Peptides and combination of peptides of non-cancer derivations

Abstract

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention also relates to tumor-associated T cell peptide epitopes, alone or in combination with other tumor-associated peptides (active pharmaceutical ingredients of vaccine complexes that stimulate anti-tumor immune responses or stimulate T cells ex vivo and transfer into patients). Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, may also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Description

[0001] This application is a divisional application of an invention patent application with an application date of February 8, 2019, application number 201980013690.5, and invention name “Peptides and peptide combinations of non-classical origin for immunotherapy of different types of cancer”.

[0002] The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapy methods. In particular, the present invention relates to immunotherapy for cancer. The present invention also relates to tumor-associated T cell (CTL) peptide epitopes used alone or in combination with other tumor-associated peptides (active pharmaceutical ingredients of vaccine complexes that stimulate anti-tumor immune responses or stimulate T cells in vitro and transfer to patients). Peptides that bind to major histocompatibility complex (MHC) molecules or peptides of the same class may also be targets for antibodies, soluble T cell receptors and other binding molecules.

[0003] The present invention relates to several novel peptide sequences and variants thereof, which are derived from HLA class I molecules of human tumor cells and can be used in vaccine compositions for eliciting anti-tumor immune responses or as targets for developing pharmaceutical / immunologically active compounds and cells. Background of the Invention

[0005] According to the World Health Organization (WHO), cancer was one of the top four non-communicable killer diseases worldwide in 2012. In the same year, colorectal cancer, breast cancer and respiratory cancer were among the top 10 causes of death in high-income countries.

[0006] GBM is the most common malignant tumor of the central nervous system, with an age-adjusted incidence of 3.19 per 100,000 residents in the United States. GBM has a very poor prognosis, with a 1-year survival rate of 35% and a 5-year survival rate of less than 5%. Male sex, older age, and race appear to be risk factors for GBM (Thakkar et al., 2014).

[0007] CLL is the most common leukemia in the Western world, accounting for approximately one-third of all leukemia cases. The incidence rate is similar in the United States and Europe, with an estimated 16,000 new cases each year. CLL is more common in Caucasians than in Africans, less common in Hispanics and Native Americans, and rare in Asians. In people of Asian descent, the incidence of CLL is three times lower than in Caucasians (Gunawardana et al., 2008). The five-year overall survival rate for patients with CLL is approximately 79%.

[0008] AML is the second most common type of leukemia diagnosed in adults and children. In the United States, the estimated number of new cases is about 21,000 per year. The five-year survival rate for patients with AML is about 25%.

[0009] Lung cancer is the most common type of cancer worldwide and the leading cause of cancer death in many countries. Lung cancer is divided into small cell lung cancer and non-small cell lung cancer. NSCLC includes histological types adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, and in the United States, it accounts for about 85% of all lung cancer cases. The occurrence of NSCLC is closely related to smoking history, including current and former smokers, and the five-year survival rate is reported to be 15% (World Cancer Report, 2014; MoIina et aI., 2008).

[0010] Given the serious side effects and costs associated with treating cancer, there is a need to identify factors that can be used to treat cancer in general, and in particular for AML (acute myeloid leukemia), BRCA (breast cancer), CCC (cholangiocellular carcinoma), CLL (chronic lymphocytic leukemia), CRC (colorectal cancer), GBC (gallbladder cancer), GBM (glioblastoma), GC (gastric cancer), GEJC (gastroesophageal junction cancer), HCC (hepatocellular carcinoma), HNSCC (head and neck squamous cell carcinoma), MEL (melanoma), NHL (non-Hodgkin lymphoma), NSCLC (non-small cell lung cancer), OC (ovarian cancer), OSCAR (esophageal cancer), PACA (pancreatic cancer), PRCA (prostate cancer), RCC (renal cell carcinoma), SCLC (small cell lung cancer), UBC (bladder cancer), and UEC (endometrial cancer). There is also a need to identify factors that represent biomarkers for cancer in general, and for acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer, so that cancer can be better diagnosed, prognosis can be assessed, and treatment success can be predicted.

[0011] Cancer immunotherapy represents an option for specific targeting of cancer cells while minimizing side effects. Cancer immunotherapy exploits the presence of tumor-associated antigens.

[0012] The current classification of tumor-associated antigens (TAAs) mainly includes the following groups:

[0013] a) Cancer-testis antigens: The first TAAs recognized by cells belong to this class of antigens, and were initially 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 testicular cells do not express HLA class I and II molecules, these antigens cannot be recognized by T cells in normal tissues and can therefore be considered immunologically tumor-specific. Well-known examples of CT antigens are members of the MAGE family and NY-ESO-1.

[0014] b) Differentiation antigens: Both tumors and normal tissues (the tissues from which the tumor originates) contain TAAs. Most known differentiation antigens are found in melanoma and normal melanocytes. Many of these melanocyte lineage-related proteins are involved in the biosynthesis of melanin, so these proteins are not tumor-specific, but are still widely used in cancer immunotherapy. Examples include, but are not limited to, tyrosinase and Melan-A / MART-1 for melanoma or PSA for prostate cancer.

[0015] c) Overexpressed TAAs: TAAs encoded by genes that are widely expressed have been detected in histologically distinct tumors as well as in many normal tissues, generally at low expression levels. It is possible that many epitopes processed and potentially presented by normal tissues are below the threshold level for T cell recognition, and their overexpression in tumor cells can trigger an anti-cancer response by breaking previously established tolerance. Typical examples of such TAAs are Her-2 / neu, survivin, telomerase or WT1.

[0016] 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 can generally induce a strong immune response without the risk of autoimmune reactions to normal tissues. On the other hand, these TAAs are in most cases only associated with the exact tumor on which the TAA is identified, and are usually not shared between many individual tumors. In the case of proteins containing tumor-specific (associated) isoforms, peptide tumor specificity (or association) may also occur if the peptide is derived from a tumor (associated) exon.

[0017] e) TAAs resulting from aberrant post-translational modifications: These TAAs may be generated from proteins that are neither specific nor abundantly expressed in tumors, but are still tumor-associated due to post-translational processing that is primarily active on tumors. These TAAs arise from altered glycosylation patterns, leading to the production of novel epitopes for MUC1 by tumors or events such as protein splicing during degradation, which may or may not be tumor-specific.

[0018] f) Oncovirus proteins: These TTAs are viral proteins that play a key role in the carcinogenesis process and because they are foreign (non-human) proteins, they are able to stimulate T cell responses. Examples of such proteins are the human papillomavirus type 16 proteins, E6 and E7, which are expressed in cervical cancer.

[0019] A large portion of the human proteome is derived from non-canonical sources, such as alternative open reading frames (altORFs (Vanderperre et al., 2013)), endogenous retroviral elements, or involves additional post-transcriptional (alternative splicing of RNA (Nilsen and Graveley, 2010)) or post-translational processing (post-translational modifications (Khoury et al., 2011), proteasomal splicing (Liepe et al., 2016)) steps. Since many cellular processes involved in the generation of these non-canonical proteins and peptides are often altered in cancer cells, this part of the proteome provides a rich source of TAAs (Laumont and Perreault, 2018).

[0020] Many messenger RNAs (mRNAs) contain non-traditional alternative open reading frames (altORFs, (de Klerk and 't Hoen, 2015)) in addition to a reference open reading frame (ORF). These additional coding sequences can be of a small number of codons, i.e., usually less than 100 (small open reading frames; sORFs (Olexiouk et al., 2016)) to hundreds of codons in size, and can be located upstream, downstream, or even overlapping with the reference ORF. Translation of these altORFs occurs at different translation start sites and may occur in a different reading frame as the reference ORF. Small open reading frames exist not only in mRNAs, but have also been described in other regulatory RNAs (ncRNAs, (Nam et al., 2016)) that were previously considered non-coding. Studies have shown that many long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) can encode and widely translate small peptides (Anderson et al., 2015; Aspden et al., 2014).

[0021] Human endogenous retroviruses (HERVs) make up a large portion of the human genome (~8%). These viral elements were integrated into the genome millions of years ago and have been vertically transmitted from generation to generation since then. The vast majority of HERVs have lost their functional activity through mutation or truncation, but some endogenous retroviruses (such as members of the HERV-K clade) still encode functional genes and have been shown to form retrovirus-like particles (Subramanian et al., 2011). The transcription of HERV proviruses is controlled by epigenetic programs and remains silent under normal physiological conditions. However, for some diseases, especially various types of cancer, reactivation and overexpression have been described, resulting in active translation of viral proteins (Gonzalez-Cao et al., 2016; Kassiotis and Stoye, 2017). This tumor-specific expression of HERV-derived proteins can be used for different types of cancer immunotherapy (Krishnamurthy et al., 2015; Schiavetti et al., 2002).

[0022] T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins presented by major histocompatibility complex (MHC) molecules. The antigens recognized by tumor-specific T lymphocytes, i.e., their epitopes, can be molecules derived from all types of proteins, such as enzymes, receptors, transcription factors, etc., which are expressed in cells of the corresponding tumor and their expression is usually upregulated compared with homologous unaltered cells.

[0023] There are two types of MHC molecules: MHC I and MHC II. MHC I molecules consist of an α heavy chain and β-2-microglobulin, while MHC II molecules consist of an α and a β chain. Its three-dimensional structure forms a binding groove for non-covalent interaction with peptides.

[0024] MHC class I molecules can be found on most nucleated cells. They present peptides that are mainly endogenous proteins, defective ribosomal products (DRIPs) and peptides generated by cleavage of larger peptides. However, peptides originating from endosomal structures or exogenous sources are also often found on MHC class I molecules. This non-classical presentation of class I molecules is referred to in the literature as cross-presentation (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules are mainly found on professional antigen presenting cells (APCs) and mainly present peptides of exogenous or transmembrane proteins that are taken up by APCs during endocytosis and subsequently processed.

[0025] The complex of peptide and MHC I class is recognized by CD8 positive T cells loaded with corresponding T cell receptor (TCR), while the complex of peptide and MHC II class molecule is recognized by CD4 positive helper T cells loaded with corresponding TCR. It is well known in the art that TCR, peptide and MHC are thus present in a 1:1:1 stoichiometric amount.

[0026] CD4-positive helper T cells play an important role in inducing and maintaining effective responses of CD8-positive cytotoxic T cells. The identification of CD4-positive T cell epitopes derived from tumor-associated antigens (TAAs) may be very important for the development of drug products that can elicit anti-tumor immune responses (Gnjatic et al., 2003). At the tumor site, T helper cells maintain a cytokine environment that is friendly to cytotoxic T cells (CTLs) (Mortara et al., 2006) and attract effector cells such as CTLs, natural killer (NK) cells, macrophages and granulocytes (Hwang et al., 2007).

[0027] In the absence of inflammation, the expression of MHC class II molecules is mainly limited to cells of the immune system, especially professional antigen presenting cells (APCs), such as monocytes, monocyte-derived cells, macrophages, and dendritic cells. The expression of MHC class II molecules has been found in tumor cells of cancer patients (Dengjel et al., 2006).

[0028] The elongated (longer) peptides of the present invention can serve as MHC class II active epitopes.

[0029] Helper T cells activated by MHC class II epitopes play an important role in orchestrating CTL effector functions in anti-tumor immunity. Helper T cell epitopes that trigger TH1 cell responses support the effector functions of CD8-positive killer T cells, including cytotoxic functions directed against tumor cells that display tumor-associated peptide / MHC complexes on their surfaces. Thus, tumor-associated T helper cell epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients in vaccine compounds that stimulate anti-tumor immune responses.

[0030] Mammalian (e.g. mouse) models have shown that even in the absence of CD8-positive T lymphocytes, CD4-positive T cells can suppress angiogenesis by secreting interferon-γ (IFNγ) and thus be sufficient to suppress tumor expression (Beatty and Paterson, 2001; Mumberg et al., 1999). There is no evidence that CD4 T cells act as direct anti-tumor effectors (Braumuller et al., 2013; Tran et al., 2014).

[0031] Since the constitutive expression of HLA class II molecules is usually limited to immune cells, it was previously considered impossible to isolate class II peptides directly from primary tumors. However, Dengjel et al. successfully identified multiple MHC class II epitopes directly in tumors (WO 2007 / 028574, EP 1 760 088 B1).

[0032] Since both CD8-dependent and CD4-dependent responses contribute jointly and synergistically to antitumor effects, identifying and characterizing tumor-associated antigens recognized by CD8+ T cells (ligands: MHCl class molecules + peptide epitopes) or CD4-positive T helper cells (ligands: MHC class II molecules) is very important for the development of tumor vaccines.

[0033] For a peptide of the MHCl class to trigger (initiate) a cellular immune response, it must also bind to an MHC molecule. This process depends on the allele of the MHC molecule and the specific polymorphism of the peptide's amino acid sequence. MHC-1 class-binding peptides are typically 8-12 amino acid residues in length and typically contain two conserved residues ("anchors") in their sequence that interact with the corresponding binding groove of the MHC molecule. In this way, each MHC allele has a "binding motif" that determines which peptides can specifically bind to the binding groove.

[0034] In the MHC class I-dependent immune response, peptides must not only bind to certain MHC class I molecules expressed by tumor cells, but they must then be recognized by specific T cell receptors (TCRs) carried by T cells.

[0035] For proteins that are recognized by T lymphocytes as tumor-specific antigens or associated antigens and used for treatment, special conditions must be met. The antigen should be mainly expressed by tumor cells, not by normal healthy tissues, or the expression amount is relatively small. In a preferred embodiment, the peptide should be over-presented in tumor cells compared with normal healthy tissues. More preferably, the corresponding antigen not only appears in a tumor, but also has a high concentration (i.e., the number of copies of the corresponding peptide per cell). Tumor-specific antigens and tumor-associated antigens are often derived from proteins that are directly involved in the transformation of normal cells into tumor cells due to their functions in cell cycle control or apoptosis inhibition. In addition, the downstream targets of these proteins that directly cause transformation events may be upregulated and may therefore be indirectly related to tumors. These indirect tumor-associated antigens may also be targets for vaccination methods (Singh-Jasuja et al., 2004). It is crucial that the epitope is present in the antigen amino acid sequence to ensure that such peptides from tumor-associated antigens ("immunogenic peptides") can lead to in vitro or in vivo T cell responses.

[0036] Basically, any peptide that can bind to an MHC molecule can potentially serve as a T cell epitope. The prerequisite for inducing a T cell response in vitro or in vivo is the presence of T cells with the corresponding TCR and the absence of immune tolerance to the specific epitope.

[0037] Therefore, TAAs are the starting point for the development of T cell-based therapies (including but not limited to tumor vaccines). Methods for identifying and characterizing TAAs are usually based on the use of T cells from patients or healthy subjects, or on the generation of differential transcriptional properties or differential expression patterns between tumor and normal tissue peptides. However, the identification of genes that are highly expressed or selectively expressed in tumor tissues or human tumor cell lines does not provide accurate information for the use of antigens transcribed by these genes in immunotherapy. This is because T cells with corresponding TCRs must be present and immune tolerance to this specific epitope must be absent or at a minimal level, so only a part of the epitopes of these antigens are suitable for this application. Therefore, in a very preferred embodiment of the present invention, it is very important to select only those over-presented or selectively presented peptides for which functional and / or proliferative T cells can be found. Such functional T cells are defined as T cells that can be cloned and expanded after stimulation with specific antigens and can perform effector functions ("effector T cells").

[0038] In case of peptide-MHC targeting via specific TCRs (e.g. soluble TCRs) and antibodies or other binding molecules (scaffolds) according to the invention, the immunogenicity of the underlying peptide is secondary. In these cases, presentation is the determining factor.

[0039] Introduction of the invention

[0040] In the first aspect of the present invention, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, or a variant sequence thereof having at least 77%, preferably at least 88% homology (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 101 (wherein the variant binds to MHC and / or induces T cells to cross-react with the peptide), or a pharmaceutically acceptable salt thereof (wherein the peptide is not the potential full-length polypeptide).

[0041] The present invention further relates to a peptide of the present invention, comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, or a variant thereof having at least 77%, preferably at least 88% homology (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 101, wherein the total length of the peptide or its variant is 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0042] The following table shows the peptides according to the present invention, their respective SEQ ID NOs, and possible source (potential) genes of these peptides. The peptides in Table 3 are peptides derived from so-called "alternative" or "short" open reading frames. Each peptide sequence proposes a representative source transcript ID (Ensemble (Aken et al., 2016) or RefSeq (O'Leary et al., 2016) annotation). These peptides may further be derived from other additional or alternative transcripts not listed herein.

[0043] In Table 3, peptides containing SEQ ID NO: 1 to SEQ ID NO: 71 bind to HLA-A*02. The peptides in Table 4 are peptides derived from human endogenous retroviruses. A representative chromosomal location is listed for each peptide. These peptides may be further mapped to additional or alternative chromosomal locations not listed herein. In Table 4, peptides containing SEQ ID NO: 72 to SEQ ID NO: 74 bind to HLA-A*02, and peptides containing SEQ ID NO: 75 to SEQ ID NO: 95 bind to different HLA-1 classes (see HLA alleles). The peptides in Table 5 are peptides that have no direct reference in the human genome. In Table 5, peptides containing SEQ ID NO: 96 to SEQ ID NO: 101 bind to HLA-A*02.

[0044] Table 3: Peptides of the invention from alternative or short open reading frames.

[0045]

[0046]

[0047]

[0048] Table 4: Additional peptides of the invention from human endogenous retroviruses.

[0049]

[0050]

[0051] Table 5: Additional peptides of the invention that have no direct reference in the human genome.

[0052] Serial ID Number sequence Peptide Code 96 KIIEDLANTV KRT18-001 97 GLIDDKGTIKL CDC2-006 98 SLMEVTHDL LARS-001 99 ALMDGSESRFFV BSG-001 100 SLGPPPVGV ClZ1-001 101 KLPEGHLPEV AHNAK2-003

[0053] Table 6: Peptides of the invention for personalized cancer therapy.

[0054] Serial ID Number sequence Peptide Code 102 GLDPTQFRV POLAl-003 103 SLVSYLDKV KRT16P-001

[0055] In the context of the present invention, alternative open reading frames were surprisingly found to be a source of effective tumor-associated antigens. To date, only a few reports have described such antigens, such as those derived from gp75 (Wang et al., Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen. J Exp Med. 1996 Mar 1; 183(3): 1131-40) and NY-ESO-1 / LAGE-1 ORF2 (Mandic, et al.. The alternative open reading frame of LAGE-1 gives rise to multiple promiscuous HLA-DR-restricted epitopes recognized by T-helper 1-type tumor-reactive CD4+T cells, Cancer Res. 2003 Oct 1; 63(19): 6506-15). Similarly, only a few reports have discussed endogenous retrovirus (HERV) sequences as the actual source of tumor-associated antigens (Mullins CS and Linnebacher M. Endogenous retrovirus sequences as a novel class of tumor-specific antigens: an example of HERV-H env encoding strong CT Lepitopes. Cancer Immunol lmmunother. 2012 Jul; 61(7): 1093-100; and Attermann AS, et al., Human endogenous retroviruses and their implication for immunotherapeutics of cancer. Ann Onc0l. 2018 Nov 1; 29(11): 2183-2191).HERVs have been proposed to act as "intrinsic adjuvants," potentially sensitizing cancer cells to immune recognition, or as self-antigens that can induce autoimmunity in neuropsychiatric diseases such as multiple sclerosis and schizophrenia (Tu X, et al., Human leukemia antigen-A*0201-restricted epitopes of human endogenous retrovirus W family envelope (HERV-W env) induce strong cytotoxic T lymphocyte responses. Virol Sin. 2017 Aug; 32(4): 280-289).

[0056] The present invention also generally relates to peptides according to the invention for use in the treatment of proliferative diseases, for example, acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer.

[0057] Particularly preferred are peptides (alone or in combination) according to the present invention selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101. More preferred are peptides (alone or in combination) selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 14, SEQ ID No. 72 to SEQ ID No. 81, SEQ ID No. 96 to SEQ ID No. 101 (see Table 3, Table 4 and Table 5) and their use in immunotherapy of acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer.

[0058] Therefore, another aspect of the present invention relates to the use of the peptides according to the present invention - preferably in combination for the treatment of a proliferative disease selected from the group consisting of acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer.

[0059] The present invention also relates to the peptides of the present invention, which have the ability to bind to major histocompatibility complex (MHC) class I or MHC class II molecules in an elongated form, such as a variable length.

[0060] The present invention further relates to the peptides of the present invention, wherein said peptide (each peptide) consists of or essentially consists of an amino acid sequence according to SEQ ID NO 1 to SEQ ID NO 101.

[0061] The present invention further relates to the peptides of the present invention, wherein the peptides are modified and / or comprise non-peptide bonds.

[0062] The present invention further relates to the peptides of the present invention, wherein the peptides are part of a fusion protein, in particular fused to the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (li), or to an antibody (e.g. a dendritic cell-specific antibody) or a sequence of an antibody.

[0063] The present invention further relates to a nucleic acid encoding the peptide of the present invention. The present invention further relates to a nucleic acid of the present invention, which is DNA, cDNA, PNA, RNA, or a combination thereof.

[0064] The present invention further relates to an expression vector capable of expressing and / or expressing the nucleic acid of the present invention.

[0065] The present invention further relates to a peptide of the present invention, a nucleic acid of the present invention or a pharmaceutical expression vector of the present invention for treating a disease, in particular for treating cancer.

[0066] The present invention further relates to antibodies specific for the peptides of the present invention or complexes of the peptides of the present invention and MHC and methods for producing these antibodies.

[0067] The present invention further relates to the T cell receptors (TCRs) of the present invention, in particular soluble TCRs (sTCRs) and cloned TCRs processed into autologous or allogeneic T cells, as well as methods for producing these TCRs and methods for producing NK cells carrying the TCRs or cross-reacting with the TCRs.

[0068] Antibodies and TCRs are further embodiments of existing immunotherapeutic uses of the peptides according to the invention.

[0069] The present invention further relates to a host cell comprising the nucleic acid of the present invention or the aforementioned expression vector. The present invention further relates to the host cell of the present invention, which is an antigen presenting cell, preferably a dendritic cell.

[0070] The present invention further relates to a method of formulating a peptide of the present invention, said method comprising culturing the host cell of the present invention and isolating the peptide from said host cell or its culture medium.

[0071] The present invention further relates to the method of the present invention, wherein the antigen is loaded onto class I or II MHC molecules expressed on the surface of suitable antigen-presenting cells or artificial antigen-presenting cells by binding to a sufficient amount of antigen-containing antigen-presenting cells.

[0072] The present invention further relates to the method of the present invention, wherein the antigen presenting cell comprises an expression vector capable of expressing or expressing the peptide or variant amino acid sequence comprising SEQ ID NO.1 to SEQ ID NO.101, preferably comprising SEQ ID No.1 to SEQ ID No.14, SEQ ID No.72 to SEQ ID No.81, SEQ ID No.96 to SEQ ID No.101.

[0073] The present invention further relates to activated T cells produced by the method of the present invention, wherein the T cells selectively recognize a cell expressing a polypeptide comprising an amino acid sequence of the present invention.

[0074] The present invention further relates to a method for killing target cells of a patient, wherein the target cells of the patient abnormally express a polypeptide comprising any amino acid sequence of the present invention, the method comprising administering to the patient an effective amount of T cells produced by the method of the present invention.

[0075] The present invention further relates to the use of any of the peptides, nucleic acids of the present invention, expression vectors of the present invention, cells of the present invention, activated T lymphocytes, T cell receptors or antibodies or other peptide- and / or peptide-MHC binding molecules of the present invention as a medicament or in the manufacture of a medicament. The medicament preferably has anticancer activity.

[0076] Preferably, the agent is a cell therapy drug, vaccine or protein based on soluble TCR or antibody.

[0077] The present invention further relates to the use according to the present invention, wherein the cancer cells are acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer, preferably acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer cells.

[0078] The present invention further relates to a biomarker based on the peptides of the present invention, referred to herein as a "target", which can be used to diagnose cancer, preferably acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer. The marker can be over-presented by the peptide itself or over-expressed by the corresponding gene. The marker can also be used to predict the likelihood of success of treatment, preferably immunotherapy, most preferably 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 of the relevant peptide complexed with MHC.

[0079] Optionally, the antibodies have further effector functions, such as immunostimulatory domains or toxins.

[0080] The present invention also relates to the use of these novel targets in cancer treatment.

[0081] Detailed description of the invention

[0082] Whether an immune response can be stimulated depends on the presence of antigens that are considered foreign by the host immune system. The discovery of tumor-associated antigens has raised the possibility of using the host immune system to intervene in tumor growth. Various mechanisms that exploit both the humoral and cellular aspects of the immune system are currently being explored for cancer immunotherapy.

[0083] Specific elements of the cellular immune response can specifically recognize and destroy tumor cells. T cells isolated from tumor-infiltrating cell populations or peripheral blood have shown that these cells play an important role in the natural immune defense against cancer. In particular, CD8-positive T cells play an important role in this response, which recognize class I molecules contained in peptides carried by the major histocompatibility complex (MHC) of usually 8 to 10 amino acid residues derived from proteins or defective ribosomal products (DRIPs) located in the cytoplasm. Human MHC molecules are also called human leukocyte-antigens (HLA).

[0084] Unless otherwise stated, all terms used herein are defined below.

[0085] The term "T cell response" refers to the specific proliferation and activation of effector functions induced in vitro or in vivo by a peptide. For MHC class I restricted cytotoxic T cells, the effector function may be lysis of target cells pulsed with peptide, pulsed with peptide precursor or presented with native peptide, secretion of cytokines, preferably interferon-γ, TNF-α or IL-2 induced by peptide, secretion of effector molecules, preferably granzymes or perforins induced by peptide, or degranulation.

[0086] As used herein, the term "peptide" refers to a series of amino acid residues, usually linked by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. These peptides are preferably 9 amino acids in length, but can be as short as 8 amino acids in length, as long as 10, 11 or 12 amino acids in length or longer, and in the case of MHC class II peptides (elongated variants of the peptides of the present invention), can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length or longer.

[0087] Thus, the term "peptide" shall include salts of a series of amino acid residues, usually linked by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. Preferably, the salt is a pharmaceutically acceptable salt of the peptide, for example, a chloride or acetate (trifluoroacetate) salt. It must be noted that the salts of the peptides of the present invention are significantly different from the peptides in their in vivo state, as the peptides are not in salt form or associated with an in vivo counterion.

[0088] The term "peptide" shall also include "oligopeptide". The term "oligopeptide" as used herein refers to a series of amino acid residues, usually connected by peptide bonds between the α-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. Typically, the oligopeptide is less than about 30 amino acid residues in length and is longer than about 15 amino acids.

[0089] The term "polypeptide" refers to a series of amino acid residues, usually connected by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not critical to the present invention as long as the correct epitope is maintained. In contrast to the terms peptide or oligopeptide, the term polypeptide refers to a molecule containing more than about 30 amino acid residues.

[0090] A peptide, oligopeptide, protein or nucleotide encoding the molecule is "immunogenic" (and therefore an "immunogen" in the present invention) if it is able to induce 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. Thus, an "immunogen" is 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 enhancing specific antibody or TCR resistance.

[0091] A class I T cell "epitope" requires a short peptide that binds to the MHC class I receptor, forming a ternary complex (MHC class I alpha chain, beta-2-microglobulin, and peptide) that can be recognized by a T cell loaded with a matching T cell receptor bound to the MHC / peptide complex with appropriate affinity. The typical length of peptides that bind to MHC class I molecules is 8-14 amino acids, with 9 amino acids being the most typical.

[0092] In humans, there are three different gene loci encoding MHC class I molecules (human MHC molecules are also designated human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02, and HLA-B*07 are examples of different MHC class I alleles that can be expressed from these gene loci.

[0093] Table 7: Expression frequency F of HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 serotypes. The haplotype frequency Gf is derived from a study that used HLA typing data from more than 6.5 million volunteer donors registered in the United States (Gragert et al., 2013). The haplotype frequency is the frequency of a unique allele on an individual chromosome. Due to the diploid chromosome set in mammalian cells, the genotype of the allele has a high frequency of occurrence, which can be calculated using the Hardy-Weinberg law (F=1-(1-Gf)2).

[0094]

[0095]

[0096] The peptides of the invention preferably bind to A*02 when incorporated into the vaccines of the invention as described herein. Vaccines may also include pan-binding MHC class II peptides. Thus, the vaccines of the invention can be used to treat cancer in A*02 positive patients, but do not require selection of MHC class II allotypes because these peptides have pan-binding properties.

[0097] If the A*02 peptide of the present 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 compared to the MHC class I allele alone. Although in most populations, less than 50% of patients can be solved by a single allele, a vaccine containing HLA-A*24 and HLA-A*02 epitopes in the present invention can treat at least 60% of patients in any relevant population. Specifically, in each region, the following proportions of patients have a positive effect on at least one of these alleles: 61% in the United States, 62% in Western Europe, 75% in China, 77% in South Korea, and 86% in Japan (calculated according to www.allelefrequencies.net).

[0098] Table 8: HLA allele coverage in the European white population (calculated according to the method described in (Gragert et al., 2013)).

[0099]

[0100] In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.

[0101] The nucleotide sequence encoding a specific peptide, oligopeptide or polypeptide may be a natural nucleotide sequence or a synthetic nucleotide sequence. In general, the DNA fragment encoding the peptide, polypeptide and protein of the present invention is composed of a cDNA fragment and a short oligonucleotide linker, or a series of oligonucleotides, to provide a synthetic gene that can be expressed in a recombinant transcription unit containing regulatory elements derived from a microorganism or a viral operon.

[0102] The term "nucleotide encoding a peptide" as used herein refers to a nucleotide sequence encoding a peptide, which includes artificial (man-made) start and stop codons that are compatible with a biological system for expressing the sequence, such as a dendritic cell or another cell system for producing a TCR.

[0103] The nucleic acid sequences mentioned herein include both single-stranded nucleic acids and double-stranded nucleic acids.Therefore, unless otherwise indicated herein, for example, for DNA, a specific sequence is a single-stranded DNA of the sequence, a duplex (double-stranded DNA) of the sequence and its complementary sequence, and the complementary sequence of the sequence.

[0104] 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, ie, the region that encodes the native expression product of the gene in vivo.

[0105] The coding region can be derived from a non-mutated ("normal") gene, a mutant gene, or an abnormal gene, and can even be derived from a DNA sequence that can be synthesized entirely in the laboratory using DNA synthesis methods well known in the art.

[0106] The term "expression product" refers to a polypeptide or protein which is the product of translation of a gene and any nucleic acid sequence encoding the equivalent resulting from the degeneration of the genetic code and thereby encoding the same amino acids.

[0107] The term "fragment", when referring to a coding sequence, means a portion of DNA including 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.

[0108] The term "DNA fragment" refers to a DNA polymer, either in the form of an individual fragment or as a component of a larger DNA structure, obtained from DNA that has been isolated at least once in a substantially pure form, i.e., free of contaminating endogenous materials, and in quantities or concentrations that allow for the identification, manipulation, and recovery of the fragment and its component nucleotide sequences using standard biochemical methods, such as the use of cloning vectors. Such fragments are present in the form of open reading frames (not interrupted by internal untranslated sequences) or introns (commonly present in eukaryotic genes). Untranslated DNA sequences may be present downstream of the open reading frame, where they do not interfere with the manipulation or expression of the coding region.

[0109] The term "primer" refers to a short nucleic acid sequence that pairs with a DNA strand and provides a free 3'-OH terminus at which DNA polymerase begins to synthesize a DNA strand.

[0110] The term "promoter" refers to a region of DNA that participates in the binding of RNA polymerase and thereby initiates transcription.

[0111] The term "isolated" means that a material is removed from its original environment (e.g., its natural environment if naturally occurring). For example, a native nucleotide or polypeptide in a living animal is not isolated, but a nucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector and / or such polynucleotides and polypeptides may be part of a composition and still be isolated in that the vector or composition is not part of its natural environment.

[0112] The polynucleotides and recombinant or immunogenic polypeptides disclosed in the present invention may also exist in a "purified" form. The term "purified" does not require absolute purity; it is only a relative definition and can include highly purified or partially purified preparations, and these terms are understood by technicians in the relevant fields. For example, various clones isolated from a cDNA library that has been purified to electrophoretic homogeneity by traditional methods. It is explicitly considered that the starting material or natural substance is purified by at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude. In addition, it is explicitly contemplated that the purity of the polypeptide is preferably 99.999%, or at least 99.99% or 99.9%; even preferably 99% or more by weight.

[0113] The nucleic acid and polypeptide expression products disclosed in accordance with the present invention, as well as expression vectors comprising such nucleic acids and / or polypeptides, may be present in a "concentrated form". As used herein, the term "concentrated" refers to a material having a concentration of at least about 2, 5, 10, 100 or 1000 times its natural concentration, advantageously 0.01% by weight, preferably at least 0.1%. Concentrated preparations of about 0.5%, 1%, 5%, 10% and 20% by weight are also expressly contemplated. The sequences, configurations, vectors, clones and other materials comprising the present invention may advantageously be present in a concentrated or isolated form. The term "active fragment" refers to a fragment that produces an immune response (i.e., has immunogenic activity), generally a fragment of a peptide, polypeptide or nucleic acid sequence, whether administered alone or optionally with a suitable adjuvant or in a carrier to an animal, such as a mammal, for example a rabbit or mouse, including a human; the immune response takes the form of stimulating a T cell response in the recipient animal (e.g., a human). Alternatively, the "active fragment" may also be used to induce a T cell response in vitro.

[0114] As used herein, the terms "portion", "segment" and "fragment" when used in relation to polypeptides refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide is treated with any endopeptidase (such as trypsin or chymotrypsin), the oligopeptides obtained from the treatment will represent a portion, segment or fragment of the starting polypeptide. When used in relation to polynucleotides, these terms refer to the products produced by treating the polynucleotide with any endonuclease.

[0115] According to the present invention, the term "percent identity" or "percent equivalence", if referring to a sequence, means that the sequence to be compared ("compared sequence") is compared with the sequence or sequence of the claim ("reference sequence") after the sequence to be compared ("compared sequence") is aligned with the sequence or sequence of the claim. The percent identity is then calculated according to the following formula:

[0116] Percentage of equivalence = 100[1-(C / R)]

[0117] Where 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,

[0118] (i) Each base or amino acid sequence in the reference sequence has no corresponding aligned base or amino acid in the compared sequence;

[0119] (ii) each gap in the reference sequence, and

[0120] (iii) each aligned base or amino acid in the reference sequence that differs from an aligned base or amino acid in the compared sequence constitutes a difference; and

[0121] (iiii) Alignment must start at the first position of the alignment sequence;

[0122] And R is the number of bases or amino acids in the reference sequence over the length of the alignment between the reference sequence and the compared sequence, with any gaps in the reference sequence also counted as one base or amino acid.

[0123] If there is an alignment between the "compared sequence" and the "reference sequence" where the percent identity calculated as above is approximately equal to or greater than the specified minimum percent identity, then the compared sequence has the specified minimum percent identity to the reference sequence, although there may be alignments where the percent identity calculated as above in this document is lower than the specified percent identity.

[0124] Therefore, as described above, the present invention provides a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101 or a variant thereof having 88% homology with SEQ ID NO: 1 to SEQ ID NO: 101, or a variant thereof inducing T cells to cross-react with the peptide. The peptide of the present invention has the ability to bind to a major histocompatibility complex (MHC) class I or a class II molecule of an elongated version of the peptide.

[0125] In the present invention, the term "homology" refers to the degree of identity between two amino acid sequences (see the percentage of identity above, such as peptide or polypeptide sequences. The "homology" mentioned above is determined by aligning the two sequences adjusted under ideal conditions with the sequences to be compared. Such sequence homology can be calculated by creating an alignment using algorithms such as CIustalW. It can also be used using general sequence analysis software, more specifically Vector NTI, GENETYX or other tools provided by public databases.

[0126] One skilled in the art is able to assess whether T cells induced by a particular peptide variant can cross-react with the peptide itself (Appay et al., 2006; Colombetti et al., 2006; Fong et al., 2001; Zaremba et al., 1997).

[0127] By "variant" of a given amino acid sequence, the inventors mean that the side chains of one or two amino acid residues, etc., are altered (e.g., by substitution with the side chain of another naturally occurring amino acid residue or some other side chain), such that the peptide is still able to bind to an HLA molecule in substantially the same manner as a peptide consisting of the given amino acid sequence (consisting of SEQ ID NO: 1 to SEQ ID NO: 101). For example, a peptide may be modified so as to at least maintain (if not improve) its ability to interact with and bind to the binding groove of a suitable MHC molecule, such as HLA-A*02 or -DR, and so as to at least maintain (if not improve) its ability to bind to the TCR of an activated T cell.

[0128] Subsequently, these T cells can cross-react with cells and kill cells that express polypeptides (including the natural amino acid sequence of the homologous peptide defined in the present invention). As described in scientific literature and databases (Rammensee et al., 1999; Godkin et al., 1997), certain positions of HLA-A binding peptides are usually anchor residues that can form a core sequence that is compatible with the binding motif of the HLA binding groove, which is defined by the polarity, electrophysical, hydrophobicity and spatial properties of the polypeptide chains that constitute the binding groove. Therefore, those skilled in the art can modify the amino acid sequences set forth in SEQ ID No: 1 to SEQ ID NO: 101 by maintaining the 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 present invention retain the ability to bind to the TCR of activated T cells, and subsequently, these T cells can cross-react with cells expressing a polypeptide including the natural amino acid sequence of the homologous peptide defined in the present invention and kill these cells.

[0129] If not otherwise stated, the original (unmodified) peptides disclosed herein may be modified by replacing one or more residues at different (possibly selective) sites within the peptide chain. Preferably, these substitutions are located at the termini of the amino acid chain. The substitution may be conservative, for example, where one amino acid is replaced by another amino acid of similar structure and characteristics, such as where one hydrophobic amino acid is replaced by another hydrophobic amino acid. More conservative substitutions are substitutions between amino acids of the same or similar size and chemical properties, for example, leucine is replaced by isoleucine. In studies of sequence variation in natural homologous protein families, substitutions of certain amino acids are often more tolerated than others, and these amino acids often show similarities with the original amino acids in size, charge, polarity and hydrophobicity, which is the basis for determining "conservative substitutions".

[0130] In this article, conservative substitutions are defined as exchanges within one of the following five groups: Group 1 - small aliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2 - polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3 - polar, positively charged residues (His, Arg, Lys); Group 4 - large aliphatic nonpolar residues (Met, Leu, Ile, VaI, Cys) and Group 5 - large aromatic residues (Phe, Tyr, Trp).

[0131] In one aspect, conservative substitutions may include substitutions described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5", Natl. Biomedical Research, the contents of which are incorporated herein by reference in their entirety. For example, in one aspect, amino acids belonging to one of the following groups may be exchanged with each other, thus constituting a conservative exchange: Group 1: Alanine (A), Proline (P), Glycine (G), Asparagine (N), Serine (S), Threonine (T); Group 2: Cysteine ​​(C), Serine (S), Tyrosine (Y), Threonine (T); Group 3: Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Phenylalanine (F); Group 4: Lysine (K), Arginine (R), Histidine (H); Group 5: Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Histidine (H); and Group 6: Aspartic acid (D), Glutamic acid (E). In one aspect, conservative amino acid substitutions can be selected from the following substitutions: T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G and / or T→S.

[0132] In one aspect, conservative amino acid substitutions may include replacing one amino acid with another amino acid of the same class, for example, (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative amino acid substitutions may also be made as follows: (1) aromatic: Phe, Tyr, His; (2) proton donors: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptors: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, e.g., U.S. Pat. No. 10,106,805, the contents of which are incorporated herein by reference in their entirety).

[0133] On the other hand, conservative substitutions can be made according to Table A. Methods for predicting protein modification tolerance can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety.

[0134] Table A

[0135]

[0136] On the other hand, conservative substitutions may be those shown under the heading "Conservative Substituents" in Table B. If such substitutions result in a change in biological activity, then major changes designated as "Representative Substituents" in Table B may be introduced and the products screened, if desired.

[0137] Table B

[0138]

[0139] Less conservative substitutions may involve the replacement of one amino acid with another of similar characteristics but different in size, such as the replacement of an alanine with an isoleucine residue. Highly non-conservative substitutions may involve the replacement of an acidic amino acid with another with polar or even basic properties. However, such "radical" substitutions should not be dismissed as ineffective, as chemistry is not entirely predictable and radical substitutions may bring about unforeseen effects that could not be foreseen in their simple chemistry.

[0140] Of course, such substitutions may involve other structures besides common L-amino acids. Thus, D-amino acids may be substituted with common L-amino acids in the antigenic peptides of the present invention, and still be within the scope of the present disclosure. In addition, non-standard amino acids (i.e., in addition to common naturally occurring proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.

[0141] If substitutions at more than one position are found to result in an antigenic activity of the peptide substantially equal to or greater than the value defined below, combinations of these substitutions are tested to determine whether the combined substitutions produce an additive or synergistic effect on the antigenicity of the peptide. No more than 4 positions may be substituted simultaneously within the peptide.

[0142] A peptide consisting essentially of the amino acid sequence as described herein may have one or two non-anchor amino acids (see below for anchor motifs) exchanged, without the situation that the ability to bind to human major histocompatibility complex (MHC) class I or II molecules is substantially altered or adversely affected compared to the unmodified peptide. In another embodiment, in a peptide consisting essentially of the amino acid sequence as described herein, one or two amino acids may be exchanged with their conservative exchange partners (see below), without the situation that the ability to bind to human major histocompatibility complex (MHC) class I or II molecules is substantially altered or adversely affected compared to the unmodified peptide.

[0143] These amino acid residues that do not substantially interact with T cell receptors can be modified by substituting other amino acids that have little effect on T cell response and do not hinder binding to relevant MHC. Therefore, except for specific limiting conditions, the peptide of the present invention may be any peptide (the term used by the inventors includes oligopeptides or polypeptides) comprising a given amino acid sequence or a portion or variant thereof.

[0144] Table 9: Peptide variants and motifs according to SEQ ID NO: 4, 8, 72, 74, 96 and 97.

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] Longer (elongated) peptides may also be suitable. MHC class I epitopes (usually 8 to 11 amino acids in length) may be produced by processing of the peptide from a longer peptide or protein containing the actual epitope. The residues flanking the actual epitope are preferably residues that do not affect the protein cleavage required to expose the actual epitope during processing.

[0152] The peptides of the present invention can be elongated by up to four amino acids, i.e., 1, 2, 3 or 4 amino acids, and can be added to either end in any combination between 4:0 and 0:4. The elongation combinations of the present invention can be seen in Table 10.

[0153] Table 10: Elongated combinations of peptides of the present invention

[0154]

[0155] The stretched / extended amino acids can be the original sequence peptide of the protein or any other amino acids. Stretching can be used to enhance the stability or solubility of the peptide.

[0156] Therefore, the epitope described in the present invention may be the same as a natural tumor-associated epitope or tumor-specific epitope, or may include a peptide that differs by no more than four residues from a reference peptide, as long as they have substantially the same antigenic activity.

[0157] In an alternative embodiment, one or both sides of the peptide are extended by more than 4 amino acids, preferably up to a total length of 30 amino acids. This can form an MHC-II class binding peptide. Binding to MHC class II peptides can be tested by methods known in the art.

[0158] Therefore, the present invention provides peptides and variants of MHC class I epitopes, wherein the total length of the peptide or antibody is 8 to 100, preferably 8 to 30, and most preferably 8 to 14 amino acids in length (i.e., 10, 11, 12, 13, 14 amino acids, and if it is an elongated class II binding peptide, the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids).

[0159] Of course, the peptides or variants of the present invention can bind to human major histocompatibility complex (MHC) class I or II molecules. The binding of the peptides or variants to the MHC complex can be tested by methods known in the art.

[0160] Preferably, when the peptide-specific T cells of the present invention are tested in comparison with the substituted peptide, if the substituted peptide increases the solubility relative to the background peptide to half of the maximum value, the peptide concentration is no more than about 1 mM, preferably no more than about 1 μM, more preferably no more than about 1 nM, even more preferably no more than about 100 pM, and most preferably no more than about 10 pM. It is also preferred that the substituted peptide is recognized by more than one T cell, at least 2, more preferably 3.

[0161] In a particularly preferred embodiment of the present invention, the peptide consists or consists essentially of an amino acid sequence selected according to SEQ ID NO: 1 to SEQ ID NO: 101.

[0162] "Essentially consisting of..." means that the peptide of the present invention, in addition to being composed of any one of SEQ ID NO: 1 to SEQ ID NO: 101 or a variant thereof, also contains amino acids located at other N- and / or C-terminal extensions, which may not necessarily form a peptide that serves as an MHC molecule epitope.

[0163] However, these extended regions play an important role in effectively introducing the peptides of the present invention into cells. In one embodiment of the present invention, the peptide is part of a fusion protein, containing 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (p33, hereinafter referred to as "li") from NCBI, GenBank Accession No. X00497. In other fusions, the peptides of the present invention can be fused to the antibodies described herein, or functional parts thereof, in particular, to sequences of antibodies, so that the antibodies perform specific targeting, or, for example, enter the dendritic cell-specific antibodies described herein.

[0164] In addition, the peptide or variant can be further modified to improve stability and / or binding to MHC molecules, thereby eliciting a stronger immune response. Such optimization methods of peptide sequences are well known in the art, including, for example, the introduction of trans-peptide bonds and non-peptide bonds.

[0165] In a reverse peptide bond, the amino acid residues are not linked by a peptide (-CO-NH-) linkage, but the peptide bond is reversed. Such retro-inverso peptidomimetics can be prepared by methods known in the art, such as the method described by Meziere et al. (Meziere et al., 1997), which is incorporated herein by reference. This method involves preparing peptidomimetics that contain changes to the backbone (rather than the side chains). Studies by Meziere et al. (Meziere et al., 1997) showed that these peptidomimetics facilitate MHC binding and helper T cell responses. Retro-inverso peptides that replace CO-NH peptide bonds with NH-CO bonds have greatly improved resistance to hydrolysis.

[0166] Non-peptide bonds are -CH2-NH, -CH2S-, -CH2CH2-, -CH=CH-, -COCH2-, -CH(OH)CH2- and -CH2SO-, etc. U.S. Patent No. 4897445 proposes a non-solid phase synthesis method for non-peptide bonds (-CH2-NH) in polypeptide chains, which involves a polypeptide synthesized according to a standard procedure and a non-peptide bond synthesized through the interaction of an aminoaldehyde and an amino acid containing NaCNBH3.

[0167] Peptides containing the above sequences can be synthesized with other chemical groups at their amino and / or carboxyl termini to improve the stability, bioavailability, and / or affinity of the peptides. For example, hydrophobic groups such as benzyloxycarbonyl, dansyl, or tert-butyloxycarbonyl groups can be added to the amino terminus of the peptide. Similarly, acetyl or 9-fluorenylmethoxycarbonyl groups may be located at the amino terminus of the peptide. In addition, hydrophobic groups, tert-butyloxycarbonyl groups, or amino groups may be added to the carboxyl terminus of the peptide.

[0168] In addition, all peptides of the present invention may be synthesized to change their spatial configuration. For example, the dextrorotatory form of one or more amino acid residues of these peptides may be used, usually not the levorotatory form. Further, at least one amino acid residue of the peptide of the present invention may be substituted by a well-known non-natural amino acid residue. Such changes may help to increase the stability, bioavailability and / or binding effect of the peptide of the present invention.

[0169] Likewise, the peptides or variants of the invention may be chemically modified by reaction of specific amino acids before or after peptide synthesis. Examples of such modifications are well known in the art and are summarized, for example, in R. Lundblad, Chemical Reagents for Protein Modification (3rd ed. CRC Press, 2004) (Lundblad, 2004), which is incorporated herein by reference. Although the methods for chemical modification of amino acids are not limited, they include (but are not limited to) modification by the following methods: acylation, amidination, lysine pyridoylation, reductive alkylation, trinitrophenylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amino modification of carboxyl groups and sulfhydryl groups by performic acid oxidation of cysteine ​​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, and methylaminoacylation with cyanate at alkaline pH. In this regard, the skilled person is referred to Chapter 15 of Current Protocols In Protein Science (Eds. Coligan et al. (John Wiley & '26 Sons NY 1995-2000) (Coligan et al., 1995) for extensive methods related to chemical modification of proteins.

[0170] In brief, modification of arginyl residues of proteins is often based on the reaction of ortho-dicarbonyl compounds such as benzyl carboxaldehyde, 2,3-butanedione and 1,2-hexanedione to form adducts. Another example is the reaction of methylglyoxal with arginine residues. Cysteine ​​can be modified without concomitant modification of nucleophilic sites such as lysine and histidine. Therefore, there are a large number of reagents for the modification of cysteine. The websites of companies such as Sigma-Aldrich (http: / / www.sigma-aldrich.com) contain information on specific reagents.

[0171] Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during heat treatment of biopharmaceuticals. Woodward's reagent K can be used to modify specific glutamic acid residues. N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide can be used to form intramolecular crosslinks between lysine residues and glutamic acid residues. For example, diethyl pyrocarbonate is a reagent for modifying histidine residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. Reaction of lysine residues with other α-amino groups, for example, facilitates peptide binding to the surface or crosslinking of proteins / peptides. Lysine is the attachment point for poly(ethylene) glycol and is also the main modification site for protein glycosylation. Methionine residues of proteins can be modified using iodoacetamide, bromoethylamine, chloramine T, etc.

[0172] Tetranitromethane and N-acetylimidazole can be used to modify tyrosine residues. Cross-linking via dityrosine can be accomplished with hydrogen peroxide / copper ions.

[0173] Recent studies on tryptophan modification have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, or 3-bromo-3-methyl-2-(2-nitrophenylthio)-3H-indole (BPNS-skatole).

[0174] Successful modification of therapeutic proteins and peptides containing polyethylene glycol often results in extended circulation half-lives when cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is used to formulate hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamidomethylation with potassium cyanate.

[0175] A peptide or variant, wherein the peptide is modified or contains non-peptide bonds, is a preferred embodiment of the present invention.

[0176] Another embodiment of the present invention relates to a non-natural peptide, wherein the peptide consists of or consists essentially of an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 101 and is synthetically produced (i.e., synthesized) as a pharmaceutically acceptable salt. Methods for synthetically producing peptides are well known in the art. Salts of the peptides of the present invention are substantially different from the peptides in their in vivo state, because these in vivo produced peptides are not salts. The non-natural salt form of the peptide mediates the solubility of the peptide, particularly in the case of a pharmaceutical composition comprising the peptide, for example, a peptide vaccine disclosed herein. In order to effectively provide the peptide to a subject in need of treatment, it is necessary for the peptide to have sufficient, at least substantial, solubility. Preferably, the salt is a pharmaceutically acceptable salt of the peptide. These salts of the present invention include benzene and alkaline earth salts, such as salts of the Hofmeister series, containing an anion PO4 3- 、SO4 2- 、CH3COO - , Cl - Br - 、NO3 -、ClO4 - ,I - 、SCN - and cation NH4 + , Rb + , K + 、Na + , Cs + , Li + 、Zn 2+ Mg 2+ , Ca 2+ , Mn 2+ , Cu 2+ and Ba 2+In particular, the salt is selected from (NH4)3PO4, (NH4)2HPO4, (NH4)H2PO4, (NH4)2SO4, NH4CH3COO, NH4Cl, NH4Br, NH4NO3, NH4ClO4, NH4I, NH4SCN, Rb3PO4, Rb2HPO4, RbH2PO4, Rb2SO4, Rb4CH3COO, Rb4Cl, Rb4Br, Rb4NO3, Rb4ClO4, Rb4I, Rb4SCN, K3PO4, K2HP O4, KH2PO4, K2SO4, KCH3COO, KCI, KBr, KNO3, KCIO4, KI, KSCN, Na3PO4, Na2HPO4, NaH2PO4, Na2SO4, NaCH3COO, NaC l, NaBr, NaNO3, NaClO4, Nal, NaSCN, ZnCl2Cs3PO4, Cs2HPO4, CsH2PO4, Cs2SO4, CsCH3COO, CsCl, CsBr, CsNO3, CsCl O4, Csl, CsSCN, Li3PO4, Li2HPO4, LiH2PO4, Li2SO4, LiCH3COO, LiCl, LiBr, LiNO3, LiClO4, Lil, LiSCN, Cu2SO4, M g3(PO4)2, Mg2HPO4, Mg(H2PO4)2, Mg2SO4, Mg(CH3COO)2, MgCl2, MgBr2, Mg(NO3)2, Mg(ClO4)2, Mgl2, Mg(SCN)2, Mn Cl2, Ca3(PO4), Ca2HPO4, Ca(H2PO4)2, CaSO4, Ca(CH3COO)2, CaCl2, CaBr2, Ca(NO3)2, Ca(ClO4)2, Cal2, Ca(SCN)2, Ba3(PO4)2, Ba2HPO4, Ba(H2PO4)2, BaSO4, Ba(CH3COO)2, BaCl2, BaBr2, Ba(NO3)2, Ba(ClO4)2, Bal2 and Ba(SCN)2. Particularly preferred are NH acetate, MgCl2, KH2PO4, Na2SO4, KCI, NaCl and CaCl2, for example chloride or acetate (trifluoroacetic acid) salts.

[0177] In general, peptides and variants (at least containing peptide linkages between amino acid residues) can be synthesized using the Fmoc-polyamide model of solid phase peptide synthesis as disclosed by Lukas et al. (Lukas et al., 1981) and references cited therein. Temporary protection of the N-amino groups is provided by the 9-fluorenylmethoxycarbonyl (Fmoc) group. Repeated cleavage of this highly base-labile protecting group is performed using 20% ​​dimethylpiperidinium in N,N-dimethylformamide. Side chain functions may be protected by their butyl ethers (in the case of serine, threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), tert-butyloxycarbonyl derivatives (in the case of lysine and histidine), trityl derivatives (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (in the case of arginine). Whenever glutamine and asparagine are C-terminal residues, side chain amino functions are protected by 4,4′-dimethoxydiphenyl groups. The solid support is based on polydimethylacrylamide polymer, which is composed of three monomers dimethylacrylamide (backbone monomer), bis(acrylethylenediamine) (crosslinker) and N-acryloylsarcosine methyl ester (functional agent). The peptide-resin linker used is an acid-sensitive 4-hydroxymethylphenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives, except asparagine and glutamine, which are added using a reversed N,N-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, nitrobenzenesulfonic acid or isotin test procedures. After the synthesis is completed, the peptide is cleaved from the resin support with the concomitant removal of the side chain protecting groups using a 95% trifluoroacetic acid solution containing 50% scavenger mixture. Commonly used scavenger mixtures include ethanedithiol, phenol, anisole and water, the exact choice depending on the amino acid composition of the synthesized peptide. Furthermore, it is possible to synthesize peptides using a combination of solid and solution phase methods (see, for example, (Bruckdorfer et al., 2004) and references cited therein).

[0178] Trifluoroacetic acid was removed by evaporation in vacuo followed by titration with diethyl ether loaded with crude peptide. Any scavenger compounds present were removed by a simple extraction procedure which yielded scavenger compound-free peptides after lyophilization of the aqueous phase. Peptide synthesis reagents are generally available from Calbiochem-Novabiochem (Nottingham, UK).

[0179] Purification may be performed by any one or a combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and (usually) reversed-phase high performance liquid chromatography (e.g. using an acetonitrile / water gradient separation).

[0180] Peptide analysis can be performed using thin layer chromatography, electrophoresis, in particular, capillary electrophoresis, solid phase extraction (CSPE), reversed phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectrometry, and MALDI, ESI-Q-TOF mass spectrometry.

[0181] To select over-presented peptides, a presentation plot was calculated that shows the amount of bit presentation in the sample and the variation in replication. The features parallel the baseline values ​​of samples of the relevant tumor entity and normal tissue samples. Each of the above features can be incorporated into the over-presentation score by calculating the p-value of the adjusted linear mixed effects model (Pinheiro et al., 2015), thereby adjusting multiple tests by the false discovery rate (Benjamini and Hochberg, 1995) (see Example 1, Figure 1).

[0182] For identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shock-frozen tissue samples were purified and HLA-associated peptides were isolated. The isolated peptides were separated and identified by on-line nano-electrospray-ionization (nanoESI) liquid chromatography-spectroscopy (LC-MS) experiments. The resulting peptide sequences were validated by comparing the fragmentation patterns of natural tumor-associated peptides (TUMAPs) recorded in acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer samples (N = 490 samples) with the fragmentation patterns of corresponding synthetic reference peptides of the same sequence. Since the peptides were directly identified as ligands for HLA molecules of primary tumors, these results provide direct evidence for the natural processing and presentation of the identified peptides on primary cancer tissues obtained from patients with acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer.

[0183] Discovery pipeline v2.1 (see, e.g., US2013-0096016, which is hereby incorporated by reference in its entirety) allows for the identification and selection of relevant over-presented peptide vaccine candidates based on direct relative quantification of HLA-restricted peptide levels in cancer or other infected tissues compared to several different non-cancerous tissues and organs. This was achieved by developing a label-free differential quantification method using LC-MS acquisition data processed using a proprietary data analysis pipeline, combining sequence recognition algorithms, spectral clustering, counting ions, retention time adjustment, charge state convolution, and normalization.

[0184] Additional sequence information from public sources (Olexiouk et al., 2016; Subramanian et al., 2011) was incorporated into Discovery pipeline to identify TUMAPs of non-classical origin. De novo sequencing was used in an orthogonal database-independent search strategy to identify peptide sequences of tumor-specific clusters (by This allows the identification of novel TUMAPs that have no direct reference in human genomic or proteomic databases. Presentation levels, including error estimates, were established for each peptide and sample. Peptides exclusively presented by tumor tissues as well as peptides over-presented in tumor versus non-tumor tissues and organs have been identified.

[0185] HLA peptide complexes from tissue samples of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer were purified, and HLA-associated peptides were separated and analyzed using LC-MS (see Example 1). All TUMAPs included in this application were identified using this approach on samples of acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer, confirming their presentation on acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer.

[0186] TUMAPs identified on multiple acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer and normal tissues were quantified using an ion counting method for label-free LC-MS data. The method assumes that the LC-MS signal area of ​​a peptide is related to its abundance in the sample. All quantified signals of peptides in various LC-MS experiments are normalized on the basis of central tendency, averaged for each sample, and merged into a histogram (called a presentation plot). The presentation plot integrates different analytical methods such as protein database search, spectral clustering, charge state convolution (de-charge) and retention time calibration and normalization.

[0187] In addition, the pipeline was found Direct absolute quantification of MHC-peptides, preferably HLA-restricted peptides, on cancer or other infected tissues can be performed. Briefly, the total cell count is calculated based on the total DNA content of the tissue sample being analyzed. The total peptide content of TUMAP in the tissue sample is measured by nanoLC-MS / MS as the ratio between the natural TUMAP and the known amount of isotope-labeled form of TUMAP (the so-called internal standard). The peptide:MHC complex of all selected TUMAPs is incorporated into the tissue lysate at the earliest possible time point of the TUMAP separation procedure and the efficiency of TUMAP separation is determined by nanoLC-MS / MS detection after the completion of the peptide separation procedure. The total cell count and total peptide amount are calculated based on three measurements per tissue sample. The peptide-specific separation efficiency is calculated as the average value of 9 spike experiments with three replicates each (see Example 6 and Table 15).

[0188] In addition to over-presented peptides, mRNA expression of potential genes was also tested. mRNA data was obtained by RNA sequencing analysis of normal tissues and cancer tissues (see Example 2, Figure 2). An additional source of normal tissue data is a database of RNA expression data publicly available from 3000 normal tissue samples (Lonsdale, 2013). Peptides obtained from proteins show high expression of encoding mRNA in cancer tissues, but are very low or absent in important normal tissues. These peptides are included in the present invention as preferred peptides.

[0189] The present invention proposes peptides that are beneficial for treating cancer / tumors, preferably for treating acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer that over-present or exclusively present the peptides of the present invention. These peptides were shown by mass spectrometry to be naturally presented by HLA molecules in primary human acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer samples.

[0190] Compared with normal tissues, many source genes / proteins (also called "full-length proteins" or "potential proteins") from which peptides are derived are highly overexpressed in cancer - "normal tissues" related to the present invention should refer to healthy blood, brain, heart, liver, lung, adipose tissue, adrenal gland, bile duct, bladder, bone marrow, esophagus, eye, gall bladder, head and neck, large intestine, small intestine, kidney, lymph node, central nervous system, peripheral nervous system, pancreas, parathyroid, peritoneum, pituitary, pleura, skeletal muscle, skin, spinal cord, spleen, stomach, thyroid, trachea and ureter cells or other normal tissue cells, which shows that tumors are highly associated with these source genes (see Example 2). In addition, these peptides themselves are also over-presented in large quantities in tumor tissues (the "tumor tissue" related to the present invention should refer to samples from patients with acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer), but are not over-presented in large quantities in normal tissues (see Example 1).

[0191] HLA-bound peptides can be recognized by the immune system, especially T lymphocytes. T cells can destroy cells presenting the recognized HLA / peptide complex, such as acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer cells presenting the derived peptide.

[0192] All peptides of the present invention have been shown to have the ability to stimulate T cell responses and / or over-presentation, and can therefore be used to prepare antibodies and / or TCRs of the present invention, such as soluble TCRs (see Example 3). In addition, the peptides can also be used to prepare antibodies and / or TCRs of the present invention, in particular sTCRs, when combined with the corresponding MHC. The various methods are well known to the skilled person and can be found in various documents (see also below). Therefore, the peptides of the present invention can be used to generate an immune response in a patient, thereby destroying tumor cells. The patient's immune response can be induced by directly administering the peptide or precursor substance (e.g., elongated peptides, proteins or nucleic acids encoding these peptides) to the patient, preferably in combination with an immunogenicity-enhancing agent. The immune response derived from the therapeutic vaccine is expected to be highly specific against tumor cells, because the target peptides of the present invention are presented in a low number of copies on normal tissues, preventing the risk of adverse autoimmune reactions against normal cells in the patient.

[0193] The present invention also relates to a T cell receptor (TCR) comprising an alpha chain and a beta chain (alpha / beta TCR). Also provided are peptides of the present invention that can bind to TCR and antibodies when presented by MHC molecules.

[0194] The present specification also relates to a TCR fragment of the present invention that is presented by an HLA molecule and is capable of binding to a peptide antigen of the present invention. The term particularly relates to a soluble TCR fragment, such as a TCR that has lost a transmembrane portion and / or a constant region, a single-chain TCR, and a fusion thereof with, for example, lg.

[0195] The present specification also relates to nucleic acids, vectors, and host cells for expressing TCR and peptides of the present specification; and methods of using the same.

[0196] The term "T cell receptor" (abbreviation 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 to peptide antigens presented by HLA molecules. The term also includes so-called gamma / delta TCRs.

[0197] 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 the TCR under conditions suitable for promoting expression of the TCR.

[0198] In another aspect, the present description relates to a method according to the present description, wherein the antigen is loaded onto a class I or II MHC molecule expressed on the surface of a suitable antigen-presenting cell or an artificial antigen-presenting cell by binding to a sufficient amount of antigen-containing antigen-presenting cells, or the antigen is loaded onto a class I or II MHC tetramer / class I or II MHC complex monomer by tetramerization.

[0199] The α and β chains of α / β TCR and the γ and δ chains of γ / δ TCR are generally considered to have two "domains", namely variable and constant domains. The variable domain consists of a combination of a variable region (V) and a joining region (J). The variable domain may also include a leader region (L). The β and δ chains may also include a diversity region (D). The α and β constant domains may also include a C-terminal transmembrane (TM) domain that anchors the α and β chains to the cell membrane.

[0200] With respect to γ / δ TCR, as used herein, the term "TCRγ variable domain" refers to a combination of a TCRγ V (TRGV) region without a leader region (L) and a TCRγ (TRGJ) region, and the term TCRγ constant domain refers to an extracellular TRGC region or a C-terminal truncated TRGC sequence. Similarly, the term "TCR δ variable domain" refers to a combination of a TCR δ V (TRDV) region without a leader region (L) and a TCR δ D / J (TRDD / TRDJ) region, and the term "TCR δ constant domain" refers to an extracellular TRDC region or a C-terminal truncated TRDC sequence.

[0201] The TCR of the present specification preferably binds to a peptide HLA molecule complex with a binding affinity (KD) of about 100 μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM or less. A more preferred situation is a high affinity TCR with a binding affinity of about 1 uM or less, about 100 nm or less, about 50 nM or less, or about 25 nM or less. Non-limiting examples of preferred binding affinity ranges of the TCR of the present invention include about 1 nM to about 10 nM; about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; and about 90 nM to about 100 nM.

[0202] As used herein, "specifically binds" and its grammatical variations when used in connection with the TCRs of this specification are used to indicate a TCR that has a binding affinity (KD) for a peptide-HLA molecule complex of 100 μM or less.

[0203] The α / β heterodimeric TCRs of the present specification may have introduced disulfide bonds 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, except that threonine 48 of TRAC and serine 57 of TRBC1 or TRBC2 are substituted with cysteine ​​residues that form disulfide bonds between the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

[0204] With or without the above-mentioned introduced interchain bond, the α / β heterodimeric TCR of the present specification 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 via a native disulfide bond between Cys4 of TRAC exon 2 and Cys4 of TRBC1 or TRBC2 exon 2.

[0205] The TCR of the present specification may include a detectable label selected from the group consisting of a radionuclide, a fluorophore and biotin. The TCR of the present specification may be conjugated to a therapeutically active agent, such as a radionuclide, a chemotherapeutic agent or a toxin.

[0206] In one embodiment, a TCR of the present disclosure having at least one mutation in the α chain and / or having at least one mutation in the β chain has modified glycosylation compared to a non-mutated TCR.

[0207] In one embodiment, a TCR comprising at least one mutation in a TCR alpha chain and / or a TCR beta chain has a binding affinity and / or binding half-life for a peptide HLA molecule complex that is at least twice that of a TCR comprising an unmutated TCR alpha chain and / or an unmutated TCR beta chain. Tumor-specific TCR affinity enhancement and its development rely on the existence of a window of optimal TCR affinity. The existence of such a window is based on the observation that specific TCRs such as HLA-A2 restricted pathogens generally have KD values ​​that are approximately 10 times lower than specific TCRs such as HLA-A2 restricted tumor-associated self-antigens. It is now known that although tumor antigens may be immunogenic, because the tumor comes from the individual's own cells, only mutant proteins or proteins with altered translation processing will be considered foreign by the immune system. Antigens that are upregulated or overexpressed (so-called self-antigens) do not necessarily induce a functional immune response against tumors: T cells expressing TCRs that are highly reactive to these antigens are unfavorably selected in the thymus in a process called central tolerance, that is, only cells with low-affinity TCRs for self-antigens remain. Therefore, the affinity of the TCR or variant of the present specification for peptides can be enhanced by methods well known in the art.

[0208] The present specification also relates to a method for identifying and isolating the TCR of the present specification, the method comprising: incubating PBMCs from HLA-A*02 negative healthy donors with A2 / peptide monomers, incubating PBMCs with tetramer-phycoerythrin (PE) and isolating high-affinity T cells through fluorescence activated cell sorting (FACS) method-Calibur analysis.

[0209] The present specification also relates to a method for identifying and isolating the TCR of the present specification, the method comprising: obtaining transgenic mice containing the entire human TCRαβ gene locus (1.1 and 0.7 Mb) (whose T cells express diversified human TCRs to compensate for the TCR deficiency of mice), immunizing the mice with a peptide, incubating PBMc obtained from the transgenic mice with tetramer-phycoerythrin (PE), and isolating high-affinity T cells through fluorescence activated cell sorting (FACS) method-Calibur analysis.

[0210] On the one hand, in order to obtain T cells expressing the TCR of the present specification, nucleic acids encoding the TCR-α and / or TCR-β chains of the present specification are cloned into expression vectors, such as gamma retroviruses or lentiviruses. Recombinant viruses are produced and then tested for function, such as antigen specificity and functional affinity. Then, aliquots of the final product are used to transduce target T cell populations (generally purified from the patient's PBMc) and are launched before being introduced into the patient.

[0211] On the other hand, in order to obtain T cells expressing the TCR of the present specification, TCR RNA is synthesized by techniques known in the art (e.g., in vitro transcription system). Then, the in vitro synthesized TCR RNA is introduced into primary CD8+ T cells obtained from healthy donors by electroporation to re-express tumor-specific TCR-α and / or TCR-β chains.

[0212] To increase expression, the nucleic acid encoding the TCR of the present specification can be operably linked to a strong promoter, such as retroviral long terminal repeat (LTR), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin, ubiquitin and simian virus 40 (SV40) / CD43 composite promoter, elongation factor (EF)-1a and spleen focus forming virus (SFFV) promoter. In a preferred embodiment, the promoter is heterologous to the nucleic acid being expressed.

[0213] In addition to a strong promoter, the TCR expression cassette of the present specification may contain additional elements that can enhance transgene expression, including the central polypurine tract (cPPT), which promotes nuclear translocation of lentiviral constructs (FolIenzi et al., 2000), and woodchuck hepatitis virus posttranscriptional regulatory elements (wPRE), which increase transgene expression levels by improving RNA stability (Zufferey et al., 1999).

[0214] The α and β chains of the TCR of the present invention may be encoded by nucleic acids located on separate vectors, or may be encoded by polynucleotides located on the same vector.

[0215] Realizing high-level TCR surface expression requires the introduction of TCR-α and TCR-β chain high-level transcription of TCR. To achieve it, the TCR-α and TCR-β chain of this specification can be cloned into a bicistronic construct in a single vector, which has been shown to overcome this obstacle. The use of the viral ribosome entry site (lRES) between TCR-α and TCR-β chains causes the coordinated expression of the two chains, because TCR-α and TCR-β chains are all produced by a single transcript that is divided into two proteins during translation, thereby ensuring the equal molar ratio of producing TCR-α and TCR-β chains.

[0216] The nucleic acid encoding the TCR of the present specification may be a codon optimized to increase expression from a host cell. Genetic code redundancy allows some amino acids to be encoded by more than one codon, but some codons are not as "ideal" as other codons because of the relative availability of matching tRNA and other factors (Gustafsson et al., 2004). Modifying the TCR-α and TCR-β gene sequences so that each amino acid is encoded by the optimal codon for mammalian gene expression, as well as eliminating mRNA instability motifs or cryptic splicing sites, has been shown to significantly increase TCR-α and TCR-β gene expression (Scholten et al., 2006).

[0217] In addition, mispairing between introduced and endogenous TCR chains may result in the acquisition of specificities that pose a significant risk for autoimmunity. For example, the formation of mixed TCR dimers may reduce the number of CD3 molecules available to form correctly paired TCR complexes and, therefore, can significantly reduce the functional avidity of cells expressing the introduced TCR (Kubal et al., 2007).

[0218] To reduce mispairing, the C-terminal domains of the TCR chains introduced in the present specification can be modified to promote interchain affinity while reducing the ability of the introduced chains to pair with endogenous TCRs. These strategies may include replacing the human TCR-α and TCR-β C-terminal domains with mouse counterparts (murinized C-terminal domains); generating a second interchain disulfide bond in the C-terminal domain by introducing a second cysteine ​​residue into the TCR-a and TCR-β chains of the introduced TCR (cysteine ​​modification); exchanging the interacting residues of the C-terminal domains of the TCR-a and TCR-β chains ("knob-in-hole structure"); directly fusing the variable domains of the TCR-α and TCR-β chains to CD3ζ (CD3ζ fusion) (Schmitt et al., 2009).

[0219] In one embodiment, the host cell is structurally altered to express the TCR of this specification. In a preferred embodiment, the host cell is a human T cell or a T cell progenitor cell. In some embodiments, the T cell or T cell progenitor cell is obtained from a cancer patient. In other embodiments, the T cell or T cell progenitor cell is obtained from a healthy donor. The host cell of this specification can be allogeneic or autologous relative to the patient to be treated. In one embodiment, the host is a γ / δ T cell transformed to express an α / β TCR.

[0220] A "pharmaceutical composition" refers to a composition suitable for administration to humans in a medical setting. Preferably, the pharmaceutical composition is sterile and manufactured according to GMP guidelines.

[0221] The pharmaceutical composition includes 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 an acid or pharmaceutically acceptable salt of thiazolidine. For example, acid salts are prepared by reacting free thiazolidine (generally neutral drugs having a neutral -NH2 group) with a suitable acid. Suitable acids for preparing acid salts include organic acids such as acetic acid, propionic acid, hydroxy acids, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, methanesulfonic acid, benzenesulfonic acid, salicylic acid, and the like, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Conversely, thiazolidine salt preparations of acidic groups present on a peptide are prepared using pharmaceutically acceptable thiazolidine groups such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

[0222] In particularly preferred embodiments, the pharmaceutical composition comprises the peptide in the form of acetic acid (acetate), trifluoroacetate, or hydrochloric acid (chloride).

[0223] The agent of the present invention is preferably an immunotherapeutic agent, for example, a vaccine. The vaccine may be administered directly to the affected organ of the patient, or systemically by id, im, sc, ip and iv injection, or applied in vitro to cells from the patient or human cell line (which are then injected into the patient), or used in vitro to select immune cell subsets 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 immunostimulatory cytokines (such as interleukin-2). The peptide may be substantially pure or combined with an immunostimulatory adjuvant (see below), or used in combination with an immunostimulatory cytokine, or administered in an appropriate delivery system (such as liposomes). The peptide may also be conjugated to a suitable carrier (such as keyhole limpet hemocyanin (KLH) or mannan) to a suitable carrier (see WO 95 / 18145 and (Longenecker et al., 1993)). The peptide may also be labeled, or may be a fusion protein or hybrid molecule. The peptides of the present invention, whose peptide sequences are given, are expected to stimulate CD4 or CD8 T cells. However, CD8 T cell stimulation is more effective with the help of CD4 T-helper cells. Therefore, for MHC class I epitopes that stimulate CD8 T cells, a fusion partner or fragment of a hybrid molecule provides an appropriate epitope for stimulating CD4 positive T cells. CD4- and CD8 stimulatory epitopes are well known in the art and include those identified in the present invention.

[0224] In one aspect, the vaccine comprises at least one peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 101 and at least one additional 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. The peptides may be derived from one or more specific TAAs and may bind to MHC class I molecules.

[0225] In another aspect, the present invention provides a nucleic acid (e.g., a polynucleotide) encoding a peptide or peptide variant of the present invention. The polynucleotide may be, for example, DNA, cDNA, PNA, RNA, or a combination thereof, which may be single-stranded and / or double-stranded, or a native or stable form of a polynucleotide (e.g., a polynucleotide having a thiophosphate backbone), and may or may not contain introns as long as it encodes a peptide. Of course, a polynucleotide can only encode a peptide that incorporates a natural peptide bond and contains natural amino acid residues. In another aspect, the present invention provides an expression vector that can express a polypeptide according to the present invention.

[0226] A variety of methods have been developed for joining polynucleotides, especially for DNA, by adding ligatable ends to the vector. For example, complementary homopolymer tracks can be added to the DNA fragment, which is then inserted into the vector DNA. The vector and DNA fragment are then joined by hydrogen bonding of the complementary homopolymer tails, thereby forming a recombinant DNA molecule.

[0227] Synthetic linkers containing one or more restriction endonucleases provide another method for ligating DNA fragments to vectors. Synthetic linkers containing various restriction endonucleases can be purchased from a variety of sources, including International Biotechnologies Inc. (New Haven, Connecticut, USA).

[0228] The DNA encoding the polypeptide of the present invention is preferably modified by the polymerase chain reaction method disclosed by Saiki RK et al. (Saiki et al., 1988). This method can be used to introduce DNA into a suitable vector (for example, by designing suitable restriction sites), and can also be used to modify DNA by other useful methods known in the art. If a viral vector is used, a poxvirus vector or an adenovirus vector is preferred.

[0229] Afterwards, the DNA (or in the case of a retroviral vector, RNA) may be expressed in a suitable host, thereby producing a polypeptide containing the peptide or variant of the present invention. Thus, the DNA encoding the peptide or variant of the present invention may be used according to known techniques, after appropriate modification using the methods described herein, to construct an expression vector, which is then used to transform a suitable host cell, thereby expressing and producing the polypeptide of the present 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.

[0230] The DNA (or RNA in the case of retroviral vectors) encoding the polypeptide containing the compound of the invention may be added to a variety of other DNA sequences and introduced 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 is necessary to maintain it as an episome or to be combined with each other.

[0231] In general, the DNA can be attached to an expression vector (such as a plasmid) in the proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the corresponding transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such control functions are generally present in the expression vector. 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 includes inserting a DNA sequence into the expression vector with any necessary control elements, which encodes a selectable attribute (such as antibiotic resistance) in the transformed cell.

[0232] Alternatively, the gene conferring such a selectable trait may be on another vector which is used to co-transform the desired host cell.

[0233] The host cells transformed with the recombinant DNA of the present invention are then cultured under appropriate conditions familiar to those skilled in the art as described herein and for a sufficient period of time to express the peptide which can then be recovered.

[0234] There are many known expression systems, including bacteria (such as E. coli and Bacillus subtilis), yeast (such as Saccharomyces cerevisiae), filamentous fungi (such as Aspergillus), plant cells, animal cells and insect cells. The system can be preferably mammalian cells, such as CHO cells from ATCC Cell Biology Collection.

[0235] Typical mammalian cell constitutive expression vector plasmids include CMV or SV40 promoters containing a suitable poly A tail and resistance markers (such as neomycin). An example is pSVL obtained from Pharmacia (Piscataway, New Jersey, USA). An example of an inducible mammalian expression vector is pMSG, which can also be obtained from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, which are generally available from Stratagene Cloning Systems (La Jolla, California 92037, USA). Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integration plasmids (YIp), and yeast selectable markers HIS3, TRP1, LEU2 and URA3 are inserted. pRS413-416 plasmids are yeast centromere plasmids (Ycp). Vectors based on the CMV promoter (e.g., from Sigma-Aldrich) provide transient or stable expression, cytoplasmic expression or secretion, and N-terminal or C-terminal tagging in different combinations of FLAG, 3xFLAG, c-imyc or MATN. These fusion proteins can be used to detect, purify and analyze recombinant proteins. Dual-label fusions provide flexibility for detection. The strong human cytomegalovirus (CMV) promoter regulatory region allows constitutive protein expression levels in COS cells to reach up to 1 mg / L. For weaker cell lines, protein levels are generally less than 0.1 mg / L. The presence of the SV40 replication origin will result in high-level replication of DNA in SV40 replication-tolerant COS cells. For example, CMV vectors can contain the pMB1 (derivative of pBR322) replication origin in bacterial cells, the β-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA and f1 origins. Vectors containing the preproinsulin leader (PPT) sequence can use anti-FLAG antibodies, resins and plates to direct the secretion of FLAG fusion proteins into the culture medium for purification. Other vectors and expression systems for use with a variety of host cells are well known in the art.

[0236] In another embodiment, two or more peptides or peptide variants of the invention are encoded and thus expressed in a continuous order (similar to a "beaded structure" construct). Upon reaching the target, the peptides or peptide variants may be linked or fused together via a stretch of linker amino acids (e.g., LLLLLL) or may be linked without any additional peptides between them. These constructs may also be used in cancer therapy to induce an immune response involving MHCl and MHCII.

[0237] The present invention also relates to a host cell, which is transformed with the polynucleotide vector construction of the present invention. The host cell can be a prokaryotic cell or a eukaryotic cell. In some cases, bacterial cells are preferred prokaryotic host cells, typically Escherichia coli strains, for example, Escherichia coli strain DH5 (obtained from Bethesda Research Laboratories, Bethesda, Maryland, USA) and RR1 (obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA), ATCC No. 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells, such as cells in mouse, rat, monkey or human fibroblasts and colon cancer cell lines. Yeast host cells include YPH499, YPH500 and YPH501, which are generally available from Stratagene Cloning Systems, LaJolla, California 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells, CCL61 cells in ATCC, NIH Swiss mouse embryonic cells NlH / 3T3 cells, CRL 1658 cells in ATCC, monkey kidney-derived COS-1 cells, CRL 1650 cells in ATCC, and human embryonic kidney cells No. 293 cells. Preferred insect cells are Sf9 cells, which can be transfected with baculovirus expression vectors. An overview of the selection of appropriate host cells for expression can be found in the textbook (Paulina and Argelia Lorence "Methods in Molecular Biology Recombinant Gene Expression, Reviews and Protocols" Part One, Second Edition, ISBN 978-1-58829-262-9) and other documents known to those skilled in the art.

[0238] Transformation of appropriate host cells containing the DNA construct of the present invention can be accomplished using well-known methods, generally depending on the type of vector used. For 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 method described in Beggs (Beggs, 1978) is also useful.

[0239] For vertebrate cells, reagents for transfecting these cells, such as calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, Inc. or Life Technologies, Inc. (Gaithersburg, Maryland 20877, USA). Electroporation can also be used to transform and / or transfect cells and is a well-known method in the art for transforming yeast cells, bacterial cells, insect cells, and vertebrate cells.

[0240] Successfully transformed cells (i.e. cells containing the DNA construct of the present invention) can be identified by well-known methods (e.g. PCR). In addition, proteins present in the supernatant can be detected using antibodies.

[0241] It should be understood that certain host cells of the present invention are used to prepare the peptides of the present invention, such as bacterial cells, yeast cells and insect cells. However, other host cells may be useful for certain therapeutic methods. For example, antigen presenting cells (such as dendritic cells) can be used to express the peptides of the present invention so that they can be loaded into the corresponding MHC molecules. Therefore, the present invention provides a host cell containing the nucleic acid or expression vector of the present invention.

[0242] In a preferred embodiment, the host cell is an antigen presenting cell, in particular a dendritic cell or an antigen presenting cell. On April 29, 2010, the U.S. Food and Drug Administration (FDA) approved a recombinant fusion protein containing prostatic acid phosphatase (PAP) for the treatment of asymptomatic or mildly symptomatic metastatic HRPC (Rini et al., 2006; Small et al., 2006).

[0243] In another aspect, the present invention provides a method for preparing a peptide and variants thereof, the method comprising culturing host cells and isolating the peptide from the host cells or their culture medium.

[0244] In another embodiment, the peptide, nucleic acid or expression vector of the present invention is used in medicine. For example, the peptide or its variant can be prepared as an intravenous (iv) injection, a subcutaneous (sc) injection, an intradermal (id) injection, an intraperitoneal (ip) injection, or an intramuscular (im) injection. Preferred methods for peptide injection include sc, id, ip, im and iv injection. Preferred methods for DNA injection are id, im, sc, ip and iv injection. For example, 50 μg to 1.5 mg, preferably 125 μg to 500 μg of peptide or DNA is administered, depending on the specific peptide or DNA. The above dosage range has been successfully used in previous experiments (WaIter et al., 2012).

[0245] The polynucleotides used for active immunization can be substantially pure or contained in a suitable carrier or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, or a combination thereof. The design and introduction of such nucleic acids are well known in the art. For example, there is an overview of this in the literature (Teufel et al., 2005). Polynucleotide vaccines are easy to prepare, but the mode of action of these vectors in inducing immune responses is not yet fully understood. Suitable carriers 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 include cationic liposomes and cationic polymers, which are well-known systems in the field of DNA delivery. Physical delivery systems can also be used, such as through a "gene gun". The peptide or nucleic acid-encoded peptide can be a fusion protein, for example, containing epitopes that stimulate T cells to carry out the above-mentioned CDRs.

[0246] The medicaments of the present invention may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or strengthen immune responses (e.g., immune responses to an antigen mediated by CD8-positive T cells and helper T (TH) cells) and are therefore considered useful for the medicaments of the present invention. Suitable adjuvants include (but are not limited to) 1018ISS, aluminum salts, AS15, BCG, CP-870,893, CPG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod , resiquimod, ImuFactIMP321, interleukin IL-2, IL-13, IL-21, interferon α or β, or their PEGylated derivatives, IS Patch, ISS, ISCOMATRIX, ISCOMs, LipoVac, MALP2, MF59, monophosphatidylserine A, Montanide lMS1312, 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, Vector systems, polylactide-co-glycolide [PLG] and dextran-based microparticles, recombinant human lactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulator derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, QuiI or Superfos. Preferred adjuvants are Freund's adjuvant or GM-CSF. Some dendritic cell-specific immune adjuvants (such as MF59) and their preparation have been described by previous researchers (Allison and Krummel, 1995). Cytokines can also be used. Some cytokines have been directly linked to influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into potent antigen-presenting cells of T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, specifically incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich et al., 1996).

[0247] CpG immunostimulatory oligonucleotides have been reported to enhance the effects of adjuvants in vaccines. Without being bound by theory, CpG oligonucleotides may act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. 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 maturation and differentiation of dendritic cells, leading to enhanced activation of TH1 cells and enhanced generation of cytotoxic T lymphocytes (CTLs), even in the absence of CD4 T cell help. The TH1 shift induced by TLR9 stimulation can be maintained even in the presence of vaccine adjuvants, such as alum or Freund's incomplete adjuvant (IFA), which normally promote TH2 shifts. CpG oligonucleotides exhibit stronger adjuvant activity when prepared or co-administered with other adjuvants or formulations, such as microparticles, nanoparticles, fat emulsions or similar preparations, which are particularly necessary to induce strong responses when the antigen is relatively weak. They can also accelerate immune responses, reducing the antigen dose by about two orders of magnitude, and in some experiments, similar antibody responses can be generated to full-dose vaccines without CpG (Krieg, 2006). U.S. Patent No. 6406705 B1 describes the use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens in combination to promote antigen-specific immune responses. A CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules, such as RNA binding to TLR7, TLR8 and / or TLR9, can also be used.

[0248] Other useful adjuvant examples include (but are not limited to) chemically modified CpG (such as CpR, Idera), dsRNA mimics, such as Poly (1: C) and its derivatives (such as: Poly-(ICLC), poly(IC-R), poly(I:C12U)), non-CpG bacterial DNA or RNA, and immunoreactive small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting major immune system structures (e.g., anti-CD40, anti-TGF β, anti-TNF α receptor), and SC58175, all of which may have therapeutic effects and / or act as adjuvants. The amount and concentration of adjuvants and additives useful in the present invention can be readily determined by the skilled artisan without undue experimentation.

[0249] Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon α, CpG oligonucleotides and derivatives, poly (l: C) and derivatives, RNA, sildenafil, and microparticle preparations of PLG or virus particles.

[0250] In a preferred embodiment of the pharmaceutical composition of the present invention, the adjuvant is selected from a preparation containing colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod and interferon-α.

[0251] In a preferred embodiment of the pharmaceutical composition of the present invention, the adjuvant is selected from a preparation containing colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod and resimiquimod. In a preferred embodiment of the pharmaceutical composition of the present invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. More preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA50V, Montanide ISA-51, polyisocyanate ... and anti-CD40 mAb or a combination thereof.

[0252] This combination drug is used for parenteral injection, such as subcutaneous, intradermal, intramuscular injection, and can also be taken orally. For this reason, the peptide and optionally other molecules are decomposed or suspended in a pharmaceutical carrier, preferably an aqueous carrier. In addition, the composition may contain excipients, such as buffers, binders, shock agents, diluents, spices, lubricants, etc. These peptides can also be used in combination with immunostimulants, such as cytokines. More excipients that can be used for such compositions can be found in books such as Handbook of Pharmaceutical Excipients (Kibbe, 2000) written by A.Kibbe. This combination drug can be used to prevent, prevent and / or treat adenomas or cancerous diseases. For example, representative preparations are available in EP2112253.

[0253] It is important to realize that the immune response elicited by the vaccine 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 is an advantage over other vaccines, which target only one or a few targets, which may cause the tumor to easily adapt to the attack (tumor escape). In addition, not all individual tumors express the same pattern of antigens. Therefore, the combination of several tumor-associated peptides ensures that each tumor bears at least some targets. The composition is designed in such a way that it is expected that each tumor may express several antigens and cover several independent pathways required for tumor growth and maintenance. Therefore, the vaccine can be easily "off-the-shelf" for a larger patient population. This means that pre-selection of patients receiving vaccine treatment can be limited to HLA typing, without any additional biomarker assessment of antigen expression, but still ensures that multiple targets are attacked by the induced immune response at the same time, which is important for efficacy (Banchereau et al., 2001; Walter et al., 2012).

[0254] As used herein, the term "scaffold" refers to a molecule that specifically binds to a (e.g., antigen) determinant. In one embodiment, the scaffold is capable of directing the entity to which it is attached (e.g., a (second) antigen binding moiety) to a target of interest, for example, to a specific type of tumor cell or a tumor stroma bearing an antigenic determinant (e.g., a complex of a peptide and MHC according to the current 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 an antibody heavy chain variable region and an antibody light chain variable region, bound proteins comprising at least one ankyrin repeat motif and a single domain antigen binding (SDAB) molecule, an aptamer, a (soluble) TCR, and (modified) cells, such as allogeneic or autologous T cells. In order to assess whether a molecule is a scaffold that binds to a target, a binding assay may be performed.

[0255] "Specific" binding means that the scaffold binds better to the peptide-MHC complex of interest than to other natural peptide-MHC complexes, to the extent that a scaffold with active molecules capable of killing cells bearing the specific target is not able to kill another cell without the specific target but presenting one or more other peptide-MHC complexes. If the peptide of the cross-reactive peptide-MHC is not natural, i.e., not from the human HLA-peptide group, binding to other peptide-MHC complexes is irrelevant. Tests to assess target cell killing are well known in the art. They should be performed with target cells (primary cells or cell lines) presenting unaltered peptide-MHC or with cells loaded with the peptide so as to reach the level of natural peptide-MHC.

[0256] Each scaffold may include a label that can detect the presence or absence of a signal provided by the label. For example, the scaffold can be labeled with a fluorescent dye or any other suitable cell 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 by fluorescent or laser scanning microscopy or flow cytometry.

[0257] Each scaffold can be conjugated to a second active molecule (eg, IL-21, anti-CD3, anti-CD28).

[0258] For further information on polypeptide scaffolds, see, for example, the background section of WO 2014 / 071978 A1 and the references cited therein.

[0259] The present invention also relates to aptamers. Aptamers (e.g., see WO 2014 / 191359 and documents cited therein) are short single-stranded nucleic acid molecules that can fold into a defined three-dimensional structure and recognize a specific target structure. They appear to be a suitable alternative for developing targeted therapies. Aptamers have been shown to selectively bind to complex targets with high affinity and specificity.

[0260] Aptamers that recognize cell surface molecules have been identified over the past decade and provide a means for developing diagnostic and therapeutic approaches. Aptamers have been shown to be virtually non-toxic and non-immunogenic, and therefore, are promising candidates for biomedical applications. Aptamers, such as prostate-specific membrane antigen-recognizing aptamers, have been successfully used for targeted therapy and have been shown to function in xenografts in in vivo models. In addition, aptamers that recognize specific tumor cell lines have also been identified.

[0261] DNA aptamers can be selected to reveal broad-spectrum recognition properties for various cancer cells, especially those derived from solid tumors, while non-tumorigenic and predominantly healthy cells are not recognized. If the identified aptamers not only recognize tumor-specific subtypes but also interact with a range of tumors, this makes the aptamers suitable as so-called broad-spectrum diagnostics and therapeutics.

[0262] Furthermore, investigation of the cell binding behavior using flow cytometry revealed that the aptamers displayed good affinity in the nanomolar range.

[0263] Aptamers are used for diagnostic and therapeutic purposes. Furthermore, it could also be shown that some aptamers are taken up by tumor cells and thus can serve as molecular vehicles for the targeted delivery of anticancer agents, such as siRNA into tumor cells.

[0264] Aptamers can be selected against complex targets such as cells and tissues and complexes of peptides according to the current invention comprising (preferably consisting of) a sequence according to any of SEQ ID NO 1 to SEQ ID NO 101 and MHC molecules using the cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology.

[0265] The peptides of the present invention can be used to generate and develop specific antibodies against MHC / peptide complexes. These antibodies can be used therapeutically to target toxins or radioactive substances to diseased tissue. Another use of these antibodies is to target radionuclides to diseased tissue for imaging purposes (such as PET). This can help detect small metastases or determine the size and exact location of diseased tissue.

[0266] Therefore, another aspect of the present invention is to propose a method for producing a recombinant antibody that specifically binds to class I or II human major histocompatibility complex (MHC) complexed with an HLA-restricted antigen (preferably a peptide according to the present invention), the method comprising: immunizing a genetically engineered non-human mammal containing cells expressing the major histocompatibility complex (MHC) class I or II with a soluble form of the (MHC) class I or II molecule complexed with the HLA-restricted antigen; separating mRNA molecules from antibody-producing cells of the non-human mammal; producing a phage display library that displays protein molecules encoded by the mRNA molecules; and isolating at least one phage from the phage display library, the at least one phage displaying the antibody specifically binding to the human major histocompatibility complex (MHC) class I or II complexed with the HLA-restricted antigen.

[0267] Therefore, another aspect of the present invention provides an antibody which specifically binds to a class I or II human major histocompatibility complex (MHC) complexed with an HLA-restricted antigen, wherein the antibody is preferably a polyclonal antibody, a monoclonal antibody, a bispecific antibody and / or a chimeric antibody.

[0268] Corresponding methods for producing such antibodies and single-chain class I major histocompatibility complexes, as well as other tools for producing these antibodies are disclosed in WO 03 / 068201, WO 2004 / 084798, WO 01 / 72768, WO 03 / 070752 and in publications (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), all of which are incorporated herein by reference in their entirety for the purposes of the present invention.

[0269] Preferably, the antibody binds to the complex with an affinity lower than 20 nanomolar, preferably lower than 10 nanomolar, which is also considered "specific" in the context of the present invention.

[0270] The present invention relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101 or a variant thereof having at least 88% homology (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 101, or a variant thereof that induces T cells to cross-react with the peptide, wherein the peptide is not a potential full-length polypeptide.

[0271] The present invention further relates to a peptide comprising a sequence selected from the group of SEQ ID NO: 1 to SEQ ID NO: 101, or a variant having at least 88% homology (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 101, wherein the total length of the peptide or variant is 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0272] The present invention further relates to the peptides of the present invention, which have the ability to bind to major histocompatibility complex (MHC) class I or II molecules.

[0273] The present invention further relates to the peptides of the present invention, wherein the peptide consists of or essentially consists of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 101.

[0274] The present invention further relates to the peptides of the present invention, wherein said peptide is (chemically) modified and / or comprises non-peptide bonds.

[0275] The present invention further relates to the peptides of the present invention, wherein the peptide is part of a fusion protein, in particular comprising the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii), or wherein the peptide is fused to an antibody (e.g. a dendritic cell-specific antibody).

[0276] The present invention further relates to a nucleic acid encoding a peptide according to the present invention, with the proviso that the peptide is not a complete (full) human protein.

[0277] The present invention further relates to a nucleic acid according to the present invention, which is DNA, cDNA, PNA, RNA, or a combination thereof.

[0278] The present invention further relates to an expression vector capable of expressing the nucleic acid of the present invention.

[0279] The present invention further relates to the use of a peptide of the present invention, a nucleic acid of the present invention or an expression vector of the present invention in medicine, in particular for the treatment of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer.

[0280] The present invention further relates to a host cell comprising the nucleic acid according to the invention or the expression vector according to the invention.

[0281] The present invention further relates to the host cell of the present invention, which is an antigen presenting cell, preferably a dendritic cell.

[0282] The present invention further relates to a method of formulating a peptide of the present invention, said method comprising culturing the host cell of the present invention and isolating the peptide from said host cell or its culture medium.

[0283] The present invention further relates to the method of the present invention, wherein the antigen is loaded onto class I or II MHC molecules expressed on the surface of suitable antigen presenting cells by binding to a sufficient amount of antigen containing antigen presenting cells.

[0284] The present invention further relates to the method of the present invention, wherein the antigen presenting cell comprises an expression vector capable of expressing the peptide or variant amino acid sequence comprising SEQ ID NO: 1 to SEQ ID NO: 101.

[0285] The present invention further relates to activated T cells produced by the method of the present invention, wherein the T cells selectively recognize a cell that abnormally expresses a polypeptide comprising an amino acid sequence of the present invention.

[0286] The present invention further relates to a method for killing target cells of a patient, wherein the target cells of the patient abnormally express a polypeptide comprising any amino acid sequence of the present invention, the method comprising administering an effective amount of T cells of the present invention to the patient.

[0287] The present invention further relates to the use of any of the peptides, a nucleic acid of the present invention, an expression vector of the present invention, a cell of the present invention, an activated cytotoxic T lymphocyte of the present invention as a medicament or for the manufacture of a medicament. The present invention further relates to a use of the present invention, wherein the medicament is effective against cancer.

[0288] The present invention further relates to a use of the present invention, wherein the medicament is a vaccine. The present invention further relates to a use of the present invention, wherein the medicament is effective against cancer.

[0289] The present invention further relates to the use according to the present invention, wherein the cancer cells are acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer cells, or other solid or blood tumor cells, such as: acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer.

[0290] The present invention further relates to a specific marker protein and biomarker based on the peptide of the present invention, referred to herein as a "target", which can be used to diagnose and / or judge the prognosis of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer. The present invention also relates to these new targets for cancer treatment.

[0291] The term "antibody" is defined in a broad sense herein, including both polyclonal and monoclonal antibodies. In addition to complete 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, as long as they exhibit any desired attributes of the present invention (e.g., acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer marker (poly) peptide specific binding, delivery of toxins to cancer marker gene expression levels increased acute myeloid leukemia disease, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer cells and / or inhibit the activity of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer marker polypeptides)o

[0292] Whenever possible, the antibodies of the present invention may be purchased from commercial sources. The antibodies of the present invention may also be made using known methods. The skilled artisan will appreciate that full-length acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer marker polypeptides or fragments thereof may be used to prepare the antibodies of the present invention. The polypeptides used to generate the antibodies of the present invention may be purified in part or in whole from natural sources, or may be produced using recombinant DNA technology.

[0293] For example, cDNA encoding a peptide of the present invention (e.g., a peptide according to a polypeptide of SEQ ID NO: 1 to SEQ ID NO: 101) or a variant or fragment thereof can be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect or mammalian cells), after which the recombinant protein can be purified and used to produce a monoclonal or polyclonal antibody preparation that specifically binds to an acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer marker polypeptide used to produce the antibody of the present invention.

[0294] Those skilled in the art will recognize that two or more different sets of monoclonal antibodies or polyclonal antibodies can maximize the possibility of obtaining an antibody with the specificity and affinity required for the intended use (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy). Depending on the use of the antibody, its desired activity is tested using known methods (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance on the generation and testing of antibodies, see, for example, Greenfield, 2014 (Greenfield, 2014). For example, the antibody can be tested by ELISA or immunoblotting, immunohistochemical staining of formalin-fixed cancer tissue or frozen tissue sections. After initial in vitro characterization, antibodies for therapeutic or in vivo diagnostic use are tested according to known clinical testing methods.

[0295] The term "monoclonal antibody" as used herein refers to an antibody obtained from a large number of homogeneous antibodies, i.e., a population of antibodies consisting of identical antibodies, except for natural mutations that may be present in small amounts. The monoclonal antibodies described herein specifically include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical (homogeneous) to the corresponding sequence of antibodies obtained from a specific species or belonging to a specific antibody type and subtype, while the remaining chain is identical (homogeneous) to the corresponding sequence of antibodies obtained from other species or belonging to a specific antibody type and subtype, as well as fragments of these antibodies, as long as they exhibit the expected antagonistic activity (U.S. Patent No. 4,816,567, which is incorporated herein in its entirety).

[0296] The monoclonal antibodies of the invention may be prepared using a hybridoma method. In the hybridoma method, mice or other appropriate host animals are typically immunized with an immunizing agent to elicit or produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0297] Monoclonal antibodies can also be made by recombinant DNA methods, such as described in U.S. Patent No. 4816567. DNA encoding the monoclonal antibodies of the invention can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding mouse antibody heavy and light chains).

[0298] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments of antibodies, particularly Fab fragments, can be accomplished using conventional techniques known in the art. For example, digestion can be accomplished using papain. Examples of papain digestion are described in WO 94 / 29348 and U.S. Patent No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments (each with one antigen binding site) and a residual Fc fragment. Pepsin treatment produces aF(ab′)2 fragment and pFc′ fragment.

[0299] Antibody fragments, whether or not attached to other sequences, may include insertions, deletions, substitutions, or other selective modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications may provide additional properties, such as deletion / addition of amino acids that can bind to disulfide bonds to increase their biological lifespan, alter their secretory properties, etc. In any case, antibody fragments must possess biologically active properties, such as binding activity, modulation of binding domain binding, etc. The functional or active regions of antibodies can be determined by genetic mutations in specific regions of the protein, followed by expression and testing of the expressed polypeptides. These methods are well known to those skilled in the art and may include site-specific genetic mutations of nucleic acids encoding antibody fragments.

[0300] The antibodies of the present invention may further include humanized antibodies or human antibodies. The humanized form of non-human (e.g., mouse) antibodies is a chimeric antibody immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab' or other antigen-binding sequences of antibodies), which contains minimal sequences obtained from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (receptor antibodies) in which residues from the complementary determining regions (CDRs) of the receptor are replaced by residues from CDRs of non-human species (donor antibodies) (e.g., mice, rats or rabbits with specificity, affinity and ability). In some cases, Fv framework (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are found neither in the receptor antibody nor in the imported CDR or framework sequences. In general, humanized antibodies will include nearly all of at least one, usually two, variable domains, wherein all or nearly all of the CDR regions correspond to regions of non-human immunoglobulins and all or nearly all of the FR regions are regions of the same sequence of human immunoglobulins. Ideally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0301] Methods for humanizing non-human antibodies are well known in the industry. In general, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are usually obtained from an "import" variable domain. Humanization can be essentially accomplished by replacing rodent CDRs or CDR sequences with corresponding human antibody sequences. Therefore, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which much less than the complete human variable domain is replaced by the 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 replaced by residues from analogous sites in rodent antibodies.

[0302] Transgenic animals (e.g., mice) that can produce complete human antibodies after immunization when endogenous immunoglobulin production is absent can be used. For example, it has been described that homozygous deletion of the antibody heavy chain joining region gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in this germline mutant mouse will result in the generation of human antibodies after antigen challenge. Human antibodies can also be produced in phage display libraries.

[0303] The antibodies of the present invention are preferably administered to a subject in the form of a pharmaceutical carrier. Typically, an appropriate amount of a pharmaceutical salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutical 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, carriers also include sustained-release preparations, such as a solid hydrophobic polymer semipermeable matrix containing the antibody, wherein the matrix is ​​in the form of a tangible article, 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 on, for example, the route of administration and concentration of the antibody.

[0304] The antibodies can be administered to the subject, patient or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular) or by other methods such as infusion to ensure that they are 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. Local or intravenous injection is preferred.

[0305] The effective dosage and schedule of antibody administration 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 specific type of antibody used, and other drugs being used. A typical daily dosage of the antibody used alone may be from about 1 μg / kg to up to 100 mg / kg of body weight or more, depending on the above factors. After administration of the antibody, preferably for the treatment of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer, the efficacy of the therapeutic antibody can be assessed by various methods well known to the skilled artisan. For example, the size, number, and / or distribution of cancer in a subject receiving treatment can be monitored using standard tumor imaging techniques. An antibody administered therapeutically that prevents tumor growth, causes tumor shrinkage, and / or prevents the development of new tumors compared to the course of the disease without administration of the antibody is an effective antibody for treating cancer.

[0306] Another aspect of the present invention proposes a method for preparing a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex. Such soluble T cell receptors can be generated from specific T cell clones, and their affinity can be increased by targeted mutagenesis of the complementary determining region. For the purpose of T cell receptor selection, phage display can be used (US 2010 / 0113300, (Liddy et al., 2012)). For the purpose of stabilizing T cell receptors during phage display and when actually used as drugs, the α and β chains can be connected through, for example, non-natural disulfide bonds, other covalent bonds (single-chain T cell receptors) or through dimerization domains (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999). T cell receptors can be linked to toxins, drugs, cytokines (see US2013 / 0115191), domains to recruit effector cells, such as anti-CD3 domains, etc., in order to perform specific functions on target cells. Furthermore, it may be expressed in T cells for adoptive transfer. Further information can be found in WO 2004 / 033685A1 and WO 2004 / 074322A1. WO 2012 / 056407A1 describes combinations of sTCRs. WO 2013 / 057586A1 discloses further production methods.

[0307] Furthermore, the peptides and / or TCRs or antibodies or other binding molecules of the invention can be used to confirm a pathologist's diagnosis of cancer based on a biopsy sample.

[0308] The antibodies or TCRs can also be used in in vivo diagnostic experiments. Generally, antibodies are labeled with radionuclides (e.g. 111 In, 99 Tc, 14 C. 131 I. 3 H. 32 P or 35 S), thereby localizing the tumor by immunoscintigraphy. In one embodiment, the antibody or fragment thereof binds to the extracellular domain of two or more protein targets selected from the group comprising the above proteins with an affinity value (Kd) of less than 1 x 10 μM.

[0309] Diagnostic antibodies can be labeled with probes suitable for detection by various imaging methods. Probe detection methods include, but are not limited to, fluorescence, light, confocal and electron microscopy methods; magnetic resonance imaging and spectroscopy techniques; fluoroscopy, computer tomography and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other rare earths, paramagnetic magnets, fluorine-18 and other positron-emitting radionuclides. In addition, the probes may be bifunctional or multifunctional and can be detected by more than one of the above methods. These antibodies can be labeled directly or indirectly with the probes. The connection of antibody probes includes covalent connection of probes, fusion of probes into antibodies, and covalent connection of chelating compounds to bind probes, as well as other methods well known in the industry. For immunohistochemistry methods, disease 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 the expression of proteins in situ.

[0310] Another aspect of the present invention includes a method for preparing activated T cells in vitro, the method comprising connecting T cells in vitro with human MHC molecules loaded with antigens, which are expressed on the surface of suitable antigen presenting cells for a sufficient period of time to activate T cells in an antigen-specific manner, wherein the antigen is a peptide according to the present invention. Preferably, a sufficient amount of antigen is used together with the antigen presenting cells.

[0311] Preferably, the mammalian cell lacks or has a reduced level or reduced function of the TAP peptide transporter. Suitable cells lacking the TAP peptide transporter include T2, RMA-S and Drosophila cells. TAP is a transporter associated with antigen processing.

[0312] The human peptide loading-deficient cell line T2 is available from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, USA) catalog number CRL1992; the Drosophila cell line Schneider 2 is available from ATCC catalog number CRL19863; the mouse RMA-S cell line was described by Ljunggren et al. (Ljunggren and Karre, 1985).

[0313] Preferably, the host cells do not substantially express MHCl class molecules before transfection. The stimulatory factor cells are also preferably expressed in molecules that play an important role in T cell co-stimulatory signals, such as any of B7.1, B7.2, ICAM-1 and LFA 3. Nucleic acid sequences of a large number of MHC class I molecules and co-stimulatory molecules are publicly available from GenBank and EMBL databases.

[0314] When an MHC class I epitope is used as an antigen, the T cell is a CD8-positive T cell.

[0315] If the antigen presenting cell is transfected to express such an epitope, preferably the cell comprises an expression vector capable of expressing a peptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 101 or a variant thereof.

[0316] Other methods can be used to generate T cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used to generate CTLs. PIebanski et al. (PIebanski et al., 1995) used autologous peripheral blood lymphocytes (PLB) to generate T cells. In addition, it is also possible to pulse dendritic cells with peptides or polypeptides or to generate 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 can be used to prepare autologous T cells. S. Walter et al. (Walter et al., 2003) described the in vitro priming of T cells by using artificial antigen presenting cells (aAPC), which is also a suitable method for generating T cells that act on selected peptides. In the present invention, aAPCs are generated by coupling preformed MHC:peptide complexes to polystyrene particles (microspheres) according to the biotin:streptavidin biochemical method. The system enables precise regulation of the MHC density on aAPCs, which allows for the selective induction of high- or low-affinity, efficient antigen-specific T cell responses in blood samples. In addition to the MHC:peptide complex, aAPCs should also carry other proteins with co-stimulatory activity, such as anti-CD28 antibodies coupled to their surface. In addition, such aAPC-based systems often require the addition of appropriate soluble factors, for example, cytokines such as interleukin-12.

[0317] T cells can also be prepared using allogeneic cells, a method described in detail in WO 97 / 26328, which is incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells can be used to present peptides, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, and vaccinia-infected target cells. In addition, plant viruses can also be used (for example, see the article by Porta et al. (Porta et al., 1994), which describes the development of cowpea mosaic virus as a high-yield system for presenting foreign peptides).

[0318] The activated T cells are directly targeted to the peptides of the present invention, which is helpful for treatment. Therefore, another aspect of the present invention provides activated T cells prepared by the aforementioned method of the present invention.

[0319] The activated T cells prepared by the above method will selectively recognize cells that abnormally express a polypeptide containing the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 101.

[0320] Preferably, the T cell recognizes the cell by interacting (e.g., binding) with its TCR containing the HLA / peptide complex. The T cell is a useful cell in a method of killing a target cell in a patient, wherein the target cell abnormally expresses a polypeptide containing the amino acid sequence of the present invention, wherein the patient is 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, preferably, the individual is a healthy person. The inventors use "healthy individual" to refer to a person who is generally in good health, preferably has a competent immune system, and more preferably does not have any disease that can be easily tested or detected.

[0321] According to the present invention, the in vivo target cells of CD8-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)).

[0322] The T cells of the present invention can be used as an active ingredient in a therapeutic composition. Therefore, the present invention also provides a method for killing a patient's target cells, wherein the patient's target cells abnormally express a polypeptide containing the amino acid sequence of the present invention, the method comprising administering the above-mentioned effective amount of T cells to the patient.

[0323] The "abnormal expression" used by the inventors also means that the polypeptide is overexpressed compared to the expression level in normal tissues, or the gene is not expressed in the tissue from which the tumor originates, but is expressed in the tumor. "Overexpression" means that the polypeptide level is at least 1.2 times that in normal tissues; preferably at least 2 times that in normal tissues, and more preferably at least 5 or 10 times that in normal tissues.

[0324] T cells can be prepared by methods known in the art (eg, as described above).

[0325] T cell adoptive transfer protocols are well known in the art. Reviews can be found in: Gattioni et al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006).

[0326] Another aspect of the invention includes the use of peptides complexed with MHC to generate T cell receptors, the nucleic acid of which is cloned and introduced into a host cell, preferably a T cell. The genetically engineered T cell can then be transferred to a patient for cancer treatment.

[0327] Any molecule of the present invention (i.e., peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor or encoding nucleic acid) is useful for treating a disease, and its characteristic is that the cell escapes the attack of the immune response. Therefore, any molecule of the present invention can be used as a medicament or used to manufacture a medicament. Such a molecule can be used alone or in combination with other molecules of the present invention or known molecules.

[0328] The present invention also relates to a kit comprising:

[0329] (a) a container comprising the pharmaceutical composition in the form of a solution or lyophilized powder;

[0330] (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized dosage form; and

[0331] (c) optionally, instructions for (i) using the solution or (ii) reconstitution and / or use of the lyophilized dosage form.

[0332] 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, a vial, a syringe or a test tube, and may be a multi-purpose container. The pharmaceutical composition is preferably a lyophilized powder.

[0333] The kit of the present invention preferably comprises a lyophilized formulation placed in a suitable container and instructions for reconstitution and / or use. Suitable containers include, for example, bottles, vials (such as dual chamber bottles), syringes (such as 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 instructions on or about the container indicating directions for reconstitution and / or use. For example, the label may indicate that the lyophilized dosage form will be reconstituted into the above-mentioned peptide concentration. The label may further indicate that the formulation is for subcutaneous injection.

[0334] The container for storing the preparation may be a multi-use vial, so that the reconstituted dosage form can be administered repeatedly (eg, 2-6 times). The kit may further include a second container containing a suitable diluent (eg, sodium bicarbonate solution).

[0335] After the diluent and the lyophilized formulation are mixed, the final peptide concentration in the reconstituted formulation is preferably at least 0.15 mg / mL / peptide (=75 μg) and no more than 3 mg / mL / peptide (=1500 μg). The kit may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filtrates, needles, syringes, and package inserts with instructions for use.

[0336] The kit of the present invention may have a single container containing the pharmaceutical composition preparation of the present invention, which may have other ingredients (eg, other compounds or pharmaceutical compositions thereof), or no other ingredients, or each ingredient may have its own different container.

[0337] Preferably, the kit of the invention comprises a formulation of the invention packaged for use in combination with a second compound such as an adjuvant (e.g., GM-CSF), a chemotherapeutic drug, a natural product, a hormone or antagonist, an anti-angiogenic agent or inhibitor, an apoptosis inducer or a chelating agent, or a pharmaceutical combination thereof. The components of the kit may be pre-complexed or each component may be placed in a separate container prior to administration to the 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 liquids upon addition of a suitable solvent, preferably placed in another separate container.

[0338] The container of the treatment kit may be a vial, test tube, flask, bottle, syringe, or any other tool for holding solids or liquids. Typically, when there is more than one ingredient, the kit will contain a second vial or other container so that it can be quantified separately. The kit may also contain another container for loading a medicinal liquid. Preferably, the treatment kit will contain a device (e.g., one or more needles, syringes, eye droppers, pipettes, etc.) that allows the injection of the drug of the present invention (the composition of the present kit).

[0339] The pharmaceutical formulation of the present invention is suitable for peptide administration by any acceptable route, such as oral (enteral), intranasal, intraocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal administration. Preferably, it is administered subcutaneously, and most preferably, it is administered intradermally through an infusion pump.

[0340] Since the peptides of the present invention are isolated from acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer, the agent of the present invention is preferably used to treat acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer.

[0341] The present 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 library, wherein the at least one peptide used in the pharmaceutical composition is selected to be suitable for the 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 isolates or soluble antibodies and other therapeutic options.

[0342] “Personalized medicine” refers to treatments that are tailored to an individual patient and will be used only for that individual patient, including personalized active cancer vaccines and adoptive cell therapy using autologous tissue.

[0343] As described herein, "repository" shall refer to a group or series of peptides that have been pre-screened for immunogenicity and / or over-presented in a specific tumor type. The term "repository" does not imply that the specific peptides included in the vaccine have been pre-manufactured and stored in a physical device, although this possibility is expected. It is clearly expected that the peptides can be used to newly manufacture each personalized vaccine, and may also be pre-manufactured and stored. The repository (e.g., in the form of a database) is composed of tumor-associated peptides, which are highly over-expressed in tumor tissues of patients with acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer with various HLA-A HLA-B and HLA-C alleles. It may contain peptides including MHC class I and MHC class II or elongated MHC class I peptides. In addition to tumor-associated peptides collected from several acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial tissue cancer tissues, the repository may also contain HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 labeled peptides. These peptides allow for the quantitative comparison of T cell immunity induced by TUMAPs, which can draw important conclusions about the ability of vaccines to respond to tumors. Second, they serve as important positive control peptides from "non-self" antigens when no vaccine-induced T cell responses are observed from TUMAPs derived from the patient's "self" antigens. Third, it also allows conclusions to be drawn about the patient's immune function status.

[0344] The repository's TUMAPs were identified using a functional genomics approach that combines gene expression analysis, mass spectrometry, and T cell immunology. This approach ensures that only TUMAPs that are truly present in a high percentage of tumors but are not expressed or only expressed in very low amounts in normal tissues are selected for further analysis. For the initial peptide selection, patient samples of acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer and blood from healthy donors were analyzed in a step-by-step approach:

[0345] 1. Determination of HLA ligands of malignant material by mass spectrometry

[0346] 2. Genome-wide messenger RNA (mRNA) expression analysis was used to identify genes that are overexpressed in malignant tumor tissues (acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer) compared with a range of normal organs and tissues

[0347] 3. Comparison of the identified HLA ligands with gene expression data. Peptides that are over- or selectively presented on tumor tissues, preferably those encoded by the selectively expressed or over-expressed genes detected in step 2, are considered to be suitable candidate TUMAPs for polypeptide vaccines.

[0348] 4. Literature search to identify additional evidence to support the relevance of the peptide identified as TUMP

[0349] 5. The relevance of overexpression at the mRNA level is determined by re-profiling of the TUMAPs selected in step 3 on tumor tissues and the lack (or infrequent) detection on healthy tissues.

[0350] 6. To evaluate whether it is feasible to induce T cell responses in vivo by the selected peptides, in vitro immunogenicity assays were performed using human T cells from healthy donors and patients with acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer.

[0351] In one aspect, the peptides are screened for immunogenicity before being added to the library. For example (but not limited to), the method for determining the immunogenicity of the peptides included in the library includes in vitro T cell priming, specifically: repeatedly stimulating CD8+ T cells from healthy donors with artificial antigen presenting cells loaded with peptide / MHC complexes and anti-CD28 antibodies.

[0352] This approach is preferred for rare cancers and patients with rare expression profiles. In contrast to peptide cocktails containing currently developed fixed components, the library allows a higher degree of matching of the actual expression of antigens in the tumor to the vaccine. In a multi-target approach, each patient will receive a selected single peptide or a combination of several "off-the-shelf" peptides. Theoretically, an approach based on the selection of, for example, 5 different antigenic peptides from a library of 50 antigenic peptides could provide approximately 1.7 million possible drug product (DP) components.

[0353] In one aspect, the peptides are selected for use in a vaccine based on suitability for an individual patient and using the methods of the invention as described herein or hereinafter.

[0354] HLA phenotype, transcriptional and peptidomic data are collected from patient tumor material and blood samples to identify peptides that are most appropriate for each patient and contain both the "reservoir" and the patient's unique (i.e., mutation) TUMAP. Those peptides will be selected to be selectively or overexpressed in the patient's tumor and, where possible, to demonstrate strong in vitro immunogenicity when tested with the patient's individual PBMCs.

[0355] Preferably, a method for determining the peptides included in the vaccine comprises: (a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from an individual patient; (b) comparing the peptides identified in (a) with a repository (database) of the peptides; and (c) selecting at least one peptide from the repository (database) associated with the tumor-associated peptides identified in the patient. For example, the method for identifying TUMAPs presented by a tumor sample comprises: (a1) comparing expression data from a tumor sample with expression data from 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 tissue; and (a2) correlating the expression data with the sequence of MHC ligands bound to class I MHC and / or class II molecules in the tumor sample to determine the MHC ligands derived from proteins that are overexpressed or abnormally expressed in the tumor. Preferably, the sequence of the MHC ligand is determined by eluting MHC molecule-bound peptides separated from the tumor sample and sequencing the eluted ligands. Preferably, the tumor sample and the normal tissue are obtained from the same patient.

[0356] In addition to or as an alternative to using a repository (database) model to select peptides, TUMAPs may be identified in new patients and then included in vaccines. As an example, candidate TUMAPs in patients may be identified by: (a1) comparing expression data from a tumor sample with expression data from 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 tissue; and (a2) correlating the expression data with the MHC ligand sequences that bind to class I MHC and / or class II molecules in the tumor sample to determine the MHC ligands derived from proteins that are overexpressed or abnormally expressed by the tumor. As another example, the protein identification method may include mutations that are 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 mutation. For example, the genome of the tumor and the corresponding normal tissue may be sequenced by a whole genome sequencing method: in order to discover non-synonymous mutations in the protein coding region of the gene, genomic DNA and RNA are extracted from the tumor tissue, and normal non-mutated genomic germline DNA is extracted from peripheral blood mononuclear cells (PBMC). The NGS method used is limited to the resequencing of protein coding regions (exome resequencing). For this purpose, a target sequence enrichment kit provided by the supplier is used to capture exon DNA from human samples, followed by sequencing using HiSeq2000 (Illumina). In addition, the mRNA of the tumor is sequenced to directly quantify gene expression and confirm that mutant genes are expressed in patient tumors. The millions of sequence reads obtained are processed by software algorithms. The output list contains mutations and gene expression. Tumor-specific somatic mutations are determined by comparison with PBMC-derived germline changes and optimized. Then, the newly determined peptides may be tested for immunogenicity as described above for the library, and candidate TUMAPs with suitable immunogenicity are selected for vaccines.

[0357] In one exemplary embodiment, the peptides included in the vaccine are identified by the following method: (a) identifying tumor-associated peptides (TUMAP) presented by a tumor sample from an individual patient using the above method; (b) comparing the peptides identified in (a) with a library of peptides pre-screened for immunogenicity and over-presentation in tumors (compared to corresponding normal tissues); (c) selecting at least one peptide from the library that is related to the tumor-associated peptides identified in the patient; and (d) optionally selecting at least one newly identified peptide in (a) and confirming its immunogenicity.

[0358] In one exemplary embodiment, the peptides included in the vaccine are identified by: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; and (b) selecting at least one newly identified peptide in (a) and confirming its immunogenicity.

[0359] Once the peptides for the personalized 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, for example about 33% DMSO.

[0360] Each peptide included in the product is dissolved in DMSO. The choice of the concentration of the single peptide solution depends on the number of peptides to be included in the product. The single peptide-DMSO solutions are mixed equally to achieve a solution containing all peptides at a concentration of ~2.5 mg / ml per peptide. The mixed solution is then diluted with water for injection at a ratio of 1:3 to achieve a concentration of 0.826 mg / ml per peptide in 33% DMSO. The diluted solution is filtered through a 0.22μm sterile filter. The final bulk solution is obtained.

[0361] The final bulk solution was filled into vials and stored at -20°C before use. One vial contained 700 μL of solution, containing 0.578 mg of each peptide, of which 500 μL (about 400 μg of each peptide) would be used for intradermal injection.

[0362] In addition to being used to treat cancer, the peptides of the present invention can also be used for diagnosis. Since the peptides are produced by acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer cells, and it has been determined that these peptides are absent or at low levels in normal tissues, these peptides can be used to diagnose the presence or absence of cancer.

[0363] Tissue biopsies in blood samples contain the claimed peptides, which can help pathologists diagnose cancer. Detection of certain peptides using antibodies, mass spectrometry or other methods known in the art can enable pathologists to determine whether the tissue sample is malignant or inflammatory or general lesions, and can also be used as biomarkers for acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer and endometrial cancer. The presentation of peptide groups enables the classification or further subdivision of diseased tissues.

[0364] Detection of peptides in disease specimens allows the benefit of immune system therapeutic approaches to be determined, especially if T-lymphocytes are known or expected to be involved in the mechanism of action. Loss of MHC expression is a well-characterized mechanism by which infected malignant cells escape immune surveillance. Therefore, presentation of peptides indicates that the analyzed cells do not utilize this mechanism.

[0365] The peptides of the present invention can be used to analyze lymphocyte responses to peptides (such as T cell responses), or antibody responses to peptides or peptides complexed with MHC molecules. These lymphocyte responses can be used as prognostic indicators to determine whether further treatment should be taken. These responses can also be used as alternative response indicators in immunotherapy, which aims to induce lymphocyte responses in different ways, such as protein vaccines, nucleic acids, autologous materials, and adoptive transfer of lymphocytes. In gene therapy, the response of lymphocytes to peptides can be considered in the assessment of side effects. Lymphocyte response monitoring may also become a valuable tool in follow-up examinations of transplantation therapy, such as for detecting graft-versus-host and host-versus-graft diseases.

[0366] The present invention will be illustrated by the following examples which describe preferred embodiments and with reference to, but not limitation to, the accompanying drawings. For the purposes of the present invention, all references are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0367] Figures 1A to 1FThe over-presentation of various peptides in different cancer tissues is shown (black dots). Upper part: The median MS signal intensity according to the technical replicate measurements is plotted as a dot, the single HLA-A*02-positive normal tissues where the peptide was detected are gray dots (left panel), and the tumor samples are black dots (right panel). The box shows the median, 25th and 75th percentiles of the normalized signal intensity, and the whiskers extend to the lowest data point that is still within 1.5 interquartile range (IQR) of the lower quartile and the highest data point that is still within 1.5 IQR of the upper quartile. Normal organs are ranked according to the risk category (blood cells, blood vessels, brain, liver, lung: high risk, gray dots; reproductive organs, breast, prostate: low risk, gray dots; all other organs: medium risk; gray dots). Lower part: The relative peptide detection frequency of each organ is shown as a spine diagram. The numbers below the graphs indicate the number of samples in which the peptide was detected out of the total number of samples analyzed for each organ (normal samples N = 440, tumor samples N = 490). If a peptide was detected on a sample but could not be quantified for technical reasons, the sample was included in the detection frequency graph, but no points were shown on the upper part of the graph. Tissues (from left to right): Normal sample: blood cells; bloodvess (blood vessels); brain; heart; liver; lung; monocytes; T cells; adipose (fat tissue); adrenal gl (adrenal gland); bile duct; bladder; bone marrow; esoph (esophagus); eye; gall bl (gall bladder); head and neck; intest.la (large intestine); intest.sm (small intestine); kidney; lymph node; nerve cent (central nervous system); nerve perith (peripheral nervous system); pancreas; parathyr (parathyroid gland); perit (peritoneum); pituitary (pituitary gland); pleura; skeI.mus skeI (skeletal muscle); skin; spinal cord; spleen; stomach; thyroid; trachea; ureter; breast; ovary; placenta; prostate; testis; thymus; uterus. Tumor samples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); GEJC (gastroesophageal junction cancer); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin lymphoma); NSCLCadeno (non-small cell lung cancer adenocarcinoma); NSCLCother (NSCLC samples that could not be clearly assigned as NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (bladder cancer), and UEC (endometrial cancer). Figure 1A) peptide: KLLDFSTRI (SEQ ID NO.: 1), Figure 1B ) peptide: ALLDVLVKL (SEQ ID NO.: 2), Figure 1C ) peptide: FLLVPSPIWQL (SEQ ID NO.: 3), Figure 1D ) Peptide: LVWEVVESV (SEQ ID NO.: 5), Figure 1E ) peptide: SLLDKLSGI (SEQ ID NO.: 10). Figure 1F Over-presentation of various peptides in different cancer tissues is shown (black dots). Upper part: The median MS signal intensity according to technical replicate measurements is plotted as dots, with single HLA-A*03-positive normal tissues where the peptide was detected as gray dots (left panel) and tumor samples as black dots (right panel). The box shows the median, 25th and 75th percentiles of the normalized signal intensity, and the whiskers extend to the lowest data point that is still within 1.5 interquartile range (IQR) of the lower quartile and the highest data point that is still within 1.5 IQR of the upper quartile. Normal organs are ranked according to risk category (blood cells, blood vessels, brain, liver, lung: high risk, gray dots; reproductive organs, breast, prostate: low risk, gray dots; all other organs: intermediate risk; gray dots). Lower part: The relative peptide detection frequency of each organ is shown as a spine diagram. The numbers below the graphs indicate the number of samples in which the peptide was detected out of the total number of samples analyzed for each organ (normal samples N = 36, tumor samples N = 107). If a peptide was detected on a sample but could not be quantified for technical reasons, that sample is included in the detection frequency graph, but no points are shown in the upper part of the graph. Tissues (from left to right): Normal sample: blood cells; bloodvess; brain; heart; liver; lung; adrenal gl (adrenal glands); bladder; gall bl (gall bladder); intest.sm (small intestine); lymph nodes; pancreas; skin; trachea. Tumor samples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin lymphoma); NSCLCadeno (non-small cell lung cancer adenocarcinoma); NSCLCother (NSCLC samples that cannot be clearly assigned as NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (bladder cancer); UEC (endometrial cancer) Figure 1F) Peptide: SLLGAATVEPPK (SEQ ID NO.: 81; A*03).

[0368] Figures 2A to 2F Representative expression signatures of the source genes of the present invention are shown, which are overexpressed in different cancer samples. Tumor (black dots) and normal ( Dots) Samples are grouped according to organ of origin. Box and whisker plots represent median FPKM values, 25th and 75th percentiles (box) + whiskers, with whiskers extending to the lowest data point still within 1.5 interquartile range (IQR) of the lower quartile and the highest data point still within 1.5 IQR of the upper quartile. Normal organs are ranked according to risk category. FPKM: fragments per kilobase per million mapped reads. Tissues (from left to right): Normal sample: blood cells; bloodvess (blood vessels); brain; heart; liver; lung; adipose (fat tissue); adrenal gl (adrenal gland); bile duct; bladder; bone marrow; esoph (esophagus); eye; gall bl (gall bladder); head and neck; intest.la (large intestine); intest.sm (small intestine); kidney; lymph node; nerve perith (peripheral nerve); pancreas; parathyr (parathyroid gland); perit (peritoneum); pituitary (pituitary gland); pleura; skel.mus skel (skeletal muscle); skin; spleen; stomach; thyroid gland; trachea; ureter; breast; ovary; placenta; prostate; testis; thymus; uterus. Tumor samples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin lymphoma); NSCLCadeno (non-small cell lung cancer adenocarcinoma); NSCLCother (NSCLC samples that could not be clearly assigned as NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (bladder cancer); UEC (endometrial cancer). Figure 2A )Ensembl ID: ENST00000225964, Peptide: ALLDVLVKL (SEQ ID No.: 2), Figure 2B )Ensembl ID: ENST00000374472, peptide: SLLDKLSGI (SEQ ID No 10), Figure 2C)Ensembl ID: ENST00000617924, peptide: FASERPPSV (SEQ ID No.: 33), Figure 2D )Ensembl ID: ENST00000603198, Peptide: YIYEDEVRL (SEQ ID No.: 39), Figure 2E )Ensembl ID: ENST00000420453, peptide: AIWSTILIA (SEQ ID No.: 43), Figure 2F )Ensembl ID: ENST00000473984, peptide: IAISQLTFV (SEQ ID No.: 65), Figure 2G )Ensembl ID: ENST00000375105.7, peptide: LLLALRLSL (SEQ ID No.: 64).

[0369] Figure 3 Representative results of peptide-specific CD8+ T cell in vitro responses from healthy HLA-A*02+ donors are shown. CD8+ T cells were primed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 complexed with SeqID No 102 peptide (GLDPTQFRV, peptide code: POLA1-003) (A, left panel) and SeqID No 103 peptide (SLVSYLDKV, peptide code: KRT1 6P-001) (B, left panel). After 3 cycles of stimulation, peptide-reactive cells were detected using 2D multimer staining of A*02 / SeqID No 102 (A) and A*02 / SeqID No 103 (B). The right panels (A and B) show control staining of cells stimulated with irrelevant A*02 / peptide complexes. Live single cells were gated on CD8+ lymphocytes. Boolean gating helps exclude false positive events detected with multimers specific for different peptides. The frequencies of specific multimer+ cells and CD8+ lymphocytes are indicated.

[0370] Figure 4Representative results of peptide-specific CD8+ T cell responses in vitro from healthy HLA-A*02+ donors are shown. CD8+ T cells were primed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 complexed with SeqID No 18 peptide (KMMTFFQGL) (A, left), SeqID No 68 peptide (KLLADAFKV) (B, left), SeqID No 40 peptide (FTLPFLVNL) (C, left), SeqID No 19 peptide (MLLPWLPKL) (D, left), or SeqID No 48 peptide (MLAEIHPKA) (E, left). After 3 cycles of stimulation, peptide reactive cells were detected by 2D multimer staining with A*02 / SeqID No 18 (A), A*02 / SeqID No 68 (B), A*02 / SeqID No 40 (C), A*02 / SeqID No 19 (D), or A*02 / SeqID No 48 (E). The right panels (A, B, C, D, and E) show control staining of cells stimulated with irrelevant A*02 / peptide complexes. Live single cells were gated on CD8+ lymphocytes. Boolean gating helps exclude false positive events detected with multimers specific for different peptides. The frequencies of specific multimer+ cells and CD8+ lymphocytes are indicated. Example

[0371] Example 1

[0372] Identification and quantification of tumor-associated peptides presented on the cell surface

[0373] Tissue samples

[0374] Tumor tissues from patients were obtained from: Asterand (Detroit, Michigan, USA & Royston, Hertfordshire, UK), Bio-Options Inc. (Brea, California, USA), Geneticist Inc. (Glendale, California, USA), Heidelberg University Hospital (Heidelberg, Germany), ProteoGenex lnc. (Culver City, California, USA), Tissue Solutions Ltd (Glasgow, UK), University Hospital Munich (Munich, Germany). Normal tissues were obtained from Asterand (Detroit, Michigan, USA & Royston, Hertfordshire, UK), BioCat GmbH (Heidelberg, Germany), Bio-Options lnc. (Brea, California, USA), Bio-Options Inc. (Brea, California, USA), BioServe (Beltsville, Maryland, USA), Capital BioScience Inc. (Rockville, Maryland, USA), Clinical Transfusion Medicine Center Tübingen (Tübingen, Germany), Geneticist Inc. (Glendale, California, USA), Kyoto Prefectural University of Medicine (KPUM) (Kyoto, Japan), Osaka City University (OCU) (Osaka, Japan), ProteoGenex lnc. (Culver City, California, USA), Tissue Solutions Ltd (Glasgow, UK), Geneva University Hospital (Geneva, Switzerland), Heidelberg University Hospital (Heidelberg, Germany), Tübingen University Hospital (Tübingen, Germany), Munich University Hospital (Munich, Germany).

[0375] Written informed consent was obtained from all patients before surgery or autopsy. Tissues were snap-frozen immediately after resection and stored at -70°C or below before TUMAP isolation.

[0376] Isolation of HLA peptides from tissue samples

[0377] According to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999), the HLA peptide library of cold-shocked tissue samples was obtained by immunoprecipitation of solid tissue using HLA-A*02-specific antibody BB7.2, HLA-A, HLA-B, HLAC-specific antibody W6 / 32, HLA-DR-specific antibody L243 and HLA DP-specific antibody B7 / 21, CNBr-activated agarose gel, acid treatment and ultrafiltration.

[0378] Mass spectrometry analysis

[0379] The obtained HLA peptide library was separated according to its hydrophobicity by reverse phase chromatography (nanoAcquity UPLC system, Waters), and the eluted peptides were analyzed by LTQ-velos fusion hybrid mass spectrometer (ThermoElectron) equipped with an electrospray source. The peptide library was directly loaded into an analytical fused silica microcapillary column (75 μm inner diameter x 250 mm) filled with 1.7 μm C18 reverse phase material (Waters), and a flow rate of 400 nL per minute was applied. Subsequently, the peptides were separated using a two-step 180-minute binary gradient method from a flow rate of 300 nL per minute and a concentration of 10% to 33% solvent B. The gradient consisted of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). A gold-coated glass capillary (PicoTip, New Objective) was used to introduce into the nanoliter electrospray source. The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode using the top 5 (TOP5) strategy. Briefly, a scan cycle was started with a full scan in orbitrap with high quality accuracy (R = 30,000), followed by an MS / MS scan of the five most abundant precursor ions in orbitrap (R = 7500) using dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted by SEQUEST with a fixed false discovery rate (q ≤ 0.05) and additional manual controls. In cases where the determined peptide sequence was uncertain, further validation was performed by comparing the fragmentation pattern of the generated natural peptide with that of a synthetic reference peptide of the same sequence.

[0380] Label-free relative LC-MS quantification was performed by ion counting (i.e., by LC-MS feature extraction and analysis) (Mueller et al., 2007). This method assumes that the LC-MS signal area of ​​a peptide is related to its abundance in the sample. The extracted features were further processed by charge state deconvolution and retention time alignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MS features were cross-referenced with sequence identification results to combine quantitative data from different samples and tissues with peptide presentation features. Quantitative data were normalized in a two-layer manner based on the pooled data to account for technical and biological replicate variations. Therefore, each identified peptide can be associated with quantitative data, allowing relative quantification between samples and tissues. In addition, all quantitative data obtained for candidate peptides were manually checked to ensure data consistency and verify the accuracy of the automated analysis. For each peptide, a presentation plot was calculated, which shows the sample average presentation as well as replicate variation. These features juxtapose acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer samples with baseline values ​​for normal tissue samples. Presentation profiles of exemplary over-presented peptides are shown in Figure 1. Peptide presentation of exemplary peptides on tumors is shown in Table 11.

[0381] Table 11 shows the presentation of selected peptides on various cancer entities, thus showing the particular relevance of the mentioned peptides for the diagnosis and / or treatment of the indicated cancers (e.g., peptide SEQ ID No. 1 for acute myeloid leukemia, colorectal cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, endometrial cancer, peptide SEQ ID No. 2 for breast cancer, colorectal cancer, gallbladder cancer, gastroesophageal junction cancer, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, small cell lung cancer, endometrial cancer).

[0382] Table 11: Summary of the presentation of selected tumor-associated peptides of the present invention in various entities.

[0383] Cancer types: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); GEJC (gastroesophageal junction cancer); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin lymphoma); NSCLCadeno (non-small cell lung cancer adenocarcinoma); NSCLCother (NSCLC samples that could not be clearly assigned as NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (bladder cancer) and UEC (endometrial cancer)

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390] Example 2

[0391] Expression profile of the gene encoding the peptide of the present invention

[0392] Over- or specific presentation of a peptide on tumor cells compared to normal cells is sufficient for its effective use in immunotherapy, and some peptides are tumor-specific despite the presence of their source protein also present in normal tissues. However, mRNA expression profiles add an additional level of safety in the selection of immunotherapy target peptides. Especially 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 not present in normal tissues.

[0393] RNA source and preparation

[0394] Surgical resection tissue specimens were provided as described above (see Example 1) after written informed consent was obtained from each patient. Tumor tissue specimens were snap-frozen immediately after surgery and then homogenized in liquid nitrogen with a pestle and mortar. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, Germany) followed by RNeasy (QIAGEN, Hilden, Germany) cleanup; both methods were performed according to the manufacturer's protocol.

[0395] Total RNA from healthy human tissues for RNASeq experiments was obtained from: Asterand (Detroit, Michigan, USA & Royston, Hertfordshire, UK), Bio-Options Inc. (Brea, California, USA), Geneticist Inc. (Glendale, California, USA), ProteoGenex lnc. (Culver City, California, USA), Tissue Solutions Ltd (Glasgow, UK).

[0396] Total RNA from tumor tissues for RNASeq experiments was obtained from: Asterand (Detroit, Michigan, USA & Royston, Hertfordshire, UK), BioCat GmbH (Heidelberg, Germany), BioServe (Beltsville, Maryland, USA), Geneticist Inc. (Glendale, California, USA), Istituto Nazionale Tumori "Pascale" (Naples, Italy), ProteoGenex lnc. (Culver City, California, USA), Heidelberg University Hospital (Heidelberg, Germany).

[0397] The quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent, Walterbrunn, Germany) using the RNA 6000 Pico LabChip Kit (Agilent).

[0398] RNAseq experiments

[0399] Gene expression analysis of RNA samples from tumors and normal tissues was performed by CeGaT (Tübingen, Germany) using next-generation sequencing (RNAseq). Briefly, sequencing libraries were prepared using the Illumina HiSeq v4 kit according to the supplier's protocol (Illumina Inc., San Diego, CA, USA), which included RNA fragmentation, cDNA conversion, and addition of sequencing adapters. Libraries obtained from multiple samples were mixed in equimolar amounts according to the manufacturer's instructions and sequenced on an Illumina HiSeq2500 sequencer, generating 50 bp single-end reads. The processed reads were mapped to the human genome (GRCh38) using STAR software. Expression data were provided as RPKM (reads per kilobase per million mapped reads, generated by Cufflinks software) at the transcript level and as total reads, generated by Bedtools software, according to the ENSEMBL sequence database (Ensembl77). Exonic reads were normalized to exon length and calibrated size to obtain RPKM values.

[0400] Representative expression profiles of the source genes of the present invention that are highly overexpressed or exclusively expressed in acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer are shown in Figure 1. The expression scores of further representative genes are shown in Table 12.

[0401] Table 12: Expression scores. This table lists peptides from genes that are very highly overexpressed (+++) on tumors compared to a range of normal tissues, highly overexpressed (++) on tumors compared to a range of normal tissues, or overexpressed (+) on tumors compared to a range of normal tissues. This baseline score is calculated based on measurements of the following relevant normal tissues: blood cells; blood vessels; brain; heart; liver; lung; adipose tissue; adrenal glands; bile ducts; bladder; bone marrow; cartilage; esophagus; eye; gallbladder; head and neck; large intestine; small intestine; kidney; lymph nodes; central nervous system; peripheral nervous system; pancreas; parathyroid glands; peritoneum; pituitary gland; pleura; skeletal muscle; skin; spinal cord; spleen; stomach; thyroid gland; trachea; ureter.

[0402] If expression data of several samples of the same tissue type were obtained, the arithmetic mean of each sample was used for calculation.

[0403]

[0404]

[0405]

[0406] Example 3

[0407] In vitro immunogenicity of MHC class I presented peptides

[0408] In order to obtain information about the immunogenicity of the TUMAPs of the invention, the inventors conducted studies using an in vitro T cell priming assay based on repeated stimulation with artificial antigen presenting cells (aAPCs) loaded with peptide / MHC complexes and anti-CD28 antibodies. Using this method, the inventors could demonstrate that the HLA-A*02:01 restricted TUMAPs of the invention are immunogenic, indicating that these peptides are T cell epitopes present in human CD8+ precursor T cells (Tables 13a and 13b).

[0409] In vitro priming of CD8+ T cells

[0410] For in vitro stimulation with artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD28 antibodies, the inventors first isolated CD8+ T cells from fresh HLA-A*02 leukapheresis products by positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) from healthy donors with informed consent obtained from the University Clinic Mannheim, Germany.

[0411] PBMC and isolated CD8+ lymphocytes were cultured in T cell medium (TCM) before use, which included RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U / ml penicillin / 100 μg / ml streptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany) and 20 μg / ml gentamicin (Cambrex). At this step, 2.5 ng / ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U / ml IL-2 (Novartis Pharma, Nuremberg, Germany) were also added to TCM.

[0412] Generation of pMHC / anti-CD28 coated beads, stimulation of T cells and readout 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.

[0413] Purified co-stimulatory mouse IgG2a anti-human CD28 antibody 9.3 (Jung et al., 1987) was chemically biotinylated using sulfo-N-hydroxysuccinimide biotin as recommended by the manufacturer (Perbio, Bonn, Germany). The beads used were 5.6 μm streptavidin-coated polystyrene particles (Bangs Laboratories, IL, USA).

[0414] The pMHC used for positive and negative control stimulators were A*0201 / MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 104) modified from Melan-A / MART-1) and A*0201 / DDX5-001 (YLLPAIVHI (SEQ ID NO. 105) obtained from DDX5). 800.000 beads / 200 μl were coated in a 96-well plate containing 4 x 12.5 ng of different biotin-pMHC, washed, and then 600 ng of biotin anti-CD28 was added in a volume of 200 μl. Stimulation was initiated in 96-well plates by co-culturing 1x106 CD8+ T cells with 2x105 washed coated beads in 200 μl TCM containing 5 ng / ml IL-12 (PromoCell) at 37°C for 3 days. Afterwards, half of the culture medium was exchanged with fresh TCM supplemented with 80 U / ml IL-2 and culture was continued at 37°C for 4 days. This stimulation cycle was performed 3 times in total. For pMHC multimer readout using 8 different pMHC molecules per condition, the 2D combinatorial encoding method was used as described previously (Andersen et al., 2012) with slight modifications to include coupling to 5 different fluorescent dyes. Finally, multimer analysis was performed using Live / dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany) and fluorescent pMHC multimers. Analysis was performed using a BD LSRII SORP cytometer equipped with an appropriate laser and screening program. Peptide-specific cells were calculated as a percentage of total CD8+ cells. Multimer analysis evaluation was performed using FlowJo software (Tree Star, OR, USA). In vitro priming of specific multimer+CD8+ lymphocytes was tested in comparison with negative control stimulation groups. The immunogenicity of a given antigen was tested if at least one evaluable in vitro stimulated well from a healthy donor was found to contain a specific CD8+ T cell line after in vitro stimulation (i.e., the well contained at least 1% of a specific multimer+ CD8+ T cells and the percentage of specific multimer+ cells was at least 10 times the median of negative control stimulations).

[0415] Acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, Glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma cancer, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and In vitro immunogenicity of endometrial cancer peptides.

[0416] For the tested HLA-1 class peptides, their in vitro immunogenicity can be demonstrated by the generation of peptide-specific T cell lines. Representative results of flow cytometry detection after TUMAP-specific multimer staining of the seven peptides of the present invention are shown in FIG. Figure 3 A and 3B and Figure 4 The results of the 30 peptides of the present invention are summarized in Tables 13a and 13b.

[0417] Table 13a: In vitro immunogenicity of HLA class I peptides of the present invention

[0418] Representative results of in vitro immunogenicity experiments conducted by the applicant on the peptides of the present invention. <20% = +; 20%-49% = ++; 50%-69% = +++; > = 70% = ++++

[0419] Serial ID Number sequence Positive wells [%] 102 GLDPTQFRV +-+-+-+- 103 SLVSYLDKV +

[0420] Table 13b: In vitro immunogenicity of HLA class I peptides of the present invention

[0421] Representative results of in vitro immunogenicity experiments conducted by the applicant on the peptides of the present invention. <20% = +; 20-49% = ++; 50-69% = +++: ≥70% = ++++

[0422]

[0423]

[0424] Example 4

[0425] Peptide synthesis

[0426] All peptides were synthesized by standard, well-established solid phase peptide synthesis using the Fmoc strategy. The identity and purity of each peptide was confirmed using mass spectrometry and RP-HPLC analysis. Lyophilization (trifluoroacetate) yielded white to off-white peptides with a purity of >50%. All TUMAPs are preferably administered as trifluoroacetate or acetate salts, although other salt forms are possible.

[0427] Example 5

[0428] MHC binding assay

[0429] The candidate peptides for T cell therapy of the present invention were further tested for their MHC binding ability (affinity). The results of the 79 peptides of the present invention are summarized in Table 14.

[0430] Single peptide-MHC complexes are generated by UV-ligand exchange, where UV-sensitive peptides are cleaved after UV irradiation and exchanged with the relevant peptides to be analyzed. Only candidate peptides that can effectively bind to and stabilize the peptide-receiving MHC molecule can prevent the dissociation of the MHC complex. In order to determine the yield of the exchange reaction, an ELISA assay based on the detection of the light chain (β2m) that stabilizes the MHC complex is performed. The assay is generally performed according to the method described by Rodenko et al. (Rodenko et al., 2006).

[0431] 96-well MAXlSorp plates (NUNC) were coated with 2ug / ml streptavidin in PBS at room temperature overnight, washed 4 times and blocked in 2% BSA in blocking buffer at 37°C for 1 hour. Folded HLA-A*02:01 / MLA-001 monomers were used as standards, covering the range of 15-500ng / ml. UV exchange-reacted peptide-MHC monomers were diluted 100-fold in blocking buffer. Samples were incubated at 37°C for 1 hour, washed four times, incubated with 2ug / ml HRP-conjugated anti-β2m at 37°C for 1 hour, washed again, and detected with TMB solution blocked with NH2SO4. Absorption was measured at 450nm. In the generation and production of antibodies or fragments thereof and / or T-cell receptors or fragments thereof, candidate peptides showing a high exchange yield (preferably greater than 50%, most preferably greater than 75%) are generally preferred because they exhibit sufficient affinity for MHC molecules and can prevent dissociation of the MHC complex.

[0432] Table 14: MHC class I binding scores. Binding of HLA class I restricted peptides to HLA-A*02:01 is classified according to peptide exchange yield: ≥10% = +; ≥20% = ++; ≥50 = +++; ≥75% = ++++

[0433]

[0434]

[0435]

[0436] Example 6

[0437] Absolute quantification of tumor-associated peptides presented on the cell surfaceThe generation of binders such as antibodies and / or TCRs is a laborious process that can be performed only against some selected targets. In the case of tumor-associated and specific peptides, the selection criteria include, but are not limited to, the exclusivity of presentation and the density of presented peptides on the cell surface. In addition to the separation and relative quantification of peptides as described in Example 1, the inventors also analyzed the absolute peptide copy number per cell as described in WO 2016 / 107740. Quantification of TUMAP copy number per cell in solid tumor samples requires absolute quantification of isolated TUMAPs, efficiency of the TUMAP separation process, and cell count of the analyzed tissue samples.

[0438] nanoLC-MS / MS peptide quantification

[0439] In order to accurately quantify peptides by mass spectrometry, a calibration curve for each peptide was generated using two different isotopically labeled peptide variants (including one or two isotopically labeled amino acids during TUMAP synthesis). These isotopically labeled variants differed from the tumor-associated peptide only in mass and did not differ in other physicochemical properties (Anderson et al., 2012). For the peptide calibration curve, a series of nanoLC-MS / MS measurements were performed to determine the MS / MS signal ratio of the titrated (monoisotopically labeled peptide) and constant (doubly isotopically labeled peptide) isotopically labeled peptides.

[0440] Doubly isotopically labeled peptides (also called internal standards) are further spiked into each MS sample, and all MS signals are normalized to the internal standard MS signal, thereby balancing potential technical differences between MS experiments.

[0441] Calibration curves were prepared using at least three different matrices, i.e. HLA peptide eluates from native samples (similar to conventional MS samples), and two MS measurements were run after each preparation. For evaluation, the MS signals were normalized to the internal standard signal and the calibration curves were calculated by logistic regression.

[0442] For quantification of tumor-associated peptides from tissue samples, each sample was also spiked with an internal standard; the MS signal was normalized to the internal standard and quantified using this peptide calibration curve.

[0443] Efficiency of peptide / MHC separation

[0444] As in any protein purification process, the isolation of proteins from tissue samples is associated with some loss of the protein of interest. To determine the efficiency of TUMAP isolation, peptide / MHC complexes were generated for all TUMAPs selected for absolute quantification. To be able to distinguish native peptide / MHC complexes from spike-ins, monoisotopically labeled versions of the TUMAPs were used, i.e. 1 isotopically labeled amino acid was incorporated during TUMAP synthesis. These complexes were spiked into freshly prepared tissue lysates, i.e. at the earliest possible time point during TUMAP isolation and then captured like native peptide / MHC complexes in a subsequent affinity purification. Therefore, measuring the recovery of the singly labeled TUMAPs allows conclusions to be drawn about the efficiency of the isolation of the individual TUMAPs.

[0445] The separation efficiency was analyzed using a small number of samples and these tissue samples were comparable. In contrast, the separation efficiency varied between individual peptides. This suggests that the separation efficiency, although determined in a limited number of samples, can be extrapolated to any other tissue preparation. However, since the separation efficiency may not be extrapolated from one peptide to other peptides, it is necessary to analyze each TUMAP individually.

[0446] Determination of cell counts in solid and frozen tissues

[0447] To determine the cell count of tissue samples for absolute peptide quantification, the inventors applied a DNA content analysis method. This method is applicable to a wide range of samples from different sources, most importantly, frozen samples (Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During the peptide isolation protocol, the tissue sample was processed into a homogenous lysate, from which small aliquots of the lysate were taken. The sample was divided into three aliquots, from which DNA was isolated (QiaAmpDNA Mini Kit, Qiagen, Hilden, Germany). The total DNA content of each DNA isolation was quantified at least twice using a fluorescence-based DNA quantification assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany).

[0448] To calculate cell number, a DNA standard curve of isolated healthy blood cell aliquots from several donors was generated using a range of specified cell numbers. The standard curve was used to calculate the total cell content of the total DNA content for each DNA isolation. The average total cell count of the tissue samples used for peptide isolation was then extrapolated taking into account the known volumes of the lysate aliquots and the total lysate volume.

[0449] Number of peptide copies per cell

[0450] The inventors used the data from the above experiments to calculate the TUMAP copy number per cell by dividing the total peptide amount by the total cell number of the sample and then by the separation efficiency. The cell copy number of the selected peptides is shown in Table 15.

[0451] Table 15: Absolute copy number. This table lists the results of absolute peptide quantification in tumor samples. For each peptide, the median copy number per cell is represented as: <25 = +; ≥25 = ++; ≥50 = +++; ≥75 = ++++. The number of samples in which high-quality MS data were available for evaluation is indicated.

[0452]

[0453]

[0454] References

[0455] Accardi, L. et al., Int.J Cancer 134(2014): 2742-2747

[0456] Aken, BL et al., Database. (Oxford) 2016 (2016)

[0457] Alcoser, SY et al., BMC. Biotechnol. 11 (2011): 124

[0458] Allison, JP et al., Science 270(1995): 932-933

[0459] American Cancer Society, (2015a), www.cancer.org

[0460] American Cancer Society, (2015b), www.cancer.org

[0461] Ampie, L. et al., Front Oncol. 5 (2015): 12

[0462] Andersen, RSet al., Nat.Protoc.7(2012): 891-902

[0463] Anderson, DMet al., Cell 160(2015): 595-606

[0464] Anderson,N.L.et al.,J Proteome.Res 11(2012):1868-1878

[0465] Appay,V.et al.,Eur.J Immunol.36(2006):1805-1814

[0466] Armitage,J.O.,Blood 110(2007):29-36

[0467] Aspden,J.L.et al.,Elife.3(2014):e03528

[0468] Avigan,D.et al.,Clin Cancer Res.10(2004):4699-4708

[0469] Azevedo,R.et al.,J Control Release 214(2015):40-61

[0470] Banchereau,J.et al.,Cell 106(2001):271-274

[0471] Beatty,G.et al.,J Immunol 166(2001):2276-2282

[0472] Beggs,J.D.,Nature 275(1978):104-109

[0473] Benjamini,Y.et al.,Journal of the Royal Statistical Society.Series B(Methodological),Vol.57(1995):289-300

[0474] Berman,R.S.et al.,National Cancer Institute:PDQ(R)Colon CancerTreatment(2015a)

[0475] Berman,R.S.et al.,National Cancer Institute:PDQ(R)Rectal CancerTreatment(2015b)

[0476] Boulter,JMet al.,Protein Eng 16(2003):707-711

[0477] Braumuller,H.et al.,Nature(2013)

[0478] Bray,F.et al.,Int J Cancer 132(2013):1133-1145

[0479] Bridgewater,J.et al.,J Hepatol.60(2014):1268-1289

[0480] Brossart,P.et al.,Blood 90(1997):1594-1599

[0481] Bruckdorfer,T.et al.,Curr.Pharm.Biotechnol.5(2004):29-43

[0482] Bujas,T.et al.,Eur.J Histochem.55(2011):e7

[0483] Butterfield,LHet al.,Clin.Cancer Res.12(2006):2817-2825

[0484] Butterfield,LHet al.,Clin.Cancer Res.9(2003):5902-5908

[0485] Byrd,JCet al.,N.Engl.J Med.369(2013):32-42

[0486] Carballido,E.et al.,Cancer Control19(2012):54-67

[0487] Card,KFet al.,Cancer Immunol.Immunother.53(2004):345-357

[0488] Chang,YSet al.,Cancer Chemother.Pharmacol.59(2007):561-574

[0489] Chapiro,J.et al.,Radiol.Med.119(2014):476-482

[0490] ClinicalTrials.gov,(2015), http: / / www.clinicaltrials.gov

[0491] Cohen,C.J.et al.,J Mol.Recognit.16(2003a):324-332

[0492] Cohen,C.J.et al.,J Immunol.170(2003b):4349-4361

[0493] Cohen,S.N.et al.,Proc.Natl.Acad.Sci.U.S.A 69(1972):2110-2114

[0494] Coligan,J.E.et al.,Current Protocols in Protein Science(1995)

[0495] Colombetti,S.et al.,J Immunol.176(2006):2730-2738

[0496] Coosemans,A.et al.,Anticancer Res 33(2013):5495-5500

[0497] Counter,C.M.et al.,Blood 85(1995):2315-2320

[0498] de,Klerk E.et al.,Trends Genet.31(2015):128-139

[0499] Dedes,K.J.et al.,Sci.Transl.Med.2(2010):53ra75

[0500] Dengjel,J.et al.,Clin Cancer Res 12(2006):4163-4170

[0501] Denkberg,G.et al.,J Immunol.171(2003):2197-2207

[0502] Economopoulou,P.et al.,Ann.Transl.Med.4(2016):173

[0503] Eichhorst,B.F.et al.,Blood 107(2006):885-891

[0504] Emens,L.A.,Expert.Rev.Anticancer Ther.12(2012):1597-1611

[0505] Enguita-German,M.et al.,World J Hepatol.6(2014):716-737

[0506] Estey,E.H.,Am.J Hematol.89(2014):1063-1081

[0507] Falk,K.et al.,Nature 351(1991):290-296

[0508] Ferlay et al.,GLOBOCAN 2012 v1.0,Cancer Incidence and MortalityWorldwide:IARC CancerBase No.11[Internet],(2013), http: / / globocan.iarc.fr

[0509] Follenzi,A.et al.,Nat Genet.25(2000):217-222

[0510] Fong,L.et al.,Proc.Natl.Acad.Sci.U.S.A 98(2001):8809-8814

[0511] Forsey,R.W.et al.,Biotechnol.Lett.31(2009):819-823

[0512] Fuge,O.et al.,Res Rep.Urol.7(201 5):65-79

[0513] Furman,R.R.et al.,N.Engl.J Med.370(2014):997-1007

[0514] Gabrilovich,D.I.et al.,Nat.Med 2(1996):1 096-1103

[0515] Gandhi,A.V.et al.,Ann Surg.Oncol 20Suppl 3(2013):S636-S643

[0516] Gattinoni,L.et al.,Nat.Rev.Immunol.6(2006):383-393

[0517] Giannopoulos,K.et al.,Leukemia 24(2010):798-805

[0518] Giannopoulos,K.et al.,Int.J Oncol 29(2006):95-103

[0519] Gnjatic,S.et al.,Proc Natl.Acad.Sci.U.S.A 100(2003):8862-8867

[0520] Godkin,A.et al.,Int.Immunol 9(1997):905-911

[0521] Goede,V.et al.,N.Engl.J Med.370(2014):1101-1110

[0522] Gonzalez-Cao,M.et al.,Cancer Biol Med 13(2016):483-488

[0523] Gragert,L.et al.,Hum.Immunol.74(2013):1313-1320

[0524] Granziero,L et al.,Blood 97(2001):2777-2783

[0525] Green,J.et al.,Cochrane.Database.Syst.Rev(2005):CD002225

[0526] Green,M.R.et al.,Molecular Cloning,A Laboratory Manual 4th(2012)

[0527] Greenfield,EA,Antibodies:A Laboratory Manual 2nd(2014)

[0528] Grivas,PDet al.,Semin.Cancer Biol 35(2015):125-132

[0529] Gunawardana,C.et al.,Br.J Haematol.142(2008):606-609

[0530] Gustafsson, C. et al., Trends Biotechnol. 22 (2004): 346-353

[0531] Hallek,Michael et al.,ASH Annual Meeting Abstracts 112(2008):325

[0532] Harig,S.et al.,Blood 98(2001):2999-3005

[0533] Hinrichs,CEt al.,Nat Biotechnol.31(2013):999-1008

[0534] Holtl,L.et al.,Clin.Cancer Res.8(2002):3369-3376

[0535] Horig,H.et al.,Cancer Immunol.Immunother.49(2000):504-514

[0536] Hung,CFet al.,Immunol.Rev 222(2008):43-69

[0537] Hus,I.et al.,Oncol Rep.20(2008):443-451

[0538] Hwang,MLet al.,J Immunol.179(2007):5829-5838

[0539] Inoue,H.et al.,Int.J Cancer 63(1995):523-526

[0540] Jones,R.T.et al.,Urol.Clin North Am.43(2016):77-86

[0541] Jung,G.et al.,Proc Natl Acad Sci U S A 84(1987):4611-4615

[0542] Kalos,M.et al.,Sci.Transl.Med 3(2011):95ra73

[0543] Kanthan,R.et al.,J Oncol 2015(2015):967472

[0544] Kassiotis,G.et al.,Philos.Trans.R Soc.Lond B Biol Sci.372(2017)

[0545] Kaufman,H.L.et al.,Clin Cancer Res.14(2008):4843-4849

[0546] Khoury,G.A.et al.,Sci.Rep.1(2011)

[0547] Kibbe,A.H.,Handbook of Pharmaceutical Excipients rd(2000)

[0548] Kimura,H.et al.,Int.J Oncol 30(2007):171-179

[0549] Knollman,H.et al.,Ther.Adv.Urol.7(2015a):312-330

[0550] Knollman,H.et al.,Ther.Adv.Urol.7(2015b):312-330

[0551] Koido,S.et al.,World J Gastroenterol.19(201 3):8531-8542

[0552] Kono,K.et al.,Cancer Sci.100(2009):1502-1509

[0553] Krackhardt, AMet al., Blood 100(2002): 2123-2131

[0554] Krieg,AM,Nat.Rev.Drug Discov.5(2006):471-484

[0555] Krishnamurthy, J. et al., Clin Cancer Res 21(2015): 3241-3251

[0556] Kronenberger, K. et al., J Immunother. 31(2008): 723-730

[0557] Kuball, J. et al., Blood 109(2007): 2331-2338

[0558] Laumont,CMet al.,Cell Mol.Life Sci.75(2018):607-621

[0559] Lee, WCet al., J Immunother. 28(2005): 496-504

[0560] Endometrial carcinoma guideline, 032 / 034, (2008)

[0561] Gastric carcinoma guideline, 032-009OL, (2012)

[0562] Guidelines for diagnosis and therapy in neurology, 030 / 099, (2014)

[0563] Li, Y. et al., Cancer Epidemiol. 39(2015): 8-13

[0564] Liang,Z.et al.,Zhonghua Zhong.Liu Za Zhi.27(2005):534-537

[0565] Liddy, N. et al., Nat. Med. 18(2012): 980-987

[0566] Liepe,J.et al.,Science 354(201 6):354-358

[0567] Ljunggren,H.G.et al.,J Exp.Med 162(1985):1745-1759

[0568] Llovet,J.M.et al.,N.Engl.J Med.359(2008):378-390

[0569] Longenecker,B.M.et al.,Ann N.Y.Acad.Sci.690(1993):276-291

[0570] Lonsdale,J.,Nat.Genet.45(2013):580-585

[0571] Lukas,T.J.et al.,Proc.Natl.Acad.Sci.U.S.A 78(1981):2791-2795

[0572] Lukka,H.et al.,Clin Oncol(R Coll.Radiol.)14(2002):203-21 2

[0573] Lundblad,R.L.,Chemical Reagents for Protein Modification 3rd(2004)

[0574] Mantia-Smaldone,G.M.et al.,Hum.Vaccin.Immunother.8(2012):1179-1191

[0575] Marten,A.et al.,Cancer Immunol.Immunother.51(2002):637-644

[0576] Massari,F.et al.,Cancer Treat.Rev.41(2015):114-121

[0577] Matsueda,S.et al.,World J Gastroenterol.20(2014):1657-1666

[0578] Maus,MVet al.,Blood 123(2014):2625-2635

[0579] Mayr,C.et al.,Exp.Hematol.34(2006):44-53

[0580] Mayr,C.et al.,Blood 105(2005):1566-1573

[0581] Meziere, C. et al., J Immunol 159(1997):3230-3237

[0582] Miyagi,Y.et al.,Clin.Cancer Res.7(2001):3950-3962

[0583] Molina,JRet al.,Mayo Clin Proc.83(2008):584-594

[0584] Morgan,RA et al.,Science 314(2006):126-129

[0585] Mortara, L et al., Clin Cancer Res.12(2006):3435-3443

[0586] Moulton,HMet al.,Clin Cancer Res.8(2002):2044-2051

[0587] Mueller,LNet al.,J Proteome.Res.7(2008):51-61

[0588] Mueller,LNet al.,Proteomics.7(2007):3470-3480

[0589] Muller,MRet al.,Blood 103(2004):1763-1769

[0590] Mumberg,D.et al.,Proc.Natl.Acad.Sci.USA 96(1999):8633-8638

[0591] Nam,JWet al.,Mol.Cells 39(2016):367-374

[0592] National Cancer Institute,(6-5-2015), www.cancer. g ov

[0593] National Cancer Institute(NCI),(1-19-2011), http: / / www.cancer.gov / cancertopics / wyntk / kidney / page3

[0594] Nilsen,T.W.et al.,Nature 463(2010):457-463

[0595] O′Brien,S.et al.,Lancet Oncol15(2014):48-58

[0596] O′Leary,N.A.et al.,Nucleic Acids Res 44(2016):D733-D745

[0597] Ohigashi,Y.et al.,Clin Cancer Res.11(2005):2947-2953

[0598] Okuno,K.et al.,Exp.Ther.Med.2(2011):73-79

[0599] Olexiouk,V.et al.,Nucleic Acids Res 44(2016):D324-D329

[0600] Palma,M.et al.,Cancer Immunol.Immunother.57(2008):1705-1710

[0601] Palmer,D.H.et al.,Hepatology 49(2009):124-132

[0602] Palomba,M.L.,Curr.Oncol Rep.14(2012):433-440

[0603] Parikh,S.A.et al.,Blood 118(201 1):2062-2068

[0604] Phan,G.Q.et al.,Cancer Control 20(2013):289-297

[0605] Pinheiro,J.et al.,nlme:Linear and Nonlinear Mixed Effects Models( http: / / CRAN.R-project.org / packe=nlme )(2015)

[0606] Plebanski,M.et al.,Eur.J Immunol 25(1995):1783-1787

[0607] Porta,C.et al.,Virology 202(1994):949-955

[0608] Porter,D.L.et al.,N.Engl.J Med.365(2011):725-733

[0609] Quillien,V.et al.,Anticancer Res.17(1997):387-391

[0610] Quinn,D.I.et al.,Urol.Oncol 33(2015):245-260

[0611] Rakic,M.et al.,Hepatobiliary.Surg.Nutr.3(2014):221-226

[0612] Rammensee,H.G.et al.,Immunogenetics 50(1999):213-219

[0613] Reinisch,W.et al.,J Immunother.25(2002):489-499

[0614] Reinmuth,N.et al.,Dtsch.Med.Wochenschr.140(2015):329-333

[0615] Richards,S.et al.,J Natl.Cancer Inst.91(1999):861-868

[0616] Rini,B.I.et al.,Curr.Opin.Oncol.20(2008):300-306

[0617] Rini,B.I.et al.,Cancer 107(2006):67-74

[0618] Robak,T.et al.,Expert.Opin.Biol.Ther 14(2014):651-661

[0619] Rock,KLet al.,Science 249(1990):918-921

[0620] Rodenko,B.et al.,Nat.Protoc.1(2006):1120-1132

[0621] Rouanne,M.et al.,Crit ​​Rev Oncol Hematol.98(2016):106-115

[0622] Rucki,AAet al.,World J Gastroenterol.20(2014):2237-2246

[0623] S3 Guideline Exocrine Pancreatic Cancer, 032-010OL, (2013)

[0624] S3 Guideline Lung Cancer, 020 / 007, (2011a)

[0625] S3 Guideline Lung Cancer, 020 / 007, (2011b)

[0626] S3 guideline on malignant ovarian tumors, 032-035OL, (2013)

[0627] S3 Guideline on Breast Cancer, 032-045OL, (2012)

[0628] S3 Guideline Melanoma, 032-024OL, (2013)

[0629] S3 Guideline on Prostate Cancer, 043 / 022OL, (2014)

[0630] S3 Guideline Cervical Cancer, 032 / 033OL, (2014)

[0631] Saiki,RKet al.,Science 239(1988):487-491

[0632] Salman,B.et al.,Oncoimmunology.2(2013):e26662

[0633] Blood,B.et al.,J Clin.Oncol.22(2004):1389-1397

[0634] Schetelig,J.et al.,J Clin Oncol 26(2008):5094-5100

[0635] Schiavetti,F.et al.,Cancer Res 62(2002):5510-5516

[0636] Schmidt,SMet al.,Cancer Res.64(2004):1164-1170

[0637] Schmitt,TMet al.,Hum.Gene Ther.20(2009):1240-1248

[0638] Scholten,KBet al.,Clin Immun0l.119(2006):135-145

[0639] Seeger,FHet al.,Immunogenetics 49(1999):571-576

[0640] Sherman,F.et al.,Laboratory Course Manual for Methods in YeastGenetics(1986)

[0641] Shi,M.et al.,World J Gastroenterol.10(2004):1146-1151

[0642] Showel,MMet al.,F1000Prime.Rep.6(2014):96

[0643] Siegel,S.et al.,Blood 102(2003):4416-4423

[0644] Silva,L.P.et al.,Anal.Chem.85(2013):9536-9542

[0645] Singh-Jasuja,H.et al.,Cancer Immunol.Immunother.53(2004):187-195

[0646] Small,E.J.et al.,J Clin Oncol.24(2006):3089-3094

[0647] Spaner,D.E.et al.,Cancer Immunol.Immunother.54(2005):635-646

[0648] Srivastava,N.et al.,Cancer Manag.Res.6(2014):279-289

[0649] Stahl,M.et al.,Ann.Oncol.24Suppl 6(2013):vi51-vi56

[0650] Steinberg,R.L.et al.,Urol.Oncol(2016a)

[0651] Steinberg,R.L.et al.,Urol.Oncol(2016b)

[0652] Stevanovic,S.et al.,J Clin Oncol 33(2015):1543-1550

[0653] Stintzing,S.,F1000Prime.Rep.6(2014):108

[0654] Sturm,M.et al.,BMC.Bioinformatics.9(2008):1 63

[0655] Su,Z.et al.,Cancer Res.63(2003):2127-2133

[0656] Subramanian,R.P.et al.,Retrovirology.8(2011):90

[0657] Takayama,T.et al.,Cancer 68(1991):2391-2396

[0658] Takayama,T.et al.,Lancet 356(2000):802-807

[0659] Tanaka,F.et al.,Int.J Oncol 10(1997):1113-1117

[0660] Teufel,R.et al.,Cell Mol.Life Sci.62(2005):1755-1762

[0661] Thakkar,JPet al.,Cancer Epidemiol.Biomarkers Prev.23(2014):1985-1996

[0662] Toh,U.et al.,Int.J Clin Oncol 7(2002):372-375

[0663] Toh,U.et al.,Clin Cancer Res.6(2000):4663-4673

[0664] Toomey,PGet al.,Cancer Control 20(2013):32-42

[0665] Tran,E.et al.,Science 344(2014):641-645

[0666] Vanderperre,B.et al.,PLoS.One.8(2013):e70698

[0667] Vici, P. et al., J Exp. Clin Cancer Res 33(2014):29

[0668] Von Hoff,DDet al.,N.Engl.J Med.369(2013):1691-1703

[0669] von Rundstedt,FCet al.,Transl.Androl Urol.4(2015):244-253

[0670] Walter,S.et al.,J.Immunol.171(2003):4974-4978

[0671] Walter, S. et al., Nat Med. 18 (2012): 1254-1261

[0672] Wang, N. et al., Arch. Gynecol. Obstet. 283 (2011): 103-108

[0673] Wierda, W Get al., Blood 118(2011): 5126-5129

[0674] Wilhelm, SMet al., Cancer Res. 64 (2004): 7099-7109

[0675] Willcox, BE et al., Protein Sci. 8 (1999): 2418-2423

[0676] Wilson, PMet al., Nat Rev. Clin Oncol11(2014): 282-298

[0677] Wittig, B. et al., Hum. Gene Ther. 12 (2001): 267-278

[0678] World Cancer Report, (2014)

[0679] World Health Organization, (2014), http: / / www.who.int / en /

[0680] Zaremba, S. et al., Cancer Res. 57 (1997): 4570-4577

[0681] Zufferey, R. et al., J Virol. 73(1999): 2886-2892

[0682] More specifically, the present application provides the following items.

[0683] 1. A peptide having a length of up to 30 amino acids and comprising

[0684] a) the amino acid of SEQ ID No.48 or

[0685] b) an amino acid sequence selected from SEQ ID No. 1 to SEQ ID No. 38, SEQ ID No. 40 to SEQ ID No. 47 and SEQ ID No. 49-101; or a pharmaceutically acceptable salt thereof.

[0686] 2. The peptide according to item 1, wherein the peptide can be recognized by CD4 and / or CD8 T cells when bound to MHC.

[0687] 3. A peptide according to item 1 or 2, wherein the peptide is 8 to 16 amino acids in length.

[0688] 4. The peptide according to any one of items 1 to 3, wherein the peptide is modified and / or comprises non-peptide bonds.

[0689] 5. An antibody which specifically recognizes the peptide according to any one of items 1 to 4, or which is the peptide according to any one of items 1 to 4 when bound to an MHC molecule.

[0690] 6. A T cell receptor that reacts with an HLA ligand, wherein the ligand is a peptide according to any one of items 1 to 4, or is a peptide according to any one of items 1 to 4 when bound to an MHC molecule.

[0691] 7. A T cell receptor according to item 6, wherein the ligand amino acid sequence comprises any one of SEQ ID No. 1 to SEQ ID No. 38 or SEQ ID No. 40 to SEQ ID No. 101.

[0692] 8. A T cell receptor according to any one of items 6 or 7, wherein the T cell receptor is provided as a soluble molecule and has a further effector function selected from an immunostimulatory domain or a toxin.

[0693] 9. A recombinant host cell comprising a peptide according to any one of items 1 to 4, an antibody according to item 5, or a T cell receptor according to any one of items 6 to 8, wherein the host cell is selected from an antigen presenting cell.

[0694] 10. A method for preparing activated T lymphocytes in vitro, which comprises connecting T cells in vitro with human class I or II MHC molecules carrying antigens, which are expressed on the surface of suitable antigen-presenting cells or on the surface of artificially simulated antigen-presenting cell structures for a sufficient period of time to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide described in any one of items 1 to 4.

[0695] 11. An activated T lymphocyte prepared according to the method described in item 10, which selectively recognizes a cell presenting a polypeptide comprising the amino acid sequence given in any one of items 1 to 4.

[0696] 12. A pharmaceutical composition comprising at least one active ingredient selected from the peptide described in any one of items 1 to 4, the antibody described in item 5, the T cell receptor described in items 6-8, the host cell described in item 9 or the activated T lymphocyte described in item 11, or a conjugated or labeled active ingredient and a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient and / or stabilizer.

[0697] 13. A method for preparing a peptide according to any one of items 1 to 4, an antibody according to item 5, or a T cell receptor according to any one of items 6-8, the method comprising culturing a host cell according to item 9 and isolating the peptide, the antibody, or the T cell receptor from the host cell and / or its culture medium.

[0698] 14. Use of the peptide described in any one of items 1 to 4, the antibody described in item 5, the T cell receptor described in any one of items 6-8, the host cell described in item 9 or the activated T lymphocyte described in item 11 in the preparation of a medicament for treating cancer.

[0699] 15. The use according to claim 14, wherein the cancer is selected from the group consisting of acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, endometrial cancer and other tumors, which show overexpression of the protein derived from the peptides of SEQ ID No. 1 to SEQ ID No. 38 or SEQ ID No. 40 to SEQ ID No. 101.

[0700] 16. A kit comprising:

[0701] (a) a container comprising a pharmaceutical composition in the form of a solution or a lyophilized powder, the pharmaceutical composition comprising a peptide according to any one of items 1 to 4, an antibody according to item 5, a T cell receptor according to any one of items 6 to 8, a host cell according to item 9, or an activated T lymphocyte according to item 11;

[0702] (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized powder dosage form;

[0703] (c) Optionally, at least one peptide is selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 38 or SEQ ID No. 40 to SEQ ID No. 101.

[0704] 17. The kit according to item 16, further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.

[0705] In addition, this application also includes but is not limited to the following:

[0706] 1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 101, and variant sequences thereof having at least 88% homology with SEQ ID No. 1 to SEQ ID No. 101, wherein the variant binds to the major histocompatibility complex (MHC) and / or induces T cells to cross-react with the variant peptide; and a pharmaceutically acceptable salt thereof, wherein the peptide is not a full-length polypeptide.

[0707] 2. The peptide according to item 1, wherein the peptide is capable of binding to MHC class I or -II molecules, and wherein the peptide can be recognized by CD4 and / or CD8 T cells when bound to MHC.

[0708] 3. The peptide or variant thereof according to item 1 or 2, wherein the amino acid sequence comprises a continuous stretch of amino acids in any one of the sequences SEQ ID No. 1 to SEQ ID No. 101.

[0709] 4. A peptide or variant thereof according to any one of items 1 to 3, wherein the total length of the peptide or variant thereof is 8 to 100 amino acids, preferably 8 to 30 amino acids, more preferably 8 to 16 amino acids, and most preferably the peptide consists of or essentially consists of any amino acid sequence according to SEQ ID No.1 to SEQ ID No.101.

[0710] 5. The peptide or variant thereof according to any one of items 1 to 4, wherein the peptide is modified and / or comprises non-peptide bonds.

[0711] 6. A peptide or variant thereof according to any one of items 1 to 5, wherein the peptide is part of a fusion protein, in particular comprising the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (li).

[0712] 7. An antibody, particularly a soluble or membrane-bound antibody, preferably a monoclonal antibody or a fragment thereof, which specifically recognizes a peptide or a variant thereof according to any one of items 1 to 5, and when bound to an MHC molecule, is preferably a peptide or a variant according to any one of items 1 to 5.

[0713] 8. A T cell receptor, preferably a soluble or membrane-bound receptor or a fragment thereof, which reacts with an HLA ligand, wherein the ligand is a peptide or variant thereof according to any one of items 1 to 5, and when bound to an MHC molecule, it is preferably a peptide or variant according to any one of items 1 to 5.

[0714] 9. A T cell receptor according to item 8, wherein the ligand amino acid sequence is at least 88% identical to any one of SEQ ID No. 1 to SEQ ID No. 101, or wherein the ligand amino acid sequence comprises any one of SEQ ID No. 1 to SEQ ID No. 101.

[0715] 10. A T cell receptor according to any one of items 8 or 9, wherein the T cell receptor is provided as a soluble molecule and optionally has further effector functions, such as an immunostimulatory domain or a toxin.

[0716] 11. An aptamer that specifically recognizes the peptide or variant thereof according to items 1 to 5, preferably the peptide or variant according to any one of items 1 to 5 that binds to an MHC molecule.

[0717] 12. A nucleic acid encoding a peptide or variant thereof according to any one of items 1 to 5, an antibody or fragment thereof according to item 7, a T cell receptor or fragment thereof according to item 8 or 9, optionally linked to a heterologous promoter sequence or an expression vector for expressing said nucleic acid.

[0718] 13. A recombinant host cell comprising a peptide according to any one of items 1 to 6, an antibody or a fragment thereof according to item 7, a T cell receptor or a fragment thereof according to item 8 or 9, or a nucleic acid or an expression vector according to item 12, wherein the host cell is preferably selected from antigen presenting cells, such as dendritic cells, T cells or NK cells.

[0719] 14. A method for preparing activated T lymphocytes in vitro, the method comprising contacting T cells with human class I or II MHC molecules loaded with antigens for a sufficient period of time in vitro to activate the T cells in an antigen-specific manner, wherein these molecules are expressed on the surface of suitable antigen-presenting cells or on the surface of artificially simulated antigen-presenting cell structures, wherein the antigen is a peptide as described in any one of items 1 to 4.

[0720] 15. An activated T lymphocyte prepared according to the method described in Item 14, which selectively recognizes a cell presenting a polypeptide comprising the amino acid sequence given in any one of Items 1 to 4.

[0721] 16. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of a peptide according to any one of items 1 to 6, an antibody or a fragment thereof according to item 7, a T cell receptor or a fragment thereof according to item 8 or 9, an aptamer according to item 11, a nucleic acid or an expression vector according to item 12, a host cell according to item 13, or an activated T lymphocyte according to item 15, or a conjugated or labeled active ingredient and a pharmaceutically acceptable carrier and optionally a pharmaceutically acceptable excipient and / or stabilizer.

[0722] 17. A method for preparing a peptide or variant thereof according to any one of items 1 to 6, an antibody or fragment thereof according to item 7, or a T cell receptor or fragment thereof according to item 8 or 9, the method comprising culturing a host cell according to item 13 and isolating the peptide or variant thereof, the antibody or fragment thereof, or the T cell receptor or fragment thereof from the host cell and / or its culture medium.

[0723] 18. Use of the peptide described in any one of items 1 to 6, the antibody or fragment thereof described in item 7, the T cell receptor or fragment thereof described in item 8 or 9, the aptamer described in item 11, the nucleic acid or expression vector described in item 12, the host cell described in item 13 or the activated T lymphocyte described in item 15 in medicine.

[0724] 19. A method for killing targeted cells in a patient, wherein the targeted cells present a polypeptide comprising the amino acid sequence given in any one of items 1 to 4; the method comprises administering to the patient an effective amount of activated T cells as defined in item 15.

[0725] 20. Use of the peptide described in any one of items 1 to 6, the antibody or fragment thereof described in item 7, the T cell receptor or fragment thereof described in item 8 or 9, the aptamer described in item 11, the nucleic acid or expression vector described in item 12, the host cell described in item 13 or the activated T lymphocyte described in item 15 in cancer diagnosis and / or treatment or in the manufacture of anti-cancer drugs.

[0726] 21. The use according to claim 20, wherein the cancer is selected from the group consisting of acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, endometrial cancer and other tumors, which show overexpression of the protein derived from the peptides of SEQ ID No. 1 to SEQ ID No. 101.

[0727] 22. A kit comprising:

[0728] (a) a container comprising a pharmaceutical composition in the form of a solution or a lyophilized powder, the pharmaceutical composition comprising the peptide or variant according to any one of items 1 to 6, the antibody or fragment thereof according to item 7, the T cell receptor or fragment thereof according to item 8 or 9, the aptamer according to item 11, the nucleic acid or expression vector according to item 12, the host cell according to item 13, or the activated T lymphocyte according to item 15;

[0729] (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized powder dosage form;

[0730] (c) optionally, at least one peptide selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 103, and (d) optionally, instructions for (i) use in solution or (ii) reconstitution and / or use in lyophilized powder dosage form.

[0731] 23. The kit according to item 22, further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.

[0732] 24. A method for producing a personalized anti-cancer vaccine or compound-based and / or cell therapy for an individual patient, the method comprising:

[0733] a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from said individual patient;

[0734] b) comparing the peptides identified in a) to a pool of peptides that have been pre-screened for immunogenicity and / or are over-presented in tumors compared to normal tissues;

[0735] c) selecting at least one peptide in the repository that matches a TUMAP identified in the patient; and

[0736] d) manufacturing and / or formulating a personalized vaccine or compound-based or cell therapy based on step c).

[0737] 25. The method according to item 24, wherein the TUMAP is identified by:

[0738] a1) comparing the expression data from the tumor sample with the 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 tissue; and

[0739] a2) Correlate the expression data with the sequences of MHC ligands bound by MHC class I and / or class II molecules in tumor samples to identify protein-derived MHC ligands that are overexpressed or aberrantly expressed by the tumor.

[0740] 26. The method according to item 24 or 25, wherein the sequence of the MHC ligand is determined by eluting the MHC molecule-bound peptides isolated from the tumor sample and sequencing the eluted ligands.

[0741] 27. The method according to any one of items 24 to 26, wherein the normal tissue sample corresponding to the tumor sample of the same type is obtained from the same patient.

[0742] 28. A method according to any one of items 24 to 27, wherein the peptides contained in the storage library are identified based on the following steps:

[0743] aa. Genome-wide messenger RNA (mRNA) expression analysis by highly parallel methods, such as microarray or sequencing-based expression profiling, including identification of genes overexpressed in malignant tissue compared to normal tissue;

[0744] ab. selecting a peptide encoded by a gene that is specifically expressed or overexpressed as detected in step aa, and

[0745] ac. Determining the induction of an in vivo T cell response by a selected peptide, including an in vitro immunogenicity assay using human T cells from a healthy donor or said patient; or

[0746] ba. identifying HLA ligands from the tumor sample by mass spectrometry;

[0747] bb. Genome-wide messenger RNA (mRNA) expression analysis by highly parallel methods, such as microarray or sequencing-based expression profiling, including identification of genes overexpressed in malignant tissue compared to normal tissue;

[0748] bc. comparing the identified HLA ligands with the gene expression data;

[0749] bd. Selecting a peptide encoded by a gene with specific expression or high expression detected in step bc;

[0750] be. re-detecting the selected TUMAPs from step bd on tumor tissue that are absent or infrequently detected on healthy tissue and determining the relevance of overexpression at the mRNA level;

[0751] as well as

[0752] bf. Determination of in vivo T cell responses induced by selected peptides, including in vitro immunogenicity assays using human T cells from healthy donors or said patients.

[0753] 29. A method according to any one of items 24 to 28, wherein the immunogenicity of the peptides included in the repository is determined by a method comprising in vitro immunogenicity experiments, individual HLA binding patient immune monitoring, MHC multimer staining, ELISPOT analysis and / or intracellular cytokine staining.

[0754] 30. A method according to any one of items 24 to 29, wherein the storage library comprises a plurality of peptides selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 103.

[0755] 31. A method according to any one of items 24 to 30, further comprising identifying at least one mutation that is unique to the tumor sample compared to corresponding normal tissue of the individual patient, and selecting a peptide associated with the mutation for inclusion in a vaccine or for use in producing a cell therapy.

[0756] 32. The method of claim 31 , wherein the at least one mutation is identified by whole genome sequencing.

Claims

1. A peptide or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the total length of the peptide is at most 14 amino acids.

2. A nucleic acid encoding the peptide according to claim 1.

3. A recombinant host cell comprising the peptide according to claim 1 or the nucleic acid according to claim 2.

4. A method for preparing activated T lymphocytes in vitro, the method comprising contacting T cells with human class I or class II MHC molecules loaded with antigens for a period of time sufficient to activate the T cells in an antigen-specific manner, wherein the human class I or class II MHC molecules are expressed on the surface of a suitable antigen-presenting cell or on the surface of an artificial structure that simulates an antigen-presenting cell, wherein the antigen is the peptide according to claim 1.

5. The activated T lymphocyte prepared by the method according to claim 4, wherein the activated T lymphocyte selectively recognizes cells presenting the peptide according to claim 1.

6. A method for preparing the peptide according to claim 1, the method comprising: Cultivating the recombinant host cell according to claim 3, and isolating the peptide from the recombinant host cell or the culture medium of the recombinant host cell.

7. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of the peptide or pharmaceutically acceptable salt thereof according to claim 1, the nucleic acid according to claim 2, the recombinant host cell according to claim 3, or the activated T lymphocyte according to claim 5.

8. Use of the peptide or pharmaceutically acceptable salt thereof according to claim 1, the nucleic acid according to claim 2, the recombinant host cell according to claim 3 or the activated T lymphocyte according to claim 5 in the preparation of a medicament for diagnosing, preventing and / or treating cancer.

9. A kit comprising: (a) a container comprising a pharmaceutical composition in a solution or lyophilized form, wherein the pharmaceutical composition comprises the peptide or a pharmaceutically acceptable salt thereof according to claim 1, the nucleic acid according to claim 2, the recombinant host cell according to claim 3, or the activated T lymphocyte according to claim 5; (b) optionally, a second container comprising a diluent or reconstitution solution for the lyophilized dosage form; (c) optionally, at least one other peptide selected from SEQ ID NOs: 1 to 103, and (d) optionally, one or more of (i) a buffer, (ii) a diluent, (iii) a filter, (iv) a needle, (v) a syringe, or (vi) an adjuvant.

10. An in vitro method for providing data for diagnosing cancer in a sample, the sample comprising cells from a subject, the method comprising: (a) adding antibodies or T cell receptors to a tissue sample of the subject, and (b) detecting the binding of the antibody or the T cell receptor to cells from the tissue sample from the subject, thereby identifying the presentation of the tumor-associated peptide of claim 1 on cells from the tissue sample from the subject, wherein the antibody or T cell receptor specifically recognizes (i) the peptide according to claim 1 or (ii) the peptide when bound to an MHC molecule.

Images