Immunotherapy with b*07 restricted peptides and combination of peptides against cancers and related methods

Abstract

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention 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 to stimulate T cells ex vivo and transfer 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.

1 . A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 383 and SEQ ID NO. 448 to SEQ ID NO. 499, and variant sequences thereof which are at least 88% homologous to SEQ ID NO. 1 to SEQ ID NO. 383 and SEQ ID NO. 448 to SEQ ID NO. 499, and wherein said variant binds to molecule(s) of the major histocompatibility complex (MHC) and/or induces T cells cross-reacting with said variant peptide; and a pharmaceutical acceptable salt thereof, wherein said peptide is up to 16 amino acids in length. 
     
     
         2 . The peptide or variant according to  claim 1 , wherein said peptide has the ability to bind to an MHC class-I or -II molecule, and wherein said peptide, when bound to said MHC, is capable of being recognized by CD4 and/or CD8  T cells. 
     
     
         3 . The peptide or variant thereof according to  claim 1 , wherein the amino acid sequence thereof comprises a continuous stretch of amino acids according to any one of SEQ ID NO: 1 to SEQ ID NO: 383 and SEQ ID NO: 448 to SEQ ID NO: 499. 
     
     
         4 . The peptide or variant thereof according to  claim 1 , wherein said peptide or variant thereof has an overall length of from 8 to 30, and optionally from 8 to 16 amino acids, optionally wherein the peptide consists or consists essentially of an amino acid sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 383 and SEQ ID NO: 448 to SEQ ID NO: 499. 
     
     
         5 . The peptide or variant thereof according to  claim 1 , wherein said peptide is modified and/or includes non-peptide bonds. 
     
     
         6 . The peptide or variant thereof according to  claim 1 , wherein said peptide is part of a fusion protein, optionally comprising N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii). 
     
     
         7 . An antibody, optionally a soluble or membrane-bound antibody, optionally a monoclonal antibody or fragment thereof, that specifically recognizes said peptide or variant thereof of  claim 1 , optionally the peptide or variant thereof when bound to an MHC molecule. 
     
     
         8 . A T-cell receptor, optionally soluble or membrane-bound, or a fragment thereof, that is reactive with an HLA ligand, wherein said ligand is the peptide or variant thereof according to  claim 1 , optionally the peptide or variant thereof when bound to an MHC molecule. 
     
     
         9 . The T-cell receptor according to  claim 8 , wherein said ligand amino acid sequence is at least 88% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 383 and SEQ ID NO: 448 to SEQ ID NO: 499, or wherein said ligand amino acid sequence consists of any one of SEQ ID NO: 1 to SEQ ID NO: 383 and SEQ ID NO: 448 to SEQ ID NO: 499. 
     
     
         10 . The T-cell receptor according to  claim 8 , wherein said T-cell receptor is provided as a soluble molecule and optionally carries a further effector function optionally an immune stimulating domain or toxin. 
     
     
         11 . An aptamer that specifically recognizes the peptide or variant thereof according to  claim 1 , optionally the peptide or variant thereof that is bound to an MHC molecule. 
     
     
         12 . A nucleic acid, encoding for a peptide or variant thereof according to  claim 1 , an antibody or fragment thereof that specifically recognizes said peptide or variant, a T-cell receptor or fragment thereof that is reactive with said peptide or variant, optionally linked to a heterologous promoter sequence, or an expression vector expressing said nucleic acid. 
     
     
         13 . A recombinant host cell comprising the peptide or variant according to  claim 1 , the antibody or fragment thereof that specifically recognizes said peptide or variant, a T-cell receptor or fragment thereof that is reactive with said peptide or variant or a nucleic acid encoding for the peptide or variant thereof or an expression vector expressing said nucleic acid, wherein said host cell optionally is selected from an antigen presenting cell, optionally a dendritic cell, a T cell or an NK cell. 
     
     
         14 . An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro T cells with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell for a period of time sufficient to activate said T cells in an antigen specific manner, wherein said antigen is a peptide or variant according to  claim 1 . 
     
     
         15 . An activated T lymphocyte, produced by the method according to  claim 14 , that selectively recognizes a cell which presents a polypeptide comprising an amino acid sequence given in  claim 1 . 
     
     
         16 . A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of the peptide or variant according to  claim 1 , an antibody or fragment thereof that specifically recognizes said peptide or variant, a T-cell receptor or fragment thereof that is reactive with said peptide or variant, an aptamer that specifically recognizes the peptide or variant thereof, a nucleic acid encoding for the peptide or variant thereof or expression vector expressing said nucleic acid, a host cell comprising the peptide or variant, or an activated T lymphocyte that selectively recognizes a cell which presents said peptide or variant, or a conjugated or labelled active ingredient, and a pharmaceutically acceptable carrier, and optionally, one or more pharmaceutically acceptable excipients and/or stabilizers. 
     
     
         17 . A method for producing the peptide or variant thereof according to  claim 1 , an antibody or fragment thereof that specifically recognizes said peptide or variant, or a T-cell receptor or fragment thereof that is reactive with said peptide or variant, the method comprising culturing a host cell and isolating the peptide or variant thereof, the antibody or fragment thereof or the T cell receptor or fragment thereof from said host cell and/or a culture medium thereof. 
     
     
         18 . The peptide or variant according to  claim 1 , an antibody or fragment thereof that specifically recognizes said peptide or variant, a T-cell receptor or fragment thereof that is reactive with said peptide or variant, an aptamer that specifically recognizes the peptide or variant thereof, a nucleic acid encoding for the peptide or variant thereof or expression vector expressing said nucleic acid, a host cell comprising the peptide or variant or an activated T lymphocyte that selectively recognizes a cell which presents said peptide or variant for use in medicine. 
     
     
         19 . A method for killing target cells in a patient which target cells present a polypeptide or variant thereof of  claim 1 , the method comprising administering to the patient an effective number of activated T cells. 
     
     
         20 . The peptide or variant according to  claim 1 , an antibody or fragment thereof that specifically recognizes said peptide or variant, a T-cell receptor or fragment thereof that is reactive with said peptide or variant, an aptamer that specifically recognizes the peptide or variant thereof, a nucleic acid encoding for the peptide or variant thereof or expression vector expressing said nucleic acid, a host cell comprising the peptide or variant, or an activated T lymphocyte that selectively recognizes a cell which presents said peptide or variant for use in diagnosis and/or treatment of cancer, and/or for use in the manufacture of a medicament against cancer. 
     
     
         21 . The peptide or variant according to  claim 20 , wherein said cancer is selected from the group of acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer (including non-small cell lung cancer adenocarcinoma, squamous cell non-small cell lung cancer, and small cell lung cancer), ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, urinary bladder carcinoma, uterine and endometrial cancer and other tumors that show an overexpression of a protein from which a peptide SEQ ID NO: 1 to SEQ ID NO: 383 and SEQ ID NO: 448 to SEQ ID NO: 499 is derived from. 
     
     
         22 . A kit comprising:
 (a) a container comprising a pharmaceutical composition containing the peptide(s) or the variant according to  claim 1 , an antibody or fragment thereof that specifically recognizes said peptide or variant, a T-cell receptor or fragment thereof that is reactive with said peptide or variant, an aptamer that specifically recognizes the peptide or variant thereof, a nucleic acid encoding for the peptide or variant thereof or expression vector expressing said nucleic acid, a host cell comprising the peptide or variant, or an activated T lymphocyte that selectively recognizes a cell which presents said peptide or variant, in solution or in lyophilized form;   (b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation;   (c) optionally, at least one more peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 445 and SEQ ID NO: 448 to SEQ ID NO: 499, and (d) optionally, instructions for (i) use of the solution or (ii) reconstitution and/or use of the lyophilized formulation.   
     
     
         23 . The kit according to  claim 22 , further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe. 
     
     
         24 . A method for producing a personalized anti-cancer vaccine or a compound-based and/or cellular therapy for an individual patient, said method comprising:
 a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from said individual patient;   b) comparing the peptides as identified in a) with a warehouse of peptides that have been pre-screened for immunogenicity and/or over-presentation in tumors as compared to normal tissues;   c) selecting at least one peptide from the warehouse that matches a TUMAP identified in the patient wherein said at least one peptide is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 383 and SEQ ID NO: 448 to SEQ ID NO: 499; and   d) manufacturing and/or formulating the personalized vaccine or compound-based or cellular therapy based on c).   
     
     
         25 . The method according to  claim 24 , wherein said TUMAPs are identified by:
 a1) comparing expression data from the tumor sample to expression data from a sample of normal tissue corresponding to the tissue type of the tumor sample to identify proteins that are over-expressed or aberrantly expressed in the tumor sample; and   a2) correlating the expression data with sequences of MHC ligands bound to MHC class I and/or class II molecules in the tumor sample to identify MHC ligands derived from proteins over-expressed or aberrantly expressed by the tumor.   
     
     
         26 . The method according to  claim 24 , wherein the sequences of MHC ligands are identified by eluting bound peptides from MHC molecules isolated from the tumor sample and sequencing eluted ligands. 
     
     
         27 . The method according to  claim 24 , wherein the normal tissue corresponding to the tissue type of the tumor sample is obtained from the same patient. 
     
     
         28 . The method according to  claims 24 , wherein the peptides included in the warehouse are identified based on the following steps:
 aa. Performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identify genes that over-expressed in a malignant tissue, compared with a normal tissue or tissues;   ab. Selecting peptides encoded by selectively expressed or over-expressed genes as detected in step aa, and   ac. Determining an induction of in vivo T-cell responses by the peptides as selected comprising in vitro immunogenicity assays using human T cells from healthy donors or said patient; or   ba. Identifying HLA ligands from said tumor sample using mass spectrometry;   bb. Performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identify genes that over-expressed in a malignant tissue, compared with a normal tissue or tissues;   bc. Comparing the identified HLA ligands to said gene expression data;   bd. Selecting peptides encoded by selectively expressed or over-expressed genes as detected in bc;   be. Re-detecting of selected TUMAPs from step bd on tumor tissue and lack of or infrequent detection on healthy tissues and confirming the relevance of over-expression at the m RNA level; and   bf. Determining an induction of in vivo T-cell responses by the peptides as selected comprising in vitro immunogenicity assays using human T cells from healthy donors or said patient.   
     
     
         29 . The method according to  claim 24 , wherein the immunogenicity of the peptides included in the warehouse is determined by a method comprising in vitro immunogenicity assays, patient immunomonitoring for individual HLA binding, MHC multimer staining, ELISPOT assays and/or intracellular cytokine staining. 
     
     
         30 . The method according to  claim 24 , wherein said warehouse comprises a plurality of peptides selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 445 and SEQ ID NO: 448 to SEQ ID NO: 499. 
     
     
         31 . The method according to  claim 24 , further comprising identifying at least one mutation that is unique to the tumor sample relative to normal corresponding tissue from the individual patient and selecting a peptide that correlates with the mutation for inclusion in the vaccine or for the generation of cellular therapies. 
     
     
         32 . The method according to  claim 31 , wherein said at least one mutation is identified by whole genome sequencing.