阅读说明：本技术 用于各种肿瘤免疫治疗的新型肽和肽组合物 (Novel peptides and peptide compositions for immunotherapy of various tumors ) 是由 安德里亚·马尔 莉·斯蒂夫 托尼·温斯切尼克 奥利弗·施尔 延斯·弗里切 哈普瑞特·辛格 于 2016-03-24 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。与主要组织兼容性复合体(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 or are cognate to Major Histocompatibility Complex (MHC) molecules may also be targets for antibodies, soluble T cell receptors, and other binding molecules.)

1. A peptide comprising an amino acid sequence selected from the group consisting of: SEQ ID No.264, SEQ ID Nos. 1-13, SEQ ID Nos. 15-156, SEQ ID No.158-263 and SEQ ID No. 265-288; and a variant sequence thereof which is at least 88% homologous to any one of SEQ ID Nos. 264, 1-13, 15-156, 158-263 and 265-288, and wherein said variant binds to a Major Histocompatibility Complex (MHC) molecule and/or induces T cells cross-reacting with the variant peptide; and pharmaceutically acceptable salts thereof,

wherein the peptide is not a full-length polypeptide.

2. The peptide of claim 1, wherein the peptide has the ability to bind to an MHC-I or MHC-II molecule, and wherein the peptide, when bound to the MHC, is capable of being recognized by CD4 and/or CD 8T cells.

3. The peptide or variant thereof according to claim 1 or 2, wherein the amino acid sequence thereof comprises a continuous stretch of amino acids of any one of SEQ ID Nos. 264, 1-13, 15-156, 158-263 and 265-288.

4. The peptide or variant thereof according to any of claims 1 to 3, wherein said peptide or variant thereof has an overall length of from 8 to 100 amino acids, preferably from 8 to 30 amino acids, more preferably from 8 to 16 amino acids, most preferably wherein the peptide consists or essentially consists of any one of the amino acid sequences SEQ ID No.264, SEQ ID No.1-13, SEQ ID No.15-156, SEQ ID No.158-263 and SEQ ID No. 265-288.

5. The peptide or variant thereof according to any of claims 1 to 4, wherein said peptide is modified and/or comprises non-peptide bonds.

6. The peptide or variant thereof according to any of claims 1 to 5, wherein said peptide is part of a fusion protein, in particular comprising the N-terminal amino acid of the HLA-DR antigen associated invariant chain (li).

7. A nucleic acid encoding the peptide or variant thereof according to any one of claims 1 to 6, optionally linked to a heterologous promoter sequence.

8. An expression vector expressing the nucleic acid of claim 7.

9. A recombinant host cell comprising a peptide according to claims 1 to 6, a nucleic acid according to claim 7, or an expression vector according to claim 8, wherein said host cell is preferably an antigen presenting cell, such as a dendritic cell.

10. The peptide or variant thereof according to any one of claims 1 to 6, the nucleic acid according to claim 7, the expression vector according to claim 8 or the host cell according to claim 9 for use in medicine.

11. A method of preparing a peptide or variant thereof according to any one of claims 1 to 6, the method comprising: culturing a host cell according to claim 9 which presents a peptide according to claims 1 to 6, or expresses a nucleic acid according to claim 7, or comprises an expression vector according to claim 8; and isolating the peptide or variant thereof from the host cell or the culture medium thereof.

12. A method for producing activated T lymphocytes in vitro, the method comprising contacting T cells in vitro with antigen loaded human MHC-I or MHC-II molecules expressed on the surface of a suitable antigen-presenting cell or 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 any one of claims 1 to 4.

13. Activated T lymphocytes produced according to the method of claim 12, which selectively recognize cells presenting a polypeptide comprising an amino acid sequence given in any one of claims 1 to 4.

14. A method of killing target cells in a patient, said target cells presenting a polypeptide comprising an amino acid sequence given in any one of claims 1 to 4, the method comprising administering to the patient an effective amount of activated T cells as defined in claim 13.

15. Antibody, in particular soluble or membrane-bound antibody, which specifically recognizes a peptide or variant thereof according to any of claims 1 to 5, preferably a peptide or variant thereof according to any of claims 1 to 5 when bound to an MHC molecule.

16. The peptide according to any one of claims 1 to 6, the nucleic acid according to claim 7, the expression vector according to claim 8, the cell according to claim 9, the activated T lymphocyte according to claim 13 or the antibody according to claim 15 for use in the diagnosis and/or treatment of cancer or for the manufacture of an anti-cancer agent.

17. The peptide according to any one of claims 1 to 6, the nucleic acid according to claim 7, the expression vector according to claim 8, the cell according to claim 9, the activated T lymphocyte according to claim 13 or the antibody according to claim 15 for use according to claim 16, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), colorectal cancer (CRC), Glioblastoma (GB), Gastric Cancer (GC), esophageal cancer, non-small cell lung cancer (NSCLC), Pancreatic Cancer (PC), Renal Cell Cancer (RCC), Benign Prostatic Hyperplasia (BPH), Prostate Cancer (PCA), Ovarian Cancer (OC), melanoma, breast cancer, Chronic Lymphocytic Leukemia (CLL), Merkel Cell Cancer (MCC), Small Cell Lung Cancer (SCLC), non-hodgkin lymphoma (NHL), Acute Myelogenous Leukemia (AML), Gallbladder and bile duct cancer (GBC, CCC), bladder cancer (UBC), uterine cancer (UEC) and other tumors overexpressing proteins from which peptides of any one of SEQ ID No.264, SEQ ID Nos. 1-13, SEQ ID Nos. 15-156, SEQ ID No.158-263 and SEQ ID No.265-288 are derived.

18. A kit, comprising:

(a) a container comprising a pharmaceutical composition containing the peptide or variant according to any one of claims 1 to 6, the nucleic acid according to claim 7, the expression vector according to claim 8, the cell according to claim 10, the activated T lymphocyte according to claim 13 or the antibody according to claim 15 in solution or in lyophilized form;

(b) optionally, a second container containing a diluent or reconstitution solution for a lyophilized dosage form;

(c) optionally, at least one further peptide selected from the group consisting of SEQ ID No.264, SEQ ID Nos. 1-13, SEQ ID Nos. 15-156, SEQ ID No.158-263 and SEQ ID No.265-288, and

(d) optionally, instructions for (i) using the solution or (ii) reconstituting and/or using the lyophilized formulation.

19. The kit of claim 18, further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.

20. The kit according to claim 18 or 19, wherein the peptide is selected from the group consisting of SEQ ID No.264, SEQ ID Nos. 1-13, SEQ ID Nos. 15-156, SEQ ID No.158-263 and SEQ ID No. 265-288.

21. A method for preparing a personalized anti-cancer vaccine, the method comprising:

a) identifying a tumor associated peptide (TUMAP) presented from a tumor sample from an individual patient;

b) comparing the peptides identified in a) to a repository of peptides that have been subjected to an immunogenic pre-screening, said pre-screening being a pre-screening for immunogenicity and/or for over-presentation in a tumor as compared to normal tissue;

c) selecting from a repository at least one peptide that matches a TUMAP identified in a patient; and

d) formulating a personalized vaccine based on step c).

22. The method of claim 21, wherein the TUMAPs are identified by:

a1) comparing the expression data of the tumor sample with expression data of a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or abnormally expressed in the tumor sample; and

a2) the expression data is correlated with MHC ligand sequences that bind MHC class I and/or MHC class II molecules in the tumor sample to identify protein-derived MHC ligands that are overexpressed or abnormally expressed by the tumor.

23. The method of claim 21 or 22, wherein the sequence of the MHC ligand is identified by eluting the bound peptide from MHC molecules isolated from the tumor sample and sequencing the eluted ligand.

24. The method according to any one of claims 21 to 23, wherein the normal tissue corresponding to the tissue type of the tumor sample is obtained from the same patient.

25. The method according to any one of claims 21 to 24, wherein the peptides contained in the repository are identified based on the following steps:

performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identifying genes that are overexpressed in malignant tissue compared to normal tissue;

ab. selecting the peptide encoded by the selectively expressed or overexpressed gene detected in step aa, and ac determining the induction of an in vivo T cell response by the selected peptide, including in vitro immunogenicity assays using human T cells of a healthy donor or the patient; or

ba. identifying HLA ligands from the tumor sample by mass spectrometry;

performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identifying genes that are overexpressed in malignant tissue compared to normal tissue;

comparing the identified HLA ligands to the gene expression data;

bd. selecting the peptide encoded by the gene specifically expressed or overexpressed and detected in step bc; be. retesting TUMAP detected on tumor tissue but not or infrequently detected on healthy tissue selected from step bd and determining the correlation of overexpression at the mRNA level; and

bf. the induction of a T cell response in vivo is determined by the selected peptide, including in vitro immunogenicity assays using human T cells from healthy donors or the patient.

26. The method according to any one of claims 21 to 25, wherein the immunogenicity of the peptides comprised in the repository is determined by a method comprising an in vitro immunogenicity test, patient individual HLA binding immune monitoring, MHC multimer staining, ELISPOT testing and/or intracellular cytokine staining.

27. The method according to any one of claims 21 to 26, wherein said repertoire comprises a plurality of peptides selected from the group consisting of SEQ ID nos. 264, 1-13, 15-156, 158-263 and 265-288.

28. The method of any of claims 21 to 27, further comprising: identifying at least one mutation on the tumor sample that is characteristic of the corresponding normal tissue of the individual patient, and selecting a peptide associated with the mutation for inclusion in a vaccine or for use in generating cell therapy.

29. The method of claim 28, wherein the at least one mutation is identified by whole genome sequencing.

A T cell receptor, preferably a recombinant soluble or membrane bound T cell receptor, which is reactive with an HLA ligand, wherein said ligand has at least 75% identity to an amino acid sequence selected from the group consisting of SEQ ID No.264, SEQ ID nos. 1-13, SEQ ID nos. 15-156, SEQ ID nos. 158-263 and SEQ ID No. 265-288.

31. The T cell receptor according to claim 30, wherein the amino acid sequence is at least 88% identical to any one of SEQ ID nos. 264, 1-13, 15-156, 158-263 and 265-288.

32. The T cell receptor according to claim 30 or 31, wherein the amino acid sequence consists of any one of SEQ ID nos. 264, 1-13, 15-156, 158-263 and 265-288.

33. The T cell receptor according to any one of claims 30 to 32, wherein said T cell receptor is provided as a soluble molecule and optionally has a further effector function, such as an immunostimulatory domain or a toxin.

34. A nucleic acid encoding a TCR according to any one of claims 30 to 33, optionally linked to a heterologous promoter sequence.

35. An expression vector expressing the nucleic acid of claim 34.

36. A recombinant host cell comprising: the nucleic acid according to claim 34, or the nucleic acid encoding the antibody according to claim 15, or the expression vector according to claim 35, wherein the host cell is preferably a T cell or an NK cell.

37. A method of producing a T cell receptor according to any one of claims 30 to 33, the method comprising culturing a host cell according to claim 36 and isolating the T cell receptor from the host cell and/or its culture medium.

38. An aptamer specifically recognizing a peptide or a variant thereof according to any of claims 1 to 5, preferably a peptide or variant thereof according to any of claims 1 to 5 bound to an MHC molecule.

39. Pharmaceutical composition comprising at least one active ingredient selected from the group consisting of a peptide according to any one of claims 1 to 6, a nucleic acid according to claim 7, an expression vector according to claim 8, a cell according to claim 9, an activated T lymphocyte according to claim 13 or an antibody according to claim 15 or a T cell receptor according to any one of claims 30-32 or an aptamer according to claim 38, and optionally further pharmaceutically acceptable excipients and/or stabilizers.

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 /).

Epidemiology

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).

Within the brain, leukemia and lung cancer populations, the present invention focuses particularly on Glioblastoma (GB), Chronic Lymphocytic Leukemia (CLL) and Acute Myelogenous Leukemia (AML), non-small cell and small cell lung cancer (NSCLC and SCLC), respectively.

GB is the most common central nervous system malignancy, with an age-adjusted incidence of 3.19 per 10 million residents in the united states. The prognosis of GB is very poor, the survival rate of 1 year is 35%, and the survival rate of 5 years is lower than 5%. Male gender, older age, and race appear to be risk factors for GB (Thakkar et al, 2014).

CLL is the most common leukemia in the western world, with approximately one-third of all cases of leukemia. The incidence of disease is similar in the united states and europe, with an estimated 16,000 new cases per year. CLL is more common in caucasians than in africans, is less common in hispanic and indigenous americans, and is less common in asia. In people of asian descent, CLL occurs at a 3-fold lower rate than caucasians (Gunawardana et al, 2008). The five-year overall survival rate of CLL patients is approximately 79% (http:// www.cancer.net/cancer-types/leukamia-chronic-lymphocytic-CLL/statics).

Lung cancer is the most common type of cancer worldwide and is the leading cause of cancer death in many countries. Lung cancer is classified into small cell lung cancer and non-small cell lung cancer. NSCLC includes histological types of adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and accounts for approximately 85% of all lung cancer cases in the united states. The occurrence of NSCLC is closely related to the history of smoking, including current and previous smokers, and has been reported to have a five-year survival rate of 15% (World Cancer Report, 2014; Molina et al, 2008).

Treatment of

Breast cancer

Standard treatment of breast cancer patients depends on different parameters: tumor stage, hormone receptor status and HER2 expression pattern. Standard treatment includes surgical total resection of the tumor followed by radiation therapy. Anthracycline-and taxane-based chemotherapy may be initiated before or after resection. HER2 positive tumor patients received the anti-HER 2 antibody trastuzumab in addition to the chemotherapeutic drug (S3-leitline mammakarazinom, 2012). Breast cancer is an immunogenic tumor entity, and different types of infiltrating immune cells in the primary tumor show different prognostic and predictive significance. A number of early immunization trials have been conducted in breast cancer patients. Clinical data for modulation of immune steps using yipima and other T cell activating antibodies in breast cancer patients is emerging (emers, 2012).

Chronic lymphocytic leukemia

Although CLL is currently incurable, many patients show only slow disease progression or symptom worsening. For patients with symptoms or with rapidly progressing disease, several treatment options are available. These include chemotherapy, targeted therapy, immune-based therapies such as monoclonal antibodies, Chimeric Antigen Receptors (CARS) and active immunotherapy, and stem cell transplantation.

Some of the experiments that have been completed and are ongoing are based on engineered autologous Chimeric Antigen Receptor (CAR) modified T cells with specificity for CD19 (Maus et al, 2014). To date, only a few patients have shown detectable or persistent CAR. Porter et al and Kalos et al detected one Partial Response (PR) and 2 Complete Responses (CR) in the CAR T cell assay (Kalos et al, 2011; Porter et al, 2011).

Active immunotherapy includes the following strategies: gene therapy, bulk modified tumor cell vaccines, DC-based vaccines, and Tumor Associated Antigen (TAA) -derived peptide vaccines.

Several TAAs are overexpressed in CLL and suitable for vaccination. These include the fibromodulins (Mayr et al, 2005), RHAMM/CD168(Giannopoulos et al, 2006), MDM2(Mayr et al, 2006), hTERT (Counter et al, 1995), the carcinoembryonic antigen immature laminin receptor protein (OFAiLRP) (Siegel et al, 2003), the adipose differentiation associated proteins (Schmidt et al, 2004), survivin (Granziero et al, 2001), KW1 to KW14 (krackdt et al, 2002) and the tumor-derived vhcdr3 regions (Harig et al, 2001; Carballido et al, 2012). One phase I clinical trial used the RHAMM-derived R3 peptide as a vaccine. R3-specific CD8+ T cell responses were detectable in 5 out of 6 patients (Giannopoulos et al, 2010).

Colorectal cancer

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

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 (Koido et 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), wilms tumor antigen 1(WT1), survivin-2B, MAGE3, p53, cyclic finger protein 43, and mitochondrial outer membrane translocase 34(TOMM34) or mutant KRAS. In several phase I and phase II 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 tumor RNA or DC tumor cell fusion products pulsed with TAA-derived peptides, tumor cell lysates, apoptotic tumor cells. Although many patients in phase I/II trials exhibit specific immune responses, only a few receive clinical benefit (Koido et al, 2013).

Esophageal cancer

The primary treatment strategy for esophageal cancer depends on the stage and location of the tumor, the histological type, and the patient's condition. Chemotherapeutic regimens include oxaliplatin + fluorouracil, carboplatin + paclitaxel, cisplatin + fluorouracil, FOLFOX and cisplatin + irinotecan. Patients positive for tumor HER2 should be treated according to the stomach cancer treatment guidelines, since the randomized data for esophageal cancer targeted therapy is very limited (Stahl et al, 2013).

Esophageal cancer immunotherapy approaches have little data because only a limited number of early stage clinical trials have been conducted. In a phase I trial, patients with advanced esophageal cancer were given a vaccine containing peptides from three different cancer-testis antigens (TTK protein kinase, lymphocyte antigen 6 complex locus K and insulin-like growth factor (IGF) -II mRNA binding protein 3), with modest results. In one phase I/II study, intratumoral injection of activated T cells into tumors after in vitro stimulation of autologous tumor cells in 4 out of 11 patients elicited complete or partial tumor responses (tomey et al, 2013).

Gastric Cancer (GC) occurs first on the inner wall of the mucosal layer and spreads to the outer layer as it grows. There are four standard treatments available for gastric cancer. Methods of gastric cancer treatment include endoscopic or surgical resection, chemotherapy, radiation therapy, or chemoradiotherapy (Leitlinie magenkrazinom, 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. Adoptive transfer of tumor-associated lymphocytes and cytokine-induced killer cells, peptide-based HER 2/neu-targeted vaccines, MAGE-3 or vascular endothelial growth factor receptors 1 and 2, and dendritic cell-based HER 2/neu-targeted vaccines showed promising results in clinical trials of gastric cancer. Immune step suppression and engineering of T cells may represent more treatment options, currently being evaluated in preclinical and clinical studies (Matsueda and Graham, 2014).

Glioblastoma (WHO grade IV) treatment options are very limited. Different immunotherapeutic approaches were investigated against Glioblastoma (GB), including immune step suppression, vaccination and engineered T cell adoptive transfer.

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 stimulated 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 transgenic T cells is another immunotherapeutic approach to GB therapy. Different clinical trials currently evaluate the safety and efficacy of chimeric antigen receptors, which carry T cells against HER2, IL-13 receptor α 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. 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 (Chang et 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 this type of patient (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 in vitro experiments using peptides derived from alpha-fetoprotein (AFP) or AFP peptide-loaded DCs (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.

Melanoma (MEA)

The standard therapy for melanoma is surgical total resection and removal of surrounding healthy tissue. Treatment options include single-drug chemotherapy, multi-drug chemotherapy, and specific inhibitor targeted therapy (S3-Leitlinie Melanom, 2013).

Several different vaccination methods have been evaluated in advanced melanoma. To date, phase III clinical trials have shown quite disappointing results and vaccination strategies clearly need to be improved. Therefore, new clinical trials, such as the Oncovex GM-CSF trial or the DERMA trial, were aimed at improving clinical efficacy without reducing tolerability (http:// www.cancerresearchuk.org).

Adoptive transfer of T cells shows great potential for treatment of advanced melanoma. Autologous tumors that have been expanded in vitro by infiltrating lymphocytes and T cells containing T cell receptors with high affinity for the cancer-testis antigen NY-ESO-1 have significant beneficial and less toxic effects when transferred to melanoma patients. Unfortunately, T cells containing high affinity T cell receptors for the melanocyte specific antigens MART1 and gp100 and the cancer-testis antigen MAGEA3 induced comparable toxic effects in clinical trials. Thus, adoptive transfer of T cells has a higher therapeutic potential, but the safety and tolerability of these treatments needs to be further improved (Phan and Rosenberg, 2013; Hinrichs and Restifo, 2013).

Non-small cell lung cancer treatment options are determined by the type (small cell and non-small cell lung cancer) and stage of the cancer, including surgery, radiation therapy, chemotherapy, targeted biological treatments such as bevacizumab, erlotinib, and gefitinib (S3-Leitlinie Lungenkarzinom, 2011).

In order to expand the therapeutic regimens for NSCLC, different immunotherapeutic approaches have been or are being investigated. Although vaccination with L-BLP25 or MAGEA3 failed to demonstrate the vaccine-mediated survival advantage in NSCLC patients, allogeneic cell-derived vaccines have shown promising results in clinical studies. In addition, further vaccination trials against gangliosides, epidermal growth factor receptor and several other antigens are currently in progress. Another strategy to enhance a patient's anti-tumor T cell response involves the use of specific antibodies to block inhibitory T cell receptors or their ligands. Several of these antibodies, including ipilimumab, nivolumab, pembrolizumab, MPDL3280A, and MEDI-4736, are currently being evaluated in clinical trials for their therapeutic potential in NSCLC (Reinmuth et al, 2015).

Ovarian cancer

Surgical resection is the primary treatment for early and late stage ovarian cancer (S3-Leitlinie maligne Ovarialtumore, 2013).

Immunotherapy appears to be a promising strategy for improving the treatment of ovarian cancer patients, since the presence of pro-inflammatory tumor infiltrating lymphocytes, especially CD8 positive T cells, is associated with a good prognosis, and tumor-associated peptide-specific T cells can be isolated from cancer tissues.

Therefore, a great deal of scientific work has been devoted to the study of different immunotherapies for ovarian cancer. Considerable preclinical and clinical studies have been performed, with further studies currently in progress. There are currently clinical data for cytokine therapy, vaccination, monoclonal antibody therapy, adoptive transfer of cells, and immunomodulation.

Phase I and phase II vaccination studies using single or multiple peptides from several tumor associated proteins (HER2/neu, NY-ESO-1, p53, Wilms tumor-1) or whole tumor antigens from autologous tumor cells showed good safety and tolerability profiles, but the clinical effect was only low to moderate.

Adoptive transfer of immune cells achieved heterogeneous results in clinical trials. Adoptive transfer of autologous, in vitro expanded tumor-infiltrating T cells has proven to be a promising approach in pilot trials. In contrast, transfer of T cells containing folate receptor alpha specific chimeric antigen receptors did not induce significant clinical responses in phase I trials. Dendritic cells stimulated in vitro with tumor cell lysates or tumor-associated proteins were shown to enhance anti-tumor T cell responses after metastasis, but the extent of T cell activation was not relevant to clinical efficacy. In phase II studies, transfer of natural killer cells caused significant toxicity.

Intrinsic anti-tumor immunity as well as immunotherapy is affected by immunosuppressive tumor microenvironment. To overcome this obstacle, immunomodulatory drugs (such as cyclophosphamide, anti-CD 25 antibodies, and pegylated liposomal doxorubicin) were tested in combination with immunotherapy. The anti-CTLA 4 antibodies, which are capraloma, which enhance T cell activity, are currently the most reliable data. Lypima was shown to produce significant anti-tumor effects in ovarian cancer patients (Mantia-Smaldone et al, 2012).

Pancreatic cancer

Treatment options for pancreatic cancer patients are very limited. One major problem with effective treatment is that it is often in an advanced stage of the tumor when diagnosed.

Vaccination strategies have been investigated as a further innovative and promising alternative to the treatment of pancreatic cancer. Targeting for KRAS mutations, active telomerase, gastrin, survivin, CEA, and MUC1 have been evaluated in clinical trials, and have shown promising results in part. In addition, dendritic cell vaccines, allogeneic GM-CSF secretory vaccines and algenpancute-L clinical trials for pancreatic cancer patients have also shown beneficial effects of immunotherapy. Clinical trials to further study the efficacy of different vaccination protocols are currently in progress (Salman et al, 2013).

Prostate cancer

The treatment strategy for prostate cancer depends largely on the stage of the cancer. For locally confined non-metastatic prostate cancer, the treatment regimen included active monitoring (waiting and observation), surgical total resection of the prostate, and local high-dose radiation therapy with or without brachytherapy (S3-leitliie prostatakararzinom, 2014).

Dendritic cell-based vaccine sipuleucel-T is the first FDA-approved anti-cancer vaccine. Because of its positive effect on the survival of CRPC patients, much effort has been devoted to the development of further immunotherapy. With respect to vaccination strategies, the peptide vaccine Prostate Specific Antigen (PSA) -TRICOM, the personalized peptide vaccine PPV, the DNA vaccine pTVG-HP, and the whole cell vaccine expressing GM-CSF GVAX all showed promising results in different clinical trials. Furthermore, in addition to sipuleucel-T, dendritic cell-based vaccines, i.e. BPX-101 and DCVAC/Pa, have been shown to elicit clinical responses in prostate cancer patients. Immune step inhibitors such as, for example, Yiprioman and nivolumab are currently being evaluated in clinical studies as monotherapy and in combination with other therapies including androgen deprivation therapy, local radiation therapy, PSA-TRICOM and GVAX. The immunomodulatory substance tasquinimod, which significantly slows progression and increases progression-free survival in phase II clinical trials, is currently being further studied in phase III clinical trials. Lenalidomide, another immunomodulator, induced a promising effect in early clinical studies, but failed to improve survival in phase III clinical trials. Despite these disappointing results, further trials of lenalidomide are ongoing (Quinn et al, 2015).

Renal cell carcinoma

Initial treatment, most often partial or complete resection of the diseased kidney, remains the primary effective 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 specified 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).

The known immunogenicity of RCC represents the basis to support the use of immunotherapy and cancer vaccines in advanced RCC. The interesting correlation between lymphocyte PD-1 expression and RCC late stage staging, staging and prognosis, as well as RCC tumor cell selective PD-L1 expression, and its potential association with poor clinical outcome, led to the development of new anti-PD-1/PD-L1 formulations for the treatment of RCC, either alone or in combination with anti-angiogenic drugs or other immunotherapeutic approaches (Massari et al, 2015). In advanced RCC, a phase III cancer vaccine trial named the triso study evaluated whether TroVax (a vaccine using the tumor associated antigen 5T4, and a poxvirus vector) added to first-line standard therapy extended survival in locally advanced or mRCC patients. Mean 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 and that 5T 4-specific antibody response intensity correlates with survival improvement. In addition, several studies have sought epitope-using peptide vaccines that are overexpressed in RCC. Various approaches to tumor vaccines have been investigated. Studies using whole tumor approaches (including tumor cell lysates, dendritic cell fusions to tumor cells or whole tumor RNA) were performed in RCC patients, and remission of tumor lesions was reported in some trials (Avigan et al, 2004; Holtl et al, 2002; Marten et al, 2002; Su et al, 2003; Wittig et al, 2001).

Small cell lung cancer

Treatment and prognosis of Small Cell Lung Cancer (SCLC) is highly dependent on the diagnosed staging of the cancer. SCLC staging based on clinical outcome is more common than pathological staging. The results of the physical examination, various imaging tests and biopsy are used in clinical stages. Standard chemotherapy for SCLC uses etoposide or irinotecan in combination with cisplatin or carboplatin (American Cancer Society, 2015; S3-Leitlinie Lungenkarzinom, 2011).

Immunotherapy is an area of over-research for cancer treatment. Various approaches have been investigated for the treatment of SCLC. One approach is targeted blockade of CTLA-4, a natural human immunosuppressive. Inhibition of CTLA-4 is intended to enhance the immune system against cancer. Recently, promising immune step inhibitors for the treatment of SCLC have begun to be developed. Another approach is based on Cancer-preventative vaccines, which are currently available as SCLC therapeutics for clinical studies (American Cancer Society, 2015; National Cancer Institute, 2015).

Acute myelogenous leukemia

Treatment of AML is divided into two phases: induction therapy and post remission/"consolidation therapy". Induction therapy is induction of remission and includes combination chemotherapy. Consolidation therapy includes additional chemotherapy or hematopoietic stem cell transplantation (HCT) (Showel and Levis, 2014).

Clinical trials were recommended for patients belonging to the adverse and moderate-2 prognosis groups. Therapeutic options include demethylating drugs (HMAs), such as azacitidine or decitabine, CPX-351 (liposomal formulations of daunorubicin and cytarabine in a 1:5 "optimal" molar ratio), and volasertib (a Polo kinase inhibitor). Volasertib is administered in combination with LDAC (low dose cytarabine). Several different FLT3 inhibitors may be administered in the presence of FLT3 mutations. These include sorafenib (administered in combination with 3+ 7), quinzartinib (a more selective inhibitor of FLT3 ITD, also inhibiting CKIT), crenolanib and midostaurin (a non-selective FLT3 ITD inhibitor). Another therapeutic option is targeting CD33 (Estey,2014) with antibody-drug conjugates (anti-CD 33+ caclechiamicin, SGN-CD33a, anti-CD 33+ actinium-225), bispecific antibodies (recognizing CD33+ CD3(AMG 330) or CD33+ CD16), and Chimeric Antigen Receptors (CARs).

Non-hodgkin lymphoma

There are more than 60 subtypes of non-hodgkin lymphoma (NHL). The three most common subtypes are diffuse large B-cell lymphoma (DLBCL, the most common subtype), follicular lymphoma (FL, the second most common subtype), and small/chronic lymphocytic lymphoma (SLL/CLL, the third most common subtype). DLBCL, FL and SLL/CLL account for around 85% of NHL (Li et al, 2015). Treatment of NHL depends on histological type and stage (National Cancer Institute, 2015).

Spontaneous tumor regression was observed in lymphoma patients. Thus, active immunotherapy is a treatment option (Palomba, 2012).

One important vaccination protocol includes the Id vaccine. B lymphocytes express surface immunoglobulins with specific amino acid sequences in their heavy and light chain variable regions, each cell clone being unique (idiotype, Id). The idiotype serves as a tumor-associated antigen. Active immunization involves injection of a recombinant protein (Id) coupled to adjuvant (KLH) which is administered as an immune adjuvant with GM-CSF. Tumor specific Ids are produced through hybridoma cultures or through bacterial, insect or mammalian cell cultures using recombinant DNA technology (plasmids).

Uterine cancer

More than 80% of endometrial cancers develop in the form of endometrial adenocarcinoma (type I), which is associated with estrogen exposure and differentiates well to moderately. Treatment of endometrial and cervical Cancer is associated with staging (World Cancer Report, 2014).

Several immunotherapeutic approaches are also being tested. In a phase I/II clinical trial, uterine cancer patients were inoculated with autologous Dendritic Cells (DCs) electroporated with Wilms tumor gene 1 (WT1) mRNA. Except for one case of local allergic reaction to adjuvant, no adverse reaction was observed, 3 of 6 patients showed an immune response (Coomans et al, 2013).

Gallbladder adenocarcinoma and bile duct cancer

Cholangiocarcinoma (CCC) is difficult to treat and often fatal. The only effective treatment is complete resection (R0). The curative effect of the biological therapy of the biliary tract cancer is scurfy ginseng. For the treatment of CCC, vascular growth targeting drugs such as sorafenib, bevacizumab, pazopanib and regorafenib are now being investigated. In addition, drugs targeting EGFR, such as cetuximab and panitumumab drugs, are used in combination with chemotherapy in clinical studies (American Cancer Society, 2015). For most drugs tested to date, disease control and overall survival did not improve significantly, but further clinical trials are ongoing.

Gallbladder cancer (GBC) is the most common and most aggressive malignancy of the biliary tract worldwide. Since biliary cancer is rare, there are generally only a few GBC or CCC specific studies, most of which include all biliary cancers. This is why treatment has not improved over the last decades and R0 ablation remains the only effective treatment option.

Cancer of the bladder

Standard treatments for bladder Cancer include surgery, radiotherapy, chemotherapy and immunotherapy (National Cancer Institute, 2015).

An effective immunotherapy approach was identified in the treatment of invasive non-muscle invasive bladder cancer (NMIBC). Thus, an attenuated form of the bacterium mycobacterium Bovis (BCG) is used as an intravesical solution. The primary role of BCG treatment is to prevent cancer recurrence and reduce the rate of progression for a long period (up to 10 years). In principle, treatment with BCG can induce a local inflammatory response that stimulates a cellular immune response. The BCG immune response is based on the following key steps: infection of urothelial and bladder cancer cells by BCG, followed by increased expression of antigen presenting molecules, induction of immune responses by cytokine release mediation, induction of anti-tumor activity by participation of various immune cells (cytotoxic T lymphocytes, neutrophils, natural killer cells and macrophages) (Fuge et al, 2015; Gandhi et al, 2013). In view of the serious side effects and costs associated with the treatment of cancer, there is a need to identify factors that can be used for the treatment of cancer in general, in particular hepatocellular carcinoma (HCC), colorectal cancer (CRC), Glioblastoma (GB), Gastric Cancer (GC), esophageal cancer, non-small cell lung cancer (NSCLC), Pancreatic Cancer (PC), Renal Cell Carcinoma (RCC), Benign Prostatic Hyperplasia (BPH), Prostate Cancer (PCA), Ovarian Cancer (OC), melanoma, breast cancer, Chronic Lymphocytic Leukemia (CLL), Merkel Cell Carcinoma (MCC), Small Cell Lung Cancer (SCLC), non-hodgkin's lymphoma (NHL), Acute Myelogenous Leukemia (AML), gallbladder and bile duct cancer (GBC, CCC), bladder cancer (UBC), uterine cancer (UEC). It is also often necessary to determine factors that represent biomarkers of cancer, particularly the types of cancer described above, in order to better diagnose cancer, assess prognosis, and predict treatment success.

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 usually shared between 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 presentation of class I molecules is known in the literature as cross-presentation (Brossart and 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 may be 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 elongated peptides of the invention can be used as MHC-II active epitopes.

MHC-II epitope-activated helper T cells play an important role in orchestrating CTL effector functions of anti-tumor immunity. Triggering TH₁The helper T cell epitope of the cell response supports the effector functions of CD8 positive killer T cells, including cells directly acting on tumor cells Toxic function (tumor-associated peptide/MHC complex is displayed on the surface of the 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 CD 4T 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.

Brief introduction to the invention

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:288, 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:288 (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).

Although the most important criterion for a peptide as a target for cancer therapy is its over-presentation in primary tumor tissues compared to normal tissues, the RNA expression profile of the corresponding gene may also help to select an appropriate peptide. In particular, due to their chemical nature or low copy number on cells, some peptides are difficult to detect by mass spectrometry and screening methods that focus on peptide presentation detection may fail to recognize these targets. However, these targets can be detected by another method, starting with analysis of normal tissue for gene expression, and second to assess tumor peptide presentation and gene expression. This method is implemented in the present invention using mRNA data from publicly available databases (Lonsdale,2013) in combination with further gene expression data (including tumor samples) and peptide presentation data. If the mRNA of a gene is scarcely present in normal tissues, especially in vital organ systems, targeting the corresponding peptide through very efficient strategies (e.g.bispecific affinity optimized antibodies or T cell receptors) is more likely to be safe. Although such peptides are found in only a small fraction of tumor tissue, they represent interesting targets. Conventional mass spectrometry is not sensitive enough to assess target range at the peptide level. In contrast, tumor mRNA expression can be used to assess target range. To detect the peptide itself, a more sensitive targeted mass spectrometry approach than in conventional screening may be necessary, and may better assess the target range at the level of peptide presentation.

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 288 or a variant having at least 77%, preferably at least 88% homology (preferably at least 77% or 88% identical) to SEQ ID NO 1 to SEQ ID NO 288, 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. All peptides in tables 1 and 2 bound to HLA-a 02. 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.

Table 1: peptides of the invention

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

Serine phosphate J ═ phospho

Particularly preferred are peptides of the invention (alone or in combination) selected from the group comprising SEQ ID NO:1 to SEQ ID NO: 288. More preferred are the peptides (alone or in combination) selected from the group comprising SEQ ID NO:1 to SEQ ID NO:126 (see table 1) and their use for immunotherapy of hepatocellular carcinoma (HCC), colorectal cancer (CRC), Glioblastoma (GB), Gastric Cancer (GC), esophageal cancer, non-small cell lung cancer (NSCLC), Pancreatic Cancer (PC), Renal Cell Cancer (RCC), Benign Prostatic Hyperplasia (BPH), Prostate Cancer (PCA), Ovarian Cancer (OC), melanoma, breast cancer, Chronic Lymphocytic Leukemia (CLL), Merkel Cell Cancer (MCC), Small Cell Lung Cancer (SCLC), non-hodgkin's lymphoma (NHL), Acute Myelogenous Leukemia (AML), gallbladder and bile duct cancer (GBC, CCC), bladder cancer (UBC), uterine cancer (UEC).

Most preferred are said peptides-alone or in combination-selected from the group consisting of SEQ ID NO:274, 14, 21, 23, 25, 157, 168, 11, 253, 85, 89, 40, 264, 155, 233 and 245 (see tables 1, 2 and 10) and their use in HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC and CLL immunotherapy.

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

The invention further relates to a peptide according to the invention, wherein said peptide(s) (each peptide) consists or essentially consists of an amino acid sequence according to SEQ ID NO 1 to SEQ ID NO 288.

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 present immunotherapeutic uses of the peptides according to the 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.288, preferably SEQ ID No.1 to SEQ ID No.126, or a variant amino acid sequence.

The invention further relates to activated T cells made by the methods of the invention, wherein the T cells selectively recognize 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, activated 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 suitable for use in a soluble TCR-or antibody-based cell therapy drug, vaccine or protein.

The invention further relates to a use according to the invention, wherein the cancer cell is an HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC and CLL cell.

The invention further relates to a biomarker, herein referred to as "target", based on a peptide of the invention, which can be used for the diagnosis of cancer, preferably HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC and CLL. 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.

CABYR encodes a protein located in the major flagellar segment of fiber sheath-related sperm that exhibits calcium binding when phosphorylated during capacitation (RefSeq, 2002). Knockdown of CABYR isoforms CABYR-a and CABYR-b in non-small cell lung cancer cell lines NCI-H460 and A549 was shown to result in inhibition of proliferation and attenuation of constitutively active Akt phosphate (Qian et al, 2014). Silencing of CABYR expression was shown to affect downstream components of the Akt pathway, such as phosphorylated GSK-3 β and p53 and p27 proteins (Qian et al, 2014). In addition, knockout of CABYR has been shown to significantly increase sensitivity in response to chemotherapy drugs and drug-induced apoptosis in vitro and in vivo, and thus may be a new approach to improve lung cancer apoptotic responses and chemosensitivity (Qian et al, 2014). CABYR is described as an initial testis-specific protein, which was subsequently shown to be present in brain tumors, pancreatic and lung cancers (Hsu et al, 2005; Luo et al, 2007; Li et al, 2012). CABYR was demonstrated to be upregulated in hepatocellular carcinoma, possibly serving as an oncogenic role in liver cancer formation and its progression (Li et al, 2012). COL6a3 encodes one of the three alpha chains of collagen, type VI, alpha 3, collagen type VI, which is a beaded silk collagen found in most connective tissues and is important for the stromal component tissue (RefSeq, 2002). COL6A3 encodes the alpha-3 chain of collagen type VI, a beaded silk collagen found in most connective tissues, and plays an important role in the organization of the stromal component (RefSeq, 2002). COL6a3 was alternatively spliced in colon, bladder and prostate cancers. The long isoform of COL6a3 is almost exclusively expressed in cancer samples and could potentially serve as a new cancer marker (Thorsen et al, 2008). COL6a3 was highly expressed in pancreatic ductal adenocarcinoma tissue and underwent tumor-specific alternative splicing (Kang et al, 2014). COL6a3 has been shown to be associated with high grade ovarian cancer and contributes to the development of cisplatin resistance. COL6a3 was observed to be frequently overexpressed in gastric cancer tissues (Xie et al, 2014). The COL6a3 mutation could clearly predict a better overall survival rate in colorectal cancer patients, independent of tumor differentiation and TNM staging (Yu et al, 2015). COL6a3 expression was reported to be increased in pancreatic, colon, gastric, mucoepidermoid, and ovarian cancers. Cancer-associated transcript variants including exons 3, 4 and 6 were detected in colon, bladder, prostate and pancreatic cancers (aracat et al, 2011; Smith et al, 2009; Yang et al, 2007; Xie et al, 2014; Leivo et al, 2005; Sherman-Baust et al, 2003; Gardina et al, 2006; Thorsen et al, 2008). In ovarian cancer, COL6A3 levels were associated with higher tumor grade, and in pancreatic cancer, COL6A3 was shown to present a suitable diagnostic serum biomarker (Sherman-Baust et al, 2003; Kang et al, 2014).

CXorf61, also known as CT83, encodes cancer/testis antigen 83, located on chromosome Xq23 (RefSeq, 2002). Expression of CXorf61 has been described in different cancer types, including breast and lung cancer (Yao et al, 2014; Hanagiri et al, 2013; Baba et al, 2013). CXorf61 was shown to be an immunogenic cancer-testis antigen for lung cancer. It may therefore represent a promising candidate for anti-tumor immunotherapy (Fukuyama et al, 2006).

CYP4Z1 encodes a member of the cytochrome P450 superfamily of enzymes. Cytochrome P450 proteins are monooxygenases that catalyze many reactions involving drug metabolism and synthesis of cholesterol, steroids, and other lipids (RefSeq, 2002). In breast cancer, CYP4Z1 overexpression is associated with higher tumor grade and poorer prognosis. Functionally, CYP4Z1 promotes tumor angiogenesis and growth of breast cancer in part through PI3/Akt and ERK1/2 signaling (Yu et al, 2012; Murray et al, 2010). In addition, CYP4Z1 was described to play a role in the progression of non-small cell lung cancer (Bankovic et al, 2010). CYP4Z1 has been identified as an independent predictive marker in prostate and ovarian cancers (Transkyy et al, 2012; Downie et al, 2005). CYP4Z2P is a pseudogene located on chromosome 1p33 (RefSeq, 2002).

DCAF4L2 encodes DDB1 and CUL4 related factor 4-like 2. The specific function of this protein has yet to be elucidated; however, the DCAF4L2 gene was shown to be involved in disc morphology and cleft lip development (Springelkamp et al, 2015; Beaty et al, 2013).

ESR1 encodes an estrogen receptor, a ligand-activated transcription factor, important for hormone binding, DNA binding and transcriptional activation, and essential for sexual development and reproductive function (RefSeq, 2002). Mutations and single nucleotide polymorphisms of ESR1 are associated with risk for different cancer types, including liver, prostate, gall bladder, and breast cancer. Upregulation of ESR1 expression was associated with cell proliferation and tumor growth, however, patients with ESR 1-positive tumors experienced better overall survival due to successful treatment with selective estrogen receptor modulators (Sun et al, 2015; Hayashi et al, 2003; Bogush et al, 2009; Miyoshi et al, 2010; Xu et al, 2011; Yakimchuk et al, 2013; Fuqua et al, 2014). ESR1 signaling interferes with different pathways responsible for cell transformation, growth and survival, such as the EGFR/IGFR, PI3K/Akt/mTOR, p53, HER2, NF κ B and TGF- β pathways (Frasor et al, 2015; Band and Laiho, 2011; Berger et al, 2013; Skandalis et al, 2014; Mehta and Tripathy, 2014; Ciruelos Gil, 2014).

FMN1 encodes formazan 1, a protein that plays a role in adhesion knot formation and linear actin cord polymerization (RefSeq, 2002). Single nucleotide polymorphisms of FMN1 are associated with increased risk of prostate cancer (lisitskaria et al, 2010).

HAVCR1, also known as hepatitis A virus cell receptor 1 or KIM-1, encodes a membrane receptor protein of human hepatitis A virus and TIMD4, and is likely involved in the stabilization of asthma and allergic diseases (RefSeq, 2002). HAVCR1 has been described as a new candidate biomarker associated with clear cell carcinoma of the ovary and renal cell carcinoma (Bonventre, 2014; Kobayashi et al, 2015). HAVCR1 was shown to activate the IL-6/STAT-3/HIF-1A axis in clear cell renal cell carcinoma-derived cell lines and to determine tumor progression and patient outcome (Cuadros et al, 2014). Constitutive expression of HAVCR1 in the kidney is described as a potentially sensitive feature of clear cell renal cell carcinogenesis (Cuadros et al, 2013). Furthermore, enhanced shedding of the HAVCR1 ectodomain was demonstrated to promote an invasive phenotype in vitro and a more invasive tumor in vivo (Cuadros et al, 2013). HAVCR1 is described as being up-regulated in kidney, ovarian clear cell, and colorectal cancers (Wang et al, 2013 b). HAVCR1 upregulation is described as a potential diagnostic biomarker for colorectal cancer, a prognostic marker for long disease-free intervals after surgery (and may also be involved in the colorectal cancer metastasis cascade) (Wang et al, 2013 b). HAVCR1 was shown to be associated with T-cell large granular lymphocytic leukemia (Wlodarski et al, 2008).

HORMAD1 (also known as CT46) encodes a HORMA domain-containing protein that may play a role in meiosis. The HORMA domain is involved in chromatin binding and cell cycle regulation (RefSeq, 2002). HORMAD1 is a cancer/testis antigen that is overexpressed in different cancer types, including breast, gastric and ovarian cancers, and thus is a potential biomarker and immunotherapeutic target (Yao et al, 2014; Shahzad et al, 2013; Chen et al, 2005; ang et al, 2006; Adelaide et al, 2007). Down-regulation of HORMAD1 resulted in decreased infiltration, migration and tumor weight, as well as decreased VEGF protein levels (Shahzad et al, 2013).

HSF2BP encodes an HSF2 binding protein associated with HSF2, and may be involved in regulating HSF2 activation (RefSeq, 2002).

HSF4 encodes heat shock transcription factor 4, which activates the heat shock response gene under heat or other stress conditions (RefSeq, 2002). HSF4 was shown to be down-regulated in glioblastoma (Mustafa et al, 2010).

HTR3A encodes a 5-hydroxytryptamine (serotonin) receptor belonging to the ligand-gated ion channel receptor superfamily, which leads to a rapid depolarization response of neurons upon activation (RefSeq, 2002). HTR3A (also known as 5-HT3) is deregulated in several cancer types, e.g., down-regulated in mantle cell lymphoma, differentially expressed in different B cell tumors, and reduced in breast cancer cell lines (Pai et al, 2009; Rinaldi et al, 2010; Ek et al, 2002).

IGF2BP1, also known as CRD-BP, encodes a member of the insulin-like growth factor 2mRNA binding protein family that functions by binding to mrnas of certain genes and regulating their translation (RefSeq, 2002). Two members of the IGF 2mRNA binding protein family, including IGF2BP1, are described as true oncofetal proteins, are synthesized de novo in various human cancers, and are likely to be powerful post-transcriptional oncogenes of tumor growth, drug resistance, and metastasis (Lederer et al, 2014). Expression of IGF2BP1 is reported to be associated with overall poor prognosis and metastasis in various human cancers (Lederer et al, 2014). Thus, IGF2BP1 is considered to be a potent biomarker and a candidate target for cancer therapy (Lederer et al, 2014). IGF2BP family members are described as being highly correlated with cancer metastasis and expression of oncogenic factors such as KRAS, MYC, and MDR1 (Bell et al, 2013). IGF2BP1 was shown to interact with C-MYC and was found to be expressed in the vast majority of colon and breast tumors and sarcomas, as well as benign tumors such as breast fibroadenoma and meningioma (ioanidis et al, 2003). IGF2BP1 was shown to be upregulated in hepatocellular carcinoma and basal cell carcinoma (Noubissi et al, 2014; Zhang et al, 2015 a). Upregulation of IGF2BP1 and other genes proved to be significantly associated with poor prognosis after hepatocellular carcinoma surgery (Zhang et al, 2015 a). IGF2BP1 was shown to be a target for miR-9 and miR-372 tumor suppressor in hepatocellular carcinoma and renal cell carcinoma, respectively (Huang et al, 2015; Zhang et al, 2015 a). A decrease in the matrix IGF2BP1 was demonstrated to promote the tumorigenic microenvironment in the colon, suggesting that IGF2BP1 plays a tumor-inhibiting role in colon stromal cells (Hamilton et al, 2015). IGF2BP1 was shown to be associated with stage 4 tumors, decreased patient survival, and neuroblastoma MYCN gene amplification, and thus may be a potential oncogene and an independent negative prognostic factor for neuroblastoma (Bell et al, 2015). IGF2BP1 is described as a direct target of the WNT/β -catenin signaling pathway that regulates GLI1 expression and activity in the development of basal cell carcinoma (noubishi et al, 2014).

IGF2BP3 encodes insulin-like growth factor II mRNA binding protein 3, a oncofetal protein, that suppresses translation of insulin-like growth factor II (RefSeq, 2002). Several studies have shown that IGF2BP3 plays a role in various important aspects of cellular function, such as cellular polarization, migration, morphology, metabolism, proliferation, and differentiation. In vitro studies have shown that IGF2BP3 promotes proliferation, adhesion and invasion of tumor cells. Furthermore, IGF2BP3 has been shown to be associated with aggressive and advanced cancers (Bell et al, 2013; Gong et al, 2014). Overexpression of IGF2BP3 has been described in many tumor types and is associated with poor prognosis, high tumor stage, and metastasis, for example, in neuroblastoma, colorectal cancer, intrahepatic bile duct cancer, hepatocellular carcinoma, prostate cancer, and renal cell carcinoma (Bell et al, 2013; Findeis-Hosey and Xu, 2012; Hu et al, 2014; Szarvas et al, 2014; Jeng et al, 2009; Chen et al, 2011; Chen et al, 2013; Hoffmann et al, 2008; Lin et al, 2013; Yuan et al, 2009).

MAGEA3 encodes the melanoma-associated antigen family member A3. MAGEA3 is broadly known as a cancer-testis antigen (RefSeq, 2002; Pineda et al, 2015; De et al, 1994). MAGEA3 has long been known for use in therapeutic vaccination trials for metastatic melanoma cancer. Transdermal peptide immunization with MAGEA3 and 4 other antigens of advanced melanoma patients currently has been shown to significantly contribute to the extension of overall survival of full responders compared to incomplete responders (Coulie et al, 2002; Fujiyama et al, 2014). In NSCLC, MAGEA3 was shown to be frequently expressed. Expression of MAGEA3 was associated with a higher number of tumor necroses in NSCLC tissue samples and was shown to inhibit proliferation and invasion and promote apoptosis in lung cancer cell lines. For adenocarcinoma patients, expression of MAGEA3 correlated with better survival. Currently, whole cell anti-MAGEA 3 vaccines are being studied in a promising phase III clinical trial for the treatment of NSCLC (Perez et al, 2011; Reck, 2012; Hall et al, 2013; Grah et al, 2014; Liu et al, 2015). MAGEA3 was shown to be frequently expressed in HCC along with the other 4 genes. Expression of those genes correlates with circulating tumor cell numbers, high tumor grade, and later stages in HCC patients. The frequency of liver metastases proved to be significantly higher in the case of tumor samples expressing MAGE3 than in those not expressing the gene (Bahnassy et al, 2014; Hasegawa et al, 1998). Cancer stem-like side population cells isolated from bladder cancer cell lines as well as lung, colon or breast cancer cell lines were shown to express MAGEA3 among other cancer-testis antigens. In general, cancer stem cells are known to develop resistance to current cancer treatments and to lead to cancer recurrence and progression after treatment. Accordingly, MAGEA3 can be a new target for immunotherapy, particularly for the treatment of bladder cancer (Yamada et al, 2013; Yin et al, 2014). In head and neck squamous cell carcinoma, expression of MAGEA3 was shown to be associated with better disease-free survival (Zamuner et al, 2015). In addition, MAGEA3 can be used as a prognostic marker for ovarian cancer (Szajnik et al, 2013).

MAGEA4, also known as MAGE4, encodes a member of the MAGEA gene family, located on chromosome Xq28 (RefSeq, 2002). MAGEA4 is described as a cancer testis antigen that is found expressed in a small fraction of classical seminomas, but not in non-seminal germ cell tumors, in breast, Hodgkin lymphoma EB virus negative cases, esophageal, lung, bladder, head and neck, colorectal, oral squamous cell, and hepatocellular carcinomas (Ries et al, 2005; Bode et al, 2014; Li et al, 2005; Ottagiani et al, 2006; Hennard et al, 2006; Chen et al, 2003). MAGEA4 was shown to be frequently expressed in primary mucosal melanoma in the head and neck and therefore may be a potential target for cancer testis antigen-based immunotherapy (Prasad et al, 2004). MAGEA4 was shown to be preferentially expressed in cancer stem-like cells from LHK2 lung adenocarcinoma cells, SW480 colon adenocarcinoma cells, and MCF7 breast cancer cells (Yamada et al, 2013). Overexpression of MAGEA4 in spontaneously transformed normal oral keratin indicates that growth can be promoted by preventing cell cycle arrest and by inhibiting p53 transcription target BAX and CDKN1A mediated apoptosis (Bhan et al, 2012). MAGEA4 was shown to be expressed more frequently in patients with cirrhosis and advanced hepatocellular carcinoma than in patients with early hepatocellular carcinoma infected with hepatitis c virus, making detection of MAGEA4 transcript potentially useful for predicting prognosis (hussei et al, 2012). MAGEA4 was demonstrated to be one of several cancer/testis antigens, which are expressed in lung cancer and could be potential candidate antigens for multivalent immunotherapy in lung cancer patients (Kim et al, 2012). MAGEA4 is described as being upregulated in esophageal and hepatocellular carcinoma (ZHao et al, 2002; Wu et al, 2011). The MAGEA 4-derived native peptide analogue designated p286-1Y2L9L was described as a new candidate epitope suitable for use in the development of a vaccine against esophageal cancer peptides (Wu et al, 2011). Several members of the MAGE gene family, including MAGEA4, have been shown to be frequently mutated in melanoma (Caballero et al, 2010).

MAGEA6 encodes the melanoma-associated antigen family member a 6. MAGEA3 is broadly known as a cancer-testis antigen (RefSeq, 2002; Pineda et al, 2015; De et al, 1994). MAGEA6 was shown to be frequently expressed in melanoma, advanced myeloma, rhabdomyosarcoma of children, sarcoma, lung cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, squamous cell carcinoma of the head and neck, esophageal squamous cell carcinoma, oral squamous cell carcinoma (Ries et al, 2005; Hasegawa et al, 1998; Gibbs et al, 2000; Dalerba et al, 2001; Otte et al, 2001; van der Bruggen et al, 2002; Lin et al, 2004; Tanaka et al, 1997). MAGEA6 expression is associated with shorter progression-free survival in multiple myeloma patients. In comparison, expression of MAGEA6 has been shown to correlate with better disease-free survival in head and neck squamous cell carcinomas (van et al, 2011; Zamuner et al, 2015). MAGEA6 is a member of a group of genes that are overexpressed in paclitaxel-resistant ovarian cancer cell lines. In addition, MAGEA6 transfection also increased resistance to paclitaxel-sensitive cells (Duan et al, 2003). MAGEA6 can be used as a prognostic marker for ovarian cancer (Szajnik et al, 2013). Cancer stem-like side population cells isolated from lung, colon or breast cancer cell lines were shown to express MAGEA6 among other cancer-testis antigens (Yamada et al, 2013).

MAGEA9, also known as MAGE9 or MAGE-A9, encodes a member of the MAGEA gene family, located on chromosome Xq28 (RefSeq, 2002). High expression of MAGEA9 in non-small cell lung cancer tumors and stromal cells correlates with poor survival (Zhang et al, 2015 b). MAGEA9 expression is described as an independent prognostic factor for five-year survival in non-small cell lung cancer patients (Zhang et al, 2015 b). The presence of MAGEA9 in newly diagnosed cases of multiple myeloma has been shown to be associated with a shorter overall survival (van et al, 2011). MAGEA9 is described as a renal cell carcinoma antigen, the use of which in BALB/c mouse dendritic cell vaccination has been shown to result in rejection of low-dose RENCA-MAGEA9 renal cell carcinoma transplants (Herbert et al, 2010). The MAGEA9 peptide-specific cytotoxic T lymphocyte cell line was shown to exhibit high cytotoxic activity against peptide-loaded T2 cells and the native MAGEA 9-expressing renal cell carcinoma cell line, making MAGEA9 a potentially suitable target for immunotherapy of renal cell carcinoma (Oehlrich et al, 2005). MAGEA9 was shown to be one of the most commonly expressed cancer testis antigens in uterine cancer (Risinger et al, 2007). MAGEA9 is described as a member of the MAGE family, expressed in testicular cancer (Zhan et al, 2015). High expression of MAGEA9 was shown to be associated with venous invasion and lymph node metastasis of colorectal cancer (Zhan et al, 2015). MAGEA9 expression was shown to correlate with lower survival of colorectal cancer, and high expression of MAGEA9 was described as a poor prognostic factor in patients with colorectal cancer (Zhan et al, 2015). Therefore, MAGEA9 is expected to be a new target for colorectal cancer treatment (Zhan et al, 2015). MAGEA9 overexpression has been shown to be predictive of a poor prognosis in epithelial ovarian, breast invasive ductal, laryngeal squamous cell, and hepatocellular carcinomas (Gu et al, 2014; Han et al, 2014; Xu et al, 2015). MAGEA9 was shown to be upregulated in laryngeal squamous cell carcinoma, breast invasive ductal carcinoma, epithelial ovarian carcinoma, colon carcinoma and hepatocellular carcinoma (Gu et al, 2014; Han et al, 2014; Xu et al, 2015; Zhan et al, 2015).

MAGEA9B encodes a replicate of MAGEA9 protein on chromosome X. Expression of MAGEA9B in non-small cell lung cancer stage Ib in tumor stage is correlated with patient survival (Urgard et al, 2011).

MMP1 encodes a member of the Matrix Metalloproteinase (MMP) peptidase M10 family. Proteins of this family are involved in the destruction of the extracellular matrix in normal physiological processes (e.g., embryonic development, reproductive and tissue remodeling) as well as in disease processes (e.g., arthritis and metastasis) (RefSeq, 2002). A number of authors have demonstrated a positive correlation between MMP expression patterns and tumor invasion and metastasis, including: rectal cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, thyroid cancer, and brain tumors (Velinov et al, 2010). MMP1 was identified as a biomarker for laryngeal squamous cell carcinoma, with tumor-stage-associated expression (Hui et al, 2015). Breast cancer patients with circulating tumor cells in peripheral blood with epithelial-mesenchymal transition (CTC _ EMT) had significantly increased expression of MMP1 in tumor cells (p 0.02) and tumor-associated matrix (p 0.05) compared to patients without CTC _ EMT (ciern et al, 2014). In a mouse model, expression and secretion of MMP1 was blocked by a specific anti-FGFR 3 monoclonal antibody, which substantially prevented tumor progression (Du et al, 2014).

Proteins of the Matrix Metalloprotease (MMP) family are involved in the destruction of the extracellular matrix in normal physiological processes such as embryonic development, reproductive and tissue remodeling, as well as in disease processes such as arthritis and metastasis. However, the enzyme encoded by this gene is activated intracellularly via furin in the constitutive secretory pathway. In addition, this enzyme cleaves α 1-protease inhibitors but is weak at degrading structural proteins of the extracellular matrix relative to other MMPs (RefSeq, 2002). MMP-11, also known as matrilysin-3, is a member of the matrilysin subgroup belonging to the protease superfamily, which has been detected in cancer cells, stromal cells and adjacent microenvironments. In contrast, MMP-11 has a dual role in tumors. In one aspect, MMP-11 promotes cancer progression by inhibiting apoptosis and enhancing cancer cell migration and invasion; on the other hand, MMP-11 plays a negative role in cancer development by inhibiting metastasis in animal models. MMP-11 was found to be overexpressed in the sera of cancer patients compared to normal controls, and also in various tumor tissue samples (e.g., gastric, breast, and pancreatic cancers) (Zhang et al, 2016). MMP-11 was demonstrated to be overexpressed at the mRNA and protein levels in CRC tissues compared to the corresponding normal mucosa. In addition, MMP-11 expression was associated with CRC lymph node metastasis, distant metastasis and TNM staging (Tian et al, 2015). MMP-11 overexpression has been associated with aggressive tumor phenotype and poor clinical outcome in upper urothelial cell carcinoma (UTUC) and urothelial cell carcinoma of the bladder (UBUC), suggesting that it may be a new prognostic and therapeutic target (Li et al, 2016).

MXRA5 encodes a matrix remodeling-related protein comprising 7 leucine-rich repeats associated with perlecan and 12 immunoglobulin-like C2-type domains (RefSeq, 2002). A chinese study determined MXRA5 to be the second most common mutant gene in non-small cell lung cancer (Xiong et al, 2012). In colon cancer, MXRA5 was shown to be overexpressed and likely to be a biomarker for early diagnosis and retinal metastasis (Zou et al, 2002; Wang et al, 2013 a).

RAD54 encodes a protein belonging to the DEAD-like helicase family. Saccharomyces cerevisiae RAD54 and RDH54 share similarities, both of which are involved in DNA homologous recombination and repair. The protein binds to double-stranded DNA and exhibits atpase activity in the presence of DNA. The gene is highly expressed in testis and spleen, which suggests an active role in meiotic and mitotic recombination (RefSeq, 2002). Homozygous mutations in RAD54B were observed in primary lymphomas and colon cancer (Hiramoto et al, 1999). RAD54B negates the genomic instability effect of RAD51 binding directly to dsDNA in human tumor cells (Mason et al, 2015).

ZFP42 (also known as REX1) encodes a zinc finger protein that acts as a stem cell marker and is critical for pluripotency and reprogramming (Son et al, 2013; Mongan et al, 2006). ZFP42 expression is down-regulated in prostate and renal cell carcinomas, but in contrast is up-regulated in squamous cell carcinomas (Raman et al, 2006; Lee et al, 2010; Reinisch et al, 2011). Modulation of ZFP42 inhibits the JAK/STAT signaling pathway via modulation of SOCS3 expression, thereby modulating cell differentiation (Xu et al, 2008).

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 indicate that these cells are in cancerPlays an important role in natural immune defense. In particular, CD8 positive T cells play an important role in this response, TCD8⁺Class I molecules contained in peptides carried by the Major Histocompatibility Complex (MHC) that recognize 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 activation 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, 12 or 13 amino acids in length or longer, and in the case of MHC class II peptides (elongate variants of the peptides of the invention) may be 14, 15, 16, 17, 18, 19 or 20 amino acids in length or longer.

Thus, 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 alleles that can be expressed from these gene sites.

Table 1: HLA-A02 and HLA-A24 and the frequency of expression F of the most common HLA-DR serotypes. Frequency according to Hardy-Weinberg formula F ═ 1- (1-G) used by Mori et al (Mori et al, 1997)_f)2 adapted and deduced from haplotype frequencies within the population of the united states. Due to linkage disequilibrium, a 02 or a 24 combinations within certain HLA-DR alleles may be concentrated or less frequent than their expected single frequency. For details, please refer to Chanock et al (Chanock)et al.,2004)。

The peptides of the invention, preferably when incorporated into the vaccines of the invention as described herein, bind to a x 02. 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 x 02, 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 (e.g., 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).

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 that will express the sequence 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" refers to a short nucleic acid sequence that can pair with a DNA strand and provide a free 3' -OH terminus 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 by conventional methods to have an electrophoretic isotype. 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% or more by weight.

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 NO 1 to 288, or a variant thereof having 88% homology to SEQ ID NO 1 to 288, 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 the 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 the 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: 288). 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 an activated T cell.

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 databases (Rammensee et al, 1999; Godkin et al, 1997), certain sites of HLA-A binding peptides are usually anchor residues, which form a core sequence commensurate with the binding motif of HLA-binding grooves, defined by the polarity, electrophysical, hydrophobic and steric properties of the polypeptide chains that make up the binding grooves. Thus, one skilled in the art can modify the amino acid sequences set forth in SEQ ID No. 1 through SEQ ID No. 288 by maintaining known anchor residues and can 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 activated 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 correlations between the size, charge, polarity and hydrophobicity of the original amino acid, which underlies the identification 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, gin); 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 even 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, the combination of substitutions is tested to determine whether the combined substitutions produce a 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 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 2: peptide variants and motifs according to SEQ ID NO 4, 13 and 15.

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 3.

Table 3: 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 herein may be identical to a native tumor-associated epitope or tumor-specific epitope, and may also include different peptides of no more than four residues from a reference peptide, so long as 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. 10, 11, 12, 13, 14 amino acids, and if a class II binding peptide is elongated, 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 1 nM, 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 according to SEQ ID NO 1 to SEQ ID NO 288.

Substantially consisting of means that the peptides of the invention, in addition to consisting of a sequence according to any one of SEQ ID No. 1 to SEQ ID No. 288 or a variant 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 mimetics 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 bond being-CH₂-NH、-CH₂S-、-CH₂CH₂-、-CH＝CH-、-COCH₂-、-CH(OH)CH₂-and-CH₂SO-, and the like. The U.S. Pat. No. 4897445 proposes polypeptidesNon-peptide bonds (-CH) in the chain₂-NH) which involves the synthesis of a polypeptide according to standard procedures and which permeates the aminoaldehyde and a NaCNBH₃The amino acids of (a) interact to synthesize non-peptide bonds.

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" by r.lundblad (3rd ed. crc Press,2004) (Lundblad,2004), 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, lysine pyridoxylation, reductive alkylation, trinitrophenylation of amino groups with 2,4, 6-trinitrobenzenesulfonic 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 maleimide, 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).

Briefly, 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-alkenylhexanedione) 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) etc. contains information on the 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 -amino groups, for example, facilitates peptide binding to the surface or cross-linking of proteins/peptides. 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, or the like.

Tetranitromethane and N-acetylimidazole can 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 results in prolonged circulation half-lives when crosslinking 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 carbamylation of potassium cyanate.

A peptide or variant wherein the peptide is modified or contains non-peptide bonds, preferably an embodiment of the invention. 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 (Lukas et al, 1981) and the references cited therein. The fluorenylmethyloxycarbonyl (Fmoc) group provides temporary protection for the N-amino group. The cleavage was repeated using the highly base-sensitive protecting group in 20% dimethylpiperidine 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), t-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 a 4,4' -dimethoxydiphenyl group. The solid support is based on a polydimethylacrylamide polymer, which is composed 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 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 use solid-phase and liquid-phase methods in combination for the synthesis of peptides (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.

In order to identify the peptides of the present invention, an RNA expression database (Lonsdale,2013) of about 3000 normal tissue samples publicly available was screened for genes that were nearly null expressed in important organ systems and low expressed in other important organ systems. In a second step, cancer-associated peptides derived from the protein products of these genes are obtained by using XPRESIDENT as described herein ^TMPlatform mass spectrometry identification.

In detail, for the selection of relevant genes using RNASeq data from the database, the important organ systems considered are: brain, heart, blood vessels, lung and liver. For gene selection, the median per million reads per kilobase Read (RPKM) for vital organs is required to be less than 2, and the 75% percentile is required to be less than 5 RPKM. If the organ system is covered by multiple sample types, e.g., different brain regions analyzed separately, the maximum median and maximum 75% percentiles of the multiple sample types are used for the calculation. Other important organ systems considered are: skin, nerves, pituitary, colon, kidney, adipose tissue, adrenal gland, bladder, whole blood, esophagus, muscle, pancreas, salivary gland, small intestine, stomach, breast, spleen, thyroid. The maximum median RPKM requirement for these organs is less than 10 when selecting genes. Other organs are considered unimportant and therefore no cut-off for gene expression is applied. These organs are the cervix and uterus, fallopian tubes, vagina, prostate, testis, and ovary. Using this screening method, approximately 14,000 candidate genes were selected. Subsequently, the presentation characteristics of the peptides derived from the corresponding proteins were analyzed. A peptide is considered to be a related peptide if it is presented in less than five normal samples out of a group of more than 170 normal (i.e. non-cancerous) samples analyzed, and if normal tissue presents up to 30% below the median tumor signal (all tumor samples).

To select for over-presented peptides, a presentation graph is calculated showing the amount of bit-present as well as the variation in replication in the sample. This feature allows the baseline values of the relevant tumor entity samples to be aligned with those of normal tissue samples. Each of the above features can be incorporated into the overpresentation score by calculating the p-value that adjusts the linear mixture effect model (Pinheiro et al, 2015), thereby adjusting multiple tests through false discovery rates (Benjamini and Hochberg, 1995).

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 sequence thus generated was verified by comparing the pattern of fragments of native TUMAP recorded in a sample of the primary tumor with the pattern of fragments of the corresponding synthetic reference peptide 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.

The number of samples was (all/sample through QC): PC N ═ 39(36), RCC N ═ 22(18), CRC N ═ 31(28), esophageal cancer N ═ 14(11), BPH and prostate cancer N ═ 53(43), HCC N ═ 15(15), NSCLC N ═ 96(87), GC N ═ 35(33), GB N ═ 38(27), breast cancer N ═ 2(2), melanoma N ═ 5(2), ovarian cancer N ═ 21(20), CLL N ═ 5(4), SCLC N ═ 18(17), NHL N ═ 18 (18), AML N ═ 23(18), ovarian cancer N ═ 21(20), CLL N ═ 5(4), SCLC N ═ 18(17), UEC (15), UEC ═ 19 (16). If 5 replicates of the mass spectrum are collected or the sample is completely consumed, then through QC, the peptides used to calculate the normalization factor (i.e., occurring in the same sample technique replicate with less than 50% variation, and occurring in 2 independent samples) are at least 30% of all the peptides measured in the sample. Samples with few subtype samples (e.g., A.about.02: 05, A.about.02: 06) after subtype typing were excluded from the selection of the peptides of the present invention.

Discovering a pipelinev2.1 (see, e.g., US 2013-. This is achieved by the following method: the label-free differential quantification method is developed using LC-MS acquisition data processed by proprietary data analysis pipelines, in combination with sequence recognition algorithms, spectral clustering, calculating ions, retention time adjustment, state of charge convolution and normalization.

Levels of presentation were established for each peptide and sample, including error estimates. Peptides that are abundantly presented in tumor tissues and peptides that are excessively presented in tumor and non-tumor tissues and organs have been identified.

HLA peptide complexes from primary HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC and CLL samples were purified and HLA-related peptides were isolated and analyzed using LC-MS (see examples). All TUMAPs encompassed by the present application were identified using methods for HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, and CLL samples to confirm their presentation on these tumors.

TUMAP determined on multiple tumors and normal tissues was quantified using an ion counting method of unlabeled LC-MS data. This method assumes that the LC-MS signal region of the peptide correlates with its abundance in the sample. All quantitative signals for peptides in various LC-MS experiments were normalized on a central trend basis, averaged per sample, and combined into a histogram (referred to as a presentation graph). The presentation graph integrates different analytical methods, such as: protein database retrieval, spectral clustering, state of charge convolution (neutralization) and retention time calibration and normalization.

In addition, a pipeline is foundX direct absolute quantification of MHC-peptides (preferably HLA restricted peptides) on cancer or other infected tissues. Briefly, the total cell count is calculated from the total DNA content of the tissue sample being analyzed. The total peptide amount of TUMAP in tissue samples was determined by nanoLC-MS/MS as the ratio of native TUMAP and known amount of isotopically labeled version of TUMAP, referred to as internal standard. TUMAP separation efficiency determination method: the peptide: MHC for all selected TUMAPs were added to tissue lysates at the earliest time point of the TUMAP isolation procedure and detected by nanoLC-MS/MS after completion of peptide isolation. Total cell counts and total peptide amounts were calculated from three measurements per tissue sample. The peptide-specific isolation efficiency was calculated as the average of 10 spiking experiments measured in triplicate (see example 6 and table 11).

This combined analysis of RNA expression and mass spectrometry data yielded 288 peptides of the invention. In many cases, the peptide is found on only a small number of tumors. However, due to the limited sensitivity of conventional mass spectrometry, RNA data provides a better basis for range assessment (see example 2). The invention proposes HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC and CLL in favor of the treatment of cancer/tumor, preferably presenting the peptide of the invention in excess or only. These peptides are directly revealed by mass spectrometry, whereas HLA molecules are naturally presented in human primary human HCC, CRC, GB, GC, esophageal cancer, NSCLC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC and CLL samples and/or PC samples.

Many of the genes/proteins of origin (also designated as "full-length proteins" or "potential proteins") of highly overexpressed peptides in cancer as compared to normal tissues-the "normal tissues" to which the invention relates are healthy tissues of the corresponding type of tumor (liver, colon/rectum, brain, stomach, esophagus, lung, pancreas, kidney, prostate, ovary, skin, breast and leukocytes) or other normal tissue cells, indicating a high association of the tumor with these genes of origin (see example 2). Furthermore, these peptides are also over-presented themselves in tumor tissue ("tumor tissue" in connection with the present invention means a sample from a patient with HCC, CRC, GB, GC, esophageal carcinoma, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC or CLL), but not in normal tissue (see example 1).

HLA-binding peptides are recognized by the immune system, particularly T lymphocytes. T cells can disrupt cells presenting the recognized HLA/peptide complex (e.g., HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL 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. 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"). Peptides that bind to TCRs and antibodies when presented by MHC molecules are also provided. 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 so-called γ/δ TCRs. In one embodiment, the instructions provide 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 δ chains of γ/δ TCRs are generally considered to have two structural "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). The beta and delta chains may also include 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 a TCR γ v (trgv) region without the leader region (L) and a TCR γ (TRGJ) region, and the term TCR γ constant domain refers to an extracellular TRGC region, or a C-terminally truncated TRGC sequence, relative to the TCR of γ/δ. Similarly, the term "TCR δ variable domain" refers to the combination of TCR δ v (trdv) and TCR δ D/J (TRDD/TRDJ) regions without leader (L), and the term "TCR δ constant domain" refers to the 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 the threonine 48 of TRAC and the 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, since tumors are derived from the individual's own cells, only muteins or proteins with altered translational processing will be considered as 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 specification for a peptide according to the invention 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 monomers from HLA-a × 02 negative healthy donors, PBMCs were incubated with tetramer-Phycoerythrin (PE) and high affinity T cells were isolated by Fluorescence Activated Cell Sorting (FACS) method-Calibur 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 the relevant peptides, PBMCs obtained from the transgenic mice were incubated with tetramer-Phycoerythrin (PE), and high affinity T cells were isolated via Fluorescence Activated Cell Sorting (FACS) method-Calibur 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 a TCR of the present specification, TCR RNA 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 a viral internosomal entry site (IRES) between the TCR-alpha and TCR-beta chains results in the coordinated expression of both chains, since both the TCR-alpha and TCR-beta chains are produced from a single transcript that splits into two proteins during translation, thereby ensuring that an equal molar ratio of TCR-alpha and TCR-beta 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 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 the attainment of specificities which constitute a significant risk for 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 herein 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; generating a second gap disulfide bond in the C-terminal domain (cysteine modification) 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-in-hole"); TCR-alpha and TCR-beta chain variable domains were fused directly 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/δ 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, "pharmaceutically acceptable salt" refers to a derivative of the disclosed peptide wherein the peptide is modified by making acid or base salts of pharmaceutical agents. For example, with a free base which reacts with a suitable acid (usually where the neutral drug has a neutral-NH group)₂Group) 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, base salt formulations of acidic groups that may be presented on a peptide are prepared using pharmaceutically acceptable bases, such as 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, either systemically by i.d., i.m., s.c., i.p., and i.v. injection, or applied in vitro to cells from the patient or a cell line thereof (which are then injected into the patient), or ex vivo 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 CD 8T cells. However, CD 8T cell stimulation was more effective with the help of CD 4T-helper cells. Thus, for stimulation of the MHC class I epitope of CD 8T 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 288 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.

DNA encoding the polypeptide of the present inventionModification methodThe polymerase chain reaction method disclosed by Saiki RK et al (Saiki et al, 1988) was used. 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, CA 92037, 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 as 1 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 comprise the origin of pMB1 (a derivative of pBR 322) replication in bacterial cells, the calcium-lactamase gene for ampicillin resistance selection in bacteria, the origin of 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 No. 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. (La Jolla, CA 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 Recombinant Gene Expression, Reviews and Protocols, Part One, Second Edition, ISBN 978-1-58829. sup. 262-9) and other documents known to the person skilled in the art.

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, MD 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, allowing them to be loaded with 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, recombinant fusion proteins containing Prostatic Acid Phosphatase (PAP) were approved by the U.S. Food and Drug Administration (FDA) for treatment of asymptomatic or mildly symptomatic metastatic HRPC (Rini et al, 2006; Small et 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 are also possibleOne or more adjuvants are included. Adjuvants are those substances which nonspecifically enhance or potentiate the immune response (e.g., by CD 8-positive T cells and helper T (T) _H) Cell-mediated immune response to an antigen and is therefore considered useful for the agents of the invention. Suitable adjuvants include, but are not limited to 1018ISS, aluminium salts,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF, IC30, IC31, imiquimodresiquimod, ImuFact IMP321, interleukins IL-2, IL-13, IL-21, interferon alpha or beta, or polyethylene glycol derivatives thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs,Lipovac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, oil-in-water and water-in-oil emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, and mixtures thereof,Carrier system, polylactide-based composite glycolide [ PLG]And dextran microparticles, recombinant human lactoferrin SRL172, viral and other virus-like particles, YF-17D, VEGF trap, R848, β -glucan, Pam3Cys, QS21 stimulators from Aquila corporation derived from saponin, mycobacterial extracts, and bacterial cell wall synthesis mimics, as well as other proprietary adjuvants such as: ribi's Detox, Quil or Superfos. Preferred adjuvants are, for example: freund's adjuvant or GM-CSF. Several dendritic cell-specific immunoadjuvants (e.g. MF59) and methods for their preparation have been described (Allison and Krummel, 1995). Cytokines may also be used. Some cytokines directly affect dendritic cell migration to lymphoid tissues (e.g., TNF-), accelerating dendritic cell maturation to T lymphocytes Potent antigen-presenting cells (e.g., GM-CSF, IL-1, and IL-4) (U.S. patent No. 5849589, specifically incorporated herein by reference in its entirety), and serve as immunological adjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN- α, IFN- β) (Gabrilovich et al, 1996).

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 T_H1Enhanced activation of cells and enhanced Cytotoxic T Lymphocyte (CTL) production, even the absence of CD 4T cell indications. Maintenance of TLR9 activation induced T even in the presence of vaccine adjuvants_H1Drift, such adjuvants as: normal promotion of T_H2Shifted alum or Freund's incomplete adjuvant (IFA). 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, 6406705, 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 derivatives thereof (e.g., AmpliGen, Hiltonol, Poly- (IC) LC), poly (IC-R), poly (I: C12U)), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, such as: cyclophosphamide, sumicitizumab,Celecoxib, 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 receptor) and SC58175, which may have therapeutic and/or adjuvant effects. The skilled artisan can readily determine the amounts and concentrations of adjuvants and additives useful in the present invention without undue experimentation.

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 IMS 1312, Montanide ISA 206, Montanide ISA 50V, 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 activating 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 stent may include a label that is detectable by the signal provided by the tag to determine the presence or absence of the bound stent. For example, the scaffold may be labeled with a fluorescent dye or any other suitable cellular marker molecule. Such marker molecules are well known in the art. For example, fluorescent labeling with a fluorescent dye can provide visualization of the bound aptamer through 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, for example, 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 signature properties of various cancer cells, particularly those from solid tumors, while non-tumorigenic and predominantly healthy cells are not recognized. 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 NO 1 to SEQ ID NO 288 using the cellular SELEX (systematic evolution of ligands by 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 generating 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 molecule 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; 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.

Another aspect of the invention provides an antibody that specifically binds to a 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 tools 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 is also considered to be "specific" in the context of the present invention.

The present invention relates to a peptide comprising a sequence selected from the group of SEQ ID NO 1 to SEQ ID NO 288 or a variant thereof having 88% homology (preferably the same) to SEQ ID NO 1 to SEQ ID NO 288 or a variant thereof which induces T-cell cross-reactivity with said variant peptide, wherein said peptide is not an essentially full-length polypeptide.

The invention further relates to a peptide comprising a sequence selected from the group of SEQ ID NO:1 to SEQ ID NO:288 or a variant having at least 88% homology (preferably identical) to SEQ ID NO:1 to SEQ ID NO:288, 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 consists essentially of an amino acid sequence according to SEQ ID NO 1 to SEQ ID NO 288.

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 HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC or CLL.

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 NO 1 to SEQ ID NO 288 or said variant amino acid sequence.

The invention further relates to activated T cells made by the methods of the invention, wherein the T cells selectively recognize 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 of the invention, an expression vector of the invention, a cell of the invention, a use of the invention for activating cytotoxic T lymphocytes as a medicament or in 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 an HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC or CLL cell.

The invention further relates to a specific marker protein and biomarker, herein referred to as "target", based on the peptides of the invention, which can be used for diagnosing and/or determining the prognosis of HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC or CLL. 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 desired attributes of the invention (e.g., specific binding of HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL marker (poly) peptides, HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL cells and/or inhibit HCC, CRC, esophageal GB, PC, NSCLC, HCC, CLL cancer, and/or CLL cells at increased levels of expression of the marker gene, Activity of RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL marker 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 HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL marker polypeptides, or fragments thereof, can be used to prepare an antibody 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 SEQ ID NO:1 to SEQ ID NO:288, or a variant or fragment thereof, may be expressed in prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast, insect, or mammalian) cells, after which the recombinant protein may be purified and used to produce a monoclonal or polyclonal antibody preparation that specifically binds to the HCC, CRC, GB, GC, esophageal carcinoma, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast carcinoma, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL marker polypeptides used to produce an antibody 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. Antibodies for therapeutic or in vivo diagnostic use are tested according to their use by known methods (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; further guidance in producing and testing antibodies is available, see, e.g., Greenfield,2014 (Greenfield, 2014.) for example, the antibodies can be detected by ELISA or immunoblotting, immunohistochemistry staining of formalin-fixed cancer tissue or frozen tissue sections.

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 to yield aF (ab') ₂Fragment and 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 an antibody, preferably to treat HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC or CLL, the efficacy of a therapeutic antibody can be assessed by various methods well known to the skilled artisan. 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 2004/033685a1 and 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 or³⁵S) so that immunoscintigraphy can localize tumors. In one embodiment, the antibody or fragment thereof binds to two or more target extracellular domains of proteins selected from the group consisting of the proteins described above, and has an affinity value (Kd) of less than 1X 10. mu.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, computed tomography, and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium andother rare earth, paramagnetic, 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 immunohistochemistry, diseased tissue samples may be fresh or frozen or may be embedded in paraffin and fixed with a preservative 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 the 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,12301 Parklawn Drive, Rockville, Maryland 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 NO 1 to SEQ ID NO 288.

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 in vitro activation of T cells by the use of 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.

Activated T cells are directed against the peptides of the invention, contributing to the treatment. Thus, in another aspect of the invention, activated T cells produced by the methods of the invention described above are provided.

Activated T cells made as described above will selectively recognize aberrantly expressed polypeptides comprising the amino acid sequences of SEQ ID NO 1 through SEQ ID NO 288.

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 activated T cells. The T cells administered to the patient may be derived from the patient and activated 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 the tissue derived from the tumor but is expressed in the tumor. By "overexpressed" is meant 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, activated 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 further proposes an agent for the treatment of cancer, in particular HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC or CLL and other malignancies.

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 HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC and CLL, the agent of the present invention is preferably used for the treatment of HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC and CLL.

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 repository (e.g., database format) consists of tumor-associated peptides that are highly overexpressed in tumor tissues of various HLA-AHLA-B and HLA-C alleles HCC, CRC, GB, GC, esophageal carcinoma, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL 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 tumor tissues, the pools may also contain HLA-a 02 and HLA-a 24 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 of the present invention and repository is identified through the use of a functional genomics approach that combines 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, patients' HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC or CLL samples and healthy donor blood were analyzed in a progressive manner:

1. genome-wide messenger ribonucleic acid (mRNA) expression analysis is used to determine genes that are expressed at very low levels in important normal (non-cancerous) tissues. These genes were evaluated for overexpression in malignant tissues (HCC, CRC, GB, GC, esophageal carcinoma, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, CLL) compared to a range of normal organs and tissues.

2. HLA ligands for malignant materials (HCC, CRC, GB, GC, esophageal carcinoma, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, CLL) were determined by mass spectrometry.

3. The determined HLA ligands are compared to gene expression data. Peptides over-or selectively presented on tumor tissue, preferably encoded by selectively expressed or over-expressed genes detected in step 2, are considered suitable candidates for a polypeptide vaccine.

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 a T cell response in vivo through a selected peptide, in vitro immunogenicity assays were performed using healthy donors and human T cells from HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC or CLL patients.

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 activation, 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 a 50 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) aligning the peptides identified in (a) with a repository (database) of such peptides; and (c) selecting at least one peptide from a repository (database) associated with tumor associated peptides identified in the patient. For example, TUMAPs presented in tumor samples can be identified by: (a1) comparing expression data from a tumor sample to expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or aberrantly expressed in 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 (database) 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 a tumor sample to expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or aberrantly expressed in 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 HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC or CLL 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 a tissue sample is malignant or inflammatory or a general lesion, and may also be used as a biomarker for HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL. 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

FIG. 1 shows the over-presentation of various peptides in different cancer tissues compared to normal tissues. The analysis included data from 170 normal tissue samples and 376 cancer samples. Only the samples found to present peptides are shown. FIG. 1A) genes: CENPE, peptide: KLQEKIQEL (a × 02:01) (SEQ ID No.:1) -tissue from left to right: 4 white cell carcinoma cell line, 1 pancreatic carcinoma cell line, 1 melanoma cell line, 2 normal tissue samples (1 adrenal gland, 1 spleen), 31 primary cancer tissue samples (1 brain cancer, 4 colon cancer, 1 esophageal cancer, 1 kidney cancer, 2 liver cancer, 16 lung cancer, 4 ovarian cancer, 1 rectal cancer, 1 stomach cancer), fig. 1B) genes: KIF15, peptide: QLIEKNWLL (a × 02:01) (SEQ ID No.:10) -tissue from left to right: 5 white blood cell carcinoma cell line, 1 pancreatic carcinoma cell line, 1 granulocytic leukemia cell line, 1 normal tissue sample (1 adrenal gland), 29 cancer tissue samples (4 colon cancer, 2 esophageal cancer, 1 white blood cell cancer, 1 liver cancer, 10 lung cancer, 11 ovarian cancer), fig. 1C) genes: HAVCR1, peptide: LLDPKTIFL (a × 02:01) (SEQ ID No.:11) -tissue from left to right: 1 renal cancer cell line, 13 cancer tissue samples (8 renal cancer, 1 liver cancer, 2 lung cancer, 2 rectal cancer), fig. 1D) genes: RPGRIP1L, peptide: RLHDENILL (a × 02:01) (SEQ ID No.:13) -tissue from left to right: 1 renal cancer cell line, 1 prostate cancer cell line, 1 melanoma cell line, 50 cancer tissue samples (4 brain cancer, 1 colon cancer, 2 esophageal cancer, 3 kidney cancer, 2 liver cancer, 23 lung cancer, 7 ovarian cancer, 2 pancreatic cancer, 2 prostate cancer, 3 rectal cancer, 1 stomach cancer), and fig. 1E-J show the over-presentation of various peptides in different cancer tissues compared to normal tissues. The analysis included data from 320 normal tissue samples and 462 cancer samples. Only the samples found to present peptides are shown. FIG. 1E) genes: DNAH14, peptide: SVLEKEIYSI (SEQ ID NO: 2) -from left to right tissue: 4 cell line (3 blood cells, 1 pancreas), 2 normal tissue (1 lymph node, 1 trachea), 52 cancer tissue (2 bile duct cancer, 1 myeloid cell cancer, 3 leukemia cancer, 5 breast cancer, 1 esophageal and gastric cancer, 1 gallbladder cancer, 4 colon cancer, 7 lung cancer, 6 lymph node cancer, 7 ovarian cancer, 4 prostate cancer, 4 skin cancer, 2 bladder cancer, 4 uterine cancer), fig. 1F) genes: MAGEA3, MAGEA6, peptides: KIWEELSVLEV (SEQ ID NO: 40) -from left to right tissue: 8 cancer tissue (1 liver cancer, 3 lung cancer, 2 skin cancer, 1 stomach cancer, 1 bladder cancer), fig. 1G) gene: HMX1, peptide: FLIENLLAA (SEQ ID NO: 67) -from left to right tissue: 7 cancer tissue (4 brain cancer, 2 lung cancer, 1 uterine cancer), fig. 1H) gene: CCDC138, peptide: FLLEREQLL (SEQ ID NO: 84) -from left to right tissue: 3 cell line (2 blood cells, 1 skin), 24 cancer tissue (1 myeloid cell carcinoma, 3 leukemia cancer, 1 myeloid cancer, 1 breast cancer, 1 kidney cancer, 2 colon cancer, 3 rectal cancer, 1 lung cancer, 7 lymph node cancer, 3 bladder cancer, 1 uterine cancer), fig. 1I) genes: CLSPN, peptide: SLLNQPKAV (SEQ ID NO.:235) -tissues from left to right: 13 cell line (3 blood cells, 2 kidney, 8 pancreas), 30 cancer tissue (1 myeloid cell carcinoma, 1 leukemia cancer, 2 brain cancer, 2 breast cancer, 2 esophageal cancer, 1 gall bladder cancer, 1 rectal cancer, 2 liver cancer, 4 lung cancer, 5 lymph node cancer, 2 ovarian cancer, 2 skin cancer, 4 bladder cancer, 1 uterine cancer), fig. 1J) genes: SPC25, peptide: GLAEFQENV (SEQ ID No.:243) -tissues from left to right: 3 cell lines (1 blood cell, 1 kidney, 1 pancreas), 67 cancer tissue (1 bile duct cancer, 4 leukemia cancer, 1 myeloid cell cancer, 2 brain cancer, 3 breast cancer, 4 esophageal cancer, 2 gall bladder cancer, 2 colon cancer, 1 rectal cancer, 2 liver cancer, 15 lung cancer, 8 lymph node cancer, 9 ovarian cancer, 3 skin cancer, 4 bladder cancer, 6 uterine cancer).

FIG. 2 shows representative expression profiles (relative expression compared to normal kidney) of the source genes of the invention, which are highly over-or exclusively expressed in different cancers compared to a range of normal tissues. FIG. 2A) genes: PRIM2, organized from left to right: adrenal, arterial, bone marrow, brain (total), breast, colon, esophagus, heart, kidney (triplicate), white blood cells, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, thyroid, bladder, cervix, uterus, veins (each normal sample represents a pool of several donors), 22 prostate cancer samples, fig. 2B) genes: CHEK 1-left to right organization: adrenal, arterial, bone marrow, brain (total), breast, colon, esophagus, heart, kidney (triplicate), white blood cells, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, thyroid, bladder, cervix, uterus, veins (each normal sample represents a pool of several donors), 3 normal colon samples, 10 colorectal cancer samples, fig. 2C) genes: TTC 30A-left to right organization: adrenal, arterial, bone marrow, brain (total), breast, colon, esophagus, heart, kidney (triplicate), white blood cells, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, thyroid, bladder, cervix, uterus, veins (each normal sample represents a pool of several donors), 30 brain cancer samples, fig. 2D) genes: TRIP 13-organization from left to right: adrenal, arterial, bone marrow, brain (total), breast, colon, esophagus, heart, kidney (triplicate), white blood cells, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, thyroid, bladder, cervix, uterus, vein (each normal sample represents a pool of several donors), 1 normal lung sample, 38 lung cancer samples, fig. 2E) genes: MXRA 5-organization from left to right: adrenal gland, artery, bone marrow, brain (whole), breast, colon, esophagus, heart, kidney (triplicate), white blood cells, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, thyroid, bladder, cervix, uterus, veins (each normal sample represents a pool of several donors), 9 pancreatic cancer samples. FIGS. 2F to H show representative expression profiles of the source genes of the invention, which are highly over-or exclusively expressed in a range of cancers in normal tissues (white bars) and in different cancer samples (black bars). FIG. 2F) MMP11, MMP13(Seq ID No 24) -organized from left to right: 80 normal tissue samples (6 arteries, 2 blood cells, 2 brain, 1 heart, 2 liver, 3 lung, 2 veins, 1 adipose tissue, 1 adrenal, 5 bone marrow, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gall bladder, 1 kidney, 6 lymph node, 4 pancreas, 2 peripheral nerve, 2 pituitary, 1 rectum, 2 salivary gland, 2 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid, 7 trachea, 1 bladder, 1 breast, 5 ovaries, 5 placenta, 1 prostate, 1 testis, 1 thymus, 1 uterus), 50 cancer samples (10 breast cancer, 4 bile duct cancer, 6 gall bladder cancer, 11 esophageal cancer, 10 bladder cancer, 10 uterine cancer), fig. 2G) hormada 1(SEQ ID NO 168) -tissue from left to right: 80 Normal tissue samples (6 arteries, 2 blood cells, 2 brains, 1 heart, 2 livers, 3 lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 5 bone marrow, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gall bladder, 1 kidney, 6 lymph nodes, 4 pancreases, 2 peripheral nerves, 2 pituitary gland, 1 rectum, 2 salivary glands, 2 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid, 7 trachea, 1 bladder, 1 breast, 5 ovaries, 5 placenta, 1 prostate, 1 testis, 1 thymus, 1 uterus), 41 cancer samples (10 breast cancer, 10 skin cancer, 11 non-small cell lung cancer, 10 small cell lung cancer), FIG. 2H) IGF2BP1, IGF2BP3(Seq ID No 274) -tissue from left to right: 80 normal tissue samples (6 artery, 2 blood cells, 2 brain, 1 heart, 2 liver, 3 lung, 2 vein, 1 adipose tissue, 1 adrenal gland, 5 bone marrow, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gall bladder, 1 kidney, 6 lymph node, 4 pancreas, 2 peripheral nerve, 2 pituitary gland, 1 rectum, 2 salivary gland, 2 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid, 7 trachea, 1 bladder, 1 breast, 5 ovaries, 5 placenta, 1 prostate, 1 testis, 1 thymus, 1 uterus), 53 cancer samples (4 bile duct cancer, 6 gall bladder cancer, 10 lymph node cancer, 12 ovarian cancer, 11 esophageal cancer, 10 lung cancer).

Figure 3 shows exemplary immunogenicity data: flow cytometry results after staining of peptide specific multimers.

FIGS. 4A-R show the upper part: median MS signal intensity from technical replicates was plotted as a colored spot, a single HLA-a x 02 positive normal tissue as a green or gray spot, and a tumor sample in which the peptide was detected as a red spot. Tumor and normal samples were grouped by organ origin, and box-whisker plots represent the median, 25 th and 75 th percentiles (boxes) and the minimum and maximum values (whiskers) of the normalized signal intensities for the multiple samples. Normal organs are ranked according to risk category (blood cells, cardiovascular system, brain, liver, lung: high risk, dark green spots; reproductive organs, mammary glands, prostate: low risk, grey spots; all other organs: medium risk, light green spots). The lower part: the relative peptide detection frequency for each organ is shown as a spine. The numbers below the graph represent the number of samples in which peptide was detected in the total number of samples analyzed for each organ (normal sample N298, tumor sample N461). If a peptide is detected on a sample, but cannot be quantified for technical reasons, the sample is included in the detection frequency map, but the upper part of the map does not show any points. Organization (left to right): normal samples: an artery; blood cells; a brain; a core; liver; a lung; a vein; fat: adipose tissue; gl.: the adrenal gland; BM: bone marrow; color: colon and rectum; and d: the duodenum; and (4) espph: (ii) an esophagus; and (3) a gallb: a gallbladder; LN: lymph nodes; panc: a pancreas; parathyr: parathyroid gland; pert: peritoneum; pituit: a pituitary; gland: salivary glands; mus: skeletal muscle; skin; int.sm: the small intestine; a spleen; the stomach; thyroid gland; an air tube; a ureter; the bladder; a breast; an ovary; a placenta; the prostate; a testis; thymus; the uterus. Tumor sample AML: acute myeloid leukemia; PCA: prostate cancer; BRCA: breast cancer; CLL: chronic lymphocytic leukemia; CRC: colorectal cancer; GALB: gallbladder cancer; HCC: hepatocellular carcinoma; MEL: melanoma; NHL: non-hodgkin lymphoma; OC: ovarian cancer; OSCAR: esophageal cancer; OSC _ GC: esophageal/gastric cancer; PC: pancreatic cancer; GB, glioblastoma; GC: gastric cancer; NSCLC: non-small cell lung cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC: bladder cancer; and UEC: uterine and endometrial cancer.

FIGS. 5A-R show representative expression profiles of the source genes of the invention, which are overexpressed in different cancer samples. Tumor (red dots) and normal (green or 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: an artery; blood cells; a brain; a core; liver; a lung; a vein; fat: adipose tissue; gl.: the adrenal gland; BM: bone marrow; cartilage; color: colon and rectum; and (4) espph: (ii) an esophagus; an eye; and (3) a gallb: a gallbladder; the kidney; LN: lymph nodes; a nerve; panc: a pancreas; pituit: a pituitary; gland: salivary glands; mus: skeletal muscle; skin; int.sm: the small intestine; a spleen; the stomach; thyroid gland; an air tube; the bladder; a breast; an ovary; a placenta; the prostate; a testis; thymus; the uterus. Tumor sample AML: acute myeloid leukemia; PCA: prostate cancer; BRCA: breast cancer; CLL: chronic lymphocytic leukemia; CRC: colorectal cancer; GALB: gallbladder cancer; HCC: hepatocellular carcinoma; MEL: melanoma; NHL: non-hodgkin lymphoma; OC: ovarian cancer; OSCAR: esophageal cancer; PC: pancreatic cancer; GB, glioblastoma; GC: gastric cancer; NSCLC: non-small cell lung cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC: bladder cancer; and UEC: uterine and endometrial cancer.

Fig. 6A to M show exemplary results of peptide-specific CD8+ T cell in vitro reactions of healthy HLA-a x 02+ donors. The method for preparing the CD8+ T cells comprises the following steps: artificial APCs coated with anti-CD 28 mAb and HLA-a × 02 were synthesized with, for example, SeqID No 11 peptide (a, left panel) or SeqID No 14 peptide (B, left panel), respectively (SeqID nos 157(C), 233(D), 85(E), 89(F), 155(G), 153(H), 264(I), 117(J), 253(K), 39(L), and 203 (M)). After 3 cycles of stimulation, peptide-reactive cells were detected by 2D multimer staining of the relevant multimers, e.g., A.about.02/SeqID No 11(A) or A.about.02/SeqID No 14 (B). Right panels (e.g., a and B) show control staining of cells stimulated with irrelevant a x 02/peptide complexes. Viable single cells were gated on 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.

FIGS. 7A-C show the over-presentation of various peptides in different cancer tissues compared to normal tissues. The analysis included data from 320 normal tissue samples and 462 cancer samples. Only the samples found to present peptides are shown. Fig. 7A) genes: CCR8, peptide: LLIPFTIFM (SEQ ID NO: 43) -from left to right tissue: 16 cancer tissues (1 bile duct cancer, 1 breast cancer, 1 colon cancer, 7 lung cancer, 2 lymph node cancer, 3 ovarian cancer, 1 skin cancer); fig. 7B) genes: CXCR5, peptide: ILVTSIFFL (SEQ ID NO: 152) -from left to right tissue: 6 normal tissue (1 lymph node, 5 spleen), 16 cancer tissue (8 leukemia cancer, 8 lymph node cancer); FIG. 7C) genes: CYSLTR1, peptide: VILTSSPFL (SEQ ID NO: 156) -from left to right tissue: 3 normal tissue (1 lung, 1 lymph node, 1 spleen), 11 cancer tissue (2 breast cancer, 5 leukemia cancer, 3 lymph node cancer, 1 myeloid cell cancer).

Examples

Example 1

Identification and quantification of cell surface presented tumor associated peptides

Tissue sample

Patient tumor tissue was obtained from Asterand (Detroit, USA and Royston, Herts, UK), Val d' Hebron university hospital (Barcelona), BioServe (Beltsville, MD, USA), cancer immunotherapy center (CCIT), Herlev hospital (Herlev); geneticist inc. (Glendale, CA, USA), geneva university hospital, heidelberg university hospital, munich university hospital, kyoto university of medicine (KPUM), osaka university (OCU), proteogex inc. (silver City, CA, USA), tiben university hospital. Normal tissue was obtained from Bio-optics Inc., CA, USA, BioServe, Beltsville, MD, USA, Capita Bioscience Inc., Rockville, MD, USA, Geneticist Inc., Glendale, CA, USA, Rinware university Hospital, Hedelberg university Hospital, Munich university Hospital, ProteoX Inc., silver City, CA, USA, Dibingen university Hospital. All patients received written informed consent prior to surgery or autopsy. Immediately following resection, the tissue was cold shocked and stored at-70 ℃ or below prior to isolation of the TUMAP.

Isolation of HLA peptides from tissue samples

With minor modifications to the protocol (Falk et al, 1991; Seeger et al, 1999), HLA peptide libraries from cold shock tissue samples were obtained by immunoprecipitation of solid tissue using HLA-A02-specific antibody BB7.2, HLA-A, HLA-B, HLA-C-specific antibody W6/32, CNBr-activated agarose gel, acid treatment and ultrafiltration.

Mass spectrometric analysis

The obtained HLA peptide library was separated by reverse phase chromatography (Waters) according to its hydrophobicity, and the eluted peptides were analyzed by LTQ-velos fusion hybridization Mass Spectrometry (ThermoElectron) equipped with an electrospray source. The peptide library was loaded directly into an analytical fused silica microcapillary column (75 μm internal diameter x 250mm) filled with 1.7 μm C18 reverse phase material (Waters) using a flow rate of 400nL per minute. Subsequently, the peptides were separated using a two-step 180 min binary gradient from solvent B at a concentration of 10% to 33% at a flow rate of 300nL per minute. The gradient was made from solvent a (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Gold coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoliter electrospray source. The LTQ-Orbitrap mass spectrometer was operated in a data dependent mode using the TOP 5(TOP5) strategy. In short, a scan cycle is first started at orbitrap with a high accurate mass complete scan (R30000), followed by MS/MS scanning of the 5 most abundant precursor ions in orbitrap with the dynamic exclusion technique of the previously selected ions (R7500). Tandem mass spectra were interpreted in sequence and another manual controller. The resulting fragmentation pattern of the natural peptide was compared to the fragmentation pattern of a reference peptide of the same synthetic sequence, ensuring the identified peptide sequence.

Label-free versus LC-MS quantification was performed by ion counting (i.e., by LC-MS functional extraction and analysis) (Mueller et al, 2007). This method assumes that the LC-MS signal region of the peptide correlates with its abundance in the sample. The extracted features are further processed through state-of-charge deconvolution and retention time calibration (Mueller et al, 2008; Sturm et al, 2008). Finally, all LC-MS features were cross-referenced with sequence identification results to combine the quantitative data of different samples and tissues with peptide presentation features. Quantitative data were normalized in a two-layer fashion from the pooled data to account for both technical and biological replicative variation. Thus, each identified peptide can be correlated with quantitative data, thereby allowing a relative quantification between the sample and the tissue. In addition, all quantitative data obtained for candidate peptides were manually checked to ensure data consistency and to verify the accuracy of automated analysis. For each peptide, a presentation graph is calculated showing the mean presentation of the sample as well as the variation in replication. These features parallel the baseline values of pancreatic cancer samples with normal tissue samples. The presentation profile of an exemplary over-presenting peptide is shown in fig. 1. The summary of selected peptides presented in various entities is shown in table 4.

Table 4: selected peptides are presented in summary in various entities. A peptide is considered to be associated with an entity if it is over-presented on a solid cancer sample compared to normal tissue. Mell melanoma, BRCA breast cancer, OSCAR esophageal cancer. BPH includes benign prostatic hyperplasia as well as pancreatic cancer.

Table 4B: selected peptides are presented in summary in various entities.

GB ═ glioblastoma, BRCA ═ breast cancer, CRC ═ colorectal cancer, RCC ═ renal cell carcinoma, CLL ═ chronic lymphocytic leukemia, HCC ═ hepatocellular carcinoma, NSCLC ═ non-small cell lung cancer, SCLC ═ small cell lung cancer, NHL ═ non-hodgkin lymphoma, AML ═ acute myelogenous leukemia, OC ═ ovarian cancer, PC ═ pancreatic cancer, BPH ═ prostate cancer and benign prostatic hyperplasia, OSCAR ═ esophageal cancer including gastroesophageal junction cancer, GBC _ ccch ═ gallbladder adenocarcinoma and cholangiocarcinoma, MEL melanoma, GC ═ gastric cancer, UBC ═ bladder cancer, UTC ═ uterine cancer.

Example 2

Expression profiling of genes encoding the peptides of the invention

An over-presentation or specific presentation of one peptide on tumor cells compared to normal cells is sufficient for its effective use in immunotherapy, some peptides are tumor-specific, although their source proteins are also present in normal tissues. However, mRNA expression profiling increases the safety of other levels in the selection of immunotherapeutic target peptides. Particularly for treatment options with high safety risks, such as affinity matured TCRs, the ideal target peptide will be derived from a protein that is unique to the tumor and does not appear in normal tissues. For the present invention, normal tissue expression of all source genes proved to be minimal according to the above database covering RNA expression data of about 3000 normal tissue samples. In some solid cases of cancer (HCC, CRC, GB, GC, NSCLC, PC, RCC, BPH/PCA) further RNA analysis of normal and tumor tissues was added to estimate the target range for each cancer patient population.

RNA source and preparation

Surgically excised tissue specimens were provided as described above (see example 1) after obtaining written informed consent from each patient. Tumor tissue specimens were snap frozen immediately after surgery, and then homogenized with a pestle and mortar in liquid nitrogen. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, germany) followed by clearance with RNeasy (QIAGEN, Hilden, germany); both methods were performed according to the manufacturer's protocol.

Total RNA in healthy human tissue is commercially available (Ambion, Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, the Netherlands; BioChain, Hayward, Calif., USA). The RNAs of several persons (2 to 123 persons) were mixed so that the RNAs of each person were equally weighted.

The quality and quantity of all RNA samples were evaluated on an Agilent 2100Bioanalyzer analyzer (Agilent, Waldbronn, Germany) using the RNA 6000Pico LabChip Kit (Agilent).

Total RNA in healthy human tissue is commercially available (Ambion, Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, the Netherlands; BioChain, Hayward, Calif., USA). The RNAs of several persons (2 to 123 persons) were mixed so that the RNAs of each person were equally weighted. The quality and quantity of all RNA samples were evaluated on an Agilent 2100Bioanalyzer analyzer (Agilent, Waldbronn, Germany) using the RNA 6000Pico LabChip Kit (Agilent).

Total RNA from healthy human tissue used for RNASeq experiments was obtained from: asterand (Detroit, MI, USA & Royston, Herts, UK), BioCat GmbH (Heidelberg, Germany), BioServe (Beltsville, Md, USA), Capital Bioscience Inc. (Rockville, Md., USA), Geneticist Inc. (Glendale, Calif., USA), Istituto Nazionale Tumour "Pascal" (Naples, Italy), ProteoGenex Inc. (Culver City, Calif., USA), Haidelberg university Hospital (Heidelberg, Germany).

Total RNA from tumor tissue used for RNASeq experiments was obtained from: asterand (Detroit, MI, USA & Royston, Herts, UK), Bio-optics Inc. (Brea, CA, USA), BioServe (Beltsville, MD, USA), Geneticist Inc. (Glendale, CA, USA), ProteoGenex Inc. (Culver City, CA, USA), Tissue Solutions Ltd (Glasgow, UK), Bonn university Hospital (Bonn, Germany), Heidelberg university Hospital (Heidelberg, Germany), Tissuen university Hospital (Tubingen, Germany).

Microarray experiments

The range was estimated by analyzing the RNA expression profiles (Affymetrix microarrays) of 30GB, 16CRC, 56RCC, 12HCC, 38NSCLC, 11PC, 34GC and 20 prostate cancer samples.

All tumor and normal tissue RNA samples were analyzed for gene expression using Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0Affymetrix oligonucleotide chips (Affymetrix, Inc., Santa Clara, Calif., USA). All steps were performed according to the Affymetrix manual. Briefly, double-stranded cDNA was synthesized from 5-8. mu.g of RNA using SuperScript RTII (Invitrogen) and oligo-dT-T7 primer (MWG Biotech, Ebersberg, Germany) as described in the manual. The in vitro transcription was accomplished by performing U133A assay with the BioArray High Yield RNA transcription labeling Kit (ENZO Diagnostics, Farmingdale, NY, USA) or U133 Plus 2.0 assay with the GeneChip IVT labeling Kit (Affymetrix), followed by disruption, hybridization and staining with streptavidin-phycoerythrin and biotinylated anti-streptomycin protein antibody (Molecular Probes, Leiden, Netherlands). The images were scanned using an Agilent 2500A general array Scanner (U133A) or Affymetrix Gene-Chip Scanner 3000(U133 Plus 2.0), and the data were analyzed using GCOS software (Affymetrix corporation) with all parameters set by default. To achieve standardization, 100 housekeeping genes (housekeeping genes) provided by Affymetrix were used. Relative expression values were calculated using the software given signal log ratio, with the value for normal kidney tissue samples arbitrarily set at 1.0. The expression profile of the representative source genes of the invention highly overexpressed in HCC, CRC, GB, GC, NSCLC, PC, RCC, BPH/PCA is shown in FIG. 2. The target range of selected peptide source genes is summarized in the table.

Table 5A: the target range of the peptide source gene is selected. Overexpression is defined as expression on the tumor of more than 1.5 fold compared to the associated normal tissue that most highly expresses the gene. < 19% over-expression ═ I, 20-49% ═ II, 50-69% ═ III, > 70% ═ IV. If a peptide can be obtained from multiple source genes, the minimum range of genes is crucial.

RNAseq experiment

RNA samples from tumor and normal tissues were analyzed for gene expression by CeGaT (Tubingen, Germany) using a new generation sequencing technique (RNAseq). Briefly, a sequencing library was prepared using the Illumina HiSeq v4 kit according to the supplier's protocol (Illumina inc., San Diego, CA, USA), including RNA fragmentation, cDNA transformation and addition of sequencing adaptors. Libraries obtained from multiple samples were equimolar mixed according to the manufacturer's instructions and sequenced on an Illumina HiSeq 2500 sequencer, yielding single-ended reads of 50 bp. The processed reads were mapped to the human genome using STAR software (GRCh 38). According to the instructions of the ENSEMBL sequence database (Ensembl77), expression data were set at the transcription level to RPKM (reads per million mapped per kilobase, generated by Cufflinks software) and at the exonic level (total reads, generated by Bedtools software). Exon readings were classified into exon length and calibration size to obtain RPKM values.

The expression profiles of representative source genes of the invention highly overexpressed in NHL, BRCA, GBC, CCC, MEL, OC, OSCAR, SCLC, UBC, UEC are shown in FIGS. 2F-H. Further representative gene expression scores are shown in table 5B.

Table 5B: the target range of the peptide source gene is selected.

Overexpression is defined as expression on the tumor of more than 1.5 fold compared to the associated normal tissue that most highly expresses the gene. < 19% over-expression ═ I, 20-49% ═ II, 50-69% ═ III, > 70% ═ IV. If a peptide can be obtained from multiple source genes, the minimum range of genes is crucial. The baseline included the following relevant normal tissues: adipose tissue, adrenal gland, arteries, bone marrow, brain, cartilage, colon, esophagus, gall bladder, heart, kidney, liver, lung, lymph nodes, pancreas, pituitary, rectum, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid, trachea, bladder, and veins. If expression data is obtained for several samples of the same tissue type, the arithmetic mean of each sample is used for calculation. AML ═ acute myelogenous leukemia, NHL ═ non-hodgkin lymphoma, BRCA ═ breast cancer, CLL ═ chronic lymphocytic leukemia, GBC _ CCC ═ gallbladder adenocarcinoma and cholangiocarcinoma, MEL ═ melanoma, OC ═ ovarian cancer, OSCAR ═ esophageal cancer including cancer at the gastro-esophageal junction, SCLC ═ small cell lung cancer, UBC ═ bladder cancer, UTC ═ uterine cancer.

Example 3

In vitro immunogenicity of MHC-I presenting peptides

To obtain information on the immunogenicity of the TUMAPs of the present invention, the inventors performed studies using an in vitro T cell expansion assay based on repeated stimulation with artificial antigen presenting cells (aapcs) loaded with peptide/MHC complexes and anti-CD 28 antibodies. In this way, the inventors could show that the present invention so far 47 HLA-a 0201 restricted TUMAPs were immunogenic, indicating that these peptides are T cell epitopes against human CD8+ precursor T cells (tables 6A and B).

In vitro activation of CD8+ T cells

For in vitro stimulation with artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD 28 antibodies, the inventors first isolated CD8+ T cells by actively selecting fresh HLA-A02 product after leukopheresis from a healthy donor CD8 microbead (Miltenyi Biotec, Bergisch-Gladbach, Germany) obtained from University clinics Mannheim, Germany.

PBMC and isolated CD8+ lymphocytes were cultured in T cell culture medium (TCM) before use, including RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100U/ml penicillin/100. mu.g/ml streptomycin (Cambrex, Cologne, Germany), 1mM sodium pyruvate (CC Pro, Oberdorla, Germany) and 20. mu.g/ml gentamycin (Cambrex). At this step, 2.5ng/ml IL-7(Promocell, Heidelberg, Germany) and 10U/ml IL-2(Novartis Pharma, N ü rnberg, Germany) were also added to the TCM. Generation of pMHC/anti-CD 28 coated beads, stimulation and readout of T cells were performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and 8 different pMHC molecules per readout condition.

Purified co-stimulatory mouse IgG2a anti-human CD28 antibody 9.3(Jung et al, 1987) was chemically biotinylated using N-hydroxysuccinimide biotin recommended by the manufacturer (Perbio, Bonn, Germany). The beads used were streptavidin-coated polystyrene particles (Bangs laboratories, Ill., USA) of 5.6 μm.

pMHC used for the positive and negative control stimuli were A.times.0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO:289) modified from Melan-A/MART-1) and A.times.0201/DDX 5-001 (YLLPAIVHI obtained from DDX5 (SEQ ID NO:290)), respectively.

800.000 beads/200. mu.l were packed in 96-well plates containing 4X 12.5ng of different biotin-pMHC, washed, and then 600ng of biotin anti-CD 28 was added in a volume of 200. mu.l. Co-culture of 1X10 in 200. mu.l TCM with 5ng/ml IL-12(Promocell) at 37 ℃⁶CD8+ T cells and 2x10⁵The coated beads were washed for 3 days, thereby activating the stimulation. Thereafter, half of the medium was exchanged with fresh TCM supplemented with 80U/ml IL-2 and incubated at 37 ℃ for 4 days. This stimulation cycle was performed a total of 3 times. For pMHC multimer readout using 8 different pMHC molecules per condition, the two-dimensional combinatorial coding approach was used as described previously (Andersen et al, 2012), slightly modified, encompassing coupling to 5 different fluorescent dyes. Finally, multimer analysis was performed with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1(BD, Heidelberg, Germany) and fluorescent pMHC multimers. For the analysis, a BD LSRII SORP cytometer equipped with a suitable laser and screening procedure was used. Peptide-specific cells were calculated as a percentage of total CD8+ cells. Multimer analysis results were evaluated using FlowJo software (Tree Star, Oregon, usa). The in vitro priming of specific multimers + CD8+ lymphocytes was tested in comparison to the negative control stimulation group. Immunogenicity of a given antigen is tested if at least one evaluable in vitro stimulated well in a healthy donor is found to contain a specific CD8+ T cell strain after in vitro stimulation (i.e. the well contains at least 1% of specific multimers + CD8+ T cells and the percentage of specific multimers + is at least 10 times the median of negative control stimulation).

Pancreatic cancer peptide in vitro immunogenicity

For the tested HLA class I peptides, their immunogenicity in vitro can be demonstrated by the generation of peptide-specific T cell lines. Exemplary results of flow cytometry assays of the TUMAP-specific multimers after staining the 15 peptides of the invention are shown in FIGS. 3 and 6, along with corresponding negative control information. The results for 2 peptides of the invention are summarized in tables 6A and B.

Table 6A: in vitro immunogenicity of HLA class I peptides of the invention exemplary results of in vitro immunogenicity experiments performed by the applicant on the peptides of the invention. + < 20% >; 21% -49% ++; 50% -69% ++; 70% + 70 +++

Serial number	Hole(s)	Donor
			288	SLYKGLLSV	++
287	KIQEILTQV	+

Table 6B: in vitro immunogenicity of HLA class I peptides of the invention.

Exemplary results of in vitro immunogenicity experiments performed by the applicant on HLA-a 02 restricted peptides of the invention. The results of in vitro immunogenicity experiments are suggested. The percentage of positive wells and donors (other evaluable) was summarized as < 20% >; 20% -49% ++; 50% -69% ++; 70% + 70 +++

Example 4

Synthesis of peptides

All peptides were synthesized by standard, well-accepted solid-phase peptide synthesis using the Fmoc strategy. The identity and purity of each peptide has been determined using mass spectrometry and RP-HPLC analysis. White to off-white peptides were obtained with > 50% purity by lyophilization (trifluoroacetate). All of the TUMAPs are preferably administered as trifluoroacetate or acetate salts, although other salt forms are possible.

Example 5

MHC binding assay

The candidate peptides of the present invention based on T cell therapy were further tested for their MHC binding capacity (affinity). The individual peptide-MHC complexes are generated by UV-ligand exchange, wherein the UV-sensitive peptides are cleaved after UV irradiation and exchanged with the relevant peptide for analysis. Only candidate peptides that can effectively bind and stabilize a peptide-accepting MHC molecule can prevent dissociation of the MHC complex. To determine the yield of the exchange reaction, an ELISA assay based on the detection of the stable MHC complex light chain (. beta.2m) was performed. The assays were generally performed according to the method described by Rodenko et al (Rodenko et al, 2006).

96-well Maxisorp plates (NUNC) were coated overnight at 2ug/ml pronase in PBS at room temperature, washed 4-fold and blocked in 2% BSA with blocking buffer for 1 hour at 37 ℃. The folded HLA-A02: 01/MLA-001 monomer is used as a standard substance and covers the range of 15-500 ng/ml. The peptide-MHC monomer of the uv exchange reaction was diluted 100-fold in blocking buffer. The samples were incubated at 37 ℃ for 1 hour, washed four times, incubated at 37 ℃ for 1 hour with 2ug/ml HRP conjugated anti-. beta.2m, washed again, and washed with NH₂SO₄The blocked TMB solution was tested. The absorption was measured at 450 nm. Candidate peptides that exhibit high exchange yields (preferably greater than 50%, most preferably greater than 75%) are generally preferred when generating and producing antibodies or fragments thereof and/or T cell receptors or fragments thereof, because they exhibit sufficient affinity for MHC molecules and prevent dissociation of MHC complexes.

Table 7: MHC class I binding score.

Binding of HLA class I restricted peptides to HLA-a 02:01 was assessed by peptide exchange yield: more than or equal to 10% +; more than or equal to 20% +; 20% -49% ++; percent ++; not less than 50% - + +; more than or equal to 75 percent ++; 75% ═ 75% +++

Example 6

Table 8: preferred peptides of the invention

Absolute quantification of cell surface presented tumor associated peptides

The production of binders such as antibodies and/or TCRs is a laborious process that canOnly for some selected targets. In the case of tumor-associated and specific peptides, selection criteria include, but are not limited to, exclusion of the presentation and concentration of peptides presented on the cell surface. In addition to the isolation and relative quantification of the peptides described herein, the inventors also analyzed the absolute peptide copy number per cell. Quantitation of the copy number of TUMAPs per cell in solid tumor samples requires absolute quantitation of the isolated TUMAPs, efficiency of TUMAP isolation, and cell counts of the analyzed tissue samples.NanoLC-MS/MS peptide quantitation

For accurate quantification of peptides by mass spectrometry, an internal standard method was used to generate a calibration curve for each peptide. Internal standards are dual isotopically labeled variants of each peptide, i.e., 2 isotopically labeled amino acids were incorporated in the TUMAP synthesis. It differs only qualitatively from tumor-associated peptides, but not in other physicochemical properties (Anderson et al, 2012). An internal standard was incorporated into each MS sample and all MS signals were normalized to the internal standard MS signal to balance potential technical differences between MS experiments.

The calibration curves were plotted with at least three different matrices, i.e., HLA peptide eluate from a natural sample similar to a conventional MS sample, and each preparation was measured in duplicate MS runs. For evaluation, MS signals were normalized to internal standard signals and calibration curves were calculated by logistic regression.

For quantification of tumor-associated peptides from tissue samples, each sample was also spiked with an internal standard; MS signals were normalized to internal standards and quantified using the peptide calibration curve.

Efficiency of peptide/MHC separation

For any protein purification process, the isolation of proteins from a tissue sample is associated with some loss of the relevant proteins. To determine the efficiency of the TUMAP separation, peptide/MHC complexes were generated for all TUMAPs selected as absolute quantification. To enable native peptide MHC/complex and loading, a monoisotopically labeled version of TUMAP was used, i.e. 1 isotopically labeled amino acid was incorporated during TUMAP synthesis. These complexes are incorporated into freshly prepared tissue lysates, e.g., at the earliest possible time point during the TUMAP isolation, and then captured as native peptide MHC/complexes in subsequent affinity purifications. Therefore, measuring the recovery of single-marker TUMAPs can lead to conclusions regarding the separation efficiency of individual TUMAPs.

Separation efficiency was analyzed using a small number of samples, and these tissue samples were comparable. In contrast, the separation efficiency differs between individual peptides. This indicates that the separation efficiency, although measured in only a limited number of samples, can be extrapolated to any other tissue preparation. However, since separation efficiency cannot be extrapolated from one peptide to the other, it is necessary to analyze each TUMAP separately.

Determination of cell count in solid, frozen tissue

To determine the cell number of tissue samples quantified by absolute peptides, the inventors used DNA content analysis. This method is applicable to a wide range of samples from different sources, and most importantly, frozen samples (Alcoser et al, 2011; Forsey and Chaudhuri, 2009; Silva et al, 2013). During the peptide isolation protocol, tissue samples were processed into homogeneous lysates from which a small aliquot of the lysate was taken. The samples were aliquoted in triplicate and DNA was isolated therefrom (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). The total DNA content of each DNA isolation was quantified using a fluorescence-based DNA quantification Assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) at least twice in duplicate.

To calculate the cell number, a standard curve of DNA from a single aliquot of healthy blood cells was generated, using a series of assigned cell numbers. The standard curve was used to calculate the total cell content of the total DNA content per DNA isolation. The average total cell count of the tissue samples used for peptide isolation was calculated taking into account the known volume of lysate aliquots and the total lysate volume.

Each timeAnPeptide copy number of cells

Using the data from the previous experiments, the inventors calculated the number of copies of TUMAP per cell as the total peptide amount divided by the total cell count of the sample, and then divided by the separation efficiency. The cell copy number of the selected peptides is shown in table 9.

Table 9: absolute copy number. The table lists the results of absolute peptide quantification in tumor samples. Median copy number per cell for each peptide is expressed as: <100 ═ +; 100 ═ + +; 1,000+ + +; 10,000 ═ + + + + +. Sample numbers are prompted in which to provide the evaluated high quality MS data.

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<110> Imamatix Biotechnology Ltd

<120> novel peptides and peptide compositions for immunotherapy of various tumors

<130> I32799WO

<150> GB1505305.1

<151> 2015-03-27

<150> US62/139,189

<151> 2015-03-27

<160> 290

<170> PatentIn version 3.5

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Lys Leu Gln Glu Lys Ile Gln Glu Leu

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Ser Val Leu Glu Lys Glu Ile Tyr Ser Ile

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Arg Val Ile Asp Asp Ser Leu Val Val Gly Val

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Val Leu Phe Gly Glu Leu Pro Ala Leu

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Gly Leu Val Asp Ile Met Val His Leu

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Phe Leu Asn Ala Ile Glu Thr Ala Leu

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Ala Leu Leu Gln Ala Leu Met Glu Leu

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Ala Leu Ser Ser Ser Gln Ala Glu Val

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Ser Leu Ile Thr Gly Gln Asp Leu Leu Ser Val

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Gln Leu Ile Glu Lys Asn Trp Leu Leu

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Leu Leu Asp Pro Lys Thr Ile Phe Leu

1 5

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Arg Leu Leu Asp Pro Lys Thr Ile Phe Leu

1 5 10

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Arg Leu His Asp Glu Asn Ile Leu Leu

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Tyr Thr Phe Ser Gly Asp Val Gln Leu

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Gly Leu Pro Ser Ala Thr Thr Thr Val

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Ser Leu Ala Asp Leu Ser Leu Leu Leu

1 5

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Gly Leu Leu Pro Ser Ala Glu Ser Ile Lys Leu

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Lys Thr Ala Ser Ile Asn Gln Asn Val

1 5

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Lys Val Phe Glu Leu Asp Leu Val Thr Leu

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Ala Leu Val Glu Lys Gly Glu Phe Ala Leu

1 5 10

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<400> 21

Tyr Leu Met Asp Asp Phe Ser Ser Leu

1 5

<210> 22

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<213> Intelligent people

<400> 22

Leu Met Tyr Pro Tyr Ile Tyr His Val

1 5

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<400> 23

Ala Leu Leu Ser Pro Leu Ser Leu Ala

1 5

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<400> 24

Lys Val Trp Ser Asp Val Thr Pro Leu

1 5

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<400> 25

Leu Leu Trp Gly His Pro Arg Val Ala Leu Ala

1 5 10

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Val Leu Asp Gly Lys Val Ala Val Val

1 5

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Gly Leu Leu Gly Lys Val Thr Ser Val

1 5

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<400> 28

Ile Lys Val Thr Asp Pro Gln Leu Leu Glu Leu

1 5 10

<210> 29

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<400> 29

Lys Met Ile Ser Ala Ile Pro Thr Leu

1 5

<210> 30

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<213> Intelligent people

<400> 30

Ile Ile Thr Glu Val Ile Thr Arg Leu

1 5

<210> 31

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<213> Intelligent people

<400> 31

Gly Leu Leu Glu Thr Thr Gly Leu Leu Ala Thr

1 5 10

<210> 32

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<400> 32

Val Val Met Val Leu Val Leu Met Leu

1 5

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<400> 33

Thr Leu Asp Arg Asn Ser Leu Tyr Val

1 5

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<213> Intelligent people

<400> 34

Thr Leu Asn Thr Leu Asp Ile Asn Leu

1 5

<210> 35

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<400> 35

Val Ile Ile Lys Gly Leu Glu Glu Ile

1 5

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<213> Intelligent people

<400> 36

Thr Val Leu Gln Glu Leu Ile Asn Val

1 5

<210> 37

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<213> Intelligent people

<400> 37

Gln Ile Val Glu Leu Ile Glu Lys Ile

1 5

<210> 38

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<213> Intelligent people

<400> 38

Val Leu Gln Gln Glu Ser Asn Phe Leu

1 5

<210> 39

<211> 9

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<213> Intelligent people

<400> 39

Tyr Leu Glu Asp Gly Phe Ala Tyr Val

1 5

<210> 40

<211> 11

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<213> Intelligent people

<400> 40

Lys Ile Trp Glu Glu Leu Ser Val Leu Glu Val

1 5 10

<210> 41

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<213> Intelligent people

<400> 41

Ile Val Thr Glu Ile Ile Ser Glu Ile

1 5

<210> 42

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<213> Intelligent people

<400> 42

Lys Gln Met Ser Ile Ser Thr Gly Leu

1 5

<210> 43

<211> 9

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<213> Intelligent people

<400> 43

Leu Leu Ile Pro Phe Thr Ile Phe Met

1 5

<210> 44

<211> 9

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<213> Intelligent people

<400> 44

Ala Val Phe Asn Leu Val His Val Val

1 5

<210> 45

<211> 9

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<213> Intelligent people

<400> 45

Phe Leu Pro Val Ser Val Val Tyr Val

1 5

<210> 46

<211> 10

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<213> Intelligent people

<400> 46

Ile Ser Leu Asp Glu Val Ala Val Ser Leu

1 5 10

<210> 47

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<213> Intelligent people

<400> 47

Gly Leu Asn Gly Phe Asn Val Leu Leu

1 5

<210> 48

<211> 10

<212> PRT

<213> Intelligent people

<400> 48

Lys Ile Ser Asp Phe Gly Leu Ala Thr Val

1 5 10

<210> 49

<211> 11

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<213> Intelligent people

<400> 49

Lys Leu Ile Gly Asn Ile His Gly Asn Glu Val

1 5 10

<210> 50

<211> 9

<212> PRT

<213> Intelligent people

<400> 50

Ile Leu Leu Ser Val Leu His Gln Leu

1 5

<210> 51

<211> 9

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<213> Intelligent people

<400> 51

Leu Asp Ser Glu Ala Leu Leu Thr Leu

1 5

<210> 52

<211> 9

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<213> Intelligent people

<400> 52

Thr Ile Gly Ile Pro Phe Pro Asn Val

1 5

<210> 53

<211> 9

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<213> Intelligent people

<400> 53

Ala Gln His Leu Ser Thr Leu Leu Leu

1 5

<210> 54

<211> 9

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<213> Intelligent people

<400> 54

Tyr Leu Val Pro Gly Leu Val Ala Ala

1 5

<210> 55

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<213> Intelligent people

<400> 55

His Leu Phe Asp Lys Ile Ile Lys Ile

1 5

<210> 56

<211> 13

<212> PRT

<213> Intelligent people

<400> 56

Val Leu Gln Glu Asn Ser Ser Asp Tyr Gln Ser Asn Leu

1 5 10

<210> 57

<211> 10

<212> PRT

<213> Intelligent people

<400> 57

Thr Leu Tyr Pro Gly Arg Phe Asp Tyr Val

1 5 10

<210> 58

<211> 11

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<213> Intelligent people

<400> 58

His Leu Leu Gly Glu Gly Ala Phe Ala Gln Val

1 5 10

<210> 59

<211> 11

<212> PRT

<213> Intelligent people

<400> 59

Ala Leu Ala Asp Gly Ile Lys Ser Phe Leu Leu

1 5 10

<210> 60

<211> 10

<212> PRT

<213> Intelligent people

<400> 60

Tyr Leu Phe Ser Gln Gly Leu Gln Gly Leu

1 5 10

<210> 61

<211> 9

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<213> Intelligent people

<400> 61

Ala Leu Tyr Pro Lys Glu Ile Thr Leu

1 5

<210> 62

<211> 9

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<213> Intelligent people

<400> 62

Ser Leu Val Glu Asn Ile His Val Leu

1 5

<210> 63

<211> 9

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<213> Intelligent people

<400> 63

Lys Leu Leu Pro Met Val Ile Gln Leu

1 5

<210> 64

<211> 10

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<213> Intelligent people

<400> 64

Ser Leu Tyr Ala Gly Ser Asn Asn Gln Val

1 5 10

<210> 65

<211> 9

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<213> Intelligent people

<400> 65

Ser Leu Ser Glu Lys Ser Pro Glu Val

1 5

<210> 66

<211> 10

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<213> Intelligent people

<400> 66

Ala Met Phe Pro Asp Thr Ile Pro Arg Val

1 5 10

<210> 67

<211> 9

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<213> Intelligent people

<400> 67

Phe Leu Ile Glu Asn Leu Leu Ala Ala

1 5

<210> 68

<211> 9

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<213> Intelligent people

<400> 68

Gln Leu Met Asn Leu Ile Arg Ser Val

1 5

<210> 69

<211> 10

<212> PRT

<213> Intelligent people

<400> 69

Leu Lys Val Leu Lys Ala Asp Val Val Leu

1 5 10

<210> 70

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<400> 70

Gly Leu Thr Glu Lys Thr Val Leu Val

1 5

<210> 71

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<400> 71

His Met Ser Gly Lys Leu Thr Asn Val

1 5

<210> 72

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<213> Intelligent people

<400> 72

Val Leu Ser Thr Arg Val Thr Asn Val

1 5

<210> 73

<211> 8

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<213> Intelligent people

<400> 73

Ser Val Pro Lys Thr Leu Gly Val

1 5

<210> 74

<211> 9

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<213> Intelligent people

<400> 74

Gly Leu Ala Phe Leu Pro Ala Ser Val

1 5

<210> 75

<211> 9

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<213> Intelligent people

<400> 75

Ala Leu Leu Asp Gly Ala Leu Gln Leu

1 5

<210> 76

<211> 9

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<213> Intelligent people

<400> 76

Phe Thr Ala Glu Phe Leu Glu Lys Val

1 5

<210> 77

<211> 9

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<213> Intelligent people

<400> 77

Ala Leu Tyr Gly Asn Val Gln Gln Val

1 5

<210> 78

<211> 9

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<213> Intelligent people

<400> 78

Leu Phe Gln Ser Arg Ile Ala Gly Val

1 5

<210> 79

<211> 10

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<213> Intelligent people

<400> 79

Thr Val Leu Glu Glu Ile Gly Asn Arg Val

1 5 10

<210> 80

<211> 9

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<213> Intelligent people

<400> 80

Val Leu Thr Gly Gln Val His Glu Leu

1 5

<210> 81

<211> 9

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<213> Intelligent people

<400> 81

Ile Leu Ala Glu Glu Pro Ile Tyr Ile

1 5

<210> 82

<211> 11

<212> PRT

<213> Intelligent people

<400> 82

Ile Leu Ala Glu Glu Pro Ile Tyr Ile Arg Val

1 5 10

<210> 83

<211> 9

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<213> Intelligent people

<400> 83

Gly Leu Leu Glu Asn Ser Pro His Leu

1 5

<210> 84

<211> 9

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<213> Intelligent people

<400> 84

Phe Leu Leu Glu Arg Glu Gln Leu Leu

1 5

<210> 85

<211> 10

<212> PRT

<213> Intelligent people

<400> 85

Lys Leu Leu Asp Lys Pro Glu Gln Phe Leu

1 5 10

<210> 86

<211> 9

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<213> Intelligent people

<400> 86

Ser Leu Phe Ser Asn Ile Glu Ser Val

1 5

<210> 87

<211> 9

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<213> Intelligent people

<400> 87

Lys Leu Leu Ser Leu Leu Glu Glu Ala

1 5

<210> 88

<211> 10

<212> PRT

<213> Intelligent people

<400> 88

Leu Leu Leu Pro Leu Glu Leu Ser Leu Ala

1 5 10

<210> 89

<211> 9

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<213> Intelligent people

<400> 89

Ser Leu Ala Glu Thr Ile Phe Ile Val

1 5

<210> 90

<211> 11

<212> PRT

<213> Intelligent people

<400> 90

Ala Ile Leu Asn Val Asp Glu Lys Asn Gln Val

1 5 10

<210> 91

<211> 9

<212> PRT

<213> Intelligent people

<400> 91

Leu Leu Pro Ser Ile Phe Leu Met Val

1 5

<210> 92

<211> 9

<212> PRT

<213> Intelligent people

<400> 92

Arg Leu Phe Glu Glu Val Leu Gly Val

1 5

<210> 93

<211> 9

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<213> Intelligent people

<400> 93

Arg Leu Tyr Gly Tyr Phe His Asp Ala

1 5

<210> 94

<211> 9

<212> PRT

<213> Intelligent people

<400> 94

Tyr Leu Asp Glu Val Ala Phe Met Leu

1 5

<210> 95

<211> 11

<212> PRT

<213> Intelligent people

<400> 95

Lys Leu Ile Asp Glu Asp Glu Pro Leu Phe Leu

1 5 10

<210> 96

<211> 9

<212> PRT

<213> Intelligent people

<400> 96

Ala Leu Asp Thr Thr Arg His Glu Leu

1 5

<210> 97

<211> 9

<212> PRT

<213> Intelligent people

<400> 97

Lys Leu Phe Glu Lys Ser Thr Gly Leu

1 5

<210> 98

<211> 9

<212> PRT

<213> Intelligent people

<400> 98

Phe Val Gln Glu Lys Ile Pro Glu Leu

1 5

<210> 99

<211> 10

<212> PRT

<213> Intelligent people

<400> 99

Thr Leu Phe Gly Ile Gln Leu Thr Glu Ala

1 5 10

<210> 100

<211> 9

<212> PRT

<213> Intelligent people

<400> 100

Ala Leu Gln Ser Phe Glu Phe Arg Val

1 5

<210> 101

<211> 11

<212> PRT

<213> Intelligent people

<400> 101

Ser Leu Leu Glu Val Asn Glu Ala Ser Ser Val

1 5 10

<210> 102

<211> 10

<212> PRT

<213> Intelligent people

<400> 102

Gly Leu Tyr Pro Val Thr Leu Val Gly Val

1 5 10

<210> 103

<211> 9

<212> PRT

<213> Intelligent people

<400> 103

Tyr Leu Ala Asp Thr Val Gln Lys Leu

1 5

<210> 104

<211> 12

<212> PRT

<213> Intelligent people

<400> 104

Asp Leu Pro Thr Gln Glu Pro Ala Leu Gly Thr Thr

1 5 10

<210> 105

<211> 9

<212> PRT

<213> Intelligent people

<400> 105

Ala Met Leu Ala Ser Gln Thr Glu Ala

1 5

<210> 106

<211> 10

<212> PRT

<213> Intelligent people

<400> 106

Val Leu Leu Gly Ser Val Val Ile Phe Ala

1 5 10

<210> 107

<211> 11

<212> PRT

<213> Intelligent people

<400> 107

Arg Val Leu Pro Gly Gln Ala Val Thr Gly Val

1 5 10

<210> 108

<211> 11

<212> PRT

<213> Intelligent people

<400> 108

Phe Ile Ala Asn Leu Pro Pro Glu Leu Lys Ala

1 5 10

<210> 109

<211> 9

<212> PRT

<213> Intelligent people

<400> 109

Ile Leu Gly Ser Phe Glu Leu Gln Leu

1 5

<210> 110

<211> 9

<212> PRT

<213> Intelligent people

<400> 110

Gln Ile Gln Gly Gln Val Ser Glu Val

1 5

<210> 111

<211> 10

<212> PRT

<213> Intelligent people

<400> 111

Ala Gln Leu Glu Gly Lys Leu Val Ser Ile

1 5 10

<210> 112

<211> 9

<212> PRT

<213> Intelligent people

<400> 112

Ile Leu Ala Gln Asp Val Ala Gln Leu

1 5

<210> 113

<211> 9

<212> PRT

<213> Intelligent people

<400> 113

Phe Leu Phe Leu Lys Glu Val Lys Val

1 5

<210> 114

<211> 10

<212> PRT

<213> Intelligent people

<400> 114

Leu Leu Phe Pro Ser Asp Val Gln Thr Leu

1 5 10

<210> 115

<211> 9

<212> PRT

<213> Intelligent people

<400> 115

Ile Leu His Gly Glu Val Asn Lys Val

1 5

<210> 116

<211> 9

<212> PRT

<213> Intelligent people

<400> 116

Ala Leu Leu Ser Ser Val Ala Glu Ala

1 5

<210> 117

<211> 9

<212> PRT

<213> Intelligent people

<400> 117

Thr Leu Leu Glu Gly Ile Ser Arg Ala

1 5

<210> 118

<211> 10

<212> PRT

<213> Intelligent people

<400> 118

Ile Ala Tyr Asn Pro Asn Gly Asn Ala Leu

1 5 10

<210> 119

<211> 9

<212> PRT

<213> Intelligent people

<400> 119

Ser Leu Ile Glu Glu Ser Glu Glu Leu

1 5

<210> 120

<211> 11

<212> PRT

<213> Intelligent people

<400> 120

Leu Gln Leu Ser Pro Leu Lys Gly Leu Ser Leu

1 5 10

<210> 121

<211> 9

<212> PRT

<213> Intelligent people

<400> 121

Ala Leu Tyr Val Gln Ala Pro Thr Val

1 5

<210> 122

<211> 9

<212> PRT

<213> Intelligent people

<400> 122

Ser Ile Ile Asp Thr Glu Leu Lys Val

1 5

<210> 123

<211> 11

<212> PRT

<213> Intelligent people

<400> 123

Gln Thr Ala Pro Glu Glu Ala Phe Ile Lys Leu

1 5 10

<210> 124

<211> 9

<212> PRT

<213> Intelligent people

<400> 124

Ala Leu Leu Leu Arg Leu Phe Thr Ile

1 5

<210> 125

<211> 9

<212> PRT

<213> Intelligent people

<400> 125

Ala Ala Leu Glu Val Leu Ala Glu Val

1 5

<210> 126

<211> 9

<212> PRT

<213> Intelligent people

<400> 126

Gln Leu Arg Glu Ala Phe Glu Gln Leu

1 5

<210> 127

<211> 11

<212> PRT

<213> Intelligent people

<400> 127

Ile Met Lys Ala Thr Gly Leu Gly Ile Gln Leu

1 5 10

<210> 128

<211> 9

<212> PRT

<213> Intelligent people

<400> 128

Ser Ile Leu Thr Asn Ile Ser Glu Val

1 5

<210> 129

<211> 9

<212> PRT

<213> Intelligent people

<400> 129

Lys Met Ala Ser Lys Val Thr Gln Val

1 5

<210> 130

<211> 9

<212> PRT

<213> Intelligent people

<400> 130

Gln Leu Tyr Gly Ser Ala Ile Thr Leu

1 5

<210> 131

<211> 9

<212> PRT

<213> Intelligent people

<400> 131

Ser Leu Tyr Pro His Phe Thr Leu Leu

1 5

<210> 132

<211> 9

<212> PRT

<213> Intelligent people

<400> 132

Ala Leu Leu Asn Asn Val Ile Glu Val

1 5

<210> 133

<211> 9

<212> PRT

<213> Intelligent people

<400> 133

Phe Leu Asp Gly Arg Pro Leu Thr Leu

1 5

<210> 134

<211> 9

<212> PRT

<213> Intelligent people

<400> 134

Ser Leu Tyr Lys Ser Phe Leu Gln Leu

1 5

<210> 135

<211> 9

<212> PRT

<213> Intelligent people

<400> 135

His Leu Asp Thr Val Lys Ile Glu Val

1 5

<210> 136

<211> 9

<212> PRT

<213> Intelligent people

<400> 136

Leu Leu Trp Asp Ala Pro Ala Lys Cys

1 5

<210> 137

<211> 10

<212> PRT

<213> Intelligent people

<400> 137

Lys Leu Ile Tyr Lys Asp Leu Val Ser Val

1 5 10

<210> 138

<211> 9

<212> PRT

<213> Intelligent people

<400> 138

Gly Ile Ile Asn Lys Leu Val Thr Val

1 5

<210> 139

<211> 9

<212> PRT

<213> Intelligent people

<400> 139

Ile Ile Leu Glu Asn Ile Gln Ser Leu

1 5

<210> 140

<211> 9

<212> PRT

<213> Intelligent people

<400> 140

Phe Leu Asp Ser Gln Ile Thr Thr Val

1 5

<210> 141

<211> 9

<212> PRT

<213> Intelligent people

<400> 141

Asn Ile Asp Ile Asn Asn Asn Glu Leu

1 5

<210> 142

<211> 9

<212> PRT

<213> Intelligent people

<400> 142

Leu Leu Asp Ala Ala His Ala Ser Ile

1 5

<210> 143

<211> 9

<212> PRT

<213> Intelligent people

<400> 143

Met Leu Trp Glu Ser Ile Met Arg Val

1 5

<210> 144

<211> 9

<212> PRT

<213> Intelligent people

<400> 144

Phe Leu Ile Ser Gln Thr Pro Leu Leu

1 5

<210> 145

<211> 9

<212> PRT

<213> Intelligent people

<400> 145

Ala Leu Glu Glu Lys Leu Glu Asn Val

1 5

<210> 146

<211> 9

<212> PRT

<213> Intelligent people

<400> 146

Val Val Ala Ala His Leu Ala Gly Ala

1 5

<210> 147

<211> 9

<212> PRT

<213> Intelligent people

<400> 147

Gly Leu Leu Ser Ala Leu Glu Asn Val

1 5

<210> 148

<211> 9

<212> PRT

<213> Intelligent people

<400> 148

Tyr Leu Ile Leu Ser Ser His Gln Leu

1 5

<210> 149

<211> 10

<212> PRT

<213> Intelligent people

<400> 149

Asn Met Ala Asp Gly Gln Leu His Gln Val

1 5 10

<210> 150

<211> 9

<212> PRT

<213> Intelligent people

<400> 150

Val Leu Leu Asp Met Val His Ser Leu

1 5

<210> 151

<211> 9

<212> PRT

<213> Intelligent people

<400> 151

Asp Ile Ser Lys Arg Ile Gln Ser Leu

1 5

<210> 152

<211> 9

<212> PRT

<213> Intelligent people

<400> 152

Ile Leu Val Thr Ser Ile Phe Phe Leu

1 5

<210> 153

<211> 9

<212> PRT

<213> Intelligent people

<400> 153

Lys Leu Val Glu Leu Glu His Thr Leu

1 5

<210> 154

<211> 9

<212> PRT

<213> Intelligent people

<400> 154

Ala Ile Ile Lys Glu Ile Gln Thr Val

1 5

<210> 155

<211> 9

<212> PRT

<213> Intelligent people

<400> 155

Thr Leu Asp Ser Tyr Leu Lys Ala Val

1 5

<210> 156

<211> 9

<212> PRT

<213> Intelligent people

<400> 156

Val Ile Leu Thr Ser Ser Pro Phe Leu

1 5

<210> 157

<211> 9

<212> PRT

<213> Intelligent people

<400> 157

Ile Leu Gln Asp Gly Gln Phe Leu Val

1 5

<210> 158

<211> 9

<212> PRT

<213> Intelligent people

<400> 158

Tyr Leu Asp Pro Leu Trp His Gln Leu

1 5

<210> 159

<211> 9

<212> PRT

<213> Intelligent people

<400> 159

Gln Leu Gly Pro Val Pro Val Thr Ile

1 5

<210> 160

<211> 9

<212> PRT

<213> Intelligent people

<400> 160

Thr Leu Gln Glu Trp Leu Thr Glu Val

1 5

<210> 161

<211> 9

<212> PRT

<213> Intelligent people

<400> 161

Asn Leu Leu Asp Glu Asn Val Cys Leu

1 5

<210> 162

<211> 10

<212> PRT

<213> Intelligent people

<400> 162

Gly Leu Leu Gly Asn Leu Leu Thr Ser Leu

1 5 10

<210> 163

<211> 9

<212> PRT

<213> Intelligent people

<400> 163

Gly Leu Glu Glu Arg Leu Tyr Thr Ala

1 5

<210> 164

<211> 9

<212> PRT

<213> Intelligent people

<400> 164

Met Leu Ile Ile Arg Val Pro Ser Val

1 5

<210> 165

<211> 9

<212> PRT

<213> Intelligent people

<400> 165

Ser Leu Leu Asp Tyr Glu Val Ser Ile

1 5

<210> 166

<211> 9

<212> PRT

<213> Intelligent people

<400> 166

Leu Leu Gly Asp Ser Ser Phe Phe Leu

1 5

<210> 167

<211> 11

<212> PRT

<213> Intelligent people

<400> 167

Leu Val Val Asp Glu Gly Ser Leu Val Ser Val

1 5 10

<210> 168

<211> 10

<212> PRT

<213> Intelligent people

<400> 168

Val Ile Phe Glu Gly Glu Pro Met Tyr Leu

1 5 10

<210> 169

<211> 9

<212> PRT

<213> Intelligent people

<400> 169

Ala Leu Ala Asp Leu Ser Val Ala Val

1 5

<210> 170

<211> 9

<212> PRT

<213> Intelligent people

<400> 170

Phe Ile Ala Ala Val Val Glu Lys Val

1 5

<210> 171

<211> 9

<212> PRT

<213> Intelligent people

<400> 171

Leu Leu Leu Leu Asp Val Pro Thr Ala

1 5

<210> 172

<211> 12

<212> PRT

<213> Intelligent people

<400> 172

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu

1 5 10

<210> 173

<211> 9

<212> PRT

<213> Intelligent people

<400> 173

Arg Leu Ile Asp Ile Tyr Lys Asn Val

1 5

<210> 174

<211> 10

<212> PRT

<213> Intelligent people

<400> 174

Ala Leu Tyr Ser Gly Asp Leu His Ala Ala

1 5 10

<210> 175

<211> 9

<212> PRT

<213> Intelligent people

<400> 175

Ser Leu Leu Asp Leu Val Gln Ser Leu

1 5

<210> 176

<211> 9

<212> PRT

<213> Intelligent people

<400> 176

Val Gln Ser Gly Leu Arg Ile Leu Leu

1 5

<210> 177

<211> 9

<212> PRT

<213> Intelligent people

<400> 177

Ala Leu Ile Asn Val Leu Asn Ala Leu

1 5

<210> 178

<211> 9

<212> PRT

<213> Intelligent people

<400> 178

Ser Leu Val Ser Trp Gln Leu Leu Leu

1 5

<210> 179

<211> 9

<212> PRT

<213> Intelligent people

<400> 179

Thr Leu Gly Glu Ile Ile Lys Gly Val

1 5

<210> 180

<211> 9

<212> PRT

<213> Intelligent people

<400> 180

Arg Leu Tyr Glu Glu Glu Ile Arg Ile

1 5

<210> 181

<211> 9

<212> PRT

<213> Intelligent people

<400> 181

Leu Leu Trp Ala Pro Thr Ala Gln Ala

1 5

<210> 182

<211> 10

<212> PRT

<213> Intelligent people

<400> 182

Gly Leu Gln Asp Gly Phe Gln Ile Thr Val

1 5 10

<210> 183

<211> 9

<212> PRT

<213> Intelligent people

<400> 183

Ala Leu Ser Tyr Ile Leu Pro Tyr Leu

1 5

<210> 184

<211> 9

<212> PRT

<213> Intelligent people

<400> 184

Ala Leu Asp Ser Thr Ile Ala His Leu

1 5

<210> 185

<211> 10

<212> PRT

<213> Intelligent people

<400> 185

Thr Leu Tyr Gln Gly Leu Pro Ala Glu Val

1 5 10

<210> 186

<211> 9

<212> PRT

<213> Intelligent people

<400> 186

Ser Leu Leu Ser Leu Glu Ser Arg Leu

1 5

<210> 187

<211> 9

<212> PRT

<213> Intelligent people

<400> 187

Ser Ile Leu Lys Glu Asp Pro Phe Leu

1 5

<210> 188

<211> 9

<212> PRT

<213> Intelligent people

<400> 188

Val Leu Gly Glu Glu Gln Glu Gly Val

1 5

<210> 189

<211> 9

<212> PRT

<213> Intelligent people

<400> 189

Met Ala Val Ser Asp Leu Leu Ile Leu

1 5

<210> 190

<211> 9

<212> PRT

<213> Intelligent people

<400> 190

Ser Leu Ser Thr Glu Leu Phe Lys Val

1 5

<210> 191

<211> 9

<212> PRT

<213> Intelligent people

<400> 191

Ala Ala Ile Glu Ile Phe Glu Lys Val

1 5

<210> 192

<211> 11

<212> PRT

<213> Intelligent people

<400> 192

Thr Leu Leu Pro Ser Ser Gly Leu Val Thr Leu

1 5 10

<210> 193

<211> 9

<212> PRT

<213> Intelligent people

<400> 193

Ala Leu Phe His Met Asn Ile Leu Leu

1 5

<210> 194

<211> 9

<212> PRT

<213> Intelligent people

<400> 194

Lys Leu Leu Glu Glu Val Gln Leu Leu

1 5

<210> 195

<211> 9

<212> PRT

<213> Intelligent people

<400> 195

Val Ile Ile Gln Asn Leu Pro Ala Leu

1 5

<210> 196

<211> 9

<212> PRT

<213> Intelligent people

<400> 196

Thr Leu His Gln Trp Ile Tyr Tyr Leu

1 5

<210> 197

<211> 10

<212> PRT

<213> Intelligent people

<400> 197

Leu Gly Gly Pro Thr Ser Leu Leu His Val

1 5 10

<210> 198

<211> 9

<212> PRT

<213> Intelligent people

<400> 198

Ile Leu Thr Asn Lys Val Val Ser Val

1 5

<210> 199

<211> 9

<212> PRT

<213> Intelligent people

<400> 199

Ser Val Ala Asp Leu Ala His Val Leu

1 5

<210> 200

<211> 10

<212> PRT

<213> Intelligent people

<400> 200

Ile Met Pro Thr Phe Asp Leu Thr Lys Val

1 5 10

<210> 201

<211> 9

<212> PRT

<213> Intelligent people

<400> 201

Leu Leu Phe Ser Leu Leu Cys Glu Ala

1 5

<210> 202

<211> 9

<212> PRT

<213> Intelligent people

<400> 202

Ala Leu Ala Lys Asp Glu Leu Ser Leu

1 5

<210> 203

<211> 9

<212> PRT

<213> Intelligent people

<400> 203

Phe Leu Phe Val Asp Pro Glu Leu Val

1 5

<210> 204

<211> 11

<212> PRT

<213> Intelligent people

<400> 204

Ser Glu Trp Gly Ser Pro His Ala Ala Val Pro

1 5 10

<210> 205

<211> 9

<212> PRT

<213> Intelligent people

<400> 205

Leu Ala Phe Gly Tyr Asp Asp Glu Leu

1 5

<210> 206

<211> 9

<212> PRT

<213> Intelligent people

<400> 206

Gly Leu Asp Ala Phe Arg Ile Phe Leu

1 5

<210> 207

<211> 9

<212> PRT

<213> Intelligent people

<400> 207

Lys Leu Phe Glu Thr Val Glu Glu Leu

1 5

<210> 208

<211> 9

<212> PRT

<213> Intelligent people

<400> 208

His Leu Asn Asn Asp Arg Asn Pro Leu

1 5

<210> 209

<211> 10

<212> PRT

<213> Intelligent people

<400> 209

Val Leu Gln Thr Glu Glu Leu Val Ala Asn

1 5 10

<210> 210

<211> 9

<212> PRT

<213> Intelligent people

<400> 210

Gly Leu Ala Gly Asp Asn Ile Tyr Leu

1 5

<210> 211

<211> 9

<212> PRT

<213> Intelligent people

<400> 211

Leu Leu Thr Thr Val Leu Ile Asn Ala

1 5

<210> 212

<211> 9

<212> PRT

<213> Intelligent people

<400> 212

Met Thr Leu Ser Glu Ile His Ala Val

1 5

<210> 213

<211> 10

<212> PRT

<213> Intelligent people

<400> 213

Ile Leu Ala Val Asp Gly Val Leu Ser Val

1 5 10

<210> 214

<211> 9

<212> PRT

<213> Intelligent people

<400> 214

Ala Leu Phe Glu Thr Leu Ile Gln Leu

1 5

<210> 215

<211> 9

<212> PRT

<213> Intelligent people

<400> 215

Gln Ile Ala Asp Ile Val Thr Ser Val

1 5

<210> 216

<211> 9

<212> PRT

<213> Intelligent people

<400> 216

Ala Leu Ser Thr Val Thr Pro Arg Ile

1 5

<210> 217

<211> 9

<212> PRT

<213> Intelligent people

<400> 217

Leu Leu Trp Pro Ser Ser Val Pro Ala

1 5

<210> 218

<211> 9

<212> PRT

<213> Intelligent people

<400> 218

Ser Leu Thr Gly Ala Asn Ile Thr Val

1 5

<210> 219

<211> 9

<212> PRT

<213> Intelligent people

<400> 219

Gly Val Val Pro Thr Ile Gln Lys Val

1 5

<210> 220

<211> 9

<212> PRT

<213> Intelligent people

<400> 220

Ala Leu Ser Glu Leu Glu Arg Val Leu

1 5

<210> 221

<211> 9

<212> PRT

<213> Intelligent people

<400> 221

Ile Met Leu Asn Ser Val Glu Glu Ile

1 5

<210> 222

<211> 9

<212> PRT

<213> Intelligent people

<400> 222

Leu Leu Thr Gly Val Phe Ala Gln Leu

1 5

<210> 223

<211> 9

<212> PRT

<213> Intelligent people

<400> 223

Ala Leu His Pro Val Gln Phe Tyr Leu

1 5

<210> 224

<211> 13

<212> PRT

<213> Intelligent people

<400> 224

Leu Leu Phe Asp Trp Ser Gly Thr Gly Arg Ala Asp Ala

1 5 10

<210> 225

<211> 10

<212> PRT

<213> Intelligent people

<400> 225

Phe Leu Pro Gln Pro Val Pro Leu Ser Val

1 5 10

<210> 226

<211> 9

<212> PRT

<213> Intelligent people

<400> 226

Ser Leu Ala Gly Asn Leu Gln Glu Leu

1 5

<210> 227

<211> 9

<212> PRT

<213> Intelligent people

<400> 227

Ser Glu Met Glu Glu Leu Pro Ser Val

1 5

<210> 228

<211> 12

<212> PRT

<213> Intelligent people

<400> 228

Ser Leu Leu Glu Leu Asp Gly Ile Asn Leu Arg Leu

1 5 10

<210> 229

<211> 9

<212> PRT

<213> Intelligent people

<400> 229

Tyr Leu Tyr Glu Leu Glu His Ala Leu

1 5

<210> 230

<211> 9

<212> PRT

<213> Intelligent people

<400> 230

Lys Leu Leu Asn Met Ile Phe Ser Ile

1 5

<210> 231

<211> 9

<212> PRT

<213> Intelligent people

<400> 231

Leu Leu Asp Asp Ile Phe Ile Arg Leu

1 5

<210> 232

<211> 9

<212> PRT

<213> Intelligent people

<400> 232

Leu Val Val Gly Gly Ile Ala Thr Val

1 5

<210> 233

<211> 9

<212> PRT

<213> Intelligent people

<400> 233

Ser Leu Phe Glu Ser Leu Glu Tyr Leu

1 5

<210> 234

<211> 10

<212> PRT

<213> Intelligent people

<400> 234

Val Leu Leu Asn Glu Ile Leu Glu Gln Val

1 5 10

<210> 235

<211> 9

<212> PRT

<213> Intelligent people

<400> 235

Ser Leu Leu Asn Gln Pro Lys Ala Val

1 5

<210> 236

<211> 9

<212> PRT

<213> Intelligent people

<400> 236

Lys Met Ser Glu Leu Gln Thr Tyr Val

1 5

<210> 237

<211> 11

<212> PRT

<213> Intelligent people

<400> 237

Ala Leu Leu Glu Gln Thr Gly Asp Met Ser Leu

1 5 10

<210> 238

<211> 9

<212> PRT

<213> Intelligent people

<400> 238

His Leu Gln Glu Lys Leu Gln Ser Leu

1 5

<210> 239

<211> 11

<212> PRT

<213> Intelligent people

<400> 239

Val Ile Ile Lys Gly Leu Glu Glu Ile Thr Val

1 5 10

<210> 240

<211> 9

<212> PRT

<213> Intelligent people

<400> 240

Ser Val Gln Glu Asn Ile Gln Gln Lys

1 5

<210> 241

<211> 9

<212> PRT

<213> Intelligent people

<400> 241

Lys Gln Phe Glu Gly Thr Val Glu Ile

1 5

<210> 242

<211> 9

<212> PRT

<213> Intelligent people

<400> 242

Lys Leu Gln Glu Glu Ile Pro Val Leu

1 5

<210> 243

<211> 9

<212> PRT

<213> Intelligent people

<400> 243

Gly Leu Ala Glu Phe Gln Glu Asn Val

1 5

<210> 244

<211> 9

<212> PRT

<213> Intelligent people

<400> 244

Asn Val Ala Glu Ile Val Ile His Ile

1 5

<210> 245

<211> 9

<212> PRT

<213> Intelligent people

<400> 245

Ala Leu Leu Glu Glu Glu Glu Gly Val

1 5

<210> 246

<211> 9

<212> PRT

<213> Intelligent people

<400> 246

Ala Leu Ala Gly Ile Val Thr Asn Val

1 5

<210> 247

<211> 12

<212> PRT

<213> Intelligent people

<400> 247

Asn Leu Leu Ile Asp Asp Lys Gly Thr Ile Lys Leu

1 5 10

<210> 248

<211> 10

<212> PRT

<213> Intelligent people

<400> 248

Val Leu Met Gln Asp Ser Arg Leu Tyr Leu

1 5 10

<210> 249

<211> 9

<212> PRT

<213> Intelligent people

<400> 249

Tyr Leu Tyr Gln Ile Leu Gln Gly Ile

1 5

<210> 250

<211> 9

<212> PRT

<213> Intelligent people

<400> 250

Leu Met Gln Asp Ser Arg Leu Tyr Leu

1 5

<210> 251

<211> 9

<212> PRT

<213> Intelligent people

<400> 251

Leu Leu Trp Gly Asn Leu Pro Glu Ile

1 5

<210> 252

<211> 9

<212> PRT

<213> Intelligent people

<400> 252

Ser Leu Met Glu Lys Asn Gln Ser Leu

1 5

<210> 253

<211> 9

<212> PRT

<213> Intelligent people

<400> 253

Lys Leu Leu Ala Val Ile His Glu Leu

1 5

<210> 254

<211> 10

<212> PRT

<213> Intelligent people

<400> 254

Ala Leu Gly Asp Lys Phe Leu Leu Arg Val

1 5 10

<210> 255

<211> 11

<212> PRT

<213> Intelligent people

<400> 255

Phe Leu Met Lys Asn Ser Asp Leu Tyr Gly Ala

1 5 10

<210> 256

<211> 9

<212> PRT

<213> Intelligent people

<400> 256

Phe Leu Asn Asp Ile Phe Glu Arg Ile

1 5

<210> 257

<211> 10

<212> PRT

<213> Intelligent people

<400> 257

Lys Leu Ile Asp His Gln Gly Leu Tyr Leu

1 5 10

<210> 258

<211> 9

<212> PRT

<213> Intelligent people

<400> 258

Gln Leu Val Gln Arg Val Ala Ser Val

1 5

<210> 259

<211> 12

<212> PRT

<213> Intelligent people

<400> 259

Gly Pro Gly Ile Phe Pro Pro Pro Pro Pro Gln Pro

1 5 10

<210> 260

<211> 9

<212> PRT

<213> Intelligent people

<400> 260

Ala Leu Asn Glu Ser Leu Val Glu Cys

1 5

<210> 261

<211> 9

<212> PRT

<213> Intelligent people

<400> 261

Gly Leu Ala Ala Leu Ala Val His Leu

1 5

<210> 262

<211> 9

<212> PRT

<213> Intelligent people

<400> 262

Leu Leu Leu Glu Ala Val Trp His Leu

1 5

<210> 263

<211> 9

<212> PRT

<213> Intelligent people

<400> 263

Ser Ile Ile Glu Tyr Leu Pro Thr Leu

1 5

<210> 264

<211> 9

<212> PRT

<213> Intelligent people

<400> 264

Thr Leu His Asp Gln Val His Leu Leu

1 5

<210> 265

<211> 11

<212> PRT

<213> Intelligent people

<400> 265

Phe Leu Leu Asp Lys Pro Gln Asp Leu Ser Ile

1 5 10

<210> 266

<211> 9

<212> PRT

<213> Intelligent people

<400> 266

Phe Leu Leu Asp Lys Pro Gln Asp Leu

1 5

<210> 267

<211> 10

<212> PRT

<213> Intelligent people

<400> 267

Tyr Leu Leu Asp Met Pro Leu Trp Tyr Leu

1 5 10

<210> 268

<211> 9

<212> PRT

<213> Intelligent people

<400> 268

Ser Leu Asp Lys Asp Ile Val Ala Leu

1 5

<210> 269

<211> 9

<212> PRT

<213> Intelligent people

<400> 269

Gly Leu Leu Asp Cys Pro Ile Phe Leu

1 5

<210> 270

<211> 9

<212> PRT

<213> Intelligent people

<400> 270

Thr Leu Leu Thr Phe Phe His Glu Leu

1 5

<210> 271

<211> 9

<212> PRT

<213> Intelligent people

<400> 271

Val Leu Ile Glu Tyr Asn Phe Ser Ile

1 5

<210> 272

<211> 10

<212> PRT

<213> Intelligent people

<400> 272

Phe Val Met Glu Gly Glu Pro Pro Lys Leu

1 5 10

<210> 273

<211> 9

<212> PRT

<213> Intelligent people

<400> 273

Ser Leu Asn Lys Gln Ile Glu Thr Val

1 5

<210> 274

<211> 11

<212> PRT

<213> Intelligent people

<400> 274

Thr Leu Tyr Asn Pro Glu Arg Thr Ile Thr Val

1 5 10

<210> 275

<211> 9

<212> PRT

<213> Intelligent people

<400> 275

Ala Val Pro Pro Pro Pro Ser Ser Val

1 5

<210> 276

<211> 9

<212> PRT

<213> Intelligent people

<400> 276

Arg Met Pro Thr Val Leu Gln Cys Val

1 5

<210> 277

<211> 9

<212> PRT

<213> Intelligent people

<400> 277

Lys Leu Gln Glu Glu Leu Asn Lys Val

1 5

<210> 278

<211> 9

<212> PRT

<213> Intelligent people

<400> 278

Val Leu Glu Asp Lys Val Leu Ser Val

1 5

<210> 279

<211> 11

<212> PRT

<213> Intelligent people

<400> 279

Val Leu Met Asp Glu Gly Ala Val Leu Thr Leu

1 5 10

<210> 280

<211> 9

<212> PRT

<213> Intelligent people

<400> 280

His Leu Trp Gly His Ala Leu Phe Leu

1 5

<210> 281

<211> 12

<212> PRT

<213> Intelligent people

<400> 281

Leu Leu Leu Glu Ser Asp Pro Lys Val Tyr Ser Leu

1 5 10

<210> 282

<211> 9

<212> PRT

<213> Intelligent people

<400> 282

Ser Leu Tyr Ala Leu His Val Lys Ala

1 5

<210> 283

<211> 9

<212> PRT

<213> Intelligent people

<400> 283

Ala Leu Ser Glu Leu Leu Gln Gln Val

1 5

<210> 284

<211> 11

<212> PRT

<213> Intelligent people

<400> 284

Lys Leu Met Asp Pro Gly Ser Leu Pro Pro Leu

1 5 10

<210> 285

<211> 9

<212> PRT

<213> Intelligent people

<400> 285

Met Leu Leu Asp Thr Val Gln Lys Val

1 5

<210> 286

<211> 9

<212> PRT

<213> Intelligent people

<400> 286

Phe Leu Thr Glu Met Val His Phe Ile

1 5

<210> 287

<211> 9

<212> PRT

<213> Intelligent people

<400> 287

Lys Ile Gln Glu Ile Leu Thr Gln Val

1 5

<210> 288

<211> 9

<212> PRT

<213> Intelligent people

<400> 288

Ser Leu Tyr Lys Gly Leu Leu Ser Val

1 5

<210> 289

<211> 10

<212> PRT

<213> Intelligent people

<400> 289

Glu Leu Ala Gly Ile Gly Ile Leu Thr Val

1 5 10

<210> 290

<211> 9

<212> PRT

<213> Intelligent people

<400> 290

Tyr Leu Leu Pro Ala Ile Val His Ile

1 5