Novel cancer antigens and methods

Abstract

To provide polypeptides which are useful in the treatment, prevention and diagnosis of cancer, particularly ovarian cancer, and nucleic acids encoding the polypeptides, vectors, compositions, T cells, and the like.SOLUTION: The present invention provides an isolated polypeptide comprising a sequence selected from (a) a specific amino acid sequence; (b) an immunogenic variant of the sequence of (a), being at least 80% identical to the specific amino acid sequence; and (c) an immunogenic fragment of the sequence of (a), comprising at least 9 consecutive amino acids of the specific amino acid sequence. The present invention also provides an isolated nucleic acid encoding the polypeptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for the treatment or prevention of cancer, in particular ovarian cancer (particularly ovarian carcinoma, and more particularly ovarian cancer), The present invention relates to an antigenic polypeptide for use in the treatment or prevention of liquid ovarian carcinoma, such as ovarian serous cystadenocarcinoma. The present invention further relates in particular to said nucleic acids and to the corresponding polynucleotides. Polypeptides, loaded with said polypeptides and polynucleotides and / or Immune cells stimulated with said polypeptide, antibodies specific for said polypeptide, and A pharmaceutical composition comprising cells (derived from autologous or otherwise) genetically engineered with a molecule that recognizes The present invention relates to compositions and immunogenic compositions. [Background technology]

[0002] BACKGROUND OF THEINVENTION As part of normal immune surveillance against pathogenic microorganisms, all cells secrete intracellular proteins It cleaves the peptides to produce major histocompatibility complexes (MCCs) that are expressed on the surface of all cells. These peptides derived from the host cell are loaded onto major histocompatibility complex (MHC) class I molecules. Most are recognized as self and remain unrecognized by the adaptive immune system. Non-self peptides can stimulate the proliferation of naive CD8+ T cells, which are expressed in MHC I- These cells encode T cell receptors (TCRs) that tightly bind peptide complexes. The resulting T cell population is then enriched for effector CD8+ T cells (cytotoxic T cells) that can eliminate foreign antigen-tagged cells. Furthermore, the foreign antigen-tagged cells can be produced throughout the life of the animal. These antibodies can also generate memory CD8+ T cells that can be re-expanded if they subsequently emerge.

[0003] Expression of MHC class II molecules is usually observed in professional antigen-presenting cells such as dendritic cells (DCs) ( MHC class II molecules are normally restricted to antigen-specific peptides (APCs) that are internalized from the exogenous environment. T cell adhesion molecules (CD54, CD48) and costimulatory molecules (CD40, CD80, CD In the presence of various factors, including naive CD4+ T cells, complementary TCRs bind to MHC II-peptides. Upon binding to the endothelial complex, CD4+ T cell 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 B cells. It can promote differentiation into immune-specific plasma cells and also promote differentiation of antigen-specific CD8+ CTLs, This allows the immune system to confer immunity to foreign antigens, including both short-term effector functions and long-term immunological memory. DCs help induce adaptive immune responses that target exogenously derived antigens (e.g., released by pathogens or tumor cells). By delivering a peptide or protein (e.g., a peptide or protein that has been administered to the host) onto those MHC I molecules, It is capable of carrying out the cross-presentation process of peptide antigens and is involved in the expression of naive CD8+ T cells. Contributes to the generation of immune memory by providing an alternative pathway to stimulate cell proliferation. do.

[0004] Immunological memory (specifically antigen-specific B cells / antibodies and antigen-specific CTLs) controls microbial infections Immunologic memory plays an important role in preventing diseases caused by important pathogenic microorganisms. Immunological memory has also been used to develop a number of vaccines to prevent cancer. Although it is known that cancer plays an important role in the control of cancer, few effective cancer vaccines have been developed. It has not been done.

[0005] Cancer is the second most common cause of morbidity and accounts for nearly one in six of all deaths worldwide.2 Of the 8.8 million cancer deaths in 2015, the most deadly cancers were lung cancer (1.69 million) ), liver (788,000), colorectal (774,000), stomach (754,000) and breast (571,000) cancers. The economic impact of cancer in 2010 was estimated at US$1.16 trillion, with the number of new cases now estimated at It is expected to increase by approximately 70% over the next 20 years (World Health Organization, Cancer Facts, 2017).

[0006] Ovarian cancers or tumors include a group of different types of lesions, the most common of which are epithelial cell types. These are called carcinomas derived from the inflammatory bowel disease (Kurman RJ, Carcangiu ML, Herrington CS, et al., 2013). Dedicated to "WHO classification of tumours of female reproductive system" uctive organs)Lyon: IARC Press; 2014). Serous ovarian carcinoma accounts for the majority of ovarian carcinomas. , accounting for up to 80% of all gynecological cancer deaths and is the most common cause of cancer death in the gynecological field (Jayson GC, Kohn EC, Kitchener HC, Ledermann JA (October 2014) "Ovarian cancer." Lance 384(951): 1376-88). Ovarian serous cystadenocarcinoma is a histopathological subtype of serous ovarian carcinoma. It is similar.

[0007] Current therapies for ovarian cancer are diverse and depend largely on the stage of the disease. One is surgery to remove the tumor and surrounding tissue. For late-stage ovarian cancer, lymph node dissection, radiation therapy, and chemotherapy are also available. Treatment may require radiation therapy or chemotherapy. Immune checkpoint blockade strategies These include the use of antibodies targeting negative immune regulators such as PD-1 / PD-L1 and CTLA4, Recently, it has revolutionized the treatment of various malignant tumors (Ribas, A. and Wolchok, J.D., 2 018)Science, 359:1350-1355). The extraordinary value of checkpoint inhibitor therapy and its Clinical benefit and the patient's adaptive immune response (especially T cell-based immune responses) to the patient's own cancer antigens ) is a well-known link to effective cancer vaccines, vaccine modalities and cancer vaccines. This stimulated the search for antigens.

[0008] Human endogenous retroviruses (HERVs) are exogenous infectious retroviruses that infect ancestral germ cells. HERVs are relics of integration into the cell lineage. They contain long terminal repeats flanking the viral genome. It belongs to a group of endogenous retroelements characterized by the presence of long terminal repeats (LTRs). In addition, it also contains mammalian apparent LTR retrotransposons (MaLR), which are collectively referred to as LTR elements. (Herein, we refer to them collectively as ERVs to mean all LTR elements. ERVs constitute a significant proportion (8%) of the mammalian genome and are divided into approximately 100 subunits based on sequence homology. Many ERV sequences encode defective proviruses. They are prototypic retroviruses consisting of gag, pro, pol, and env genes flanked by long terminal repeats. Several intact ERV ORFs are expressed in exogenous pathogenic viruses such as HIV-1. retroviruses that share characteristics with proteins encoded by infectious retroviruses These proteins act as antigens to induce a strong immune response. (Hurst and Magiorkinis, 2015, J. Gen. Virol 96:1207-121 8) It has been shown that ERV-encoded polypeptides mediate the selection of T cell and B cell receptors. This suggests that the ERV process and central and peripheral tolerance can be circumvented. Immune reactivity to ERV products can arise spontaneously in infections and cancers, and some ERV products It has been implicated as a cause of several autoimmune diseases (Kassiotis and Stoye, 201 6, Nat. Rev. Immunol. 16:207-219).

[0009] Due to the accumulation of mutations and recombination events during evolution, most ERVs have loss of some or all functional open reading frames in the offspring, resulting in infectious virus However, these ERV elements have lost their ability to produce viruses. are maintained in the germline DNA of the offspring, and at least some of these genes In fact, HERV-encoded proteins have the potential to produce , which have been detected in a variety of human cancers. For example, a splice variant of the HERV-K env gene, Rec and Np9 are found only in malignant testicular germ cells and not in healthy cells (Ruprecht (2008, Cell Mol Life Sci 65:3366-3382). Increased levels of HERV transcripts have been observed in human tissues compared to human tissues (Wang-Johanning 2003, Cancer 98:187-197; Andersson et al., 1998, Int. J. Oncol 12:309-313). Overexpression of ERV-E and HERV-H has been shown to be immunosuppressive and is also a risk factor for cancer. This could contribute to the progression of However, the actual mechanisms by which HERVs can contribute to cancer progression or pathogenesis remain unclear. is still unknown.

[0010] In addition to deregulating the expression of surrounding adjacent host genes, ERV regulatory elements Activation and translocation to new genomic sites can lead to the generation of novel transcripts, Some of them may be carcinogenic (Babaian and Mager, Mob. DNA, 2016 , Lock et al., PNAS 2014, 111:3534-3543).

[0011] A wide range of vaccine modalities are known. One well-described approach aims to enhance immune responses (including B cell and T cell responses) and stimulate immunological memory. Alternatively, the polynucleotide may be delivered to a subject via a In order to express in vivo the immunogenic polypeptide encoded by the polynucleotide of It may be administered to a subject by a vector. The use of immunizers has potential applications in both preventive vaccination and therapeutic treatment strategies against cancer. It has been extensively explored for the delivery of antigens (Wold et al., Current Gene Therapy, 2013, " Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy ctors for Gene Therapy, Vaccination and Cancer Gene Therapy)” 13:421-433). immunity The cytotoxic peptides, polypeptides, or polynucleotides encoding them may also be administered from a patient. These can be used to load native antigen-presenting cells (APCs), which can then be used to administer therapeutic or prophylactic immunity. The antibody can then be injected into the subject as a vaccine to elicit an immune response. An example of this approach is The vaccine, Provenge, is the only anti-cancer vaccine currently approved by the FDA.

[0012] Cancer antigens also have potential applications for their use in creating a variety of non-vaccine therapeutic modalities. These therapies include: 1) antigen-binding biologics; 2) adoptive cell transfer; Therapies are divided into two different classes.

[0013] Antigen-binding biologics usually recognize cancer cells decorated with antigens and promote their destruction. These biologics are composed of multivalent polypeptides engineered to bind to the antigen. The components may also consist of TCR-based biologics, which can be delivered by a variety of techniques. TCRs, high affinity TCRs and TCRs based on monoclonal antibody technology Mimetics of the cytolytic components of these types of multivalent biologics The targeting and activation of cytotoxic chemicals, biological toxins, and immune cells is facilitated by It may also comprise targeting motifs and / or immunostimulatory motifs, both of which may be targeted to tumor cells. It promotes the therapeutic destruction of

[0014] Adoptive cell therapy may be based on a patient's own T cells, which are harvested and then transplanted into the body. stimulated by vaccine antigen preparations outside the (JCI Insight. October 2018, 4;3(19) pii: 122467. doi:10.1172 / jci.insight.122467). Alternatively, adoptive cell therapy could be used to target cancer antigens. Cells (patient-derived or non-patient-derived) deliberately engineered to express an antigen-binding polypeptide These antigen-binding polypeptides can also be based on antigens. These fall into the same class as those described above for conjugated biologics. Lymphocytes (autologous or non-autologous) genetically engineered to express a polypeptide , can be administered to a patient as adoptive cellular therapy to treat the patient's cancer.

[0015] The use of ERV-derived antigens to mount effective immune responses against cancer has been shown to be useful in the treatment of cancer in rodents. Promising results show improved tumor regression and better prognosis in patients with rheumatoid arthritis (Kershaw et al., 2001, Cancer Res. 61:7920-7924; Slansky et al., 2000, Immunity 13:529-538). Thus, due to the strict restriction of the identified tumor-specific ERV antigens, Despite limited progress in research, HERV antigen-based immunotherapy trials are underway. Al has been studied in humans (Sacha et al., 2012, J. Immunol 189:1467-1479).

[0016] WO 2007 / 137279 describes, for example, the use of HERV-K+ binding antibodies to prevent or inhibit cancer cell proliferation. The present invention discloses methods and compositions for the use of the HERV-K+ antibody to detect, prevent and treat HERV-K+ cancers. WO 2006 / 103562 discloses a method for the expression of the immunosuppressive Np9 protein from the env gene of HERV-K. The present invention also discloses a method for treating or preventing cancer caused by the protein. A pharmaceutical composition containing a nucleic acid or an antibody capable of inhibiting the activity of the protein, or an antibody against the protein It relates to an immunogenic or vaccine composition capable of inducing an immune response. WO 2007 / 109583 describes a method for treating tumors comprising the steps of: providing ... The present invention relates to a method for preventing or treating a neoplastic disease in a mammalian subject by providing a composition comprising: Compositions and methods are provided.

[0017] Cancer, particularly ovarian cancer, especially ovarian carcinoma, more especially serous ovarian carcinoma, e.g. ovarian serous cyst There is a need to identify additional HERV-associated antigenic sequences that can be used for immunotherapy of adenocarcinoma. do. Summary of the Invention

[0018] (Summary of the invention) The inventors have surprisingly found that the LTR element is a nucleotide sequence that contains or is adjacent to the LTR element. It is derived from a genomic sequence that is found at high levels in ovarian cancer cells but not in normal healthy tissue. We found certain RNA transcripts that were either undetectable or found at very low levels in tissues ( See Example 1. Such transcripts are referred to herein as cancer-specific LTR-element spanning transcripts. Furthermore, the present inventors have identified the latent transcription products (CLTs) encoded by these CLTs. A subset of typical polypeptide sequences (i.e., open reading frames (ORFs)) are identified in the cancer It is translated within the cell, processed by components of the antigen processing machinery, and In particular, the class I and class II major histocompatibility complexes (MHC class I and MHC class II) and In tumor tissue, together with class I and class II human leukocyte antigen (HLA class I, HLA class II) molecules These findings suggest that the IL-16 protein is expressed on the surface of cells that can be seen in vivo (see Example 2). The present invention shows that these polypeptides (herein referred to as CLT antigens) are antigenic in nature. Thus, cancer cell presentation of CLT antigens may signal the ability of these cells to mediate cognate T cell reception to these antigens. These cognate receptors are expected to be more susceptible to elimination by T cells that have the TCR. CLT antigen-based vaccination methods / regimens that expand TCR-bearing T cells are useful for the treatment of cancer cells (and and tumors containing same), in particular ovarian cancer, especially ovarian carcinoma, more especially serous ovarian carcinoma, more especially It is also expected to elicit an immune response against ovarian serous cystadenocarcinoma tumors.

[0019] The CLT and CLT antigens that are the subject of the present invention are derived from known tumor genomes found in The Cancer Genome Atlas. This CLT is not a canonical sequence that can be easily derived from the ERV-derived transcriptional regulatory sequence. The transcription factors are transcriptional complexes driven by transcription and splicing events. These CLTs are expressed at high levels, and the CLT antigen polypeptide sequences are similar to those of normal human tandems. Since the sequence is not that of a protein, it is likely to elicit a strong and specific immune response, making it a promising candidate for cancer immunotherapy. It is expected that they will be suitable for therapeutic use in therapeutic settings.

[0020] This CLT antigen was found in a highly expressed transcript that characterizes tumor cells. Before the Meiji Restoration, there were no known proteins that existed in the human body and could produce protein products. and can be used in several forms. First, the CLT antigen polypeptide of the present invention is Delivered directly to a subject as a vaccine to elicit a therapeutic or prophylactic immune response against tumor cells Second, the nucleic acids of the present invention can enhance the expression of the CLT antigens they encode. and codon-optimized to provide therapeutic or prophylactic immunity against tumor cells. As a vaccine to elicit an immune response, the vector may be administered directly or alternatively may be formulated for in vivo delivery. The vector can be inserted into a subject to allow production of the encoded protein product in the subject. Third, the polynucleotides and / or polypeptides of the present invention can be used to infect antigen-presenting cells derived from a patient. These can be used to load anti-tumor cells (APCs), which can then be used to deliver therapeutic or prophylactic targets to tumor cells. The vaccine can be injected into a subject's body as a vaccine to induce a protective immune response. The polynucleotides and / or polypeptides of the present invention stimulate T cells in a subject in vitro. and generate stimulated T cell preparations that can be used as treatments for cancer. Fifth, it recognizes CLT antigens that are in complex with MHC I molecules and inhibits cancer cells. T cell receptors (TCRs) or TCR mimetics that have been further modified to kill (or promote killing) Sixth, biological molecules such as IgG1 and IgG2A, which are complexed with MHC cells, may be administered to a subject as a method for treating cancer. Chimeric versions of biological molecules that recognize the CLT antigens that form the body (autologous or The antibody may be introduced into (or into non-autologous) T cells, and the introduced cells may be administered to a subject as a method of treating cancer. These and other applications are described in more detail below.

[0021] In view of the above, the present invention particularly relates to: (a) any one of SEQ ID NOs: 1 to 2; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); An isolated polypeptide comprising a sequence selected from the group consisting of (Provide The present invention also relates to nucleic acid molecules encoding the polypeptides of the invention (hereinafter "nucleic acids of the invention"). ").

[0022] The polypeptides of the invention and the nucleic acids of the invention, as well as related aspects of the invention, are described in more detail below. As described below, the immunotherapy and prevention of cancer, particularly ovarian cancer, and especially ovarian carcinoma, as well as Particularly in the field of embodiments in immunotherapy and prevention of serous ovarian carcinoma, e.g., ovarian serous cystadenocarcinoma. It is expected to be useful in [Brief description of the drawings]

[0023] Description of the drawings For each of Figures 1-2, the top panel shows peptides isolated from patient tumor samples. The extracted MS / MS spectrum of the 1H-NMR spectroscopy (with assigned fragment ions) is shown in the upper panel, and the lower panel shows the The figure shows a rendering of the linear peptides mapped to fragment ions. The position of the chit sequence is displayed. [Figure 1] FIG. 1: Spectra of the peptide of SEQ ID NO:3 isolated from tumor samples of patients OvCa53 and OvCa65. [Diagram 2] Figure 2: Spectra of peptide of SEQ ID NO: 4 isolated from tumor samples of patients OvCa66 and OvCa59. Each of Figures 3-4 shows an alignment of the native MS / MS spectrum of a peptide isolated from a patient's tumor sample to the native spectrum of a synthetic peptide corresponding to the same sequence. [Diagram 3] Figure 3: Spectra of the peptide of SEQ ID NO:3 isolated from tumor samples of patients OvCa53 and OvCa65. [Figure 4] Figure 4: Spectra of the peptide of SEQ ID NO:4 isolated from tumor samples of patients OvCa66 and OvCa59. [Diagram 5] FIG. 5 shows the results of a qRT-PCR assay assessing the transcription of CLT (SEQ ID NO: 6), encoding CLT antigen 2, in tumor samples from ovarian cancer patients.

[0024] (Array Description) SEQ ID NO:1 is the polypeptide sequence of CLT antigen 1. SEQ ID NO:2 is the polypeptide sequence of CLT antigen 2. SEQ ID NO:3 is a CLT antigen 1-derived peptide sequence. SEQ ID NO:4 is a CLT antigen 2-derived peptide sequence. SEQ ID NO:5 is the cDNA sequence of CLT encoding CLT antigen 1. SEQ ID NO:6 is the cDNA sequence of CLT encoding CLT antigen 2. SEQ ID NO:7 is the cDNA sequence encoding CLT antigen-1. SEQ ID NO:8 is the cDNA sequence encoding CLT antigen 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Invention (Polypeptide) The terms "protein," "polypeptide," and "peptide" are used interchangeably herein. It is used to detect peptide-linked amino acid chains, regardless of length, co-translational or post-translational modifications. Refers to either one.

[0026] The term "amino acid" refers to naturally occurring amino acids as well as amino acids that are similar to naturally occurring amino acids. It refers to any one of the amino acid analogs and amino acid mimetics that function according to the formula: The amino acids are the 20 L-amino acids encoded by the genetic code and the 20 L-amino acids that are later modified. Amino acids such as hydroxyproline, γ-carboxyglutamic acid and O-phosphoryl The term "amino acid analog" refers to an amino acid that has the same basic chemical structure as a naturally occurring amino acid. That is, a compound having a hydrogen, a carboxyl group, an amino group, and an α carbon bonded to an R group. However, compared to natural amino acids, they have modified R groups or modified peptide backbones. Examples include homoserine, norleucine, methionine sulfoxide, Amino acid mimetics include the amino acids methylsulfonium, thionine, and norleucine. have a structure different from the general chemical structure of the amino acids, but are similar to naturally occurring amino acids Preferably, the amino acid is a naturally occurring amino acid or an amino acid. Acid analogues, particularly naturally occurring amino acids, and furthermore, amino acids encoded by the genetic code. It is one of the 20 L-amino acids that can be synthesized.

[0027] Amino acids are referred to herein by either their commonly known three letter symbols or by their one letter symbols. This is recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are also sometimes represented by the commonly accepted single-letter codes: It may be represented by a do.

[0028] Thus, the present invention provides: (a) any one of SEQ ID NOs: 1 to 2; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a). .

[0029] The present invention also provides: (a) any one of SEQ ID NOs: 1 to 2 with the first methionine residue removed; To, (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a). .

[0030] Generally, variants of the polypeptide sequences of the invention will have a high degree of sequence identity thereto. For example, variants preferably have at least one or more sequences that are identical to the corresponding sequences in the entire length of the relevant reference sequence. at least about 80% identity, more preferably at least about 85% identity, and most preferably at least and about 90% identity (e.g., at least about 95%, at least about 98%, or at least about 99%). .

[0031] Preferably, the variant is an immunogenic variant. A variant is an immunogenic variant that is a variant of a reference sequence (i.e. at least 20%, preferably at least 50%, of the activity of the sequence of which the variant is a mutant; and In particular, it is considered to be an immunogenic variant that elicits a response that is at least 75% (such as at least 90%). This response can be achieved, for example, by in vivo immunohistochemistry of PBMCs or whole blood using the polypeptide as an antigen. Tro restimulation assay (e.g., from a few hours up to 1 year, up to 6 months, etc., from 1 day to 1 month, or 1- 2-week restimulation) to stimulate lymphocyte proliferation (e.g., T cell proliferation) activation of T cells, production of cytokines (e.g., IFN-γ) in the culture supernatant (measured by ELISA, etc.), or Characterization of cellular responses was performed using intracellular and extracellular staining (e.g., CD3, CD4, CD8, IL2, TNF-α, I (using antibodies specific for immune markers such as FNg, type 1 IFN, CD40L, and CD69) and subsequent Measured by analysis on a flow cytometer.

[0032] A variant can be, for example, a conservatively modified variant. The modification may be a substitution of an amino acid with a functionally similar amino acid or a modification of the biology of the mutant. These modifications result in substitutions / deletions / additions of residues that do not substantially affect the target protein function. The biological function of such a variant would be to induce an immune response against ovarian cancer. Conservative substitution tables providing functionally similar amino acids are well known in the art. , may include homologs of polypeptides found in other species.

[0033] Variants of the polypeptides of the invention may contain a number of substitutions, e.g., conservative substitutions compared to the reference sequence. Substitutions (e.g., 1-25, 1-10, etc., particularly 1-5, and particularly 1 amino acid residue is altered) The number of substitutions, e.g., the number of conservative substitutions, may be calculated based on the number of residues in the reference sequence. It may be at most 20%, for example at most 10%, for example at most 5%, for example at most 1%. Conservative substitutions may fall within one of the amino acid classes identified below, but in some cases Other substitutions are possible, so long as they do not substantially affect the immunogenic properties of the antigen. Each of the eight groups contains amino acids that are usually conservative substitutions for one another. .

[0034] 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, for example, Creighton, Proteins 1984).

[0035] Preferably, such substitutions do not alter the immunological structure of the epitopes (e.g., they The substitutions do not occur within the epitope region that maps to the primary sequence, so the antigen does not significantly affect the immunogenic properties of

[0036] Polypeptide variants also include those in which additional amino acids are inserted compared to a reference sequence. For example, such insertions can be at positions 1 to 10 (e.g., positions 1 to 5, preferably positions 1 or 2). , particularly at position 1), the insertions may be, for example, up to 50 (e.g., up to 20) at each position. The amino acid sequence may include the addition of up to 10 amino acids, particularly up to 5 amino acids. Such insertions do not occur in epitope regions and therefore do not significantly affect the immunogenic properties of the antigen. An example of an insert is a humanized polypeptide to aid in the expression and / or purification of an antigen of interest. It contains a short stretch of staphylococcal residues (e.g., 2 to 6 residues).

[0037] Polypeptide variants include those with multiple amino acid deletions compared to a reference sequence; For example, such deletions may be at positions 1 to 10 (e.g. positions 1 to 5, preferably positions 1 or 2, particularly The deletions may occur at up to 50 positions (e.g., up to 20 positions, In particular, the deletion of up to 10 amino acids, more preferably up to 5 amino acids, can be included. The deletions do not occur in the epitope region and therefore do not significantly affect the immunogenic properties of the antigen. Don't blur.

[0038] Those skilled in the art will appreciate that particular protein variants may include substitutions, deletions, and additions (or any combination thereof). For example, substitutions / deletions / additions may be made to the desired patient's H Enhances binding to LA molecules (or has a neutral effect) and enhances immunogenicity (or reduces immunogenicity) (their gender remains unchanged).

[0039] The immunogenic fragments of the present invention may be any fragment of the full length polypeptide sequence depending on the length of the CLT antigen. Usually at least 9 (e.g., at least 9 or 10) consecutive amino acids, at least 12 consecutive amino acids (e.g., at least 15 or at least 20 consecutive amino acids), particularly at least At least 50 contiguous amino acids, at least 100 contiguous amino acids (e.g., at least 200 contiguous amino acids), Preferably, the immunogenic fragment will contain the full length polypeptide sequence. At least 10% of the length of the queue, e.g. at least 20%, e.g. at least 50%, e.g. at least It would be at least 70%, or at least 80%.

[0040] An immunogenic fragment typically contains at least one epitope. Preferably, the immunogenic fragment comprises a T cell epitope, such as a CD4+ or CD8+ T cell epitope. The antibody comprises at least one T cell epitope selected from the group consisting of

[0041] T cell epitopes are those that, when bound to an HLA molecule, are targeted to T cells (e.g., CD4+ or CD8+ T cells). T cell epitopes are short, consecutive stretches of amino acids that are recognized by the This may be achieved by epitope mapping experiments well known to those skilled in the art (see, e.g., Paul, , Fundamental Immunology, 3rd ed., pp. 243-247 (1993); Beiβbarth et al., 2005, Bioinfo (see rmatics, 21(Suppl 1):i29-i37).

[0042] As a consequence of the crucial involvement of T cell responses in cancer, at least one T cell epitope Fragments of the full-length polypeptides of SEQ ID NOs: 1-9, including the It is quite clear that the potential for

[0043] In diverse outbred populations, such as humans, different HLA types represent populations of specific epitopes. It will be understood that this means that the information contained herein is not necessarily recognized by all members of the The result is to maximize the level of recognition and magnitude of the immune response to a given polypeptide. To achieve this, an immunogenic fragment typically comprises multiple epitopes from the full-length sequence, preferably multiple epitopes from the CLT. It is preferable to include all epitopes within the antigen.

[0044] Particular fragments of the polypeptides of SEQ ID NOs: 1-2 that may be useful include those that bind to at least one CD8+ T Preferably, the epitope is at least two CD8+ T cell epitopes, and more preferably all CD8+ T Those that contain cellular epitopes, particularly those that are associated with multiple HLA class I alleles, e.g. Sequence numbers that may be useful include those associated with 2, 3, 4, 5 or more alleles. Particular fragments of the polypeptides of Nos. 1-2 contain at least one CD4+ T cell epitope, preferably It is believed that the IgG antibody contains at least two CD4+ T cell epitopes, and even all CD4+ T cell epitopes. of HLA class II alleles (especially those associated with multiple HLA class II alleles, e.g., 2, 3, 4, 5 or more thereof). However, those skilled in the art of vaccine design should be aware of the possibility of The CD4+ T cell epitopes of the present invention can be combined with the CD8+ T cell epitopes of the present invention to The desired response against 8+ T cell epitopes could be achieved.

[0045] When individual fragments of a full-length polypeptide are used, such fragments may be That is, the fragment has at least 20%, preferably at least 10%, of the activity of the sequence of which it is a fragment. An antibody is considered immunogenic if it elicits a response that is at least 50%, particularly at least 75% (such as at least 90%). This response is believed to be due to, for example, PBMC or in vitro restimulation assays of whole blood (e.g., from a few hours up to a year, up to 6 months, etc., from 1 day The activity in lymphocyte proliferation (e.g., T cell proliferation) was evaluated by restimulation over a period of 1 month or 1-2 weeks. Activation of cells through cell proliferation, production of cytokines (e.g., IFN-γ) in the culture supernatant (ELISA) A, etc.) or characterization of T cell responses using intracellular and extracellular staining (e.g., CD3, CD4, We used antibodies specific to immune markers such as CD8, IL2, TNF-α, IFN-γ, type 1 IFN, CD40L, and CD69. The antibody is measured by a flow cytometer (using a ELISA kit) and subsequent analysis on a flow cytometer.

[0046] In some cases, multiple fragments of a full-length polypeptide (which may or may not overlap, (which may or may not span the entire full-length sequence) to the full-length sequence itself For example, at least two (3, 4, or Immunogenic fragments of the antibodies (e.g., 5) are combined and assayed in vitro in PBMC or whole blood restimulation assays (e.g., Preferably, the sequence matches at least 50% of the reference sequence in a T cell proliferation and / or IFN-γ production assay. Preferably, it provides at least 75% and even at least 90% activity.

[0047] Examples of immunogenic fragments of the polypeptides of SEQ ID NOs: 1-2, and therefore examples of peptides of the invention, are The polypeptides include those having sequences of SEQ ID NOs. 3 to 4 or consisting of the sequences. The sequence was confirmed to bind to HLA class I molecules by immunopeptidome analysis ( See Example 2).

[0048] (nucleic acid) The present invention relates to isolated nucleic acids encoding the polypeptides of the invention (referred to as nucleic acids of the invention). For example, the nucleic acid of the present invention may include a sequence selected from SEQ ID NOs: 5 to 6 or 7 to 8. , or an array thereof.

[0049] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to a nucleic acid. Nucleotide monomers, particularly deoxyribonucleotide monomers or ribonucleotide monomers The term refers to a polymeric macromolecule made up of known nucleotide analogs or includes nucleic acids containing modified backbone residues or linkages, which are naturally occurring and those of non-natural origin, which have similar properties to the reference nucleic acid and have similar nucleotide sequences to the reference nucleic acid. are intended to be metabolized in a similar manner or to have an extended half-life in the system Examples of such analogs include, but are not limited to, However, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl These include phosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). The term "nucleic acid" refers to a nucleic acid sequence consisting of deoxyribonucleotide or ribonucleotide monomers. It refers to a polymer of natural origin. Preferably, the nucleic acid molecule of the present invention is recombinant. is a nucleic acid molecule that has been isolated by a cloning, restriction or ligation step or by natural other manipulations which result in nucleic acid molecules that are distinct from those found in In one embodiment, the nucleic acid of the invention is an artificial product of at least one of A nucleic acid sequence (e.g., a cDNA sequence or a nucleic acid sequence that includes non-natural codon usage). In the present invention, the nucleic acid is DNA. Alternatively, the nucleic acid is RNA.

[0050] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are It refers to a nucleic acid molecule having a ribosyl moiety and a sugar moiety backbone that is a ribosyl moiety. The four natural bases in DNA (adenine (A), guanine (G), cytosine (C), thymine (T), and R Binding to the bases adenine (A), guanine (G), cytosine (C), and uracil (U) in NA As used herein, a "corresponding RNA" is a RNA having the same sequence as a reference DNA. However, it is an RNA with a sequence in which the thymine (T) in DNA is replaced by uracil (U) in RNA. The components also include inosine, xanthosine, 7-methylguanosine, dihydrouridine and 5-methyl It may be linked to an unnatural base such as lucitidine. Sugar (deoxyribosyl / ribosyl) moiety The natural phosphodiester bond between the Preferably, the nucleic acids of the present invention are deoxyribonucleic acids having phosphodiester bonds between the sugar moieties. It consists of a natural base attached to a bosyl or ribosyl sugar backbone.

[0051] In one embodiment, the nucleic acid of the present invention is DNA. For example, the nucleic acid is a sequence represented by SEQ ID NO: 5-6 or 7-8. Also provided are sequences selected from SEQ ID NOs. A nucleic acid comprising or consisting of a variant of a sequence selected from Nos. 5-6 or 7-8, Variants of the gene encode the same amino acid sequence but produce different nucleic acids based on the degeneracy of the genetic code. It is something that has.

[0052] Thus, because of the degeneracy of the genetic code, a large number of different but functionally identical nucleic acids are possible. For example, the codons GCA, GCC, GCG, and GCU can all be used to code for any given polypeptide. The codon 1 encodes the amino acid alanine. At every position designated, the codon modifies the encoded polypeptide. Such a nucleic acid mutation can be modified to any of the above codons without modifying the corresponding codon. , resulting in "silent" (sometimes referred to as "degenerate" or "synonymous") mutations, It is one type of conservatively modified variant. Every nucleic acid sequence disclosed herein also includes all possible silent variations in that nucleic acid. The skilled artisan will recognize each codon in a nucleic acid (usually the only codon for methionine). and UGG, which is normally the only codon for tryptophan) It will be appreciated that one can generate functionally identical molecules by using the same sequence. Each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. and is provided as one embodiment of the present invention.

[0053] Degenerate codon substitution also refers to the substitution of one or more (or all) of the third positions of a selected codon with a mixed base pair. This can also be achieved by generating sequences substituted with cyclic 3'-amino acids 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).

[0054] The nucleic acid of the present invention includes or consists of a sequence selected from SEQ ID NOs: 5 to 6 or 7 to 8. The sequence has many silent variants (e.g., 1-50, 1-25, etc., especially 1-5, may contain one codon in , and one codon in may be modified.

[0055] The nucleic acids of the invention have the sequence of SEQ ID NO: 1 excluding the first codon for methionine (i.e., ATG or AUG). 7 to 8 or a variant thereof as described above, or consisting of the sequence selected from the sequence This sometimes happens.

[0056] In one embodiment, the nucleic acid of the invention is RNA. The RNA sequences provided herein are The DNA sequence is then converted to a ribonucleotide backbone instead of a deoxyribonucleotide backbone. It has a cytoplasmic base, and the side chain base is uracil (U) instead of thymine (T).

[0057] Thus, the nucleic acid of the present invention is an RNA equivalent of a cDNA sequence selected from SEQ ID NOs: 5-6 or 7-8. It comprises or consists of a number of silent variants (e.g. For example, 1 to 50, 1 to 25, etc., particularly 1 to 5, and even 1 codon may be modified. An "RNA equivalent" is one that contains the same genetic information as a reference cDNA sequence (i.e., It has a ribonucleotide backbone instead of a nucleotide backbone, and the side chain base is thymine (T). "RNA sequences" refers to RNA sequences that contain identical codons with uracil (U) instead.

[0058] The present invention also includes sequences complementary to the above cDNA and RNA sequences. In one embodiment, the nucleic acid of the invention is a polypeptide that is optimized for expression in a human host cell. It's Don. The nucleic acid of the present invention, in the case of a DNA nucleic acid, can be transcribed and translated into a polypeptide of the present invention. and, in the case of an RNA nucleic acid, can be translated into a polypeptide of the invention.

[0059] (Polypeptides and Nucleic Acids) Preferably, the polypeptides and nucleic acids used in the present invention are isolated. A "removed" polypeptide or nucleic acid is one that has been removed from its original environment. For example, a naturally occurring The original polypeptide or nucleic acid is separated from some or all of the materials with which it coexists in the natural system. A nucleic acid is isolated when it is isolated from a vector that is not part of its natural environment. A gene is considered isolated when it is cloned into a target cell.

[0060] "Naturally occurring," when used in reference to a polypeptide or nucleic acid sequence, means a polypeptide that is not found in nature. By this is meant a sequence which has been produced and has not been synthetically modified. "Artificial" when used in reference to a polypeptide or nucleic acid sequence refers to, e.g., a naturally occurring It refers to a sequence that is not found in nature, including synthetic modifications of a sequence or non-naturally occurring sequences. .

[0061] The term "heterologous" refers to the relationship of one nucleic acid or polypeptide to another nucleic acid or polypeptide. When used in the context of, two or more sequences are not found in the same relationship to each other in nature. A "heterologous" sequence also refers to a nucleic acid sequence that is naturally occurring or found within a host organism. is not isolated from, derived from, or based on a polypeptide sequence It can also mean:

[0062] As mentioned above, the polypeptide variants preferably have at least one amino acid sequence which is at least 100% amino acid sequence longer than the overall length of the relevant reference sequence. At least about 80% identity, more preferably at least about 85% identity, and most preferably at least at least about 90% identical (e.g., at least about 95%, at least about 98%, or at least about 99%) has.

[0063] For purposes of comparing two closely related polypeptide or polynucleotide sequences, first The "% sequence identity" between the sequence of the first sequence and the second sequence may be calculated. 0% sequence identity, the polypeptide sequence is identical to the other polypeptide sequence. The residues in the sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus of the polypeptide. The terms "identical" or percent "identity" refer to two or more polypeptides. In the context of a field array, they are compared and aligned for maximum correspondence over a comparison window. When the amino acid sequence is modified, it is determined that the amino acid sequence is identical or has a certain percentage of identical amino acid residues. (i.e., 70% identity within a specified region, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99%) The comparison refers to a comparison of two or more sequences or subsequences (percent identity). Preferably, the comparison is performed over the entire length of the reference sequence. It is executed in the corresponding window.

[0064] In sequence comparison, one sequence acts as a reference sequence to which test sequences are compared. When using a comparison algorithm, the test and reference sequences are input into a computer and, if necessary, Specify the sub-array coordinates according to the above, and then specify the array algorithm program parameters. Default program parameters may be used, or alternative parameters may be designated. The sequence comparison algorithm then performs a comparison of the matched sequences with respect to the reference sequence, based on the program parameters. The percentage sequence identity of the test sequences is calculated.

[0065] As used herein, a "comparison window" refers to a window within which one sequence and the same number of contiguous A comparison of the two sequences is a segment of a sequence that can be compared to a reference sequence at a position that matches the position of the two sequences. This is done after the sequences have been optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal sequence alignment for comparison is well known in the art, for example, Smith and Waterman, "Local Homology Algorithm," Adv. Appl. Math. 2:482, 1981, by Needleman and W. by the search for similarity algorithm of Pears and Unsch, 1970, J. Mol. Biol. 48:443. According to the similarity search method of On and Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444, These (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Gen The Department of Computer Science, 575 Science Dr., Madison, WI by a filtered implementation or by manual alignment and visual inspection (e.g., "Current Trends in Molecular Biology" "Current Protocols in Molecular Biology," edited by Ausubel et al., 1995, supplemented; This can be done by

[0066] One example of a useful algorithm is PILEUP, which performs progressive pairwise Use alignment to create multiple sequence alignments from related sequences and compare them The relationship and percent sequence identity are shown. This is also used to create the alignment. PILEUP plots a tree or dendrogram showing the clustering relationships found in Feng and A simple implementation of Doolittle's progressive alignment method (1987, J. Mol. Evol. 35:351-360) The method used is that described in Higgins and Sharp, 1989, CABIOS 5:151-153. The program is similar to the method described in . The maximum length of each is 5,000 nucleotides. A maximum of 300 sequences can be aligned by amino acid sequence or amino acid sequence. The alignment procedure begins with a pairwise alignment of the two most similar sequences and then This cluster is then divided into two groups based on the most related sequences. Align two clusters of sequences by using two The alignment is made by a simple extension of the pairwise alignment of the individual sequences. The alignment is achieved by a series of progressive, pairwise alignments. The program allows you to specify specific sequences and their amino acid coordinates for a region of sequence comparison. and by specifying the program parameters. Use UP to compare the reference sequence with the other test sequence using the following parameters: Default Gy Gap weight (3.00), default gap length weight (0.10), and weighted end gap The percent sequence identity relationship is determined using PILEUP, a GCG sequence analysis software. Software packages, e.g., version 7.0 (Devereaux et al., 1984, Nuc. Acids Res. 12:387-395).

[0067] Other suitable algorithms for determining percent sequence identity and sequence similarity Examples of such algorithms are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., 1977 and 1980, respectively. Nuc. Acids Res. 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol. 215:403- Software for performing BLAST analyses is available from the National Center for Publicly available via the Biotechnology Information website at www.ncbi.nlm.nih.gov / This algorithm aligns words of the same length in the database sequence. If the query is matched with or satisfies some positive threshold score T, then the query High-scoring sequence pairs (HSPs) are first identified by identifying short word lengths W in the sequences. T refers to the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits initiate searches to find longer HSPs containing them. The word hits act as seeds for the search. The cumulative score is calculated based on the nucleotide sequence. The parameters M (reward score for a matching residue pair; always >0) and N (reward score for mismatching residues) are For amino acid sequences, the score is calculated using the penalty score (always <0). A cumulative score is calculated using a matching matrix. Extension stops when: the cumulative alignment score falls by X amount from its maximum achieved value; The product score became 0 or less due to accumulation of 1 or more negative scoring residue alignments; Or, the end of either sequence is reached. For amino acid sequences, use the BLASTP program. The program was set as default, with a word length of 3, an expectation (E) of 10, and the BLOSUM62 score. The approximation matrix (Henikoff and Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89 :10915) with an alignment (B) of 50, expectation (E) of 10, M=5, N=-4, and both strands Comparison is used.

[0068] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and A. (See, for example, Ltschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by algorithms is the minimum sum probability (P(N)), which is the sum of two provides an indication of the probability that a match between two nucleotide sequences or between two amino acid sequences would occur by chance do.

[0069] A "difference" between sequences is a difference of one residue at one position in the second sequence compared to the first sequence. This refers to an insertion, deletion, or substitution. Two sequences may have one, two, or more such differences. An insertion in a second sequence that is otherwise identical to the first sequence (100% sequence identity). For example, if the identical sequence is 9 residues long, A substitution of 1 in the second sequence results in a sequence identity of 88.9%. In the long case, two substitutions in the second sequence result in 88.2% sequence identity.

[0070] Alternatively, a second sequence may be generated for the purpose of comparing a first reference sequence to a second comparison sequence. In order to identify the number of additions, substitutions and / or deletions that have been made to the first sequence, one may An addition is the addition of one residue to the first sequence (at either end of the first sequence). A substitution is defined as replacing one residue in a first sequence with a different residue. A deletion of one residue is a deletion of one residue from the first sequence (first sequence (including deletions at either end of the gene).

[0071] (Production of Polypeptides of the Invention) The polypeptides of the present invention can be prepared, for example, as described in Green and Sambrook, 2012, "Molecular Cloning. Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring It can be obtained and manipulated using techniques disclosed in Harbour Laboratory Press. In particular, polynucleotides can be generated using artificial gene synthesis techniques. (Nambiar et al., 1984, Science, 223:1299-1301; Sakamar and Khorana, 1988, Nucl. Acids Res., 14:6361-6372; Wells et al., 1985, Gene, 34:315-323; and Grund Strom et al., 1985, Nucl. Acids Res., 13:3305-3316), followed by expression in a suitable organism. The gene encoding the polypeptide of the present invention may be, for example, For example, the entire gene can be synthetically produced by solid-phase DNA synthesis. To obtain the desired oligonucleotide, it may be synthesized de novo without the need for NA. The building blocks are then assembled into a growing oligonucleotide in the order required by the product sequence. Once chain assembly is complete, the product is released from the solid phase into solution. The product is isolated by high performance liquid chromatography (HPLC) to give the desired product. The oligonucleotides can be obtained in high purity (Verma and Eckstein, 1998, An nu. Rev. Biochem. 67:99-134). These relatively short segments can be amplified by a variety of gene amplification techniques. (Methods Mol Biol., 2012;834:93-109) can be used to easily assemble longer DNA molecules. These compounds are suitable for use in a myriad of recombinant DNA-based expression systems. In the clear context, the skilled artisan can easily derive a polynucleotide encoding a polypeptide antigen according to the present invention. The nucleotide sequence is suitable for use in a variety of vaccine production systems, including, for example, viral vectors. You will find it easy to use.

[0072] For purposes of producing the polypeptides of the invention in microbial hosts (e.g., bacterial or fungal), To this end, the nucleic acid of the present invention may be provided with suitable regulatory and control sequences (promoters, termination signals, etc. ), as well as a polypeptide for promoting secretion suitable for protein production in a host. Similarly, the polypeptides of the present invention may be expressed in eukaryotic cells (e.g., Chinese The culture of the cells (Hamster ovary cells or Drosophila S2 cells) was transfected with the nucleic acid of the present invention. In order to achieve this, suitable regulatory and control sequences (including promoters, termination signals, etc.) and their In combination with a sequence for promoting polypeptide secretion suitable for protein production in cells. Therefore, it could be generated by transduction.

[0073] Improved isolation of the polypeptides of the invention produced by recombinant techniques can optionally be achieved by isolating the polypeptides A stretch of histidine residues towards one end of the peptide (commonly known as a His tag) This would be facilitated by adding Polypeptides may also be prepared synthetically.

[0074] (vector) In additional embodiments, a genetic construct comprising one or more of the nucleic acids of the invention is When introduced into cells, the polypeptide of the present invention is produced in vivo and elicits an immune response. Nucleic acids (e.g., DNA) can be expressed in a variety of nucleic acid expression systems, including bacterial and some viral expression systems, as known to those skilled in the art. The gene delivery system may be present in any of a variety of delivery systems known to those skilled in the art. See, for example, Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems. 15:143-198, and references cited therein. Some of these approaches are outlined below for illustrative purposes.

[0075] From the above, vectors (also referred to herein as "DNA expression constructs" or In one embodiment, a method for producing a nucleic acid molecule comprising the steps of: Preferably, the vector is capable of transcribing a translationally active RNA molecule in a human host cell. The gene encoding the appropriate regulatory elements (such as a suitable promoter and termination signal) to enable "Translationally active RNA molecules" include nucleic acids that are capable of being converted into proteins by the translational apparatus of human cells. It is an RNA molecule that can be translated into protein. From the above, a vector containing the nucleic acid of the present invention (hereinafter referred to as the "vector of the present invention") " is provided.

[0076] In particular, the vector may be a viral vector. adeno-associated viruses (AAV) (e.g., AAV types 5 and 2), alphaviruses (e.g., vector Venezuela equine encephalitis virus (VEEV), Sindbis virus (SIN), Semliki Forest virus (SF V), herpesviruses, arenaviruses (e.g., lymphocytic choriomeningitis virus (LCMV) ), measles virus, poxvirus (modified vaccinia virus (MVA), etc.), paramyxovirus Vectors such as vesicular stomatitis virus (VSV) can also be used. Well, the vector can be derived from any of the above viruses. The virus is characterized by its medium genome size, ease of manipulation, high titer, wide target cell range, and high Due to its rapid infectivity, this virus is particularly suitable for use as a gene transfer vector. Both ends of the genome contain inverted repeats (ITRs) of 100 to 200 base pairs, which are The early (E) and later (L) regions of the genome are cis elements necessary for the synthesis and packaging of ribosomal RNA. The late (L) region contains different transcription units, which are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) regulates the transcription of the viral genome and some cellular genes. Expression of the E2 region (E2A and E2B) is required for viral DNA replication. These proteins are involved in DNA replication, expression of late genes, and and host cell shutoff (Renan, 1990). The products of most late genes are driven by a single major late promoter (MLP). It is expressed only after significant processing of the primary transcript. MLP is expressed less efficiently at later stages of infection. Well, all mRNAs transcribed from this promoter contain a 5'-tripartite linker. It contains a transcriptional activator (TPL) sequence, making it the preferred mRNA for translation. Particularly useful are replication-deficient adenoviruses, which are made from viral genomes that have been deleted for The reason is that they have limited replication in vaccinated hosts and thus limit the spread of the pathogen. This is because the likelihood of infection is low, and the likelihood of pathogenic contact in a vaccinated host is also low. .

[0077] (Other Polynucleotide Delivery) In certain embodiments of the invention, an expression construct comprising one or more polynucleotide sequences comprises: It may simply consist of a naked recombinant DNA plasmid. Ulmer et al., 1993 , Science 259:1745-1749 and reviewed by Cohen, 1993, Science 259: Introduction of the construct can be achieved, for example, by physically or chemically modifying the cell membrane. This can be achieved by any method that renders the cells permeable. The method is applicable to the introduction of a gene of interest into a host cell, but is equally applicable for in vivo use. It has been shown that DNA encoding such a gene can also be introduced in vivo in a similar manner to express the gene product. A number of delivery systems are used to deliver DNA molecules to animal models and humans. Several products based on this technology have been approved for use in animals and have been shown to be effective in humans. There are others in Phase II and III clinical trials.

[0078] (RNA delivery) In another embodiment of the invention, an expression construct comprising one or more polynucleotide sequences is Alternatively, the RNA may be derived from recombinant DNA that is soluble in the endothelial cells (Ulmer et al., 2012, Vaccine 30:4414-4418). For DNA-based expression constructs, various methods have been used to in vitro express RNA molecules. RNA-based constructs can be used for delivery into cells either in vivo or in vivo. By mimicking messenger RNA (mRNA) molecules, the introduced biological molecule is able to interact with the host cell's translation machinery. to produce the polypeptide it codes for in the cell into which it is introduced. Alternatively, the RNA molecule can be designed to act as a transcription factor for viral RNA-dependent RNA polymerase. In order to do so, they are incorporated into the structural genes of the target cells, allowing them to self-regulate in the cells into which they are introduced. The mRNA may be designed in such a way that it can be amplified. Thus, self-amplifying mRNA (SAM™) This type of RNA molecule, known as the ribozyme molecule (Geall et al., 2012, PNAS, 109:14604-14609), is It shares characteristics with some RNA-based viral vectors. mRNA-based RNA or SAM®R Any of the NAs may be modified (e.g., by alteration of their sequence or by the use of modified nucleotides). Thus, further modifications can be made to enhance stability and translation (Schlake et al., RNA Biol. logy, 9:1319-1330), and both types of RNA can be formulated (e.g., in emulsions (Brito (Kranz et al., Molecular Therapy, 2014 22:2118-2129) or in lipid nanoparticles (Kranz et al., Molecular Therapy, 2014 22:2118-2129). (2006, Nature, 534:396-401) and in vitro or in vivo stability and / or Myriad formulations of modified (and unmodified) RNA may facilitate cell entry. A number of RNA-based vaccines have been tested as vaccines in models and humans. is being used in ongoing clinical trials.

[0079] Pharmaceutical Composition The polypeptides, nucleic acids and vectors of the present invention can be used in immunogenic and vaccine compositions, etc. It may be formulated for delivery in a pharmaceutical composition (hereinafter all referred to as the "composition of the invention"). The composition of the present invention preferably comprises a polypeptide, a nucleic acid or a vector of the present invention as a medicine. together with an acceptable carrier. Thus, in one embodiment, the polypeptide, nucleic acid or vector of the invention is used as a medicament. Immunogenic pharmaceutical compositions comprising the compound in association with an acceptable carrier are provided.

[0080] In another embodiment, the polypeptide, nucleic acid or vector of the invention is The vaccine composition is generally prepared by the steps of: See, e.g., Powell and Newman, eds., Vaccine Design (Subunit and Adjuvant Approaches), " (Vaccine Design (the subunit and adjuvant approach)), 1995. The compositions of the invention can also contain other compounds, which may be biologically active or inactive. Preferably, the compositions of the present invention are sterile compositions suitable for parenteral administration.

[0081] In certain preferred embodiments of the present invention, the pharmaceutical compositions of the present invention are The present invention also includes one or more (e.g., one) polypeptides of the present invention in combination with a carrier. Provided. In certain preferred embodiments of the present invention, the compositions of the present invention comprise a pharma- ceutically acceptable cysteine. One or more (e.g., one) nucleic acids of the invention in combination with a carrier or one or more (e.g., one) nucleic acids of the invention The vectors of the invention are provided as including.

[0082] In one embodiment, the compositions of the invention comprise one or more (e.g., one) polynucleotides and one or more Alternatively, the composition may include the above (e.g., one) polypeptide component. The article may comprise one or more (e.g., one) vector and one or more (e.g., one) polypeptide components. Alternatively, the composition may comprise one or more (e.g., one) vector and one The polynucleotide may include one or more (e.g., one) polynucleotide components. The composition may be provided to enhance the immune response.

[0083] (Pharmaceutical acceptable salts) The compositions of the invention comprise pharma- ceutically acceptable versions of the nucleic acids or polypeptides provided herein. It will be apparent that the present invention may include salts thereof which are pharma- ceutical acceptable. The bases can be prepared from non-toxic bases, such as organic bases (e.g., primary, secondary and tertiary amines, and salts of basic amino acids) and inorganic bases (e.g., sodium, potassium , lithium, ammonium, calcium and magnesium salts).

[0084] Pharmaceutically acceptable carriers Many pharma- ceutically acceptable carriers known to those of skill in the art may be used in the compositions of the present invention. However, 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 administration includes, for example, intramuscular, subcutaneous or intravenous administration. For parenteral administration, The carrier preferably comprises water, and may also contain buffers for adjusting pH, stabilizers (e.g., surfactants, etc. and amino acids), and tonicity adjusting agents (e.g., salts and sugars). If the composition is to be provided in lyophilized form for dilution at the time of use, the formulation may contain a lyoprotectant. For oral administration, the carriers may include sugars such as trehalose. or any solid carrier, mannitol, lactose, starch, magnesium stearate Nesium, sodium saccharin, talcum, cellulose, glucose, sucrose, and and magnesium carbonate, etc. can be used.

[0085] Thus, the compositions of the invention may be prepared using a buffer (e.g., neutral buffered saline or phosphate buffered saline). buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrose), strun), mannitol, proteins, polypeptides, or amino acids such as glycine , antioxidants, bacteriostatic agents, chelating agents such as EDTA or glutathione, and preparations Contains solutes, suspending agents, thickening agents, and / or preservatives that render the solution isotonic, hypotonic, or slightly hypertonic with blood Alternatively, the compositions of the present invention may be formulated as a lyophilizate.

[0086] (Immunostimulant) The compositions of the present invention may also include one or more immunostimulants. Any agent that enhances or strengthens the immune response (antibody-mediated and / or cell-mediated) to an antigen. Immunostimulants are often referred to as adjuvants in the context of vaccine formulations, Examples include aluminum hydroxide gel (alum) or aluminum phosphate. salts, saponins such as QS21, immunostimulatory oligonucleotides such as CPG, oil-in-water emulsions ion (for example, when the oil is squalene), aminoalkyl glucosaminide 4-phosphate, lipopolysaccharide Sugars or derivatives thereof (e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPL (registered trademark) and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and In view of the above, the one or more immunostimulants of the composition of the present invention are preferably Suitable are aluminum salts, saponins, immunostimulatory oligonucleotides, oil-in-water emulsions. phosphates, aminoalkyl glucosaminide 4-phosphates, lipopolysaccharides and their derivatives, The immunostimulatory agent is selected from other TLR4 ligands, TLR7 ligands, TLR8 ligands and TLR9 ligands. Active agents may also be monoclonal antibodies that specifically interact with other immune components, e.g. A monoclonal antibody that blocks the interaction of immune checkpoint receptors including PD-1 and CTLA4 It may also include antibodies.

[0087] For recombinant nucleic acid delivery methods (e.g., DNA, RNA, viral vectors), protein-based The gene encoding the immunostimulant of the present invention is combined with the gene encoding the polypeptide of the present invention. The drug may be conveniently delivered to the patient.

[0088] (Sustained Release) The compositions described herein are sustained release formulations that result in sustained / sustained release of the compound after administration. Part of a drug (i.e. a preparation such as a capsule, sponge, patch or gel (e.g. made of a polysaccharide) It can be administered as a

[0089] (Storage and Packaging) The compositions of the invention may be contained in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be hermetically sealed to ensure sterility of the formulation until use. The formulations are generally prepared as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, the compositions of the present invention may be stored in a lyophilized state. Simply add a sterile liquid carrier (such as water for injection or saline) immediately before use. It may be whatever you need.

[0090] (Dosage) The amount of nucleic acid, polypeptide or vector in each composition of the invention may vary depending on the therapeutic or prophylactic use. The composition may be formulated in such a way that a suitable dosage for the intended use is obtained. Factors such as lability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations. Considerations should be taken into account by those skilled in the art in preparing such compositions, and therefore various dosage forms may be used. Positivity and treatment regimens will be as desired.

[0091] Typically, a composition containing a therapeutically or prophylactically effective amount is about 0.1 ug to about 1000 ug of the compound per administration. The polypeptides of the invention, more typically, are delivered in an amount of about 2.5 ug to about 100 ug per dose. When delivered in the form of short synthetic long chain peptides, the dosage is 1-200ug / peptide / dose. For polynucleotide compositions, these typically range from about 10 ug to about 10 ug per dose. Approximately 20 mg of the nucleic acid of the invention is delivered, more typically from about 0.1 mg to about 10 mg of the nucleic acid of the invention per administration. Reach.

[0092] (Disease to be treated or prevented) As described elsewhere in the specification, SEQ ID NOs: 1-2 are intended to be used in the treatment of ovarian serous cystadenocarcinoma. Polypeptide sequences corresponding to overexpressed CLT antigens. In one embodiment, the present invention provides a polypeptide, a nucleic acid, or a polypeptide of the present invention for use in medicine. A vector or composition is provided.

[0093] A further aspect of the invention is a method of increasing an immune response in a human, comprising administering to the human The method comprises administering a polypeptide, a nucleic acid, a vector or a composition of the invention. do. The present invention also relates to a method for the production of a polypeptide, nucleic acid, or nucleic acid fragment of the present invention for use in raising an immune response in humans. The present invention provides an acid, vector or composition. A polypeptide of the invention for the manufacture of a medicament for use in raising an immune response in humans. Uses of the nucleic acid, vector or composition are also provided.

[0094] Preferably, the immune response is directed to SEQ ID NOs: 1-2 and variants thereof, or any one of them. The immunogenic fragment of the present invention is a fragment of the present invention. "Corresponding" in this context means that the tumor corresponds to, for example, SEQ ID NO: A (A corresponds to SEQ ID NO: 1-2). The polypeptide of the invention is one of the polypeptides shown in The peptide, nucleic acid, vector or composition and pharmaceuticals containing them are This means that the antibody will be based on the antibody or an immunogenic fragment thereof.

[0095] Suitably, the immune response is a CD8+ T cell response, a CD4+ T cell response and / or an antibody response, in particular a CD8 + cytolytic T cell responses and CD4+ helper T cell responses. Preferably, the immune response is directed against tumors, particularly against SEQ ID NOs: 1-2 and variants thereof and immunogenic fragments thereof. The vector is induced to express a sequence selected from the fragment.

[0096] In a preferred embodiment, the tumor is an ovarian cancer, in particular an ovarian carcinoma, more in particular a serous ovarian carcinoma, e.g. For example, ovarian serous cystadenocarcinoma. The tumor may be a primary tumor or a metastatic tumor.

[0097] A further aspect of the invention is a method of treating a human patient suffering from cancer, comprising administering to said patient a therapeutically effective amount of The cell is selected from SEQ ID NO: 1 to 2 and any one of immunogenic fragments and variants thereof. The method of claim 1, further comprising expressing a sequence comprising administering to a subject a therapeutically effective amount of a therapeutically effective amount of a medicament for the treatment of a cancer. A sequence selected from SEQ ID NOs: 1 to 2 and any one of their immunogenic fragments and variants. The method will express the corresponding polypeptides, nucleic acids, vectors of the invention. or administering the composition to said human.

[0098] The present invention also relates to a method for the treatment or prevention of cancer in humans, comprising administering to said humans a polypeptide, a nucleic acid, A vector or composition, wherein the cancer cells are selected from SEQ ID NOs: 1 to 2 and any one of them. A polypeptide, nucleic acid, or vector that expresses a corresponding sequence selected from the immunogenic fragments. The present invention provides a method and composition for the treatment of cancer. The terms "prevention" and "prophylaxis" are used interchangeably herein.

[0099] (Treatment and vaccination regimens) The therapeutic regimen comprises: (i) a polypeptide, nucleic acid or vector of the invention; and (ii) one or more Further polypeptides, nucleic acids or vectors of the invention and / or (iii) various other therapeutic and optionally further components such as compounds useful for the treatment of Coadministration with adjuvant, either at the same time (e.g., simultaneous administration) or sequentially (e.g., prime-boost) Examples of simultaneous administration include simultaneous administration to the same external site. Simultaneous administration includes administration to opposite, contralateral sites. This refers to all components being delivered during the same treatment session. The components are administered simultaneously (e.g., both DNA and protein are administered simultaneously), but even if the drugs are administered within minutes (e.g., during the same appointment or doctor's visit) or even within a few hours. good.

[0100] In some embodiments, "priming" a polypeptide, nucleic acid or vector of the invention or following the first administration, one or more "boosters" of the polypeptides, nucleic acids or vectors of the invention. In one embodiment, a second "prime and boost" or booster dose is administered (a "prime and boost" regimen). The polypeptide, nucleic acid or vector of In one embodiment, both the prime and boost are polypeptides of the invention. and in each case the same polypeptide of the invention. Both the gene and the booster are nucleic acids or vectors of the invention, and in each case Alternatively, the priming may be performed using a nucleic acid or vector of the present invention. The boost may be performed using a polypeptide of the present invention, and the prime may be performed using the polypeptide of the present invention. The boosting is carried out using the nucleic acid or vector of the invention. Typically, this involves a first or "priming" administration and a second or "boosting" administration. The next booster dose is administered after an interval of about 1 to 12 weeks, or up to 4 to 6 months. Doses may be given as frequently as every 1-6 weeks, or later (up to several years later). This is also fine.

[0101] (Combination of antigens) The polypeptide, nucleic acid or vector of the invention may be synthesized in combination with one or more other polypeptides or vectors of the invention. or in combination with a nucleic acid or vector, and / or to enhance immunity against ovarian cancer. Other antigenic polypeptides (or polynucleotides encoding same) that elicit a response These other antigenic polypeptides can be obtained from a variety of sources. The sources may be well-described proteins such as MUC16, CRABP1 / 2, FOLR1, and KLK10. These sources may include ovarian cancer tumor-associated antigens (TAAs). The resulting antigenic peptide may also be a non-specific immunostimulant / adjuvant species, and / or a For example, it contains universal CD4 helper epitopes and induces strong CD4 helper T cells. The antigens (as polypeptides or as polynucleotides encoding these CD4 antigens) are known to In combination with co-administered antigens (delivered as peptides or vectors), It may also be possible to amplify the anti-ovarian cancer specific response elicited by the antibody.

[0102] The different polypeptides, nucleic acids or vectors may be formulated in the same formulation or in separate formulations. Alternatively, the polypeptide may be a polypeptide of the invention, in which the polypeptide of the invention is a second polypeptide. or may be provided as a fusion protein fused to a further polypeptide (see below). (see ).

[0103] A nucleic acid may be provided that encodes the fusion protein. More generally, when two or more components are used in combination, For example, (1) As two or more individual antigenic polypeptide components, (2) as a fusion protein containing both (or additional) polypeptide components , (3) as one or more polypeptide and one or more polynucleotide components, (4) as two or more individual polynucleotide components, (5) As a single polynucleotide encoding two or more individual polypeptide components. Or, (6) Encoding a fusion protein containing both (or further) polypeptide components. As a single polynucleotide to be encoded, There could be.

[0104] For convenience, where multiple components are present, they may be expressed as a single fusion protein or a single It is often desirable to include the nucleotide sequence of the fusion protein in a single polynucleotide encoding the fusion protein. In one embodiment of the invention, all components are polypeptides. In another embodiment of the invention, the entire All components may be polynucleotides (e.g., those encoding a single fusion protein). The nucleic acid sequence is provided as a single polynucleotide such as a single polynucleotide.

[0105] (Fusion Protein) As an embodiment of the above discussion of antigen combinations, the present invention also provides a method for producing a combination of antigens encoding individual antigens. The second or further nucleic acid constructs of the invention can be prepared by fusing together sequences which correspond to the nucleic acid sequences of the present invention. The isolated polypeptide of the present invention fused to a polypeptide (hereinafter, "the combination of the present invention") The combination polypeptides of the present invention are The peptides are predicted to have the utility described herein and to have excellent immunity. Epidemiogenic or vaccine activity, or prophylactic or therapeutic effect (increasing the breadth and depth of response) These may have advantages including improved cloning and cloning ability (including improved cloning ability) and may be of particular value in outbred populations. Fusion of the polypeptides of the present invention may also be used as vaccine antigens and / or vectored vaccines (nucleic acid vaccines). It would also provide the advantage of increasing the efficiency of construction and manufacturing of composite materials (including cutins).

[0106] As described above in the "Combination of Antigens" section, the polypeptide of the present invention and the Combination polypeptides may also include polypeptide sequences that are not polypeptides of the invention. , may be fused to include one or more of the following: (a) an ovarian cancer-associated antigen and therefore potentially useful as an immunogenic sequence in a vaccine; other polypeptides of interest (e.g., MUC16, CRABP1 / 2, FOLR1 and KLK10, mentioned above); Beauty (b) A polypeptide sequence capable of enhancing an immune response (ie, an immunostimulatory sequence). (c) For example, it contains a universal CD4 helper epitope and can provide strong CD4+ help. and augmenting CD8+ T cell responses to CLT antigen epitopes. Petite arrangement.

[0107] The present invention also relates to a nucleic acid encoding the fusion protein and to a polypeptide of the invention. Mutatis mutandis, other aspects of the invention (vectors, compositions, cells, etc.) are provided.

[0108] (CLT antigen-binding polypeptide) An antigen-binding polypeptide that is immunospecific for a tumor-expressed antigen (the polypeptide of the present invention) The IL-1 receptor agonist (IL-1 receptor agonist) is a cytolytic agent that recruits cytolytic cells to antigen-modified tumor cells to mediate their destruction. The mechanism of recruitment of cytolytic cells by such antigen-binding polypeptides may be One of the mechanisms is known as antibody-dependent cell-mediated cytotoxicity (ADCC). The present invention provides antigen-binding polypeptides immunospecific for the polypeptides of the invention. Monoclonal antibodies and fragments thereof, such as domain antibodies, Fab fragments, Fv fragments, and VHH fragments. In some embodiments, antigen-binding polypeptides, including antibodies, are expressed in non-human animal species (e.g., rodents or camelids). The antibodies may be produced in a non-human species (e.g., modified to have a human immune system) and humanized. It may also be produced in genetically engineered rodents.

[0109] Antigen-binding polypeptides can be produced by methods well known to those skilled in the art. Clonal antibodies are produced by using hybridoma technology to culture specific antibody-producing B cells in tissue culture. Fuse with myeloma (B cell cancer) cells selected for their proliferation potential and lack of antibody chain synthesis. (Kohler and Milstein, 1975, Nature 256(5517):495-49 7 and Nelson et al., June 2000, Mol Pathol. 53(3):111-7, which are hereby incorporated by reference in their entirety. (The body of this application is incorporated herein.)

[0110] Monoclonal antibodies against a desired antigen can be prepared, for example: a) Lymphocytes obtained from the peripheral blood of animals (including humans) that have been previously immunized / exposed to the desired antigen. immortalizing the spheres with immortal cells, preferably myeloma cells, to form hybridomas; b) Cultivating the resulting immortalized cells (hybridomas) to produce antibodies with the desired specificity and recovering the cells. can be obtained by

[0111] Monoclonal antibodies can be obtained by a process comprising the following steps: (a) vectors, particularly phages, more particularly filamentous bacteriophages, (preferably DNA or other nucleic acid obtained from lymphocytes, particularly peripheral blood lymphocytes, of an animal previously immunized with a desired antigen. Cloning step of cDNA sequence (b) transforming a prokaryotic cell with the vector under conditions allowing antibody production; (c) selecting the antibodies by subjecting them to antigen affinity selection; (d) recovering antibodies having the desired specificity; (e) derived from B cells of patients exposed to an antigen or from animals immunized in the laboratory with the antigen Also, expressing the antibody-encoding nucleic acid molecule. The selected antibodies can then be produced using conventional recombinant protein production techniques (e.g., The antibody may be produced from engineered CHO cells.

[0112] The present invention provides isolated antigen-binding polypeptides immunospecific for the polypeptides of the invention. Preferably, the antigen-binding polypeptide is a monoclonal antibody or a fragment thereof. It is. In certain embodiments, the antigen-binding polypeptide is conjugated to a cytotoxic moiety. An example of a cytotoxic moiety includes the Fc domain of an antibody, which binds to Fc receptor-bearing cells to promote ADCC. Alternatively, the antigen-binding polypeptide may be a biological toxin or a cytotoxic agent. The compound may be linked to a chemical substance.

[0113] Another important class of antigen-binding polypeptides is the polypeptides that bind to HLA-presenting fragments of the antigens of the invention. In this embodiment, the CLT antigen (or its derivatives) on the surface of a tumor cell is TCR-based biologics (TCRs derived directly from patients or specifically engineered TCRs) that recognize The TCRs (containing the high affinity TCRs) also reside on T cells (or other types of immune cells) and mediate these immune responses. and a targeting moiety that recognizes a component that attracts immune cells to tumors. In some embodiments, the targeting moiety may also provide a redirecting effect. They may also stimulate beneficial activities (including cytolytic activity) of stimulated immune cells.

[0114] Thus, in one embodiment, the antigen-binding polypeptide is a polypeptide of the invention or or a portion thereof. The polypeptide is a T cell receptor. In one embodiment, the antigen-binding polypeptides of the invention are administered to a subject via the administration of cytotoxic cells or other The antibody may be linked to another polypeptide capable of binding to an immune component of the antibody.

[0115] In one embodiment, the antigen-binding polypeptide is for use in medicine. In one embodiment, the antigen-binding polypeptides of the invention are administered in a pharma- ceutical acceptable carrier. Such compositions are in a sterile formulation suitable for parenteral administration. For example, see the disclosure of pharmaceutical compositions above.

[0116] According to the present invention, there is provided a method of treating a human suffering from cancer, the method comprising administering to said human a cancer cell comprising the nucleic acid sequence identified as SEQ ID NO: The present invention relates to a method for expressing a sequence selected from the group consisting of sequences 1 to 2 and immunogenic fragments and variants thereof. or a method for preventing a human from contracting cancer, wherein the cancer cells are The present invention relates to a method for expressing a sequence selected from the group consisting of sequences 1 to 2 and immunogenic fragments and variants thereof. The method, which comprises administering to the patient an antigen-binding polypeptide of the present invention or an antigen-binding polypeptide thereof. The method includes administering to the human a composition comprising the peptide.

[0117] In one embodiment, the present invention provides a method for the treatment or prevention of cancer in humans, comprising administering to said patient a cytotoxic moiety conjugated to said patient a cytotoxic moiety. or the antigen-binding polypeptide of the present invention. A composition comprising the above-mentioned compound, wherein the cancer cells are immunogenic in the presence of SEQ ID NO: 1 to 2 and any one of them. the antigen-binding polypeptide or a corresponding sequence selected from the A composition is provided.

[0118] Suitably, in any of the above embodiments, the cancer is ovarian cancer, particularly ovarian carcinoma, especially serous ovarian carcinoma. Carcinoma, especially ovarian serous cystadenocarcinoma.

[0119] The antigen-binding polypeptide (antibody or a fragment thereof, etc.) is, for example, 5 to 1000 mg, for example, 25 to 500 mg For example, it may be administered in a dose of 100 to 300 mg, for example about 200 mg.

[0120] (Cell therapy to enhance antigen presentation in vivo) Any of a variety of cellular delivery vehicles can be used within the pharmaceutical composition to enhance the induction of an antigen-specific immune response. Thus, the present invention provides a method for loading the polypeptide of the present invention ex vivo. or genetically engineered to express a polypeptide of the invention. providing an isolated cell that is an antigen-presenting cell (hereinafter referred to as "APC of the present invention"); Antigen-presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes, and efficient Other cells that may be engineered to become APCs. Such cells do not necessarily have to be. It is not necessary to use a T cell antibody to enhance antigen presentation, improve activation and / or persistence of T cell responses, and / or and / or genetically modified to be immunocompatible (i.e., HLA haplotype-matched) with the receptor. APCs can generally be isolated from any of a variety of body fluids and organs, and may be autologous. The cells may be autologous, allogeneic, syngeneic or xenogeneic.

[0121] In certain preferred embodiments of the invention, dendritic cells or their precursors are used as APCs. Thus, in one embodiment, the APC of the present invention is a dendritic cell. APC (Banchereau and Steinman, 1998, Nature 392:245-251) and may be used prophylactically or therapeutically. It has been shown to be effective as a physiological adjuvant for eliciting therapeutic immunity (T (See Immerman and Levy, 1999, Ann. Rev. Med. 50:507-529.) In general, dendritic cells The vesicles were characterized by their typical shape (stellate in situ and prominent cytoplasmic projections visible in vitro). They have dendrites, the ability to take up, process and present antigens efficiently, and Dendritic cells may also be identified based on their ability to activate naive T cell responses. Of course, specific cell surface expression not normally found on dendritic cells in vivo or in vitro may be involved. The modified dendritic cells may be engineered to express a receptor or ligand. Instead of dendritic cells, antigen-loaded secretory vesicles (Evessels) are also contemplated by the present invention. These xenosomes may be used in immunogenic compositions (Zitvogel et al., 1998, (See Nature Med. 4:594-600.) Thus, in one embodiment, the polypeptides of the invention are The loaded exosomes are provided.

[0122] Dendritic cells and precursor cells may be derived from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood or other sources. Dendritic cells may be obtained from any suitable tissue or body fluid. For example, dendritic cells may be obtained from monocytes obtained from peripheral blood. A combination of cytokines, such as GM-CSF, IL-4, IL-13, and / or TNFα, was added to the culture. Alternatively, cells may be collected from peripheral blood, umbilical cord blood, or bone marrow. The CD34 positive cells were treated with GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and and / or other compounds that induce differentiation, maturation and proliferation of dendritic cells. The cells may be differentiated into dendritic cells by incubating them with water.

[0123] Dendritic cells are conveniently categorized into "immature" and "mature" cells, which allows easy differentiation between two well-characterized phenotypes, however. This nomenclature should not be construed as excluding all possible intermediate steps in differentiation. Immature dendritic cells are characterized as APCs with high antigen uptake and processing capabilities. This correlates with high expression of Fcγ receptors and mannose receptors. In general, expression of these markers is low, but expression of T cell activation markers such as class I and class II MHC is low. Cell surface molecules that mediate cell proliferation, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD4 These cells are characterized by high expression of CD40, CD80, CD86 and 4-1BB.

[0124] Alternatively, APCs can be genetically engineered to express, for example, proteins (or portions thereof or other modifications thereof). A polynucleotide encoding a polypeptide is transfected into the cells. Such transfection may be performed in vitro. The transfected cells may then be transfected into a pharmaceutical composition as described herein. Alternatively, gene transfer vectors can be used to target dendritic cells or other antigen-presenting cells. The vehicle may be administered to a patient to allow transfection to occur in vivo. Transfection of dendritic cells in vivo and in vitro is described, for example, in WO97 / 24447. or the method described in Mahvi et al., 1997, Immunology and Cell Biology 75:456-460. This can be done using any method commonly known in the art, such as the gene gun approach that has been used Antigen loading into dendritic cells may involve injecting dendritic cells or precursor cells with a polypeptide, with DNA (e.g., plasmid vectors) or RNA; or with recombinant bacteria expressing the antigen Viruses (e.g., adenoviruses, adeno-associated viruses (AAV) (e.g., AAV types 5 and 2) , alphaviruses (e.g., Venezuelan equine encephalitis virus (VEEV)), Sindbis virus ( SIN), Semliki Forest virus (SFV), herpesviruses, arenaviruses (e.g., lymphoproliferative LCMV), measles virus, poxvirus (modified vaccinia virus Viruses such as varicella (MVA) or fowlpox), paramyxoviruses, lentiviruses, or rhabdoviruses ( This may be achieved by incubation with a virus such as vesicular stomatitis virus (VSV). Prior to loading the polypeptide, the polypeptide is coupled to an immunological receptor that provides T cell help. The dendritic cells may be covalently attached to a conjugate (e.g., a carrier molecule). Conjugated immunological partners, either individually or in the presence of a polypeptide or vector It may be pulsed in the presence of

[0125] The present invention relates to the preparation of specifically designed, short, chemically synthesized epitope constructs for polypeptide antigens. The encoded fragments are provided for delivery to antigen presenting cells. Those skilled in the art will appreciate that such molecules are Known as growing chain peptides (SLPs), the antigenic polypeptides of the present invention may be used to For stimulating (or loading) cells in vitro (Gornati et al., 2018, Front. Immunol. 9:1484), or as a method for introducing polypeptide antigens into antigen-presenting cells in vivo ( Melief and van der Burg et al., 2008, Nat Rev Cancer, 8:351-60) Therapeutic Platform will understand that you will be provided with a form.

[0126] In one embodiment, the antigen-presenting cells of the invention, preferably dendritic cells, are administered as medicaments. In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising the compound of formula (I) in combination with a carrier suitable for parenteral administration. It may also be a sterile composition, see, for example, the disclosure of pharmaceutical compositions above. In one embodiment, an antigen-presenting cell, preferably a dendritic cell, according to the invention for use in medicine. is provided.

[0127] Similarly, there is provided a method of treating a human suffering from cancer, the method comprising the steps of: and immunogenic fragments and variants thereof. The method or the method for preventing a human from developing cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOs: 1 to 2. and immunogenic fragments and variants thereof. The method further comprises using an antigen-presenting cell of the present invention, preferably a dendritic cell, or an antigen-presenting cell of the present invention. Such methods are provided that include administering to the human a composition comprising antigen presenting cells.

[0128] In one embodiment, the antigen-presenting cells of the invention for use in the treatment or prevention of human cancer, Preferably, the composition comprises a dendritic cell or an antigen-presenting cell of the present invention, The present invention relates to a method for producing a nucleic acid sequence comprising the steps of: The antigen-presenting cell or composition thereof is provided.

[0129] In one embodiment, the exosomes of the present invention are combined with a pharma- ceutical acceptable carrier. Such compositions may be sterile compositions suitable for parenteral administration. See, for example, the disclosure of pharmaceutical compositions above. The compositions may optionally contain an immunostimulant. See the immunostimulant disclosure above. In one embodiment, there is provided an exosome of the invention for use in medicine.

[0130] Similarly, there is provided a method of treating a human suffering from cancer, the method comprising the steps of: and immunogenic fragments and variants thereof. The method or the method for preventing a human from developing cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOs: 1 to 2. and immunogenic fragments and variants thereof. The exosome of the present invention or a composition comprising the exosome of the present invention, to the human.

[0131] In one embodiment, the exosomes or antigen-binding fragments of the present invention are provided for use in the treatment or prevention of cancer in a human. The composition of the present invention contains the exosome, and the cancer cells are selected from SEQ ID NOs: 1 to 2 and the like. The exosome expresses a corresponding sequence selected from any one of the immunogenic fragments above. A method or composition is provided.

[0132] In any one of the above embodiments, suitably the cancer is ovarian cancer, particularly ovarian carcinoma, especially serous ovarian carcinoma. Ovarian carcinoma, for example ovarian serous cystadenocarcinoma.

[0133] (Stimulated T cell therapy) APC-Mediated Generation, In Vivo or Ex Vivo, of T Cells Immunospecific for the Polypeptides of the Invention In addition, autologous or non-autologous T cells can be obtained from a subject, e.g., from peripheral blood, umbilical cord blood, and and / or tumor cells isolated by apheresis and loaded onto the MHC molecule (signal 1) of APC cells. The tumor-associated antigen stimulates the proliferation of T cells with immune-specific TCR for the tumor-associated antigen. This is also fine.

[0134] For successful T cell activation, the costimulatory surface molecules B7 and CD28 are required to bind to antigen-presenting cells, respectively. To achieve optimal T cell activation, the IL-1 receptor must bind to the T cell surface (signal 1). Both signals 1 and 2 are required. In contrast, the anti- Antigenic peptide stimulation (signal 1) fails to induce complete T cell activation and T cell tolerance. In addition to costimulatory molecules, there are inhibitory molecules, such as CTLA-4 and PD-1, that mediate the proliferation and proliferation of T cells. It induces signals to inhibit cellular activation.

[0135] Thus, autologous or non-autologous T cells are stimulated in the presence of a polypeptide of the invention, and the cancer cells express the corresponding polypeptide of the present invention. The antibody may be reintroduced into patients at risk for or suffering from cancer, but only if the The antigen-specific TCR of the patient recognizes the antigen presented by the MHC of the patient and synthesizes the corresponding polypeptide. This is only possible if the compound will target cancer cells that express the gene and induce their death.

[0136] In one embodiment, the ex vivo stimulation and / or expansion of T cells from a human suffering from cancer is and then administering the stimulated and / or expanded T cells to the human for use in treating cancer. and reintroducing the polypeptide, nucleic acid, vector or composition of the invention into said human for the purpose of Things are provided.

[0137] The present invention relates to a method for treating human cancer, the method comprising the steps of: The method comprises expressing a sequence selected from any one of the immunogenic fragments and variants, and removing a population of leukocytes, including at least T cells, optionally together with antigen-presenting cells, from the subject. in the presence of a corresponding polypeptide, nucleic acid, vector or composition of the invention. and stimulating and / or expanding at least the stimulated and / or expanded T cells. reintroducing some or all of the leukocytes, including the cells, into the human. provide.

[0138] In any one of the above embodiments, suitably the cancer is ovarian cancer, particularly ovarian carcinoma, especially serous ovarian carcinoma. Ovarian carcinoma, specifically ovarian serous cystadenocarcinoma.

[0139] In one embodiment, SEQ ID NOs: 1-2 and immunogenic fragments and variants of any one of them. To prepare a T cell population that is cytotoxic against cancer cells expressing a sequence selected from (a) obtaining T cells and antigen-presenting cells from a cancer patient; and (ii) administering to the patient a live T cell. and incubating the T cell population ex vivo with a corresponding polypeptide, nucleic acid, vector or composition of the invention. stimulating and amplifying.

[0140] "Corresponding" in this context means that the cancer cells correspond to, for example, SEQ ID NO: A (A corresponds to SEQ ID NO: 1-2). or a variant or an immunogenic fragment thereof, The group may be in the form of a polypeptide, a nucleic acid or a vector, or a composition comprising one of the above. In vitro stimulation and amplification by No. A or its variants or immunogenic fragments. means.

[0141] For example, in such a process, the culture and expansion is carried out in the presence of dendritic cells. Dendritic cells can be transfected with the nucleic acid molecules or vectors of the invention to deliver the polypeptides of the invention. The peptide will be expressed.

[0142] The present invention relates to a T cell population obtainable by any of the above processes (hereinafter referred to as the present invention The T cell population is provided by the antibody. In one embodiment, T cells stimulated with a polypeptide, nucleic acid, vector or composition of the invention are In one embodiment, a cell that is a T cell of the present invention is provided, hereinafter referred to as a T cell of the present invention.

[0143] In one embodiment, the T cell population or T cells of the invention are administered in a medicament comprising ... Such compositions are suitable for parenteral administration, e.g. It may be a sterile composition. In one embodiment there is provided a T cell population or a T cell of the invention for use in medicine.

[0144] Similarly, there is provided a method of treating a human suffering from cancer, the method comprising the steps of: and immunogenic fragments and variants thereof. The method or the method for preventing a human from developing cancer, wherein the cancer cells are selected from the group consisting of SEQ ID NOs: 1 to 2. and immunogenic fragments and variants thereof. The method further comprises administering to said patient a T cell population or a T cell of the present invention or a T cell population of the present invention. Alternatively, the method comprises administering to the human a composition comprising the T cells.

[0145] In one embodiment, the T cell population of the invention for use in the treatment or prevention of cancer in a human. A composition comprising a T cell of the invention or a T cell population or T cell of the invention, The cell is a cell that contains a corresponding sequence selected from SEQ ID NO: 1 to 2 and an immunogenic fragment of any one of them. The T cell population of the present invention, the T cell of the present invention, or the T cell population or T cell of the present invention, In any one of the above embodiments, suitably the cancer is ovarian cancer, particularly ovarian carcinoma, especially serous ovarian carcinoma, especially ovarian serous cystadenocarcinoma.

[0146] (Therapy using genetically engineered immune cells) All of the above-mentioned CLT antigen-binding polypeptide derivatives form complexes with human HLA molecules. TCR or TCR mimics that recognize peptides derived from CLT antigens (Dubrovsky et al., 2016, Oncoi (see immunology) and can be genetically engineered to be expressed on the surface of T cells (autologous or non-autologous). The antibodies may be expressed in a manner that is then administered as an adoptive T cell therapy for the treatment of cancer.

[0147] These derivatives fall into the "chimeric antigen receptor (CAR)" category and are used herein. For example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor may be used. may also refer to receptors that have been engineered to confer artificial specificity to particular immune effector cells. CARs can be used to confer the specificity of monoclonal antibodies to T cells. and thereby generate large numbers of specific T cells for use in, for example, adoptive cell therapy. CAR is a polypeptide of the present invention that binds to HLA and is capable of regulating cell specificity. The antibody will be directed against a tumor-associated antigen.

[0148] Another approach to treating cancer in patients involves targeting antigens expressed on tumor cells. The goal is to genetically modify T cells to target the immune system through the expression of chimeric antigen receptors (CARs). This technique is described in Wendell and June, 2017, Cell, 168:724-740, which is incorporated herein by reference in its entirety. (which is incorporated herein).

[0149] Such CAR T cells can be prepared by administering to a subject a cell sample comprising T cells or T cell precursors, e.g., For example, cells obtained from peripheral blood, umbilical cord blood and / or by apheresis are bound to HLA. A chimeric T cell receptor (CAR) encoding a chimeric T cell receptor (CAR) that is immunospecific for the polypeptide of the present invention to which it is linked. Such nucleic acids may be produced by transfection of cells. and administering to the subject an effective amount of the cells It is also possible to provide a T cell response against cells expressing the polypeptides of the invention. If desired, a cell sample may be obtained from the subject.

[0150] The cells used to generate the CAR-expressing T cells can be autologous or non-autologous. It is understood that Transgenic CAR-expressing T cells are targeted to endogenous T cell receptor inactivation and / or endogenous HLA inactivation. For example, cells may be modified to eliminate expression of endogenous α / β T cell receptors (TCRs). The device may be operated in such a way that

[0151] Methods for transfecting cells are well known in the art, including electroporation, For example, the highly efficient transfection method of the CAR construct can be used. The nucleic acid or vector of the invention expressing the The gene may be introduced into a cell using a gene expression vector.

[0152] The cell population for CAR-expressing T cells may be enriched following transfection of the cells. For example, CAR-expressing cells can be identified by the use of an antigen bound by the CAR or a CAR-binding antibody. Alternatively, the enrichment step can be to separate (e.g., by FACS) non-expressing cells from non-expressing cells. This includes depleting T cells and depleting non-CAR expressing cells. 56+ cells can be depleted from the culture population.

[0153] The population of transfected CAR-expressing cells is cultured in a medium that selectively enhances the proliferation of CAR-expressing T cells. They may also be cultured ex vivo. Thus, CAR-expressing T cells can be expanded ex vivo.

[0154] A sample of the CAR cells may be stored (or maintained in culture). For example, the sample may be The cells may be frozen for propagation or analysis. CAR-expressing T cells are a promising candidate for other therapies, such as checkpoint blockade, including PD-L1 antagonists. It may also be used in combination with a toxic agent.

[0155] In one embodiment, the antibody is adapted to express any of the above antigen-binding polypeptides on its surface. In one embodiment, a cytotoxic cell is provided which is engineered to be a T cell. Suitably, the cytotoxic cell is a T cell.

[0156] In one embodiment, a pharmaceutical composition comprising the antigen-binding polypeptide on its surface for use in medicine. Cytotoxic cells, preferably T cells, engineered to express either do. The present invention provides a pharmaceutical composition comprising a cytotoxic cell of the present invention, which is preferably a T cell. do.

[0157] A method of treating a human patient suffering from cancer, the method comprising administering to said patient a therapeutically effective amount of the cancer cell line comprising the sequence of SEQ ID NO: 1-2 and SEQ ID NO: 3. The method of the present invention further comprises expressing a sequence selected from any one of the immunogenic fragments and variants thereof in a mammalian animal. or a method for preventing a human from developing cancer, the cancer being a cancer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-2 and the sequences thereof. The method of the present invention will express a sequence selected from the immunogenic fragments and variants of any one of the above. The method comprises administering to the human a cytotoxic cell, preferably a T cell, of the invention. The method is provided.

[0158] In one embodiment, the cytotoxic cells, preferably T cells, of the invention are used to treat or prevent cancer in humans. The cancer cells are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 2 and any one of them. The immunogenic fragment of the present invention expresses a corresponding sequence selected from the following:

[0159] (Combination therapy) The cancer treatment methods of the present invention may be used in combination with other therapies, particularly checkpoint inhibitors and interferon. This may be performed in combination with Polypeptides, nucleic acids, vectors, antigen-binding polypeptides, and (APC and T cell-based) Adoptive cell therapy may increase their immunogenicity (e.g., the magnitude of the immune response they elicit and / or or to improve range), or other activity (e.g., innate or adaptive immune response). Other components engineered to provide other aspects of the activation of the IL-1 receptor, such as activation of other aspects of the IL-1 receptor, or destruction of tumor cells. It can be used in combination with

[0160] Thus, the present invention relates to a composition (i.e., an immunogenic, vaccine or pharmaceutical composition) of the invention. or a kit of several of these compositions together with a pharma- ceutical acceptable carrier. a polypeptide, nucleic acid, or vector of the invention; and (i) one or more further immunogenic or or immunostimulatory polypeptides (e.g., interferon, IL-12, checkpoint inhibitors) (ii) a small molecule (e.g., For example, HDAC inhibitors or other drugs that alter the epigenetic profile of cancer cells) or biologics ( The polypeptide or a nucleic acid encoding the polypeptide, or a vector containing the nucleic acid and (c) enhancing the translation and / or presentation of the polypeptide product that is the subject of the present invention. Provides services that include:

[0161] Checkpoint inhibitors block normal proteins on cancer cells or the T proteins that respond to them. It blocks proteins on cells, and these inhibitors help protect cancer cells from immune system attacks. to combine with CLT antigen-based therapy to try to overcome one of the main defenses of These could be a particularly important class of drugs.

[0162] Thus, one aspect of the present invention is the use of a polypeptide, a nucleic acid, a vector, an antigen-binding polypeptide, The peptide, composition, T cell, T cell population, or antigen presenting cell is administered in combination with a checkpoint inhibitor. Examples of checkpoint inhibitors include pembrolizumab, PD-1 inhibitors such as nivolumab (Opdivo) and atezolizumab (Teccent PD-L1 inhibitors such as ribavirin (Riku), avelumab (Bavencio) and durvalumab (Imfinzi), as well as and CTLA-4 inhibitors such as ipilimumab (Yervoy).

[0163] Interferons (such as alpha, beta, and gamma) are a family of proteins that the body produces in very small amounts. Interferon slows or stops cancer cell division, helping cancer cells defend themselves against the immune system. It may reduce the ability of the body to defend itself and / or enhance multiple aspects of the adaptive immune system. -Ferron is usually administered by subcutaneous injection, for example in the thigh or abdomen.

[0164] Thus, one aspect of the present invention is the use of a polypeptide, a nucleic acid, a vector, an antigen-binding polypeptide, Administration of the peptide or composition in combination with an interferon, e.g., interferon alpha Includes giving.

[0165] Also, different forms of the invention may be combined, e.g., the polypeptides, nucleic acids, and vectors may be combined with the APCs, T cells or T cell populations of the invention (described below). can be). One or more modalities of the present invention may also be used in combination with conventional anti-cancer chemotherapy and / or radiation therapy. Good too.

[0166] (diagnosis) In another aspect, the present invention relates to a method for diagnosing ovarian cancer, or a method for administering a polypeptide, a nucleic acid, or a nucleic acid fragment of the present invention to a patient. Treating with an acid, vector, antigen-binding polypeptide, adoptive cell therapy, or composition One or more of the polypeptides or nucleic acids of the invention can be used to diagnose a human subject suitable for This provides a method for

[0167] Accordingly, the present invention provides a method for diagnosing a human suffering from cancer, comprising: The cell is a polypeptide selected from SEQ ID NO: 1 to 2 and any one of their immunogenic fragments or variants. A peptide sequence (e.g., selected from the sequences of SEQ ID NOs: 3 to 4), or a polypeptide sequence thereof Expressing a nucleic acid encoding the nucleic acid (e.g., selected from the sequences of SEQ ID NOs: 5-6 and 7-8) and determining whether the polypeptide or the corresponding nucleic acid is present in the cancer cell. diagnosing the human as suffering from cancer if the human is overexpressed in the The method is provided.

[0168] The present invention relates to ovarian carcinoma, particularly serous ovarian carcinoma, e.g., ovarian serous cystadenocarcinoma. A method for diagnosing a human having a cancer, comprising: detecting a cancer cell line comprising SEQ ID NOs: 1 and 2 and or a polypeptide sequence selected from any of the immunogenic fragments or variants thereof, determining whether the nucleic acid encoding the polypeptide sequence is expressed; When the polypeptide or corresponding nucleic acid of is overexpressed in the cancer cells, the human suffers from ovarian carcinoma, particularly serous ovarian carcinoma, e.g. ovarian serous cystadenocarcinoma and diagnosing the condition as described above. As used herein, "overexpressed" in a cancer cell refers to a gene that is expressed at a level that is higher than the expression level of the gene in the cancer cell. This means that the level is higher than in normal cells.

[0169] Overexpression is determined by expression of a nucleic acid or polypeptide of the invention in control human subjects known to be cancer-free. Overexpression can be determined by reference to the level of the polypeptide of the present invention. The nucleic acid or polypeptide is detected at a significantly higher level (e.g., 3 The control human subjects are shown to be detected at levels (0%, 50%, 100% or 500% higher) of the present invention. When a nucleic acid or polypeptide of the present invention is present at an undetectably low level, A diagnosis can be made upon detection of the peptide.

[0170] The present invention also provides a method of treating a human suffering from cancer, comprising: (a) The cancer cells are characterized in that they contain SEQ ID NO: 1 to 2 and any one of their immunogenic fragments or variants. A polypeptide sequence selected from the following (for example, the sequences of SEQ ID NOs: 3 to 4), or and expressing a nucleic acid encoding a polypeptide (e.g., a sequence selected from SEQ ID NOs: 5 to 6 and 7 to 8). and, if expressed, determining whether (b) administering to said human a corresponding polypeptide, nucleic acid, vector, composition, T cell population, or a step of administering a fusion protein, a T cell, an antigen-presenting cell, an antigen-binding polypeptide or a cytotoxic cell to the subject. and

[0171] Similarly, isolated from tumors in humans suffering from cancer, (a) any one of SEQ ID NOs: 1 to 2; or (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); or the use of a nucleic acid encoding the polypeptide comprising a sequence selected from The use of the corresponding polypeptides, nucleic acids, vectors, compositions, Vaccines comprising T cell populations, T cells, antigen-presenting cells, antigen-binding polypeptides or cytotoxic cells as a biomarker to determine whether a patient would be suitable for treatment with chinese drugs , said uses are provided. Suitably the cancer is ovarian cancer, particularly ovarian carcinoma, especially serous ovarian carcinoma, e.g. ovarian serous cystoma. It's cancer.

[0172] Preferably, the polypeptide of the present invention is SEQ ID NO: 1-2, or a fragment thereof, such as an immunogenic fragment thereof. The fragment has a sequence selected from the fragments (for example, selected from the sequences of SEQ ID NOs: 3 to 4). Preferably, the nucleic acid of the present invention has a sequence selected from any one of SEQ ID NOs: 5 to 6 or 7 to 8. Has or contains.

[0173] Kits for detecting the presence of nucleic acids are well known. A kit containing at least two hybridizing oligonucleotides is used for real-time PCR. It may also be used in RT-PCR reactions, allowing the detection and semi-quantification of specific nucleic acids. The kit enables the generation of a fluorescent signal as a result of Förster resonance energy transfer (FRET). by (e.g., TaqMan® kits) or in binding of double-stranded DNA (e.g., SYBR (Registered Trademark Green Kit), which will allow detection of PCR products. , which includes TaqMan® probes that span multiple exons of the target DNA, For example, the present invention provides a method for detecting and quantifying a nucleic acid encoding a transcript thereof. Assays using the ELISA kit are set up in a multiplex format to simultaneously detect multiple ELISAs in one reaction. It will detect nucleic acids that are active (i.e., DNA that has a particular epigenetic signature that indicates expression). Kits for detection may also be used. Additional components that may be present in such kits include: The components include diagnostic reagents or reporters that facilitate detection of the nucleic acids of the invention. do.

[0174] The nucleic acids of the invention may also be detected in a liquid biopsy, using a blood sample from the patient. Such a procedure provides a non-invasive alternative to surgical biopsy. Plasma from the samples can be isolated and analyzed for the presence of the nucleic acids of the invention.

[0175] The polypeptides of the invention can be detected using antigen-specific antibodies in ELISA-type assays. may be used, which involves detecting the polypeptides of the invention in homogenized preparations of patient tumor samples. Alternatively, the polypeptides of the present invention can be used in immunohistochemical analysis. The tumor may be detected by staining patient tumors using appropriately labeled antibody preparations. Sections of the samples are examined using a light microscope to identify the presence of polypeptide antigens. As a further alternative, the polypeptides of the present invention can be identified by immunohistochemical analysis. may be detected by staining patient tumor samples using appropriately labeled antibody preparations. Sections of the sample are examined using a light microscope to identify the presence of polypeptide antigens. It is.

[0176] The polypeptides of the present invention also have the advantage that they increase the reactivity to the polypeptide. This may be detected by determining whether it is able to stimulate T cells.

[0177] Method for treating human ovarian cancer, particularly ovarian carcinoma, particularly serous ovarian cancer, e.g. ovarian serous cystadenocarcinoma The method comprises (i) detecting the presence of a nucleic acid or polypeptide of the invention, and (ii) administering to the subject: A nucleic acid, polypeptide, vector, cell, T cell or T cell population, or composition of the invention is administered. administering to the subject a nucleic acid or polypeptide (preferably the same nucleic acid or polypeptide or fragment thereof as that detected); (administering a piece of the drug).

[0178] Method for treating human ovarian cancer, particularly ovarian carcinoma, particularly serous ovarian cancer, e.g. ovarian serous cystadenocarcinoma The method also includes the step of detecting the presence of (preferably the same) nucleic acid or polypeptide of the invention in a subject. to the nucleic acid, polypeptide, vector, cell, T cell or T cell population or composition of the invention The method includes administering

[0179] In particular, the cancer to be diagnosed and, if possible, treated is ovarian cancer, particularly ovarian carcinoma, especially stromal. Liquid ovarian cancer, for example ovarian serous cystadenocarcinoma. If a polypeptide of the present invention is detected that is SEQ ID NO: 1 or 2 or a fragment thereof, The cancer may be ovarian cancer, particularly ovarian carcinoma, especially serous ovarian cancer, e.g., ovarian serous cystadenocarcinoma. do.

[0180] (Special embodiments) In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:1. An exemplary fragment comprises or consists of SEQ ID NO:3. Exemplary nucleic acids include or consist of SEQ ID NO: 3 or SEQ ID NO: 7. nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, The nucleic acid (e.g., D NA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes are useful in the treatment of ovarian cancer, particularly ovarian carcinoma, especially serous ovarian cancer, e.g., ovarian serous cysts. It may be used in the treatment of adenocarcinoma. Related diagnostic methods are also provided.

[0181] In one embodiment, the CLT antigen polypeptide comprises or consists of SEQ ID NO:2. An exemplary fragment comprises or consists of SEQ ID NO: 4. Exemplary nucleic acids include or consist of SEQ ID NO:6 or SEQ ID NO:8. nucleic acids (e.g., DNA or RNA), T cells, T cell populations, cytotoxic cells, The nucleic acid (e.g., D NA or RNA), T cells, T cell populations, cytotoxic cells, antigen-binding polypeptides, antigen-presenting cells and exosomes are useful in the treatment of ovarian cancer, particularly ovarian carcinoma, especially serous ovarian cancer, e.g., ovarian serous cysts. It may also be used in the treatment of adenocarcinoma. Related diagnostic methods are also provided. EXAMPLES

[0182] (Example) (Example 1 - CLT specific) The aim is to identify cancer-specific transcripts that consist entirely or partially of LTR elements. That was the case. First step: de novo assembly of comprehensive, cross-cancer transcriptomes To achieve this, we obtained data from The Cancer Genome Atlas (TCGA) Consortium, The data were collected from a wide variety of cancer types (32 cancer types (31 primary and 1 metastatic melanoma)). From 768 patient samples, representing 24 gender-balanced samples (Table S1), RNA sequencing reads were used for genome-guided assembly. The isolated samples (excluding sex-specific tissues) were then used as adaptors using cutadapt (v1.13) (Marcel M (2011, EMBnet J., 17:3), quality (Q20) trimmed and length filtered ( Both reads were ≥35 nucleotide pairs long) and were generated using khmer (v2.0) (Crusoe et al., 2015, F1000Res., 4:900) with kmer normalization with maximum and minimum depths of 200 and 3, respectively. The reads were analyzed using STAR (2.5.2b) with the same settings as those used for the entire TCGA. The genome was then mapped to GRCh38 using Trinity (v2.2.0) (Grabherr, MG et al., 201 1, Nat. Biotechnol., 29:644-52) with built-in in silico depth normalization. The majority of the assembly process was performed on a 32-core HP The failed process was completed within the 256GB RAM on the C node and rerun using the 1.5TB RAM node. The resulting contigs were poly(A) trimmed (using trimpoly in SeqClean v110222) and Entropy filtering (≥0.7) was used to remove low-quality and artificial contigs (b in BBMap v36.2). bduk was removed. For each cancer type, the original 24 samples were run on Salmon (v0.8.2 or v0.9.2) (Patro, R. ,et al., 2017, Nat. Methods, 14:417-419) to the cleaned assembly. Contigs that were submapped and showed expression of <0.1 transcripts per million (TPM) were The remaining ones were reconstituted using GMAP(v161107) (Wu et al., 2005, Bioinf., 21:1859-187 5) to GRCh38, and found to have 85% or more identity over 85% or more of its length. Contigs that did not align with the original sequence were removed from the assembly. The resulting assembly was flattened and run using gffread (Cufflinks v2.2.1) (Trapnell et al., 2010, Na The longest continuous transcript was obtained by merging the assemblies using the ELISA kit (J. Biotech., 28:511-515). The Bry process was specifically designed to allow for the evaluation of repetitive elements, Sonogenic transcripts were retained but flagged. Completeness and quality of transcript assemblies The sequences were analyzed using GENCODE v24basic and MiTranscriptome1 (Iyer et al., 2015, Nat. Genet., 47:199 The list of unique splice sites in GENCODE was compared to the The splice site is aligned within a 2-nucleotide window between the transcript and the target site. This process resulted in 1,001,931 We identified 1,006 transcripts, of which 771,006 were spliced ​​and 230,925 were monoexos. The results showed that the antibody was soluble in water.

[0183] Separately, the assembled contigs were overlaid with genomic repeat annotations. We identified transcripts containing LTR elements. LTR and non-LTR elements were identified using the The sequences were annotated as described previously (Attig et al., 2017, Front. In Microbiol., 8:2489). Briefly, we used a hidden Markov model (HMM) representing known human repeat families ( Dfam 2.0 library v150923), RepeatMasker Open-3.0 (Smit, A., R. Hubley and P. Gree Annotate GRCh38 using the repeatmasker.n literature (http: / / www.repeatmasker.org,1996-2010). and constructed it using nhmmer (Wheeler et al., 2013, Bioinform., 29:2487-2489). The scan was performed using a BLAST-based method (Hubley et al., 2016, Nuc. Acid. Res., 44:81-89). The annotation accuracy is improved compared to RepeatMasker. RepeatMasker separates LTR and internal regions. To annotate, the tabular output is parsed to find adjacent annotations for the same element. The annotations were merged. In this process, one or more complete or partial LTR elements were merged. We obtained 181,967 transcripts, including

[0184] Salmon was used to estimate transcripts per million (TPM) abundance for all transcripts and within each cancer type. Expression of α- and β-terminal α-terminal β ... Genotype-Tissue Expression Consortium, 2015, Science, 348:648-60) Expression was compared to that in tissue-matched healthy controls for all cancer types. If a gene was detected in a sample at >1 TPM, it was considered to be expressed in the cancer. A gene was considered cancer-specific if it met the following criteria: (i) among 24 samples of each cancer type, (ii) expressed at less than 10 TPM in more than 90% of all healthy tissue samples; (iii) is expressed in the target cancer type at 3-fold or greater than the median expression in any control tissue type; and and (iv) for the targeted cancer type, at least three times the 90th percentile of each available healthy tissue. is expressed on

[0185] The list of cancer-specific transcripts was then analyzed for transcripts containing complete or partial LTR elements. We crossed the list of transcripts with the 1,000 transcripts in the control group to generate a list of 5,923 transcripts that met all criteria (Cancer (termed CLT for specific LTR element-spanning transcript).

[0186] Further curation was performed on 390 CLTs specifically expressed in ovarian serous cystadenocarcinoma, and corresponding to the assembly of potential contigs and cellular genes Additional manual evaluation was performed to determine whether the splicing patterns were Supported by original RNA sequencing reads from cancers determined to be differentially expressed The CLT was further triaged to determine whether it was a normal GTEx tissue. Genes with median expression greater than 1 TPM were discarded. Of 390 CLTs for ovarian serous cystadenocarcinoma, 40 CLTs passed these filters. .

[0187] Example 2 - Immunopeptidome Analysis Mass spectrometry (MS)-based immunopeptidome analysis identifies peptides that bind to HLA molecules (HLAp) and are expressed on the cell surface. It is a powerful technique that allows the direct detection of specific peptides being presented. This technology detects HLAp by anti-HLA antibody capture from biological samples such as cells or tissues. The isolated HLA molecules and bound peptides are then separated from each other. The isolated and eluted peptides were analyzed by nano-ultra-performance liquid chromatography-mass spectrometry (nUPLC-MS). (Freudenmann et al., 2018, Immunology 154(3):331-345). Specific peptides with a defined mass-to-charge ratio (m / z) are selected, isolated, fragmented, and then The second mass spectrometry (MS / MS) reveals the m / z of the resulting fragment ions. Collating the fragmentation spectra (MS / MS) identified selected ions that generated the detected fragment ions. The amino acid sequence of the peptide can be precisely identified.

[0188] MS / MS spectrum interpretation and subsequent peptide sequence assignment were based on experimental data and reference data. The theoretical spectra generated from the peptide sequences contained in the database matched well with those of the original data. It depends on all the known transcriptomes, or even the entire genome. Analyze MS data using a predefined list of ORFs corresponding to open reading frames (ORFs). Although it is possible to search for 5) Matching these very large sequence databases leads to very high false discovery rates (FDRs). In addition, technical issues (e.g., leucine content) limit the identification of the presented peptides. amount = isoleucine mass), and theoretical issues (e.g., peptide splicing (Liepe et al., (2016, Science 354(6310):354-358)) is a method to identify known transcriptomes or whole genomes. Increase the limitations associated with the use of very large databases, such as those created from human bodies. Thus, in effect, it refers to a well-defined set of possible polypeptide sequences. Without the need for a single reference, it is extremely difficult to perform accurate immunopeptidome analysis to identify novel antigens. It is difficult to identify the genetic variants of genomic DNA (Li et al., 2016, BMC Genomics 17(Suppl 13):1031).

[0189] Therefore, the present inventors have identified 10 or more residues derived from the 40 ovarian serous cystadenocarcinoma CLTs of Example 1. We have constructed a database of all predicted polypeptide sequences (ORFs) of We obtained 1,111 ORFs ranging in length from 10 to 147 amino acids.

[0190] Schuster et al. reported 38 serous ovarian carcinomas, 3 endometrioid carcinomas, and 1 mixed differentiation (predominantly endometrioid). HLA class I and HLA class II binding pairs derived from tumors of 42 ovarian cancer patients, including ovarian carcinoma. MS / MS data was generated from the peptide sample (Schuster et al., 2017, PNAS, 114(46), E 9942-E9951. http: / / doi.org / 10.1073 / pnas.1707658114; PXD007635 Database - http http: / / proteomecentral.proteomexchange.org / cgi / GetDataset?ID=PXD007635). Schuster et al. compiled these data using polypeptide sequences reported for the entire human proteome. The analysis revealed tens of thousands of peptides that matched known human proteins. As expected, these peptides were expressed in various proteins, including MUC16, CRABP1 / 2, FOLR1, and KLK10. The sequences included sequences derived from various ovarian cancer tumor-associated antigens (TAAs) (Schuster et al., 2013). 17).

[0191] By applying detailed knowledge of immunopeptidome evaluation, the inventors have shown in Example 1 The HLA class I dataset includes ORFs derived from the 40 discovered CLTs (Schuster et al., 2013). 017, PNAS, 114(46), E9942-E9951; PXD007635 database) , which uses PEAKS™ software (v8.5 and vX, Bioinformatics Solutions Inc.) and aligned with all polypeptide sequences found in the human proteome (UniProt) (each CLT The majority of HLA class I HLA-binding peptides found in cells Since the proteins are constitutively expressed, the UniProt profiles of these databases Simultaneous matching with the genome confirmed that our assignment of CLT ORF sequences to MS / MS spectra was correct. The PEAKS software, like other MS / MS matching software, Similarly, we assigned a significance probability (p-value) (-10lgP; see Table 1) to each spectral assignment and compared the results. Quantify the allocation.

[0192] The results of these studies identified several individual peptides, which were identified by Schuster et al. The antibody bound to HLA class I molecules immunoprecipitated from tumor samples from 42 patients examined. These correspond to the amino acid sequences of the CLT-derived ORFs and are known to be related to the human proteome (UniProt). The polypeptide sequences did not correspond to those present in

[0193] The peptide spectra assigned to these CLT-derived ORF sequences by the PEAKS software were The sequences were further manually evaluated and the spectra were compared to the peptides that were mapped to the two CLT-derived ORFs. As a result of confirming the assignment of the antigens, these were defined as CLT antigens (Table 1; SEQ ID NOs: 1 to 2).

[0194] Detection of these peptides binding to HLA class I molecules was based on the two ORFs from which they were derived. It is first translated in ovarian tumors, processed via the HLA class I pathway, and finally transduced into the HLA class II pathway. The results confirm that the CLT antibodies are presented to the immune system as a complex with the CLT antibody I molecule. The characteristics of peptides found in the original samples are shown in Table 1. All four peptides identified by Schuster et al. as being associated with serous ovarian carcinoma. 1 and 2 show representative MS / MS spectra of each of the peptides shown in Table 1. The top panel of the figure shows the MS / MS peptide fragment profile along with standard MS / MS annotation. (b: N-terminal fragment ion; y: C-terminal fragment ion; -H2O: water loss; -NH3: ammonia loss) ; [2+]: doubly charged peptide ion; pre: unfragmented precursor peptide ion; a n -n:in The most abundant fragment ion peaks are shown above, which are the PEAKS The software was used to create an ovarian tumor MS / MS database (Schuster et al., 2017, PNAS, 11 4(46), E9942-E9951; PXD007635 database). The panels are spectral panels showing the positions of linear peptide sequences mapped to fragment ions. This is consistent with the −101gP score assigned to the peptide in Table 1, so The spectrum exactly matches the peptide sequences (SEQ ID NOs: 3-4) found in these analyses. Contains multiple fragments.

[0195] Both peptides detected as binding to HLA class I in Table 1 were analyzed using the NetMHCpan 4.0 prediction software. The patient's HLA class I was assessed using the NetMHCpan (http: / / www.cbs.dtu.dk / services / NetMHCpan / ) The predicted strengths of their binding to type A molecules were determined (Schuster et al.). The results of the study showed that both peptides were ranked by at least one of the HLA types found in the patients. Most patient peptides were predicted to bind to the core 5% and had better rank scores (2 As expected, the eight amino acids were linked to the 1.0% carboxyl group (Table 2). The acid peptide (ISKPLIYY, SEQ ID NO: 3) has a lower binding prediction than longer peptides. It should be noted that both of the peptides detected were predicted to be present in the patient population. The fact that the antibody was predicted to bind to the selected HLA type is not inconsistent with these findings.

[0196] Further assignment of MS spectra from tumor tissue to the peptide sequences we discovered To provide certainty that these sequences are consistent with those in the original study, peptides with these discovered sequences were synthesized and nUPLC-MS was performed using the same conditions as those applied to the tumor samples. 2 (Schuster et al., 2017, PNAS, 114(46), E9942-E9951; PXD007635 database). Synthetic peptides and endogenous A comparison of the spectra of the specific (i.e., tumor) peptides is shown in Figures 3-4. In each figure, the upper spectrum is Corresponding to tumor samples (Schuster et al., 2017, PNAS, 114(46), E9942-E9951; PXD0076 35 database), the lower spectrum corresponds to a synthetically prepared peptide with the same sequence. The selected m / z values ​​of the detected ion fragments are then used to determine the upper and lower cutoffs of these MS / MS spectra. These figures clearly show the correct alignment of the fragments (tumor-derived The small differences between the experimentally determined m / z values ​​of the fragment ions from the and synthetic peptides were (within the m / z tolerance range of <0.5 Daltons) Verify the authenticity of the assignments to the encoded peptides.

[0197] In summary, the peptide data presented in Tables 1 and 2, Figures 1-2, and Figures 3-4 indicate that ovarian cancer patients This provides very strong support for the translation, processing and presentation of the corresponding CLT antigens.

[0198] In summary, peptide identification of the immunopeptidome, derived from predicted ORFs, was performed using these The CLT is translated into a polypeptide (SEQ ID NO: 1-2; also referred to as CLT antigen) in tumor tissue. They are then processed by the cellular immune surveillance machinery and The peptide is loaded onto HLA class I molecules and recognizes the resulting peptide / class I complex. These CLT antibodies allow the cells to be targeted for cytolysis. The antigens and fragments thereof are intended for the treatment of ovarian cancer in patients whose tumors express these antigens. , are predicted to be useful in a variety of therapeutic modalities.

[0199] (Table 1: List of peptides identified by immunopeptidome analysis of ovarian tumor samples. , and cross-reference of CLT antigen names and sequence numbers) [Table 1] 1: HLA class I peptides identified by mass spectrometry. 2 :Schuster et al., 2017, PNAS, 114(46), E9942-E9951; PXD007635 Database 3 : Calculated peptide mass. 4 PEAKS™ program - 10 lgP values ​​were calculated for peptides / patients with more than 1 detected spectrum. The peptides with the highest match to the target peptide are shown. 5 : Number of spectra in which the peptide was detected. 6 : Deviation between observed and calculated mass; the selected ppm value is used if a spectrum exceeding 1 was obtained. Peptides are shown.

[0200] (Table 2) Peptides identified by mass spectrometry reported for the patient from whom the tumor was derived Predicted NetMHCpan 4.0 binding to HLA types and cross-reference of CLT antigen names and sequence numbers (see) [Table 2] 1 :Schuster et al., 2017, PNAS, 114(46), E9942-E9951; PXD007635 Database 2 : Prediction of the HLA class I type reported for the patient with rank scores of ≦5.0% score The combined 3 : Number of patient HLA class I types predicted to bind with ≦5.0% rank score. 4 : Number of patient class I types predicted to bind with ≦2.0% rank score.

[0201] The results presented in Examples 1 and 2 herein may be used in whole or in part in support of The Cancer Genome Atlas (TCGA) research. Cancer Research Network (http: / / cancergenome.nih.gov / ) and the Genotype-Tissue Expression (GTEx) Project The National Institutes of Health (NIH) Office of the Director's Common Fund, and the NCI, NHGRI, NHLBI, NIDA, NIMH, and NIND This work is based on data generated by the National Institute of Standards and Technology (NIS), which is supported by the National Institute of Standards and Technology (NIST).

[0202] Example 3 - Assay to demonstrate T cell specificity for CLT antigens in ovarian cancer patients (a) Staining of reactive T cells with CLT antigen peptide pentamer) The presence and activity of circulating CD8 T cells specific for CLT antigens in ovarian cancer patients is related to HLA class I / peptide Measured using tidopentamer ("pentamer") staining and / or in vitro killing assays Thus, the compounds discovered using the methods described in Examples 1 and 2 (Tables 1 and 2, Figures 1 to 31) Application of these techniques to selected CLT antigens will allow the identification of therapeutically relevant T cell responses to CLT antigens in cancer patients. can be used to indicate the presence of

[0203] For these studies, CD8 T cells isolated from patients' blood were cultured using a variety of culture methods, e.g. For example, anti-CD3 and anti-CD28 coated microbeads and interleukin-2 were used. The expanded cells then use the CLT peptide pentamer to bind to their T cell receptors. It can be stained for specific CLT antigen reactivity, and the pentamer is a peptide of the HLA molecule. It consists of a pentamer of HLA class I molecules that binds to related CLT antigenic peptides in the tide-binding groove. The binding is specific to the coiled-coil multimerization domain of the pentameric structure. Detection using antibody fragments conjugated with erythroerythrin or allophycocyanin In addition to this pentamer staining, the memory markers CD45RO and lysosomal The pentamer and specific surface markers, such as the CD107a release marker, can be used to identify Positive binding to specific surface markers was used to determine the number and status of pentamer-reactive T cell populations. It can be used to infer both the memory state and the naive / stem state.

[0204] Pentamer-stained cells were also sorted and purified using a fluorescence-activated cell sorter (FACS). The selected cells may then be subjected to an in vitro killing assay to determine whether they can kill target cells. These assays can further test for the ability of CD8 T cell populations and In this case, the CD8 population is a population of CLT antigen-specific cells or CD8 T cells that are pentamer-selected and known to induce potent killing responses, such as Mart-1. The cells targeted in these studies are specific for the positive control antigens that have been HLA-A * T2 cells expressing HLA-A 02 were pulsed with peptides * 02, 03 or B * C1R cells transfected with 07 or previously shown to express the CLT / CLT antigen The present invention can include ovarian cancer cell lines or tumor cells of patients. The peptides used for pulsing included the CLT antigen peptide or a positive control peptide. The target cells are stained with a red nuclear dye (nuclight rapid reactivation) that is incorporated into the nuclei of healthy cells. d) may be double-labeled with a vital dye such as erythrocyte tyrosine kinase (YK), which is a marker for the tracking of target cell attack by specific T cells. Additional evidence was provided by the green caspase 3 / 7 activity indicator, which indicates caspase 3 / 7-mediated apoptosis. Thus, CD8 T cell-mediated apoptosis can be shown to be a potent factor in the pathogenesis of inflammatory bowel disease. When target cells are killed by apoptosis, the target cells are activated by caspase 3 / 7 activity specific to apoptosis. They lose their red fluorescence and emit green fluorescence. Thus, pentamer-selected CLT antigen-specific C Application of such a killing assay to D8 T cells has been shown to be useful in the detection of C-terminal cytoplasmic leukemia in ex vivo cultures of ovarian cancer patient T cells. It can be used to demonstrate the cytotoxic activity of LT antigen-specific T cells.

[0205] (b) HERVfest analysis of T cell specificity in ovarian cancer patients The specific T cell functional expansion (fest) technology is a promising tool to detect and treat tumors in patients who respond to checkpoint blockade therapy. Tumor-specific mutations present in the "mutation-associated neoantigen" (MANA) repertoire found in tumor cells have been used to identify derived epitopes (Anagnostou et al., Cancer Discover (Le et al., Science 2017). The methods described in Examples 1 and 2 (Tables 1 and 2, Figures 1 to 31) Applying this technology to CLT antigens discovered using the method, it is possible to develop treatments for CLT antigens in cancer patients. The presence of therapy-associated T cell responses can be confirmed.

[0206] Other assays to identify epitope-specific T cells in immune-exposed subjects (e.g., Similar to ELISA (e.g., ELISPOT), the "fest" technique involves the use of antigen-presenting cells and suitable antigenic peptides. In vitro culture, cognate T cells are activated to reveal their specificity. This technique Unlike other immunological assays, these expanded cultures contain T cell receptors Next-generation sequencing of TCR mRNA (specifically, TCRseq, which targets the TCR-Vβ CDR3 region) Utilizing standard HLA binding algorithms to match the patient's HLA type Detect specific TCRs propagated in cells cultured with a preselected target peptide Tumor tissue from the same patient taken after successful checkpoint blockade therapy The application of TCRseq to TCR-seq then allowed us to characterize which TCRs were detected in ex vivo peptide-stimulated cultures. This could be used to determine whether TCR / T cells are also present at immune-suppressing sites in cancer. In the case of MANAfest, this method uses T cells produced in each patient's tumor and Used to identify specific TCRs that recognize MHC-presented neoantigenic peptides found within were discovered by whole-exome sequencing of normal and tumor tissues from each patient. Identifying functionally relevant neo-antigenic peptides among thousands of possible variant peptides (Le et al., Science 2017).

[0207] The application of MANAfest technology (Anagnostou et al., 2017, Cancer Discovery) to CLT antigens: The procedure is as follows: Step 1: Select an epitope that efficiently binds the selected HLA supertype. Identify peptides within the CLT antigen that are predicted to contain the tope. Step 2: Suitable patients? PBMCs were selected from these mice and matched by HLA type with the peptide library selected in step 1. Step 4: PBMCs from these patients are separated into T cell and non-T cell fractions. The non-T cells are irradiated (to prevent proliferation) and then reintroduced into the patient's T cells, then split into 20-50 samples. Divide and mix 10-10 with T cell growth factors and individual CLT-specific synthetic peptides (selected in step 1). Grow for 4 days. Step 4: TCRseq (sequencing of epitope-specific TCR-Vβ CDR3 sequences) A multiplication test (multiplication of cognate T cells / TCR) was performed for all wells to measure the cognate T cells / TCR amplified in the presence of the test peptide. Identify the specificities of these TCRs detected in non-expanded / expanded T cells. The TCR is determined by comparing it with TCRseq. The data obtained from this step The data can then be used to determine which peptides elicited an immune response in the patient. Step 5: TCRs eq was performed on tumor samples to determine which of the specifically amplified TCRs were involved in checkpoint inhibition. We then determined which T cells with this TCR homed to the tumors of patients who responded to anti-tumor therapy, and which T cells with this TCR were identified as T cells that could be used to treat tumors. This study provides evidence that may contribute to the efficacy of checkpoint inhibitor therapy.

[0208] Example 4 - High affinity T cells specific for CLT antigens are expressed in the T cell repertoire of normal subjects Assay to show that the lysate has not been removed from the lysate) The ELISPOT assay demonstrated that CLT antigen-specific CD8 T cells are present in the normal T cell repertoire of healthy individuals. It may be used to show that naïve and thymic tissue from these patients The expression of cancer-specific CLT antigens in the tumor cells was not abrogated by central tolerance. This type of ELISPOT assay involves several steps. Step 1: CD8 T cells and CD14 monocytes were isolated from peripheral blood of normal blood donors and these cells were then cultured in a HL CD8 T cells can be classified by type and matched to the specific CLT antigen being tested. Using a magnetically labeled antibody against the memory marker CD45RO, naïve and memory subtypes were identified. Step 2: CD14 monocytes are cultured with individual or pooled CLT antibodies. Pulse with the original peptide for 3 hours, then co-culture with CD8 T cells for 14 days. Step 3: Expanded CD8 T Cells are isolated from these cultures and restimulated overnight with fresh peptide-pulsed monocytes. These peptides included individual CLT antigen peptides, unrelated control peptides, or infectious (e.g. For example, CMV, EBV, influenza, HCV) or self (e.g., Mart-1) antigens. Restimulation may include peptides known to elicit a response. The assay is carried out on plates coated with an antibody against IFN-gamma, which binds the peptide It captures any IFNγ secreted by stimulated T cells. After overnight activation, cells were The plate was washed and the captured IFNγ was then eluted with additional anti-IFNγ antibody and a standard ratio. The cells were detected by a colorimetric dye. The IFN-γ producing cells were detected in the areas where they were originally present on the plate. The data obtained from this assay include spot number, spot size, These include the median spot size and median spot intensity. These are used to estimate the frequency and and the amount of IFNγ per cell. The standard values ​​were calculated based on the specific response measured as the number of spots or the median spot size. The stimulation index was calculated by dividing the background response to peptide-free monocytes. By this method, the response to the CLT antigen and the control antigen can be calculated. Comparison of responses to antigens revealed that naïve subjects had a strong population of CLT antigen-reactive T cells. It has been shown that the IL-1 antibody contains a IL-1 antibody, which is a IL-1 antibody that is immunized with a CLT antigen-based immunogenic formulation. It can be expanded by seeds.

[0209] Example 5 - Validation assay of CLT expression in ovarian cancer cells a) qRT-PCR validation of CLT expression in ovarian cancer cells Quantitative real-time polymerase chain reaction (qRT-PCR) is a method for determining the amount of DNA fragments (DNA fragments) in a given biological sample. It is a widely used technique for determining the amount of specific transcripts present in extracted RNA. Specific nucleic acid primer sequences are designed for the transcript of interest, and then subeqeuntly The intermer region is amplified through a series of thermal cycling reactions to generate an intercalator colorimeter. The primer pairs were engineered against CLT and quantified fluorescently using SYBR Green. Assayed on RNA extracted from ovarian cancer patient tumor tissue. Non-ovarian cancer cell lines were used as negative controls. RNA was extracted from each sample and reverse transcribed into cDNA according to standard procedures. qRT-PCR analysis with SYBR Green detection was performed according to standard techniques to identify transcripts overlapping with CLT antigen 2. The experiments were performed using primers designed against the region of the chromosome 11 and the reference gene. (RQ) was calculated as follows: RQ=2[Ct(reference)-Ct(target)] .

[0210] The results of these experiments are shown in Figure 5. Panel A shows the qRT-PCR assay using primer set (166+167). - PCR assay results from 10 ovarian tissue samples and one non-ovarian cancer cell line (Jurkat). 1 shows a targeting of CLT (SEQ ID NO: 6), which encodes CLT antigen 2 on RNA extracted from These results demonstrate that the IFN-γ expression level in RNA extracted from ovarian tissue samples is significantly higher than in non-ovarian cancer cell lines. CLT antigen 2 was specifically expressed in more than 50% of the analyzed tissue samples. Expression was detected in ovarian cancer control cell lines and no expression was detected in non-ovarian cancer control cell lines.

[0211] b) RNAScope validation of CLT expression in ovarian cancer cells in situ Transcript expression analysis by in situ hybridization (ISH) is a method to detect expression of transcripts in histopathological specimens. It allows visualization of the presence and expression level of a given transcript in a specific context. The assay also uses oligonucleotide probes specific for short stretches of the desired RNA sequence. The method involves the in situ recognition of native RNA molecules, which can be achieved by using antibodies or enzymes. The signal is visualized by a combination of colorimetric reactions based on RNAScope is a new class of in s It is a technique based on itu hybridization, ensuring the specificity of the signal generated. and allows for sensitive, single-molecule visualization of target transcripts (Wang et al., 2013). (2012 J Mol Diagn. 14(1):22-29). Positive staining of transcript molecules indicates that small These appear as multiple red dots, with multiple dots indicating the presence of multiple transcripts.

[0212] RNAScope probes were designed against CLT antigen 2 and were analyzed using 12 formalin-fixed, paraffin-embedded Sections of tumor cores from ovarian cancer patients were assayed. Expression signals were scored according to the following: As described above, this was performed on a representative image from each core. The assessment of % of cells with positive staining for the CLT probe was rounded up to the nearest 10. The estimated per cell levels of expression across a given section are: 0 = no staining 1 = 1-2 dots per cell 2 = 2-6 dots per cell 3 = 6-10 dots per cell · 4 = more than 10 dots per cell.

[0213] CLT antigen 2 expression was detected in tumor cores from multiple different patients and was independently determined to be of tumor origin. To validate CLT findings from previous RNAseq data and to determine their occurrence in tumor tissue across multiple samples. The uniformity of the results was confirmed (Table 3).

[0214] Table 3 - RNAScope scoring in ovarian cancer patient tissue cores [Table 3]

[0215] Throughout this specification and the claims that follow, unless the context indicates otherwise, the term "includes" " and variations such as "included" and "comprising" are intended to include any integer, step, integer combination, or combination of steps. includes any integer, step, group of integers or group of steps, but excludes any other integer, step, group of integers or group of steps. It will be understood to mean that the term is not intended to be used interchangeably.

[0216] All patents, patent applications and literature references cited throughout this specification are hereby incorporated by reference. No. 6,399,433, which is incorporated herein in its entirety. The present invention relates to the preferred and more preferred groups and the preferred and more preferred groups enumerated above. It is intended to include all suitable groups and combinations of groups of embodiments.

[0217] (Sequence Listing) [ka] TIFF2025032114000005.tif248170TIFF2025032114000006.tif244170TIFF2025032114000007.tif245170TIFF2025032114000008.tif246170TIFF2025032114000009.tif119170

Claims

1. (a) any one of SEQ ID NOs: 1-2; and (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); An isolated polypeptide comprising a sequence selected from:

2. Claim 1, comprising or consisting of a sequence selected from any one of SEQ ID NOs: 3 to 4.

2. An isolated peptide according to claim 1.

3. A second or further polypeptide comprising: (i) a polypeptide according to claim 1 or claim 2; (ii) another polypeptide which is an ovarian cancer associated antigen; (iii) (iv) a polypeptide sequence capable of enhancing an immune response (i.e., an immunostimulatory sequence); and These antibodies can provide strong CD4+ help (e.g., uniclonal antibodies) that increase the CD8+ T cell response to the antibody. a polypeptide sequence comprising a universal CD4 helper epitope; 3. The isolated polypeptide of claim 1 or claim 2, fused to

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

5. The nucleic acid of claim 4, which is DNA.

6. A sequence selected from any one of SEQ ID NOs: 5 to 6 and 7 to 8, or consisting of said sequence.

6. The nucleic acid of claim 5.

7. 7. The nucleic acid of claim 6, which is codon optimized for expression in a human host cell.

8. The nucleic acid of claim 4, which is RNA.

9. 9. The nucleic acid of claim 4, 5, 7 or 8, which is an artificial nucleic acid sequence.

10. A vector comprising the nucleic acid according to any one of claims 4 to 9.

11. A regulatory element suitable for enabling transcription of a translationally active RNA molecule in a human host cell. The vector of claim 10, comprising DNA encoding the cleavage gene.

12. 12. The vector according to claim 10 or 11, which is a viral vector.

13. Adenovirus vector, adeno-associated virus (AAV), alphavirus, herpes simplex viruses, arenaviruses, measles viruses, poxviruses, paramyxoviruses, 13. The vector of claim 12, which is a tivirus or rhabdovirus vector.

14. The polypeptide, nucleic acid or vector according to any one of claims 1 to 13 is approved as a pharmaceutical. An immunogenic pharmaceutical composition comprising the compound of formula (I) and / or the compound of formula (I) together with an acceptable carrier.

15. The polypeptide, nucleic acid or vector according to any one of claims 1 to 13 is approved as a pharmaceutical. A vaccine composition comprising the vaccine composition together with an acceptable carrier.

16. 16. The composition of claim 14 or claim 15, further comprising one or more immunostimulants.

17. The immunostimulant is an aluminum salt, a saponin, an immunostimulatory oligonucleotide, and water. Oil-based emulsion, aminoalkyl glucosaminide 4-phosphate, lipopolysaccharide and its derivatives and other TLR4 ligands, TLR7 ligands, TLR8 ligands, TLR9 ligands, IL-12, and 17. The composition of claim 16, wherein the compound is selected from the group consisting of interferon.

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

19. A polypeptide, nucleic acid or vector according to any one of claims 1 to 18 for use in medicine. tar or composition.

20. A method for increasing an immune response in a human, comprising administering to said human a compound according to any one of claims 1 to 18. The method comprises administering a polypeptide, nucleic acid, vector or composition described in.

21. The immune response is characterized in that the antibody is an antibody to SEQ ID NO: 1-2 and variants and immunogenic fragments of any one of them. The method of claim 20, wherein the expression is elevated for a cancerous tumor expressing a sequence selected from the group consisting of:

22. A method for the production of a polypeptide according to any one of claims 1 to 18 for use in raising an immune response in humans. A polypeptide, a nucleic acid, a vector or a composition.

23. The immune response is directed to SEQ ID NOs: 1-2 and immunogenic fragments or variants of any one of them. The polypeptide of claim 22 is elevated in a cancerous tumor expressing a corresponding sequence selected from the group consisting of: A peptide, a nucleic acid, a vector or a composition.

24. A method of treating a human patient suffering from cancer, the cells of the cancer comprising the sequence of SEQ ID NOs: 1-2 and expressing a sequence selected from any one of the immunogenic fragments and variants thereof. or a method for preventing a human from suffering from cancer, the cancer being a cancer comprising the sequence of SEQ ID NO: 1-2 and their derivatives. The method will express a sequence selected from any one of the immunogenic fragments and variants. The corresponding polypeptide, nucleic acid, vector according to any one of claims 1 to 18. or administering the composition to said human.

25. A polypeptide according to any one of claims 1 to 18 for use in the treatment or prevention of cancer in humans. a peptide, nucleic acid, vector or composition, wherein the cancer cells are The polypeptide, nucleic acid, or nucleic acid fragment expressing a corresponding sequence selected from any one of the immunogenic fragments. An acid, vector or composition.

26. For use in the in vitro stimulation and / or expansion of T cells from humans suffering from cancer A polypeptide, a nucleic acid, a vector or a composition according to any one of claims 1 to 18. The stimulated and / or expanded T cells are then administered to the human to treat the cancer in the human. The polypeptide, nucleic acid, vector or composition for reintroduction.

27. A method for treating a human cancer, the method comprising the steps of: and expressing a sequence selected from the immunogenic fragments and variants of said human, said method comprising: and extracting a leukocyte population containing at least T cells together with the original presentation cells. The presence of a corresponding polypeptide, nucleic acid, vector or composition according to any one of paragraphs 1 to 18. and stimulating and / or amplifying at least some or all of the leukocytes under and reintroducing the stimulated and / or expanded T cells into the human. 。

28. The cancer may be ovarian cancer, particularly ovarian carcinoma, more particularly serous ovarian carcinoma, such as ovarian serous cystoma. The method or use according to any one of claims 21 and 23 to 27, wherein the polypeptide is a cancer. A peptide, nucleic acid, vector or composition.

29. A sequence selected from SEQ ID NO: 1-2 and immunogenic fragments and variants of any one of them. The present invention relates to a process for preparing a T cell population that is cytotoxic against cancer cells expressing the (a) obtaining T cells, optionally together with antigen-presenting cells, from a cancer patient; and (ii) performing an ex vivo The T cell population is then subjected to a corresponding polypeptide, nucleic acid or vector according to any one of claims 1 to 18. and stimulating and amplifying with a vector or composition.

30. 30. A T cell population obtainable from the process of claim 29.

31. Stimulated with a polypeptide, nucleic acid, vector or composition according to any one of claims 1 to 18. , T cells.

32. A method for producing a polypeptide, a nucleic acid, a vector or a composition according to any one of claims 1 to 18. A method for producing a polypeptide according to any one of claims 1 to 3, which is modified by loading in vitro. Antigen-presenting cells that have been genetically engineered to express

33. 33. The antigen-presenting cell of claim 32, which is a dendritic cell.

34. A method for the preparation of a polypeptide, a nucleic acid, a vector or a composition according to any one of claims 1 to 18.

4. An exosome loaded with a polypeptide produced from a cell genetically engineered to express the polypeptide of any one of claims 1 to 3.

35. The T cell population, T cell, antigen-presenting cell or exosome according to any one of claims 30 to 34. A pharmaceutical composition comprising the compound of formula (I) in combination with a pharma- ceutical acceptable carrier.

36. A T cell population, T cell or antigen according to any one of claims 30 to 34 for use in medicine. Presenting cells or exosomes.

37. A method of treating a human suffering from cancer, the cancer comprising administering to said human a cancer cell line comprising SEQ ID NOs: 1-2 and The method of claim 1, further comprising expressing a sequence selected from the group consisting of immunogenic fragments and variants of any one of the above. The present invention relates to a method for preventing a human from suffering from cancer, the method comprising the steps of: The method will express a sequence selected from any one of the immunogenic fragments and variants of A method for the treatment of a T cell population, a T cell, an antigen-presenting ... The method further comprises administering the xosome or composition to the human.

38. A T cell according to any one of claims 30 to 35 for use in the treatment or prevention of cancer in a human. A population, T cell, antigen presenting cell, exosome or composition, wherein the cells of the cancer are SEQ ID NO:

1. to 2, and an immunogenic fragment of any one of them, A T cell population, a T cell, an antigen presenting cell, an exosome or a composition.

39. The cancer may be ovarian cancer, particularly ovarian carcinoma, more particularly serous ovarian carcinoma, such as ovarian serous cystoma. The process, method or use of any one of claims 29, 37 and 38, wherein the treatment is cancer. The present invention relates to a T cell population, a T cell, an antigen-presenting cell, an exosome or a composition for the treatment of a patient with a pulmonary artery disease.

40. An isolated antibody that is immunospecific for the polypeptide of any one of claims 1 to 3. An antigen-binding polypeptide.

41. 41. The antigen-binding polypeptide of claim 40 which is a monoclonal antibody or a fragment thereof.

42. 42. An antigen-binding polypeptide according to claim 40 or claim 41, which is conjugated to a cytotoxic moiety. 。

43. 43. An antigen-binding polypeptide according to any one of claims 40 to 42 for use in medicine.

44. The antigen-binding polypeptide of any one of claims 40 to 42 is a medicament that is acceptable for use as a medicament. A pharmaceutical composition comprising the compound in combination with a carrier.

45. A method of treating a human suffering from cancer, the cancer comprising administering to said human a cancer cell line comprising SEQ ID NOs: 1-2 and The method of claim 1, further comprising expressing a sequence selected from the group consisting of immunogenic fragments and variants of any one of the above. The present invention relates to a method for preventing a human from suffering from cancer, the method comprising the steps of: The method will express a sequence selected from any one of the immunogenic fragments and variants of A method comprising administering to a subject an antigen-binding polypeptide or composition according to any one of claims 40 to 42 and 44. administering to said human an

46. A composition according to any one of claims 40 to 42 and 44 for use in the treatment or prevention of cancer in humans. The antigen-binding polypeptide or composition of claim 1, wherein the cells of the cancer are selected from the group consisting of SEQ ID NOs: 1-2 and any of their respective The antigen-binding polypeptide expressing a corresponding sequence selected from any one of the immunogenic fragments. Tide or composition.

47. The cancer may be ovarian cancer, particularly ovarian carcinoma, more particularly serous ovarian carcinoma, such as ovarian serous cystoma.

47. The method, antigen-binding polypeptide or composition of claim 45 or claim 46, which is cancer.

48. An HLA-binding polypeptide which is a polypeptide according to any one of claims 1 to 3 or a part thereof. An isolated antigen-binding polypeptide immunospecific for a IL-16 polypeptide.

49. 49. The antigen-binding polypeptide of claim 48 which is a T cell receptor or a fragment thereof.

50. to another polypeptide capable of binding to cytotoxic cells or other immune components within the subject.

50. An antigen-binding polypeptide as described in claim 48 or claim 49, which is conjugated to said antigen-binding polypeptide.

51. expressing on its surface an antigen-binding polypeptide according to any one of claims 48 to 50 Cytotoxic cells engineered to

52. The cytotoxic cell of claim 51, which is a T cell.

53. 53. A cytotoxic cell according to claim 51 or claim 52 for use in medicine.

54. 53. A pharmaceutical composition comprising a cell according to claim 51 or claim 52.

55. A method of treating a human patient suffering from cancer, the cells of the cancer comprising the sequence of SEQ ID NOs: 1-2 and expressing a sequence selected from any one of the immunogenic fragments and variants thereof. or a method for preventing a human from suffering from cancer, the cancer being a cancer comprising the sequence of SEQ ID NO: 1-2 and their derivatives. The method will express a sequence selected from any one of the immunogenic fragments and variants.

53. The method of claim 52, comprising administering to said human a cell according to claim 51. 。

56. A cytotoxic agent according to claim 51 or claim 52 for use in the treatment or prevention of cancer in humans. A cell, wherein the cancer cell is selected from SEQ ID NOs: 1-2 and any one of the immunogenic fragments thereof. The cytotoxic cell expressing the corresponding sequence of choice.

57. 1. A method for diagnosing a human being suffering from cancer, comprising: The cancer cells are selected from SEQ ID NO: 1 to 2 and any one of their immunogenic fragments or variants. Whether the polypeptide sequence to be detected or the nucleic acid encoding the polypeptide sequence is expressed determining If the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, the human has cancer. The method comprises the step of diagnosing the patient as suffering from the disease.

58. A human having ovarian cancer, particularly ovarian carcinoma, more particularly serous ovarian carcinoma, e.g., ovarian serous cystadenocarcinoma.

1. A method for diagnosing a cancer, comprising: The cancer cells are characterized in that they are either SEQ ID NO: 1 or SEQ ID NO: 2 or an immunogenic fragment or variant thereof. Determining whether to express a polypeptide sequence or a nucleic acid encoding the polypeptide sequence and If the polypeptide or the corresponding nucleic acid is overexpressed in the cancer cells, the human is suffer from cancer, particularly ovarian carcinoma, more particularly serous ovarian carcinoma, e.g. ovarian serous cystadenocarcinoma diagnosing the patient as suffering from the disease.

59. 1. A method of treating a human suffering from cancer comprising: (a) the cancer cells are selected from SEQ ID NOs: 1-2 and immunogenic fragments or variants of any one of them; A selected polypeptide sequence, or a nucleic acid encoding the polypeptide (e.g., SEQ ID NO:5- determining whether the gene expresses a sequence selected from sequences 6 and 7-8; and, if so, If it appears, (b) to said human, any one of claims 1 to 18, 30 to 35, 40 to 42, 44, 50, 51 and 53 Corresponding polypeptides, nucleic acids, vectors, compositions, T cell populations, T cells, and antigen-presenting cells of administering an exosome, an antigen-binding polypeptide or a cytotoxic cell; The method comprising:

60. isolated from tumors of humans suffering from cancer, (a) any one of SEQ ID NOs: 1-2; or (b) a variant of the sequence of (a); and (c) an immunogenic fragment of the sequence of (a); Use of a polypeptide comprising a sequence selected from the group consisting of: The method according to any one of claims 1 to 18, 30 to 35, 40 to 42, 44, 51, 52 and 54, The corresponding polypeptides, nucleic acids, vectors, compositions, T cell populations, T cells, antigen presentations, by a vaccine comprising a demonstrator cell, an exosome, an antigen-binding polypeptide or a cytotoxic cell The use as a biomarker for determining whether a patient will be suitable for treatment. 。

61. The cancer may be ovarian cancer, particularly ovarian carcinoma, more particularly serous ovarian carcinoma, such as ovarian serous cystoma.

61. The method or use of claim 59 or claim 60, wherein the cancer is cancer.

62. The cancer may be ovarian cancer, particularly ovarian carcinoma, more particularly serous ovarian carcinoma, such as ovarian serous cystoma.

57. The method or cytotoxic cell of claim 55 or claim 56, which is cancer.

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