阅读说明：本技术 用于癌症免疫治疗的肽及其肽组合物 (Peptides and peptide compositions thereof for cancer immunotherapy ) 是由 M·迪·马尔科 S·海顿 D·科瓦勒斯基 M·夫勒 A·内尔德 H·拉姆森 S·斯蒂凡诺 于 2018-04-10 设计创作，主要内容包括：本发明涉及用于免疫治疗方法的肽、蛋白质、核酸和细胞。特别是,本发明涉及癌症的免疫疗法。本发明还涉及单独使用或与其他肿瘤相关肽(刺激抗肿瘤免疫反应或体外刺激T细胞和转入患者的疫苗复合物的活性药物成分)联合使用的肿瘤相关T细胞(CTL)肽表位。与主要组织相容性复合体(MHC)分子结合的肽或与此同类的肽也可能是抗体、可溶性T细胞受体和其他结合分子的靶标。(The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the invention relates to immunotherapy of cancer. The invention also relates to tumor-associated T Cell (CTL) peptide epitopes, used alone or in combination with other tumor-associated peptides that stimulate an anti-tumor immune response or stimulate T cells in vitro and the active pharmaceutical ingredient of the vaccine composition transferred into the patient. Peptides that bind to Major Histocompatibility Complex (MHC) molecules or peptides of the same may also be targets for antibodies, soluble T cell receptors and other binding molecules.)

1. A peptide comprising the amino acid sequence of SEQ ID No.101, and pharmaceutically acceptable salts thereof, wherein the total length of the peptide is less than 30 amino acids.

2. The peptide of claim 1, wherein said peptide has the ability to bind to an MHC class-I or-II molecule, wherein said peptide, when bound to said MHC, is capable of being recognized by CD4 and/or CD8T cells.

3. The peptide of claim 1 or 2, wherein the peptide is modified and/or comprises non-peptide bonds.

4. The peptide according to any one of claims 1 to 3, wherein the peptide is part of a fusion protein fused to the N-terminal amino acid of HLA-DR antigen associated invariant chain (Ii).

5. A binding molecule which specifically recognizes a peptide according to any one of claims 1 to 3, or a peptide according to any one of claims 1 to 3 when bound to an MHC molecule.

6. The binding molecule of claim 5 which is an antibody, antibody fragment, T Cell Receptor (TCR), soluble TCR, or TCR fragment.

7. An aptamer that specifically recognizes the peptide according to any one of claims 1 to 3, the peptide according to any one of claims 1 to 3 bound to an MHC molecule.

8. A nucleic acid encoding the peptide according to any one of claims 1 to 3.

9. The nucleic acid of claim 8, which is DNA, PNA, RNA, or a combination thereof.

10. The nucleic acid of claim 8 or 9 linked to a heterologous promoter sequence.

11. An expression vector capable of expressing a nucleic acid according to any one of claims 8-10.

12. A recombinant host cell comprising a peptide according to any one of claims 1-4, a nucleic acid according to any one of claims 8-10, or an expression vector according to claim 11.

13. The recombinant host cell of claim 12, wherein the host cell is an antigen presenting cell.

14. The recombinant host cell according to claim 13, wherein the antigen presenting cell is a dendritic cell, a T cell, or an NK cell.

15. An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro T cells with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell for a period of time sufficient to activate said T cells in an antigen-specific manner, wherein said antigen is a peptide according to claim 1 or 2.

16. An activated T lymphocyte, produced by the method of claim 15, that selectively recognizes a cell that presents a polypeptide that is less than 30 amino acids in length and comprises the amino acid sequence of SEQ ID No. 101.

17. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of a peptide according to any one of claims 1 to 4, a binding molecule according to claim 6, an aptamer according to claim 7, a nucleic acid according to any one of claims 8 to 10, an expression vector according to claim 11, a host cell according to any one of claims 12 to 14, or an activated T lymphocyte according to claim 16, or a conjugated or labeled active ingredient and a pharmaceutically acceptable carrier.

18. A method for preparing a peptide according to any one of claims 1 to 4, comprising culturing a host cell according to claim 12 and isolating the peptide from the host cell and/or its culture medium.

19. Use of a peptide according to any one of claims 1 to 4, a binding molecule according to claim 5 or 6, an aptamer according to claim 7, a nucleic acid according to any one of claims 8 to 10, an expression vector according to claim 11, a host cell according to any one of claims 12 to 14, or an activated T lymphocyte according to claim 16 in the manufacture of a medicament for the treatment of cancer.

20. The use of claim 19, wherein the medicament is a vaccine.

21. Use according to claim 19 or 20, wherein the cancer is selected from colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia and other lymphomas (e.g. non-Hodgkin's lymphoma), post-transplant lymphoproliferative disease (PTLD) and other myelomas (e.g. primary myelofibrosis), primary thrombocytopenia, polycythemia vera and other tumors, such as: esophageal cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, melanoma, breast cancer, gallbladder and bile duct cancer, bladder cancer, uterine cancer, head and neck squamous cell carcinoma, mesothelioma and other tumors that show overexpression of a protein from which a peptide of SEQ ID No.101 is derived.

22. A kit, comprising:

a) a container comprising the pharmaceutical composition of claim 17 in solution or lyophilized form; and

b) a second container containing a diluent or reconstitution solution in the form of a lyophilized powder.

Background

According to the World Health Organization (WHO) data, cancer is one of four non-infectious fatal diseases worldwide in 2012. Colorectal, breast and respiratory cancers were listed as the top 10 causes of death in high income countries (http:// www.who.int/media/videos/fs 310/en /).

In 2012, 1410 ten new cancer cases, 3260 cancer patients (diagnosed within 5 years) and 820 cancer death cases were estimated worldwide (Ferlay et al, 2013; Bray et al, 2013).

The estimated number of different cancer types worldwide in 2012 (adult population, male and female) was (Ferlay et al, 2013; Bray et al, 2013): brain and nervous system cancers: 256213, colorectal cancer: 1360602, kidney cancer: 337860, liver cancer: 782451, gastric cancer: 951594 cases.

The estimated number of different cancer types (adult population, male and female) in the us, eu 28, china and japan in 2012 (Ferlay et al, 2013; Bray et al, 2013): brain and nervous system cancers: 135884, colorectal cancer: 845797, kidney cancer: 226733, liver cancer: 513172, gastric cancer: 615641 cases.

The estimated number of deaths (adult population, male and female) for different cancer types worldwide in 2012 was (Ferlay et al, 2013; Bray et al, 2013): brain and nervous system cancers: 189382, colorectal cancer: 693933, kidney cancer: 143406, liver cancer: 745533, gastric cancer: 723073 cases.

The estimated number of deaths (adult population, male and female) for the different cancer types in the us, eu 28, china and japan in 2012 was (Ferlay et al, 2013; Bray et al, 2013): brain and nervous system cancers: 100865, colorectal cancer: 396066, kidney cancer: 83741, liver cancer: 488485, gastric cancer: 447735 cases.

Within the brain cancer population, the present invention is particularly focused on Glioblastoma (GBM). GBM is the most common central nervous system malignancy, with an age-adjusted incidence of 3.19 per 10 million residents in the united states. GBM prognosis is very poor, with a 1-year survival rate of 35% and a 5-year survival rate of less than 5%. Male gender, older age, and race appear to be risk factors for GBM (Thakkar et al, 2014).

Colorectal cancer-depending on the colorectal cancer (CRC) stage, there are different standard therapies available for colon and rectal cancer. Standard procedures include surgery, radiotherapy, chemotherapy and targeted therapy for CRC (Berman et al, 2015 a; Berman et al, 2015 b).

Resection of the tumor is essential for the treatment of CRC. Because the rectum is located within the pelvis, the anatomical environment of rectal cancer is different from other CRCs and tumor access may be difficult. Highly differentiated small rectal tumors (stage T1) require resection but no further chemotherapy. Patients with rectal tumors of higher T stage received fluoropyrimidine neoadjuvant chemotherapy prior to total rectal resection (TME) and adjuvant chemotherapy. For chemotherapy, the drugs capecitabine or 5-fluorouracil (5-FU) are used. For combination chemotherapy, cocktail therapy (FOLFOX) with 5-FU, folinic acid and oxaliplatin is recommended (Stintzing, 2014; Berman et al, 2015 b).

Treatment of colon cancer includes radical hemicolectomy and lymphadenectomy. Early (UICC phase I) requires no additional treatment. UICC stage II tumor patients received 5-FU or capecitabine. Treatment of UICC phase III patients included drug combination regimens FOLFOX and XELOX (capecitabine + oxaliplatin) (Berman et al, 2015 a; Stintzing, 2014).

Metastatic unresectable CRC can be treated with cocktail chemotherapy such as FOLFIRI (5-FU, leucovorin, irinotecan), FOLFOX, FOLFOXIRI (5-FU, irinotecan, oxaliplatin), FOLFOX/capecitabine, FOLFOX/oxaliplatin, FOLFIRI/capecitabine and irinotecan or UFT (5-FU, tegafur-uracil) (sting, 2014).

In addition to chemotherapeutic drugs, several monoclonal antibodies targeting epidermal growth factor receptors (EGFR, cetuximab, panitumumab) or vascular endothelial growth factor-a (VEGF-a, bevacizumab) are administered to patients with high disease stage. For second line and subsequent treatments, the VEGF inhibitor Abbescept, the tyrosine kinase inhibitor Regorafenib, the thymidylate synthase inhibitor TAS-102 and the deoxyuridine triphosphate hydrolase inhibitor TAS-114(Stintzing, 2014; Wilson et al, 2014) may be used.

Recent clinical trials have analyzed active immunotherapy as a treatment option for CRC. These therapeutic strategies include vaccination with peptides of Tumor Associated Antigens (TAAs), whole tumor cells, Dendritic Cell (DC) vaccines and viral vectors (Koidoet al, 2013).

Peptide vaccines to date are directed against carcinoembryonic antigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen 3 recognized by T cells (SART3), β -human chorionic gonadotropin (β -hCG), wilm's tumor antigen 1(WT1), survivin-2B, MAGE3, p53, cyclic finger protein 43, and mitochondrial outer membrane translocase 34(TOMM34) or mutant KRAS. In several primary and secondary clinical trials, patients exhibit antigen-specific CTL responses or produce antibodies. In contrast to immune responses, many patients do not derive clinical-level benefit from peptide vaccines (Koido et al, 2013; Miyagi et al, 2001; Moulton et al, 2002; Okuno et al, 2011).

Dendritic cell vaccines include DCs or DC-tumor cell fusion products that are pulsed with TAA-derived peptides, tumor cell lysates, apoptotic tumor cells, or tumor RNA. Although many patients in phase I/II trials exhibit specific immune responses, only a few receive clinical benefit (Koido et al, 2013).

Whole tumor cell vaccines consist of autologous tumor cells that are modified to secrete GM-CSF, are modified by irradiation or viral infection, and are irradiated. Most patients did not show clinical benefit in several phase II/III clinical trials (Koido et al, 2013).

Vaccinia virus or replication-defective avipox virus encoding CEA and B7.1, ICAM-1 and LFA-3 has been used as an excipient in viral vector vaccines for phase I clinical trials. Different studies used non-replicating canarypox viruses encoding CEA and B7.1. In addition to inducing CEA-specific T cell responses, 40% of patients exhibited objective clinical responses (Horiget al., 2000; Kaufman et al., 2008).

Gastric cancer-the gastric wall is composed of 3 layers of tissue: mucosal (innermost), muscular (middle) and serosal (outermost). Gastric Cancer (GC) first occurs in cells lining the mucosal layer and spreads to the outer layer as it grows. There are four standard treatments available for gastric cancer. Methods of treatment for gastric cancer include endoscopic or surgical resection, chemotherapy, radiation therapy, or chemoradiotherapy. Surgery is the primary treatment for gastric cancer and is the only curative treatment. Since gastric cancer is mostly asymptomatic in the early stages, the disease is usually diagnosed in the late stages. For metastatic gastric cancer, no globally accepted standard chemotherapy combination regimen has been established. However, while 5-FU can be replaced by capecitabine or irinotecan and cisplatin by oxaliplatin, the combination of 5-FU and platinum analog remains the most widely accepted reference scheme worldwide. Furthermore, triple therapy comprising cisplatin, 5-FU and docetaxel may be applied, or cisplatin, 5-FU and trastuzumab for HER-2 overexpressing tumors (Leitlinie magenkarrzinom, 2012).

For advanced gastric cancer, current treatment regimens are all poorly effective, resulting in a low 5-year survival rate. Immunotherapy may be another approach to improve survival in gastric cancer patients. In clinical trials with gastric cancer, adoptive transfer of tumor-associated lymphocytes and cytokine-induced killer cells, peptide-based vaccines targeting HER2/neu, MAGE-3 or vascular endothelial growth factor receptors 1and 2, and dendritic cell-based vaccines targeting HER2/neu showed promising results. Immune checkpoint suppression and engineering T cells may represent more treatment options, currently being evaluated in preclinical and clinical studies (Matsueda and Graham, 2014).

The treatment options for glioblastoma-glioblastoma (WHO grade iv) are very limited. Standard treatment for young patients includes tumor resection or biopsy, focal radiotherapy, and chemotherapy with temozolomide, according to guidelines issued by the german academy of neurology. Alternative chemotherapeutic regimens consist of CCNU/lomustine or procarbazine in combination with CCNU and vincristine (PCV). For elderly patients, resection or biopsy of the tumor is not recommended. Depending on the methylation status of the O6-methylguanine-DNA-methyltransferase- (MGMT) -promoter, these patients received chemotherapy or radiation therapy. Methylation state negative is an indication of focal radiotherapy, while methylation state positive is an indication of temozolomide treatment with or without focal radiotherapy. Relapsing treatment includes re-ablation as well as chemotherapy and radiation therapy. Bevacizumab (anti-VEGF-antibody) is also approved for the treatment of relapses in the united states, canada and switzerland (Leitlinien fur diagnostic and therapy in der Neurologie, 2014).

Different immunotherapeutic approaches were investigated for the treatment of Glioblastoma (GB), including immune checkpoint suppression, vaccination and engineered T cell adoptive transfer.

Antibodies directed against inhibitory T cell receptors or their ligands have been shown to be effective in enhancing T cell-mediated anti-tumor immune responses in different cancer types, including melanoma and bladder cancer. Thus, the effect of T cell activating antibodies such as yipimema and nivolumab was evaluated in clinical GB trials, but preliminary data show autoimmune related adverse events.

Different vaccination strategies for GB patients are currently under investigation, including peptide-based vaccines, heat shock protein vaccines, autologous tumor cell vaccines, dendritic cell-based vaccines and viral protein-based vaccines. In these methods, peptides derived from GB-related proteins, such as epidermal growth factor receptor variant iii (egfrviii) or heat shock proteins or dendritic cells pulsed with autologous tumor cell lysate or cytomegalovirus components are used to induce an anti-tumor immune response in GB patients. Some of these studies show good safety and tolerability as well as promising efficacy data.

Adoptive transfer of genetically modified T cells is another immunotherapeutic approach to GB therapy. Different clinical trials are currently evaluating the safety and efficacy of T cells loaded with chimeric antigen receptors for HER2, IL-13 receptor alpha 2 and EGFRvIII (Ampie et al, 2015).

Liver cancer-disease management depends on the stage of the tumor at the time of diagnosis and the overall condition of the liver. If possible, a portion of the liver (hepatectomy) or the entire organ (hepatectomy) may be surgically removed. In particular patients with small tumors or completely resectable tumors are eligible for liver transplantation.

If surgery is not a treatment option, other different therapies are available. In the case of tumor ablation, the probe is injected into the liver and the tumor is destroyed by radio or microwave or cryotherapy. In embolization procedures, the blood supply to a tumor is blocked mechanically or chemically. In radiation therapy, high energy radio waves can be used to destroy tumors.

Chemotherapy for HCC includes a combination of doxorubicin, 5-fluorouracil and cisplatin for systemic treatment and doxorubicin, floxuridine and mitomycin C for hepatic arterial infusion. However, most HCC cases show high resistance to chemotherapeutic drugs (Enguita-German and Fortes, 2014).

Treatment options for advanced unresectable HCC are limited to sorafenib, a multiple tyrosine kinase inhibitor (Changet al, 2007; Wilhelm et al, 2004). Sorafenib is the only systemic drug demonstrated to extend survival by approximately 3 months and is currently the only experimental treatment for such patients (Chapiro et al, 2014; lovet et al, 2008).

Recently, a small number of immunotherapy trials have been conducted on HCC. Cytokines have been used to activate subpopulations of immune cells and/or increase tumor immunogenicity (Reinisch et al, 2002; Sangro et al, 2004). Other experiments have focused on the infusion of tumor infiltrating lymphocytes or activated peripheral blood lymphocytes (Shi et al, 2004; Takayama et al, 1991; Takayama et al, 2000).

To date, a small number of therapeutic vaccination trials have been conducted. Butterfield et al performed two experiments using peptides derived from alpha-fetoprotein (AFP) as vaccines or AFP peptide-loaded DCs in vitro (Butterfield et al, 2003; Butterfield et al, 2006). In two different studies, autologous Dendritic Cells (DCs) were pulsed in vitro with autologous tumor lysates (Lee et al, 2005) or lysates of the hepatoblastoma cell line HepG2 (Palmer et al, 2009). To date, vaccination trials have shown only limited improvement in clinical outcome.

Renal cell carcinoma-the initial treatment, most often partial or complete resection of the diseased kidney, remains the primary curative treatment (Rini et al, 2008). For first line treatment of patients with poor prognostic scores, several cancer organizations and guidelines set by the institute recommend receptor Tyrosine Kinase Inhibitors (TKI) sunitinib and pazopanib, the monoclonal antibody bevacizumab in combination with interferon alpha (IFN- α) and the mTOR inhibitor temsirolimus. TKI sorafenib, pazopanib, or more recently axitinib, were recommended as second line therapy for RCC patients who had failed prior cytokine (IFN- α, IL-2) therapy, according to guidelines established by the US NCCN and European EAU and ESMO. The NCCN guidelines also suggest sunitinib for this case (high level of evidence according to NCCN classification I).

Everolimus and acitinib are recommended as second line therapy for patients who do not benefit from VEGF-targeted therapy for TKIs according to current guidelines.

The known immunogenicity of RCC represents the basis to support the use of immunotherapy and cancer vaccines in advanced RCC. Interesting correlations between lymphocyte PD-1 expression and late stage staging, staging and prognosis of RCC, as well as RCC tumor cell selective PD-L1 expression and its potential association with poor clinical outcome, have led to the development of new anti-PD-1/PD-L1 formulations, whether used alone or in combination with anti-angiogenic drugs or other immunotherapeutic approaches to treat RCC (Massari et al, 2015). In advanced RCC, a phase III cancer vaccine trial named the triso study evaluated whether TroVax (vaccine using tumor associated antigen 5T4, containing poxvirus vector) added to first line standard therapy extended survival in locally advanced or mRCC patients. Median survival was not achieved in both groups and 399 patients (54%) continued to participate in the study, but data analysis confirmed previous clinical efficacy, suggesting that TroVax is immunologically active, with a correlation between 5T 4-specific antibody response intensity and survival improvement. In addition, there are several studies looking for peptide vaccines that use epitopes that are overexpressed in RCC.

Various approaches to tumor vaccines have been investigated. Studies using whole tumor approaches (including tumor cell lysates, dendritic cell fusion with tumor cells or whole tumor RNA) were performed in RCC patients, and remission of tumor lesions was reported in some of these experiments (Avigan et al, 2004; Holtl et al, 2002; Marten et al, 2002; Su et al, 2003; Wittig et al, 2001).

In view of the severe side effects and costs associated with the treatment of cancer, it is necessary to identify factors that can be used to treat cancer in general and colorectal, glioblastoma, gastric, hepatocellular, and renal cell carcinomas. There is also a need to determine factors representing biomarkers of cancer in general and colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, and renal cell carcinoma in particular, to better diagnose cancer, assess prognosis, and predict whether treatment was successful.

Cancer immunotherapy represents one option for cancer cell-specific targeting, while minimizing side effects. Cancer immunotherapy utilizes the presence of tumor-associated antigens.

The current classification of Tumor Associated Antigens (TAAs) mainly includes the following groups:

a) cancer-testis antigen: the first identified TAA that T cells can recognize belongs to this class of antigens, which were originally called cancer-testis (CT) antigens because their members are expressed in histologically distinct human tumors, in normal tissues, only in spermatocytes/spermatogonia of the testis, and occasionally in the placenta. Since testis cells do not express HLA class I and class II molecules, these antigens are not recognized by T cells in normal tissues and are therefore immunologically considered to be tumor-specific. Well known examples of CT antigens are members of the MAGE family and NY-ESO-1.

b) Differentiation antigen: both tumor and normal tissues (from which the tumor originates) contain TAAs. Most known differentiation antigens are found in melanoma and normal melanocytes. Many of these melanocyte lineage associated proteins are involved in melanin biosynthesis, and thus these proteins are not tumor specific, but are still widely used in cancer immunotherapy. Examples include, but are not limited to, tyrosinase for melanoma and PSA for Melan-A/MART-1 or prostate cancer.

c) Overexpressed TAA: genes encoding widely expressed TAAs have been detected in histologically distinct tumors as well as in many normal tissues, generally at low levels. It is likely that many epitopes processed and potentially presented by normal tissues are below the threshold level of T cell recognition, and their overexpression in tumor cells can trigger an anti-cancer response by breaking the previously established tolerance. Typical examples of such TAAs are Her-2/neu, survivin, telomerase or WT 1.

d) Tumor specific antigens: these unique TAAs arise from mutations in normal genes (e.g., β -catenin, CDK4, etc.). Some of these molecular changes are associated with neoplastic transformation and/or progression. Tumor specific antigens generally induce strong immune responses without risking autoimmune responses in normal tissues. On the other hand, these TAAs are in most cases only associated with the exact tumor on which they are confirmed, and are not commonly shared among many individual tumors. Peptide tumor specificity (or relatedness) may also occur if the peptide is derived from a tumor (associated) exon, in the case of proteins containing tumor-specific (associated) isoforms.

e) TAA resulting from aberrant post-translational modifications: such TAAs may be produced by proteins that are neither specific nor overexpressed in tumors, but which still have tumor-related properties (the association results from post-translational processing that is primarily active against tumors). Such TAAs arise from alterations in the variant glycosylation pattern, resulting in the tumor developing a novel epitope for MUC1 or in degradation processes resulting in events such as protein splicing, which may or may not be tumor specific.

f) Oncoviral proteins: these TTAs are viral proteins that may play a key role in carcinogenesis and, since they are foreign proteins (non-human proteins), are capable of triggering T cell responses. Examples of such proteins are human papilloma virus type 16 proteins, E6 and E7, which are expressed in cervical cancer.

T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins presented by Major Histocompatibility Complex (MHC) molecules. The antigen recognized by tumor-specific T lymphocytes, i.e. the epitope thereof, can be molecules derived from all protein types, such as enzymes, receptors, transcription factors, etc., which are expressed in the cells of the respective tumor and whose expression is usually upregulated compared to homologous unaltered cells.

MHC molecules are of two classes: MHC class I and MHC class II. MHC class I molecules consist of an alpha heavy chain and beta-2-microglobulin, and MHC class II molecules consist of an alpha chain and a beta chain. Its three-dimensional configuration forms a binding pocket for non-covalent interaction with the peptide.

MHC class I molecules are found on most nucleated cells. They presented peptides generated by cleavage of mainly endogenous proteins, defective ribosomal products (DRIP) and larger peptides. However, peptides derived from endosomal structures or from exogenous sources are also often found on MHC class I molecules. This non-classical way of presenting class I molecules is known in the literature as cross-presentation (Brossartand Bevan, 1997; Rock et al, 1990). MHC class II molecules are found predominantly on professional Antigen Presenting Cells (APCs) and present predominantly, for example, peptides of exogenous or transmembrane proteins that are occupied by APCs during endocytosis and subsequently processed.

Complexes of peptides and MHC class I are recognized by CD8 positive T cells bearing the corresponding T Cell Receptor (TCR), while complexes of peptides and MHC class II molecules are recognized by CD4 positive helper T cells bearing the corresponding TCR. Thus, it is well recognized that TCR, peptide and MHC are presented at a 1:1:1 stoichiometry.

CD 4-positive helper T cells play an important role in inducing and maintaining an effective response of CD 8-positive cytotoxic T cells. The recognition of a Tumor Associated Antigen (TAA) derived CD4 positive T cell epitope is important for the development of pharmaceutical products capable of eliciting anti-tumor immune responses (Gnjatic et al, 2003). At the tumor site, T helper cells maintain a cytokine environment friendly to cytotoxic T Cells (CTL) (Mortara et al, 2006) and attract effector cells such as CTL, Natural Killer (NK) cells, macrophages and granulocytes (Hwang et al, 2007).

In the absence of inflammation, MHC class II molecule expression is primarily restricted to immune system cells, especially professional Antigen Presenting Cells (APCs), e.g., monocytes, monocyte derived cells, macrophages, dendritic cells. Expression of MHC class II molecules is found in tumor cells of cancer patients (Dengjel et al, 2006).

The longer (elongated) peptides of the invention may serve as MHC class II active epitopes. MHC-II epitope-activated helper T cells play an important role in orchestrating CTL effector functions of anti-tumor immunity. Helper T cell epitopes that trigger TH1 cell responses support effector functions of CD8 positive killer T cells, including cytotoxic functions that act directly on tumor cells (tumor-associated peptide/MHC complexes are displayed on the surface of such tumor cells). Thus, the tumor-associated T helper cell epitopes, used alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients of vaccine compounds for stimulating anti-tumor immune responses.

Mammalian (e.g., mouse) models have shown that CD4 positive T cells can inhibit angiogenesis sufficiently to inhibit tumor expression by secreting interferon-gamma (IFN γ) even in the absence of CD8 positive T lymphocytes (Beatty and Paterson, 2001; Mumberg et al, 1999). There is no evidence for CD4T cells as direct anti-tumor effectors (Braumuller et al, 2013; Tran et al, 2014).

Since constitutive expression of HLA class II molecules is usually restricted to immune cells, it was previously considered impossible to isolate class II peptides directly from primary tumors. However, Dengjel et al succeeded in directly recognizing multiple MHC class II epitopes in tumors (WO 2007/028574, EP 1760088B 1).

Since both CD 8-dependent and CD 4-dependent responses together and synergistically contribute to anti-tumor effects, the identification and characterization of tumor-associated antigens recognized by CD8+ T cells (ligand: MHC class I molecule + peptide epitope) or CD4 positive T helper cells (ligand: MHC class II molecule) is important for the development of tumor vaccines.

For a peptide to trigger (elicit) a cellular immune response by an MHC class I peptide, it must also bind to an MHC molecule. This process relies on alleles of MHC molecules and specific polymorphisms of peptide amino acid sequences. MHC class I-binding peptides are typically 8-12 amino acid residues in length and typically comprise two conserved residues ("anchors") in their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele has a "binding motif" to determine which peptides are capable of specifically binding to the binding groove.

In an MHC class I dependent immune response, peptides not only bind to certain MHC class I molecules expressed by tumor cells, but they must then be recognized by a T cell-loaded specific T Cell Receptor (TCR).

Special conditions must be met for the proteins recognized by T lymphocytes as tumor-specific or related antigens and for therapy. The antigen should be expressed predominantly by tumor cells and not by normal healthy tissue, or in relatively small amounts. In a preferred embodiment, the peptide should be over-represented in tumor cells compared to normal healthy tissue. Preferably, the corresponding antigen is not only present in a tumor, but also in high concentrations (i.e., the number of copies of the corresponding peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins that are directly involved in the transformation of normal cells into tumor cells, which occurs due to their function in cell cycle control or apoptosis inhibition. In addition, downstream targets of these proteins that directly lead to a transformation event may be upregulated and thus may be indirectly associated with the tumor. These indirect tumor-associated antigens may also be targets for vaccination methods (Singh-Jasuja et al, 2004). It is essential that epitopes are present in the amino acid sequence of the antigen to ensure that such peptides from tumour associated antigens ("immunogenic peptides") can cause T cell responses in vitro or in vivo.

Basically, any peptide that binds to an MHC molecule may serve as a T cell epitope. The prerequisite for inducing an in vitro or in vivo T cell response is the presence of T cells with the corresponding TCR and the absence of immune tolerance to this particular epitope.

Thus, TAAs are the starting point for development based on T cell therapies, including but not limited to tumor vaccines. Methods for identifying and characterizing TAAs are generally based on the use of T cells in patients or healthy subjects, or on the generation of differential transcriptional profiles or differential expression patterns between tumor and normal tissue peptides. However, the identification of genes that are overexpressed or selectively expressed in tumor tissues or human tumor cell lines does not provide accurate information on the antigens transcribed using these genes in immunotherapy. This is because only a portion of the epitopes of these antigens are suitable for this application, since T cells with the corresponding TCR must be present and immune tolerance to this particular epitope must be absent or minimal. Therefore, in a very preferred embodiment of the invention, it is important to select only those peptides that are over-or selectively presented for situations where functional and/or proliferative T cells are found. Such functional T cells are defined as T cells that are clonally expanded upon stimulation with a specific antigen and are capable of performing effector functions ("effector T cells").

Where targeting is to a peptide-MHC through a particular TCR (e.g. a soluble TCR) and an antibody or other binding molecule (scaffold) according to the invention, immunogenicity of the potential peptide is of minor importance. In these cases, presentation is the determining factor.

In a first aspect of the invention, the invention relates to a peptide comprising an amino acid sequence selected from the group comprising SEQ ID NO:1 to SEQ ID NO:268, or a variant sequence thereof which is at least 77%, preferably at least 88% homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO:1 to SEQ ID NO:268 (wherein said variant binds to MHC and/or induces T cells to cross-react with said peptide), or a pharmaceutically acceptable salt thereof (wherein said peptide is not a potentially full-length polypeptide).

The invention further relates to a peptide of the invention comprising a sequence selected from the group comprising SEQ ID No.1 to SEQ ID No.268 or a variant having at least 77%, preferably at least 88% homology (preferably at least 77% or at least 88% identical) to SEQ ID No.1 to SEQ ID No.268, wherein the total length of said peptide or variant thereof is from 8 to 100, preferably from 8 to 30, most preferably from 8 to 14 amino acids.

The following table shows the peptides according to the invention, their respective SEQ ID NOs, and possible source (potential) genes for these peptides. In Table 1, the peptides containing SEQ ID NO1 to SEQ ID NO 37 were bound to HLA-A01, the peptides containing SEQ ID NO 38 to SEQ ID NO 61 were bound to HLA-A02, the peptides containing SEQ ID NO 62 to SEQ ID NO 112 were bound to HLA-A03, the peptides containing SEQ ID NO 113 to SEQ ID NO 142 were bound to HLA-A24, the peptides containing SEQ ID NO 143 to SEQ ID NO 175 were bound to HLA-B07, the peptides containing SEQ ID NO 176 to SEQ ID NO 194 were bound to HLA-B08, and the peptides containing SEQ ID NO 195 to SEQ ID NO 241 were bound to HLA-B44. The peptides in table 2 were previously disclosed in a large list as high-throughput screening results with high error rates, or calculated using algorithms, but had no previous association with cancer. In Table 2, the peptides containing SEQ ID NO:242 to SEQ ID NO:248 were bound to HLA-A01, the peptides containing SEQ ID NO:249 to SEQ ID NO:251 were bound to HLA-A02, the peptides containing SEQ ID NO:252 to SEQ ID NO:254 were bound to HLA-A03, the peptides containing SEQ ID NO:255 to SEQ ID NO:259 were bound to HLA-B07, and the peptides containing SEQ ID NO:260 to SEQ ID NO:266 were bound to HLA-B44. The peptides in table 3 are other peptides that may be useful, particularly in combination with other peptides of the invention. In Table 3, the peptide containing SEQ ID NO. 267 bound to HLA-A02 and the peptide containing SEQ ID NO.268 bound to HLA-A24.

Table 1: the peptide of the present invention.

Table 2: other peptides of the invention, have not previously been known to be associated with cancer.

Table 3: peptides of the invention for use in, e.g., personalized cancer therapy

Sequence ID number	Sequence of	Formal gene symbol	HLA allotype
				267	KTLGKLWRL	SOX10,SOX8,SOX9	A*02
268	DYIPYVFKL	APOB	A*24

The present invention relates generally to peptides according to the invention for use in the treatment of proliferative diseases, such as chronic lymphocytic leukemia, chronic myelogenous leukemia and acute myelogenous leukemia and other lymphomas (e.g., non-hodgkin lymphoma), such as post-transplant lymphoproliferative disease (PTLD) and myeloma (e.g., primary myelofibrosis), primary thrombocytopenia, polycythemia vera, and other tumors, such as: esophageal cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, melanoma, breast cancer, gallbladder and bile duct cancer, bladder cancer, uterine cancer, head and neck squamous cell carcinoma, mesothelioma.

Particularly preferred are peptides according to the invention (alone or in combination) selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 268. More preferred are said peptides (alone or in combination) selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:241 (see Table 1) and their use for immunotherapy of colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia and other lymphomas (e.g. non-Hodgkin's lymphoma), post-transplant lymphoproliferative disorder (PTLD) and other myelomas, such as: primary myelofibrosis, primary thrombocytopenia, polycythemia vera, and other tumors, such as: esophageal cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, melanoma, breast cancer, gallbladder and bile duct cancer, bladder cancer, uterine cancer, head and neck squamous cell carcinoma, mesothelioma, preferably colorectal cancer, glioblastoma, gastric cancer, hepatocellular cancer, renal cell carcinoma.

Another aspect of the invention relates to the use of the peptides according to the invention, preferably in combination, for the treatment of proliferative diseases selected from colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia and acute myelogenous leukemia and other lymphomas (e.g. non-Hodgkin's lymphoma), post-transplant lymphoproliferative disease (PTLD) and other myelomas (e.g. primary myelofibrosis), primary thrombocytopenia, polycythemia vera and other tumors, such as: esophageal cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, melanoma, breast cancer, gallbladder and bile duct cancer, bladder cancer, uterine cancer, head and neck squamous cell carcinoma, mesothelioma.

The invention also relates to peptides of the invention having the ability to bind to the Major Histocompatibility Complex (MHC) I or to molecules present in elongated form, e.g.of varying length-MHC-class II.

The invention further relates to the peptides of the invention, wherein the (each) peptide consists or consists essentially of an amino acid sequence according to SEQ ID NO1 to SEQ ID NO 268.

The invention further relates to a peptide according to the invention, wherein said peptide is modified and/or comprises non-peptide bonds.

The invention further relates to a peptide of the invention, wherein the peptide is part of a fusion protein, in particular fused to the N-terminal amino acid of the invariant chain (Ii) associated with the HLA-DR antigen, or to an antibody (e.g. a dendritic cell specific antibody) or a sequence of an antibody.

The invention further relates to a nucleic acid encoding a peptide according to the invention. The invention further relates to a nucleic acid according to the invention, being DNA, cDNA, PNA, RNA, and possibly a combination thereof.

The invention further relates to an expression vector capable of expressing and/or expressing the nucleic acid of the invention.

The invention further relates to a peptide according to the invention, a nucleic acid according to the invention or a pharmaceutically acceptable expression vector according to the invention for the treatment of a disease, in particular for the treatment of cancer.

The invention further relates to specific antibodies to the peptides of the invention or to the peptide complexes (containing MHC) described in the invention and to methods for producing these antibodies.

The invention further relates to T Cell Receptors (TCRs) of the invention, in particular soluble TCRs (stcrs) and cloned TCRs processed into autologous or allogeneic T cells, as well as methods of making these TCRs and methods of making NK cells bearing said TCRs or said TCR cross-reactivity.

Antibodies and TCRs are further embodiments of the peptide immunotherapeutic uses according to the current invention.

The invention further relates to a host cell comprising a nucleic acid according to the invention or an expression vector as described above. The invention further relates to a host cell of the invention which is an antigen presenting cell, preferably a dendritic cell.

The invention further relates to a method for formulating a peptide of the invention, said method comprising culturing a host cell of the invention and isolating the peptide from said host cell or its culture medium.

The invention further relates to the methods of the invention wherein the antigen is loaded into class I or II MHC molecules expressed on the surface of a suitable antigen presenting cell or artificial antigen presenting cell by binding to a sufficient amount of antigen containing antigen presenting cells.

The invention further relates to the method of the invention wherein the antigen presenting cell consists of an expression vector capable of expressing a peptide comprising SEQ ID No.1 to SEQ ID No.268, preferably comprising SEQ ID No.1 to SEQ ID No.241, or a variant amino acid sequence.

The invention further relates to a T-cell promoter produced by the method of the invention, wherein the T-cell selectively recognizes a cell that expresses a polypeptide comprising an amino acid sequence of the invention.

The invention further relates to a method of killing target cells in a patient, wherein the target cells in the patient abnormally express a polypeptide comprising any of the amino acid sequences of the invention, the method comprising administering to the patient an effective amount of T cells produced by the method of the invention.

The invention further relates to the use of any of said peptides, nucleic acids of the invention, expression vectors of the invention, cells of the invention, priming T lymphocytes of the invention as a medicament or in the manufacture of a medicament, T cell receptors or antibodies or other peptide-and/or peptide-MHC binding molecules. The agent preferably has anti-cancer activity.

Preferably, the agent is a soluble TCR or antibody based cell therapy drug, vaccine or protein.

The invention further relates to a use of the invention, wherein the cancer cell is colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia and acute myelogenous leukemia and other lymphomas (e.g., non-hodgkin lymphoma), post-transplant lymphoproliferative disease (PTLD) and other myelomas (e.g., primary myelofibrosis), primary thrombocytopenia, polycythemia vera and other tumors, such as: esophageal cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, melanoma, breast cancer, gallbladder and bile duct cancer, bladder cancer, uterine cancer, head and neck squamous cell carcinoma, mesothelioma, preferably chronic lymphocytic leukemia, chronic myelogenous leukemia, and acute myelogenous leukemia cells.

The invention further relates to a biomarker, herein referred to as "target", based on a peptide according to the invention, which can be used for the diagnosis of cancer, preferably colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma. The marker may be over-presented by the peptide itself or over-expressed by the corresponding gene. The marker may also be used to predict the likelihood of success of a treatment, preferably an immunotherapy, most preferably an immunotherapy targeting the same target recognized by the biomarker. For example, antibodies or soluble TCRs can be used to stain tumor sections to detect the presence or absence of the relevant peptide complexed to MHC.

Alternatively, the antibody has further effector functions, such as an immunostimulatory domain or a toxin.

The invention also relates to the use of these novel targets in the treatment of cancer.

Whether or not an immune response is stimulated depends on the presence of antigens that are recognized as foreign by the host immune system. The discovery that the presence of tumor associated antigens increases the likelihood of using the host immune system to interfere with tumor growth. Currently, various mechanisms of utilizing the humoral and cellular immune systems for immunization are being explored for cancer immunotherapy.

Specific elements of the cellular immune response specifically recognize and destroy tumor cells. T-cells isolated from tumor infiltrating cell populations or peripheral blood suggest that these cells play an important role in the natural immune defense of cancer. In particular, CD8 positive T cells play an important role in this response, TCD8+ recognizes class I molecules contained in peptides carried by the Major Histocompatibility Complex (MHC) of typically 8 to 10 amino acid residues derived from proteins or defective ribosomal products (DRIP) located in the cytoplasm. Human MHC molecules are also known as human leukocyte-antigens (HLA).

The term "T cell response" refers to the specific spread and initiation of effector functions induced by a peptide in vitro or in vivo. For MHC class I-restricted cytotoxic T cells, the effector function may be lysis of peptide pulsed, peptide precursor pulsed or native peptide presented target cells, secretion of cytokines, preferably peptide induced interferon- γ, TNF- α or IL-2, secretion of effector molecules, preferably peptide induced granzyme or perforin, or degranulation.

The term "peptide" as used herein refers to a series of amino acid residues, typically joined by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. These peptides are preferably 9 amino acids in length, but may be 8 amino acids in length to as short as 10, 11, or 12 amino acids in length or longer, and if MHC class II peptides (elongate variants of the peptides of the invention) may be 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length or longer.

Furthermore, the term "peptide" shall include salts of a series of amino acid residues, typically linked through peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. Preferably, the salt is a pharmaceutically acceptable salt of the peptide, for example: chloride or acetic acid (trifluoroacetic acid) salt. It must be noted that the salts of the peptides of the invention are substantially different from the peptides in their in vivo state, since they are not salts in vivo.

The term "peptide" shall also include "oligopeptides". The term "oligopeptide" as used herein refers to a series of amino acid residues, typically linked through peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not critical to the present invention, as long as the correct epitope is maintained in the oligopeptide. Generally, oligopeptides are less than about 30 amino acid residues in length and longer than about 15 amino acids in length.

The term "polypeptide" refers to a series of amino acid residues, typically joined through peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not critical to the present invention, so long as the correct epitope is maintained. The term "polypeptide" as opposed to the term peptide or oligopeptide refers to a molecule comprising more than about 30 amino acid residues.

A peptide, oligopeptide, protein or nucleotide encoding such a molecule is "immunogenic" (and thus an "immunogen" in the present invention) if it induces an immune response. In the context of the present invention, a more specific definition of immunogenicity is the ability to induce a T cell response. An "immunogen" is therefore a molecule capable of inducing an immune response, and in the context of the present invention, a molecule capable of inducing a T cell response. In another aspect, the immunogen may be a peptide, a complex of a peptide with MHC, and/or a protein for increasing specific antibody or TCR resistance.

A class I T cell "epitope" requires a short peptide that binds to the MHC class I receptor, thereby forming a ternary complex (MHC class I α chain, β -2-microglobulin and peptide) that can be recognized by T cell load matching T cell receptor binding to MHC/peptide complexes with appropriate affinity. Peptides that bind to MHC class I molecules are typically 8-14 amino acids in length, most typically 9 amino acids in length.

In humans, there are three distinct genetic loci that encode MHC class I molecules (human MHC molecules are also designated Human Leukocyte Antigens (HLA)): HLA-A, HLA-B and HLA-C. HLA-A01, HLA-A02 and HLA-B07 are examples of different MHC class I isobaric genes that can be expressed from these gene sites.

Table 4: HLA-A02, HLA-A01, HLA-A03, HLA-A24, HLA-B07, HLA-B08, and HLA-B44. Haplotype frequency Gf was derived from a study using HLA typing data from over 650 million volunteer donor enrolled in the united states (Gragert et al, 2013). The haplotype frequency is the frequency of unique allelic genes on the individual's chromosomes. The frequency of genotype for these bitgenes is high due to the diploid genome in mammalian cells and can be calculated using Hardy-Weinberg's law (F ═ 1- (1-Gf) 2).

The peptides of the invention, preferably when incorporated into the vaccines of the invention as described herein, are conjugated to a x 02, a 01, a 03, a 24, B07, B08 or B44. Vaccines may also include pan-bound MHC class II peptides. Thus, the vaccines of the invention can be used to treat cancer in patients positive for a × 02, a × 01, a × 03, a × 24, B × 07, B × 08 or B × 44, but not because of the extensive tuberculous nature of these peptides, MHC class II allotypes must be selected.

If an a 02 peptide of the invention is combined with a peptide that binds to another allele, for example a 24, a higher proportion of the patient population can be treated than the MHC class I allele alone. Although less than 50% of patients in most populations are resolved by individual alleles, a vaccine of the invention containing both HLA-A24 and HLA-A02 epitopes can treat at least 60% of patients in any relevant population. In particular, in each region, at least one of these alleles of the patient in the following ratios has a positive effect: 61% in the united states, 62% in western europe, 75% in china, 77% in korea, and 86% in japan (calculated according to www.allelefrequencies.net).

Table 5: HLA allele gene coverage (calculated according to the method described in (Gragert et al, 2013)) in the european white population.

In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxynucleotides.

The nucleotide sequence encoding a particular peptide, oligopeptide or polypeptide may be a natural nucleotide sequence or a synthetic nucleotide sequence. Generally, DNA fragments encoding peptides, polypeptides, and proteins of the invention are composed of cDNA fragments and short oligonucleotide linkers, or a series of oligonucleotides, to provide a synthetic gene that can be expressed in a recombinant transcription unit comprising regulatory elements derived from a microbial or viral operon.

The term "nucleotide encoding of a peptide" as used herein refers to encoding a peptide with a nucleotide sequence, wherein the peptide comprises artificial (man-made) start and stop codons compatible with the biological system in which the sequence will be expressed by the dendritic cell or another cellular system used to generate the TCR.

Reference herein to a nucleic acid sequence includes both single-stranded and double-stranded nucleic acids. Thus, unless otherwise indicated herein, for example with respect to DNA, a particular sequence is a single-stranded DNA of that sequence, a duplex (double-stranded DNA) of that sequence with its complement, and the complement of that sequence.

The term "coding region" refers to that portion of a gene that naturally or normally encodes the expression product of the gene in its natural genomic environment, i.e., the region that encodes the natural expression product of the gene in vivo.

The coding region may be derived from a non-mutated ("normal") gene, a mutated gene or an aberrant gene, and may even be derived from a DNA sequence, and may well be synthesized in the laboratory using DNA synthesis methods well known in the art.

The term "expression product" refers to a polypeptide or protein that is the translation product of any nucleic acid sequence encoded equivalent by the degeneracy of genes and genetic codes and thus encoding the same amino acids.

The term "fragment," when referring to a coding sequence, refers to a portion of DNA that contains a non-complete coding region, the expression product of which has substantially the same biological function or activity as the expression product of the complete coding region.

The term "DNA fragment" refers to a DNA polymer, either in the form of individual fragments or as a component of a larger DNA construct, which is obtained in substantially pure form, i.e., free of contaminating endogenous material, from DNA that has been isolated at least once, and in quantities or concentrations that enable the identification, manipulation and recovery of the fragment and its component nucleotide sequences using standard biochemical methods, e.g., using cloning vectors. Such fragments exist in the form of open reading frames (not interrupted by internal untranslated sequences) or introns (usually present in eukaryotic genes). The untranslated DNA sequence may be present downstream of the open reading frame where it does not interfere with the manipulation or expression of the coding region.

The term "primer" denotes a short nucleic acid sequence that can pair with a DNA strand and provide a free 3' -OH end where DNA polymerase begins to synthesize a strand of deoxyribonucleic acid.

The term "promoter" refers to a region of DNA that is involved in the binding of RNA polymerase to initiate transcription.

The term "isolated" means that a substance is removed from its original environment (e.g., the natural environment if it occurs naturally). For example, a native nucleotide or polypeptide in a living animal is not isolated, but a nucleotide or polypeptide isolated from some or all of the coexisting materials in the native system is isolated. Such polynucleotides may be part of a vector and/or such polynucleotides and polypeptides may be part of a composition, and as the vector or composition is not part of its natural environment, it remains isolated.

The polynucleotides and recombinant or immunogenic polypeptides disclosed in the present invention may also be present in "purified" form. The term "purified" does not require absolute purity; it is a relative definition and may include highly purified or partially purified preparations, as those skilled in the relevant art will understand. For example, each clone isolated from a cDNA library that has been purified to electrophoretic homogeneity by conventional methods. It is expressly contemplated that the starting material or natural substance may be purified by at least one order of magnitude, preferably two or three orders of magnitude, more preferably four or five orders of magnitude. Furthermore, it is expressly contemplated that the purity of the polypeptide is preferably 99.999%, or at least 99.99% or 99.9%; even more suitably 99% by weight or more.

Nucleic acid and polypeptide expression products disclosed according to the invention, as well as expression vectors comprising such nucleic acids and/or polypeptides, may exist in "concentrated form". The term "concentrated" as used herein means that the concentration of a material is at least about 2, 5, 10, 100 or 1000 times its natural concentration, advantageously 0.01%, preferably at least 0.1% by weight. Concentrated formulations of about 0.5%, 1%, 5%, 10% and 20% by weight are also specifically contemplated. The sequences, configurations, vectors, clones, and other materials comprising the present invention may advantageously be present in concentrated or isolated form. The term "active fragment" refers to a fragment that generates an immune response (i.e., has immunogenic activity), typically a fragment of a peptide, polypeptide or nucleic acid sequence, whether administered alone or optionally together with a suitable adjuvant or in a carrier to an animal, such as a mammal, e.g., a rabbit or mouse, also including a human; this immune response takes the form of stimulating a T cell response in a recipient animal (e.g., a human). Alternatively, an "active fragment" may also be used to induce an in vitro T cell response.

The terms "portion", "segment" and "fragment" as used herein when used in relation to a polypeptide refer to a contiguous sequence of residues, such as amino acid residues, the sequence of which forms a subset of a larger sequence. For example, if a polypeptide is treated with any endopeptidase (e.g., trypsin or chymotrypsin), the oligopeptide resulting from the treatment will represent a portion, segment, or fragment of the starting polypeptide. When used in relation to a polynucleotide, these terms refer to the product resulting from treatment of the polynucleotide with any endonuclease.

According to the present invention, the term "percent identity" or "percent identity", if referring to a sequence, means that the sequence to be compared ("the compared sequence") is compared to the sequence or sequences of claims after alignment of the sequence to be compared ("the reference sequence"). The percent equivalence is then calculated according to the following formula: percent equivalence of 100[1- (C/R) ]

Wherein C is the number of differences between the reference sequence and the compared sequence over the alignment length between the reference sequence and the compared sequence, wherein

(i) Each base or amino acid sequence in the reference sequence has no corresponding aligned base or amino acid in the compared sequence;

(ii) each gap in the reference sequence, an

(iii) Each aligned base or amino acid in the reference sequence is different from the aligned base or amino acid in the aligned sequence, i.e., a difference is formed and

(iiii) alignment must begin at position 1 of the alignment sequence;

and R is the number of bases or amino acids in the reference sequence that produce any gaps in the reference sequence over the length of alignment of the reference sequence to the compared sequence, also counted as one base or amino acid.

If there is an alignment between the "aligned sequence" and the "reference sequence" that is approximately equal to or greater than the specified minimum percent of identity as calculated above, then the aligned sequence has the specified minimum percent of identity with the reference sequence, although there may be alignments where the percent of identity as calculated above herein is less than the specified percent of identity.

Thus, as mentioned above, the present invention proposes a peptide comprising a sequence selected from the group of SEQ ID NO1 to SEQ ID NO268, or a variant thereof having 88% homology to SEQ ID NO1 to SEQ ID NO268, or a variant inducing T cell cross-reactivity with the peptide. The peptides of the invention have the ability to bind to a class II molecule of Major Histocompatibility Complex (MHC) I or an elongated version of the peptide.

In the present invention, the term "homology" refers to the degree of identity between two amino acid sequences (see percent identity above, e.g., peptide or polypeptide sequences. the "homology" described above is determined by aligning two sequences adjusted under ideal conditions with the sequences to be compared.

One skilled in the art can assess whether T cells induced by a particular peptide variant cross-react with the peptide itself (Apay et al, 2006; Colombetti et al, 2006; Fong et al, 2001; Zaremba et al, 1997).

By "variant" of a given amino acid sequence, the inventors mean that the side chain of one or two amino acid residues, etc., is altered by substitution with the side chain of another natural amino acid residue or other side chain, so that such a peptide can still bind to an HLA molecule in substantially the same manner as a peptide comprising the given amino acid sequence (consisting of SEQ ID NO:1 to SEQ ID NO: 268). For example, a peptide may be modified to at least maintain (e.g., not increase) its ability to interact with and bind to the binding groove of a suitable MHC molecule such as HLA-a 02 or-DR, and to at least maintain (e.g., not increase) its ability to bind to a TCR of a T-cell promoter.

These T cells can then cross-react with cells expressing the polypeptide (which comprises the natural amino acid sequence of the homologous peptide as defined in the present invention) and with killer cells. As described in the scientific literature and data banks (Rammensee et al, 1999; Godkin et al, 1997), certain sites of HLA-A binding peptides are usually anchor residues, which form a core sequence that is commensurate with the binding pattern of HLA-binding grooves, whose definition is determined by the polarity, electrophysical, hydrophobic and steric properties of the polypeptide chains that make up the binding grooves. Thus, the skilled person is able to modify the amino acid sequences set forth in SEQ ID No.1 to SEQ ID No.268 by retaining known anchor residues and to determine whether these variants retain the ability to bind to MHC class I or II molecules. The variants of the invention retain the ability to bind to the TCR of initiating T cells which can then cross-react with and kill cells expressing a polypeptide comprising the native amino acid sequence of the cognate peptide as defined herein.

The original (unmodified) peptides disclosed herein may be modified by substitution of one or more residues at different (possibly selective) positions within the peptide chain, if not otherwise indicated. Preferably, these substitutions are at the end of the amino acid chain. Such substitutions may be conservative, for example, where one amino acid is substituted for another amino acid of similar structure and characteristics, such as where one hydrophobic amino acid is substituted for another. More conservative substitutions are those between amino acids of the same or similar size and chemical nature, for example, leucine substituted with isoleucine. In the study of sequence variations in the native homologous protein family, certain amino acid substitutions tend to be more tolerant than others, and these amino acids tend to show similarity in size, charge, polarity and hydrophobicity with respect to the original amino acids, which underlies the determination of "conservative substitutions".

Herein, conservative substitutions are defined as exchanges within one of the following five groups: group 1-small aliphatic, non-polar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); group 2-polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); group 3-polar, positively charged residue (His, Arg, Lys); group 4-a bulky aliphatic nonpolar residue (Met, Leu, Ile, Val, Cys) and group 5-a bulky aromatic residue (Phe, Tyr, Trp).

Less conservative substitutions may involve the substitution of one amino acid with another having similar characteristics but differing in size, such as: alanine is substituted with an isoleucine residue. Highly non-conservative substitutions may involve the substitution of one acidic amino acid with another amino acid having polar or basic properties. However, such "aggressive" substitutions cannot be considered ineffective and are not considered because the chemical action is not fully predictable, and aggressive substitutions may bring about unexpected contingent effects in their simple chemical principles.

Of course, such substitutions may involve structures other than the normal L-amino acid. Thus, D-amino acids may be substituted by L-amino acids commonly found in the antigenic peptides of the present invention and remain within the scope of this disclosure. In addition, non-standard amino acids (i.e., in addition to the common natural proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.

If substitutions at more than one position are found to result in a peptide having an antigenic activity substantially equal to or greater than the value defined below, combinations of these substitutions are tested to determine whether the combined substitutions produce an additive or synergistic effect on the antigenicity of the peptide. The number of positions within the peptide that are simultaneously substituted cannot exceed 4 at the most.

A peptide consisting essentially of the amino acid sequence referred to herein may have one or two non-anchor amino acids (see below in relation to the anchor motif) exchanged, without the situation that the ability to interact with human Major Histocompatibility Complex (MHC) -class I or II molecules is substantially altered or adversely affected compared to the unmodified peptide. In another embodiment, one or two amino acids may be exchanged with their conserved exchange partners (see below) in a peptide consisting essentially of the amino acid sequences described herein, without the situation that the ability to bind to human Major Histocompatibility Complex (MHC) -class I or II molecules is substantially altered or adversely affected compared to the unmodified peptide.

These amino acid residues that do not substantially interact with the T cell receptor can be modified by substituting other amino acids that do not substantially affect the T cell response and do not interfere with the binding to the relevant MHC. Thus, except for certain limiting conditions, a peptide of the invention may be any peptide comprising a given amino acid sequence or portion or variant thereof (this term as used by the inventors includes oligopeptides or polypeptides).

Table 6: variants and motifs of the peptides according to SEQ ID NO1, 21, 48, 250, 91, 253, 117, 125, 160, 255, 178, 183, 229 and 232

Longer (elongated) peptides may also be suitable. MHC class I epitopes (typically 8 to 11 amino acids in length) may result from processing of peptides from longer peptides or proteins containing the actual epitope. Residues flanked by actual epitopes are preferably residues that hardly affect the proteolytic cleavage required to expose the actual epitope during processing.

The peptides of the invention may be elongated by up to four amino acids, i.e. 1,2, 3 or 4 amino acids, and may be added to either end in any combination between 4:0 and 0: 4. The inventive combinations of elongations are shown in table 7.

Table 7: elongated combinations of the peptides of the invention

The stretched/extended amino acid may be the pro-sequence peptide of the protein or any other amino acid. Elongation may be used to enhance the stability or solubility of the peptide.

Thus, the epitope described in the present invention may be identical to a native tumor-associated epitope or a tumor-specific epitope, and may also include different peptides of no more than four residues from the reference peptide if they have substantially the same antigenic activity.

In an alternative embodiment, one or both sides of the peptide are elongated by more than 4 amino acids, preferably up to a total length of 30 amino acids. This can result in MHC class II binding peptides. Binding to MHC class II peptides can be tested by methods known in the art.

Thus, the invention proposes peptides and variants of MHC class I epitopes, wherein the total length of the peptide or antibody is between 8 and 100, preferably between 8 and 30, most preferably between 8 and 14 amino acids in length (i.e. 8, 9, 10, 11, 12, 13, 14 amino acids, and if a class II binding peptide is elongated, also 15, 16, 17, 18, 19, 20, 21 or 22 amino acids in length).

Of course, the peptides or variants of the invention are capable of binding to human Major Histocompatibility Complex (MHC) class I or II molecules. Binding of the peptide or variant to the MHC complex can be tested using methods known in the art.

Preferably, when peptide-specific T cells of the invention are tested in comparison to the substituted peptide, the peptide concentration is no more than about 1mM, preferably no more than about 1. mu.M, more preferably no more than about 1nM, even more preferably no more than about 100pM, and most preferably no more than about 10pM, if the solubility of the substituted peptide increases to half of the maximum relative to the background peptide. Also preferably, the substituted peptide is recognized by more than one T cell, a minimum of 2, more preferably 3.

In a particularly preferred embodiment of the invention, the peptide consists of or essentially of an amino acid sequence selected from the group consisting of SEQ ID NO1 to SEQ ID NO 268.

Essentially consisting of "peptides of the invention, which in addition to a sequence according to any one of SEQ ID No.1 to SEQ ID No.268 or variants thereof, contain amino acids in other N-and/or C-terminal extensions, which are not necessarily capable of forming peptides as epitopes of MHC molecules.

However, these extended regions are important for efficiently introducing the peptide of the present invention into cells. In one embodiment of the present invention, the peptide is a part of a fusion protein, and contains 80N-terminal amino acids of HLA-DR antigen-associated invariant chain (p33, hereinafter referred to as "Ii") derived from NCBI and GenBank accession number X00497, and the like. In other fusions, the peptides of the invention may be fused to the antibodies described herein, or functional portions thereof, in particular to the sequences of the antibodies, so that the antibodies perform a specific targeting action, or, for example, into dendritic cell-specific antibodies as described herein.

In addition, the peptide or variant may be further modified to improve stability and/or binding to MHC molecules, thereby eliciting a stronger immune response. Such optimization methods for peptide sequences are well known in the art and include, for example, the introduction of trans-peptide bonds and non-peptide bonds.

In the trans peptide bond amino acid, the peptide (-CO-NH-) is not linked to its residue, but its peptide bond is reversed. Such reverse-reverse mimetics can be prepared by methods known in the art, for example: the method described in Meziere et al (Meziere et al, 1997) is incorporated herein by reference. This method involves the preparation of a mimetic peptide comprising a change in the backbone (rather than in the side chains). Studies by Meziere et al (Meziere et al, 1997) show that these analogical peptides are favorable for MHC binding and helper T cell responses. The reverse peptide with NH-CO bond to replace CO-NH peptide bond has greatly raised hydrolysis resistance.

Non-peptide bonds are-CH 2-NH, -CH2S-, -CH2CH2-, -CH ═ CH-, -COCH2-, -CH (oh) CH2-, and-CH 2SO-, etc. U.S. patent No. 4897445 proposes a non-solid phase synthesis of non-peptide bonds (-CH2-NH) in polypeptide chains involving polypeptides synthesized according to standard procedures and non-peptide bonds synthesized through the interaction of an amino aldehyde and an amino acid containing NaCNBH 3.

Peptides containing the above sequences may be synthesized with other chemical groups at the amino and/or carboxy terminus of the peptide, thereby improving stability, bioavailability, and/or affinity of the peptide. For example, a hydrophobic group such as benzyloxycarbonyl or dansyl, or a tert-butoxycarbonyl group may be added to the amino terminus of the peptide. Similarly, acetyl or 9-fluorenylmethyloxycarbonyl may be located at the amino terminus of the peptide. Furthermore, a hydrophobic group, a t-butyloxycarbonyl group, or an amino group may be added to the carboxy terminus of the peptide.

In addition, all peptides of the present invention may be synthesized to change their spatial configuration. For example, it is possible to use the right-hand forms of one or more amino acid residues of these peptides, usually not the left-hand forms thereof. Further, at least one amino acid residue of the peptide of the present invention may be substituted with a known non-natural amino acid residue. Such changes may contribute to increased stability, bioavailability and/or binding of the peptides of the invention.

Also, the peptides or variants of the invention may be chemically modified by reaction with specific amino acids before or after synthesis of the peptide. Examples of such modifications are well known in the art, and are summarized, for example, in "chemical reagents for Protein Modification" (3rd ed. crc Press,2004) (Lundblad,2004) by r.lundblad, which is incorporated herein by reference. Although there is no limitation on the chemical modification method of amino acids, it includes (but is not limited to) modification by the following methods: acylation, amidination, lyme pyridolation, reductive alkylation, trinitrophenylation of amino groups with 2,4, 6-trinitrobenzenesulphonic acid (TNBS), amino modification of carboxyl groups and sulfhydryl groups by oxidation of cysteine performic acid to cysteic acid, formation of labile derivatives, formation of mixed disulfide compounds with other sulfhydryl compounds, reaction with maleimides, carboxymethylation with iodoacetic acid or iodoacetamide, carbamoylation with cyanate at basic pH. In this connection, the skilled worker refers to the extensive methods In connection with the chemical modification of proteins described In chapter 15 of Current Protocols In Protein Science (eds. Coligan et al (John Wiley and Sons NY 1995-2000)) (Coligan et al, 1995).

In short, the arginyl residues of modified proteins and the like are often based on the reaction of ortho-dicarbonyl compounds (e.g., benzaldehyde, 2, 3-butanedione, and 1, 2-cyclohexenedione) to form adducts. Another example is the reaction of methylglyoxal with an arginine residue. Cysteine can be modified without concomitant modification at nucleophilic sites such as lysine and histidine. Thus, there are a number of reagents available for cysteine modification. The website of Sigma-Aldrich (http:// www.sigma-Aldrich. com) contains information about specific reagents.

Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds may be formed and oxidized in biopharmaceutical heat treatments. Wood wade reagent K can be used to modify specific glutamic acid residues. N- (3-dimethylaminopropyl) -N' -ethyl-carbodiimide can be used to form intramolecular cross-links of lysine and glutamic acid residues. For example: diethylpyrocarbonate is an agent that modifies histidine residues in proteins. Histidine can also be modified with 4-hydroxy-2-nonenal. The reaction of lysine residues with other alpha-amino groups, for example, facilitates peptide binding to the surface or cross-linking of the protein/peptide. Lysine poly is the attachment point for poly (ethylene) glycol and is also the primary modification site for protein glycosylation. The methionine residue of the protein can be modified by iodoacetamide, bromoethylamine, chloramine T, etc.

Tetranitromethane and N-acetylimidazole may be used for the modification of tyrosine residues. The crosslinking via the di-tyrosine can be accomplished via hydrogen peroxide/copper ions.

Recent studies on tryptophan modification have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-methyl-2- (2-nitrobenzenesulfo-l) -3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides containing polyethylene glycol often can extend the loop half-life when cross-linking of the protein with glutaraldehyde, polyethylene glycol diacrylate and formaldehyde is used to formulate hydrogels. Chemical modification of allergens for immunotherapy is often achieved through carbamoylation of potassium cyanate.

A peptide or variant wherein the peptide is modified or contains non-peptide bonds, preferably an embodiment of the invention.

Another embodiment of the invention relates to a non-natural peptide, wherein said peptide consists or consists essentially of an amino acid sequence according to SEQ ID No.1 to SEQ ID No.268, synthetically produced (i.e. synthesized) as a pharmaceutically acceptable salt. Methods for synthetically producing peptides are well known in the art. The salts of the peptides of the invention are substantially different from peptides in their in vivo state, since these in vivo produced peptides are not salts. The non-native salt form of the peptide mediates the solubility of the peptide, particularly in the case of pharmaceutical compositions comprising the peptide, e.g., the peptide vaccines disclosed herein. In order to effectively provide a peptide to a subject in need of treatment, it is desirable that the peptide has sufficient, at least substantial, solubility. Preferably, the salt is a pharmaceutically acceptable salt of the peptide. These salts of the invention include alkali and alkaline earth salts, such as salts of the Hofmeister series, comprising anionic PO₄ ^3-、SO₄ ^2-、CH₃COO^-、Cl^-、Br^-、NO₃ ^-、ClO₄ ^-、I^-、SCN^-And a cation NH₄ ⁺、Rb⁺、K⁺、Na⁺、Cs⁺、Li⁺、Zn²⁺、Mg²⁺、Ca²⁺、Mn²⁺、Cu²⁺And Ba²⁺. In particular, the salt is selected from (NH)₄)₃PO₄、(NH₄)₂HPO₄、(NH₄)H₂PO₄、(NH₄)₂SO₄、NH₄CH₃COO、NH₄Cl、NH₄Br、NH₄NO₃、NH₄CIO₄、NH₄I、NH₄SCN、Rb₃PO₄、Rb₂HPO₄、RbH₂PO₄、Rb₂SO₄、Rb₄CH₃COO、Rb₄Cl、Rb₄Br、Rb₄NO₃、Rb₄CIO₄、Rb₄I、Rb₄SCN、K₃PO4、K₂HPO₄、KH₂PO₄、K₂SO₄、KCH₃COO、KCl、KBr、KNO₃、KClO₄、KI、KSCN、Na₃PO₄、Na₂HPO₄、NaH₂PO₄、Na₂SO₄、NaCH₃COO、NaCl、NaBr、NaNO₃、NaCIO₄、NaI、NaSCN、ZnCI₂Cs₃PO₄、Cs₂HPO₄、CsH₂PO₄、Cs₂SO₄、CsCH₃COO、CsCl、CsBr、CsNO₃、CsCIO₄、CsI、CsSCN、Li₃PO₄、Li₂HPO₄、LiH₂PO₄、Li₂SO₄、LiCH₃COO、LiCl、LiBr、LiNO₃、LiClO₄、LiI、LiSCN、Cu₂SO₄、Mg₃(PO₄)₂、Mg₂HPO₄、Mg(H₂PO₄)₂、Mg₂SO₄、Mg(CH₃COO)₂、MgCl₂、MgBr₂、Mg(NO₃)₂、Mg(ClO₄)₂、MgI₂、Mg(SCN)₂、MnCl₂、Ca₃(PO₄),、Ca₂HPO₄、Ca(H₂PO₄)₂、CaSO₄、Ca(CH₃COO)₂、CaCl₂、CaBr₂、Ca(NO₃)₂、Ca(ClO₄)₂、CaI₂、Ca(SCN)₂、Ba₃(PO₄)₂、Ba₂HPO₄、Ba(H₂PO₄)₂、BaSO₄、Ba(CH₃COO)₂、BaCl₂、BaBr₂、Ba(NO₃)₂、Ba(ClO₄)₂、BaI₂And Ba (SCN)₂. NH acetic acid and MgCl are particularly preferable₂、KH₂PO₄、Na₂SO₄KCl, NaCl and CaCl₂For example: chloride or acetate (trifluoroacetic acid) salts.

In general, peptides and variants (containing at least peptide linkages between amino acid residues) can be synthesized using the solid phase peptide synthesis Fmoc-polyamide model disclosed by Lukas et al (Lukaset al, 1981) and the references cited therein. The fluorenylmethyloxycarbonyl (Fmoc) group provides temporary protection for the N-amino group. The highly sensitive protecting group is repeatedly cleaved using 20% dimethyl piperidine in N, N-dimethylformamide. The side chain function may be protected due to their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), tert-butyloxycarbonyl derivatives (in the case of lysine and histidine), trityl derivatives (in the case of cysteine) and 4-methoxy-2, 3, 6-trimethylbenzenesulfonyl derivatives (in the case of arginine). As long as glutamine and asparagine are C-terminal residues, the side chain amino function protection is provided by using a4, 4' -dimethoxydiphenyl group. The solid support is based on a polydimethylacrylamide polymer consisting of three monomers, dimethylacrylamide (backbone monomer), bisacryloylethylene diamine (crosslinker) and N-acryloylsarcosine methyl ester (functional agent). The peptide-resin coupling agent used is an acid-sensitive 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives were added as their preformed symmetrical anhydride derivatives, except for asparagine and glutamine, which were added using a reversed N, N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions were monitored using ninhydrin, nitrobenzenesulfonic acid, or isotin test procedures. After completion of the synthesis, the peptide was cleaved from the resin support with concomitant removal of the side chain protecting groups using trifluoroacetic acid at a concentration of 95% containing 50% scavenger mix. Commonly used scavenger mixtures include ethanedithiol, phenol, anisole and water, the exact choice being based on the amino acid composition of the synthetic peptide. Furthermore, it is possible to synthesize peptides using a combination of solid-phase and liquid-phase methods (see, for example, Bruckdorfer et al, 2004 and references cited therein)

Trifluoroacetic acid was removed by evaporation in vacuo, followed by titration with diethyl ether bearing the crude peptide. Any scavenger mixture present was purged using a simple extraction procedure (after lyophilization of the aqueous phase, which produced peptides without scavenger mixture). Peptide synthesis reagents are generally available from Calbiochem-Novabiochem (Nonburg, England).

Purification can be carried out by any one or a combination of the following techniques, such as: recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (typically) reversed phase high performance liquid chromatography (e.g., using an acetonitrile/water gradient separation).

Peptide analysis can be performed using thin layer chromatography, electrophoresis, particularly capillary electrophoresis, solid phase extraction (CSPE), reverse phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, Fast Atom Bombardment (FAB) mass spectrometry, and MALDI and ESI-Q-TOF mass spectrometry.

For the identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shock frozen tissue samples are purified and HLA-related peptides are isolated. The isolated peptides were separated and identified by on-line nano-electrospray-ionization (nanoESI) liquid chromatography-spectroscopy (LC-MS) experiments. The peptide sequences thus generated were verified by comparing the fragment patterns of the natural tumor associated peptide (TUMAP) recorded in colorectal, glioblastoma, gastric, hepatocellular, and renal cell carcinoma samples (N ═ 35CRC, 12GBM, 10GC, 22HCC, and 79RCC samples) with the fragment patterns of the corresponding synthetic reference peptides of the same sequence. Since these peptides were directly identified as ligands for HLA molecules of primary tumors, these results provide direct evidence for the natural processing and presentation of certain peptides on primary cancer tissues from 158 patients with colorectal, glioblastoma, gastric, hepatocellular, renal cell carcinoma (see example 1).

Discovering a pipelinev2.1 (see, e.g., US 2013-. This is achieved by the following method: LC-MS data collection, integration using proprietary data analysis pipeline processingSequence identification algorithms, spectral clustering, ion calculation, retention time adjustment, charging state convolution and normalization.

HLA peptide complexes from colorectal, glioblastoma, gastric, hepatocellular, and renal cell carcinoma tissue samples were purified and HLA-related peptides were isolated and analyzed using LC-MS (see example 1). All TUMAPs contained in the present application were identified using the present method on primary colorectal, glioblastoma, gastric, hepatocellular, and renal cell carcinoma samples, confirming their presentation on primary colorectal, glioblastoma, gastric, hepatocellular, and renal cell carcinoma.

In addition to over-presenting the peptides, mRNA expression of potential genes was also tested. mRNA data were obtained by RNA sequencing analysis of normal and cancerous tissues (see example 2, FIG. 1). Peptides obtained from the protein show high expression of the coding mRNA in cancer tissues, but are very low or absent in important normal tissues, and these peptides are preferably peptides incorporated into the present invention.

The present invention proposes a therapeutic benefit for cancer/tumors, preferably for colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma that are either over-presented or presented with the peptides of the invention alone. These peptides were directly shown by mass spectrometry and were naturally presented by HLA molecules in primary human colorectal, glioblastoma, gastric, hepatocellular, and renal cell carcinoma samples.

Many of the source genes/proteins (also designated as "full-length proteins" or "potential proteins") from which peptides are highly overexpressed in cancer compared to normal tissue-the "normal tissue" to which the invention relates is colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma cells, or other normal tissue cells, indicating a high association of the tumor with these source genes (see example 2). In addition, these peptides are presented in tumor tissues themselves (the "tumor tissues" related to the present invention means samples from patients with colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma).

HLA-binding peptides are recognized by the immune system, particularly T lymphocytes. T cells can destroy cells presenting the recognized HLA/peptide complex (e.g., colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma cells presenting the derived peptide).

All peptides of the invention have been shown to have the ability to stimulate a T cell response and are presented in excess and thus can be used to prepare antibodies and/or TCRs of the invention, e.g., soluble TCRs (see example 3 and example 4). Furthermore, peptides, when combined with the corresponding MHC, may also be used to prepare antibodies and/or TCRs, particularly stcrs, of the invention. The respective methods are well known to the skilled worker and can be found in the respective literature (see also below). Thus, the peptides of the invention can be used to generate an immune response in a patient, thereby enabling the destruction of tumor cells. The immune response in a patient can be induced by direct administration of the peptide or precursor (e.g., an elongated peptide, protein, or nucleic acid encoding such peptide) to the patient, preferably in combination with an agent that enhances immunogenicity. The immune response derived from this therapeutic vaccine is expected to be highly specific against tumor cells, since the target peptides of the invention present a smaller number of replications on normal tissues, preventing the risk of the patient of an adverse autoimmune response against normal cells.

The present specification also relates to T Cell Receptors (TCRs) comprising one alpha chain and one beta chain ("alpha/beta TCRs"). Also provided are peptides of the invention that bind to TCR and antibodies when presented by MHC molecules.

The present specification also relates to the presentation of TCR fragments of the invention by HLA molecules capable of binding to peptide antigens of the invention. The term particularly relates to soluble TCR fragments, such as TCRs lacking transmembrane portions and/or constant regions, single chain TCRs and fusions thereof with, for example, Ig.

The specification also relates to nucleic acids, vectors, and host cells for expressing the TCR and the peptides of the specification; and methods of using them.

The term "T cell receptor" (abbreviated TCR) refers to a heterodimeric molecule comprising one alpha polypeptide chain (α chain) and one beta polypeptide chain (β chain), wherein the heterodimeric receptor is capable of binding a peptide antigen presented by an HLA molecule. The term also includes the so-called γ/TCR.

In one embodiment, the present specification provides a method of producing a TCR as described herein, the method comprising culturing a host cell capable of expressing a TCR under conditions suitable to promote TCR expression.

In another aspect, the present disclosure relates to a method according to the present disclosure, wherein the antigen is loaded into class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell by binding to a sufficient amount of antigen containing the antigen-presenting cell, or the antigen is loaded into class I or II MHC tetramer/class I or II MHC complex monomer by tetramerization. The α and β chains of α/β TCRs and the γ and chains of γ/TCRs are generally considered to have two "domains" each, namely variable and constant domains. The variable domain consists of a combination of a variable region (V) and a linking region (J). The variable domain may also comprise a leader region (L). Beta and the strand may also comprise a diversity region (D). The alpha and beta constant domains may also include a C-terminal Transmembrane (TM) domain that anchors the alpha and beta chains to the cell membrane.

The term "TCR γ variable domain" as used herein refers to the combination of TCR γ v (trgv) regions without leader region (L) and TCR γ (TRGJ) regions, and the term TCR γ constant domain refers to extracellular TRGC regions, or C-terminally truncated TRGC sequences, relative to the TCR of γ/. Similarly, the term "TCR variable domain" refers to the combination of a TCRV (TRDV) region without a leader (L) and a TCRD/J (TRDD/TRDJ) region, and the term "TCR constant domain" refers to an extracellular TRDC region, or a C-terminally truncated TRDC sequence.

The TCRs of the present disclosure preferably bind to a peptide HLA molecule complex having a binding affinity (KD) of about 100 μ Μ or less, about 50 μ Μ or less, about 25 μ Μ or less or about 10 μ Μ or less. More preferred are high affinity TCRs having a binding affinity of about 1 μ M or less, about 100nM or less, about 50nM or less, or about 25nM or less. Non-limiting examples of preferred binding affinity ranges for the inventive TCR include from about 1nM to about 10 nM; about 10nM to about 20 nM; about 20nM to about 30 nM; about 30nM to about 40 nM; about 40nM to about 50 nM; about 50nM to about 60 nM; about 60nM to about 70 nM; about 70nM to about 80 nM; about 80nM to about 90 nM; and about 90nM to about 100 nM.

In connection with the TCRs of the present specification, "specific binding" and grammatical variants thereof are used herein to refer to TCRs having a binding affinity (KD) for a peptide-HLA molecule complex of 100 μ M or less.

The α/β heterodimeric TCRs of the present specification may have an introduced disulfide bond between their constant domains. Preferred TCRs of this type include those having a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, unless threonine 48 of TRAC and serine 57 of TRBC1 or TRBC2 are substituted with cysteine residues which form a disulfide bond between the TRAC constant domain sequence and the TRBC1 or TRBC2 constant region sequence of the TCR.

The α/β heterodimeric TCRs of the present specification, with or without the introduction of interchain linkages as described above, may have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked through the native disulfide bond between Cys4 of TRAC exon 2 and Cys4 of TRBC1 or TRBC2 exon 2.

The TCRs of the present disclosure may include a detectable label selected from the group consisting of a radionuclide, a fluorophore, and a biotin. The TCRs of the present disclosure may be conjugated to a therapeutically active agent, such as a radionuclide, chemotherapeutic agent, or toxin.

In one embodiment, a TCR having at least one mutation in the alpha chain and/or having at least one mutation in the beta chain has modified glycosylation compared to the non-mutated TCR.

In one embodiment, a TCR comprising at least one mutation in the TCR α chain and/or the TCR β chain has a binding affinity and/or binding half-life for the peptide HLA molecule complex that is at least twice the binding affinity of a TCR comprising an unmutated TCR α chain and/or an unmutated TCR β chain. Tumor-specific TCR affinity enhancement and its development rely on the window where the optimal TCR affinity exists. The presence of such windows is based on observations: HLA-A2-restricted pathogen-specific TCRs generally have KD values approximately 10-fold lower than HLA-A2-restricted tumor-associated autoantigen-specific TCRs. It is now known that, although tumor antigens may be immunogenic, because tumors are derived from the individual's own cells, only mutant proteins or proteins with altered translational processing will be considered foreign by the immune system. Antigens that are up-regulated or overexpressed (so-called autoantigens) do not necessarily induce a functional immune response against the tumor: t cells expressing TCRs that are highly reactive against these antigens are selected against the thymus in a procedure called central tolerance, i.e. only cells with low affinity TCRs for self-antigens remain. Thus, the affinity of the TCRs or variants of the disclosure for a peptide can be enhanced by methods well known in the art.

The present specification also relates to a method of identifying and isolating a TCR of the present invention, the method comprising: PBMCs were incubated with a 2/peptide monomer from HLA-a × 02 negative healthy donors, PBMCs were incubated with tetramer-Phycoerythrin (PE) and high affinity T cells were isolated by fluorescence-initiated cell sorting (FACS) -Calibur method analysis.

The present specification also relates to a method of identifying and isolating a TCR of the present invention, the method comprising: transgenic mice containing the entire human TCR α β gene locus (1.1and 0.7Mb) were obtained (T cells expressing diversified human TCRs to compensate for mouse TCR deficiency), mice were immunized with peptides, PBMCs obtained from transgenic mice were incubated with tetramer-Phycoerythrin (PE), and high affinity T cells were isolated by fluorescence-initiated cell sorting (FACS) -Calibur method analysis.

In one aspect, to obtain a T cell expressing a TCR of the specification, a nucleic acid encoding a TCR-a and/or a TCR- β chain of the specification is cloned into an expression vector, such as a gamma retrovirus or lentivirus. Recombinant viruses are produced and then tested for functions such as antigen specificity and functional avidity. Aliquots of the final product are then used to transduce a population of target T cells (typically PBMCs purified from the patient) and are expanded prior to infusion into the patient. On the other hand, to obtain T cells expressing TCRs of the present specification, TCRRNA is synthesized by techniques known in the art (e.g., in vitro transcription systems). In vitro synthesized TCR RNA is then introduced into primary CD8+ T cells obtained from healthy donors by electroporation to re-express tumor specific TCR-alpha and/or TCR-beta chains.

To increase expression, the nucleic acid encoding the TCR of the specification may be operably linked to strong promoters, such as retroviral Long Terminal Repeats (LTR), Cytomegalovirus (CMV), Murine Stem Cell Virus (MSCV) U3, phosphoglycerate kinase (PGK), beta actin, ubiquitin protein, and simian virus 40(SV40)/CD43 complex promoters, Elongation Factor (EF) -1a, and Spleen Focus Forming Virus (SFFV) promoters. In a preferred embodiment, the promoter is heterologous to the nucleic acid being expressed. In addition to a strong promoter, the TCR expression cassettes of the present specification may contain additional elements that enhance transgene expression, including a central polypurine tract (CPPT) that promotes nuclear translocation of lentiviral constructs (Follenzi et al, 2000), and woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE) that increase transgene expression levels by enhancing RNA stability (Zufferey et al, 1999).

The α and β chains of the inventive TCR may be encoded by separate vector nucleic acids, or may be encoded by polynucleotides located on the same vector.

Achieving high levels of TCR surface expression requires high levels of transcription of the TCR-a and TCR- β chains into which the TCR is introduced. To achieve this, the TCR- α and TCR- β chains of the present specification can be cloned into a bicistronic construct in a single vector, which has been shown to overcome this obstacle. The use of TCR- α and TCR- β chains with an entry site (IRES) between the viral ribosomes results in the coordinated expression of both chains, since both TCR- α and TCR- β chains are produced from a single transcript that splits into two proteins during translation, thereby ensuring that an equal molar ratio of TCR- α and TCR- β chains is produced. (Schmitt et al 2009).

The nucleic acid encoding the TCR of the specification may be codon optimized for increased expression from the host cell. The genetic code redundancy allows some amino acids to be encoded by more than one codon, but some codons are not "optimized" by others because of the relative availability of matching trnas and other factors (gusfsson et al, 2004). Modification of TCR-a and TCR- β gene sequences such that each amino acid is encoded by the optimal codon for mammalian gene expression, as well as elimination of mRNA instability motifs or cryptic splice site sites, has been shown to significantly increase TCR-a and TCR- β gene expression (Scholten et al, 2006).

Furthermore, mismatches between the introduced and endogenous TCR chains may lead to specificity being obtained, which constitutes a significant risk of autoimmunity. For example, the formation of mixed TCR dimers may reduce the number of CD3 molecules available to form the correct paired TCR complex, and thus, may significantly reduce the functional avidity of cells expressing the introduced TCR (Kuball et al, 2007).

To reduce mismatches, the C-terminal domain of the TCR chains introduced in the present specification can be modified to promote interchain affinity while reducing the ability of the introduced chain to pair with endogenous TCRs. These strategies may include replacement of the human TCR- α and TCR- β C-terminal domains (the murinized C-terminal domains) with murine counterparts; a second interchain disulfide bond (cysteine modification) in the C-terminal domain is created by introducing a second cysteine residue into the TCR- α and TCR- β chains of the introduced TCR; exchanging interacting residues of the C-terminal domains of the TCR-alpha and TCR-beta chains ("knob and hole structures"); direct fusion of TCR-alpha and TCR-beta chain variable domains to CD3 zeta (CD3 zeta fusion) (Schmitt et al 2009).

In one embodiment, the host cell is structurally altered to express a TCR of the specification. In a preferred embodiment, the host cell is a human T cell or T cell progenitor cell. In some embodiments, the T cells or T cell progenitors are obtained from a cancer patient. In other embodiments, the T cells or T cell progenitors are obtained from a healthy donor. The host cells of the present specification may be allogeneic or autologous with respect to the patient to be treated. In one embodiment, the host is a γ/T cell transformed to express an α/β TCR.

"pharmaceutical composition" refers to a composition suitable for use in a medical facility for the human body. Preferably, the pharmaceutical compositions are sterile and manufactured according to GMP guidelines.

Pharmaceutical compositions include the peptide in free form or in the form of a pharmaceutically acceptable salt (see also above). As used herein, a "pharmaceutically acceptable salt" refers to a derivative of the disclosed peptide that is modified by a base salt of an acid or pharmaceutical agent. For example, the free base by reaction with a suitable acid (usually whereinThe neutral drug has a neutral-NH₂Group) to prepare the acid salt. Suitable acids for preparing the acid salts include organic acids such as: acetic, propionic, hydroxy, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, benzenesulfonic, salicylic, and the like, as well as inorganic acids such as: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. In contrast, a preparation of a salt of an acidic group that can be present on a peptide is prepared using a pharmaceutically acceptable base, e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

In a particularly preferred embodiment, the pharmaceutical composition comprises the peptide in the form of acetic acid (acetate), trifluoroacetate or hydrochloric acid (chloride).

The agent of the invention is preferably an immunotherapeutic agent, for example, a vaccine. The vaccine may be administered directly to the affected organ of the patient, or systemically by id, im, sc, ip and iv injection, or applied in vitro to cells from the patient or a cell line thereof (which are then injected into the patient), or in vitro to a subpopulation of cells from immune cells from the patient (which are then re-administered to the patient). If the nucleic acid is injected into the cells in vitro, it may be beneficial to transfect the cells to co-express an immunostimulatory cytokine (e.g., interleukin-2). The peptides may be administered entirely alone, in combination with an immunostimulating adjuvant (see below), or in combination with an immunostimulating cytokine, or in a suitable delivery system (e.g. liposomes). The peptide may also be conjugated to form a suitable carrier, such as Keyhole Limpet Hemocyanin (KLH) or mannoprotein (see WO 95/18145 and (Longenecker et al, 1993)). The peptide may also be labeled, may be a fusion protein, or may be a hybrid molecule. Peptides of the sequences given in the present invention are expected to stimulate CD4 or CD8T cells. However, CD8T cell stimulation was more effective with the help of CD 4T-helper cells. Thus, for stimulation of the MHC class I epitope of CD8T cells, a fusion partner or fragment of a hybrid molecule provides an appropriate epitope for stimulation of CD4 positive T cells. CD 4-and CD 8-stimulating epitopes are well known in the art and include the epitopes identified in the present invention.

In one aspect, the vaccine comprises at least one peptide set forth in SEQ ID No.1 to SEQ ID No.268 and at least one further peptide, preferably 2 to 50, more preferably 2 to 25, even more preferably 2 to 20, most preferably 2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 peptides. Peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

In another aspect, the invention features a nucleic acid (e.g., a polynucleotide) encoding a peptide or peptide variant of the invention. A polynucleotide may be, for example, DNA, cDNA, PNA, RNA, or combinations thereof, which may be single-and/or double-stranded, or a native or stabilized form of a polynucleotide (e.g., a polynucleotide having a phosphorothioate backbone), and which may or may not contain an intron so long as it encodes a peptide. Of course, polynucleotides can only encode peptides that incorporate natural peptide bonds and contain natural amino acid residues. In another aspect, the invention features an expression vector for expressing a polypeptide according to the invention.

For the ligation of polynucleotides, various methods have been developed, and in particular, ligation can be performed by a method of supplementing a vector with a ligatable terminus, or the like, particularly for DNA. For example, a complementary homopolymer track can be added to the DNA fragment, after which the DNA fragment is inserted into the vector DNA. The vector and DNA fragments are then bound via hydrogen bonding of the complementary homopolymer tail, thereby forming a recombinant DNA molecule.

Synthetic linkers containing one or more cleavage sites provide an alternative method for ligating DNA fragments into vectors. Synthetic linkers containing various restriction endonucleases are commercially available through a variety of tubes, including International Biotechnology Inc., New Haven, CN, USA.

A desirable modification method for DNA encoding a polypeptide of the present invention is the polymerase chain reaction method employed by Saiki et al (Saiki et al, 1988). This method can be used to introduce the DNA into a suitable vector (e.g., by designing appropriate cleavage sites), and can also be used to modify the DNA by other useful methods known in the art. If viral vectors are used, either poxvirus vectors or adenoviral vectors are preferred.

The DNA (or RNA in the case of retroviral vectors) may then be expressed in a suitable host to produce a polypeptide comprising a peptide or variant of the invention. Thus, a DNA encoding a peptide or variant of the invention may be used according to known techniques, suitably modified by the methods described herein, to construct an expression vector, which is then used to transform a suitable host cell, thereby expressing and producing a polypeptide of the invention. Such techniques include those disclosed in, for example, U.S. patents 4,440,859, 4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

DNA (or RNA in the case of retroviral vectors) encoding a polypeptide comprising a compound of the invention may be added to a variety of other DNA sequences for introduction into a suitable host. The companion DNA will depend on the nature of the host, the manner in which the DNA is introduced into the host, and whether it needs to be maintained episomally or bound to each other.

In general, the DNA can be attached to an expression vector (e.g., a plasmid) in the proper orientation and correct expression reading frame. If necessary, the DNA may be linked to corresponding transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such control functions are typically present in expression vectors. The vector is then introduced into the host by standard methods. In general, not all hosts will be transformed by the vector. Therefore, it is necessary to select transformed host cells. The selection method involves the insertion of a DNA sequence encoding a selectable attribute (e.g., antibiotic resistance) in the transformed cell into the expression vector using any necessary control elements.

Alternatively, the gene having such selective properties may be on another vector which is used to co-transform the desired host cell.

Host cells transformed with the recombinant DNA of the invention are then cultured under suitable conditions, as described herein, familiar to those skilled in the art, for a time sufficient to express the peptide that can be recovered thereafter.

There are many known expression systems, including bacteria (e.g., E.coli and Bacillus subtilis), yeasts (e.g., yeast), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The system may preferably be mammalian cells, such as CHO cells from the ATCC Cell Biology Collection (Cell Biology Collection).

Typical mammalian cell constitutive expression vector plasmids include the CMV or SV40 promoter with a suitable poly-A tail, and resistance markers (e.g., neomycin). An example is pSVL obtained from Pharmacia (Piscataway, New Jersey, USA). An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, generally available from Stratagene cloning Systems, La Jolla, 92037, Calif., USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrative plasmids (YIp) with the insertion of the yeast selectable markers HIS3, TRP1, LEU2 and URA 3. The pRS413-416 plasmid is a yeast centromere plasmid (Ycp). The CMV promoter-based vector (e.g., from Sigma-Aldrich) provides transient or stable expression, cytoplasmic expression or secretion, as well as N-terminal or C-terminal markers in various combinations of FLAG, 3xFLAG, C-myc, or MATN. These fusion proteins can be used for detection, purification and analysis of recombinant proteins. Dual label fusion provides flexibility for detection.

The strong human Cytomegalovirus (CMV) promoter regulatory region resulted in expression levels of constitutive proteins in COS cells as high as1 mg/L. For weaker cell lines, protein levels are generally below 0.1 mg/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication competent COS cells. For example, CMV vectors may contain the origin of pMB1 (a derivative of pBR 322) replication in bacterial cells, the origin of the ca-lactase gene for ampicillin resistance breeding in bacteria, hGH polyA, and f 1. Vectors containing preproinsulin leader (PPT) sequences can be secreted into the medium for purification using anti-FLAG antibodies, resins and plate-directed FLAG fusion proteins. Other vectors and expression systems for use with a variety of host cells are well known in the art.

In another embodiment, two or more peptides or peptide variants of the invention are encoded and, thus, expressed in a sequential order (similar to a "string of beads" construct). To achieve this, the peptides or peptide variants may be linked or fused together via an extension of a linker amino acid (e.g., LLLLLL), or may be linked without any additional peptide therebetween. These constructs are also useful in cancer therapy, and induce immune responses involving MHC class I and MHC class II molecules.

The invention also relates to a host cell transformed with the polynucleotide vector construct of the invention. The host cell may be a prokaryotic cell or a eukaryotic cell. In some instances, bacterial cells are preferred prokaryotic host cells, typically E.coli strains, e.g., E.coli strain DH5 (obtained from Bethesda Research Laboratories Inc. [ Bethesda, Md., USA.) and RR1 (obtained from American type culture Collection [ ATCC, Rockville, Md., USA ], ATCC number 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells, such as: mouse, rat, monkey or human fibroblasts and colon cancer cell lines. Yeast host cells include YPH499, YPH500 and YPH501, and are generally available from Stratagene Cloning Systems, Inc. (LaJolla, Calif. 92037, USA). The preferred mammalian host cells include CCL61 cells from ATCC as Chinese Hamster Ovary (CHO) cells, CRL 1658 cells from ATCC as NIH Swiss mouse embryo cells NIH/3T3, CRL 1650 cells from ATCC as monkey kidney-derived COS-1 cells, and 293 cells from human embryonic kidney cells. The preferred insect cell is Sf9 cell, which can be transfected with baculovirus expression vector. A summary of the selection of suitable host cells for Expression can be found in textbooks (Paulina Balb. sup. s and Argelia Lorence. Methods in Molecular Biology recombined Gene Expression, Reviews and Protocols, Part One, Second Edition, ISBN 978-1-58829. sup. 262-9) and other documents known to the skilled worker.

Transformation of a suitable host cell containing a DNA construct of the invention may be accomplished using well known methods, generally depending on the type of vector used. For the transformation of prokaryotic host cells, see, for example, Cohen et al (Cohen et al, 1972) and (Green and Sambrook, 2012). Transformation of yeast cells is described in Sherman et al (Sherman et al, 1986). The methods described in Beggs (Beggs,1978) are also useful. For vertebrate cells, reagents for transfecting these cells, etc., e.g., calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene cloning Systems, Inc. or Life Technologies, Inc. (Gaithersburg, Maryland 20877, USA). Electroporation may also be used to transform and/or transfect cells, and is a well-known method used in the art for transforming yeast cells, bacterial cells, insect cells, and vertebrate cells.

Successfully transformed cells (i.e.cells containing the DNA construct of the invention) can be identified by well-known methods, such as PCR. Alternatively, proteins present in the supernatant may be detected using antibodies.

It will be appreciated that certain host cells of the invention are useful for the production of peptides of the invention, such as bacterial cells, yeast cells and insect cells. However, other host cells may be useful for certain therapeutic approaches. For example, antigen presenting cells (e.g., dendritic cells) can be used to express the peptides of the invention so that they can be loaded into the corresponding MHC molecules. Thus, the present invention provides a host cell comprising a nucleic acid or expression vector of the invention.

In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell or an antigen-presenting cell. On 29/4/2010, the U.S. Food and Drug Administration (FDA) approved recombinant fusion proteins loaded with Prostate Acid Phosphatase (PAP) for use in the treatment of asymptomatic or mildly symptomatic metastatic HRPC (Rini et al, 2006; Smallet al, 2006).

In another aspect, the invention features a method of formulating a peptide and variants thereof, the method including culturing a host cell and isolating the peptide from the host cell or culture medium thereof.

In another embodiment, the peptide, nucleic acid or expression vector of the invention is used in medicine. For example, the peptide or variant thereof may be prepared as an intravenous (i.v.) injection, a subcutaneous (s.c.) injection, an intradermal (i.d.) injection, an intraperitoneal (i.p.) injection, an intramuscular (i.m.) injection. Preferred methods of peptide injection include s.c., i.d., i.p., i.m., and i.v. injections. Preferred methods of DNA injection are i.d., i.m., s.c., i.p., and i.v. injection. For example, 50. mu.g to 1.5mg, preferably 125. mu.g to 500. mu.g, of peptide or DNA is administered, depending on the particular peptide or DNA. The above dosage ranges were successfully used in previous trials (Walter et al, 2012).

Polynucleotides used for active immunization may be in substantially purified form, or may be coated onto a carrier or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, or a combination thereof. Methods for the design and introduction of such nucleic acids are well known in the art. For example, there is a summary thereof in the literature (Teufel et al, 2005). Polynucleotide vaccines are readily prepared, but the mode of action of these vectors to induce an immune response is not fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as systems based on adenovirus, vaccinia virus, retrovirus, herpes virus, adeno-associated virus, or mixed viruses containing more than one viral element. Non-viral delivery systems, including cationic liposomes and cationic polymers, are well known in the art for DNA delivery. Physical delivery systems, such as through a "gene gun," may also be used. The peptide or nucleic acid encoding the peptide can be a fusion protein, e.g., containing an epitope that stimulates T cells to perform the above-described CDRs.

The agents of the invention may also include one or more adjuvants. Adjuvants are those substances that non-specifically enhance or potentiate an immune response (e.g., an immune response to an antigen mediated by CD 8-positive T cells and helper T (TH) cells and are therefore considered useful for the agents of the present inventionAS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF, IC30, IC31, imiquimodresiquimod, ImuFactIMP321, interleukins IL-2, IL-13, IL-21, interferon α or β, or polyethylene glycol derivatives thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs,Lipovac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA 206, Montanide ISA50V, Montanide ISA-51, oil-in-water and water-in-oil emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, Yttrium, Mydrimum, Mylar,carrier system, polylactide-based composite glycolide [ PLG]And dextran microparticles, recombinant human lactoferrin SRL172, viral and other virus-like particles, YF-17D, VE GF trap, R848, β -dextran, Pam3Cys, QS21 stimulators from Aquila company derived from saponins, mycobacterial extracts and bacterial cell wall synthesis mimics, as well as other proprietary adjuvants such as Ribi's Detox, Quil or Superfos, preferably adjuvants such as Freund's adjuvant or GM-CSF. predecessors described some dendritic cell-specific immune adjuvants (e.g., MF59) and methods of preparation thereof (Allison and Krummel,1995) could also use cytokines.

CpG immunostimulatory oligonucleotides are reported to enhance the effect of adjuvants in vaccines. Without being bound by theory, CpG oligonucleotides can act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), mainly TLR 9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and enhanced Cytotoxic T Lymphocyte (CTL) production, even with the loss of CD4T cell repertoire. The activation-induced TH1 bias of TLR9 was maintained even in the presence of vaccine adjuvants such as: alum or Freund's incomplete adjuvant (IFA) which normally promotes TH2 migration. CpG oligonucleotides, when prepared or co-administered with other adjuvants or formulations such as microparticles, nanoparticles, fat emulsions or the like, exhibit enhanced adjuvant activity, which is particularly necessary to induce a strong response when the antigen is relatively weak. They also accelerated the immune response, reducing antigen dose by about two orders of magnitude, and in some experiments, produced similar antibody responses to full dose vaccines without CpG (Krieg, 2006). U.S. Pat. No. 4, 7726705, 1 describes the use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens in combination to elicit an antigen-specific immune response. One CpG TLR9 antagonist is dlim (dual stem-loop immunomodulator) from Mologen, berlin, germany, which is a preferred ingredient of the pharmaceutical composition of the present invention. Other TLR-binding molecules, such as: the RNA binds TLR7, TLR8, and/or TLR 9.

Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), dsRNA mimetics, e.g., Poly (I: C) and its derivatives (e.g., AmpliGen, Hiltonol, Poly- (ICLC), Poly (IC-R), Poly (I: C12U)), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, e.g.: cyclophosphamide, sumicizumab,Xilepubao, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA 4, other antibody-targeting major structures of the immune system (e.g., anti-CD 40, anti-TGF β, anti-TNF α receptors), and SC58175, all of which may have therapeutic and/or adjuvant effectsThe amount and concentration of adjuvants and additives useful in the present invention can be readily determined.

Preferred adjuvants are particulate formulations of anti-CD 40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon alpha, CpG oligonucleotides and derivatives, poly (I: C) and derivatives, RNA, sildenafil and PLG or viral particles.

In a preferred embodiment of the pharmaceutical composition of the invention, the adjuvant is selected from the group consisting of colony stimulating factor-containing preparations, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargrastim), cyclophosphamide, imiquimod, resiquimod and interferon- α.

In a preferred embodiment of the pharmaceutical composition of the invention, the adjuvant is selected from the group consisting of colony stimulating factor-containing preparations, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargrastim), cyclophosphamide, imiquimod and resimiquimod. In a preferred embodiment of the pharmaceutical composition of the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. More preferred adjuvants are Montanide IMS1312, Montanide ISA 206, Montanide ISA50V, Montanide ISA-51, poly-ICLCAnd anti-CD 40 mAB or a combination thereof.

The composition can be administered by parenteral injection, such as subcutaneous injection, intradermal injection, intramuscular injection, or oral administration. For this purpose, the peptides and other selective molecules are dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. In addition, the composition may comprise adjuvants such as: buffers, binders, impactors, diluents, flavorants, lubricants, and the like. These peptides may also be used in combination with immunostimulatory substances, such as: a cytokine. Further adjuvants which can be used in such compositions are known from Handbook of pharmaceutical excipients (Kibbe,2000) et al, by a. The combination is useful for the prevention, prophylaxis and/or treatment of adenoma or cancerous disease. For example, there are exemplified formulations in EP 2112253.

It is important to recognize that the immune response elicited by the vaccines of the present invention attacks cancer at different cellular stages and at different stages of development. And different cancer-related signaling pathways are attacked. This has the advantage over other vaccines that target only one or a few targets, which may lead to easy adaptation of the tumor to attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Thus, the combination of several tumor-associated peptides ensures that each tumor bears at least some of the targets. The composition is designed in such a way that it is expected that each tumor can express several antigens and cover several independent pathways required for tumor growth and maintenance. Thus, the vaccine can be readily "off-the-shelf" for use in a larger patient population. This means that patients pre-selected for vaccine treatment can be restricted to HLA typing without any additional biomarker assessment of antigen expression, but still ensure that multiple targets are simultaneously challenged by an induced immune response, which is important for therapeutic efficacy (Banchereau et al, 2001; Walter et al, 2012).

The term "scaffold" as used herein refers to a molecule that specifically binds to a (e.g. antigenic) determinant. In one embodiment, the scaffold is capable of directing the entity (e.g., the (second) antigen-binding moiety) to which it is attached to a target of interest, e.g., to a specific type of tumor cell or tumor substrate bearing an antigenic determinant (e.g., a complex of a peptide and MHC according to the present application). In another embodiment, the scaffold is capable of initiating a signaling pathway through its target antigen (e.g., a T cell receptor complex antigen). Scaffolds include, but are not limited to, antibodies and fragments thereof, antigen-binding regions of antibodies comprising antibody heavy chain variable regions and antibody light chain variable regions, bound proteins including at least one ankyrin repeat motif and Single Domain Antigen Binding (SDAB) molecules, aptamers, (soluble) TCRs, and (modified) cells, such as allogeneic or autologous T cells. To assess whether a certain molecule is a scaffold bound to a target, a binding assay can be performed.

By "specific" binding is meant that the scaffold binds better to the peptide-MHC complex of interest than to other native peptide-MHC complexes to the extent that a scaffold possessing an active molecule capable of killing cells bearing a particular target is not capable of killing another cell not bearing the particular target but presenting one or more other peptide-MHC complexes. If the cross-reactive peptide-MHC peptide is not native, i.e., not from the human HLA-polypeptide group, binding to other peptide-MHC complexes is not critical. Assays to assess target cell killing are well known in the art. They should be carried out with target cells (primary cells or cell lines) or cells loaded with peptides, which contain unaltered peptide-MHC presentation, in order to reach the level of native peptide-MHC. Each scaffold may include a label that is detectable by a signal provided by determining the presence or absence of a label. For example, the scaffold may be labeled with a fluorescent dye or any other suitable cell labeling molecule. Such marker molecules are well known in the art. For example, fluorescent labeling with a fluorescent dye can provide visualization of the binding aptamer by fluorescence or laser scanning microscopy or flow cytometry.

Each scaffold can be conjugated to a second active molecule (e.g., IL-21, anti-CD 3, anti-CD 28).

For further information on polypeptide scaffolds, see, e.g., the background section of WO 2014/071978A1, incorporated herein by reference.

The invention also relates to aptamers. Aptamers (see, for example, WO 2014/191359 and the references cited therein) are short single-stranded nucleic acid molecules that can fold into a defined three-dimensional structure and recognize specific target structures. They appear to be suitable alternatives for the development of targeted therapies. Aptamers have been shown to selectively bind complex targets with high affinity and specificity.

Aptamers that recognize cell surface molecules have been identified over the past decade and provide a means for developing diagnostic and therapeutic methods. Aptamers are promising candidates for biomedical applications, since they have been shown to be almost non-toxic and immunogenic. In fact, aptamers, such as prostate specific membrane antigen recognition aptamers, have been successfully used for targeted therapy and have been shown to function in xenografts in vivo models. In addition, it is recognized that aptamers to specific tumor cell lines have also been identified.

DNA aptamers can be selected to reveal a broad spectrum of recognition attributes of various cancer cells, particularly those from solid tumors, but not tumorigenic and predominantly healthy cells. If the aptamers identified not only recognize tumor-specific subtypes, but also interact with a range of tumors, this makes the aptamers suitable as so-called broad-spectrum diagnostic and therapeutic tools.

Furthermore, studies of cell binding behavior with flow cytometry showed that aptamers displayed good affinity in the nanomolar range.

Aptamers are used for diagnostic and therapeutic purposes. In addition, it may also be shown that some aptamers are taken up by tumor cells and thus can enter tumor cells as molecular excipients for targeted delivery of anticancer agents, such as siRNA.

Aptamers can be selected against targets of complexes such as cells and tissues and peptide complexes and MHC molecules according to the current invention comprising, preferably including, a sequence according to any of SEQ ID NO1 to SEQ ID NO268, using the cellular SELEX (systematic evolution of ligands through exponential enrichment) technique.

The peptides of the invention are useful for the generation and development of specific antibodies against MHC/peptide complexes. These antibodies are useful in therapy to target toxins or radioactive substances to diseased tissue. Another use of these antibodies is to target radionuclides to diseased tissue for imaging purposes (e.g., PET). This may help to detect small metastases or to determine the size and accurate location of diseased tissue.

Thus, another aspect of the invention is directed to a method of producing a recombinant antibody that specifically binds to class I or II human Major Histocompatibility Complex (MHC) complexed with an HLA-restricted antigen, the method comprising: immunizing a genetically engineered non-human mammal comprising a protein expressing said Major Histocompatibility Complex (MHC) class I or II with a soluble form of a (MHC) class I or II molecule complexed with an HLA-restricted antigen, preferably a peptide according to the invention; separating the mRNA molecules from antibodies raised to said non-human mammalian cells; generating a phage display library displaying protein molecules encoded by said mRNA molecules; and separating at least one bacteriophage from said phage display library, said at least one bacteriophage displaying said antibody specifically binding to said human Major Histocompatibility Complex (MHC) class I or II complexed to an HLA-restricted antigen.

Thus, in another aspect the invention provides an antibody that specifically binds to class I or II human Major Histocompatibility Complex (MHC) complexed to an HLA-restricted antigen, wherein the antibody is preferably a polyclonal antibody, a monoclonal antibody, a bispecific antibody and/or a chimeric antibody.

Corresponding methods for producing such antibodies and single chain class I major histocompatibility complexes, as well as other means for producing such antibodies, are disclosed in WO 03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and publications (Cohen et al, 2003 a; Cohen et al, 2003 b; Denkberg et al, 2003), all references being incorporated herein by reference in their entirety for the purposes of the present invention.

Preferably, the binding affinity of the antibody to the complex is less than 20 nanomolar, preferably less than 10 nanomolar, which in the context of the present invention is also considered to be "specific".

The present invention relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NO1 to SEQ ID NO268 or a variant thereof having 88% homology (preferably the same) to SEQ ID NO1 to SEQ ID NO268 or a variant thereof which induces T cell cross-reactivity with said variant peptide, wherein said peptide is not a substantially full length polypeptide.

The invention further relates to a peptide comprising a sequence selected from the group consisting of SEQ ID No.1 to SEQ ID No.268 or a variant having at least 88% homology (preferably identical) to SEQ ID No.1 to SEQ ID No.268, wherein the total length of said peptide or variant is from 8 to 100, preferably from 8 to 30, most preferably from 8 to 14 amino acids.

The invention further relates to peptides of the invention having the ability to bind to a Major Histocompatibility Complex (MHC) class I or II molecule.

The invention further relates to a peptide according to the invention, wherein the peptide consists or essentially consists of an amino acid sequence according to SEQ ID NO1 to SEQ ID NO 268.

The invention further relates to a peptide of the invention, wherein the peptide is (chemically) modified and/or comprises non-peptide bonds.

The invention further relates to a peptide of the invention, wherein the peptide is part of a fusion protein, in particular comprising the N-terminal amino acid of HLA-DR antigen associated invariant chain (Ii), or wherein the peptide is fused to an antibody, e.g., a dendritic cell specific antibody.

The invention further relates to a nucleic acid encoding a peptide according to the invention, with the proviso that the peptide is not a complete (fully) human protein.

The invention further relates to a nucleic acid according to the invention, being DNA, cDNA, PNA, RNA, and possibly a combination thereof.

The invention further relates to an expression vector capable of expressing the nucleic acid of the invention.

The invention further relates to a peptide according to the invention, a nucleic acid according to the invention or a pharmaceutical expression vector according to the invention, in particular for the treatment of colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma.

The invention further relates to a host cell comprising a nucleic acid according to the invention or an expression vector according to the invention.

The invention further relates to a host cell of the invention which is an antigen presenting cell, preferably a dendritic cell.

The invention further relates to a method for formulating a peptide of the invention, said method comprising culturing a host cell of the invention and isolating the peptide from said host cell or its culture medium.

The invention further relates to methods of the invention wherein an antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell by binding to a sufficient amount of antigen comprising the antigen-presenting cell.

The invention further relates to the method of the invention wherein the antigen presenting cell comprises an expression vector capable of expressing the peptide comprising SEQ ID NO1 to SEQ ID NO268 or said variant amino acid sequence.

The invention further relates to a T-cell promoter produced by the method of the invention, wherein the T-cell selectively recognizes a cell that aberrantly expresses a polypeptide comprising an amino acid sequence of the invention.

The invention further relates to a method of killing target cells in a patient, wherein the target cells in the patient abnormally express a polypeptide comprising any of the amino acid sequences of the invention, the method comprising administering to the patient an effective amount of a T cell of the invention.

The invention further relates to any of said peptides, a nucleic acid according to the invention, an expression vector according to the invention, a cell according to the invention, a use of a T lymphocyte according to the invention for initiating cytotoxicity as a medicament or for the manufacture of a medicament. The invention further relates to a use according to the invention, wherein the medicament is effective against cancer.

The invention further relates to a use according to the invention, wherein the medicament is a vaccine. The invention further relates to a use according to the invention, wherein the medicament is effective against cancer.

The invention further relates to a use according to the invention, wherein the cancer cell is a colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma cell or other solid or hematological tumor cell, such as: chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia and other lymphomas (e.g., non-hodgkin's lymphoma), post-transplant lymphoproliferative disease (PTLD) and other myelomas (e.g., primary myelofibrosis), primary thrombocytopenia, polycythemia vera, and other tumors such as: esophageal cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, melanoma, breast cancer, gallbladder and bile duct cancer, bladder cancer, uterine cancer, head and neck squamous cell carcinoma, mesothelioma.

The invention further relates to specific marker proteins and biomarkers, herein referred to as "targets", based on the peptides of the invention, which can be used for the diagnosis and/or prognosis of colorectal, glioblastoma, gastric, hepatocellular, renal cell carcinoma. The invention also relates to these novel targets for use in cancer therapy.

The term "antibody" is defined herein in a broad sense to include both polyclonal and monoclonal antibodies. In addition to intact or "whole" immunoglobulin molecules, the term "antibody" also includes fragments (e.g., CDR, Fv, Fab, and Fc fragments) or polymers of these immunoglobulin molecules and humanized immunoglobulin molecules, so long as they exhibit any of the desirable attributes of the present invention (e.g., specific binding of colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma marker (poly) peptide, colorectal cancer when increased levels of expression of genes that deliver toxins to cancer markers, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma cells, and/or inhibition of the activity of colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma polypeptide).

Antibodies of the invention may be purchased from commercial sources whenever possible. The antibodies of the invention may also be prepared using known methods. The skilled artisan will appreciate that full-length colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma marker polypeptides, or fragments thereof, may be used to prepare the antibodies of the invention. The polypeptides used to produce the antibodies of the invention may be partially or wholly purified from natural sources or may be produced using recombinant DNA techniques.

For example, a cDNA of the invention encoding a peptide, e.g., a peptide according to the polypeptides of SEQ ID NO:1 to SEQ ID NO:268, or a variant or fragment thereof, can be expressed in prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast, insect, or mammalian) cells, after which the recombinant protein can be purified and used to produce a monoclonal or polyclonal antibody preparation that specifically binds to the colorectal, glioblastoma, gastric, hepatocellular, renal cell carcinoma marker polypeptides used to produce the antibodies of the invention.

One skilled in the art will recognize that two or more different sets of monoclonal or polyclonal antibodies maximize the likelihood of obtaining an antibody with the specificity and affinity (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy) required for its intended use. Depending on the use of the antibody, its desired activity is tested by known methods (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance in producing and testing antibodies, see, e.g., Greenfield, 2014). For example, the antibody can be detected by ELISA or immunoblotting, immunohistochemical staining of formalin fixed cancerous tissue or frozen tissue sections. After initial in vitro characterization, antibodies for therapeutic or in vivo diagnostic use are detected according to known clinical test methods.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a large homogeneous population of antibodies, i.e., a population of antibodies consisting of identical antibodies, except for natural mutations that may be present in minor amounts. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chains are identical (homogeneous) to the corresponding sequences of antibodies obtained from a particular species or antibodies belonging to a particular antibody type and class, while the remaining chains are identical (homogeneous) to the corresponding sequences of antibodies obtained from other species or antibodies belonging to a particular antibody type and sub-class, and fragments of such antibodies, so long as they exhibit the desired antagonistic activity (U.S. patent No. 4816567, which is incorporated herein in its entirety).

The monoclonal antibodies of the invention may be made using hybridoma methods. In the hybridoma method, a mouse or other appropriate host animal is typically primed with an immunizing agent to elicit or produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

Monoclonal antibodies can also be made by recombinant DNA methods, such as: as described in U.S. patent No. 4816567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies).

In vitro methods are also suitable for the production of monovalent antibodies. Digestion of antibodies to produce fragments of antibodies, particularly Fab fragments, can be accomplished by using conventional techniques known in the art. Digestion can be accomplished, for example, by using papain. Examples of papain digestion are described in WO 94/29348 and U.S. Pat. No. 4342566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments (each having an antigen binding site) and a residual Fc fragment. Pepsin treatment produced an F (ab ')2 fragment and a pFc' fragment.

Antibody fragments, whether attached to other sequences or not, may include insertions, deletions, substitutions, or other selective modifications of particular regions or particular amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide some additional attributes, such as: amino acids that can bind to disulfide bonds are deleted/added to increase their biological life, alter their secretory properties, etc. In any case, the antibody fragment must possess bioactive properties such as: binding activity, modulating binding capacity of the binding domain, and the like. The functional or active region of an antibody can be determined by genetic mutation of a particular region of the protein, subsequent expression and testing of the expressed polypeptide. Such methods are well known to those skilled in the art and may include site-specific genetic mutations in the nucleic acids encoding the antibody fragments.

The antibody of the present invention may further include a humanized antibody or a human antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibody immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab' or other antigen binding sequences of an antibody) which contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are substituted with residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit having the specificity, affinity and capacity therefor. In some cases, Fv Framework (FR) residues of the human immunoglobulin are substituted for corresponding non-human residues. Humanized antibodies may also include residues found in neither the recipient antibody nor the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of the same sequence of a human immunoglobulin. Ideally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. In general, humanized antibodies have one or more amino acid residues introduced from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, and are typically obtained from an "import" variable domain. Humanization can be essentially accomplished by substituting rodent CDRs or CDR sequences with corresponding human antibody sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567) in which substantially less than an entire human variable domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are immunized to produce fully human antibodies in the absence of endogenous immunoglobulin can be used. For example, it is described that homozygous deletion of antibody heavy chain junction region genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays in such germline variant mice will result in the production of human antibodies following antigen challenge. Human antibodies can also be produced in phage display libraries.

The antibodies of the invention are preferably administered to a subject in the form of a pharmaceutically acceptable carrier. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include physiological saline, ringer's solution and dextrose solution. The pH of the solution is preferably about 5 to 8, more preferably about 7 to 7.5. In addition, the carrier may also include sustained release formulations such as: semipermeable matrices of solid hydrophobic polymers containing the antibody, wherein the matrices are in the form of shaped articles, such as: a film, liposome, or microparticle. It is well known to those skilled in the art that certain carriers may be more preferred depending, for example, on the route of administration and the concentration of the antibody.

The antibody can be administered to a subject, patient, or cell by other means such as injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular) or by infusion, to ensure that it is delivered to the blood in an effective form. These antibodies can also be administered via intratumoral or peritumoral routes to exert local and systemic therapeutic effects. Topical or intravenous injection is preferred.

Effective dosages and schedules for administration of the antibodies can be determined empirically, and making such determinations is within the skill of the art. Those skilled in the art will appreciate that the dosage of antibody that must be administered will vary depending on factors such as: the subject receiving the antibody, the route of administration, the antibody used, and the particular type of drug being used. Typical daily dosages of antibody used alone may range from about 1. mu.g/kg up to 100mg/kg body weight or more, depending on the factors mentioned above. After administration of the antibody, preferably colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, the efficacy of the therapeutic antibody can be assessed by different methods well known to the skilled person. For example: the size, amount, and/or distribution of cancer in a subject receiving treatment can be monitored using standard tumor imaging techniques. An antibody administered as a result of treatment prevents tumor growth, causes tumor shrinkage, and/or prevents the development of new tumors, as compared to the course of disease in the absence of administration of the antibody, and is an antibody effective for the treatment of cancer.

In another aspect of the invention, a method of making a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex is provided. Such soluble T cell receptors can be generated from specific T cell clones, and their affinity can be increased by complementarity determining region-targeted mutagenesis. For the purpose of T cell receptor selection, phage display may be used (us 2010/0113300, (likdy et al, 2012)). For the purpose of stabilizing the T cell receptor during phage display and when actually used as a drug, the alpha and beta chains can be linked via non-native disulfide bonds, other covalent bonds (single chain T cell receptors) or via a dimerization domain (Boulter et al, 2003; Card et al, 2004; Willcox et al, 1999). T cell receptors may be linked to toxins, drugs, cytokines (see US 2013/0115191), domain recruitment effector cells such as anti-CD 3 domains, etc., in order to perform specific functions on target cells. In addition, it may be expressed in T cells for adoptive transfer. Further information can be found in WO 2004/033685A 1and WO 2004/074322A 1. Combinations of stcrs are described in WO 2012/056407a 1. Further methods of preparation are disclosed in WO 2013/057586a 1.

In addition, the peptides and/or TCRs or antibodies or other binding molecules of the invention can be used to validate the pathologist's diagnosis of cancer on the basis of biopsy samples.

The antibodies or TCRs may also be used in vivo diagnostic assays. Generally, antibodies are labeled with radionuclides (e.g., 111In, 99Tc, 14C, 131I, 3H, 32P, or 35S) to localize tumors by immunoscintigraphy. In one embodiment, the antibody or fragment thereof binds to the extracellular domain of two or more protein targets selected from the group comprising the proteins described above and has an affinity value (Kd) of less than 1x10 μ M.

Diagnostic antibodies can be labeled with probes suitable for detection by various imaging methods. Probe detection methods include, but are not limited to, fluorescence, light, confocal and electron microscopy methods; magnetic resonance imaging and spectroscopy techniques; fluoroscopy, computer tomography, and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other rare earths, paramagnetic iron, fluorine-18 and other positron emitting radionuclides. In addition, the probe may be bifunctional or multifunctional, and detection may be performed using more than one of the methods described above. These antibodies can be labeled with the probes directly or indirectly. The linking of antibody probes, including covalent attachment of probes, fusion of probes into antibodies, and covalent attachment of chelating compounds to bind probes, among other methods well known in the art. For immunohistochemical methods, diseased tissue samples may be fresh or frozen or may be embedded in paraffin and fixed with preservatives such as formalin. The fixed or embedded sections include samples contacted with labeled primary and secondary antibodies, wherein the antibodies are used to detect in situ protein expression.

Another aspect of the invention includes a method for preparing activated T cells in vitro, comprising contacting T cells in vitro with antigen loaded human MHC molecules expressed on the surface of a suitable antigen-presenting cell for a period of time sufficient to activate T cells in an antigen-specific manner, wherein the antigen is a peptide according to the invention. Preferably, a sufficient amount of antigen is used with the antigen presenting cells.

Preferably, the TAP peptide transporter is absent or reduced in level or function in mammalian cells. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter associated with antigen processing.

Human peptide-loaded defective cell line T2, catalog number CRL1992, from American type culture Collection (ATCC,12301ParklawnDrive, Rockville, Md. 20852, USA); the ATCC catalogue CRL 19863, which is subordinate to the Drosophila cell line Schneider No.2 strain; mouse RMA-S cell line Ljunggren et al (Ljunggren and Karre, 1985).

Preferably, the host cell does not substantially express MHC class I molecules prior to transfection. The stimulator cells also preferably express molecules that play an important role in T cell costimulatory signaling, e.g., any of B7.1, B7.2, ICAM-1, and LFA 3. Nucleic acid sequences for a number of MHC class I molecules and co-stimulatory molecules are publicly available from GenBank and EMBL databases.

When MHC class I epitopes are used as one antigen, the T cells are CD8 positive T cells.

If the antigen presenting cell is transfected to express such an epitope, preferred cells include an expression vector capable of expressing the peptide or variant amino acid sequence comprising SEQ ID NO1 to SEQ ID NO 268.

Several other methods can be used to generate T cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used to generate CTLs. Plebanski et al (Plebanski et al, 1995) used autologous peripheral blood lymphocytes (PLB) to make T cells. Alternatively, dendritic cells may be pulsed with peptides or polypeptides or made autologous T cells by infection with recombinant viruses. In addition, B cells can be used to prepare autologous T cells. In addition, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses may be used to formulate autologous T cells. Walter et al (Walter et al, 2003) describe the in vitro priming of T cells by using artificial antigen presenting cells (aapcs), which is also a suitable method for generating T cells that act on selected peptides. In the present invention, according to biotin: streptomycin biochemical methods are performed by contacting preformed MHC: peptide complexes were coupled to polystyrene particles (microspheres) to generate aapcs. The system enables precise regulation of MHC density on aapcs, which allows for the selective priming of high-potency antigen-specific T cell responses of high or low avidity in blood samples. In addition to MHC: in addition to peptide complexes, aapcs should also carry other proteins containing co-stimulatory activity, such as anti-CD 28 antibodies coupled to the surface. In addition, such aAPC-based systems often require the addition of appropriate soluble factors, e.g., cytokines such as interleukin 12.

T cells can also be prepared from allogeneic cells, a process which is described in detail in WO 97/26328, incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present peptides, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, vaccinia-infected target cells. Furthermore, plant viruses can also be used (see, for example, Porta et al (Porta et al, 1994) which describes the development of cowpea mosaic virus as a highly productive system for presenting foreign peptides.

The activated T cells are directed against the peptides of the invention and contribute to the treatment. Thus, in another aspect of the invention, there are provided primed T-cells prepared by the methods of the invention described above.

The T-promoter cells prepared by the above method will selectively recognize the abnormally expressed polypeptides having the amino acid sequences of SEQ ID NO1 to SEQ ID NO 268.

Preferably, the T cell recognizes the cell by interacting with (e.g., binding to) its TCR comprising HLA/peptide complex. T cells are cells useful in a method of killing a target cell in a patient, wherein the target cell abnormally expresses a polypeptide comprising an amino acid sequence of the invention. Such patients are administered an effective amount of priming T cells. The T cells administered to the patient may be derived from the patient and primed as described above (i.e., they are autologous T cells). Alternatively, the T cells are not derived from the patient, but from another person. Of course, it is preferred that the donor is a healthy person. By "healthy individual" we mean a person who is generally in good condition, preferably has a qualified immune system, and more preferably is free of any disease that can be easily tested or detected.

According to the present invention, the in vivo target cells of CD 8-positive T cells may be tumor cells (sometimes expressing MHC-class II antigens) and/or stromal cells (tumor cells) surrounding the tumor (sometimes also expressing MHC-class II antigens; (Dengjel et al, 2006)).

The T cells of the invention are useful as active ingredients in therapeutic compositions. Accordingly, the present invention also provides a method of killing target cells in a subject, wherein the target cells in the subject aberrantly express a polypeptide comprising an amino acid sequence of the invention, the method comprising administering to the subject an effective amount of a T cell as described above.

The meaning of "aberrantly expressed" as used by the inventors also includes that the polypeptide is overexpressed compared to the normal expression level, or that the gene is not expressed in tumor-derived tissues but is expressed in tumors. "overexpression" refers to a level of polypeptide that is at least 1.2 fold higher than in normal tissue; preferably at least 2-fold, more preferably at least 5 or 10-fold, that of normal tissue.

T cells can be prepared by methods known in the art (e.g., as described above).

T cell secondary transfer protocols are well known in the art. A review can be found in: gattioni et al, and Morgan et al (Gattinone et al, 2006; Morgan et al, 2006).

Another aspect of the invention includes the use of peptides complexed with MHC to generate T cell receptors, the nucleic acids of which are cloned and introduced into host cells, preferably T cells. The genetically engineered T cells can then be delivered to a patient for cancer therapy.

Any of the molecules of the invention (i.e., peptides, nucleic acids, antibodies, expression vectors, cells, priming T cells, T cell receptors, or encoding nucleic acids) is useful in treating diseases characterized by cells that escape the immune response. Thus, any of the molecules of the present invention may be used as a medicament or in the manufacture of a medicament. Such molecules may be used alone or in combination with other or known molecules of the present invention.

The invention also relates to a kit comprising:

(a) a container containing the above pharmaceutical composition in the form of a solution or lyophilized powder;

(b) optionally a second container containing a diluent or reconstitution solution in the form of a lyophilized powder; and

(c) optionally (i) instructions for use of the solution or (ii) for use of the recombinant and/or lyophilized formulation.

The kit further comprises one or more of (iii) a buffer, (iv) a diluent, (v) a filtrate, (vi) a needle, or (v) a syringe. The container is preferably a bottle, vial, syringe or test tube, and may be a multi-purpose container. The pharmaceutical composition is preferably lyophilized.

The kit of the present invention preferably comprises a lyophilized formulation in a suitable container together with reconstitution and/or instructions for use. Suitable containers include, for example, bottles, vials (e.g., dual chamber bottles), syringes (e.g., dual chamber syringes), and test tubes. The container may be made of a variety of materials, such as glass or plastic. The kit and/or container preferably has a container or instructions for the container indicating the direction of reconstitution and/or use. For example, the label may indicate that the lyophilized dosage form will reconstitute to the peptide concentration described above. The label may further indicate that the formulation is for subcutaneous injection.

The container holding the formulation may use a multi-purpose vial of penicillin such that the reconstituted dosage form may be administered repeatedly (e.g., 2-6 times). The kit may further comprise a second container containing a suitable diluent, such as a sodium bicarbonate solution.

After mixing the dilution and the lyophilized formulation, the final concentration of peptide in the recombinant formulation is preferably at least 0.15 mg/mL/peptide (═ 75 μ g), and not more than 3 mg/mL/peptide (═ 1500 μ g). The kit may also include other materials that are commercially and user-friendly, including other buffers, diluents, filtrates, needles, syringes, and package inserts with instructions for use.

The kit of the present invention may have a separate container containing a formulation of a pharmaceutical composition of the present invention with or without other ingredients (e.g., other compounds or pharmaceutical compositions thereof), or with different containers for each ingredient.

Preferably, the kits of the present invention comprise a formulation of the present invention packaged for use in combination with a second compound (e.g., an adjuvant (e.g., GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic or inhibitory agent, an apoptosis inducing agent, or a chelator) or pharmaceutical composition thereof. The components of the kit may be pre-complexed or each component may be placed in a separate, distinct container prior to administration to a patient. The components of the kit may be one or more solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The components of the kit may also be in solid form, converted to a liquid upon addition of a suitable solvent, and preferably placed in a different container.

The container of the treatment kit may be a vial, test tube, flask, bottle, syringe, or any other means for holding a solid or liquid. Typically, where there is more than one component, the kit will contain a second vial or other container of penicillin, so that it can be dosed separately. The kit may also contain another container for holding a medicinal liquid. Preferably, the treatment kit will contain a device (e.g., one or more needles, syringes, eye droppers, pipettes, etc.) such that the medicament of the present invention (the composition of the present kit) can be injected.

The pharmaceutical formulations of the present invention are suitable for administration of the peptides by any acceptable route, such as oral (enteral), intranasal, intraocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal administration. Preferably, the administration is subcutaneous, most preferably intradermal, and may also be via an infusion pump.

Since the peptide of the present invention is isolated from colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, and renal cell carcinoma, the pharmaceutical agent of the present invention is preferably used for the treatment of colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, and renal cell carcinoma. The invention further relates to a method for preparing a personalized medicine for an individual patient, comprising: manufacturing a pharmaceutical composition comprising at least one peptide selected from a pre-screened TUMAP reservoir, wherein the at least one peptide used in the pharmaceutical composition is selected to be suitable for an individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can also be modified to produce T cell clones for downstream applications such as: TCR spacers or soluble antibodies and other therapeutic options.

"personalized medicine" refers to treatments that are specific to an individual patient and will only be used for that individual patient, including personalized active cancer vaccines and adoptive cell therapies using autologous tissue.

As used herein, "depot" shall refer to a group or series of peptides that have been subjected to immunogenic pre-screening and/or over-presented in a particular tumor type. The term "depot" does not imply that the particular peptide included in the vaccine has been pre-manufactured and stored in physical equipment, although such a possibility is contemplated. It is expressly contemplated that the peptides may be used in the new manufacture of each individual vaccine, and may also be pre-manufactured and stored. The repertoire (e.g., library format) consists of tumor-associated peptides that are highly overexpressed in tumor tissues of various HLA-a HLA-B and HLA-C allele genes colorectal, glioblastoma, gastric, hepatocellular, renal cell carcinoma patients. It may comprise peptides including MHC class I and MHC class II or elongated MHC class I peptides. In addition to tumor-associated peptides collected from several colorectal, glioblastoma, gastric, hepatocellular, renal cell carcinoma tissues, the pools may also contain HLA-A02, HLA-A01, HLA-A03, HLA-A24, HLA-B07, HLA-B08, and HLA-A24B 44 labeled peptides. These peptides allow quantitative comparison of the TUMAP-induced T cell immunity, leading to important conclusions about the capacity of vaccines to respond to tumors. Second, they can serve as important positive control peptides from "non-self" antigens in the absence of any vaccine-induced T cell responses from the patient's "self" antigen TUMAP. Third, it may also allow conclusions to be drawn on the immune function status of the patient.

TUMAP from the repository was identified by using a functional genomics approach combining gene expression analysis, mass spectrometry and T cell immunologyThis approach ensures that only TUMAPs that are actually present in a high percentage of tumors but are not or only rarely expressed in normal tissues are selected for further analysis. For the initial peptide selection, the patient's colorectal, glioblastoma, gastric, hepatocellular, renal cell carcinoma samples and blood of healthy donors were analyzed in a progressive manner:

1. determination of HLA ligands for malignant materials by mass spectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression assays are used to determine genes that are overexpressed in malignant tissues (colorectal, glioblastoma, gastric, hepatocellular, renal cell carcinoma) compared to a range of normal organs and tissues.

3. The determined HLA ligands are compared to gene expression data. Peptides overexpressed or selectively presented on tumor tissue, preferably the selectively expressed or overexpressed genes detected in step 2 encode suitable TUMAP candidates for polypeptide vaccines.

4. Literature search to determine more evidence to support the relevance of peptides identified as TUMP

5. The correlation of overexpression at the mRNA level was determined by the selected TUMAP retest at step 3 in tumor tissue and the absence (or infrequent) of detection in healthy tissue.

6. To assess whether it is feasible to induce T cell responses in vivo through selected peptides, in vitro immunogenicity assays were performed using healthy donors and human T cells from patients with colorectal, glioblastoma, gastric, hepatocellular, renal cell carcinoma.

In one aspect, the peptides are screened for immunogenicity prior to being added to the repository. For example, but not limited to, methods for determining the immunogenicity of peptides incorporated into a depot include in vitro T cell priming, specifically: CD8+ T cells from healthy donors were repeatedly stimulated with artificial antigen presenting cells loaded with peptide/MHC complexes and anti-CD 28 antibodies. This method is preferred for rare cancers and patients with rare expression profiles. In contrast to the cocktail containing polypeptides currently developed as fixed components, the depot allows a higher degree of matching of the actual expression of antigens in tumors to the vaccine. In a multi-objective approach, each patient will use a selected single peptide or combination of several "off-the-shelf" peptides. Theoretically, one approach based on selecting, for example, 5 different antigenic peptides from a50 antigenic peptide library could provide about 170 million possible pharmaceutical product (DP) components.

In one aspect, the peptides are selected for use in a vaccine based on the suitability of the individual patient and using the methods of the invention described herein or below.

HLA phenotypic, transcriptional and peptidological data were collected from tumor material and blood samples of patients to determine the peptides most appropriate for each patient and containing a "repository" and patient-unique (i.e., mutated) TUMAP. Those peptides selected are selectively or over-expressed in patient tumors and, possibly, exhibit strong in vitro immunogenicity if tested with patient individual PBMCs.

Preferably, a method of determining the peptide comprised by the vaccine comprises: (a) identifying a tumor associated peptide (TUMAP) presented by a tumor sample from an individual patient; (b) comparing the peptides identified in (a) to a repository (database) of said peptides; and (c) selecting at least one peptide from a repository (library) associated with the identified tumor-associated peptides in the patient. For example, TUMAPs presented in tumor samples can be identified by: (a1) comparing expression data from the tumor sample with expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are over-or aberrantly expressed in the tumor tissue; and (a2) correlating the expression data with MHC ligand sequences bound to MHC class I and/or class II molecules in the tumor sample to determine MHC ligands derived from proteins overexpressed or abnormally expressed by the tumor. Preferably, the sequence of the MHC ligand is determined by: the MHC molecules isolated from the tumor sample are eluted with the peptide and the eluted ligand is sequenced. Preferably, the tumor sample and the normal tissue are obtained from the same patient.

In addition to, or as an alternative to, using a repository (library) model for peptide selection, TUMAPs may be identified in new patients and then included in vaccines. As an example, candidate TUMAPs in a patient may be identified by: (a1) comparing expression data from the tumor sample with expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are over-or aberrantly expressed in the tumor tissue; and (a2) correlating the expression data with MHC ligand sequences bound to MHC class I and/or class II molecules in the tumor sample to determine MHC ligands derived from proteins overexpressed or abnormally expressed by the tumor. As another example, the method of identifying a protein may comprise a mutation that is unique to the tumor sample relative to the corresponding normal tissue of the individual patient, and the TUMAP may be identified by specifically targeting the variation. For example, the genome of a tumor and corresponding normal tissue can be sequenced by whole genome sequencing methods: to find non-synonymous mutations in the protein coding region of the gene, genomic DNA and RNA were extracted from tumor tissue and normal non-mutated genomic germline DNA was extracted from Peripheral Blood Mononuclear Cells (PBMC). The NGS method used is limited to the re-sequencing of protein coding regions (exome re-sequencing). For this purpose, exon DNA from human samples was captured using a target sequence enrichment kit supplied by the supplier, followed by sequencing using HiSeq2000 (Illumina). In addition, mRNA from the tumor was sequenced to directly quantify gene expression and confirm that the mutant gene was expressed in the patient's tumor. Millions of sequence reads are obtained and processed through software algorithms. The output list contains mutations and gene expression. Tumor specific somatic mutations were determined by comparison to PBMC-derived germline changes and optimized. Then, for the purpose of storage it is possible to test newly identified peptides for immunogenicity as described above and select candidate TUMAPs with appropriate immunogenicity for use in vaccines.

In an exemplary embodiment, the peptides contained in the vaccine are identified by: (a) identifying a tumor associated peptide (TUMAP) presented from a tumor sample from an individual patient using the method described above; (b) aligning the peptides identified in (a) with a repository of tumor-bearing (as compared to corresponding normal tissue) immunogenic and over-presenting pre-screening peptides; (c) selecting at least one peptide from a repository associated with identified tumor-associated peptides in a patient; and (d) optionally selecting at least one newly identified peptide from (a) for confirmation of its immunogenicity.

In an exemplary embodiment, the peptides contained in the vaccine are identified by: (a) identifying a tumor associated peptide (TUMAP) presented by a tumor sample from an individual patient; and (b) selecting at least one newly identified peptide in (a) and confirming its immunogenicity.

Once the peptides for the individualized peptide vaccine are selected, the vaccine is produced. The vaccine is preferably a liquid formulation comprising the individual peptides dissolved in between 20-40% DMSO, preferably about 30-35% DMSO, e.g., about 33% DMSO.

Each peptide listed in the product was dissolved in DMSO. The choice of the concentration of the individual peptide solutions depends on the amount of peptide to be included in the product. The single peptide-DMSO solutions were mixed equally to achieve a solution containing all peptides at a concentration of 2.5mg/ml per peptide. The mixed solution was then mixed according to a 1: 3 ratios were diluted with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution was filtered through a 0.22 μm sterile screening procedure. Thereby obtaining the final bulk solution.

The final bulk solution was filled into vials and stored at-20 ℃ prior to use. One vial contained 700 μ L of solution with 0.578mg of each peptide. Of which 500 μ L (about 400 μ g of each peptide) will be used for intradermal injection.

The peptides of the invention are useful in diagnosis as well as in the treatment of cancer. Since peptides are produced by colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma cells, and it has been determined that these peptides are absent or present at low levels in normal tissues, these peptides can be used to diagnose the presence or absence of cancer.

A tissue biopsy from a blood sample containing the peptide of claim, useful for a pathologist in diagnosing cancer. Detection of certain peptides by antibodies, mass spectrometry, or other methods known in the art allows a pathologist to determine whether the tissue sample is malignant or inflammatory or general pathological, and may also be used as a biomarker for colorectal, glioblastoma, gastric, hepatocellular, renal cell carcinoma. Presentation of the peptide groups allows for classification or further subclassification of the diseased tissue.

Detection of peptides in diseased specimens allows for the determination of the benefit of immune system treatment methods, particularly if T-lymphocytes are known or predicted to be involved in the mechanism of action. Loss of MHC expression is a mechanism that adequately accounts for which infected malignant cells evade immune surveillance. Thus, presentation of the peptide indicated that the analyzed cells did not utilize this mechanism.

The peptides of the invention can be used to assay lymphocyte responses to peptides (e.g., T cell responses), or antibody responses to peptides complexed with peptides or MHC molecules. These lymphocyte responses can be used as prognostic indicators to determine whether further treatment should be undertaken. These responses can also be used as surrogate indicators in immunotherapy aimed at inducing lymphocyte responses in different ways, such as vaccination, nucleic acids, autologous material, adoptive transfer of lymphocytes. In gene therapy, the reaction of lymphocytes to peptides can be taken into account in the assessment of side effects. Lymphocyte response monitoring may also be a valuable tool in follow-up examinations of transplantation therapies, e.g., for detecting graft-versus-host and host-versus-graft disease.

The invention will now be illustrated by the following examples describing preferred embodiments, and with reference to the accompanying drawings, but without being limited thereto. All references cited herein are incorporated by reference for the purposes of this invention.

Graph table

FIGS. 1A to 1P show representative expression profiles of the source gene of the present invention, which is overexpressed in different cancer samples. Tumor (black dots) and normal (gray dots) samples were grouped by organ origin, and box and whisker plots represent median, 25 th and 75 th percentiles (boxes), and minimum and maximum (whiskers) RPKM values. Normal organs are ranked according to risk categories. RPKM ═ per million mapped reads per kilobase read. Normal samples: blood cells; a blood vessel; a brain; a core; liver; a lung; fat: adipose tissue; gl.: the adrenal gland; a bile duct; the bladder; BM: bone marrow; cartilage; and (4) espph: (ii) an esophagus; an eye; and (3) a gallb: a gallbladder; a head and neck; a kidney; large _ int: the large intestine; LN: lymph nodes; a nerve; a pancreas; parathyr: parathyroid gland; pert: peritoneum; pituit: a pituitary; mus: skeletal muscle; skin; small _ int: the small intestine; a spleen; the stomach; thyroid gland; an air tube; a ureter; a breast; an ovary; a placenta; the prostate; a testis; thymus; the uterus. Tumor samples: CRC: colorectal cancer; GBM: glioblastoma; GC: gastric cancer; HCC: hepatocellular carcinoma; RCC: renal cell carcinoma. FIG. 1A) Gene symbol: EGFR, peptide: LPSPTDSNFY (SEQ ID No.:2), FIG. 1B) Gene symbol: PTPRZ1, peptide: LTDYINANY (SEQ ID No.:28), 1C) Gene symbol: CCDC146, peptide: KMMALVAEL (SEQ ID No.:42), 1D) gene symbol: MMP16, peptide: YLPPTDPRMSV (SEQ ID No.:54), 1E) Gene symbol: RP11-1220K2.2, peptide: GLPDFVKEL (SEQ ID No.:60), 1F) Gene symbol: GRM8, peptide: ATMQSKLIQK (SEQ ID No.:99), 1G) gene symbol: HAVCR1, peptide: GVIIAKKYFFK (SEQ ID No.:101), 1H) Gene symbol: SEMA5B, peptide: GTESGTILK (SEQ ID No.:110), 1I) Gene symbol: PIWIL1, peptide: SFDSNLLSF (SEQ ID No.:133), 1J) Gene symbol: UGT1a3, peptide: KYLSIPTVF (SEQ ID No.:138), 1K) Gene symbol: FEZF1, peptide: APAAVPSAPA (SEQ ID No.:153), 1L) Gene symbol: MMP11, peptide: RPASSLRP (SEQ ID No.:163), 1M) gene symbol: QRFPR, peptide: SPMWHVQQL (SEQ ID No.:170), 1N) Gene symbol: REG4, peptide: SRSMRLLLL (SEQ ID No.:190), 1O) gene signature: PTHLH, peptide: AEIHTAEI (SEQ ID No.:231), 1P) Gene signature: EGFR, peptide: DEYL IPQQGF (SEQ ID No.: 264).

Figure 2 shows exemplary results of peptide-specific CD8+ T cell in vitro responses from healthy HLA-a x 02+ donors. The method for preparing the CD8+ T cells comprises the following steps: artificial APC coated with anti-CD 28 mAb and HLA-a × 02 was used with SeqID No267 peptide (KTLGKLWRL, Seq ID NO:267), respectively (a, left panel). After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with A.multidot.02/SeqID No267 (A). Right panel (B) shows control staining of cells stimulated with irrelevant a × 02/peptide complexes. Viable single cells were gated by CD8+ lymphocytes. Boolean gating helps to exclude false positive events detected with different peptide-specific multimers. The frequency of specific multimer + cells and CD8+ lymphocytes is suggested.

Figure 3 shows exemplary results of peptide-specific CD8+ T cell in vitro responses from healthy HLA-a x 24+ donors. The method for preparing the CD8+ T cells comprises the following steps: the artificial APC coated with anti-CD 28 mAb and HLA-A x 24 were conjugated to the peptide SEQ ID NO:268 (A, left panel), respectively. After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with A.times.24/SEQ ID NO:268(DYIPYVFKL, SEQ ID NO:268) (A). Right panel (B) shows control staining of cells stimulated with irrelevant a x 24/peptide complexes. Viable single cells were gated by CD8+ lymphocytes. Boolean gating helps to exclude false positive events detected with different peptide-specific multimers. The frequency of specific multimer + cells and CD8+ lymphocytes is suggested.

Figure 4 shows exemplary results of peptide-specific CD8+ T cell in vitro responses from healthy HLA-a × 01+ donors. CD8+ T cells were primed using artificial APCs coated with anti-CD 28 mAb and HLA-A01 complexed with the peptide of SEQ ID NO:6 (VLDLSTNVY; A, left panel) and SEQ ID NO:245 (ITEKNSGLY; B, left panel), respectively. After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with A.01/SEQ ID NO:6(A) or A.01/SEQ ID NO:245 (B). The right panels (a and B) show control staining of cells stimulated with irrelevant a × 01/peptide complexes. Viable single cells were gated to give CD8+ lymphocytes. Boolean gating helps to exclude false positive events detected with different peptide-specific multimers. The frequency of specific multimer + cells in CD8+ lymphocytes is suggested.

Figure 5 shows exemplary results of peptide-specific CD8+ T cell in vitro responses from healthy HLA-a x 02+ donors. The method for preparing the CD8+ T cells comprises the following steps: artificial APC coated with anti-CD 28 mAb and HLA-A02 were synthesized with the peptide SEQ ID NO:42 (KMMALVAEL; A, left panel) or the peptide SEQ ID NO:250 (KMILKMVQL; B, left panel), respectively. After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with A.about.02/SEQ ID NO:42(A) or A.about.02/SEQ ID NO:250 (B). The right panels (a and B) show control staining of cells stimulated with irrelevant a x 02/peptide complexes. Viable single cells were gated by CD8+ lymphocytes. Boolean gating helps to exclude false positive events detected with different peptide-specific multimers. The frequency of specific multimer + cells and CD8+ lymphocytes is suggested.

Figure 6 shows exemplary results of peptide-specific CD8+ T cell in vitro responses from healthy HLA-a 03+ donors. CD8+ T cells were primed using artificial APCs coated with anti-CD 28 mAb and HLA-A03 complexed with the peptide SEQ ID NO:94 (KTYVGHPVKM; A, left panel) and SEQ ID NO:110 (GTESGTILK; B, left panel), respectively. After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with A03/SEQ ID NO:94(A) or A03/SEQ ID NO:110 (B). The right panels (a and B) show control staining of cells stimulated with irrelevant a × 03/peptide complexes. Viable single cells were gated to give CD8+ lymphocytes. Boolean gating helps to exclude false positive events detected with different peptide-specific multimers. The frequency of specific multimer + cells in CD8+ lymphocytes is suggested.

Figure 7 shows exemplary results of peptide-specific CD8+ T cell in vitro responses from healthy HLA-a x 24+ donors. CD8+ T cells were primed using artificial APCs coated with HLA-a x 24 complexed with the peptide SEQ ID NO:138 (KYLSIPTVF; left panel) of anti-CD 28 mAb. After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with A.sup.02/SEQ ID NO: 138. The right panel shows control staining of cells stimulated with irrelevant a x 24/peptide complexes. Viable single cells were gated to give CD8+ lymphocytes. Boolean gating helps to exclude false positive events detected with different peptide-specific multimers. The frequency of specific multimer + cells in CD8+ lymphocytes is suggested.

Figure 8 shows exemplary results of peptide-specific CD8+ T cell in vitro responses from healthy HLA-B07 + donors. The method for preparing the CD8+ T cells comprises the following steps: artificial APC coated with anti-CD 28 mAb and HLA-B07 were synthesized with SeqID No170 peptide (SPMWHVQQL; A, left panel) or SeqID NO:155 peptide (FPYPYAERL; B, left panel), respectively. After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with B07/Seq ID NO:170(A) or B07/Seq ID NO:155 (B). The right panels (a and B) show control staining of cells stimulated with irrelevant B × 07/peptide complexes. Viable single cells were gated by CD8+ lymphocytes. Boolean gating helps to exclude false positive events detected with different peptide-specific multimers. The frequency of specific multimer + cells and CD8+ lymphocytes is suggested.

Figure 9 shows exemplary results of peptide-specific CD8+ T cell in vitro responses from healthy HLA-B44 + donors. The method for preparing the CD8+ T cells comprises the following steps: artificial APC coated with anti-CD 28 mAb and HLA-B44 were synthesized with the peptide SEQ ID NO:225 (SEAFPSRAL; A, left panel) or SEQ ID NO:236 (EEKLIIQDF; B, left panel), respectively. After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining with B44/SEQ ID NO:225(A) or B44/SEQ ID NO:236 (B). Right panels (a and B) show control staining of cells stimulated with irrelevant B × 44/peptide complexes. Viable single cells were gated by CD8+ lymphocytes. Boolean gating helps to exclude false positive events detected with different peptide-specific multimers. The frequency of specific multimer + cells and CD8+ lymphocytes is suggested.

Examples