide that is part of a fusion protein. Claim 7 In claim 6, the polypeptide is an isolated polypeptide fused to a second or additional polypeptide selected from (i) one or more other polypeptides according to any one of claims 1 to 5, (ii) one or more other polypeptides that are cancer-related antigens, optionally esophageal cancer-related antigens, (iii) one or more polypeptide sequences capable of enhancing an immune response (i.e., an immunostimulator sequence), and (iv) one or more polypeptide sequences comprising a universal CD4 helper epitope. Claim 8 Isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 7. Claim 9 In paragraph 8, nucleic acid, which is DNA. Claim 10 In claim 8 or 9, a nucleic acid that is codon-optimized for expression in a human host cell. Claim 11 In paragraph 8, nucleic acid, which is RNA. Claim 12 In any one of paragraphs 8 to 11, the nucleic acid is an artificial nucleic acid sequence. Claim 13 A vector comprising a nucleic acid according to any one of paragraphs 8 to 12. Claim 14 In paragraph 13, a vector comprising a DNA-coding regulatory element suitable for allowing transcription of a translationally active RNA molecule in a human host cell. Claim 15 In paragraph 13 or 14, a vector that is a virus vector. Claim 16 A pharmaceutical composition comprising a polypeptide, nucleic acid, or vector according to any one of claims 1 to 15 together with a pharmaceutically acceptable carrier. Claim 17 A vaccine as a pharmaceutical composition comprising a polypeptide, nucleic acid, or vector according to any one of claims 1 to 15 together with a pharmaceutically acceptable carrier. Claim 18 A composition according to claim 16 or 17, comprising one or more immunostimulators. Claim 19 In paragraph 18, the above one or more immunostimulators are aluminum salts, saponins, immunostimulating oligonucleotides, oil-in-water emulsions, aminoalkyl glucosamine 4-phosphate, lipopolysaccharides and derivatives thereof and other TLR4 ligands, TLR7 ligands, TLR8 ligands and TLR9 ligands, IL12, interferon, incomplete Freund adjuvant (IFA), aluminum-based adjuvants, TLR agonists, synthetic double-stranded RNA (dsRNA), glucopyranosyl lipid α (GLA), imidazoquinoline, CPG oligodeoxynucleotides (ODN), cyclic dinucleotides (CDN), manganese-based adjuvants; A composition selected from metabolic adjuvants, nanoparticle-based adjuvants, water-in-oil nanoemulsions, micro or nanoparticle adjuvants, CGAS-STING / STING agonists, cytokines, self-assembling peptides, virus-like particles (VLPs), inorganic nanoparticles, cage protein nanoparticles, nucleoside-unmodified mRNA, PRR ligands (pattern recognition receptor ligands), mRNA-coding functional proteins, component lipid-based adjuvants, peptides and glycolipid immunostimulators, and TLR agonists. Claim 20 A composition that is a sterile composition suitable for parenteral administration, in any one of claims 16 to 19. Claim 21 A polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 20 for use in medicine. Claim 22 A polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 20 for use in inducing a human immune response. Claim 23 A polypeptide, nucleic acid, vector, or composition, wherein the immune response is induced in a cancerous tumor expressing (a) SEQ ID NOs 15, 1 to 14, 16, 17, and 57 and any one of the immunogenic fragments or variants thereof, or (b) a corresponding sequence selected from SEQ ID NOs 18 to 56, 58, and variant sequences thereof. Claim 24 A polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 20, for use in in vitro stimulation and / or amplification of T-cells derived from a human having cancer, and subsequently for reintroducing said stimulated and / or amplified T-cells into said human for the treatment of said cancer. Claim 25 A method for producing a T-cell population that is cytotoxic to cancer cells expressing a sequence selected from (a) SEQ ID NOs 15, 1 to 14, 16, 17 and 57 and any one of the immunogenic fragments and variants thereof, or (b) SEQ ID NOs 18 to 56 and 58 and variant sequences thereof, comprising: (i) optionally obtaining T-cells using antigen-presenting cells from a cancer patient; and (ii) stimulating and amplifying the T-cell population in vitro with a corresponding polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 20. Claim 26 A T-cell population obtainable by the method of paragraph 25. Claim 27 T-cells stimulated by a polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 20. Claim 28 Antigen-presenting cells modified by in vitro loading of a polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 20, or genetically engineered to express a polypeptide according to any one of claims 1 to 7. Claim 29 In paragraph 28, antigen-presenting cells, which are dendritic cells. Claim 30 An exosome loaded with a polypeptide prepared from a cell loaded with a polypeptide, nucleic acid, vector, or composition according to any one of claims 1 to 20, or genetically engineered to express a polypeptide according to any one of claims 1 to 7. Claim 31 An isolated antigen-binding polypeptide that is immunospecific to a polypeptide according to any one of claims 1 to 7. Claim 32 In paragraph 31, an antigen-binding polypeptide that is a monoclonal antibody or a fragment thereof or a T cell receptor or a fragment thereof. Claim 33 In paragraph 31 or 32, an antigen-binding polypeptide coupled to a cytotoxic moiety. Claim 34 A pharmaceutical composition comprising a T-cell population, T-cell, antigen-presenting cell, exosome, or antigen-binding polypeptide according to any one of claims 26 to 33 together with a pharmaceutically acceptable carrier. Claim 35 A T-cell population, T-cell, antigen-presenting cell, exosome, or antigen-binding polypeptide according to any one of claims 26 to 33, or a pharmaceutical composition according to claim 34, for use in medicine. Claim 36 A polypeptide, nucleic acid, vector, T-cell population, T-cell, antigen-presenting cell exosome, antigen-binding polypeptide, or composition according to any one of claims 1 to 20 and 26 to 34 for use in the treatment or prevention of human cancer, wherein the cancer cells express (a) an immunogenic fragment selected from SEQ ID NOs 15, 1 to 14, 16, 17 and 57 and any one thereof, or (b) a corresponding sequence selected from SEQ ID NOs 18 to 56 and 58 and variant sequences thereof. Claim 37 A method or polypeptide, nucleic acid, vector, T-cell population, T-cell, antigen-presenting cell, exosome, antigen-binding polypeptide, or composition for use in accordance with any one of claims 23 to 25 and 36, wherein the cancer is any of ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, stromal cancer, osteosarcoma, colon cancer, breast cancer, skin cancer, melanoma, head and neck cancer, sarcoma, rectal cancer, lung cancer (e.g., LUSC, LUAD, NSCLC), gastric cancer, cervical cancer, uterine cancer, esophageal cancer, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESSC). Claim 38 An isolated antigen-binding polypeptide that is immunospecific to and / or binds to an HLA-binding polypeptide that is a polypeptide or part thereof according to any one of claims 1 to 7. Claim 39 In paragraph 38, an antigen-binding polypeptide that is a molecule derived from a T-cell receptor or a T-cell receptor or a fragment thereof, or an antibody mimic of any of these. Claim 40 In paragraph 38 or 39, an antigen-binding polypeptide coupled to another polypeptide capable of binding to an immunoreactive cell. Claim 41 A cell engineered to express the antigen-binding polypeptide of any one of claims 38 to 40 on its surface. Claim 42 In the 41st, a cell that is an immune cell. Claim 43 A pharmaceutical composition comprising an antigen-binding fragment according to any one of claims 38 to 40 or a cell according to claim 41 or 42. Claim 44 Cells according to claim 41 or 42, antigen-binding proteins according to any one of claims 38 to 40, or pharmaceutical compositions according to claim 43 for use in medicine. Claim 45 An antigen-binding polypeptide according to any one of claims 38 to 40 or a cell according to claim 41 or 42, or a pharmaceutical composition according to claim 43 for use in treating or preventing human cancer, wherein the cancer cell expresses (a) an immunogenic fragment or variant of SEQ ID NOs 15, 1 to 14, 16, 17 and 57 and any one thereof or (b) a corresponding sequence selected from SEQ ID NOs 18 to 56 and 58 and variant sequences thereof. Claim 46 A use of polypeptide or a use of nucleic acid comprising (a) any one of SEQ ID NOs 15, 1 to 14, 16, 17 and 57 isolated from a tumor of a human suffering from cancer; or (b) a variant of the sequence of (a); and (c) a sequence selected from a fragment of the sequence of (a) or (b); or a use of nucleic acid encoding said polypeptide as a biomarker for determining whether said human is suitable for treatment by a polypeptide, nucleic acid, vector, composition, T-cell population, T-cell, antigen-presenting cell, exosome, antigen-binding polypeptide or cell according to any one of claims 1 to 20, 26 to 34 and 38 to 43.

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