Peptides and peptide combinations of non-canonical origin for use in immunotherapy against different types of cancer

Abstract

To provide peptides, pharmaceutical compositions for use in cancer immunotherapies and methods for killing cancer cells.SOLUTION: The invention relates to peptides, proteins, nucleic acids and cells for use in cancer immunotherapies. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or stimulate T cells ex vivo and transfer them into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to peptides, proteins, nucleic acids, and cells used in immunotherapy. In particular, the present invention relates to cancer immunotherapy. The present invention also relates to tumor-associated T cell peptide epitopes, alone or in combination with other tumor-associated peptides, which can serve as active pharmaceutical ingredients in vaccine compositions, for example, to stimulate anti-tumor immune responses or to stimulate T cells ex vivo and transfer them into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or the peptides themselves, can also be targeted by antibodies, soluble T cell receptors, and other binding molecules.

[0002] The present invention relates to several novel peptide sequences and their variants derived from HLA class I molecules of human tumor cells, which can be used in vaccine compositions to elicit anti-tumor immune responses or as targets for the development of pharmacologically / immunologically active compounds and cells. [Background technology]

[0003] According to the World Health Organization (WHO), cancer was one of the four leading non-communicable fatal diseases in the world in 2012. In the same year, colorectal, breast, and respiratory tract cancers were listed among the top 10 causes of death in high-income countries.

[0004] GBM is the most common central nervous system malignancy, with an age-adjusted incidence rate of 3.19 per 100,000 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 gender, older age, and ethnicity appear to be risk factors for GBM (Thakkar et al., 2014).

[0005] CLL is the most common leukemia in Western countries, comprising approximately one-third of all leukemias. Incidence rates are similar in the United States and Europe, with an estimated number of new cases of approximately 16,000 per year. CLL is more common in Caucasians than in African Americans, is rarer in Hispanics and Native Americans, and is almost never seen in Asians. Among people of Asian descent, the incidence of CLL is one-third that of Caucasians (Gunawardana et al., 2008). The 5-year overall survival rate for patients with CLL is approximately 79%.

[0006] AML is the second most common type of leukemia diagnosed in both adults and children. There are an estimated 21,000 new cases per year in the United States. The five-year survival rate for patients with AML is approximately 25%.

[0007] Lung cancer is the most common type of cancer worldwide and the leading cause of cancer death in many countries. Lung cancer is subdivided into small cell lung cancer and non-small cell lung cancer. NSCLC includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma histologies and accounts for 85% of all lung cancers in the United States. The incidence of NSCLC is closely correlated with smoking rates, including current and former smokers, and the 5-year survival rate has been reported to be 15% (World Cancer Report, 2014; Molina et al., 2008). Summary of the Invention [Problem to be solved by the invention]

[0008] Considering the serious side effects and costs associated with cancer treatment, there is a need to identify elements that can be used in cancer treatment in general, and in AML (acute myeloid leukemia), BRCA (breast cancer), CCC (cholangiocarcinoma), 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's 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 (uterine endometrial cancer) in particular. 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, squamous cell carcinoma of the head and neck, 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 in particular, to provide better cancer diagnosis, assessment of prognosis, and prediction of treatment success.

[0009] Cancer immunotherapy represents an option that specifically targets cancer cells while minimizing side effects. Cancer immunotherapy takes advantage of the presence of tumor-associated antigens.

[0010] The current classification of tumor-associated antigens (TAA) comprises the following major groups: a) Cancer-Testis Antigens: The first identified TAAs capable of being recognized by T cells belonged to this class and were originally called cancer-testis (CT) antigens because their members are expressed in histologically distinct human tumors and are present only in testicular spermatocytes / spermatogonia in normal tissues and occasionally in the placenta. Because testicular cells do not express class I and II HLA molecules, these antigens cannot be recognized by T cells in normal tissues and are therefore considered immunologically tumor-specific. Well-known examples of CT antigens are MAGE family members and NY-ESO-1. b) Differentiation antigens: These TAAs are shared between tumors and the normal tissues from which they arise. Most of the known differentiation antigens are found in melanomas and normal melanocytes. Many of these melanocyte-related proteins are involved in melanin biosynthesis and are therefore not tumor-specific, but are still widely used for cancer immunotherapy. Examples include, but are not limited to, tyrosinase and Melan-A / MART-1 for melanoma, or PSA for prostate cancer. c) Overexpressed TAAs: Genes encoding ubiquitously expressed TAAs have been detected in histologically distinct tumor types and also in many normal tissues, generally at lower expression levels. While many of the epitopes processed and potentially presented by normal tissues may be below the threshold level for T cell recognition, their overexpression in tumor cells can trigger anti-cancer responses by breaking previously established immune tolerance. Prominent examples of this class of TAAs are Her-2 / neu, survivin, telomerase, or WT1. d) Tumor-specific antigens: These unique TAAs arise from mutations in normal genes (e.g., β-catenin, CDK4). Some of these molecular alterations are associated with neoplastic transformation and / or progression. Tumor-specific antigens can usually induce a strong immune response without the risk of an autoimmune reaction against normal tissue. On the other hand, these TAAs are most often associated only with the very tumor in which they were identified and are usually not shared among many individual tumors. For proteins with tumor-specific (associated) isoforms, tumor specificity (or association) of peptides may also occur if the peptide is derived from a tumor (associated) exon. e) TAAs resulting from aberrant post-translational modifications: Such TAAs may arise from proteins that are neither specific nor overexpressed in tumors, but nevertheless become tumor-associated through post-translational processes that are primarily active in tumors. Examples of this class arise from altered glycosylation patterns that result in novel epitopes in tumors, such as MUC1, or from protein splicing events during degradation that may or may not be tumor-specific. f) Oncoviral proteins: These TAAs are viral proteins that may play an important role in the carcinogenic process and, because they are foreign (not of human origin), can induce T cell responses. Examples of such proteins are the human papillomavirus type 16 proteins E6 and E7, which are expressed in cervical cancer.

[0011] A large portion of the human proteome originates from non-canonical origins, such as alternative open reading frames (altORFs (Vanderperre et al., 2013)), endogenous retroviral elements, or involves additional post-transcriptional (RNA alternative splicing (Nilsen and Graveley, 2010)) or post-translational processing (post-translational modification (Khoury et al., 2011), proteasomal splicing (Liepe et al., 2016)) steps. This portion of the proteome presents a rich source of TAAs, as many of the cellular processes involved in the generation of these non-canonical proteins and peptides are frequently altered in cancer cells (Laumont and Perreault, 2018).

[0012] Many messenger RNAs (mRNAs) contain non-conventional alternative open reading frames (altORFs) (de Klerk and 't Hoen, 2015) in addition to the reference open reading frame (ORF). The size of these additional coding sequences can vary from typically less than 100 codons (small open reading frames; sORFs (Olexiouk et al., 2016)) to several hundred codons and can be located upstream, downstream, or even overlapping the reference ORF. Translation of these altORFs may occur from different translation start sites and in a different reading frame than the reference ORF. The presence of small open reading frames is not limited to mRNAs; it has also been described in other regulatory RNAs (ncRNAs, Nam et al., 2016) previously thought to be noncoding. Several long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been shown to encode small peptides and translate extensively (Anderson et al., 2015; Aspden et al., 2014).

[0013] Human endogenous retroviruses (HERVs) comprise a significant portion (approximately 8%) of the human genome. These viral elements integrated into the genome millions of years ago and have been transmitted vertically across generations ever since. While the majority of HERVs have lost their functional activity due to mutation or truncation, some endogenous retroviruses, such as members of the HERV-K clade, have been shown to still encode functional genes and form retrovirus-like particles (Subramanian et al., 2011). HERV proviral transcription is epigenetically regulated and remains silenced under normal physiological conditions. However, reactivation and overexpression, leading to active translation of viral proteins, have been described in certain diseases, particularly different types of cancer (Gonzalez-Cao et al., 2016; Kassiotis and Stoye, 2017). This tumor-specific expression of HERV-derived proteins can be exploited for different types of cancer immunotherapy (Krishnamurthy et al., 2015; Schiavetti et al., 2002).

[0014] T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins presented by molecules of the major histocompatibility complex (MHC). The antigens, i.e., the epitopes, recognized by tumor-specific T lymphocytes can be molecules derived from all protein classes, such as enzymes, receptors, and transcription factors, which are expressed in the respective tumor cells and are usually upregulated compared to unmodified cells of the same origin.

[0015] There are two classes of MHC molecules: MHC class I and MHC class II. MHC class I molecules are composed of α heavy chains and β2 microglobulin, while MHC class II molecules are composed of α and β chains. Their three-dimensional structure provides a binding groove that is used for non-covalent interactions with peptides.

[0016] MHC class I molecules are found on most nucleated cells. They primarily present peptides derived from endogenous proteins, defective ribosomal products (DRIPs), and proteolytic cleavage of larger peptides. However, peptides derived from endosomal compartments or exogenous sources are also frequently found on MHC class I molecules. This non-classical mode of class I presentation is referred to in the literature as cross-presentation (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules are mostly found on professional antigen-presenting cells (APCs) and primarily present peptides from exogenous or transmembrane proteins that are taken up by APCs during endocytosis and subsequently processed.

[0017] It is well known that peptide-MHC class I complexes are recognized by CD8-positive T cells bearing the appropriate T cell receptor (TCR), while peptide-MHC class II complexes are recognized by CD4-positive helper T cells bearing the appropriate TCR. As a result, TCR, peptide, and MHC exist in a 1:1:1 stoichiometric ratio.

[0018] CD4+ helper T cells play a crucial role in inducing and maintaining effective responses by CD8+ cytotoxic T cells. Identification of CD4+ T cell epitopes derived from tumor-associated antigens (TAAs) is crucial for the development of therapeutic agents that can trigger antitumor immune responses (Gnjatic et al., 2003). At tumor sites, T helper cells maintain a cytokine-friendly environment for cytotoxic T cells (CTs) (Mortara et al., 2006) and attract effector cells, such as CTLs, natural killer (NK) cells, macrophages, and granulocytes (Hwang et al., 2007).

[0019] In the absence of inflammation, expression of MHC class II molecules is primarily restricted to cells of the immune system, particularly professional antigen-presenting cells (APCs), such as monocytes, monocyte-derived cells, macrophages, and dendritic cells. In cancer patients, tumor cells have been found to express MHC class II molecules (Dengjel et al., 2006).

[0020] The extended (longer) peptides of the present invention can act as MHC class II active epitopes.

[0021] T helper cells activated by MHC class II epitopes play an important role in orchestrating the effector functions of CTLs in anti-tumor immunity. T helper cell epitopes that initiate TH1-type T helper cell responses support the effector functions of CD8-positive killer T cells, including cytotoxicity directed against tumor cells that display tumor-associated peptide / MHC complexes on their cell surface. In this way, tumor-associated T helper cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients in vaccine compositions that stimulate anti-tumor immune responses.

[0022] For example, in mammalian models such as mice, CD4+ T cells have been shown to be sufficient to inhibit tumor development through the inhibition of angiogenesis by secreting interferon-γ (IFNγ), even in the absence of CD8+ T lymphocytes (Beatty and Paterson, 2001; Mumberg et al., 1999). There is evidence that CD4+ T cells are direct antitumor effectors (Braumuller et al., 2013; Tran et al., 2014).

[0023] Because constitutive expression of HLA class II molecules is usually restricted to immune cells, it was previously thought that it might be possible to isolate class II peptides directly from primary tumors. However, Dengjel et al. succeeded in identifying several MHC class II epitopes directly from tumors (WO 2007 / 028574, EP 1760088 B1).

[0024] Because both CD8- and CD4-dependent responses synergistically contribute to antitumor effects, the identification and characterization of tumor-associated antigens recognized by either CD8+ T cells (ligand: MHC class I molecule + peptide epitope) or CD4-positive T helper cells (ligand: MHC class II molecule + peptide epitope) is important for the development of tumor vaccines.

[0025] For an MHC class I peptide to initiate (elicit) a cellular immune response, it must also bind to an MHC molecule. This process depends on the MHC molecule allele and specific polymorphisms in the peptide's amino acid sequence. MHC class I-binding peptides are typically 8-12 amino acid residues long and typically contain two conserved residues ("anchors") in their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele possesses a "binding motif" that determines which peptides can specifically bind to the binding groove.

[0026] In an MHC class I-dependent immune response, peptides must not only be able to bind to specific MHC class I molecules expressed by tumor cells, but they must also be subsequently recognized by T cells bearing specific T cell receptors (TCRs).

[0027] Certain requirements must be met for a protein to be recognized by T lymphocytes as a tumor-specific or tumor-associated antigen and utilized therapeutically. The antigen should be expressed primarily by tumor cells and not expressed or expressed in relatively low amounts by healthy tissues. In a preferred embodiment, the peptide should be over-presented by tumor cells compared to healthy tissues. It is desirable that each antigen is not only present in certain tumors but also present at high concentrations (i.e., the number of copies of each peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins directly involved in the transformation of normal cells into tumor cells, for example, due to their function in cell cycle regulation or apoptosis suppression. Furthermore, downstream targets of proteins directly responsible for transformation may be upregulated and thus indirectly tumor-associated. Such indirect tumor-associated antigens may also be targets for vaccination approaches (Singh-Jasuja et al., 2004). To ensure that such peptides ("immunogenic peptides") are derived from tumor-associated antigens and elicit in vitro or in vivo T cell responses, the presence of an epitope within the amino acid sequence of the antigen is essential.

[0028] Essentially, any peptide capable of binding to an MHC molecule may serve as a T cell epitope. A prerequisite for the induction of a T cell response in vitro or in vivo is the presence of T cells bearing the corresponding TCR and the absence of immune tolerance to this particular epitope.

[0029] 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 isolated from patients or healthy individuals, or on the generation of differential transcriptional profiles or peptide expression patterns between tumor and normal tissues. However, the identification of genes overexpressed in tumor tissues or human tumor cell lines or selectively expressed in such tissues or cell lines does not provide accurate information regarding the use of antigens transcribed from these genes in immunotherapy. This is because only individual subsets of epitopes of these antigens are suitable for such use, since T cells with the corresponding TCR must exist and immune tolerance to this particular epitope must be absent or minimal. Therefore, in a highly preferred embodiment of the present invention, it is important to select only excessively or selectively presented peptides for which functional and / or proliferative T cells exist. Such functional T cells are defined as T cells that can undergo clonally expansion upon stimulation with a specific antigen and perform effector functions ("effector T cells").

[0030] When targeting peptide-MHC with a specific TCR (e.g., a soluble TCR) and antibody or other binding molecule (scaffold) according to the present invention, the immunogenicity of the underlying peptide is secondary; in these cases, presentation is the determining factor. [Means for solving the problem]

[0031] In a first aspect, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 88 to SEQ ID NO: 101, or a variant thereof that is at least 77%, preferably at least 88% homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 101, wherein said variant binds to MHC and / or induces cross-reactivity of T cells with said peptide or a pharmaceutically acceptable salt thereof, and wherein said peptide is not the underlying full-length polypeptide.

[0032] 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 that is at least 77%, preferably at least 88% homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 101, wherein said peptide or variant thereof has an overall length of 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0033] The following table shows peptides according to the present invention, their respective SEQ ID NOs, and their predicted origin (basic) genes. The peptides in Table 3 are peptides derived from so-called "alternative" or "short" open reading frames. For each peptide sequence, one exemplary origin transcript ID (Ensemble (Aken et al., 2016) or RefSeq (O'Leary et al., 2016) annotation) is provided. Peptides may also be derived from other additional or alternative transcripts not listed here.

[0034] In Table 3, peptides having SEQ ID NO: 1 to SEQ ID NO: 71 are identified as HLA-A * 02. The peptides in Table 4 are peptides derived from human endogenous retroviruses. For each peptide, one exemplary chromosomal location is provided. Peptides may further be mapped to additional or alternative chromosomal locations not listed herein. In Table 4, peptides having SEQ ID NOs: 72 to 74 bind to HLA-A *02, and peptides having SEQ ID NO: 75 to SEQ ID NO: 95 bind to different HLA-class I (see HLA alleles). The peptides in Table 5 are peptides that are not directly referenced to the human genome. In Table 5, peptides having SEQ ID NO: 96 to SEQ ID NO: 101 bind to HLA-A * Combine with 02.

[0035] [Table 1-1] [Table 1-2] [Table 1-3]

[0036] [Table 2-1] [Table 2-2]

[0037] [Table 3]

[0038] [Table 4]

[0039] Surprisingly, in the context of the present invention, alternative open reading frames have been found to be effective sources of tumor-associated antigens. To date, only a few such antigens have been reported, 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 considered endogenous retroviral (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 CTL epitopes. Cancer Immunol Immunother. 2012 Jul;61(7):1093-100; and Attermann AS, et al., Human endogenous retroviruses and their implications for immunotherapeutics of cancer. Ann Oncol. 2018 Nov 1;29(11):2183-2191).HERVs have been proposed as "endogenous adjuvants" that possibly sensitize cancer cells to immunological recognition, or as autoantigens 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).

[0040] The present invention further relates generally to peptides according to the invention for use in the treatment of proliferative diseases such as, for example, 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. Particularly preferred are peptides according to the invention, alone or in combination, selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101. More preferred are peptides 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 Tables 3, 4, and 5), alone or in combination, and their use in the immunotherapy of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, 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. Therefore, another aspect of the present invention relates to the use of a peptide according to the invention for the combined treatment of proliferative diseases, preferably selected from the group of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, 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.

[0041] The present invention further relates to peptides according to the invention which have the ability to bind to molecules of the human major histocompatibility complex (MHC) class I, or in extended forms such as length variants, have the ability to bind to MHC class II.

[0042] The present invention further relates to peptides according to the invention, said peptides consisting of or consisting essentially of the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 101 (respectively).

[0043] The present invention further relates to a peptide according to the invention, said peptide being modified and / or comprising a non-peptide bond.

[0044] The present invention further relates to a peptide according to the invention, said peptide being part of a fusion protein, in particular fused to the N-terminal amino acid of the HLA-DR antigen-associated invariant chain (Ii) or fused to (or fused within the sequence of) an antibody, e.g. an antibody specific for dendritic cells.

[0045] The present invention further relates to a nucleic acid encoding a peptide according to the invention. The present invention further relates to a nucleic acid according to the invention which is DNA, cDNA, PNA, RNA or a combination thereof.

[0046] The present invention further relates to an expression vector capable of expressing and / or which expresses a nucleic acid according to the invention.

[0047] The present invention further relates to a peptide according to the invention, a nucleic acid according to the invention or an expression vector according to the invention for use in the treatment of diseases and in medicine, in particular in the treatment of cancer.

[0048] The present invention further relates to antibodies specific for the peptides according to the invention or for complexes of said peptides according to the invention with MHC, and to methods for their production.

[0049] The present invention further relates to T cell receptors (TCRs), in particular soluble TCRs (sTCRs) and cloned TCRs integrated into autologous or allogeneic T cells; methods for producing these; and methods for producing NK cells or other cells bearing or cross-reacting with said TCRs.

[0050] Antibodies and TCRs are further embodiments of immunotherapeutic uses of the peptides according to the invention.

[0051] The present invention further relates to a host cell comprising a nucleic acid or an expression vector according to the invention as described above.The present invention further relates to a host cell according to the invention which is an antigen-presenting cell, preferably a dendritic cell.

[0052] The present invention further relates to a method for producing a peptide according to the invention, comprising the steps of culturing a host cell according to the invention and isolating the peptide from said host cell or its culture medium.

[0053] The present invention further relates to the method according to the present invention, wherein a sufficient amount of antigen is contacted with antigen-presenting cells, thereby loading the antigen onto class I or II MHC molecules expressed on the surface of suitable antigen-presenting cells or artificial antigen-presenting cells.

[0054] The present invention further relates to a method according to the invention, wherein the antigen-presenting cells comprise an expression vector capable of expressing or expressing said peptide containing SEQ ID NO: 1 to SEQ ID NO: 101, preferably containing 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 or a variant amino acid sequence.

[0055] The present invention further relates to activated T cells produced by the method according to the invention, said T cells selectively recognizing cells expressing a polypeptide comprising an amino acid sequence according to the invention.

[0056] The present invention further relates to a method of killing target cells in a patient which aberrantly express a polypeptide comprising any amino acid sequence according to the present invention, comprising the step of administering to the patient an effective number of T cells produced according to the present invention.

[0057] The present invention further relates to the use of any of the described peptides, nucleic acids according to the invention, expression vectors according to the invention, cells according to the invention, activated T lymphocytes, T cell receptors or antibodies or other peptides and / or peptide-MHC binding molecules according to the invention as a medicament or in the manufacture of a medicament, preferably said medicament being effective against cancer.

[0058] Preferably, the agent is a cell therapy, a vaccine or a protein based on a soluble TCR or an antibody.

[0059] The present invention further relates to a use according to the present invention, wherein said cancer cells are 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 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 cells.

[0060] The present invention further relates to peptide-based biomarkers according to the present invention, referred to herein as "targets," which can be used in the diagnosis of 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 markers can be overexpression of the peptide itself or overexpression of the corresponding gene. The markers can also be used to predict the success of immunotherapy, preferably immunotherapy targeting the same target identified by the biomarker. For example, tumor sections can be stained using antibodies or soluble TCRs to detect the presence of the peptide of interest complexed with MHC.

[0061] Optionally, the antibody possesses additional effector functions, such as an immunostimulatory domain or a toxin.

[0062] The present invention also relates to the use of these novel targets in the context of cancer therapy. DETAILED DESCRIPTION OF THE INVENTION

[0063] Stimulation of an immune response depends on the presence of antigens recognized as foreign by the host immune system. The discovery of the existence of tumor-associated antigens has raised the possibility of harnessing the host's immune system to intervene in tumor growth. Various mechanisms that utilize both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

[0064] Specific elements of the cellular immune response can specifically recognize and destroy tumor cells. Isolation of T cells from tumor-infiltrating cell populations or from peripheral blood suggests that these cells play an important role in the innate immune defense against cancer. In particular, CD8+ T cells play a key role in this response, recognizing class I molecules of the major histocompatibility complex (MHC) bearing peptides, usually 8–10 amino acid residues long, derived from proteins or defective ribosomal products (DRIPS) located in the cytosol. In humans, MHC molecules are also called human leukocyte antigens (HLA).

[0065] As used herein, unless otherwise specified, all terms are defined as set forth below.

[0066] The term "T cell response" refers to the specific proliferation and activation of effector functions induced by a peptide in vitro or in vivo. For MHC class I-restricted cytotoxic T cells, the effector functions may be lysis of peptide-pulsed, peptide precursor-pulsed, or naturally peptide-presenting target cells; secretion of cytokines, preferably interferon-γ, TNF-α, or IL-2, induced by the peptide; secretion of effector molecules, preferably granzymes or perforins, induced by the peptide; or degranulation.

[0067] The term "peptide" is used herein to designate a series of amino acid residues that are typically linked together by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. Peptides are preferably nine amino acids in length, but can be as short as eight amino acids in length, and can be 10, 11, or 12 or more amino acids in length, and in the case of MHC class II peptides (extended variants of the peptides of the invention), they can be 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acids in length.

[0068] Furthermore, the term "peptide" is intended to include salts of a series of amino acid residues, typically linked together 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, such as, for example, a chloride salt or acetate (trifluoroacetate). It should be noted that the salts of the peptides according to the present invention are substantially different from their in vivo state, since the peptides are not in the form of a salt or associated with a counterion in vivo.

[0069] The term "peptide" is also intended to include "oligopeptide." The term "oligopeptide" is used herein to designate a series of amino acid residues typically linked together by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not critical to the present invention, provided the correct epitope or epitopes are retained therein. Oligopeptides are typically less than about 30 amino acid residues in length and more than about 15 amino acids in length.

[0070] The term "polypeptide" typically refers to a series of amino acid residues linked together by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not critical to the present invention, provided the correct epitope is retained. In contrast to the terms peptide or oligopeptide, the term polypeptide is intended to refer to a molecule containing more than about 30 amino acid residues.

[0071] A peptide, oligopeptide, protein, or polynucleotide encoding such a molecule is "immunogenic" if it is capable of inducing an immune response (and thus is an "immunogen" in the context of the present invention). For the purposes of the present invention, immunogenicity is more specifically defined as the ability to induce a T-cell response. An "immunogen" is therefore a molecule capable of inducing an immune response, and in the context of the present invention, a molecule capable of inducing a T-cell response. In another embodiment, the immunogen can be a peptide, a peptide-MHC complex, an oligopeptide, and / or a protein used to raise specific antibodies or TCRs thereagainst.

[0072] A class I T cell "epitope" requires a short peptide bound to a class I MHC receptor, forming a ternary complex (MHC class I α chain, β-2-microglobulin, and peptide) that can be recognized by a T cell bearing a compatible T cell receptor that binds to the MHC / peptide complex with appropriate affinity. Peptides bound to MHC class I molecules are typically 8-14 amino acids in length, most typically 9 amino acids in length.

[0073] In humans, there are three distinct genetic loci that encode MHC class I molecules (human MHC molecules also called human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. * 01. HLA-A * 02, and HLA-B * 07 are examples of different MHC class I alleles that can be expressed from these loci.

[0074] [Table 5]

[0075] The peptides of the invention, when included in the vaccines of the invention described herein, preferably comprise * 02. The vaccine may also comprise a pan-binding MHC class II peptide. Thus, the vaccine of the present invention can be used to *While cancer can be treated in O2-positive patients, the pan-binding properties of these peptides do not require selection of MHC class II allotypes.

[0076] A of the present invention * 02 peptide, for example, A * When combined with a peptide that binds to another allele, such as HLA-A 24, a higher percentage of any patient population can be treated compared to addressing any one MHC class I allele alone. In the majority of populations, any one allele alone could address less than 50% of patients, while HLA-A 24 could address fewer than 50% of patients. * 24 and HLA-A * A vaccine comprising the O2 epitope may treat at least 60% of patients in any reasonable population. Specifically, at least one of these alleles is positive in the following percentages of patients in various geographic regions: United States 61%, Western Europe 62%, China 75%, South Korea 77%, and Japan 86% (calculated from www.allelefrequencies.net).

[0077] [Table 6]

[0078] In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.

[0079] Nucleotide sequences encoding particular peptides, oligopeptides, or polypeptides may be of natural origin, or they may be synthetically constructed. Generally, DNA fragments encoding the peptides, polypeptides, and proteins of the invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide synthetic genes that can be expressed in recombinant transcription units comprising regulatory elements derived from microbial or viral operons.

[0080] As used herein, the term "nucleotides encoding a peptide" refers to a nucleotide sequence that encodes a peptide containing artificial start and stop codons that are compatible with the biological system in which the sequence is expressed, for example, by a dendritic cell or another cell line useful for producing a TCR.

[0081] As used herein, a reference to a nucleic acid sequence includes both single-stranded and double-stranded nucleic acids. Thus, for example, a specific sequence refers to the single-stranded DNA of such a sequence, the duplex of such a sequence and its complement (double-stranded DNA), and the complement of such a sequence, unless the context clearly indicates otherwise.

[0082] The term "coding region" refers to that portion of a gene that naturally or normally encodes the expression product of the gene in its natural genomic environment, i.e., the region that encodes the natural expression product of the gene in vivo.

[0083] The coding region may be derived from a non-mutated ("normal"), mutated or modified gene, or may even be derived from a DNA sequence or gene that is entirely synthesized in the laboratory using methods well known to those skilled in the art of DNA synthesis.

[0084] The term "expression product" refers to a polypeptide or protein that is the natural translation product of a gene and of any nucleic acid sequence that, due to the degeneracy of the genetic code, encodes an equivalent and therefore encodes the same amino acid.

[0085] The term "fragment," when referring to a coding sequence, means a portion of DNA comprising less than the entire coding region, the expression product of which retains essentially the same biological function or activity as the expression product of the entire coding region.

[0086] The term "DNA segment" refers to a polymer of DNA, in the form of a separate fragment or as a component of a larger DNA construct, that is derived from DNA that has been isolated at least once, in an amount or concentration that allows the segment and its constituent nucleotide sequences to be identified, manipulated, and recovered in a substantially pure form, i.e., free from contaminating endogenous material, by standard biochemical methods, for example, using a cloning vector. Such segments are provided in the form of an open reading frame, uninterrupted by internal untranslated sequences, or introns, typically present in eukaryotic genes. Non-translated 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.

[0087] The term "primer" means a short nucleic acid sequence that can pair with a single strand of DNA and provide a free 3'-OH end at which DNA polymerase initiates deoxyribonucleotide chain synthesis.

[0088] The term "promoter" refers to a region of DNA involved in RNA polymerase binding to initiate transcription.

[0089] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is of natural origin). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, but is still isolated in the sense that such a vector or composition is not part of its natural environment.

[0090] The polynucleotides and recombinant or immunogenic polypeptides disclosed by the present invention may be in "purified" form. The term "purified" does not require complete purity; rather, it is intended as a relative definition and can include highly purified or only partially purified preparations, as these terms are understood by those of skill in the art. For example, individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. Purification of the starting material or natural substance to at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude, is expressly contemplated. Furthermore, claimed polypeptides having a purity of preferably 99.999%, or at least 99.99% or 99.9%, by weight; even more desirably 99% or greater, are expressly encompassed.

[0091] The nucleic acid and polypeptide expression products disclosed by the present invention, as well as expression vectors containing such nucleic acids and / or such polypeptides, may be in "enriched form." As used herein, the term "enriched" refers to a concentration of a substance that is (for example) at least about 2, 5, 10, 100, or 1000 times its natural concentration, advantageously 0.01% by weight, and preferably at least about 0.1% by weight. Enriched preparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. The sequences, constructs, vectors, clones, and other substances comprising the present invention may advantageously be in enriched or isolated form. The term "active fragment" generally refers to a fragment of a peptide, polypeptide, or nucleic acid sequence that generates an immune response (i.e., has immunogenicity) when administered alone, optionally with a suitable adjuvant, or in a vector, to an animal, such as a mammal, including, for example, a rabbit or mouse, and also humans, where such immune response takes the form of stimulating a T-cell response in the recipient animal, such as a human. Alternatively, the "active fragment" may also be used to induce an in vitro T cell response.

[0092] As used herein, when used in reference to a polypeptide, the terms "portion," "segment," and "fragment" refer to a sequence of contiguous residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide is treated with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptide resulting from such treatment will correspond to a portion, segment, or fragment of the starting polypeptide. When used in reference to a polynucleotide, these terms refer to the product resulting from treatment of said polynucleotide with any endonuclease.

[0093] According to the present invention, when referring to a sequence, the term "percent identity" or "percent identical" means that the sequence is compared to the claimed or described sequence ("reference sequence") after alignment of the sequence being compared ("comparison sequence") with the described or claimed sequence. The percent identity is then determined according to the following formula: Percent identity=100[1-(C / R)] 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; (i) each base or amino acid in the reference sequence that does not have a corresponding aligned base or amino acid in the comparison sequence; and (ii) each gap in the reference sequence, and (iii) each aligned base or amino acid in the reference sequence that differs from an aligned base or amino acid in the comparison sequence constitutes a difference; (iiii) the alignment must start at position 1 of the aligned sequence; R is the number of bases or amino acids in the reference sequence over the alignment length with the comparison sequence, and any gaps that occur in the reference sequence are also counted as bases or amino acids.

[0094] If there is an alignment between a comparison sequence and a reference sequence for which the percent identity is calculated as above that is about or exceeds a particular minimum percent identity, then the comparison sequence has a particular minimum percent identity with the reference sequence, even if there is an alignment therein whose percent identity, as calculated as above, is less than the particular percent identity.

[0095] Thus, as noted 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 that is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 101, or a variant thereof that cross-reacts T cells with said peptide. The peptides of the present invention have the ability to bind human major histocompatibility complex (MHC) class I molecules or extended versions of said peptides to class II.

[0096] In the present invention, the term "homologous" refers to the degree of identity between two amino acid sequences, i.e., peptide or polypeptide sequences (see percentage identity above). The aforementioned "homology" is determined by comparing two sequences aligned under optimal conditions across the sequences being compared. Such sequence homology can be calculated, for example, by creating an alignment using the ClustalW algorithm. Publicly available sequence analysis software, more specifically, Vector NTI, GENETYX or other tools, are provided by public databases.

[0097] One skilled in the art would be 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).

[0098] By "variant" of a given amino acid sequence, we mean that, for example, the side chains of one or two of the amino acid residues are altered (e.g., by replacing them with the side chains of another naturally occurring amino acid residue, or with other side chains) so that the peptide can still bind to an HLA molecule substantially similarly to a peptide consisting of the given amino acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 101. For example, a peptide can be modified so that it binds to an HLA-A molecule in a manner substantially similar to a peptide consisting of SEQ ID NO: 1 to SEQ ID NO: 101. * It may be modified to at least maintain, if not improve, its ability to interact with and bind to the binding groove of an appropriate MHC molecule, such as -02 or -DR, and thus it at least maintains, if not improves, its ability to bind to the TCR of activated T cells.

[0099] These T cells can subsequently cross-react with and kill cells expressing polypeptides containing the native amino acid sequence of the cognate peptides defined in embodiments of the present invention. As can be deduced from the scientific literature and databases (Rammensee et al., 1999; Godkin et al., 1997), specific positions of HLA-binding peptides are typically anchor residues, forming core sequences that fit the binding motif of HLA receptors, defined by the polar, electrophysical, hydrophobic, and spatial properties of the polypeptide chain that constitute the binding groove. Thus, one skilled in the art could modify the amino acid sequences set forth in SEQ ID NOs: 1-101 by retaining the known anchor residues and determine whether such variants maintain the ability to bind to MHC class I or II molecules. The variants of the present invention maintain the ability to bind to the TCR of activated T cells, which can subsequently cross-react with and kill cells expressing polypeptides containing the native amino acid sequence of the cognate peptides defined in embodiments of the present invention.

[0100] The original (unmodified) peptides disclosed herein may be modified by substitution of one or more residues at different, possibly selective, positions within the peptide chain, unless otherwise specified. Preferably, these substitutions are located at the ends of the amino acid chain. Such substitutions may be conservative in nature, e.g., an amino acid is replaced by an amino acid with a similar structure and characteristics, such as a hydrophobic amino acid being replaced by another hydrophobic amino acid. Even more conservative substitutions are those with amino acids of identical or similar size and chemical properties, such as the replacement of leucine with isoleucine. In studies of sequence diversity in naturally occurring homologous protein families, certain amino acid substitutions are often more tolerated than others, and these often correlate with the similarity in size, charge, polarity, and hydrophobicity between the original amino acid and its substitute, which is the basis for the definition of a "conservative substitution."

[0101] Conservative substitutions are defined herein as exchanges within one of the following five groups: Group 1 - small aliphatic, non-polar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2 - polar negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3 - polar positively charged residues (His, Arg, Lys); Group 4 - large aliphatic non-polar residues (Met, Leu, Ile, Val, Cys); and Group 5 - large aromatic residues (Phe, Tyr, Trp).

[0102] In one embodiment, conservative substitutions may include those described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5," Natl. Biomedical Research, the entire contents of which are incorporated by reference. For example, in one embodiment, amino acids belonging to one of the following groups may be exchanged for one another, and thus constitute conservative exchanges: 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 may be selected from T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and / or T→S.

[0103] In one embodiment, conservative amino acid substitutions may include the substitution of an amino acid with another amino acid of the same class, such as, for example, (1) non-polar: 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, such as: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, e.g., U.S. Pat. No. 10,106,805, the entire contents of which are incorporated by reference).

[0104] In another embodiment, conservative substitutions may be made according to Table A. Methods for predicting tolerance to protein modifications can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the entire contents of which are incorporated by reference.

[0105] [Table 7]

[0106] In another embodiment, conservative substitutions may be those shown in Table B under the heading of "conservative substitutions." If such substitutions result in altered biological activity, more substantial changes, designated "exemplary substitutions" in Table B, may be introduced and the products screened as appropriate.

[0107] [Table 8]

[0108] A less conservative substitution might involve the substitution of another amino acid with similar characteristics but somewhat different in size, such as the substitution of an isoleucine residue for an alanine. A highly non-conservative substitution might involve the substitution of a polar amino acid with an acidic amino acid, or even a basic amino acid. However, even such "radical" substitutions cannot be dismissed as potentially ineffective, because chemical effects are not completely predictable and radical substitutions can lead to serendipitous effects that cannot be predicted from simple chemical principles.

[0109] Of course, such substitutions may involve structures other than conventional L-amino acids. Thus, D-amino acids may be substituted for L-amino acids normally found in the antigenic peptides of the invention and still be encompassed by the disclosure herein. Furthermore, non-standard amino acids (i.e., other than the common naturally occurring proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the invention.

[0110] If substitutions at two or more positions are found to result in a peptide with substantially equal or greater antigenic activity, as defined below, combinations of these substitutions are tested to determine whether the combination of substitutions results in an additive or synergistic effect on the antigenicity of the peptide. At most, no more than four positions in a peptide are substituted simultaneously.

[0111] Peptides consisting essentially of amino acid sequences as set forth herein may have one or two non-anchor amino acids (see below for anchor motifs) exchanged without substantially altering or adversely affecting their ability to bind to human major histocompatibility complex (MHC) class I or II molecules, as compared to the unmodified peptide. In another embodiment, in peptides consisting essentially of amino acid sequences as set forth herein, one or two amino acids may be exchanged with their conservative exchange partners (see below), without substantially altering or adversely affecting their ability to bind to human major histocompatibility complex (MHC) class I or II molecules, as compared to the unmodified peptide.

[0112] Amino acid residues that do not substantially contribute to interaction with the T cell receptor may be modified by substitution with other amino acids whose incorporation does not substantially affect T cell reactivity or eliminate binding to the relevant MHC. Thus, except as provided, a peptide of the invention may be any peptide (we include by that term an oligopeptide or polypeptide) comprising an amino acid sequence as given or a portion or variant thereof.

[0113] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0114] Longer (extended) peptides may also be suitable. MHC class I epitopes are typically 8-11 amino acids in length, but can be generated by peptide processing from longer peptides or proteins containing the actual epitope. Preferably, the residues flanking the actual epitope are those that do not substantially affect the proteolytic cleavage required to expose the actual epitope during processing.

[0115] The peptides of the present invention can be extended by up to four amino acids, i.e., 1, 2, 3 or 4 amino acids can be added to either end in any combination between 4:0 and 0:4. Extension combinations according to the present invention are listed in Table 10.

[0116] [Table 10]

[0117] The amino acids for extension / elongation can be the peptide of the original protein sequence or any other amino acid. Extension can be used to increase the stability or solubility of the peptide.

[0118] Thus, epitopes of the present invention may be identical to naturally occurring tumor-associated or tumor-specific epitopes, or may include epitopes that differ from the reference peptide by no more than four residues, so long as they have substantially the same antigenic activity.

[0119] In alternative embodiments, the peptide is extended on one or both sides by more than four amino acids, preferably up to a total length of 30 amino acids. This may result in an MHC class II binding peptide. Binding to MHC class II can be tested by methods known in the art.

[0120] Thus, the present invention provides peptides and variants of MHC class I epitopes, wherein the peptides or variants have an overall length of 8 to 100, preferably 8 to 30, most preferably 8 to 14, i.e., 8, 9, 10, 11, 12, 13, 14 amino acids, and for extended class II binding peptides, the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

[0121] Of course, the peptides or variants according to the invention have the ability to bind to molecules of the human major histocompatibility complex (MHC) class I or II. Binding of the peptides or variants to MHC complexes may be tested by methods known in the art.

[0122] Preferably, when T cells specific for a peptide according to the invention are tested with a substituted peptide, the peptide concentration at which the substituted peptide achieves half-maximal increase in lysis over background is about 1 mM or less, preferably about 1 μM or less, more preferably about 1 nM or less, even more preferably about 100 pM or less, and most preferably about 10 pM or less. It is also preferred that the substituted peptide be recognized by T cells from more than one individual, at least two, and more preferably three individuals.

[0123] In a particularly preferred embodiment of the invention, the peptide consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 101.

[0124] "Consisting essentially of" is intended to mean that the peptide according to the invention contains, in addition to the sequence set forth in any of SEQ ID NOs: 1 to 101 or a variant thereof, an additional N- and / or C-terminally positioned sequence of amino acids which do not necessarily constitute part of the peptide that functions as an epitope for an MHC molecule epitope.

[0125] Nevertheless, these sequences may be important for providing efficient introduction of the peptides according to the invention into cells. In one embodiment of the invention, the peptide is part of a fusion protein comprising the 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (p33, hereinafter "Ii"), e.g., from NCBI, GenBank accession number X00497. In other fusions, the peptides of the invention may be fused to antibodies, or functional parts thereof, as described herein, in particular to antibody sequences, so as to be specifically targeted by said antibodies, or may be fused to or within, for example, antibodies specific for dendritic cells, as described herein.

[0126] Furthermore, the peptides or variants may be further modified to improve stability and / or binding to MHC molecules in order to elicit a stronger immune response. Methods for such optimization of peptide sequences are well known in the art and include, for example, the introduction of reverse peptide or non-peptide bonds.

[0127] In reverse peptide bonds, amino acid residues are not linked by peptide (-CO-NH-) bonds, but the peptide bond is reversed. Such retro-inverso peptidomimetics can be produced using methods known in the art, such as those described in Meziere et al. (1997) (Meziere et al., 1997), which is incorporated herein by reference. This approach involves creating pseudopeptides that contain changes related to the main chain, rather than the direction of the side chain. Meziere et al. (Meziere et al., 1997) demonstrate that these pseudopeptides are useful for MHC binding and T helper cell response. Retro-inverse peptides that contain NH-CO bonds instead of CO-NH peptide bonds are much more resistant to proteolysis.

[0128] Examples of non-peptide bonds are -CH-NH, -CHS-, -CHCH-, -CH=CH-, -COCH-, -CH(OH)CH-, and -CHSO-. U.S. Pat. No. 4,897,445 provides a method for solid-phase synthesis of non-peptide bonds (-CH-NH) in polypeptide chains, involving polypeptides synthesized by standard procedures and non-peptide bonds synthesized by reacting amino acids with amino aldehydes in the presence of NaCNBH.

[0129] Peptides comprising the above sequences may be synthesized with additional chemical groups at their amino and / or carboxy termini to improve the stability, bioavailability, and / or affinity of the peptides. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to the amino termini of the peptides. Similarly, an acetyl group or a 9-fluorenylmethoxycarbonyl group may be placed at the amino terminus of the peptides. Furthermore, hydrophobic groups, t-butyloxycarbonyl, or amide groups may be added to the carboxy terminus of the peptides.

[0130] Furthermore, the peptides of the present invention may be synthesized to alter their configuration. For example, the D-isomer of one or more amino acid residues of the peptide may be used instead of the usual L-isomer. Still further, at least one of the amino acid residues of the peptides of the present invention may be substituted with one of the known non-naturally occurring amino acid residues. Such modifications may serve to increase the stability, bioavailability, and / or binding activity of the peptides of the present invention.

[0131] Similarly, the peptides or variants of the present invention may be chemically modified by reacting specific amino acids either 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. Chemical modifications of amino acids include, but are not limited to, acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide and sulfhydryl modification of carboxyl groups by performic acid oxidation of cysteine ​​to cysteic acid, mercury derivatization, mixed disulfide formation with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and carbamoylation with cyanate at alkaline pH. In this regard, those skilled in the art are referred to Chapter 15 of Current Protocols in Protein Science, Eds. Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995) for more detailed procedures regarding the chemical modification of proteins.

[0132] Briefly, for example, modification of arginyl residues in proteins is often based on the reaction of vicinal dicarbonyl compounds, such as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione, to form adducts. Another example is the reaction of arginine residues with methylglyoxal. Cysteines can be modified without the simultaneous modification of other nucleophilic sites, such as lysines and histidines. Consequently, numerous reagents are available for cysteine ​​modification. Company websites, such as Sigma-Aldrich (http: / / www.sigma-aldrich.com), provide information on specific reagents.

[0133] Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during heat treatment of biopharmaceuticals. Specific glutamic acid residues can be modified using Woodward's reagent K. N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide can be used to form intramolecular crosslinks between lysine and glutamic acid residues. For example, diethylpyrocarbonate is a reagent for modifying histidyl residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. Reactants of lysine residues and other α-amino groups are useful, for example, in binding peptides to surfaces or protein / peptide crosslinking. Lysine is the attachment site for poly(ethylene)glycol and is the primary modification site for protein glycosylation. Methionine residues in proteins can be modified, for example, with iodoacetamide, bromoethylamine, and chloramine T.

[0134] Modification of tyrosyl residues can be achieved with tetranitromethane and N-acetylimidazole. Crosslinking through the formation of dityrosine can be achieved with hydrogen peroxide / copper ion.

[0135] Recent studies on the modification of tryptophan have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

[0136] Successful modification of therapeutic proteins and peptides with PEG is often associated with increased circulatory half-life, while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is used for hydrogel preparation. Chemical modification of allergens for immunotherapy is often achieved by carbamylation with potassium cyanate.

[0137] Peptides, or peptide variants that are modified or contain non-peptide bonds, are preferred embodiments of the present invention.

[0138] Another embodiment of the present invention relates to non-naturally occurring peptides, wherein the peptides consist of or consist essentially of the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 101 and are synthetically produced (e.g., synthesized) as pharmaceutically acceptable salts. Methods for synthetically producing peptides are well known in the art. Because peptides produced in vivo are not salts, salts of peptides according to the present invention are substantially different from the in vivo state of the peptides. The non-naturally occurring salt form of the peptide mediates the solubility of the peptide, particularly in the context of pharmaceutical compositions comprising the peptide, such as the peptide vaccines disclosed herein. Sufficient, or at least substantial, solubility of the peptide is necessary to efficiently deliver the peptide to the subject being treated. Preferably, the salt is a pharmaceutically acceptable salt of the peptide. These salts according to the present invention include PO4 as the anion. 3- , SO4 2- , CH3COO - , Cl - , Br - , NO3 - , ClO4 - , I - , SCN - , and NH4 as the cation + , Rb + , K. + , Na + , Cs + , Li + , Zn 2+ , Mg 2+ , Ca 2+ , Mn 2+ , Cu 2+ and Ba 2+In particular, salts include alkali and alkaline earth salts such as the Hofmeister series of salts comprising (NH4)3PO4, (NH4)2HPO4, (NH4)H2PO4, (NH4)2SO4, NH4CH3COO, NH4Cl, NH4Br, NH4NO3, NH4CIO4, NH4I, NHClSCN, Rb3PO4, Rb2HPO4, RbH2PO4, Rb2SO4, Rb4CH3COO, Rb4Cl, Rb4Br, Rb4NO3, Rb4CIO4, Rb4I, Rb4SCN, K3PO4, K2HPO4, KH2PO4, K2SO4, KCH3COO, KCl, KBr, KNO3, KClO4, KI, KSCN, Na3PO4, Na2HPO4, NaH2PO4, Na2SO4, NaCH3COO, NaCl, NaB r, NaNO3, NaCIO4, NaI, NaSCN, ZnCI2Cs3PO4, Cs2HPO4, CsH2PO4, Cs2SO4, CsCH3COO, CsCl, CsBr, CsNO3, CsCIO4, CsI , CsSCN, Li3PO4, Li2HPO4, LiH2PO4, Li2SO4, LiCH3COO, LiCl, LiBr, LiNO3, LiClO4, LiI, LiSCN, Cu2SO4, Mg3(PO4) 2, Mg2HPO4, Mg(H2PO4)2, Mg2SO4, Mg(CH3COO)2, MgCl2, MgBr2, Mg(NO3)2, Mg(ClO4)2, MgI2, Mg(SCN)2, MnCl2, Ca3( PO4), Ca2HPO4, Ca(H2PO4), CaSO4, Ca(CH3COO), CaCl2, CaBr2, Ca(NO3), Ca(ClO4), CaI2, Ca(SCN), Ba3(PO4), Ba2HPO4, Ba(H2PO4), BaSO4, Ba(CH3COO), BaCl2, BaBr2, Ba(NO3), Ba(ClO4), BaI2, and Ba(SCN). Particularly preferred are NH acetates, such as chlorides or acetates (trifluoroacetates), MgCl2, KH2PO4, Na2SO4, KCl, NaCl, and CaCl2.

[0139] In general, peptides and variants (containing at least peptide bonds between amino acid residues) may be synthesized by the Fmoc-polyamide solid-phase peptide synthesis method disclosed by Lukas et al. (Lukas et al., 1981) and the references cited therein. Temporary N-amino group protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is carried out using 20% ​​piperidine in N,N-dimethylformamide. Side chain functional groups may be protected as their butyl ethers (for serine, threonine, and tyrosine), butyl esters (for glutamic acid and aspartic acid), butyloxycarbonyl derivatives (for lysine and histidine), trityl derivatives (for cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (for arginine). When glutamine or asparagine is the C-terminal residue, a 4,4'-dimethoxybenzhydryl group is utilized to protect the side-chain amide functionality. The solid-phase support is based on a polydimethyl-acrylamide polymer composed of three monomers: dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (crosslinker), and acryloylsarcosine methyl ester (functionalizer). The peptide-to-resin cleavable linker used is an acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives, except for asparagine and glutamine, which are added using a reverse N,N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or isatin test procedures. Upon completion of synthesis, the peptide is cleaved from the resin support and the side chain protecting groups are simultaneously removed by treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture. Commonly used scavengers include ethanedithiol, phenol, anisole, and water, with the exact choice depending on the constituent amino acids of the peptide being synthesized.A combination of solid and solution phase methods for the synthesis of peptides is also possible (see, for example, (Bruckdorfer et al., 2004) and references cited therein).

[0140] The trifluoroacetic acid is removed by evaporation under vacuum, followed by trituration with diethyl ether to yield the crude peptide. Any scavengers present are removed by a simple extraction procedure, which, upon lyophilization of the aqueous phase, gives the scavenger-free crude peptide. Reagents for peptide synthesis are commonly available, for example, from Calbiochem-Novabiochem (Nottingham, UK).

[0141] Purification may be achieved by any one or combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (usually) reversed-phase high performance liquid chromatography using, for example, an acetonitrile / water gradient separation.

[0142] Analysis of peptides may be carried out 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, by fast atom bombardment (FAB) mass spectrometry, and by MALDI and ESI-Q-TOF mass spectrometry.

[0143] To select over-represented peptides, a representation profile is calculated, which shows the median sample representation and the variation of repeated tests. The profile juxtaposes the sample of the tumor entity of interest to the baseline of normal samples. Then, by calculating the p-value of the linear mixed-effects model (Pinheiro et al., 2015) and correcting for multiple tests by the false discovery rate (Benjamini and Hochberg, 1995), each of these profiles can be combined into an over-representation score (see Example 1, Figure 1).

[0144] For identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shock-frozen samples were purified and HLA-associated peptides were isolated. The isolated peptides were separated and sequenced by online nanoelectrospray ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS) experiments. The resulting peptide sequences were validated by comparing the fragmentation patterns of corresponding synthetic reference peptides of identical sequences with those of native tumor-associated peptides (TUMAPs) recorded from 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 (N = 490 samples). Because the peptides were directly identified as ligands for HLA molecules in primary tumors, these results provide direct evidence of the natural processing and presentation of the identified peptides on primary cancer tissues obtained from patients with acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, 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.

[0145] The discovery pipeline XPRESIDENT® v2.1 (see, e.g., U.S. Patent No. 2013-0096016, the entire contents of which are incorporated herein by reference) enables the identification and selection of plausible over-presented peptide vaccine candidates based on direct relative quantification of HLA-restricted peptide levels on cancer tissues compared to several different non-cancerous tissues and organs. This was achieved through the development of label-free differential quantification that combines algorithms for sequence identification, spectral clustering, ion counting, retention time alignment, charge state deconvolution, and normalization using acquired LC-MS data processed through a proprietary data analysis pipeline.

[0146] Additional sequence information from public resources (Olexiouk et al., 2016; Subramanian et al., 2011) was integrated into the PRESIDENT® discovery pipeline, enabling the identification of TUMAPs from non-canonical sources. De-novo sequencing was employed in an orthogonal database-independent search strategy to identify peptide sequences in tumor-specific spectral clusters determined by XPRESIDENT®. This allowed the identification of novel TUMAPs not directly referenced in human genome or proteomics databases. Presentation levels, including error estimates, were established for each peptide and sample. Peptides exclusively presented on tumor tissues and peptides over-represented in tumors compared to non-cancerous tissues and organs were identified.

[0147] HLA-peptide complexes from 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 tissue samples were purified, and HLA-associated peptides were isolated and analyzed by LC-MS (see Example 1). All TUMAPs included in this application have been identified by this approach on 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, and their presence is confirmed on 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.

[0148] TUMAPs identified in 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, as well as normal tissues, were quantified using ion counts from label-free LC-MS data. The method assumes that the LC-MS signal area of ​​a peptide correlates with its abundance in the sample. All quantitative signals for peptides from various LC-MS experiments were normalized based on central tendency, averaged per sample, and merged into a bar graph called a presentation profile. The presentation profile integrates different analytical methods, such as protein database searching, spectral clustering, charge state deconvolution (deconvolution), and retention time alignment and normalization.

[0149] Furthermore, the discovery pipeline XPRESIDENT® allows for the direct absolute quantification of MHC-restricted, preferably HLA-restricted, peptide levels in cancer or other infected tissues. Briefly, total cell number was calculated from the total DNA content of the analyzed tissue sample. The total peptide amount of TUMAP in the tissue sample was measured by nanoLC-MS / MS as the ratio between natural TUMAP and a known amount of an isotope-labeled version of TUMAP, the so-called internal standard. The efficiency of TUMAP isolation was determined by spiking all selected TUMAP peptide:MHC complexes into the tissue lysate as early as possible during the TUMAP isolation procedure and detecting them by nanoLC-MS / MS following completion of the peptide isolation procedure. Total cell number and total peptide amount were calculated from triplicate measurements per tissue sample. Peptide isolation efficiency was calculated as the average from nine spike experiments, each measured in triplicate (see Example 6 and Table 15).

[0150] In addition to over-representation of peptides, mRNA expression of underlying genes was also examined. mRNA data was obtained through RNASeq analysis of normal and cancer tissues (see Figure 2 in Example 2). A further source of normal tissue data was a publicly available database of RNA expression data derived from approximately 3,000 normal tissue samples (Lonsdale, 2013). Peptides derived from proteins whose mRNAs are highly expressed in cancer tissues but very low or absent in vital normal tissues are preferably included in the present invention.

[0151] The present invention provides peptides useful for treating cancers / tumors, 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, which present the peptides of the present invention in excess or exclusively. These peptides were shown by mass spectrometry to be naturally presented by HLA molecules on primary human 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.

[0152] Many of the original genes / proteins (also referred to as "full-length proteins" or "basic proteins") from which the peptides are derived have been shown to be highly overexpressed in cancer compared to normal tissues, and in the context of the present invention "normal tissue" shall mean either healthy blood, brain, heart, liver, lung, adipose tissue, adrenal gland, bile duct, bladder, bone marrow, esophagus, eye, gallbladder, head and neck, large intestine, small intestine, kidney, lymph node, central nervous system, peripheral nervous system, pancreas, parathyroid gland, peritoneum, pituitary gland, pleura, skeletal muscle, skin, spinal cord, spleen, stomach, thyroid, trachea, and ureter cells, or other normal tissue cells, which show a high degree of tumor association of the original gene (see Example 2). Furthermore, the peptide itself is greatly over-represented on tumor tissue, and in the context of the present invention "tumor tissue" refers to samples from patients suffering from acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, 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 not normal tissue (see Example 1).

[0153] HLA-binding peptides can be recognized by the immune system, particularly T lymphocytes, which can destroy cells that present 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.

[0154] The peptides of the present invention have been shown to be capable of stimulating T cell responses and / or being over-presented, and therefore can be used in accordance with the present invention for the production of antibodies and / or TCRs, such as soluble TCRs (see Example 3). Furthermore, when complexed with the respective MHC, the peptides can also be used for the production of antibodies and / or TCRs, particularly TCRs, according to the present invention. The respective methods are well known to those skilled in the art and can also be found in the respective references (see also below). Thus, the peptides of the present invention are useful for generating an immune response in patients by which tumor cells can be destroyed. An immune response in patients can be induced by directly administering the described peptides, or appropriate precursors (e.g., extended peptides, proteins, or nucleic acids encoding these peptides), to the patient, ideally in combination with an agent (i.e., an adjuvant) that enhances immunogenicity. Because the target peptides of the present invention are not presented in equivalent copy numbers on normal tissues, the immune response resulting from such therapeutic vaccination can be expected to be highly specific to tumor cells, preventing the risk of unwanted autoimmune reactions against the patient's normal cells.

[0155] The present specification further relates to T cell receptors (TCRs) comprising an alpha chain and a beta chain ("alpha / beta TCR"). Also provided are peptides according to the invention that are capable of binding to TCRs and antibodies when presented by MHC molecules.

[0156] The present specification also relates to fragments of the TCRs according to the invention which are capable of binding to peptide antigens according to the invention when presented by HLA molecules. This term particularly relates to soluble TCR fragments, e.g. TCRs lacking the transmembrane portion and / or constant region, single chain TCRs and fusions thereof, e.g. Ig.

[0157] The present specification also relates to nucleic acids, vectors, and host cells for expressing the TCRs and peptides of the present specification; and methods of using the same.

[0158] The term "T cell receptor" (abbreviated as TCR) refers to a heterodimeric molecule comprising an alpha polypeptide chain (alpha chain) and a beta polypeptide chain (beta chain), which heterodimeric receptor is capable of binding peptide antigens presented by HLA molecules. The term also includes so-called gamma / delta TCRs.

[0159] In one embodiment, the description provides a method of producing a TCR as described herein, the method comprising culturing a host cell capable of expressing the TCR under conditions suitable to promote expression of the TCR.

[0160] In another embodiment, the description relates to a method described herein in which a sufficient amount of antigen is contacted with an antigen-presenting cell to load the antigen onto a class I or II MHC molecule expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell, or in which the antigen is loaded onto a class I or II MHC tetramer by tetramerizing an antigen / class I or II MHC complex monomer.

[0161] The α and β chains of α / β TCRs, and the γ and δ chains of γ / δ TCRs, are generally considered to have two "domains" each: a variable and a constant domain. The variable domain consists of a variable region (V) linked to a joining region (J). The variable domain may also contain a leader region (L). The β and δ chains may also contain a diversity region (D). The α and β constant domains may also contain a C-terminal transmembrane (TM) domain that anchors the α and β chains to the cell membrane.

[0162] With respect to gamma / delta TCRs, the term "TCRgamma variable domain" as used herein refers to the linkage between the TCRgamma V (TRGV) region and the TCRgamma J (TRGJ) region without the leader region (L), and the term TCRgamma constant domain refers to the extracellular TRGC region or a C-terminally truncated TRGC sequence. Similarly, the term "TCRdelta variable domain" refers to the linkage between the TCRdelta V (TRDV) region and the TCRdelta D / J (TRDD / TRDJ) region without the leader region (L), and the term "TCRdelta constant domain" refers to the extracellular TRDC region or a C-terminally truncated TRDC sequence.

[0163] The TCRs herein preferably bind to peptide-HLA molecule complexes with a binding affinity (KD) of about 1 μM or less, about 0.1 μM or less, about 25 μM or less, or about 10 μM or less. High-affinity TCRs with binding affinities of about 1 μM or less, about 100 nM or less, about 50 nM or less, or about 25 nM or less are more preferred. Non-limiting examples of preferred binding affinity ranges for the TCRs 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.

[0164] As used herein, in the context of TCRs herein, "specific binding" and grammatical variants thereof are used to mean a TCR that has a binding affinity (KD) of 100 μM or less for a peptide-HLA molecule complex.

[0165] The α / β heterodimeric TCRs herein may have a disulfide bond introduced 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, with Thr48 of TRAC and Ser57 of TRBC1 or TRBC2 replaced by cysteine ​​residues that form a disulfide bond between the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

[0166] In the presence or absence of the above-described introduced interchain bond, the α / β heterodimer TCR of the present specification may have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, and the TCR TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence may be linked by a natural disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

[0167] The TCRs herein may comprise a detectable label selected from the group consisting of a radionuclide, a fluorophore, and biotin. The TCRs herein may be conjugated to a therapeutically active agent such as a radionuclide, a chemotherapeutic agent, or a toxin.

[0168] In one embodiment, the TCRs herein having at least one mutation in the alpha chain and / or having at least one mutation in the beta chain have modified glycosylation compared to the non-mutated TCR.

[0169] In one embodiment, a TCR comprising at least one mutation in the TCR α chain and / or TCR β chain has at least twice the binding affinity and / or binding half-life to a peptide-HLA molecule complex as a TCR comprising an unmutated TCR α chain and / or an unmutated TCR β chain. The affinity enhancement and utilization of tumor-specific TCRs depend on the existence of a window of optimal TCR affinity. The existence of such a window is based on the observation that TCRs specific for, for example, HLA-A2-restricted pathogens generally have KD values ​​approximately 10-fold lower than TCRs specific for, for example, HLA-A2-restricted tumor-associated self-antigens. While tumor antigens have the potential to be immunogenic, because tumors arise from an individual's own cells, it is now known that only mutant proteins or proteins with altered translational processing are considered foreign by the immune system. Upregulated or overexpressed antigens (so-called self-antigens) do not necessarily induce a functional immune response against tumors; T cells expressing highly reactive TCRs against these antigens are negatively selected in the thymus by a process known as central immune tolerance, i.e., only T cells with low-affinity TCRs against self-antigens remain. Thus, the affinity of the TCRs or variants herein for peptides can be increased by methods well known in the art.

[0170] The present disclosure further relates to a method for identifying and isolating a TCR according to the present disclosure, said method comprising: * The method comprises the steps of incubating PBMCs from O2-negative healthy donors with A2 / peptide monomer, incubating the PBMCs with tetrameric phycoerythrin (PE), and isolating high avidity T cells by fluorescence-activated cell sorting (FACS)-Calibur analysis.

[0171] The present specification further relates to a method for identifying and isolating TCRs according to the present specification, comprising the steps of obtaining transgenic mice carrying the entire human TCRαβ gene locus (1.1 and 0.7 Mb) whose T cells express a diverse human TCR repertoire that compensates for the mouse TCR deficiency, immunizing the mice with peptides, incubating PBMCs obtained from the transgenic mice with tetrameric phycoerythrin (PE), and isolating high avidity T cells by fluorescence-activated cell sorting (FACS) Calibur analysis.

[0172] In one embodiment, to obtain T cells expressing the TCRs herein, nucleic acids encoding the TCR-α and / or TCR-β chains herein are cloned into an expression vector, such as a gamma retrovirus or lentivirus. Recombinant viruses are produced and then tested for functionality, such as antigen specificity and functional binding activity. An aliquot of the final product is then used to transduce a target T cell population (generally purified from the patient's PBMCs) and expanded prior to infusion into the patient.

[0173] In another embodiment, to obtain T cells expressing the TCRs herein, TCR RNA is synthesized by techniques known in the art, such as, for example, an in vitro transcription system. The in vitro synthesized TCR RNA is then introduced by electroporation into primary CD8+ T cells obtained from a healthy donor, resulting in re-expression of the tumor-specific TCR-α and / or TCR-β chains.

[0174] To increase expression, the nucleic acids encoding the TCRs herein may be operably linked to strong promoters 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 preferred embodiments, the promoter is heterologous to the nucleic acid to be expressed.

[0175] In addition to a strong promoter, the TCR expression cassettes herein may contain additional elements that can enhance transgene expression, including a central polypurine tract (cPPT), which facilitates nuclear translocation of the lentiviral construct (Follenzi et al., 2000), and a woodchuck hepatitis virus posttranscriptional regulatory element (wPRE), which increases RNA stability and thereby enhances levels of transgene expression (Zufferey et al., 1999).

[0176] The α and β chains of the TCR of the present invention may be encoded by nucleic acids on separate vectors or may be encoded by polynucleotides on the same vector.

[0177] Achieving high levels of TCR surface expression requires high levels of transcription of both the TCR-α and TCR-β chains of the introduced TCR. To achieve this, the TCR-α and TCR-β chains herein may be cloned into a bicistronic construct within a single vector, which has been shown to overcome this obstacle. Because the TCR-α and TCR-β chains are generated from a single transcript that splits into two proteins during translation, ensuring the generation of equimolar ratios of the TCR-α and TCR-β chains, the use of a viral internal ribosome entry site between the TCR-α and TCR-β chains results in coordinated expression of both chains (Schmitt et al., 2009).

[0178] The nucleic acids encoding the TCRs herein may be codon-optimized to increase expression from host cells. Redundancy in the genetic code allows some amino acids to be coded for by more than one codon, but certain codons are less "optimal" than others due to the relative availability of compatible tRNAs as well as other factors (Gustafsson et al., 2004). Modifying the TCR-α and TCR-β gene sequences so that each amino acid is coded for by the optimal codon for mammalian gene expression, as well as removing mRNA instability motifs or potential splice sites, has been shown to significantly enhance TCR-α and TCR-β gene expression (Scholten et al., 2006).

[0179] Furthermore, mispairing between the introduced and endogenous TCR chains can lead to specificity gains that pose significant autoimmune risks. For example, the formation of mixed TCR dimers can reduce the number of CD3 molecules available to form properly paired TCR complexes, which can significantly reduce the functional avidity of cells expressing the introduced TCR (Kuball et al., 2007).

[0180] To reduce mispairing, the C-terminal regions of the introduced TCR chains herein may be modified to increase interchain affinity while decreasing the ability of the introduced chains to pair with endogenous TCRs. These strategies include replacing the C-terminal regions of human TCR-α and TCR-β with their murine counterparts (murinized C-terminal regions); introducing a second cysteine ​​residue into both the TCR-α and TCR-β chains of the introduced TCR to create a second interchain disulfide bond in the C-terminal region (cysteine ​​modification); exchanging interacting residues within the C-terminal regions of the TCR-α and TCR-β chains ("knob-in-hole"); and fusing the variable regions of the TCR-α and TCR-β chains directly to CD3ζ (CD3ζ fusion) (Schmitt et al., 2009).

[0181] In one embodiment, the host cell is genetically engineered to express a TCR herein. In a preferred embodiment, the host cell is a human T cell or T cell precursor. In some embodiments, the T cell or T cell precursor is obtained from a cancer patient. In other embodiments, the T cell or T cell precursor is obtained from a healthy donor. The host cell herein can be allogeneic or autologous with respect to the patient being treated. In one embodiment, the host is a gamma / delta T cell transformed to express an alpha / beta TCR.

[0182] A "pharmaceutical composition" is a composition suitable for administration to humans in a medical setting. Preferably, pharmaceutical compositions are sterile and manufactured in accordance with GMP guidelines.

[0183] Pharmaceutical compositions comprise peptides in either free form or in the form of pharmaceutically acceptable salts (see also above). As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed peptides, wherein the peptides are modified by forming acid or base salts of the drug. For example, acid salts are prepared from the free base (typically the neutral form of the drug has a neutral -NH group) by reaction with a suitable acid. Suitable acids for preparing acid salts include both organic acids, such as acetic acid, propionic acid, glycolic acid, 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, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid, as well as inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, and nitric acid and phosphoric acid. Conversely, basic salts of acidic moieties which may be present on the peptide are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine and the like.

[0184] In one particularly preferred embodiment, the pharmaceutical composition comprises the peptide as a salt of acetic acid (acetate), trifluoroacetic acid or hydrochloric acid (chloride).

[0185] Preferably, the medicament of the present invention is an immunotherapy such as a vaccine. It can be administered to a patient directly, to an affected organ, or systemically, via id, im, sc, ip, and iv routes; or applied ex vivo to cells derived from the patient or a human cell line, which are subsequently administered to the patient; or used ex vivo to select a subpopulation of immune cells derived from the patient, which are then re-administered to the patient. When nucleic acids are administered ex vivo to cells, it may be useful to transfect the cells to co-express an immunostimulatory cytokine, such as interleukin-2. The peptides may be substantially pure, combined with an immunostimulatory adjuvant (see below), used in combination with an immunostimulatory cytokine, or administered via a suitable delivery system, such as liposomes. The peptides may also be conjugated to a suitable carrier, such as keyhole limpet hemocyanin (KLH) or mannan (see WO 95 / 18145 and (Longenecker et al., 1993)). The peptides may also be labeled, be fusion proteins, or be hybrid molecules. The peptides whose sequences are described in the present invention are predicted to stimulate CD4 or CD8 T cells. However, stimulation of CD8 T cells is more efficient in the presence of help provided by CD4 T helper cells. Therefore, for MHC class I epitopes that stimulate CD8 T cells, the fusion partner or section of the hybrid molecule suitably provides an epitope that stimulates CD4-positive T cells. CD4 and CD8 stimulating epitopes are well known in the art and include those identified in the present invention.

[0186] In one embodiment, the vaccine comprises at least one peptide having an 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, and 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.

[0187] A further aspect of the present invention provides nucleic acids (e.g., polynucleotides) encoding the peptides or peptide variants of the present invention. The polynucleotide may be, for example, either single-stranded and / or double-stranded DNA, cDNA, PNA, RNA, or a combination thereof, or a native or stabilized form of polynucleotide, such as a polynucleotide having a phosphorothioate backbone, so long as it encodes the peptide, and it may or may not contain introns. Of course, only peptides containing natural amino acid residues linked by naturally occurring peptide bonds may be encoded by the polynucleotide. A still further aspect of the present invention provides expression vectors capable of expressing the polypeptides according to the present invention.

[0188] A variety of methods have been developed for linking polynucleotides, particularly DNA, to vectors via complementary cohesive ends. For example, complementary homopolymer sequences can be added to the DNA segment to be inserted into the vector DNA. Hydrogen bonding between the complementary homopolymer tails then joins the vector and DNA segment to form a recombinant DNA molecule.

[0189] Synthetic linkers containing one or more restriction sites provide an alternative method of joining DNA segments to vectors. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from several sources, including International Biotechnologies Inc., New Haven, Connecticut, USA.

[0190] A desirable method for modifying DNA encoding a polypeptide of the present invention is to use the polymerase chain reaction as disclosed in Saiki RK, et al. (Saiki et al., 1988). This method may be used, for example, to introduce the DNA into a suitable vector by engineering appropriate restriction sites, or it may be used to modify the DNA in other useful ways known in the art. If a viral vector is used, poxvirus or adenovirus vectors are preferred.

[0191] The DNA (or RNA, in the case of retroviral vectors) may then be expressed in a suitable host to produce a polypeptide comprising the peptide or variant of the invention. Thus, DNA encoding the peptide or variant of the invention may be used to construct expression vectors, which are then used to transform suitable host cells for the expression and production of the polypeptides of the invention, according to known techniques, appropriately modified in light of the teachings contained herein. Such techniques include, for example, those disclosed in U.S. Pat. Nos. 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.

[0192] The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend on the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.

[0193] Generally, DNA is inserted into an expression vector such as a plasmid in the appropriate direction and correct reading frame for expression. If necessary, the DNA may be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, and such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all hosts will be transformed by the vector. Therefore, it is necessary to select transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence with any necessary control elements that encodes a selectable trait in transformed cells, such as antibiotic resistance.

[0194] Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

[0195] Host cells transformed with the recombinant DNA of the present invention are then cultured under appropriate conditions known to those of skill in the art, in light of the teachings disclosed herein, for a period of time sufficient to express the polypeptide, which may then be recovered.

[0196] Numerous expression systems are known, including bacteria (e.g., E. coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. Preferably, the expression system may be mammalian cells, such as CHO cells, available from the ATCC Cell Biology Collection.

[0197] Typical mammalian cell vector plasmids for constitutive expression comprise a CMV or SV40 promoter with an appropriate polyA tail and a resistance marker such as neomycin. One example is pSVL, available from Pharmacia, Piscataway, NJ, USA. An example of an inducible mammalian expression vector, pMSG, is also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, generally available from Stratagene Cloning Systems, La Jolla, CA 92037. Plasmids pRS403, pRS404, pRS405, and pRS406 are Yeast Integrating plasmids (YIps) that incorporate the yeast selectable markers HIS3, TRP1, LEU2, and URA3. Plasmid pRS413-416 is a Yeast Centromeric Plasmid (Ycps). CMV promoter-based vectors (e.g., from Sigma-Aldrich) offer transient or stable expression, cytoplasmic or secreted expression, and N- or C-terminal tagging with various combinations of FRAG, 3xFLAG, c-myc, or MAT. These fusion proteins allow recombinant proteins to be detected, purified, and analyzed. Dual-tagged fusions offer versatility in detection.

[0198] The strong human cytomegalovirus (CMV) promoter regulatory region elevates constitutive protein expression levels as high as 1 mg / L in COS cells. In less potent cell lines, protein levels are typically about 0.1 mg / L. The presence of the SV40 origin of replication results in high levels of DNA replication in SV40-permissive COS cells. CMV vectors can contain, for example, the pMB1 (a derivative of pBR322) origin of replication in bacterial cells, a b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and an f1 origin. Vectors containing a preprotrypsin leader (PPT) sequence can direct secretion of FRAG fusion proteins into the culture medium for purification using anti-FRAG antibodies, resins, and plates. Other vectors and expression systems for use with a variety of host cells are well known in the art.

[0199] In another embodiment, two or more peptides or peptide variants of the invention are encoded and thus expressed sequentially (similar to a "beads and beads" construct), whereby the peptides or peptide variants may be linked or fused together by a stretch of linker amino acids, e.g., LLLLLL, or may be linked without any additional peptide between them. These constructs may also be used for cancer therapy and may induce immune responses involving both MHC I and MHC II.

[0200] The present invention also relates to host cells transformed with the polynucleotide vector constructs of the present invention. Host cells can be either prokaryotic or eukaryotic. Bacterial cells may be preferred prokaryotic host cells in some circumstances, typically E. coli strains such as E. coli DH5, available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 (ATCC No. 31343), available from the American Type Culture Collection (ATCC), Rockville, MD, USA. Preferred eukaryotic host cells include yeast, insect, and mammalian cells, preferably vertebrate cells such as those derived from mouse, rat, monkey, or human fibroblast and colon cell lines. Yeast host cells include YPH499, YPH500, and YPH501, publicly available from Stratagene Cloning Systems, La Jolla, CA 92037. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryonic cells NIH / 3T3 available from the ATCC as CRL1658, monkey kidney-derived COS-1 cells available from the ATCC as CRL1650, and human embryonic kidney cells 293. Preferred insect cells are Sf9 cells, which can be transfected with baculovirus expression vectors. Reviews regarding the selection of appropriate host cells for expression can be found, for example, in the textbook "Methods in Molecular Biology: Recombinant Gene Expression, Reviews and Protocols," Part One, Second Edition, ISBN 978-1-58829-262-9 by Paulina Balbas and Argelia Lorence, and other references known to those skilled in the art.

[0201] Transformation of suitable cell hosts with the DNA constructs of the present invention is typically accomplished by well-known methods, which depend on the type of vector used. For the transformation of prokaryotic host cells, see, for example, Cohen et al. (Cohen et al., 1972) and (Green and Sambrook, 2012). Transformation of yeast cells is described in Sherman et al. (Sherman et al., 1986). The method of Beggs (Beggs, 1978) is also useful. For vertebrate cells, reagents useful for transfecting such cells, such as calcium phosphate and DEAE-dextran or liposome preparations, are available from Life Technologies Inc., Gaithersburg, Maryland, 20877. Electroporation is also useful for transforming and / or transfecting cells and is well known in the art for transforming yeast, bacterial, insect, and vertebrate cells.

[0202] Successfully transformed cells, i.e., cells containing the DNA construct of the present invention, can be identified by well-known techniques such as PCR. Alternatively, antibodies can be used to detect the presence of proteins in the supernatant.

[0203] It will be understood that certain host cells of the present invention, such as bacteria, yeast, and insect cells, are useful in preparing the peptides of the present invention. However, other host cells may be useful in certain therapeutic methods. For example, antigen-presenting cells such as dendritic cells may be usefully used to express the peptides of the present invention so that they may be loaded into appropriate MHC molecules. Thus, the present invention provides host cells comprising a nucleic acid or expression vector according to the present invention.

[0204] In a preferred embodiment, the host cell is an antigen-presenting cell, particularly a dendritic cell or an antigen-presenting cell. APC loaded with a recombinant fusion protein containing prostatic acid phosphatase (PAP) was approved by the US Food and Drug Administration (FDA) on April 20, 2010 (sipuleucel-T) for the treatment of asymptomatic or minimally symptomatic metastatic HRPC (Rini et al., 2006; Small et al., 2006).

[0205] A further aspect of the present invention provides a method for producing a peptide or a variant thereof, comprising the steps of culturing a host cell and isolating the peptide from the host cell or its culture medium.

[0206] In another embodiment, the peptides, nucleic acids, or expression vectors of the present invention are used in medical treatment. For example, the peptides or their variants may be formulated for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Preferred methods of peptide injection include sc, id, ip, im, and iv. Preferred methods of DNA injection include id, im, sc, ip, and iv. For example, a dose of 50 μg to 1.5 mg, preferably 125 μg to 500 μg, of peptide or DNA may be administered, depending on the peptide or DNA. Doses in this range have been successfully used in previous clinical trials (Walter et al., 2012).

[0207] The polynucleotides used for active vaccination may be substantially pure or contained in a suitable vector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, or a combination thereof. Methods for designing and introducing such nucleic acids are well known in the art. A review is provided, for example, by Teufel et al. (Teufel et al., 2005). Polynucleotide vaccines are easy to prepare, but the mechanism of action of these vectors in inducing immune responses is not fully understood. Suitable vectors and delivery systems include viral DNA and / or RNA, such as adenovirus, vaccinia virus, retrovirus, herpesvirus, adeno-associated virus, or hybrid-based systems containing components of two or more viruses. Non-viral delivery systems include cationic lipids and cationic polymers, and are well known in the DNA delivery art. Physical delivery, such as via a "gene gun," may also be used. The peptide or peptides encoded by the nucleic acid may be a fusion protein with an epitope that stimulates T cells of the respective reverse CDRs, for example, as described above.

[0208] The medicaments of the present invention may also contain one or more adjuvants. Adjuvants are substances that nonspecifically promote or enhance immune responses (e.g., immune responses to antigens mediated by CD8-positive T cells and helper T (TH) cells) and are therefore considered useful in the medicaments of the present invention. Suitable adjuvants include 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod (ALDARA®), resiquimod, ImuFact®, and the like. IMP321, interleukins such as IL-2, IL-13 and IL-21, interferon-α or -β or their PEGylated derivatives, IS patch, ISS, ISCOMATRIX, ISCOM, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, water-in-oil and Adjuvants include, but are not limited to, oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, poly(lactide-co-glycolide) [PLG]-based and dextran microparticles, talactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Several immunological adjuvants specific for dendritic cells and their preparations (e.g., MF59) have been previously described (Allison and Krummel, 1995). Cytokines may also be used.Several cytokines have been directly implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, the entire contents of which are specifically incorporated herein by reference), and acting as immune enhancers (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich et al., 1996).

[0209] CpG immunostimulatory oligonucleotides have also been reported to enhance adjuvant effects in vaccine settings. Without being bound by theory, CpG oligonucleotides 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 wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced TH1 cell activation and potent cytotoxic T lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar compositions, which is particularly necessary for inducing a strong response when the antigen is relatively weak. They also accelerate immune responses, allowing for a nearly two-order reduction in antigen dose in some experiments with antibody responses equivalent to those of the total vaccine without CpG (Krieg, 2006). U.S. Patent No. 6,406,705 B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. A CpG TLR9 antagonist is dSLIM (double stem-loop immunomodulator) manufactured by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, may also be used.

[0210] Other examples of useful adjuvants include chemically modified CpG (e.g., CpR, Idera); dsRNA analogs such as poly(I:C) and their derivatives (e.g., AmpliGen®, Hiltonol®, poly(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA; and cyclophosphamide, sunitinib, bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Immunoactive small molecules and antibodies, such as Trap, ZD2171, AZD2171, anti-CTLA4, and other antibodies that target critical structures of the immune system (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptor), and SC58175, may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of ordinary skill in the art without undue experimentation.

[0211] Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon alpha, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, and PLG or virosome microparticle formulations.

[0212] In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is selected from the group consisting of colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, and interferon alpha.

[0213] In a preferred embodiment of the pharmaceutical composition according to the present invention, the adjuvant is selected from the group consisting of colony-stimulating factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, and resiquimod. In a preferred embodiment of the pharmaceutical composition according to the present invention, the adjuvant is cyclophosphamide, imiquimod, or resiquimod. Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 20, Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®), and anti-CD40 mAB, or a combination thereof.

[0214] The composition is used for parenteral administration, such as subcutaneous, intradermal, or intramuscular administration, or for oral administration. For this purpose, the peptide and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. The composition may further contain excipients such as buffers, binders, blasting agents, diluents, flavors, lubricants, etc. The peptide may also be administered together with immune stimulants, such as cytokines. A detailed list of excipients that can be used in such compositions can be found, for example, in A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The composition can be used for the prevention, prophylaxis, and / or treatment of adenomatous or cancerous diseases. Exemplary formulations are found, for example, in EP 2112253.

[0215] It is important to understand that the immune response elicited by the vaccine of the present invention attacks cancers at different cell division stages and different developmental stages. Furthermore, different cancer-associated signaling pathways are attacked. This is an advantage over vaccines that address only one or a few targets, sometimes leading to tumors easily adapting to the attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Therefore, combining several tumor-associated peptides ensures that every possible tumor has at least some of the targets. The composition is designed with the expectation that each tumor will express several antigens, covering several independent pathways necessary for tumor growth and maintenance. Therefore, the vaccine can be easily used "off the shelf" for a larger patient population. This means that pre-selection of patients to be treated with the vaccine can be limited to HLA typing and does not require any additional biomarker assessment of antigen expression, yet it is still certain that several targets are simultaneously attacked by the induced immune response, which is important for efficacy (Banchereau et al., 2001; Walter et al., 2012).

[0216] As used herein, the term "scaffold" refers to a molecule that specifically binds to a (e.g., antigenic) determinant. In one embodiment, the scaffold can also direct the entity to which it is attached (e.g., a (second) antigen-binding moiety) to a target site, such as, for example, a specific tumor cell or tumor stroma bearing an antigenic determinant (e.g., a peptide-MHC complex described herein). In another embodiment, the scaffold can activate signaling through its target antigen, such as, for example, a T-cell receptor complex antigen. Scaffolds include, but are not limited to, antibodies and fragments thereof, antibody antigen-binding domains comprising an antibody heavy chain variable region and an antibody light chain variable region, binding proteins comprising at least one ankyrin repeat motif and a single-domain antigen-binding (SDAB) molecule, aptamers, (soluble) TCRs, and (engineered) cells such as allogeneic or autologous T cells. Binding assays can be performed to assess whether a molecule is a scaffold that binds to a target.

[0217] "Specific" binding means that the scaffold binds to the target peptide-MHC complex better than other natural peptide-MHC complexes, so that a scaffold equipped with an active molecule capable of killing cells bearing a specific target cannot kill other cells that do not have the specific target but present other peptide-MHC complexes. If the peptide of the cross-reactive peptide-MHC is not naturally occurring, i.e., not derived from the human HLA peptidome, binding to other peptide-MHC complexes is irrelevant. Tests to evaluate target cell killing are well known in the art. They should be performed using target cells (primary cells or cell lines) with unmodified peptide-MHC presentation, or cells loaded with peptides to reach naturally occurring peptide-MHC levels.

[0218] Each scaffold may comprise a label, which allows the binding scaffold to be detected by determining the presence or absence of a signal provided by the label. For example, the scaffold may be labeled with a fluorescent dye or any other applicable cell marker molecule. Such marker molecules are well known in the art. For example, the fluorescent label provided by a fluorescent dye may allow the binding aptamer to be visualized by fluorescence or laser scanning microscopy or flow cytometry.

[0219] Each scaffold can be conjugated to a second active molecule, such as, for example, IL-21, anti-CD3, and anti-CD28.

[0220] For further information regarding polypeptide scaffolds, see, e.g., the background section of WO 2014 / 071978 A1 and the references cited therein.

[0221] The present invention further relates to aptamer.Aptamer (see, for example, International Publication No. 2014 / 191359 and the documents cited therein) is a short single-stranded nucleic acid molecule, which can fold into a predetermined three-dimensional structure and recognize specific target structure.They have been considered as a suitable alternative for developing targeted therapy.Aptamer has been shown to selectively bind with various complex targets with high affinity and specificity.

[0222] Aptamers that recognize molecules located on cell surfaces have been identified within the past decade, providing a means for developing diagnostic and therapeutic approaches. Aptamers have been shown to be nearly non-toxic and immunogenic, making them promising candidates for biomedical applications. Indeed, aptamers, such as those recognizing prostate-specific membrane antigen (PSMA), have been successfully used for targeted therapy and have been shown to function in xenograft in vivo models. Furthermore, aptamers that recognize specific tumor cell lines have been identified.

[0223] DNA aptamers can be selected to exhibit broad-spectrum recognition properties against various cancer cells, particularly those derived from solid tumors, while not recognizing non-tumorigenic and primary healthy cells. If an identified aptamer not only recognizes a specific tumor subtype but also interacts with a range of tumors, this makes the aptamer applicable as a so-called broad-spectrum diagnostic and therapeutic agent.

[0224] Furthermore, investigation of cell binding behavior by flow cytometry showed that the aptamer exhibited very good apparent affinity in the nanomolar concentration range.

[0225] Aptamers are useful for diagnostic and therapeutic purposes. Furthermore, it has been shown that some aptamers are taken up by tumor cells and can therefore function as molecular vehicles for the targeted delivery of anti-cancer drugs, such as siRNA, into tumor cells.

[0226] Aptamers can be selected using cell-SELEX (extraneous evolution of proteins by evolution in vitro) technology against complex targets such as cells and tissues, and against peptide complexes comprising, preferably consisting of, any of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 101 according to the present invention and an MHC molecule.

[0227] Using the peptides of the present invention, specific antibodies against MHC / peptide complexes can be produced and developed. These can be used for therapeutic purposes to target toxins or radioactive substances to diseased tissues. Another use of these antibodies can be to target radionuclides to diseased tissues for imaging purposes such as PET. This application can help detect small metastases or determine the size and precise location of diseased tissues.

[0228] It is therefore a further aspect of the present invention to provide a method for producing a recombinant antibody that specifically binds to human major histocompatibility complex (MHC) class I or II molecule complexed with an HLA-restricted antigen (preferably a peptide according to the present invention), comprising the steps of: immunizing a genetically engineered non-human mammal comprising cells expressing said human major histocompatibility complex (MHC) class I or II with a soluble form of an MHC class I or II molecule complexed with said HLA-restricted antigen; isolating mRNA molecules from antibody-producing cells of said non-human mammal; creating a phage display library displaying protein molecules encoded by said mRNA molecules; and isolating at least one phage from said phage display library, wherein said at least one phage displays said antibody that specifically binds to said human major histocompatibility complex (MHC) class I or II complexed with said HLA-restricted antigen (preferably a peptide according to the present invention).

[0229] It is therefore a further aspect of the present invention to provide an antibody that specifically binds to human major histocompatibility complex (MHC) class I or II complexed with an HLann-restricted antigen, wherein the antibody is preferably a polyclonal, monoclonal, bispecific and / or chimeric antibody.

[0230] Respective 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 the literature (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), the contents of which are all expressly incorporated by reference in their entirety for the purposes of the present invention.

[0231] Preferably, the antibody binds to the complex with a binding affinity of less than 20 nanomolar, preferably less than 10 nanomolar, which is also considered "specific" in the context of the present invention.

[0232] 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 that is at least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 101, or a variant thereof that cross-reacts T cells with said peptide, wherein said peptide is not the underlying full-length polypeptide.

[0233] The present invention further 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 that is at least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 101, said peptide or variant having an overall length of 8 to 100, preferably 8 to 30, most preferably 8 to 14 amino acids.

[0234] The present invention further relates to a peptide according to the invention, which has the ability to bind to a molecule of the human major histocompatibility complex (MHC) class I or II.

[0235] The present invention further relates to a peptide according to the invention, wherein the peptide consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 101.

[0236] The present invention further relates to a peptide according to the invention, wherein the peptide is (chemically) modified and / or comprises non-peptide bonds.

[0237] The present invention further relates to a peptide according to the 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 the peptide is fused to (or into) an antibody, e.g., a dendritic cell-specific antibody.

[0238] The present invention further relates to nucleic acids encoding peptides according to the invention, with the proviso that the peptide is not a complete (full-length) human protein.

[0239] The present invention further relates to a nucleic acid according to the invention which is DNA, cDNA, PNA, RNA or a combination thereof.

[0240] The present invention further relates to an expression vector capable of expressing a nucleic acid according to the invention.

[0241] The present invention further relates to a peptide according to the invention, a nucleic acid according to the invention or an expression vector according to the invention for use in medicine, in particular in the treatment of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, 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.

[0242] The present invention further relates to a host cell comprising a nucleic acid according to the invention or an expression vector according to the invention.

[0243] The present invention further relates to a host cell according to the invention which is an antigen-presenting cell, preferably a dendritic cell.

[0244] The present invention further relates to a method for producing a peptide according to the invention, comprising the steps of culturing a host cell according to the invention and isolating the peptide from said host cell or its culture medium.

[0245] The present invention further relates to a method according to the invention, wherein a sufficient amount of antigen is contacted with antigen-presenting cells, thereby loading the antigen onto class I or II MHC molecules expressed on the surface of appropriate antigen-presenting cells.

[0246] The present invention further relates to a method according to the present invention, wherein the antigen-presenting cells comprise an expression vector capable of expressing the peptide containing SEQ ID NO: 1 to SEQ ID NO: 101 or the heterologous amino acid sequence.

[0247] The present invention further relates to activated T cells produced by the method according to the invention, said T cells selectively recognizing cells that aberrantly express a polypeptide comprising an amino acid sequence according to the invention.

[0248] The present invention further relates to a method of killing target cells in a patient which aberrantly express a polypeptide comprising any amino acid sequence according to the present invention, comprising the step of administering to the patient an effective number of T cells according to the present invention.

[0249] The present invention further relates to the use of any of the described peptides, nucleic acids according to the invention, expression vectors according to the invention, cells according to the invention or activated cytotoxic T lymphocytes according to the invention as a medicament or in the manufacture of a medicament. The present invention further relates to the use according to the invention, wherein the medicament is effective against cancer.

[0250] The present invention further relates to a use according to the present invention, wherein the medicament is a vaccine.The present invention further relates to a use according to the present invention, wherein the medicament is effective against cancer.

[0251] The invention further relates to the use according to the invention, wherein said cancer cells are 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, or other solid or hematological tumor cells 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.

[0252] The present invention further relates to specific peptide-based labeled proteins and biomarkers according to the present invention, referred to herein as "targets," which may be used in the diagnosis and / or 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 the use of these novel targets for cancer therapy.

[0253] The terms "antibody" or "antibodies" are used broadly herein to include both polyclonal and monoclonal antibodies. In addition to intact or "intact" immunoglobulin molecules, the term "antibody" includes any antibody having the desired properties according to the present invention (e.g., specific binding 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 (poly)peptides; and / or inhibit the activity of a marker polypeptide (e.g., acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, 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).

[0254] Whenever possible, the antibodies of the present invention may be purchased from commercial sources. They may also be produced using well-known methods. Those skilled in the art will understand 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 produce the antibodies of the present invention. The polypeptides used to produce the antibodies of the present invention may be partially or completely purified from natural sources or produced using recombinant DNA technology.

[0255] For example, cDNA encoding a peptide according to the present invention, such as the peptides set forth in SEQ ID NO: 1 to SEQ ID NO: 101 polypeptides; or variants or fragments thereof, can be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, or mammalian cells), and the recombinant protein can then be purified and used to generate antibodies according to the present invention, which can be used to generate monoclonal or polyclonal antibody preparations that specifically bind to acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, 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.

[0256] Those skilled in the art will understand that generating two or more distinct sets of monoclonal or polyclonal antibodies maximizes the likelihood of obtaining antibodies with the specificity and affinity required for their intended use (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy). Antibodies are tested for the desired activity by known methods (e.g., ELISA, immunohistochemistry, immunotherapy, etc.), depending on the purpose for which the antibodies will be used; see, for example, Greenfield, 2014 (Greenfield, 2014) for further guidance on antibody production and testing. For example, antibodies may be tested in ELISA assays, or Western blots, or immunohistochemical staining of formalin-fixed cancer or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested by known clinical testing methods.

[0257] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous antibody population; i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. As used herein, "monoclonal antibody" specifically includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (U.S. Pat. No. 4,816,567, the entire contents of which are incorporated herein by reference).

[0258] Monoclonal antibodies of the invention may be prepared using hybridoma technology. In the hybridoma technology, a mouse or other suitable host animal is typically immunized with an immunizing agent to produce lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0259] Monoclonal antibodies may also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).

[0260] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate antibody fragments, particularly Fab fragments, can be achieved using conventional techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in International Publication No. 94 / 29348 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each having a single antigen-binding site, and a remaining Fc fragment. Pepsin treatment produces an F(ab')2 fragment and a pFc' fragment.

[0261] Antibody fragments, whether attached to other sequences or not, may also contain insertions, deletions, substitutions, or other selected 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 unmodified antibody or antibody fragment. These modifications may provide additional properties, such as removing / adding amino acids capable of disulfide bonding, increasing its biological lifespan, or altering its secretion characteristics. In either case, the antibody fragment must retain biologically active properties, such as binding activity or modulation of binding in the binding region. Functional or active regions of an antibody may be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to skilled practitioners and may include site-directed mutagenesis of nucleic acids encoding the antibody fragment.

[0262] The antibodies of the present invention may further comprise humanized or human antibodies. Humanized forms, such as non-human (e.g., murine) antibodies, are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region.

[0263] Methods for humanizing non-human antibodies are well known in the art. Humanized antibodies typically have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which typically come from an "import" variable domain. Humanization can essentially be performed by substituting rodent CDR(s) or CDR(s) sequences for the corresponding human antibody sequences. Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an entire human variable domain has been substituted 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 substituted by residues from analogous sites in rodent antibodies.

[0264] Transgenic animals (e.g., mice) can be used that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production upon immunization. For example, it has been described that homozygous deletion of antibody heavy chain joining region genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen challenge. Human antibodies can also be generated in phage display libraries.

[0265] The antibody of the present invention is preferably administered to a subject in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmacologically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmacologically acceptable carriers include saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained-release semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles such as films, liposomes, or microparticles. Those skilled in the art will recognize that certain carriers may be more preferable depending, for example, on the route of administration and concentration of the antibody being administered.

[0266] The antibody can be administered to a subject, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular) or by other methods, such as infusion, that ensure delivery to the bloodstream in an effective form. The antibody can also be administered intratumorally or via a peritumoral route to exert a local as well as a systemic therapeutic effect. Local or intravenous injection is preferred.

[0267] Effective dosages and schedules for administering antibodies may be determined empirically, and making such determinations is within the skill of one in the art. Those skilled in the art will understand that the antibody dosage to be administered will vary depending, for example, on the subject receiving the antibody, the route of administration, the particular antibody type used, and other agents administered. A typical daily dosage of an antibody used alone may range from about 1 μg / kg up to 100 mg / kg body weight or more per day, depending on the factors mentioned above. Preferably, the dosage is for treating acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, 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. Following administration of the antibody, the effectiveness of the therapeutic antibody can be assessed by a variety of methods well known to the skilled practitioner. For example, standard tumor imaging techniques may be used to monitor the size, number, and / or distribution of cancer in the treated subject. A therapeutically administered antibody that halts tumor growth, causes tumor shrinkage, and / or prevents the development of new tumors, compared to the disease course that would occur in the absence of antibody administration, is an effective antibody for cancer treatment.

[0268] A further aspect of the present invention provides a method for producing soluble T cell receptors (sTCRs) that recognize specific peptide-MHC complexes. Such soluble T cell receptors can be generated from specific T cell clones, and their affinity can be increased by targeted mutagenesis of complementarity-determining regions. Phage display can be used to select T cell receptors (US Patent Publication No. 2010 / 0113300, (Liddy et al., 2012)). In phage display and for pharmaceutical applications, the α and β chains can be linked by, for example, a non-natural disulfide bond, other covalent bonds (single-chain T cell receptors), or a dimerization domain to stabilize the T cell receptor (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999). T cell receptors can be linked to toxins, drugs, cytokines (see, for example, U.S. Patent No. 2013 / 0115191), and effector cell recruiting domains such as anti-CD3 domains to exert specific functions on target cells. Furthermore, they can be expressed in T cells used for adoptive transfer. Further information can be found in WO 2004 / 033685 A1 and WO 2004 / 074322 A1. The combined use of sTCRs is described in WO 2012 / 056407 A1. Further manufacturing methods are disclosed in WO 2013 / 057586 A1.

[0269] Additionally, the peptides and / or TCRs or antibodies or other binding molecules of the present invention may be used to confirm a pathologist's cancer diagnosis based on a biopsy sample.

[0270] The antibodies or TCRs may also be used for in vivo diagnostic assays. Typically, the antibodies are coupled to radionucleotides (e.g., TCRs) using immunoscintigraphy so that tumors can be localized. 111 In, 99 Tc, 14 C. 131 I, 3 H, 32P or 35 In one embodiment, the antibody or fragment thereof binds to the extracellular domain of two or more targets of proteins selected from the group consisting of the aforementioned proteins with an affinity (Kd) of less than 1 x 10 μM.

[0271] Diagnostic antibodies may be labeled with probes suitable for detection by various imaging methods. Probe detection methods include, but are not limited to, fluorescence, optical, confocal, and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography, and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin, and other fluorophores, radioisotopes, gold, gadolinium, and other lanthanides, paramagnetic iron, fluorine-18, and other positron-emitting radionuclides. Furthermore, probes may be bifunctional or multifunctional, allowing detection by more than one of the listed methods. These antibodies may be directly or indirectly labeled with the probes. Attaching a probe to an antibody, particularly as is well-recognized in the art, includes covalent coupling of the probe, incorporation of the probe into the antibody, and covalent coupling of a chelating compound for probe binding. For immunohistochemistry, diseased tissue samples may be fresh or frozen, or may be paraffin-embedded and fixed in a preservative such as formalin. Fixed or embedded sections containing the sample are contacted with labeled primary and secondary antibodies, and the antibodies are used to detect in situ protein expression.

[0272] Another aspect of the invention includes an in vitro method of generating activated T cells, the method comprising contacting ex vivo T cells with antigen-loaded human MHC molecules expressed on the surface of suitable antigen-presenting cells for a time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide according to the invention. Preferably, a sufficient amount of antigen is used in conjunction with the antigen-presenting cells.

[0273] Preferably, the mammalian cells have no or reduced levels or function of the TAP peptide transporter. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter involved in antigen processing.

[0274] The human peptide loading-deficient cell line T2 is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA, under catalog number CRL1992; the Drosophila cell line Schneider strain 2 is available from the ATCC under catalog number CRL19863; the mouse RMA-S cell line is described in Ljunggren et al. (Ljunggren and Karre, 1985).

[0275] Preferably, before transfer, the host cells do not substantially express MHC class I molecules. It is also preferred that the stimulator cells express molecules important for providing costimulatory signals for T cells, such as B7.1, B7.2, ICAM-1, and LFA3. Nucleic acid sequences of many MHC class I molecules and costimulatory molecules are publicly available from the GenBank and EMBL databases.

[0276] When an MHC class I epitope is used as the antigen, the T cells are CD8 positive T cells.

[0277] When antigen-presenting cells are transfected to express such epitopes, the cells preferably comprise an expression vector capable of expressing a peptide containing SEQ ID NO: 1 to SEQ ID NO: 101, or a variant amino acid sequence thereof.

[0278] Several other methods can be used to generate T cells ex vivo. For example, autologous tumor-infiltrating lymphocytes can be used to generate CTLs. Plebanski et al. (Plebanski et al., 1995) utilized autologous peripheral blood lymphocytes (PLBs) to prepare T cells. Furthermore, autologous T cells can also be generated by pulsing dendritic cells with peptides or polypeptides or infecting them with recombinant viruses. B cells can also be used to produce autologous T cells. Furthermore, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses can be used to prepare autologous CTLs. S. Walter et al. (Walter et al., 2003) described ex vivo priming of T cells using artificial antigen-presenting cells (aAPCs), which is also a suitable method for producing T cells against selected peptides. In the present invention, aAPCs are generated by conjugating preformed MHC:peptide complexes to surface polystyrene particles (microbeads) using biotin:streptavidin biochemistry. This system allows for precise control of MHC density on aAPCs, which allows for selective elicitation of high- or low-avidity antigen-specific T cell responses from blood samples with high efficiency. In addition to MHC:peptide complexes, aAPCs should possess other proteins with costimulatory activity, such as anti-CD28 antibodies, conjugated to their surface. Furthermore, such aAPC-based systems often require the addition of appropriate soluble factors, such as cytokines like interleukin-12.

[0279] Allogeneic cells may also be used in the preparation of T cells, and methods are described in detail in International Publication No. 97 / 26328, which is incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present antigens, such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, and vaccinia-infected target cells. Plant viruses may also be used (see, for example, Porta et al. (Porta et al., 1994), which describes the development of cowpea mosaic virus as a high-yield system for the presentation of foreign peptides).

[0280] Activated T cells directed against the peptides of the invention are useful in therapy. Thus, a further aspect of the invention provides activated T cells obtainable by the methods of the invention described above.

[0281] The activated T cells produced by the above method selectively recognize cells that abnormally express a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 101.

[0282] Preferably, the T cells recognize cells by interaction with (e.g., binding to) an HLA / peptide complex through their TCR. The T cells are useful in a method for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention, to which an effective number of activated T cells are administered. 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 derived from another individual rather than the patient. Of course, it is preferred if the individual is a healthy individual. By "healthy individual," we mean that the individual is generally in good health, preferably has a competent immune system, and more preferably is not suffering from any disease that can be easily tested for and detected.

[0283] In vivo, target cells for CD8-positive T cells according to the present invention may be tumor cells (which sometimes express MHC class II) and / or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC class II; (Dengjel et al., 2006)).

[0284] The T cells of the present invention may be used as an active ingredient in a therapeutic composition. Accordingly, the present invention also provides a method for killing target cells in a patient whose target cells abnormally express a polypeptide comprising an amino acid sequence of the present invention, the method comprising the step of administering to the patient an effective number of T cells as defined above.

[0285] By "abnormally expressed," we also mean that the polypeptide is overexpressed compared to the expression level in normal tissue, or that the gene is silent in the tissue from which the tumor originates, but is expressed in the tumor. By "overexpressed," we mean that the polypeptide is present at a level at least 1.2 times the level present in normal tissue; preferably at least 2 times, more preferably at least 5 or 10 times the level present in normal tissue.

[0286] T cells may be obtained by methods known in the art, such as, for example, those described above.

[0287] Protocols for this so-called adoptive transfer of T cells are well known in the art and are reviewed in Gattinoni et al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006).

[0288] Another aspect of the invention involves the use of peptides that complex with MHC to generate T cell receptors, the nucleic acids of which are cloned and introduced into host cells, preferably T cells. These engineered T cells can then be transferred into patients for cancer treatment.

[0289] Any molecule of the present invention, i.e., peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor, or nucleic acid encoding same, is useful for treating disorders characterized by cells that escape the immune response. Thus, any molecule of the present invention may be used as a medicament or in the manufacture of a medicament. The molecule may be used alone or in combination with other molecules of the present invention or known molecules.

[0290] The present invention provides (a) a container containing the above-described pharmaceutical composition in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; and (c) optionally, (i) instructions for use of the solution, or (ii) instructions for reconstitution and / or use of the lyophilized formulation.

[0013] The present invention is further directed to a kit comprising:

[0291] The kit may further comprise one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe. The container is preferably a bottle, vial, syringe, or test tube; it may be a multi-use container. The pharmaceutical composition is preferably lyophilized.

[0292] The kit of the present invention preferably comprises a lyophilized formulation of the present invention in a suitable container and instructions for its reconstitution and / or use. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (such as dual-chamber syringes), and test tubes. The container may be formed from a variety of materials, such as glass or plastic. Preferably, the kit and / or container includes instructions on or associated with the container, which indicate instructions for reconstitution and / or use. For example, the label may indicate that the lyophilized formulation is to be reconstituted to a peptide concentration as described above. The label may further indicate that the formulation is useful for or intended for subcutaneous administration.

[0293] The container containing the formulation may be a multi-use vial, which allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. The kit may further comprise a second container comprising a suitable diluent (e.g., sodium bicarbonate solution).

[0294] Upon mixing of the diluent and the lyophilized formulation, the final peptide concentration in the reconstituted formulation is preferably at least 0.15 mg / mL / peptide (=75 μg) and preferably no more than 3 mg / mL / peptide (=1500 μg). The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions.

[0295] The kits of the present invention may have a single container containing a pharmaceutical composition formulation according to the present invention, with or without added other components (e.g., other compounds or pharmaceutical compositions of these other compounds), or may have separate containers for each component.

[0296] Preferably, the kits of the invention comprise a formulation of the invention packaged for use in conjunction with the co-administration of a second compound (such as an adjuvant (e.g., GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic agent or inhibitor, an apoptosis inducer or chelator, or a pharmaceutical composition thereof). The kit components may be premixed, or each component may be in a separate, distinct container prior to administration to a patient. The kit components may be provided in one or more liquid solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The kit components may also be provided as solids, which may be converted to a liquid by the addition of a suitable solvent, preferably provided in another, distinct container.

[0297] The container of the therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means for enclosing a solid or liquid. Usually, when there are two or more components, the kit will contain a second vial or another container to allow for separate administration. The kit may also contain another container for a pharmaceutically acceptable liquid. Preferably, the therapeutic kit will contain a device (for example, one or more needles, syringes, eyedroppers, pipettes, etc.) to allow for the administration of the active substance of the present invention that is a component of the kit.

[0298] The formulation is suitable for administration of the peptide by any acceptable means, such as oral (enteral), nasal, ocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably, administration is sc, and most preferably id, and may be by infusion pump.

[0299] Since the peptides of the present invention have been isolated from 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 medicament of the present invention is preferably used to treat 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.

[0300] The present invention further relates to a method for producing a personalized medicine for an individual patient, comprising the step of producing a pharmaceutical composition comprising at least one peptide selected from a reservoir of prescreened TUMAPs, wherein the at least one peptide used in the pharmaceutical composition is selected for its suitability in the individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can also be adapted to produce T cell clones for downstream applications such as TCR isolation, or soluble antibodies, and other therapeutic options.

[0301] "Personalized medicine" shall mean a therapy specifically tailored for an individual patient, used exclusively for the treatment of such individual patient, including proactive personalized cancer vaccines and adoptive cell therapy using autologous patient tissue.

[0302] As used herein, the term "reservoir" refers to a group or set of peptides that have been pre-screened for immunogenicity and / or over-presentation in a particular tumor type. The term "reservoir" is not intended to imply that the particular peptides included in the vaccine are pre-manufactured and stored in a physical facility, although this possibility is contemplated. It is expressly contemplated that peptides may be produced de novo for each personalized vaccine produced, or may be pre-manufactured and stored. The repository (e.g., in the form of a database) is composed of tumor-associated peptides highly overexpressed in tumor tissues 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, who have various HLA-A, HLA-B, and HLA-C alleles. It may contain MHC class I and MHC class II peptides or extended 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'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 tissues, the reservoir also contains HLA-A * 02. HLA-A * 01. HLA-A * 03. HLA-A * 24. HLA-B * 07. HLA-B * 08, and HLA-B *44 marker peptides may be included. These peptides allow quantitative comparison of the magnitude of T cell immunity induced by TUMAPs, thus allowing important conclusions to be drawn about the vaccine's ability to induce anti-tumor responses. Secondly, they serve as important positive control peptides derived from "non-self" antigens in cases where no vaccine-induced T cell response to TUMAPs derived from "self" antigens is observed in patients. Thirdly, they may allow conclusions to be drawn about the patient's immunocompetence status.

[0303] TUMAPs for the reservoir are identified using an integrated functional genomics approach that combines gene expression analysis, mass spectrometry, and T-cell immunology (XPresident®). The approach ensures that only TUMAPs that are truly present on a high percentage of tumors but not or only minimally expressed on normal tissues are selected for further analysis. For initial peptide selection, 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 from patients and blood from healthy donors were analyzed in a stepwise approach: 1. HLA ligands from malignant agents were identified by mass spectrometry 2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis was used to identify genes that are overexpressed in malignant 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'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) compared with a range of normal organs and tissues. 3. The identified HLA ligands were compared with gene expression data. Preferably, peptides that are over- or selectively presented on tumor tissues, encoded by selectively expressed or over-expressed genes as detected in step 2, were considered suitable TUMAP candidates for multi-peptide vaccines. 4. A literature search was conducted to identify additional evidence supporting the validity of the identified peptides as TUMAPs. 5. The association of overexpression at the mRNA level was confirmed by redetection of selected TUMAPs from step 3 on tumor tissues and their absence (or rare) detection in healthy tissues. 6. To assess the ability of selected peptides to induce T cell responses in vivo, 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.

[0304] In one embodiment, peptides are pre-screened for immunogenicity before being included in the reservoir. By way of example and not limitation, the immunogenicity of peptides included in the reservoir is determined by a method comprising ex vivo T cell priming through repeated stimulation of CD8+ T cells from healthy donors with artificial antigen-presenting cells loaded with peptide / MHC complexes and anti-CD28 antibodies.

[0305] This method is preferable for rare cancers and patients with rare expression profiles. In contrast to multiple peptide mixtures with fixed compositions, the currently developed reservoirs allow for significantly higher matching of vaccines with the actual expression of antigens in tumors. A multitarget approach utilizes several "off-the-shelf" peptides, selected individually or in combination, for each patient. In theory, an approach based on the selection of just five different antigenic peptides from a library of 50 antigenic peptides would yield approximately 17 million possible drug product (DP) compositions.

[0306] In one aspect, peptides are selected for inclusion in the vaccine based on their suitability for an individual patient based on the methods according to the invention described herein or as follows.

[0307] HLA phenotype, transcriptomic, and peptidomic data are collected from patient tumor material and blood samples to identify the most relevant peptides for each patient, containing "reservoir" and patient-specific (i.e., mutated) TUMAPs. Peptides are selected that are selectively or overexpressed in the patient's tumor and, if possible, demonstrate strong in vitro immunogenicity when tested with the patient's individual PBMCs.

[0308] Preferably, peptides to be included in the vaccine are identified by a method comprising the steps of: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; (b) comparing the peptides identified in (a) with the peptide repository (database) described above; and (c) selecting at least one peptide from the repository (database) that is related to the tumor-associated peptides identified in the patient. For example, TUMAPs presented by tumor samples are identified by the steps of: (a1) comparing expression data from the tumor sample with expression data from a normal tissue sample corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or aberrantly expressed in the tumor sample; and (a2) correlating the expression data with sequences of MHC ligands that bind to MHC class I and / or class II molecules in the tumor sample to identify MHC ligands derived from proteins overexpressed or aberrantly expressed by the tumor. Preferably, the sequences of the MHC ligands are identified by eluting bound peptides from MHC molecules isolated from the tumor sample and sequencing the eluted ligands. Preferably, the tumor sample and normal tissue are obtained from the same patient.

[0309] In addition to or as an alternative to selecting peptides using a repository (database) model, TUMAPs may be newly identified in patients and then included in vaccines. For example, candidate TUMAPs may be identified in patients by: (a1) comparing expression data from tumor samples with expression data from normal tissue samples corresponding to the tissue type of the tumor sample to identify proteins overexpressed or abnormally expressed in the tumor sample; and (a2) correlating the expression data with the sequences of MHC ligands that bind to MHC class I and / or class II molecules in the tumor sample to identify MHC ligands derived from proteins overexpressed or abnormally expressed by the tumor. For another example, proteins containing mutations specific to tumor samples may be identified by comparing with normal counterpart tissues from individual patients, and TUMAPs specifically targeting the mutations may be identified. For example, the genomes of tumors and corresponding normal tissues may be sequenced by whole genome sequencing. To discover nonsynonymous mutations in the protein-coding regions of genes, genomic DNA and RNA are extracted from tumor tissue, and normal, non-mutated genomic germline DNA is extracted from peripheral blood mononuclear cells (PBMCs). The applied NGS approach is limited to resequencing protein-coding regions (exome resequencing). For this purpose, exonic DNA from human samples is captured using supplier-provided target enrichment kits, followed by sequencing, for example, by HiSeq2000 (Illumina). Additionally, tumor mRNA is sequenced for direct quantification of gene expression and to assess the validity of mutant genes being expressed in the patient's tumor. The resulting millions of sequence reads are processed through software algorithms. The output list contains mutations and gene expression. Tumor-specific somatic mutations are identified and prioritized by comparison with PBMC-derived germline diversity. The newly identified peptides can then be tested for immunogenicity as described above for the reservoir, and candidate TUMAPs that retain appropriate immunogenicity are selected for inclusion in vaccines.

[0310] In one exemplary embodiment, peptides for inclusion in a vaccine are identified by the following steps: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients using the methods described above; (b) comparing the peptides identified in a) with a pool of peptides pre-screened for immunogenicity and over-presentation in tumors compared to matched normal tissue; (c) selecting at least one peptide from the pool 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. In one exemplary embodiment, peptides for inclusion in a vaccine are identified by the following steps: (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.

[0311] Once peptides for a personalized peptide-based vaccine are selected, the vaccine is manufactured as a liquid formulation consisting of individual peptides, preferably dissolved in 20-40% DMSO, such as about 33% DMSO, preferably about 30-35% DMSO.

[0312] Each peptide included in the product is dissolved in DMSO. The concentration of the single peptide solution must be selected depending on the number of peptides included in the product. Equal amounts of the single peptide DMSO solutions are mixed to obtain a solution containing all peptides included in the product at a concentration of approximately 2.5 mg / ml per peptide. The mixed solution is then diluted 1:3 with water for injection to obtain a concentration of 0.826 mg / ml per peptide in 33% DMSO. The diluted solution is filtered through a 0.22 μm sterile filter to obtain the final bulk solution.

[0313] The final bulk solution is filled into vials and stored at -20°C until use. Each vial contains 700 μL of solution containing 0.578 mg of each peptide, of which 500 μL (approximately 400 μg per peptide) is applied for intradermal injection.

[0314] In addition to being useful for treating cancer, the peptides of the present invention are also useful as diagnostic agents. Because the peptides were produced from 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 because these peptides were determined to be absent or present at lower levels in normal tissues, these peptides can be used to diagnose the presence of cancer.

[0315] The presence of the claimed peptides in blood samples or tissue biopsies can assist pathologists in cancer diagnosis. Detection of specific peptides by antibodies, mass spectrometry, or other methods known in the art can tell pathologists that a tissue sample is malignant, inflammatory, or generally pathological, or can be used as a biomarker 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 presence of peptide groups can enable classification or subclassification of diseased tissue.

[0316] The detection of peptides on diseased tissue samples allows the evaluation of the benefits of therapies involving the immune system, especially when T lymphocytes are known or predicted to be involved in the mechanism of action. Loss of MHC expression is a well-described mechanism by which infected malignant cells escape immune surveillance. The presence of peptides therefore indicates that this mechanism is not being utilized by the analyzed cells.

[0317] The peptides of the present invention may be used to analyze lymphocyte responses to these peptides, such as T cell or antibody responses to peptides or peptides complexed with MHC molecules. These lymphocyte responses can be used as prognostic markers to determine further treatment steps. These responses can also be used as surrogate response markers in immunotherapy approaches that aim to induce lymphocyte responses by different means, such as vaccination with proteins, nucleic acids, or autologous materials, or adoptive transfer of lymphocytes. In the setting of gene therapy, lymphocyte responses to peptides can be taken into account in the evaluation of side effects. Monitoring lymphocyte responses may also be a useful tool for follow-up testing of transplantation therapy, for example, for detecting graft-versus-host disease and host-versus-graft disease.

[0318] The present invention will now be described in the following examples which depict preferred embodiments thereof and with reference to the accompanying drawings, in which: For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0319] Figure 1A-F shows the over-representation of various peptides in different cancer tissues (black dots). Top: Median MS signal intensity from technical replicates for a single HLA-A *The normalized signal intensities are plotted as dots for normal (gray dots, left side of the figure) and tumor samples (black dots, right side of the figure) in which the peptide was detected. The boxes represent the median, 25th, and 75th percentiles of normalized signal intensity, while the whiskers extend to the lowest data point within 1.5 interquartile ranges (IQR) of the lower quartile and the highest data point within 1.5 IQR of the upper quartile. Normal organs are ordered by risk category (blood cells, blood vessels, brain, liver, lung: high risk, gray dots; genitals, breast, prostate: low risk, gray dots; all other organs: medium risk, gray dots). Bottom: The relative peptide detection frequency in each organ is shown as a bar graph. The numbers below the panels indicate the number of samples in which the peptide was detected per total number of samples analyzed for each organ (N = 440 normal samples, N = 490 tumor samples). If a peptide was detected in a sample but could not be quantified for technical reasons, the sample is included in this detection frequency display, but no dot is shown at the top of the figure. Tissues (from left to right): Normal sample: blood cells; blood vessels; brain; heart; liver; lung; monocytes; T cells; adipose tissue; adrenal gland; bile duct; bladder; bone marrow; esophagus; eye; gallbladder; head and neck; large intestine; small intestine; kidney; lymph nodes; central nervous system; peripheral nervous system; pancreas; parathyroid gland; peritoneum; pituitary gland; pleura; skeletal muscle; skin; spinal cord; spleen; stomach; thyroid gland; trachea; ureter; breast; ovary; placenta; prostate; testis; thymus; uterus.Tumor samples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocarcinoma); 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's lymphoma); NSCLCadeno (non-small cell lung adenocarcinoma); NSCLCother (NSLC samples that could not be clearly assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung carcinoma); 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 carcinoma). [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]Overrepresentation of various peptides in different cancer tissues is shown (black dots). Top: Median MS signal intensity from technical replicates is plotted as dots for single HLA-A*03-positive normals (gray dots, left side of figure) and tumor samples on which peptides were detected (black dots, right side of figure). Boxes represent the median, 25th, and 75th percentiles of normalized signal intensity, while whiskers extend to the lowest data point within 1.5 interquartile ranges (IQR) of the lower quartile and the highest data point within 1.5 IQR of the upper quartile. Normal organs are ordered by risk category (blood cells, blood vessels, brain, liver, lungs: high risk, gray dots; genitals, breast, prostate: low risk, gray dots; all other organs: medium risk, gray dots). Bottom: Relative peptide detection frequency in each organ is shown as a bar graph. The numbers below the panels indicate the number of samples in which the peptide was detected per total number of samples analyzed for each organ (N = 36 normal samples, N = 107 tumor samples). If a peptide was detected in a sample but could not be quantified for technical reasons, the sample is included in this detection frequency display but no dot is shown at the top of the figure. Tissues (from left to right): Normal samples: blood cells; blood vessels; brain; heart; liver; lung; adrenal gland; bladder; gallbladder; intestinal tract; lymph nodes; pancreas; skin; spleen; trachea. Tumor samples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocarcinoma); 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's lymphoma); NSCLCadeno (non-small cell lung adenocarcinoma); NSCLCother (NSLC samples that could not be clearly assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung carcinoma); 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 carcinoma).Figure 1F peptide: SLLGAATVEPPK (SEQ ID NO: 81; A*03). Figures 2A-2F show exemplary expression profiles of the inventive gene of origin overexpressed in different cancer samples. Tumor (black dots) and normal (gray dots) samples are grouped by organ of origin. Box plots represent the median FPKM value, the 25th and 75th percentiles (boxes), plus whiskers extending to the lowest data point still within 1.5 interquartile ranges (IQR) of the lower quartile, and the highest data point still within 1.5 IQR of the upper quartile. Normal organs are ordered by risk category. FPKM: fragments per kilobase per million mapped reads. Tissues (from left to right): Normal sample: Blood cells; Blood vessels (blood vessels); Brain; Heart; Liver; Lung; Adipose (adipose tissue); Adrenal gland (adrenal gland); Bile duct; Bladder; Bone marrow; Esophagus; Eye; Gallbladder; Head and neck; Intestinal (large intestine); Intestinal (small intestine); Kidney; Lymph node; Nerve perith (peripheral nerve); pancreas; parathyroid gland (parathyroid gland); perit (peritoneum); pituitary gland); pleura; skeletal muscle (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 (cholangiocarcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); HCC (hepatocellular carcinoma); HNSCC (head and neck cancer) epithelial carcinoma); MEL (melanoma); NHL (non-Hodgkin's lymphoma); NSCLCadeno (non-small cell lung adenocarcinoma); NSCLCother (NSLC samples that could not be clearly assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung carcinoma); 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 carcinoma). [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). [Figure 3] 1 shows exemplary results of peptide-specific in vitro CD8+ T cell responses from a healthy HLA-A*02+ donor. CD8+ T cells were primed using anti-CD28 mAb complexed with SEQ ID NO: 102 peptide (GLDPTQFRV, peptide code: POLA1-003) (A, left panel) and SEQ ID NO: 103 peptide (SLVSYLDKV, peptide code: KRT16P-001) (B, left panel) and artificial APCs coated with HLA-A*02. After three cycles of stimulation, detection of peptide-reactive cells was performed by 2D multimer staining with A*02 / SEQ ID NO: 102 (A) and A*02 / SEQ ID NO: 103 (B). The right panels (A and B) show control staining of cells stimulated with an irrelevant A*02 / peptide complex. Viable singlet cells were gated for CD8+ lymphocytes. Boolean gating helped eliminate false positive events detected by multimers specific to different peptides. The frequency of specific multimer+ cells among CD8+ lymphocytes is shown. [Figure 4-1]1 shows exemplary results of peptide-specific in vitro CD8+ T cell responses from a healthy HLA-A*02+ donor. CD8+ T cells were primed using anti-CD28 mAb complexed with SEQ ID NO:18 peptide (KMMTFFQGL) (A, left panel), SEQ ID NO:68 peptide (KLLADAFKV) (B, left panel), SEQ ID NO:40 peptide (FTLPFLVNL) (C, left panel), SEQ ID NO:19 peptide (MLLPWLPKL) (D, left panel), or SEQ ID NO:48 peptide (MLAEIHPKA) (E, left panel) and artificial APCs coated with HLA-A*02. After three cycles of stimulation, detection of peptide-reactive cells was performed by 2D multimer staining with A*02 / SEQ ID NO:18 (A), A*02 / SEQ ID NO:68 (B), A*02 / SEQ ID NO:40 (C), A*02 / SEQ ID NO:19 (D), or A*02 / SEQ ID NO:48 (E). The right panels (A, B, C, D, E, and F) show control staining of cells stimulated with an irrelevant A*02 / peptide complex. Viable single cells were gated on CD8+ lymphocytes. Boolean gating helped eliminate false-positive events detected by multimers specific for different peptides. The frequency of specific multimer+ cells among CD8+ lymphocytes is shown. [Figure 4-2] Same as above [Figure 4-3] Same as above [Example]

[0320] Example 1 Identification and quantification of tumor-associated peptides displayed on the cell surface Tissue samples Patient tumor tissues were obtained from Asterand (Detroit, Michigan, USA and Royston, Hertfordshire, UK); Bio-Options Inc. (Brea, California, USA); Geneticist Inc. (Glendale, California, USA); University Hospital Heidelberg (Heidelberg, Germany); ProteoGenex Inc. (Culver City, California, USA); Tissue Solutions Ltd (Glasgow, UK); and University Hospital Munich (Munich, Germany). Normal tissues were obtained from Asterand (Detroit, Michigan, USA and Royston, Hertfordshire, UK); Bio-Options Inc. (Brea, California, USA); BioServe (Beltsville, Maryland, USA); Capital BioScience Inc. (Rockville, Maryland, USA); Centre for Clinical Transfusion Medicine Tuebingen (Tübingen, Germany); Geneticist Inc. (Glendale, California, USA); Kyoto Prefectural University of Medicine (KPUM) (Kyoto, Japan); Osaka City University (OCU) (Osaka, Japan); ProteoGenex Inc. (Culver City, California, USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva (Geneva, Switzerland); University Hospital Heidelberg (Heidelberg, Germany); University Hospital Tubingen (Tübingen, Germany); and University Hospital Munich (Munich, Germany).

[0321] All patients gave informed consent before surgery or autopsy. Tissues were shock-frozen immediately after resection and stored below -70°C until TUMAP isolation.

[0322] Isolation of HLA peptides from tissue samples HLA peptide pools from shock-frozen tissue samples were prepared by HLA-A peptide pooling according to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999). * The O2-specific antibody BB7.2, the HLA-A, -B, -C-specific antibody W6 / 32, the HLA-DR-specific antibody L243, and the HLA DP-specific antibody B7 / 21 were obtained from solid tissue by immunoprecipitation using CNBr-activated Sepharose, acid treatment, and ultrafiltration.

[0323] mass spectrometry The resulting HLA peptide pool was separated according to their hydrophobicity by reversed-phase chromatography (nanoAcquity UPL C system, Waters), and the eluted peptides were analyzed in an LTQ-velos and fusion hybrid mass spectrometer (ThermoElectron) equipped with an ESI source. The peptide pool was directly loaded onto an analytical fused silica microcapillary column (75 μm i.d. × 250 mm) packed with 1.7 μm C18 reversed-phase material (Waters) at a flow rate of 400 nL / min. Peptides were subsequently separated using a two-step 180-min binary gradient from 10% to 33% B at a flow rate of 300 nL / min. The gradient consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Gold-coated glass capillaries (PicoTip, New Objective) were used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode using the TOP5 strategy. Briefly, a scan cycle began with a high-mass-accuracy full scan in the Orbitrap (R = 30,000), followed by an MS / MS scan of the five most abundant precursor ions, also in the Orbitrap (R = 7,500), with dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted with a fixed false detection rate (q ≤ 0.05) and additional manual controls, or by SEQUEST. In cases where the identified peptide sequence was uncertain, it was further verified by comparison of the generated native peptide fragmentation pattern with that of a synthetic sequence-identical reference peptide.

[0324] Label-free relative LC-MS quantification was performed by ion counting, i.e., by extraction and analysis of LC-MS features (Mueller et al., 2007). This method assumes that the LC-MS signal area of ​​a peptide correlates with 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 tissue-to-peptide presentation profiles. Quantitative data were normalized using a two-stage method according to central tendency, which accounts for technical and biological intra-replicate variability. In this way, each identified peptide can be correlated with quantitative data, allowing for relative quantification between samples and tissues. Furthermore, all quantitative data obtained for peptide candidates was manually inspected to ensure data consistency and confirm the accuracy of the automated analysis. Presentation profiles were calculated for each peptide, showing the average sample presentation as well as replicate variability. The profiles juxtapose baseline normal tissue samples 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 samples. The presentation profile of an exemplary over-presented peptide is shown in Figure 1. Peptide presentation on tumors for exemplary peptides is shown in Table 11.

[0325] Table 11 shows the presentation of various cancer entities for selected peptides and therefore indicates the specific relevance of the mentioned peptides with respect to 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).

[0326] [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

[0327] Example 2 Expression profiling of genes encoding the peptides of the present invention The over- or specific expression of peptides on tumor cells compared to normal cells is sufficient for their usefulness in immunotherapy, and some peptides are tumor-specific even though their origin proteins are also present in normal tissues. Nevertheless, mRNA expression profiling can increase the level of safety in selecting peptide targets for immunotherapy. In particular, for therapeutic options with high safety risks, such as affinity-matured TCRs, ideal target peptides would be derived from proteins that are specific to tumors and not found on normal tissues.

[0328] RNA origin and preparation Surgically removed tissue specimens were provided as described above after informed consent was obtained from each patient (see Example 1). Tumor tissue specimens were snap-frozen immediately after surgery and then homogenized using a mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRI Reagent (Ambion, Darmstadt, Germany), followed by purification with RNeasy (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol.

[0329] Total RNA from healthy human tissues for RNASeq experiments was obtained from Asterand (Detroit, MI, USA and Royston, Hertfordshire, UK); Bio-Options Inc. (Brea, CA, USA); Geneticist Inc. (Glendale, CA, USA); ProteoGenex Inc. (Culver City, CA, USA); and Tissue Solutions Ltd (Glasgow, UK).

[0330] Total RNA from tumor tissues for RNA-Seq experiments was obtained from Asterand (Detroit, Michigan, USA and Royston, Hertfordshire, UK); BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, Maryland, USA); Geneticist Inc. (Glendale, California, USA); Istituto Nazionale Tumori "Pascale" (Naples, Italy); ProteoGenex Inc. (Culver City, California, USA); and University Hospital Heidelberg (Heidelberg, Germany).

[0331] The quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent) using the RNA 6000 Pico LabChip kit (Agilent, Waldbronn, Germany).

[0332] RNAseq experiments Gene expression analysis of tumor and normal tissue RNA samples was performed by next-generation sequencing (RNAseq) at CeGaT (Tübingen, Germany). Briefly, sequencing libraries were generated using the Illumina HiSeq v4 reagent kit, which includes RNA fragmentation, cDNA conversion, and the addition of sequencing adapters, according to the vendor's protocol (Illumina Inc., San Diego, CA, USA). Libraries from multiple samples were mixed equimolarly and sequenced on an Illumina HiSeq 2500 sequencing instrument according to the manufacturer's instructions, generating 50-bp single-end reads. The processed reads were mapped to the human genome (GRCh38) using STAR software. Expression data are provided at the transcript level as RPKM (Reads Per Kilobase per Million mapped reads, generated by the software Cufflinks) and at the exon level (total reads, generated by the software Bedtools) based on annotations from the Ensembl sequence database (Ensembl77). Exon reads are normalized for exon length and alignment size to obtain RPKM values.

[0333] Exemplary expression profiles of source genes of the 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, squamous cell carcinoma of the head and neck, 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. Expression scores of additional exemplary genes are shown in Table 12.

[0334] [Table 12-1] [Table 12-2]

[0335] Example 3 In vitro immunogenicity of MHC class I-presented peptides To obtain information about the immunogenicity of the TUMAPs of the present invention, the inventors carried out studies using an in vitro T cell priming assay based on repeated stimulation of CD8+ T cells with artificial antigen-presenting cells (aAPCs) loaded with peptide / MHC complexes and anti-CD28 antibodies. In this way, the inventors demonstrated that the HLA-A * The immunogenicity of 02:01-restricted TUMAPs could be demonstrated, demonstrating that these peptides are T cell epitopes against which CD8+ precursor T cells are present in humans (Tables 13a and 13b).

[0336] In vitro priming of CD8+ T cells To perform ex vivo stimulation with artificial antigen-presenting cells loaded with peptide-MHC complexes (pMHC) and anti-CD28 antibodies, we first isolated fresh HLA-A antigens through positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) from healthy donors obtained from the University Clinics Mannheim, Germany, after informed consent. * CD8+ T cells were isolated from the 02 leukapheresis product.

[0337] PBMCs and isolated CD8+ lymphocytes or PBMCs were incubated in T cell medium (TCM) consisting of 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, Oberdürer, Germany), and 20 μg / ml gentamicin (Cambrex) until use. 2.5 ng / ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U / ml IL-2 (Novartis Pharma, Nuremberg, Germany) were also added to the TCM at this stage.

[0338] Generation of pMHC / anti-CD28 coated beads, T cell stimulation, and readout were performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and eight different pMHC molecules per readout condition.

[0339] Purified costimulatory mouse IgG2a anti-human CD28 Ab9.3 (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 diameter streptavidin-coated polystyrene particles (Bangs Laboratories, IL, USA).

[0340] The pMHC used for positive and negative control stimulation were A, respectively. * 0201 / MLA-001 (peptide ELAGIGILTV (SEQ ID NO: 104) derived from modified Melan-A / MART-1) and A * 0201 / DDX5-001 (YLLPAIVHI derived from DDX5, SEQ ID NO: 105).

[0341] 800,000 beads / 200 μl were coated in a 96-well plate in the presence of 4 × 12.5 ng of different biotin pMHC, washed, and subsequently added with 600 ng of biotin anti-CD28 in a volume of 200 μl. 1 × 10 beads were cultured in 200 μl of TCM supplemented with 5 ng / ml IL-12 (PromoCell). 6 2 × 10 CD8+ T cells 5 Stimulation was initiated in 96-well plates by co-incubation with 1000 washed coated beads at 37°C for 3 days. Half of the medium was then replaced with fresh TCM supplemented with 80 U / ml IL-2, and incubation continued at 37°C for 4 days. This stimulation cycle was performed a total of three times. For pMHC multimer readout using eight different pMHC molecules per condition, a two-dimensional combinatorial coding approach was used as previously described (Andersen et al., 2012) with minor modifications, including conjugation with five different fluorescent dyes. Finally, multimer analysis was performed by staining cells with Live / Dead near-infrared dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany), and fluorescent pMHC multimers. A BD LSRII SORP hemocytometer equipped with the appropriate lasers and filters was used for analysis. Peptide-specific cells were calculated as a percentage of total CD8+ cells. Multimer analysis was evaluated using FlowJo software (Tree Star, Oregon, USA). Specific multimer+ CD8+ lymphocytes were detected by in vitro stimulation compared with negative control stimulation. Immunogenicity of a given antigen was detected if at least one evaluable in vitro stimulation well from a single healthy donor was found to contain specific CD8+ T cell lines after in vitro stimulation (i.e., this well contained at least 1% specific multimer+ among CD8+ T cells, and the percentage of specific multimer+ cells was at least 10-fold higher than the median value of the negative control stimulation).

[0342] In vitro immunogenicity of peptides 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 For the HLA class I peptides tested, in vitro immunogenicity could be demonstrated by generation of peptide-specific T cell lines. Exemplary flow cytometry results after TUMAP-specific multimer staining for seven peptides of the invention, along with corresponding negative controls, are shown in Figures 3A and 3B and 4A-E. Results for 30 peptides from the invention are summarized in Tables 13a and 13b.

[0343] [Table 13a]

[0344] [Table 13b]

[0345] Example 4 Peptide synthesis All peptides were synthesized using standard, well-established solid-phase peptide synthesis using the Fmoc strategy. The identity and purity of individual peptides were determined by mass spectrometry and analytical RP-HPLC. Peptides were obtained as white to off-white lyophilizates (trifluoroacetate salts) with purity >50%. All TUMAPs are preferably administered as trifluoroacetate or acetate salts, although other salt forms are also possible.

[0346] Example 5 MHC binding assay Candidate peptides for T cell-based therapy according to the present invention were further tested for their MHC binding ability (affinity). The results for 79 peptides from the present invention are summarized in Table 14.

[0347] Individual peptide-MHC complexes were generated by UV-ligand exchange, in which UV-sensitive peptides were cleaved upon UV irradiation and exchanged with the peptide of interest to be analyzed. Only peptide candidates that could effectively bind and stabilize peptide-receptor MHC molecules prevented dissociation of the MHC complex. To determine the yield of the exchange reaction, an ELISA based on detection of the light chain (β2m) of the stabilized MHC complex was performed. The assay was performed as generally described in Rodenko et al. (Rodenko et al., 2006).

[0348] 96-well MAXISorp plates (NUNC) were coated with 2 μg / ml streptavidin in PBS overnight at room temperature, washed four times, and blocked in 2% BSA containing blocking buffer for 1 hour at 37°C. * 02:01 / MLA-001 monomer served as a standard covering a range of 15–500 ng / ml. The peptide-MHC monomer for UV exchange reactions was diluted 100-fold in blocking buffer. Samples were incubated for 1 h at 37°C, washed four times, incubated with 2 μg / ml HRP-conjugated anti-β2m for 1 h at 37°C, washed again, and detected with TMB solution quenched with NH2SO4. Absorbance was measured at 450 nm. For the generation and manufacturing of antibodies or their fragments, and / or T cell receptors or their fragments, candidate peptides that exhibit high exchange yields (preferably greater than 50%, most preferably greater than 75%) are generally preferred because they exhibit sufficient binding activity to MHC molecules and prevent dissociation of the MHC complex.

[0349] [Table 14-1] [Table 14-2] [Table 14-3]

[0350] Example 6 Absolute quantification of tumor-associated peptides displayed on the cell surface The production of binders such as antibodies and / or TCRs is a laborious process and may only be performed for a few selected targets. For tumor-associated and specific peptides, selection criteria include, but are not limited to, the exclusivity of presentation and the density of peptides presented on the cell surface. In addition to the peptide isolation and relative quantification described in Example 1, the inventors analyzed the absolute peptide copy number per cell as described in WO 2016 / 107740. Quantifying TUMAP copies per cell in solid tumor samples requires absolute quantification of the isolated TUMAPs, the efficiency of the TUMAP isolation process, and cell counting of the tissue sample being analyzed.

[0351] Peptide quantification by nanoLC-MS / MS For accurate peptide quantification by mass spectrometry, calibration curves were constructed for each individual peptide using two different isotopically labeled peptide variants (one or two isotopically labeled amino acids were included during TUMAP synthesis). These isotopically labeled variants differ from tumor-associated peptides only in their mass but do not exhibit other differences in physicochemical properties (Anderson et al., 2012). For peptide calibration curves, a series of nanoLC-MS / MS measurements were performed to determine the ratio of MS / MS signals of titrated (single-isotopically labeled peptide) to constant (dual-isotopically labeled peptide) isotopically labeled peptides.

[0352] A dual-isotopically labeled peptide, also referred to as an internal standard, was additionally spiked into each MS sample, and all MS signals were normalized to that of the internal standard to level out potential technical variance between MS experiments.

[0353] Calibration curves were generated in at least three different matrices, i.e., in HLA peptide eluates from natural samples similar to routine MS samples, and each preparation was measured in duplicate MS experiments. For evaluation, MS signals were normalized to those of the internal standard, and calibration curves were calculated by logistic regression.

[0354] For quantification of tumor-associated peptides from tissue samples, an internal standard was also added to each sample; MS signals were normalized to the internal standard and quantified using a peptide calibration curve.

[0355] Efficiency of peptide / MHC isolation As with any protein purification process, isolation of proteins from tissue samples involves some loss of target protein. To determine the efficiency of TUMAP isolation, peptide / MHC complexes were generated for all TUMAPs selected for absolute quantification. To distinguish these complexes from native peptide / MHC complexes, monoisotopically labeled versions of TUMAPs were used; i.e., one isotope-labeled amino acid was included in the TUMAP synthesis. These complexes were added to freshly prepared tissue lysates as early as possible in the TUMAP isolation procedure and then captured like native peptide / MHC complexes in the following affinity purification. Therefore, measuring the recovery of monoisotope-labeled TUMAPs allows conclusions regarding the isolation efficiency of individual native TUMAPs.

[0356] Isolation efficiency was analyzed in a small number of samples and was comparable across these tissue samples. In contrast, isolation efficiency varied between individual peptides. This suggests that isolation efficiency, although determined only in a limited number of tissue samples, may be extrapolated to any other tissue specimen. However, because isolation efficiency may not extrapolate from one peptide to another, each TUMAP must be analyzed individually.

[0357] Cell counting in solid-frozen tissue To measure cell numbers in tissue samples subjected to absolute peptide quantification, we applied DNA content analysis. This method is applicable to a wide range of samples of different origins, most importantly, frozen samples (Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During the peptide isolation protocol, tissue samples are processed into homogenous lysates, from which small lysate aliquots are taken. The aliquots are divided into three portions, from which DNA is isolated (QiaAmp DNA MiniKit, Qiagen, Hilden, Germany). Total DNA content from each DNA isolation is quantified in at least two replicates using a fluorescence-based DNA quantification assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany).

[0358] To calculate cell number, a DNA standard curve was generated from aliquots of healthy blood cells isolated from multiple donors over a defined cell number range. The standard curve was used to calculate total cell content from the total DNA content from each DNA isolate. The average total cell number 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.

[0359] Peptide copy number per cell Using the data from the previous experiments, we calculated the TUMAP copy number per cell by dividing the total peptide amount in the sample by the total cell number, and then dividing by the isolation efficiency. The cellular copy numbers of selected peptides are shown in Table 15.

[0360] [Table 15]

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Claims

1. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

2. 2. The peptide of claim 1, which has the ability to bind to a major histocompatibility complex (MHC) class I molecule and, when bound to the MHC, becomes capable of being recognized by CD8 T cells.

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

4. A fusion protein comprising a peptide according to any one of claims 1 to 3 fused to a fragment of the HLA-DR antigen-associated invariant chain (Ii), the fragment comprising the 80 amino acids closest to the N-terminus of the HLA-DR antigen-associated invariant chain (Ii).

5. An antibody or a fragment thereof which specifically binds to a peptide according to any one of claims 1 to 3, bound to an MHC molecule, or which specifically binds to a peptide according to any one of claims 1 to 3.

6. A T cell receptor (TCR) or a fragment thereof that reacts with an HLA ligand, wherein the HLA ligand is a peptide according to any one of claims 1 to 3 bound to an MHC molecule, or the HLA ligand is a peptide according to any one of claims 1 to 3.

7. the T cell receptor is provided as a soluble molecule, or The T cell receptor is provided as a soluble molecule, carrying additional effector functions, immunostimulatory domains or toxins. The T cell receptor of claim 6.

8. A nucleic acid encoding the peptide according to any one of claims 1 to 3, the antibody or fragment thereof according to claim 5, or the T cell receptor or fragment thereof according to claim 6, or A nucleic acid encoding the peptide of any one of claims 1 to 3, the antibody or fragment thereof of claim 5, or the T cell receptor or fragment thereof of claim 6, wherein the nucleic acid is linked to a heterologous promoter sequence.

9. An expression vector comprising the nucleic acid of claim 8.

10. A recombinant host cell comprising the peptide according to any one of claims 1 to 3, the fusion protein according to claim 4, the antibody or fragment thereof according to claim 5, the T cell receptor or fragment thereof according to claim 6, or the nucleic acid according to claim 8, or the expression vector according to claim 9, or 10. A recombinant host cell comprising the peptide of any one of claims 1 to 3, the fusion protein of claim 4, the antibody or fragment thereof of claim 5, the T cell receptor or fragment thereof of claim 6, the nucleic acid of claim 8, or the expression vector of claim 9, wherein the host cell is selected from a dendritic cell, a T cell, a NK cell, or an antigen-presenting cell.

11. 10. An in vitro method for producing activated T lymphocytes, comprising ex vivo contacting T cells with antigen-loaded human class I MHC molecules expressed on the surface of a suitable antigen-presenting cell or on the surface of an artificial construct that mimics an antigen-presenting cell, for a time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide described in claim 1 or 2.

12. Activated T lymphocytes that selectively recognize cells that present the peptide of claim 1.

13. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of peptides according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient and / or stabilizer.

14. the pharmaceutical composition further comprises an adjuvant, or the pharmaceutical composition further comprises an adjuvant, wherein the adjuvant is an interleukin; or the pharmaceutical composition further comprises an adjuvant, the adjuvant being an interleukin, the interleukin being IL-2 and / or IL-15; The pharmaceutical composition of claim 13.

15. A method for producing the peptide of any one of claims 1 to 3, the fusion protein of claim 4, the antibody or fragment thereof of claim 5, or the T cell receptor or fragment thereof of claim 6, comprising the steps of culturing the host cell of claim 10, and isolating the peptide, fusion protein, antibody or fragment thereof, or T cell receptor or fragment thereof from the host cell and / or its culture medium.

16. A drug comprising the peptide according to any one of claims 1 to 3, the fusion protein according to claim 4, or the activated T lymphocyte according to claim 12; or A peptide according to any one of claims 1 to 3, a fusion protein according to claim 4, or an activated T lymphocyte according to claim 12. A drug for treating a cancer selected from the group consisting of 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, and endometrial cancer.

17. 13. A drug comprising an activated T lymphocyte according to claim 12 for killing a target cell presenting a peptide according to claim 1 or 2.

18. Use of a peptide described in any one of claims 1 to 3, a fusion protein described in claim 4, or an activated T lymphocyte described in claim 12 in the manufacture of a medicament for a cancer selected from the group consisting of 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, and endometrial cancer.

19. The use according to claim 18, wherein the cancer is a cancer selected from tumors that exhibit over-expression of a peptide consisting of the amino acid sequence shown in SEQ ID NO:

2.

20. (a) a container containing a pharmaceutical composition comprising the peptide or salt thereof according to any one of claims 1 to 3, the fusion protein according to claim 4, or the activated T lymphocytes according to claim 12, in solution or lyophilized form; and the following groups: (b) a second container containing a diluent or reconstitution solution for the pharmaceutical composition in lyophilized form; and (c) instructions for (i) use of the solution, or (ii) reconstitution and / or use of the pharmaceutical composition in lyophilized form; The kit further comprises one or more selected from:

21. 21. The kit of claim 20, further comprising one or more of: (i) a buffer, (ii) a diluent, (iii) a filter, (iv) a needle, (v) a syringe, or (vi) an adjuvant.

22. 13. An in vitro method for killing cancer cells, wherein the cancer cells present a peptide according to claim 1 or 2, the method comprising contacting the cancer cells with an activated T lymphocyte according to claim 12.

23. 1. An in vitro method for aiding in the diagnosis of cancer in a sample comprising cells from a subject, comprising: The method comprises: (a) adding an antibody or T cell receptor to a tissue sample from a subject, wherein the antibody or T cell receptor is labeled with a probe or radionucleotide; and (b) detecting binding of an antibody or T cell receptor to cells derived from the subject's tissue sample, thereby identifying the presentation of a tumor-associated peptide (TUMAP) on the cells derived from the subject's tissue sample, wherein the TUMAP comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO:2; Including, The antibody, antibody fragment, or T cell receptor (i) the TUMAP comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2; or (ii) the TUMAP comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, bound to an MHC molecule; specifically recognizes method.

24. 24. The in vitro method of claim 22 or 23, wherein the cancer is selected from the group consisting of 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, and endometrial cancer.

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