Method for predicting the usefulness of proteins or protein fragments for immunotherapy

Abstract

To solve the problem in which: there is a need for a model that predicts whether epitopes, in particular, neoepitopes induce efficient immunity, and therefore are useful for immunotherapy.SOLUTION: The present invention relates to a method for predicting peptides or polypeptides such as T cell epitopes useful for immunotherapy such as for vaccination. In particular, the present invention relates to a method for predicting whether peptides or polypeptides such as tumor-related antigens or epitopes, in particular, a tumor-related neoantigens or neoepitopes are immunogenic and, in particular, useful for immunotherapy such as for vaccination. The method of the present invention can be used particularly for the provision of vaccines specific for a patient's tumor, and therefore in the context of a personalized cancer vaccines.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for predicting peptides or polypeptides, such as T cell epitopes, that are useful for immunotherapy, such as vaccination. In particular, the present invention relates to a method for predicting whether peptides or polypeptides, such as tumor-associated antigens or epitopes, particularly tumor-associated neoantigens or neoepitopes, are immunogenic and, in particular, useful for immunotherapy, such as vaccination. The method of the present invention can be used in particular for providing a patient's tumor-specific vaccine, and therefore in connection with personalized cancer vaccines. [Background technology]

[0002] The evolution of the immune system in vertebrates has resulted in a highly effective network based on two types of defense: innate and adaptive immunity. In contrast to the evolutionarily ancient innate immune system, which relies on immutable receptors that recognize common molecular patterns associated with pathogens, adaptive immunity is based on highly specific antigen receptors and clonal selection on B cells (B lymphocytes) and T cells (T lymphocytes). B cells trigger a humoral immune response by secreting antibodies, while T cells mediate a cellular immune response that leads to the destruction of recognized cells.

[0003] T cells play a central role in cell-mediated immunity in humans and animals. Recognition and binding to specific antigens are mediated by T cell receptors expressed on the surface of T cells. T cell receptors (TCRs) can bind to major histocompatibility complex (MHC) molecules and interact with immunogenic peptides (epitopes) presented on the surface of target cells. Specific binding of TCRs triggers a signaling cascade within the T cell, leading to proliferation and differentiation into mature effector T cells. To target the vast diversity of antigens, T cell receptors need to possess great diversity.

[0004] Antigen-specific immunotherapy aims to enhance or induce a specific immune response in a patient to control infectious or malignant diseases. The increasing number of identified pathogens and tumor-associated antigens has led to a broad accumulation of suitable targets for immunotherapy. Cells presenting immunogenic peptides (epitopes) derived from these antigens can be specifically targeted by either active or passive immunization strategies. Active immunization tends to induce and increase antigen-specific T cells that can specifically recognize and kill disease cells in the patient. In contrast, passive immunization relies on adoptive T cell therapy (ACT), which involves the adoption of T cells that are grown in vitro and selectively genetically engineered.

[0005] Tumor vaccines aim to induce an immune response specific to endogenous tumors through active immunization. Different antigenic forms may be used for tumor vaccination, including entire immunization vectors such as RNA, DNA, or viral vectors, which can be directly applied either in vivo or in vitro by pulsing of DCs following transfer into diseased cells, proteins, peptides, or the patient.

[0006] ACT-based immunotherapy can be broadly defined as a form of passive immunization using pre-sensitized T cells, which are then expanded ex vivo from low precursor frequencies to clinically appropriate cell numbers before being transferred to non-immune recipients or autologous hosts. An effort to overcome the limitations of ACT is adoptive transfer of autologous T cells reprogrammed to express tumor-responsive TCRs of defined specificity, which are then reinjected into the patient after a short period of ex vivo culture.

[0007] The discovery of antigens associated with multiple pathogens and tumors has provided a basis for the idea of ​​antigen-specific immunotherapy. Tumor-associated antigens (TAAs) are unusual proteins that are not expressed or are limited in normal cells, but are expressed in tumor cells due to their genetic instability. These TAAs can be linked to the specific recognition of malignant cells by the immune system.

[0008] Cancer can arise from the accumulation of genomic mutations and epigenetic changes, each of which may play a causative role. In addition to tumor-associated antigens, human cancers have an average of 100–120 non-synonymous mutations, many of which may be vaccine targets. Over 95% of tumor mutations are unique and patient-specific. The number of proteins that alter somatic mutations that can produce tumor-specific T-cell epitopes ranges from 30 to 400.

[0009] Mutations are considered ideal targets for cancer immunotherapy. As neoepitopes that completely lack expression in any healthy tissue, they are expected to be safe and can bypass central tolerance mechanisms. We recently proposed a personalized immunotherapy approach that targets the spectrum of individual mutations (Castle, JC et al., Cancer Res 72, p1081 (2012)).

[0010] Despite the increasing number of attractive target structures for immunotherapy, defining appropriate epitopes for immunotherapy remains a challenge. Therefore, there is a need for models that predict whether epitopes, particularly neoepitopes, will induce efficient immunity and thus be useful in immunotherapy.

[0011] In this specification, the inventors demonstrate that immunogenic antigens and epitopes are strongly expressed in certain intracellular compartments.

[0012] It is known that the immune response to tumor antigens, particularly mutated tumor antigens, is not carried out by the tumor cells themselves, but by antigen-presenting cells, especially dendritic cells that receive tumor antigens released from tumor cells. It is also known that, in order to achieve an effective immune response, the released tumor antigens picked up by antigen-presenting cells must be presented either by MHC class II (exogenous presentation) to induce a CD4 immune response, or by MHC class I (cross-presentation) to induce a CD8 immune response. For the latter immune response, the presence of a CD4 immune response to the same or different tumor antigens delivered to the same antigen-presenting cells is required (Bennett et al., J. Exp. Med. 186, p65-70 (1997)).

[0013] While we do not wish to be bound by any particular theory, it is thought that the cellular localization of antigens in disease cells such as tumor cells determines whether the antigen is picked up and presented by antigen-presenting cells. Exosomes released from disease cells such as tumor cells contain mRNA, proteins, and MHC peptide complexes, and therefore these components can be transferred to antigen-presenting cells. Exosomes are produced by invagination and therefore contain mainly cytoplasmic components in addition to endocytotic membrane molecules. Therefore, it is thought that cytoplasmic components such as proteins are enriched in exosomes, making them capable of being transferred to antigen-presenting cells. Exosomes can also transfer abundant mRNA, which can be translated in the cell that takes up the RNA. Therefore, without being bound by any particular theory, the present invention suggests that peptides or polypeptides contained in exosomes, particularly cytoplasmic peptides or proteins, or peptides or polypeptides, and the RNA encoding them contained in exosomes, are particularly useful for immunotherapy, because exosomes are picked up by antigen-presenting cells, and the peptides and proteins (optionally, by translation of the encoding RNA) are presented by the antigen-presenting cells. Thus, exosomes serve as a means of transport for peptides, proteins, or RNA to antigen-presenting cells and protect them from degradation by proteases and ribonucleases. Alternatively, peptides and proteins may also be picked up by antigen-presenting cells as complexes with other molecules such as antibodies via receptor-dependent mechanisms. [Overview of the project]

[0014] In one embodiment, the present invention relates to a method for predicting the usefulness of a protein or fragment thereof expressed by diseased cells for immunotherapy, comprising determining the distribution or localization of the protein or nucleic acid encoding it, or a fragment of the protein.

[0015] In one embodiment, the method includes determining whether a protein or a nucleic acid encoding it, or a protein fragment, is localized or abundant in the cytosol and / or exosome in vivo.

[0016] In one embodiment, localization or enrichment of a protein or nucleic acid encoding it, or a protein fragment, in the cytosol and / or within the exosome indicates that the protein or its fragment is useful for immunotherapy.

[0017] In a further embodiment, the present invention relates to a method for predicting the usefulness of a protein or fragment thereof expressed by disease cells for immunotherapy, comprising determining whether the protein or fragment thereof is cross-presented by antigen-presenting cells, preferably professional antigen-presenting cells.

[0018] In one embodiment, cross-presentation of a protein or its fragment by antigen-presenting cells demonstrates the usefulness of the protein or its fragment for immunotherapy.

[0019] In one embodiment, determining whether a protein or its fragment is cross-presented by antigen-presenting cells includes determining whether the protein or the nucleic acid encoding it, or the protein fragment, is localized or abundant in the cytosol and / or exosome in vivo.

[0020] In one embodiment, localization or enrichment of a protein or nucleic acid encoding it, or a protein fragment, in the cytosol and / or within the exosome indicates that the protein or its fragment is cross-presented by antigen-presenting cells.

[0021] In one embodiment, determining whether a protein or a fragment thereof is cross-presented by an antigen-presenting cell involves determining an existing antibody response against the protein or the fragment thereof.

[0022] In one embodiment, an existing antibody response against a protein or a fragment thereof indicates that the protein or the fragment thereof is cross-presented by an antigen-presenting cell.

[0023] In one embodiment, determining whether a protein or a fragment thereof is cross-presented by an antigen-presenting cell involves determining whether the protein or the fragment thereof is bound to F-actin.

[0024] In one embodiment, binding of a protein or a fragment thereof to F-actin indicates that the protein or the fragment thereof is cross-presented by an antigen-presenting cell.

[0025] In one embodiment, determining whether a protein or a fragment thereof is cross-presented by an antigen-presenting cell involves determining whether the protein or the fragment thereof is bound to RNA.

[0026] In one embodiment, binding of a protein or a fragment thereof to RNA indicates that the protein or the fragment thereof is cross-presented by an antigen-presenting cell.

[0027] In one embodiment among all aspects of the present invention, a fragment of a protein is present within exosomes as an MHC peptide complex, preferably on the surface of the exosomes.

[0028] In one embodiment of the entirety of the present invention, localization or enrichment of a protein or nucleic acid encoding it, or a protein fragment, in the cytosol preferably indicates protein processing and presentation in the MHC I pathway of diseased cells. In one embodiment, protein processing and presentation in the MHC I pathway results in recognition by CD8+ T cells of a complex formed by MHC I and the protein fragment.

[0029] In one embodiment of the entirety of the present invention, localization or enrichment of a protein or a nucleic acid encoding it, or a protein fragment within an exosome indicates the accumulation of a protein or a nucleic acid encoding it, or a protein fragment in an antigen-presenting cell, preferably a professional antigen-presenting cell. In one embodiment, the accumulation of a protein or a nucleic acid encoding it, or a protein fragment in an antigen-presenting cell preferably indicates the processing and presentation of a protein in the MHC I and / or MHC II pathway of the antigen-presenting cell. In one embodiment, the processing and presentation of a protein in the MHC I pathway results in recognition by CD8+ T cells of a complex formed by MHC I and the protein fragment.

[0030] In a further embodiment, the present invention relates to a method for predicting the usefulness of a protein or fragment thereof expressed by diseased cells for immunotherapy, the following: (a) Confirming existing antibody responses to proteins or their fragments, (b) Determine whether the protein or its fragment is bound to F-actin, and / or (c) To determine whether the protein or its fragment is bound to RNA. A method that includes verifying one or more of the following.

[0031] In one embodiment, an existing antibody response to a protein or its fragment indicates that the protein or its fragment is useful for immunotherapy.

[0032] In one embodiment, the binding of a protein or its fragment to F-actin indicates that the protein or its fragment is useful for immunotherapy.

[0033] In one embodiment, the binding of a protein or its fragment to RNA demonstrates its usefulness for immunotherapy.

[0034] In one embodiment of the present invention, the protein or fragment thereof comprises a disease-specific amino acid modification. In one embodiment, the amino acid modification is based on a disease-specific somatic cell mutation.

[0035] In one embodiment of all aspects of the present invention, the disease is cancer, and the immunotherapy is anti-cancer immunotherapy.

[0036] In one embodiment of the entirety of the present invention, the protein fragment is an MHC-binding peptide or a latent MHC-binding peptide.

[0037] In a further embodiment, the present invention relates to a method for selecting and / or rating disease-specific amino acid modifications for their usefulness in immunotherapy, (i) A step of identifying proteins expressed by diseased cells, each containing at least one disease-specific amino acid modification, and (ii) A step of confirming the distribution or localization of the protein identified in (i) or the nucleic acid encoding it, or the protein fragment, and (iii) Repeat step (ii) for at least one additional protein identified in (i). Regarding methods including

[0038] In one embodiment, step (ii) includes determining whether a protein or a nucleic acid encoding it, or a protein fragment, is localized or abundant in the cytosol and / or exosome in vivo.

[0039] In one embodiment, localization or enrichment of proteins or nucleic acids encoding them, or protein fragments, in the cytosol and / or within exosomes demonstrates that disease-specific amino acid modifications are useful for immunotherapy.

[0040] In a further embodiment, the present invention relates to a method for selecting and / or rating disease-specific amino acid modifications for their usefulness in immunotherapy, (i) A step of identifying proteins expressed by diseased cells, each containing at least one disease-specific amino acid modification, and (ii) A step to determine whether the protein or protein fragment identified in (i) is cross-presented by antigen-presenting cells, preferably professional antigen-presenting cells, and (iii) Repeat step (ii) for at least one additional protein identified in (i). Regarding methods including

[0041] In one embodiment, cross-presentation of proteins or their fragments by antigen-presenting cells demonstrates that disease-specific amino acid modifications are useful for immunotherapy.

[0042] In a further embodiment, the present invention relates to a method for selecting and / or rating disease-specific amino acid modifications for their usefulness in immunotherapy: (i) A step of identifying proteins expressed by diseased cells, each containing at least one disease-specific amino acid modification, and (ii) For the protein or protein fragment identified in (i), the following: (a) Confirming existing antibody responses to proteins or their fragments, (b) Determine whether the protein or its fragment is bound to F-actin, and / or (c) To determine whether the protein or its fragment is bound to RNA. To verify one or more of the following, (iii) Repeat step (ii) for at least one additional protein identified in (i). Regarding methods including

[0043] In one embodiment, existing antibody responses to proteins or their fragments demonstrate that disease-specific amino acid modifications are useful for immunotherapy.

[0044] In one embodiment, the binding of a protein or its fragment to F-actin demonstrates that disease-specific amino acid modifications are useful for immunotherapy.

[0045] In one embodiment, the binding of a protein or its fragment to RNA demonstrates that disease-specific amino acid modifications are useful for immunotherapy.

[0046] In one embodiment of the entirety of the present invention, the disease-specific amino acid modification is comprised of a protein fragment that is an MHC-binding peptide or a potential MHC-binding peptide.

[0047] In one embodiment of the entirety of the present invention, the method is used for the production of a vaccine. In one embodiment, the vaccine is derived from one or more proteins or fragments thereof, or one or more disease-specific amino acid modifications, which are predicted to be useful for immunotherapy.

[0048] In a further embodiment, the present invention relates to a method for providing a vaccine, comprising the step of identifying one or more proteins or fragments thereof or one or more disease-specific amino acid modifications predicted to be useful for immunotherapy by any method of the embodiments described herein. In one embodiment, the method further comprises the step of providing a vaccine comprising a peptide or polypeptide comprising one or more proteins or fragments thereof or one or more disease-specific amino acid modifications predicted to be useful for immunotherapy, or a nucleic acid encoding a peptide or polypeptide.

[0049] In a further embodiment, the present invention relates to a vaccine manufactured by any of the embodiments described herein.

[0050] In one embodiment of all aspects of the present invention, the presentation of the usefulness of a protein or fragment thereof expressed by diseased cells for immunotherapy indicates that the protein or fragment thereof will be immunogenic (in the form of a nucleic acid that optionally encodes it) when administered.

[0051] In one embodiment of the entirety of the present invention, the protein fragment described herein is an MHC-binding peptide or a potential MHC-binding peptide (for example, the prediction of MHC binding indicates that the protein fragment will bind to MHC). In one embodiment, the MHC-binding peptide is a modified peptide which is a modified protein fragment.

[0052] In one embodiment, amino acid modifications in a protein or peptide are identified by identifying non-synonymous mutations in the coding regions of one or more proteins.

[0053] In one embodiment, amino acid modifications are identified by partially or completely sequencing the genome or transcriptome of one or more cells, such as one or more cancer cells and optionally one or more non-cancerous cells, and by identifying mutations in one or more protein-coding regions. In one embodiment, the mutations are somatic mutations. In one embodiment, the mutations are cancer mutations.

[0054] In one embodiment, the method of the present invention is carried out for a patient, in particular, to provide a personalized vaccine for a patient such as a cancer patient, with one or more modifications present in the patient. In a further embodiment, the present invention provides a vaccine that can be obtained using the method according to the present invention. Preferred embodiments of such vaccines are described herein.

[0055] The vaccines provided by the present invention may contain a pharmaceutically acceptable carrier and may optionally contain one or more adjuvants, stabilizers, and other substances. The vaccines may be in the form of therapeutic or prophylactic vaccines.

[0056] Another aspect relates to a method for inducing an immune response in a patient, which includes administering a vaccine provided to the patient according to the present invention.

[0057] Another aspect is, (a) A step of providing an immunotherapy agent described herein, such as a vaccine, using a method according to the present invention; and (b) The process of administering the immunotherapy agent to the patient. Regarding methods of treating patients including [specific condition].

[0058] Another aspect relates to a method of treating a patient, which includes administering an immunotherapy agent, such as a vaccine described herein, to the patient.

[0059] In one embodiment, the patient is a cancer patient, and the vaccine is an anti-cancer vaccine, such as a vaccine in which the administration provides a cancer-specific neoepitope.

[0060] In a further embodiment, the present invention provides vaccines described herein for use in therapeutic methods described herein, particularly for use in treating or preventing cancer.

[0061] The cancer treatments described herein may be combined with surgical resection and / or radiation therapy and / or conventional chemotherapy.

[0062] Other features and advantages of the present invention will be apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0063] [Figure 1] When compared to random peptides (proteome), publicly available epitope proteins are significantly enriched in exosomes and cytosols. [Figure 2] The publicly available epitope genes are found at a significantly higher frequency in the SEREX database compared to random peptides. [Modes for carrying out the invention]

[0064] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and that these may be modified. The terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of the present invention, and it should also be understood that the scope of the present invention is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0065] The elements of the present invention will be described below. These elements are presented in specific embodiments, however, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various examples and preferred embodiments described are not to be construed as limiting the invention, but are solely for the purpose of clearly describing embodiments. This description should be understood as supporting and encompassing embodiments that combine the clearly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any substitution and combination of all elements described in this application should be considered disclosed by the description of this application unless otherwise indicated in context.

[0066] Preferably, as used herein, terms are defined as those described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," edited by HGW Leuenberger, B. Nagel, and H. Koelbl (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0067] The implementation of this invention follows conventional methods of biochemistry, cell biology, immunology, and recombinant DNA techniques described in the literature in the art unless otherwise specified (e.g., Molecular Cloning: A Laboratory Manual, 2 nd (See Edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.)

[0068] Throughout this specification and the following claims, unless otherwise indicated by context, the word “comprise,” and variations such as “comprises” and “comprising,” mean the inclusion of the stated member, integer, or process or group of members, integers, or processes, but not the exclusion of any other member, integer, or process or group of members, integers, or processes, except in some embodiments where such other members, integers, or processes or groups of members, integers, or processes may be excluded; that is, the subject matter will be understood to be the inclusion of the stated member, integer, or process or group of members, integers, or processes. In the context describing this invention (especially in the context of the claims), the terms “a,” “an,” “the,” and similar expressions should be interpreted herein as including both singular and plural, unless otherwise specified or the context makes it clearer. The listing of value ranges in this specification is intended solely as a way to shorten the reference to each individual value that falls within that range. Unless otherwise noted, each individual value is incorporated into this specification as if it were listed individually.

[0069] All methods described herein may be carried out in any suitable order unless otherwise specified herein or clearly contradicted elsewhere by context. The use of any and all examples or typical language provided herein (e.g., "etc.") is intended merely to better illustrate the invention and does not impose any limitation on the scope of the invention as claimed elsewhere. Language in the specification should not be construed as indicating any unclaimed elements essential to the carrying out of the invention.

[0070] Several documents have been cited throughout this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, user manuals, and others), whether cited prior to or following, is incorporated herein by reference in its entirety. Nothing in this specification should be construed as an acceptance that the present invention is not granted any prior rights to such disclosures due to prior art.

[0071] This invention envisions an immunotherapy for diseases, particularly cancer, by utilizing proteins or protein fragments present in diseased cells as labels for targeted diseased cells. In particular, diseased cells can be targeted by targeting protein fragments presented on the surface of diseased cells in relation to MHC. The immunotherapy according to this invention is carried out by active and / or passive immunotherapy methods.

[0072] In particular, the present invention aims to limit suitable proteins or fragments thereof for immunotherapy. Once a suitable protein is identified, the protein or fragment thereof (optionally, as part of a larger polypeptide) or the nucleic acid encoding the protein or fragment thereof (optionally, as part of a larger polypeptide) may be used as a vaccine by inducing and / or activating suitable effector cells, such as T cells that recognize the protein or fragment thereof (particularly when presented in MHC relation) through a suitable receptor (such as a T cell receptor or an artificial T cell receptor), for the purpose of enhancing or inducing an immune response against the protein or fragment thereof. Alternatively, the protein or fragment thereof (particularly when present in MHC relation) may also be administered as effector cells, such as T cells that recognize the protein or fragment thereof (particularly when present in MHC relation) through a suitable receptor (such as a T cell receptor or an artificial T cell receptor). Without wishing to be bound by any particular theory, proteins or fragments thereof predicted to be useful in immunotherapy according to the present invention are considered to have a high likelihood of being picked up by antigen-presenting cells and presented by antigen-presenting cells, particularly by cross-presentation, and therefore ultimately producing an efficient immune response against disease cells expressing the protein or fragment thereof.

[0073] In this invention, proteins defined as useful or suitable for immunotherapy are also referred to herein as “antigens.” Fragments of proteins defined as useful or suitable for immunotherapy in this invention are also referred to herein as “epitopes.”

[0074] The immunotherapy method according to the present invention is: The immunization process includes: i) a protein or peptide (natural or modified); ii) a nucleic acid encoding a protein or peptide; iii) a recombinant cell encoding a protein or peptide; iv) a recombinant virus encoding a protein or peptide; and v) antigen-presenting cells pulsed with a protein or peptide (natural or modified) or transfected with a nucleic acid encoding a protein or peptide.

[0075] The immunotherapy method according to the present invention is: vi) the transfer of T cell receptors that recognize proteins or peptides, and vii) effector cells (such as T cells) that encode receptors that recognize proteins or peptides, especially when presented in relation to MHC.

[0076] Preferred proteins and fragments according to the present invention are expressed in a disease-specific manner, for example, they are disease-related antigens or epitopes (e.g., the presence of a protein or cells expressing a protein is disease-specific), and / or include one or more disease-specific amino acid modifications, for example, they are disease-related neoantigens or neoepitopes. Preferably, the disease-specific amino acid modifications are based on one or more disease-specific somatic cell mutations. In one particularly preferred embodiment, the disease-specific amino acid modification is a cancer-specific amino acid modification, and the disease-specific somatic cell mutation is a cancer-specific somatic cell mutation. Accordingly, according to the present invention, the vaccine preferably features the patient's disease-specific amino acid modification / disease-specific somatic cell mutation and preferably provides one or more mutation-based neoepitopes when administered. Accordingly, the vaccine may include a peptide or polypeptide containing one or more mutation-based neoepitopes, or a nucleic acid encoding the peptide or polypeptide. In one embodiment, disease-specific amino acid modifications are identified by identifying disease-specific somatic cell mutations, for example, by determining the sequences of genomic DNA and / or RNA of diseased tissue or one or more diseased cells.

[0077] In the present invention, the step of identifying disease-specific amino acid modifications and / or disease-specific somatic mutations may be performed before or after the method of the present invention for predicting the usefulness of proteins or fragments thereof expressed by diseased cells for immunotherapy. In one preferred embodiment of the present invention, disease-specific amino acid modifications and / or disease-specific somatic mutations are first determined in a patient's diseased specimen, followed by a prediction of the usefulness of proteins or fragments thereof containing one or more disease-specific amino acid modifications for immunotherapy by the method of the present invention. Once identified, disease-specific amino acid modifications (proteins or fragments thereof containing one or more disease-specific amino acid modifications) can also be selected and / or rated for their usefulness in immunotherapy by the method of the present invention.

[0078] The term "exosome" refers to vesicles derived from cells that are present in biological fluids, including blood, urine, and culture media for cell cultures. Exosomes are released either from cells or directly from plasma membranes when multi-vesicle endoplasmic reticulum fuses with a plasma membrane. Exosomes, containing a variety of molecules, contain components including proteins and RNA from their original cells. The protein composition of exosomes varies depending on the original cell and tissue, but most exosomes contain a common set of evolutionarily conserved protein molecules. Exosomes can transfer molecules from one cell to another through membrane vesicle exchange, thereby influencing the immune system, such as dendritic cells and B cells, and can also play a functional role in mediating adaptive immune responses to pathogens and tumors.

[0079] As used herein, the term "cytosol" refers to the cytoplasmic portion of unbound cellular substructures within a membrane.

[0080] In this invention, the term "peptide" refers to a substance containing 2 or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more, and preferably up to 8, 10, 20, 30, 40 or 50, particularly 100 amino acids, linked by peptide bonds via covalent bonds. The terms "polypeptide" or "protein" refer to larger peptides, preferably peptides having more than 100 amino acid residues; however, generally, the terms "peptide," "polypeptide," and "protein" are synonymous and are used interchangeably herein.

[0081] In this invention, the term “modification” in relation to peptides, polypeptides, or proteins refers to a change in the sequence of a peptide, polypeptide, or protein compared to the sequence of a parent, such as the sequence of a wild-type peptide, polypeptide, or protein. The term includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and amino acid substitution variants, preferably amino acid substitution variants. All of these sequence changes according to the present invention can potentially create new epitopes.

[0082] An amino acid insertion variant contains the insertion of one or more amino acids within a specific amino acid sequence.

[0083] The amino acid addition variant comprises amino and / or carboxyl-terminated fusions of one or more amino acids, for example, 1, 2, 3, 4, or 5, or more amino acids.

[0084] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, by the removal of 1, 2, 3, 4, or 5, or more amino acids.

[0085] Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another residue in its place.

[0086] According to the present invention, modified or modified peptides can be derived from proteins containing the modifications.

[0087] In this invention, the term "induced" means that a particular entity, in particular a particular peptide sequence, is present in the entity from which it is induced. In the case of amino acid sequences, "induced" in particular a particular sequence region means that the amino acid sequence in question is derived from an existing amino acid sequence.

[0088] The present invention provides for proteins that preferably comprise one or more disease-specific amino acid modifications. In one embodiment, these one or more disease-specific amino acid modifications are located within the epitopes or potential epitopes of the protein. Therefore, the preferred proteins described herein are preferably neoantigens comprising one or more neoepitopes. Similarly, the preferred protein fragments described herein are protein fragments comprising one or more disease-specific amino acid modifications, and preferably, one or more disease-specific amino acid modifications are located within the protein fragment. Therefore, the preferred protein fragments described herein are neoepitopes.

[0089] In this invention, the term "neoantigen" refers to a peptide or protein comprising one or more amino acid modifications compared to the parent peptide or protein. For example, a neoantigen may be a tumor-related neoantigen, where the term "tumor-related neoantigen" includes a peptide or protein comprising amino acid modifications based on tumor-specific mutations.

[0090] In this invention, the term "disease-specific mutation" refers to a somatic cell mutation that is present in the nucleic acids of diseased cells but not in the nucleic acids of corresponding normal, i.e., non-disease cells.

[0091] In this invention, the terms “tumor-specific mutation” or “cancer-specific mutation” refer to somatic mutations that are present in the nucleic acids of tumor or cancer cells but not in the corresponding normal, i.e., non-tumor or non-cancer cell nucleic acids. The terms “tumor-specific mutation” and “tumor mutation,” and the terms “cancer-specific mutation” and “cancer mutation” are used interchangeably herein.

[0092] The term "immune response" refers to the reaction of the immune system, such as an immunogenic organism, e.g., bacteria or viruses, cells or substances. The term "immune response" includes innate and adaptive immune responses. Preferably, the immune response relates to the activation of immune cells, the biosynthesis of cytokines, and / or the induction of antibody production.

[0093] The immune response induced by the compositions described herein preferably includes a step of activating antigen-presenting cells, such as dendritic cells and / or macrophages; a step of the antigen-presenting cells presenting antigens or fragments thereof; and a step of activating cytotoxic T cells based on this presentation.

[0094] "Inducing an immune response" may mean that there was no immune response before induction, but this term can also mean that there was a level of immune response before induction, and that the immune response was enhanced after induction. Therefore, "inducing an immune response" also includes "enhancing an immune response." After inducing an immune response in a subject, it is preferable that the subject is protected from developing a disease such as cancer, or that the disease state is mitigated by inducing an immune response. For example, an immune response to an antigen expressed in a tumor may be induced in a patient with cancer or a subject at risk of developing cancer. In this case, inducing an immune response may mean that the disease state of the subject improves, the subject does not metastasize, or the subject at risk of developing cancer does not develop cancer.

[0095] The terms "cellular immune response" and "cellular response" or similar terms refer to an immune response directed towards cells characterized by the presentation of antigens having class I or class II MHC, including T cells or T lymphocytes that act as either "helper" or "killer" cells. Helper T cells (CD4 + T cells (also known as CD8 cells) play a central role in regulating the immune response, and killer cells (cytotoxic T cells, cytolytic T cells, CD8 cells) play a central role in regulating the immune response. + T cells (also known as CTLs) kill disease cells, such as cancer cells, and prevent the production of more disease cells. In preferred embodiments, the present invention includes stimulating an anti-disease CTL response against disease cells expressing one or more disease-associated antigens, preferably presenting antigens having such disease-associated class I MHC, and in particular, stimulating an anti-tumor CTL response against tumor cells expressing one or more tumor-expressed antigens, preferably presenting antigens having such tumor-expressed class I MHC.

[0096] In this invention, the terms “antigen” or “immunogen” encompass any substance, preferably a peptide or protein, that is a target of an immune response and / or elicits an immune response. In particular, “antigen” refers to any substance that specifically reacts with an antibody or a T lymphocyte (T cell). In this invention, the term “antigen” includes any molecule comprising at least one epitope, e.g., a T cell epitope. Preferably, in the context of this invention, an antigen is, optionally, a molecule specific to an antigen or a cell expressing an antigen, which triggers an immune response after processing. In this invention, any suitable antigen that is a candidate for an immune response may also be used, and the immune response is preferably a cellular immune response. In the context of embodiments of this invention, the antigen is, preferably, presented by a cell, preferably by an antigen-presenting cell, in relation to an MHC molecule, which elicits an immune response to the antigen. The antigen is preferably a naturally occurring antigen or a product derived therefrom. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, and the antigen may also be a tumor antigen. According to the present invention, the antigen may correspond to or be a part of a naturally occurring product, such as a viral protein. In a preferred embodiment, the antigen is a surface polypeptide, i.e., a polypeptide naturally present on the surface of a cell, a pathogen, bacteria, virus, fungus, parasite, allergen, or tumor. The antigen can elicit an immune response to a cell, pathogen, bacteria, virus, fungus, parasite, allergen, or tumor.

[0097] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to produce an antigen-specific immune response and / or humoral antibody response in cells against a disease. Therefore, disease-associated antigens can be used for therapeutic purposes. Disease-associated antigens are preferably associated with infections of the microbiome, typically microbial antigens, or with cancer, typically tumors.

[0098] The term "pathogen" refers to pathogenic biological material that can cause disease in living organisms, preferably vertebrate organisms. Pathogens include microorganisms, such as bacteria, single-celled eukaryotic organisms (protozoa), fungi, and viruses.

[0099] The terms “epitope,” “antigen peptide,” “antigen epitope,” “immunogenic peptide,” and “MHC-binding peptide” are used interchangeably herein and refer to an antigenic determinant in a molecule such as an antigen, i.e., a part or fragment of an immunologically active compound that is recognized by the immune system, for example, particularly by T cells when presented in relation to MHC molecules. Protein epitopes preferably consist of a continuous or discontinuous portion of the protein, preferably with an amino acid length between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, and most preferably between 10 and 25. For example, an epitope may also preferably have an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. By the present invention, an epitope may also bind to an MHC molecule, such as an MHC molecule on the surface of a cell, and therefore may be an “MHC-binding peptide” or “antigen peptide.”

[0100] The term “Major Histocompatibility Complex” and the abbreviation “MHC” refer to a complex of genes present in all vertebrates, comprising MHC class I and MHC class II molecules. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or disease cells in immune responses, as MHC proteins or molecules bind to peptides to present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. Preferred immunogenic moieties bind to MHC class I or class II molecules. As used herein, an immunogenic moiety is said to “bind” to an MHC class I or class II molecule if such binding is detectable using any assay known in the art. The term “MHC-bound peptide” refers to a peptide bound to an MHC class I and / or MHC class II molecule. In the case of a class I MHC / peptide complex, the bound peptide is typically 8-10 amino acids long, but longer and shorter peptides can also be effective. In the case of a class II MHC / peptide complex, the bound peptide is typically 10-25 amino acids long, particularly 13-18 amino acids long, but longer and shorter peptides can also be effective. In one preferred embodiment of all aspects of the present invention, the MHC molecule is an HLA molecule.

[0101] If the peptide is part of a larger entity, for example, a vaccine sequence or an additional sequence of polypeptide, and is to be presented after processing, particularly after cleavage, then the peptide produced by processing has a length suitable for binding to MHC molecules, particularly class I MHC molecules, preferably 7-30 amino acids long, for example, 7-20 amino acids long, more preferably 7-12 amino acids long, more preferably 8-11 amino acids long, particularly 9 or 10 amino acids long. Preferably, the sequence of the peptide to be presented after processing is derived from the amino acid sequence of the antigen or polypeptide used for vaccination, i.e., its sequence substantially corresponds to, and preferably completely identical to, a fragment of the antigen or polypeptide.

[0102] Therefore, in one embodiment, the MHC-binding peptide substantially corresponds to the antigen fragment and preferably contains an entirely identical sequence.

[0103] As used herein, the term “neoepitope” refers to an epitope that is not present in reference to normal, healthy (e.g., non-cancerous) or germline cells, but is found in diseased cells (e.g., cancer cells). This includes situations in which a corresponding epitope is found in normal, healthy or germline cells, but the sequence of the epitope has been altered to produce a neoepitope based on one or more mutations in the diseased cell.

[0104] As used herein, the term "T cell epitope" refers to a peptide bound to an MHC molecule in a configuration recognized by the T cell receptor. Typically, T cell epitopes are presented on the surface of antigen-presenting cells.

[0105] The T cell epitopes according to the present invention relate to a part or fragment of an antigen that can stimulate a cellular response to an antigen or cell characterized by an immune response, preferably by the expression of an antigen, and preferably by the presentation of an antigen such as a disease cell, particularly a cancer cell. Preferably, the T cell epitopes can stimulate a cellular response to cells characterized by the presentation of an antigen having class I MHC, and preferably can stimulate antigen-responsive cytotoxic T lymphocytes (CTLs).

[0106] In one embodiment, the vaccine according to the present invention contains an epitope suitable for vaccinating a target organism. Those skilled in the art will know that one of the principles of immunobiology and vaccination is based on the fact that an immune defense response against a disease is produced by immunizing an organism with a vaccine immunologically related to the disease to be treated. According to the present invention, the antigen is selected from the group including self-antigens and non-self-antigens. Non-self-antigens are preferably bacterial antigens, viral antigens, fungal antigens, allergens, or parasitic antigens. The antigen preferably contains an epitope capable of eliciting an immune response from a target organism. For example, the epitope can elicit an immune response against bacteria, viruses, fungi, parasites, allergens, or tumors.

[0107] In some embodiments, the non-self antigen is a bacterial antigen. In some embodiments, the antigen elicits an immune response to bacteria that infect animals, including birds, fish, and mammals, including domesticated animals. Preferably, the bacteria to which the immune response is elicited are pathogenic bacteria.

[0108] In some embodiments, the non-self antigen is a viral antigen. The viral antigen may be, for example, a peptide from a viral surface protein, such as a capsid polypeptide or spike polypeptide. In some embodiments, the antigen elicits an immune response to a virus that infects animals, including birds, fish, and mammals, including domesticated animals. Preferably, the virus to which the immune response is elicited is a pathogenic virus.

[0109] In some embodiments, the non-self antigen is a polypeptide or protein from a fungus. In some embodiments, the antigen elicits an immune response to a fungus that infects animals, including birds, fish, and mammals, including domesticated animals. Preferably, the fungus to which the immune response is elicited is a pathogenic fungus.

[0110] In some embodiments, the non-self antigen is a polypeptide or protein from a single-celled eukaryotic parasite. In some embodiments, the antigen elicits an immune response to a single-celled eukaryotic parasite, preferably a pathogenic single-celled eukaryotic parasite. Pathogenic single-celled eukaryotic parasites may be, for example, from the genus Plasmodium, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale; from the genus Leishmania; or from the genus Trypanosoma, such as Trypanosoma cruzi or Trypanosoma brucei.

[0111] In some embodiments, the non-self antigen is an allergenic polypeptide or an allergenic protein. The allergenic protein or polypeptide is also suitable for allergen immunotherapy, which is known as desensitization.

[0112] In some embodiments, the antigen is an autoantigen, particularly a tumor antigen. Tumor antigens and their determination are known to those skilled in the art.

[0113] In relation to the present invention, the terms “tumor antigen” or “tumor-associated antigen” refer to proteins that are specifically expressed under normal conditions in a limited number of tissues and / or organs, or at specific developmental stages. For example, tumor antigens may be specifically expressed under normal conditions in gastric tissue, preferably gastric mucosa; in reproductive organs, e.g., testes; in trophoblast tissue, e.g., placenta; or in germline cells; and may be expressed or abnormally expressed in one or more tumor or cancerous tissues. In this context, “limited number” means preferably three or less, more preferably two or less. In relation to the present invention, tumor antigens include, for example, differentiation antigens, preferably cell type-specific differentiation antigens, i.e., proteins that are specifically expressed under normal conditions in certain cell types at certain differentiation stages; cancer / testicular antigens, i.e., proteins that are specifically expressed in the testes, sometimes in the placenta, under normal conditions; and germline-specific antigens. In relation to the present invention, tumor antigens refer to the cell surface of cancer cells, and preferably are not expressed or are rarely expressed in normal tissues. Preferably, cancer cells are identified by the tumor antigen or the abnormal expression of a tumor antigen. In relation to the present invention, the tumor antigen expressed by cancer cells in a subject, for example, a patient suffering from cancer, is preferably an autologous protein in the subject. In a preferred embodiment, the tumor antigen is specifically expressed in, in relation to the present invention, under normal conditions, in non-essential tissues or organs, i.e., tissues or organs in which the subject does not die if damaged by the immune system, or organs or structures of the body inaccessible or barely accessible to the immune system. Preferably, the amino acid sequence of the tumor antigen is identical between the tumor antigen expressed in normal tissue and the tumor antigen expressed in cancerous tissue.

[0114] Examples of tumor antigens that may be useful in the present invention include p53, ART-4, BAGE, beta-catenin / m, Bcr-abLCAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, such as claudin-6, claudin-18.2 and claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAG These are E-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pm1 / RARa, PRAME, Proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, and WT. Particularly preferred tumor antigens include claudin-18.2 (CLDN18.2) and claudin-6 (CLDN6).

[0115] The term “immunogenicity” preferably relates to the relative efficacy of inducing an immune response associated with a therapeutic treatment, such as the treatment of cancer. As used herein, the term “immunogenicity” relates to the property of being immunogenic. For example, when the term “immunogenic modification” is used in relation to a peptide, polypeptide, or protein, it relates to the efficacy of the peptide, polypeptide, or protein in inducing an immune response caused by and / or against the modification. Preferably, an unmodified peptide, polypeptide, or protein does not induce an immune response, induces a different immune response, or induces a different level of immune response, preferably a lower level.

[0116] In this invention, the terms “immunogenicity” or “immunogenic” preferably relate to relative efficacy in inducing a biologically relevant immune response, particularly an immune response useful for vaccination. Accordingly, in a preferred embodiment, an amino acid modification or modified peptide is immunogenic if it induces an immune response that may be beneficial for therapeutic or prophylactic purposes against a targeted modification in a subject.

[0117] As used herein, the term “predicting the usefulness of a protein or its fragment for immunotherapy” refers to predicting whether a protein or one or more fragments thereof, such as an epitope, particularly a T-cell epitope, is useful for inducing or targeting an immune response. If a protein, such as a disease-related antigen, is predicted to be useful for immunotherapy, then, for example, the epitope of the protein may be used for vaccination as described herein, or effector cells targeting the epitope of the protein may be administered. It is preferable that the protein whose usefulness for immunotherapy can be predicted by the present invention be expressed in the diseased cells of the patient.

[0118] In this invention, T cell epitopes can also be present in vaccines as part of a larger entity, such as a vaccine sequence and / or a polypeptide containing more than one T cell epitope. The presented peptide or T cell epitope is produced after appropriate processing. The T cell epitope may also be modified with one or more residues that are not essential for TCR recognition or binding to MHC. Such modified T cell epitopes may be considered immunologically equivalent. Preferably, when a T cell epitope is presented by MHC and recognized by a T cell, the receptor can induce clonal proliferation of T cells carrying a T cell receptor that specifically recognizes the peptide / MHC complex in the presence of an appropriate co-stimulating signal. Preferably, the T cell epitope contains an amino acid sequence substantially corresponding to the amino acid sequence of an antigen fragment. Preferably, the antigen fragment is a peptide presented to MHC class I and / or class II.

[0119] "Antigen processing" or "processing" refers to the degradation of a peptide, polypeptide, or protein into a processed product, the product being a fragment of the peptide, polypeptide, or protein (e.g., degradation of a polypeptide into a peptide), and the association (e.g., by binding) of one or more of these fragments with MHC molecules for presentation to specific T cells by cells, preferably antigen-presenting cells.

[0120] Antigens restricted to class II enter antigen-presenting cells via the endocytosis pathway and are largely induced from exogenous proteins processed in the endosomal compartment. In contrast, antigens recognized by effector CTLs restricted to class I are typically induced from endogenously synthesized proteins. Therefore, exogenous proteins cannot provide antigenic determinants for effector CTLs restricted to class I unless they are directly introduced into the cytoplasm of target cells.

[0121] The term "cross-presentation" refers to the ability of antigen-presenting cells to pick up and process extracellular antigens and present them to CD8 T cells (cytotoxic T cells) with MHC class I molecules. Cross-priming describes the stimulation of naive cytotoxic CD8+ T cells by this process. While dendritic cells are the primary antigen-presenting cells capable of cross-presentation, macrophages, B lymphocytes, and sinusoidal endothelial cells have also been shown to be able to do so.

[0122] Cross-priming has been shown to occur against viral proteins and tumor antigens. This has led to the suggestion that cross-priming may provide the immune system with a mechanism to detect and respond to histotropic viruses that do not infect professional APCs. In the absence of such a mechanism, viruses could evade immune surveillance by avoiding professional APCs. This mechanism also provides the immune system with a means to search for neoantigens expressed by newly emerging tumor cells. Like exogenous foreign antigens, exogenous autoantigens can enter the class I presentation pathway.

[0123] Antigen-presenting cells (APCs) are cells that present peptide fragments of protein antigens on their cell surface, associated with MHC molecules. Some APCs can activate antigen-specific T cells.

[0124] Professional antigen-presenting cells highly efficiently internalize antigens, either through phagocytosis or receptor-mediated endocytosis, and then display antigen fragments bound to class II MHC molecules on their membranes. T cells recognize and interact with the antigen class II MHC molecule complex on the antigen-presenting cell membrane. Subsequently, additional costimulatory signals are produced by the antigen-presenting cells, leading to T cell activation. The expression of costimulatory molecules is a defining characteristic of professional antigen-presenting cells.

[0125] The primary types of professional antigen-presenting cells are dendritic cells, macrophages, B cells, and certain activated epithelial cells, which possess the broadest range of antigen presentation and are perhaps the most important antigen-presenting cells. Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells through both MHC class II and I antigen presentation pathways. Dendritic cells are known triggers for strong immune responses, and the activation of these cells is a critical step for inducing antitumor immunity. Dendritic cells are conveniently classified into “immature” and “mature” cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with high capacity for antigen uptake and processing, correlated with high expression of Fcγ and mannose receptors. The mature phenotype is typically characterized by lower expression of these markers, while high expression of cell surface molecules, such as class I and class II MHCs, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB), can lead to T cell activation. Dendritic cell maturation corresponds to a dendritic cell activation state, where such antigen-presenting dendritic cells induce T cell priming, in contrast to presentation by immature dendritic cells that induce tolerance. Dendritic cell maturation is primarily triggered by biomolecules with microbial characteristics detected by innate receptors (bacterial DNA, viral RNA, endodoxins, etc.), pro-inflammatory cytokines (TNF, IL-1, IFN), ligation of CD40 by CD40L on the dendritic cell surface, and substances released from cells undergoing stress-induced cell death. Dendritic cells can be induced in vitro by culturing bone marrow cells with cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.

[0126] Nonprofessional antigen-presenting cells do not constitutively express MHC class II proteins necessary for interaction with naive T cells. These proteins are expressed only when stimulated by certain cytokines, such as IFNγ.

[0127] The terms "cells characterized by antigen presentation" or "cells presenting antigens" or similar expressions refer to disease cells, such as cancer cells, or antigen-presenting cells that present an antigen or a fragment induced by antigen processing, such as an antigen-induced fragment, in the relationship of an antigen to an MHC molecule, particularly an MHC class I molecule. Similarly, the term "disease characterized by antigen presentation" refers to a disease involving cells characterized by the presentation of an antigen, particularly one having class I MHC. Antigen presentation by cells can also be performed by transfecting cells with nucleic acids, such as RNA, that encode the antigen.

[0128] The phrase "presented antigen fragment" or similar expression means that the fragment can be presented by MHC class I or class II, preferably MHC class I, when, for example, directly added to antigen-presenting cells. In one embodiment, the fragment is a fragment naturally presented by cells expressing the antigen.

[0129] "Target cells" should mean cells that are targets for an immune response, such as a cellular immune response. Target cells include cells that present an antigen, i.e., a peptide fragment derived from an antigen, and include any undesirable cells, such as cancer cells. In a preferred embodiment, target cells are cells that express the antigens described herein and, preferably, present the antigen together with a class I MHC.

[0130] The term “part” refers to a fraction. With respect to an amino acid sequence or a particular structure such as a protein, the term “part” can represent a continuous or discontinuous fraction of the structure. Preferably, a part of an amino acid sequence contains at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, preferably at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the amino acids of the amino acid sequence. Preferably, if the part is a discontinuous fraction, the discontinuous fraction consists of 2, 3, 4, 5, 6, 7, 8, or more parts of the structure, each part being a continuous element of the structure. For example, a discontinuous fraction of the amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8, or more, preferably 4 or fewer parts of the amino acid sequence, and each part preferably contains at least 5 consecutive amino acids, at least 10 consecutive amino acids, preferably at least 20 consecutive amino acids, and preferably at least 30 consecutive amino acids of the amino acid sequence.

[0131] The terms “part” and “fragment” are used interchangeably herein and refer to a contiguous element. For example, a part of a structure such as an amino acid sequence or a protein refers to a contiguous element of the structure. A part, part, or fragment of a structure preferably contains one or more functional properties of the structure. For example, a part, part, or fragment of an epitope, peptide, or protein is preferably immunologically equivalent to the epitope, peptide, or protein from which it is derived. In relation to the present invention, a “part” of a structure such as an amino acid sequence preferably includes, and preferably comprises, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the whole structure or amino acid sequence.

[0132] In relation to the present invention, the terms “effector cell,” “immune effector cell,” or “immunoreactive cell” refer to cells that exert effector function during an immune response. “Immunereactive cells” can mediate an immune response by binding to antigens or cells characterized by the presentation of antigens or their peptide fragments (e.g., T cell epitopes). For example, such cells secrete cytokines and / or chemokines and antibodies to recognize cancer cells and selectively eliminate such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in relation to the present invention, immunoreactive cells are T cells, preferably CD4 + and / or CD8 + These are T cells.

[0133] Preferably, "immunoreactive cells" recognize antigens or their peptide fragments to a certain degree of specificity when presented on the surface of antigen-presenting cells or disease cells such as cancer cells, particularly in relation to MHC molecules. Preferably, such recognition makes it possible for the cells that recognize the antigens or their peptide fragments to be responsive or reactive. Helper T cells (CD4) carry receptors that recognize antigens or their peptide fragments. + If it is a T cell, then in relation to MHC class II molecules, such responsiveness or reactivity is due to cytokine release and / or CD8 +The activation of lymphocytes (CTLs) and / or B cells may also be included. If the cell is a CTL, such responsiveness or reactivity may also include the elimination of cells presented in relation to MHC class I molecules, i.e., cells characterized by the presentation of an antigen having class I MHC, for example, by apoptosis or perforin-mediated cytolysis. By this invention, CTL responsiveness may also include a sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing the antigen. CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness. Such CTLs that are responsive or reactive in recognition of an antigen or antigenic fragment are also referred to herein as “antigen-responsive CTLs.” If the cell is a B cell, such responsiveness may also include the release of immunoglobulins.

[0134] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells.

[0135] T cells belong to the group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence of a special receptor called the T cell receptor (TCR) on their cell surface. The thymus is the main organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with distinct functions.

[0136] Among its other functions, T helper cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated when they are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist the active immune response.

[0137] Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost all cells in the body.

[0138] Most T cells possess a T cell receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor is produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and consists of two separate peptide chains called α- and β-TCR chains. Gamma delta T cells (γδT cells) represent a small subset of T cells that have T cell receptors (TCRs) with different properties on their surface. However, in γδT cells, the TCR consists of one γ-chain and one δ-chain. This group of T cells is far less common than αβT cells (2% of all T cells).

[0139] The term "antigen receptor" according to the present invention includes naturally occurring receptors such as T cell receptors, as well as engineered receptors, which confer some specificity, such as the specificity of monoclonal antibodies, to effector cells such as T cells. In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer. Therefore, the antigen receptor according to the present invention can be present on T cells, for example, in place of or in addition to the T cell receptor of the T cell itself. Such T cells do not necessarily require processing and antigen presentation to recognize target cells, but preferably they can recognize antigens of some specificity present on target cells. Preferably, the antigen receptor is expressed on the surface of the cell. For the purposes of the present invention, T cells containing engineered antigen receptors are included in the term "T cell" as used herein. In particular, the term "antigen receptor" according to the present invention includes an artificial receptor comprising a single molecule or a complex of molecules that can recognize, i.e., bind to, a target structure (e.g., an antigen) on a target cell such as a cancer cell (e.g., by binding of an antigen-binding site or antigen-binding domain to an antigen expressed on the surface of the target cell), and confer specificity to effector cells such as T cells expressing the antigen receptor on the cell surface. Preferably, recognition of the target structure by the antigen receptor leads to the activation of effector cells expressing the antigen receptor. The "antigen receptor" according to the present invention may be a "chimeric antigen receptor (CAR)", a "chimeric T cell receptor", or an "artificial T cell receptor".

[0140] The present invention allows an antigen to be recognized by an antigen receptor through an antigen-recognition domain (hereinafter also simply referred to as "domain"), and to form an antigen-binding site through an antibody and the antigen-binding portion of a T cell receptor, which may be on the same or different peptide chains. In one embodiment, the two domains forming the antigen-binding site are derived from an immunoglobulin. In another embodiment, the two domains forming the antigen-binding site are derived from a T cell receptor. Antibody variable domains, such as monoclonal antibodies and variable domains of T cell receptors, particularly single-chain variable fragments (scFv) derived from TCR alpha and beta single chains, are especially preferred. In fact, almost anything that binds to a given target with high affinity can be used as an antigen that recognizes the domain.

[0141] The primary signal for T cell activation is provided by the binding of T cell receptors to short peptides presented by MHC on another cell. This ensures that only T cells with a TCR specific to that peptide are activated. Partner cells are typically antigen-presenting cells, such as professional antigen-presenting cells, or dendritic cells in the case of a naive response, but B cells and macrophages can also be important APCs.

[0142] According to the present invention, a molecule can bind to a predetermined target if it has a significant affinity for that target, and binds to the predetermined target in a standard assay. "Affinity" or "binding affinity" is often expressed as the equilibrium dissociation constant (K). D ) is measured by ). A molecule cannot (substantially) bind to the target unless it has a significant affinity for the target, and will not significantly bind to the target in the standard assay.

[0143] Cytotoxic T lymphocytes can be generated in vivo by incorporating antigens or their peptide fragments into antigen-presenting cells in vivo. Antigens or their peptide fragments can be represented as proteins, DNA (e.g., in a vector), or RNA. Antigens can also be processed to produce peptide partners for MHC molecules, but their fragments can be presented without requiring further processing. This is especially true if they can bind to MHC molecules. Generally, administration to patients by intradermal injection is possible. However, injections can also be performed intralysed into lymph nodes (Maloy et al. (2001), Proc Natl Acad Sci USA 98: p3299-303). The resulting cells present the complex of interest, are recognized by autologous cytotoxic T lymphocytes, and then proliferate.

[0144] Specific activation of CD4+ or CD8+ T cells can be detected by various methods. Methods for detecting specific T cell activation include detecting T cell proliferation, cytokine (e.g., lymphokine) production, or the occurrence of cytolytic activity. For CD4+ T cells, a preferred method for detecting specific T cell activation is the detection of T cell proliferation. For CD8+ T cells, a preferred method for detecting specific T cell activation is the detection of the occurrence of cytolytic activity.

[0145] The term "immunologically equivalent" means that immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, exhibit the same or essentially the same immunological properties, and / or exert the same or essentially the same immunological effects with respect to, for example, the induction of humoral and / or cellular immune responses, the intensity and / or duration of the induced immune reaction, or the type of immunological effect such as the specificity of the induced immune reaction. In the context of the present invention, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of the peptides used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if the amino acid sequence elicits an immune reaction having the specificity of reaction with the reference amino acid sequence when exposed to the immune system of a subject.

[0146] In the context of the present invention, the term "immune effector function" includes any function mediated by components of the immune system that results in, for example, killing of tumor cells, or inhibition of tumor growth and / or inhibition of tumor dissemination and metastasis, including inhibition of tumor development. Preferably, the immune effector function is, in the context of the present invention, an effector function mediated by T cells. Such functions are, in the case of helper T cells (CD4 + T cells), characterized by recognition of an antigen or antigen fragment by the T cell receptor in the context of MHC class II molecules, release of cytokines and / or activation of CD8 + lymphocytes (CTLs) and / or B cells, and in the case of CTLs, recognition of an antigen or antigen fragment by the T cell receptor in the context of MHC class I molecules, elimination of presented cells, i.e., cells characterized by presentation of an antigen having class I MHC, by apoptosis or perforin-mediated cytolysis, including production of cytokines such as IFN-γ and TNF-α, and specific cytolytic killing of target cells expressing the antigen.

[0147] Generally, according to the present invention, proteins expressed by diseased cells are assessed for their usefulness in immunotherapy. Proteins with predicted usefulness in immunotherapy can be used to provide vaccines containing proteins or one or more of their peptide fragments, particularly peptides of proteins that bind one or more (potential) MHCs.

[0148] In this invention, the term "distribution" refers to a state of localization. The term "determining distribution or localization" includes, in particular, determining or predicting a state of localization, for example, determining or predicting the intracellular localization or enrichment of a peptide, protein, or nucleic acid, for example, determining or predicting whether a peptide, protein, or nucleic acid is localized or enriched in the cytosol and / or exosome in vivo.

[0149] Terms such as "predict," "predicting," or "forecast" relate to the determination of likelihood.

[0150] The present invention allows for predicting the usefulness of a protein or fragment thereof expressed by diseased cells for immunotherapy by one or more of the following: (i) determining the distribution or localization of the protein or encoding nucleic acid, or protein fragment, for example, determining whether the protein or encoding nucleic acid, or protein fragment is localized or abundant in the cytosol and / or exosome in vivo; (ii) determining whether the protein or fragment thereof is cross-presented by antigen-presenting cells, preferably professional antigen-presenting cells; (iii) determining the existing antibody response to the protein or fragment thereof; (iv) determining whether the protein or fragment thereof is bound to F-actin; and (v) determining whether the protein or fragment thereof is bound to RNA.

[0151] In one embodiment, determining whether a protein, its encoding nucleic acid, or a protein fragment is localized or abundant within an exosome in vivo is carried out by obtaining an extracellular fluid sample, separating the exosomes by, for example, fractional centrifugation, separating the protein or nucleic acid by, for example, gel electrophoresis, and identifying the protein or its fragment by, for example, mass spectrometry, ELISA, flow cytometry, antibody array, or Western blotting, or by identifying the protein-coding nucleic acid by, for example, microarray, RNA sequencing, or RT-PCR. In another embodiment, determining whether a protein, its encoding nucleic acid, or a protein fragment is localized or abundant within an exosome in vivo is carried out by extracting information from a database containing data from the experiments described in this section above, for example, ExoCarta (Keerthikumar, S. et al. J. Mol. Biol. 428, p688 (2016)).

[0152] In one embodiment, confirming an existing antibody response can be performed using SEREX. SEREX stands for serological identification of antigens by recombinant expression cloning, a method for identifying tumor antigens by screening antibodies from patient serum for recognition of a phage library transduced with cDNA derived from a tumor. This technique uses a phage display library expressing various types of patient latent antigens. The antigens are transferred to a two-dimensional surface, making it possible to map them to specific clones. The surface is incubated with autologous patient serum. The immunoreactive clones are located, cultured, and sequenced (Sahin, U. et al., PNAS 92, p11810 (1995)).

[0153] The present invention may also include decomposing a protein sequence into suitable fragments to determine MHC binding, and verifying a score for the binding of the fragments to one or more MHC molecules. The results obtained may consist of a rated list of peptides and their predicted scores indicating the likelihood of their binding. Generally, proteins according to the present invention are particularly useful for immunotherapy if they contain peptides to which one or more (potential) MHCs are bound.

[0154] The method of the present invention can also be applied to tumor specimens from patients, such as cancer patients.

[0155] The present invention provides for proteins or protein fragments that preferably contain at least one amino acid modification. Amino acid modifications whose usefulness for immunotherapy should be determined by the present invention, or which should be selected and / or graded based on their predicted immunogenicity according to the present invention, may arise from mutations in the nucleic acids of cells. Such mutations may be identified by known sequencing techniques.

[0156] In one embodiment, the mutation is a cancer-specific somatic mutation in a tumor sample from a cancer patient, which can be determined by identifying the sequence difference between the genome, exome, and / or transcriptome of the tumor sample and the genome, exome, and / or transcriptome of a non-tumoric sample.

[0157] The present invention relates to any sample, such as a body sample from a patient who has a tumor or is expected to have cancer cells. The body sample may be any tissue sample, for example, blood, a tissue sample obtained from a primary tumor or tumor metastasis, or any other sample containing tumor or cancer cells. Preferably, the body sample is blood, and cancer-specific somatic cell mutations or sequence differences are determined by one or more circulating tumor cells (CTCs) contained in the blood. In another embodiment, the tumor sample relates to a sample containing one or more isolated tumor or cancer cells, for example, circulating tumor cells (CTCs), etc.

[0158] Non-tumor-forming specimens relate to any specimen, such as a body specimen, derived from a patient or another individual, preferably of the same species as the patient, preferably from a healthy individual that does not contain or is not expected to contain tumors or cancer cells. The body specimen may be any tissue specimen, such as blood, or a specimen from non-tumor-forming tissue.

[0159] The present invention may include determining the signs of cancer mutations in a patient. The term “signs of cancer mutations” may refer to all cancer mutations present in one or more cancer cells of a patient, or it may refer to only the portion of cancer mutations present in one or more cancer cells of a patient. Therefore, the present invention may include the identification of all cancer-specific mutations present in one or more cancer cells of a patient, or it may include the identification of only the portion of cancer-specific mutations present in one or more cancer cells of a patient. Generally, the methods of the present invention provide the identification of many mutations that provide a sufficient number of modifications, or modified proteins that should be included in the methods of the present invention.

[0160] Preferably, the mutations identified by the present invention are non-synonymous mutations, and preferably non-synonymous mutations in proteins expressed in tumors or cancer cells.

[0161] In one embodiment, cancer-specific somatic mutations or sequence differences are determined in the genome of the tumor specimen, preferably the entire genome. Therefore, the present invention may include identifying cancer mutation signs in the genome, preferably the entire genome, of one or more cancer cells. In one embodiment, the step of identifying cancer-specific somatic mutations in a tumor specimen from a cancer patient includes identifying a broad cancer mutation profile of the genome.

[0162] In one embodiment, cancer-specific somatic mutations or sequence differences are determined by the exome, preferably the entire exome, of the tumor specimen. Therefore, the present invention may include identifying cancer mutation signs in the exome, preferably the entire exome, of one or more cancer cells. In one embodiment, the step of identifying cancer-specific somatic mutations in a tumor specimen from a cancer patient includes identifying a broad cancer mutation profile of the exome.

[0163] In one embodiment, cancer-specific somatic mutations or sequence differences are determined by the transcriptome, preferably the entire transcriptome of the tumor specimen. Therefore, the present invention may also include identifying cancer mutation signs in the transcriptome, preferably the entire transcriptome, of one or more cancer cells. In one embodiment, the step of identifying cancer-specific somatic mutations in a tumor specimen from a cancer patient includes identifying a broad cancer mutation profile of the transcriptome.

[0164] In one embodiment, the step of identifying cancer-specific somatic cell mutations or sequence differences includes sequencing of one or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, single-cell cancer cells. Thus, the present invention may also include identifying cancer mutation signs in the one or more cancer cells. In one embodiment, the cancer cells are circulating tumor cells. Cancer cells such as circulating tumor cells can be isolated before single-cell sequencing.

[0165] In one embodiment, the step of identifying cancer-specific somatic cell mutations or sequence differences includes the use of next-generation sequencing (NGS).

[0166] In one embodiment, the step of identifying cancer-specific somatic mutations or sequence differences includes sequencing of genomic DNA and / or RNA of a tumor specimen.

[0167] To identify cancer-specific somatic cell mutations or sequence differences, sequence information obtained from tumor specimens is preferably compared with reference sequence information obtained from sequencing nucleic acids such as DNA or RNA from normal, non-cancerous cells, such as germline cells, which can be obtained from either the patient or a different individual. In one embodiment, germline DNA of a normal genome is obtained from mononuclear cells (PBMCs) in peripheral blood.

[0168] The term "genome" refers to the total amount of genetic information contained in the chromosomes of an organism or cell.

[0169] The term "exome" refers to a portion of an organism's genome formed by exons that code for expressed gene segments. Exomes provide the genetic blueprint used in the synthesis of proteins and other functional gene products. They are the most functionally relevant part of the genome and, therefore, appear to contribute most to the organism's phenotype. The exome of the human genome is estimated to account for 1.5% of the total genome (Ng, PC et al., PLoS Gen., 4(8): p1-15, 2008).

[0170] The term "transcriptome" refers to the collection of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNAs, produced in a single cell or a group of cells. In the context of this invention, the transcriptome means the collection of all RNA molecules produced in a single cell, a group of cells, preferably a group of cancer cells, or all cells of a given individual at a given point in time.

[0171] "Nucleic acid" in this invention is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro transcribed RNA (IVTRNA) or synthetic RNA. Nucleic acid includes genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acid can exist as single-stranded or double-stranded molecules and linear or covalently cyclically closed molecules. Nucleic acid can be isolated in this invention. The term "isolated nucleic acid" means, in this invention, that nucleic acid is (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) produced by recombinant cloning, (iii) purified, for example by cleavage and separation by gel electrophoresis, or (iv) synthesized, for example by chemical synthesis. Nucleic acid can be used for cell introduction, i.e., transfection, particularly in the form of RNA that can be prepared by in vitro transcription from a DNA template. Furthermore, RNA can be modified before application through sequence stabilization, capping, and polyadenylation.

[0172] The term "genetic material" refers to isolated nucleic acids, either DNA or RNA, a section of a double helix, a section of a chromosome, or the entire genome of an organism or cell, particularly its exome or transcriptome.

[0173] The term "mutation" refers to a change or difference in a nucleic acid sequence (nucleotide substitution, addition, or deletion) compared to a reference. "Somatic mutations" can occur in any cell of the body except germ cells (sperm and egg cells) and are therefore not transmitted to offspring. These alterations may (but not necessarily) cause cancer or other diseases. Preferably, the mutation is a non-synonymous mutation. The term "non-synonymous mutation" refers to a mutation that results in an amino acid change, such as an amino acid substitution, in the translation product, preferably a nucleotide substitution.

[0174] In this invention, the term "mutation" includes point mutations, indels, fusions, chromothripsis, and RNA editing.

[0175] In this invention, the term "indel" describes a special class of mutations defined as those resulting in coexisting insertions and deletions and net gains or losses in nucleotides. In the coding regions of the genome, indels that are not multiples of 3 in length are frameshift mutations. Indels can be contrasted with point mutations; indels insert and delete nucleotides from a sequence, while point mutations are a form of substitution that replaces one nucleotide.

[0176] Fusion can generate hybrid genes formed from two originally separate genes. This can occur as a result of translocation, interstitial deletion, or chromosomal inversion. Often, fusion genes are oncogenes. Oncogenic fusion genes may produce gene products with new or different functions from the two fusion partners. Alternatively, proto-oncogenes may fuse to become strong promoters, thereby ensuring that their oncogenic function is upregulated by the strong promoter of the upstream fusion partner. Transcription of oncogenic fusions can also be caused by trans-splicing or read-through events.

[0177] In this invention, the term "chromothripsis" refers to the phenomenon in which specific regions of the genome are disrupted and then spliced ​​together through a single catastrophic event.

[0178] In this invention, the term "RNA editing" refers to a molecular process in which the information content in an RNA molecule is altered by chemical changes in the structure of the bases. RNA editing includes nucleoside modification, such as cytidine (C) to uridine (U) and adenosine (A) to inosine (I), deamination, and the addition and insertion of non-template nucleotides. RNA editing in mRNA effectively alters the amino acid sequence of the encoded protein, resulting in an amino acid sequence that differs from that predicted by the genomic DNA sequence.

[0179] The term "signs of cancer mutation" refers to a set of mutations present in cancer cells when compared to non-cancer reference cells.

[0180] In this invention, a “reference” can be used to correlate and compare results from tumor specimens. Typically, a “reference” can be obtained as a baseline for one or more normal specimens, patients, or one or more different individuals, preferably healthy individuals, especially individuals of the same species, and particularly specimens unaffected by cancer. A “reference” can be determined empirically by testing a sufficiently large number of normal specimens.

[0181] While any suitable sequencing method can be used to determine mutations according to the present invention, next-generation sequencing (NGS) technology is preferred. Third-generation sequencing methods may replace NGS technology in the future and speed up the sequencing process of said method. For the purpose of clarification: The term “next-generation sequencing” or “NGS” in relation to the present invention means all novel, high-performance sequencing technologies that read nucleic acid templates randomly and in parallel along the whole genome by breaking down the whole genome into smaller fragments, in contrast to “conventional” sequencing methods known as Sanger chemistry. Such NGS technologies (also known as large-scale parallel sequencing technologies) can deliver nucleic acid sequence information of the whole genome, exome, transcriptome (all transcribed sequences of the genome), or methylome (all methylated sequences of the genome) in a very short period of time, for example, within 1-2 weeks, preferably within 1-7 days, or most preferably less than 24 hours, enabling, in principle, a simple cell sequencing method. Several commercially available or literature-referenced NGS platforms can be used in relation to the present invention, for example, as described in detail in Zhang et al. 2011: The impact of next-generation sequencing on genomics. J. Genet Genomics 38 (3), pp. 95-109; or Voelkerding et al. 2009: Next generation sequencing: From basic research to diagnostics. Clinical Chemistry 55, pp. 641-658. Non-limiting examples of such NGS technologies / platforms are as follows: 1) For example, sequencing by a synthetic technique known as pyrosequencing, performed on the GS-FLX454 genome sequencer™ of Roche affiliate 454 Life Sciences (Branford, Connecticut), as first described in Ronaghi et al., 1998: A sequencing method based on real-time pyrophosphate. Science 281 (5375), pp. 363-365. This technique uses emulsion PCR, in which single-stranded DNA-bound beads are encapsulated in aqueous micelles containing an oil-surrounded PCR reaction product by vigorous vortexing for emulsion PCR amplification. During the pyrosequencing process, light is emitted from a phosphate molecule during nucleotide incorporation, and the synthesis of the DNA strand by polymerase is recorded.

[0182] 2) Developed by Solexa (now part of Illumina Inc., San Diego, California), this is a synthetic sequencing method based on reversible dye terminators, performed on, for example, Illumina / Solexa's Genome Analyzer™ and Illumina HiSeq2000 Genome Analyzer™. In this technique, all four nucleotides are simultaneously added to a cluster fragment using oligo-primers, along with DNA polymerase, in a fluid cell channel. For sequencing, the cluster chain is extended by bridge amplification using all four fluorescently labeled nucleotides.

[0183] 3) For example, sequencing by ligation techniques performed on the SOLid® platform of Applied Biosystems (now Life Technologies Corporation, Carlsbad, California). In this technique, a pool of all possible oligonucleotides of a fixed length is labeled according to the sequenced position. The oligonucleotides are annealed and ligated; preferential ligation with DNA ligase to match the sequences produces a signal that gives information about the nucleotide at that position. Prior to sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing only copies of the same DNA molecule, are deposited on a glass slide. As a second example, the Polonator® G.007 platform of Dover Systems (Salem, New Hampshire) also uses sequencing by ligation techniques using emulsion PCR based on randomly aligned beads to amplify DNA fragments for parallel sequencing.

[0184] 4) Single-molecule sequencing techniques performed, for example, on the PacBio RS system at Pacific Biosciences (Menlo Park, California) or the HeliScope® platform at Helicos Biosciences (Cambridge, Massachusetts). A unique feature of this technique is that it can sequence a single DNA or RNA molecule without amplification, and it is defined as Single-Molecule Real-Time (SMRT) DNA sequencing. For example, HeliScope uses a highly sensitive fluorescence detection system to detect each nucleotide directly as it is synthesized. A similar technique based on fluorescence resonance energy transfer (FRET) has been developed by Visigen Biotechnology (Houston, Texas). Other fluorescence-based single-molecule techniques are by USGenomics (GeneEngine®) and Genovoxx (AnyGene®).

[0185] 5) Nanotechnology for single-molecule sequencing, for example, using various nanostructures arranged on a chip to monitor the movement of polymerase molecules on a single strand during replication. Non-limiting examples of nanotechnology-based methods include the GridON® platform from Oxford Nanopore Technologies (Oxford, UK), the Hybridization-Assisted Nanopore Sequencing (HANS®) platform developed by Nabsys (Providence, Rhode Island), and a proprietary ligase-based DNA sequencing platform using DNA nanoball (DNB) technology called Combinatorial Probe Anchor Ligation (cPAL®).

[0186] 6) For example, electron microscopy-based techniques for single-molecule sequencing developed by LightSpeed ​​Genomics (Sunnyvale, California) and Halcyon Molecular (Redwood City, California). 7) Ion semiconductor sequencing based on the detection of hydrogen ions released during DNA polymerization. For example, Ion Torrent Systems (San Francisco, California) uses a high-density array of micromechanized wells to carry out this biochemical process in a large number of parallel methods. Each well holds a different DNA template. Below the well is an ion-sensitive layer, beneath which is a proprietary ion sensor.

[0187] Preferably, DNA and RNA preparations serve as starting materials for NGS. Such nucleic acids can be readily obtained from samples such as biological materials, for example, from fresh, flash-frozen, or formalin-fixed paraffin-embedded tumor tissue (FFPE) or from fresh isolated cells or from cytotoxic tumor cells (CTCs) present in the peripheral blood of a patient. Normal, non-mutant genomic DNA or RNA can be extracted from normal somatic cell tissue, however, germline cells are preferred in relation to the present invention. Germline DNA or RNA can also be extracted from mononuclear cells (PBMCs) in the peripheral blood of patients without hematological malignancies. Nucleic acids extracted from FFPE tissue or freshly isolated single cell populations are highly fragmented, but they are suitable for NGS application.

[0188] Several targeted NGS methods for exome sequencing have been described in the literature (see, for example, Teer and Mullikin 2010: Human Mol Genet 19(2), R145-51 for an overview), and all of them can be used in conjunction with the present invention. Many of these methods employ hybridization techniques (described, for example, as genome capture, genome distribution, genome enrichment, etc.) and include array-based (e.g., Hodges et al., 2007: Nat. Genet. 39, pp. 1522-1527) and liquid-based (e.g., Choi et al., 2009: Proc. Natl. Acad. Sci USA 106, pp. 19096-19101) hybridization methods. Commercially available kits for DNA sample preparation and subsequent exome capture are also available: for example, Illumina Inc. (San Diego, California) offers the TruSeq® DNA Sample Preparation Kit and the TruSeq® Exome Enrichment Kit.

[0189] To reduce the number of false positives in the detection of cancer-specific somatic cell mutations or sequence differences, it is preferable to determine the sequences in one or both copies of a reference sample, such as a germline sample, when comparing the sequence of a tumor sample with the sequence of a reference sample. Therefore, it is preferable that the sequence of a reference sample, such as a germline sample, be determined two, three, or more times. Alternatively, in addition to that, the sequence of the tumor sample may also be determined two, three, or more times. It may also be possible to determine the sequence of a reference sample, such as a germline sample, and / or the tumor sample more than once by determining the sequence of the genomic DNA of the reference sample and / or the tumor sample at least once, and the sequence of the RNA at least once. For example, by determining the variation between copies of a reference sample, such as a germline sample, the expected rate of false positive (FDR) somatic cell mutations can be estimated statistically. Technical repeats of a sample should produce the same result, and any mutation detected in this "same vs. same comparison" is a false positive. In particular, to determine the false detection rate for somatic mutations in tumor samples relative to a reference sample, the number of false positives can be estimated by using technical repeats of the reference sample as a reference. Furthermore, various quality-related metrics (e.g., scope or SNP quality) can be combined into a single quality score using machine learning techniques. For a given somatic variation, all other variations with exceptional quality scores can be counted, enabling the grading of all variations in the dataset.

[0190] In relation to the present invention, the term “RNA” refers to a molecule comprising at least one ribonucleotide residue, preferably composed entirely or substantially of ribonucleotide residues. “Ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term “RNA” includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinant RNA, e.g., modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may include, for example, the addition of non-nucleotide material to the terminus (one or more) of the RNA, or the addition of one or more nucleotides within the RNA, e.g., internally. Nucleotides in an RNA molecule may also include non-standard nucleotides, e.g., nucleotides that do not occur naturally, or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0191] In this invention, the term "RNA" includes, and preferably relates to, "mRNA." The term "mRNA" means "messenger RNA" and relates to a "transcript" produced by using a DNA template, encoding a peptide or polypeptide. Typically, mRNA includes a 5'-UTR, a protein-coding region, a 3'-UTR, and optionally a poly(A) tail. mRNA has a limited half-life in cells and in vitro. In relation to this invention, mRNA can be generated from a DNA template by in vitro transcription. Methods of in vitro transcription are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0192] The present invention also allows for the modification of RNA stability and translation efficiency as required. For example, RNA can be stabilized by one or more modifications having an RNA stabilizing effect and / or increased translation efficiency, thereby increasing its translation. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. To increase the expression of RNA used in the present invention, RNA can be modified to increase mRNA stability, perform codon optimization, and thus enhance translation in cells by increasing the GC- content, preferably without altering the sequence of the expressed peptide or protein, within the coding region, i.e., the sequence encoding the expressed peptide or protein.

[0193] In relation to RNA used in this invention, the term "modification" includes any modification of the RNA that does not exist in nature.

[0194] In one embodiment of the present invention, the RNA used herein does not have uncapped 5'-triphosphate esters. Removal of such uncapped 5'-triphosphate esters can be achieved by treating the RNA with a phosphatase.

[0195] The RNA according to the present invention may also have modified ribonucleotides to increase its stability and / or reduce its cytotoxicity. For example, in one embodiment, in the RNA used according to the present invention, 5-methylcytidine is partially or completely, preferably completely substituted for cytidine. Or, in addition, in one embodiment, in the RNA used according to the present invention, pseudouridine is partially or completely, preferably completely substituted for uridine.

[0196] In one embodiment, the term “modified” refers to providing RNA with a 5'-cap or a 5'-cap analog. The term “5'-cap” refers to a cap structure found at the 5'-terminus of an mRNA molecule, and generally consists of a guanosine nucleotide attached to the mRNA via an unusual 5'-to-5' triphosphate ester linkage. In one embodiment, this guanosine is methylated at the 7-position. The term “conventional 5'-cap” refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanosine cap (m 7 G) refers to the present invention. In relation to the present invention, the term "5'-cap" includes 5'-cap analogs that are similar to RNA cap structures and are modified to have the ability to stabilize RNA and / or enhance RNA translation when bound to them in vivo and / or in cells.

[0197] Providing a 5'-cap or 5'-cap analog to RNA can be achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analog, where the 5'-cap is incorporated into the RNA chain produced by co-transcription, or, for example, by in vitro transcription, RNA is generated and the 5'-cap is conjugated to the RNA after transcription using a capping enzyme, such as the capping enzyme of vaccinia virus.

[0198] The RNA may also undergo further modifications. For example, further modifications of the RNA used in the present invention may include extension or transection of the naturally occurring poly(A) tail or alteration of the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR unrelated to the coding region of the RNA, or, for example, replacement or insertion of one or more, preferably two copies, of an existing 3'-UTR with a globin gene, such as alpha-2-globin, alpha-1-globin, beta-globin, preferably beta-globin, more preferably human beta-globin.

[0199] RNA with an unshielded poly(A) sequence is translated more efficiently than RNA with a shielded poly(A) sequence. The terms "poly(A) tail" or "poly(A) sequence" typically refer to the sequence of adenyl (A) residues located at the 3' end of an RNA molecule, and "unshielded poly(A) sequence" means a poly(A) sequence that has the A of the poly(A) sequence at the 3' end of the RNA molecule and is not followed by any nucleotides other than A at the 3' end, i.e., downstream of the poly(A) sequence. Furthermore, long poly(A) sequences of approximately 120 base pairs produce optimal transcript stability and RNA translation efficiency.

[0200] Therefore, in order to increase the stability and / or expression of the RNA used in the present invention, the RNA may be modified to be present in conjunction with a polyA sequence having a length of preferably 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, and particularly 100 to 150 adenosine residues. In a particularly preferred embodiment, the polyA sequence has a length of about 120 adenosine residues. In order to further increase the stability and / or expression of the RNA used in the present invention, the polyA sequence may not be shielded.

[0201] In addition, the incorporation of an RNA molecule into a 3'-untranslated region (UTR) can result in an enhancement of translation efficiency. Synergistic effects can be achieved by incorporating two or more such 3'-untranslated regions. The 3'-untranslated regions may be autologous or heterologous to the RNA into which they are introduced. In a particular embodiment, the 3'-untranslated region is derived from the human β-globin gene.

[0202] The combinations of modifications described above, namely the incorporation of poly(A) sequences, unshielded poly(A) sequences, and one or more 3' untranslated regions, have a synergistic effect on enhancing RNA stability and translation efficiency.

[0203] The term "stability" of RNA relates to its "half-life." Half-life refers to the time required to eliminate half of the activity, quantity, or number of molecules. In the context of this invention, the half-life of RNA indicates the stability of the RNA. The half-life of RNA can affect the "duration of expression" of RNA. RNA with a long half-life can be expected to be expressed for a long period of time.

[0204] Needless to say, if it is desired to reduce the stability and / or translation efficiency of RNA according to the present invention, it is possible to enhance the stability and / or translation efficiency of RNA by modifying the RNA to interfere with the function of the elements described above.

[0205] The term "expression" is used in the present invention in its most general sense, and includes, for example, the production of RNA and / or peptides, polypeptides, or proteins by transcription and / or translation. With respect to RNA, the terms "expression" or "translation" particularly relate to the production of peptides, polypeptides, or proteins. This term also includes the partial expression of nucleic acids. Furthermore, expression can be transient or stable.

[0206] In this invention, the term "expression" also includes "atypical expression" or "abnormal expression." In this invention, "atypical expression" or "abnormal expression" means that the expression is altered, preferably increased, compared to a reference, e.g., a subject without a disease associated with atypical or abnormal expression of a protein, e.g., a tumor antigen. An increase in expression refers to an increase of at least 10%, particularly at least 20%, at least 50%, or at least 100%, or more. In one embodiment, the expression is found only in diseased tissue, while the expression in healthy tissue is suppressed.

[0207] The term "specifically expressed" means that a protein is expressed essentially only in a specific tissue or organ. For example, if a tumor antigen is specifically expressed in the gastric mucosa, it means that the protein is primarily expressed in the gastric mucosa and not expressed in other tissues or to a significant degree in other types of tissues or organs. Therefore, a protein that is expressed only in gastric mucosal cells and to a significantly lesser degree in any other tissue, such as the testes, is specifically expressed in gastric mucosal cells. In some embodiments, a tumor antigen may be specifically expressed in more than one type of tissue or organ, e.g., two or three types of tissue or organs, but preferably three or fewer different types of tissue or organs, under normal conditions. In this case, the tumor antigen is specifically expressed in these organs at that time. For example, if a tumor antigen is expressed in the lungs and stomach, preferably to about the same degree, under normal conditions, then the tumor antigen is specifically expressed in the lungs and stomach.

[0208] In relation to the present invention, the term “transcription” refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into a protein. By the present invention, the term “transcription” includes “in vitro transcription,” which refers to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied for the generation of the transcript. These cloning vectors are generally referred to as transcription vectors and are encompassed within the term “vector” according to the present invention. By the present invention, the RNA used is preferably in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter controlling the transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter according to the present invention. The DNA template for in vitro transcription can also be obtained by cloning nucleic acids, particularly cDNA, which are introduced into a suitable vector for in vitro transcription. cDNA can also be obtained by reverse transcription of RNA.

[0209] The term "translation" in this invention relates to a process in cellular ribosomes in which a chain of messenger RNA directs the assembly of amino acid sequences to produce a peptide, polypeptide, or protein.

[0210] The expression-regulating or controlling sequences that can be functionally linked to nucleic acids according to the present invention may be homologous or heterologous with respect to the nucleic acid. When a coding sequence and a regulatory sequence are covalently linked together, they are "functionally" linked together, and as a result, the transcription or translation of the coding sequence is under the control or influence of the regulatory sequence. If the coding sequence is to be translated into a functional protein by the functional linkage of the coding sequence and the regulatory sequence, then induction of the regulatory sequence results in the transcription of the coding sequence without causing a shift in the read frame in the coding sequence or the inability of the coding sequence to be translated into the desired protein or peptide.

[0211] The term “expression regulatory sequence” or “regulatory sequence” as used in this invention includes promoters, ribosome-binding sequences, and other regulatory elements that control the transcription of nucleic acids or the translation of induced RNA. In some embodiments of this invention, regulatory sequences can be controlled. The precise structure of regulatory sequences may vary depending on the species or cell type, but generally includes 5'-untranscribed and 5'- and 3'-untranslated sequences, which are involved in the initiation of transcription or translation, such as TATA boxes, capping sequences, and CAAT sequences. In particular, 5'-untranscribed regulatory sequences include promoter regions containing promoter sequences for the transcriptional control of functionally bound genes. Regulatory sequences may also include enhancer sequences or upstream activator sequences.

[0212] Preferably, according to the present invention, the RNA to be expressed in the cell is introduced into the cell. In one embodiment of the method according to the present invention, the RNA to be introduced into the cell is obtained by in vitro transcription of a suitable DNA template.

[0213] In this invention, terms such as "expressible RNA" and "RNA-encoded" are used interchangeably herein, and with respect to a particular peptide or polypeptide, RNA can be expressed and produce the peptide or polypeptide if present in a suitable environment, preferably within a cell. Preferably, RNA can interact with the cellular translation mechanism to provide an expressible peptide or polypeptide according to this invention.

[0214] Terms such as “import,” “introduce,” or “transfect” are used interchangeably herein and relate to the introduction of nucleic acids, particularly exogenous or heterologous nucleic acids, especially RNA, into cells. The present invention allows cells to form organs, tissues, and / or parts of organisms. The present invention achieves nucleic acid administration either as naked nucleic acids or in combination with an administration reagent. Preferably, nucleic acid administration is in the form of naked nucleic acids. Preferably, RNA is administered in combination with a stabilizing substance such as an RNase inhibitor. The present invention also envisions repeated introduction of nucleic acids into cells, enabling long-lasting expression.

[0215] Cells can be transfected with any carrier to which RNA may be associated, for example, by forming a complex with RNA or by forming vesicles in which RNA is sealed or encapsulated, resulting in increased RNA stability compared to naked RNA. Useful carriers according to the present invention include, for example, lipid-containing carriers, such as cationic lipids, liposomes, particularly cationic liposomes, micelles, and nanoparticles. Cationic lipids can form complexes with negatively charged nucleic acids. Any cationic lipid can be used according to the present invention.

[0216] Preferably, the introduction of RNA encoding a peptide or polypeptide into cells, particularly cells present in vivo, results in the expression of the peptide or polypeptide within the cells. In certain embodiments, it is preferable that the nucleic acid targets specific cells. In such embodiments, the carrier applied for the administration of the nucleic acid to cells (e.g., a retrovirus or liposome) indicates the target molecule. For example, molecules such as surface membrane proteins on target cells or ligand-specific antibodies against receptors on target cells can be incorporated into or bound to the nucleic acid carrier. When the nucleic acid is administered by liposomes, proteins that bind to surface membrane proteins associated with endocytosis may be incorporated into the liposomal formulation to enable targeting and / or uptake. Such proteins include capsid proteins of their fragments specific to particular cell types, antibodies against internalized proteins, proteins that target intracellular locations, and others.

[0217] The terms “cell” or “host cell” preferably refer to a complete cell, i.e., a cell having its normal intracellular components such as a complete membrane, enzymes, organelles, or genetic material that are not released. A complete cell is preferably a viable cell, i.e., a living cell capable of performing its normal metabolic functions. Preferably, the terms refer to any cell that can be transfected or transfected with exogenous nucleic acids according to the present invention. The terms “cell” according to the present invention include prokaryotic cells (e.g., Escherichia coli (E. coli)) or eukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells). Exogenous nucleic acids may be found intracellularly (i) as is, freely dispersed, (ii) incorporated into a recombinant vector, or (iii) incorporated into the host cell genome or mitochondrial DNA. Mammalian cells, such as those from humans, mice, hamsters, pigs, goats, and primates, are particularly preferred. The cells may be derived from numerous tissue types and may include primary cells and cell lineages. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In further embodiments, the cells are antigen-presenting cells, particularly dendritic cells, monocytes, or macrophages.

[0218] Cells containing nucleic acid molecules preferably express peptides or polypeptides encoded by the nucleic acid.

[0219] The term "clonal proliferation" refers to the process by which a specific entity increases. In the context of this invention, the term is preferably used in relation to an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and specific lymphocytes that recognize the antigen are amplified. Preferably, clonal proliferation leads to lymphocyte differentiation.

[0220] Terms such as “reduce” or “inhibit” preferably relate to the ability to produce an overall reduction of 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more, in terms of level. The term “inhibit” or similar phrases include complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0221] Terms such as “increase,” “enhance,” “promote,” or “prolong” preferably relate to an increase, enhancement, promotion, or prolongation of at least about 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 80%, preferably at least 100%, preferably at least 200%, and particularly at least 300%. These terms may also relate to an increase, enhancement, promotion, or prolongation from zero or an unmeasurable or undetectable level to a level greater than zero or a measurable or detectable level.

[0222] The present invention provides vaccines, such as cancer vaccines, which are preferably designed based on modified proteins or protein fragments or amino acid modifications, and are predicted to be useful in immunotherapy by the method of the present invention.

[0223] In this invention, the term "vaccine" refers to a pharmaceutical preparation (pharmaceutical composition) or product that, upon administration, induces an immune response, particularly a cellular immune response, that recognizes and attacks pathogens or disease cells, such as cancer cells. Vaccines can be used for the prevention or treatment of disease. The terms "personalized cancer vaccine" or "personalized cancer vaccine" refer to a specific cancer patient and mean that the cancer vaccine is adapted to the individual patient's needs or special circumstances.

[0224] In one embodiment, the vaccine provided by the present invention may include a peptide or polypeptide comprising one or more amino acid modifications, or one or more modified peptides predicted to be useful in immunotherapy by the method of the present invention, or a nucleic acid, preferably RNA, encoding the peptide or polypeptide.

[0225] The cancer vaccines provided by the present invention, when administered to a patient, preferably provide one or more T cells with an appropriate epitope to stimulate, prime, and / or increase T cells specific to the patient's disease cells, such as the patient's tumor. These T cells are preferably directed towards cells expressing an antigen that induces the T cell epitope. The vaccines described herein can preferably induce or enhance a cellular response to cancerous disease characterized by the presentation of one or more tumors associated with neoantigens having class I MHC, preferably cytotoxic T cell activity. Vaccines targeting cancer-specific mutations would be specific to the patient's tumor.

[0226] The vaccines provided by the present invention provide, when administered to a patient, one or more T cell epitopes incorporating amino acid modifications or modified peptides predicted to be immunogenic by the method of the present invention, for example, 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, and preferably up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, or 30 T cell epitopes. Such T cell epitopes are also referred to herein as “neoepitopes”. Presentation of these epitopes by patient cells, particularly antigen-presenting cells, preferably generates T cells that target the epitope when bound to MHC, and therefore the patient’s tumor, preferably a primary tumor and tumor metastases, express antigens that induce the T cell epitope and present the same epitope on the surface of the tumor cells.

[0227] The method of the present invention may include further steps to determine the usefulness of the identified amino acid modification or modified peptide for cancer vaccination. Accordingly, the further steps may include one or more of the following: (i) assessing whether the modification localizes to a known or predicted MHC-presented epitope; (ii) testing in vitro and / or in silico whether the modification localizes to an MHC-presented epitope, for example, testing whether the modification is part of a treated peptide sequence and / or presented as an MHC-presented epitope; and (iii) testing in vitro whether the conceived, modified epitope can stimulate T cells, such as patient T cells, with desired specificity, particularly if it exists in relation to their natural sequences, for example, if the amino acid sequences are adjacent, if it is adjacent to the epitope of a naturally occurring protein, and if it is expressed in antigen-presenting cells. Such adjacent sequences can each contain 3 or more, 5 or more, 10 or more, 15 or more, 20 or more, and preferably up to 50, up to 45, up to 40, up to 35, or 30 amino acids, and can be adjacent to an epitope sequence at the N-terminus and / or C-terminus.

[0228] The modified peptides determined by the present invention can be graded for their usefulness as epitopes for cancer vaccine administration. Accordingly, in one embodiment, the present invention includes a manual or computer-based analytical process in which the identified modified peptides are analyzed and selected for their usefulness in each vaccine to be provided. In a preferred embodiment, the analytical process is a computer algorithm-based process. Preferably, the analytical process includes determining and / or grading the epitopes according to predictions of their ability to be immunogenic.

[0229] The neoepitopes identified by the present invention and provided by the vaccine of the present invention preferably exist in the form of a polypeptide containing the neoepitope, such as a polyepitope polypeptide, or a nucleic acid encoding the polypeptide, particularly RNA. Furthermore, the neoepitope may also exist in the form of a vaccine sequence within the polypeptide, i.e., adjacent amino acid sequences adjacent to the epitope in naturally occurring proteins, for example, existing in relation to their natural sequences, may also be present. Each of these adjacent sequences may contain 5 or more, 10 or more, 15 or more, 20 or more, and preferably up to 50, up to 45, up to 40, up to 35, or 30 amino acids, and may be adjacent to the N-terminus and / or C-terminus of the epitope sequence. Thus, the vaccine sequence may contain 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, and preferably up to 50, up to 45, up to 40, up to 35, or 30 amino acids. In one embodiment, the neoepitope and / or vaccine sequence are arranged head-to-tail in the polypeptide.

[0230] In one embodiment, neoepitopes and / or vaccine sequences are separated by linkers, particularly neutral linkers. The term “linker” in this invention refers to a peptide that is added between two peptide domains, such as an epitope or vaccine sequence, and connects to the peptide domains. There are no particular restrictions on the linker sequence. However, it is preferable that the linker sequence reduces steric hindrance between the two peptide domains so that it is well translated and supports or enables the processing of the epitope. Furthermore, the linker should not have immunogenic sequence elements, or should have only very few. Preferably, the linker should not create non-endogenous neoepitopes, such as those arising from junctions of conjugations between adjacent neoepitopes, which may produce an undesirable immune response. Therefore, vaccines of polyepitopes should preferably contain linker sequences that can reduce the number of undesirable MHC-binding conjugation epitopes. Hoyt et al. (EMBO J. 25 (8), p1720-9, 2006) and Zhang et al. (J. Biol. Chem., 279 (10), p8635-41, 2004) showed that glycine-rich sequences impair proteasome processing, and therefore the use of glycine-rich linker sequences acts to minimize the number of linker-containing peptides that can be processed by the proteasome. Furthermore, it was observed that glycine inhibits strong binding at the MHC binding groove site (Abastado et al., J. Immunol. 151 (7), p3569-75, 1993). Schlessinger et al. (Proteins, 61(1), pp. 115-26, 2005) found that the inclusion of amino acids, glycine, and serine in the amino acid sequence results in a more flexible protein that is more efficiently translated and processed by the proteasome, allowing for better access to the encoded neoepitope. Each linker can contain 3 or more, 6 or more, 9 or more, 10 or more, 15 or more, 20 or more, and preferably up to 50, up to 45, up to 40, up to 35, or up to 30 amino acids. Preferably, the linkers are enriched with glycine and / or serine amino acids.Preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the linker's amino acids are glycine and / or serine. In one preferred embodiment, the linker is substantially composed of amino acids, glycine, and serine. In one embodiment, the linker is an amino acid sequence (GGS). a (GSS) b (GGG) c (SSG) d (GSG) e This includes, where a, b, c, d, and e are numbers independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and a+b+c+d+e is different from 0 and preferably 2 or more, 3 or more, 4 or more, or 5 or more. In one embodiment, the linker includes a linker sequence described herein, including an example of the sequence GGSGGGGSG.

[0231] In one particularly preferred embodiment, a polypeptide incorporating one or more neoepitopes, such as a polyepitope polypeptide, is administered to a patient by the present invention in the form of a nucleic acid, preferably RNA, such as RNA transcribed or synthesized in vitro, which can be expressed in cells, such as the patient's antigen-presenting cells, to produce polypeptides. For the purposes of the present invention, the present invention also envisions the administration of one or more multiepitope polypeptides, which are preferably included by the term "polyepitope polypeptide," preferably in the form of a nucleic acid, preferably RNA transcribed or synthesized in vitro, which can be expressed in cells, such as the patient's antigen-presenting cells, to produce one or more polypeptides. In the case of administration of more than one multiepitope polypeptide, the neoepitopes provided by the different multiepitope polypeptides may be different or partially overlapping. Once present in cells, such as the patient's antigen-presenting cells, the polypeptide is processed by the present invention to produce neoepitopes, which are then identified by the present invention. The administration of the vaccine provided by the present invention preferably provides an epitope presenting MHC class I that can elicit a CD8+ helper T cell response to cells expressing an antigen that induces the MHC-presented epitope. The administration of the vaccine provided by the present invention may also provide an epitope presenting MHC class II that can also elicit a CD4+ T cell response to cells expressing an antigen that induces the MHC-presented epitope. Furthermore, the administration of the vaccine provided by the present invention may provide one or more neoepitopes (including known neoepitopes and neoepitopes identified by the present invention) and one or more epitopes that do not contain cancer-specific somatic mutations but are expressed by cancer cells and preferably induce an immune response against cancer cells, preferably a cancer-specific immune response.

[0232] The vaccine provided by the present invention may be a recombinant vaccine.

[0233] In relation to the present invention, the term "recombinant" means "produced by genetic engineering." Preferably, "recombinant entities," such as recombinant polypeptides in relation to the present invention, are the result of combinations of entities, such as amino acids or nucleic acid sequences, that do not occur naturally and preferably do not combine naturally. For example, recombinant polypeptides in relation to the present invention may contain several amino acid sequences, such as neoepitopes or vaccine sequences derived from different proteins or different parts of the same protein fused together, for example, by peptide bonds or appropriate linkers.

[0234] As used herein, the term "naturally occurring" refers to the fact that a substance can be found in nature. For example, a peptide or nucleic acid that is present in a living organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is considered naturally occurring.

[0235] The agents and compositions described herein can be used to treat subjects having diseases characterized by the presence of disease cells expressing antigens and presenting their fragments. Cancer is a particularly preferred disease. The agents and compositions described herein can also be used for immunization or vaccination to prevent the diseases described herein.

[0236] The term “disease” refers to an abnormal condition affecting an individual’s body. A disease is often interpreted as a pathological condition associated with specific symptoms and signs. Diseases can be caused by factors originating from external sources, such as infectious diseases, or by internal dysfunctions, such as autoimmune diseases. In humans, “disease” is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the suffering individual, or similar problems for those in contact with the individual. In this broader sense, disease sometimes includes injury, incapacity, disability, syndrome, infection, solitary symptoms, abnormal behavior, and atypical changes in structure and function; however, in other relationships and for other purposes, these can also be considered distinct categories. Many diseases, and living with them, can alter a person’s outlook on life and personality; therefore, diseases usually have adverse effects on an individual not only physically but also emotionally.

[0237] The term "normal" refers to a healthy state or a state in a healthy subject or tissue, i.e., a non-pathological state, and "healthy" preferably means not cancerous.

[0238] The term “antigen-associated disease” or “antigen-involved disease” refers to any disease associated with an antigen, such as a disease characterized by the presence of an antigen or cells expressing an antigen. Antigen-involved diseases can be infectious diseases, autoimmune diseases, or cancerous diseases or simply cancer. As described above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen.

[0239] "A disease comprising cells expressing an antigen" means, according to the present invention, that the expression of an antigen is detected in cells of diseased tissue or organs. Expression in cells of diseased tissue or organs may be increased compared to the state in healthy tissue or organs. An increase refers to an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or more. In one embodiment, expression is found only in diseased tissue, while expression in healthy tissue is suppressed. According to the present invention, diseases comprising or associated with cells expressing an antigen include cancerous diseases.

[0240] The term “infectious disease” refers to any disease that can be transmitted from individual to individual or from organism to organism and is caused by a microbial factor (e.g., the common cold). Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases, which are caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases may include, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, and influenza.

[0241] The term "autoimmune disease" refers to any disease in which the body produces an immunogenic (i.e., immune system) response to some component of its own tissues. In other words, the immune system loses its ability to recognize certain tissues or systems within the body as self and attacks them as if they were foreign. Autoimmune diseases can be classified into those in which one organ is primarily affected (e.g., hemolytic anemia and anti-immunothyroiditis) and those in which the autoimmune disease process spreads to many tissues (e.g., systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells attacking the sheaths surrounding nerve fibers in the brain and spinal cord. This results in loss of coordination of muscle movements, weakness, and blurred vision. Autoimmune diseases are well known in the art and include, for example, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immunothyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes mellitus, scleroderma, psoriasis, and the like.

[0242] The term “cancer disease” or “cancer” refers to or describes a physiological condition in an individual characterized typically by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specifically, examples of such cancers include bone cancers, hematological cancers, lung cancers, liver cancers, pancreatic cancers, skin cancers, head and neck cancers, melanomas of the skin or eyes, uterine cancers, ovarian cancers, rectal cancers, anal cancers, gastric cancers, colorectal cancers, breast cancers, prostate cancers, uterine cancers, cancers of the sexual and reproductive organs, Hodgkin’s disease, esophageal cancers, small intestine cancers, endocrine cancers, thyroid cancers, parathyroid cancers, adrenal gland cancers, soft tissue sarcomas, bladder cancers, kidney cancers, renal cell carcinomas, renal pelvis carcinomas, neoplasms of the central nervous system (CNS), neuroectodermal cancers, axial tumors, gliomas, meningiomas, and pituitary adenomas. In this invention, the term "cancer" includes cancer metastasis.

[0243] In this invention, the terms “tumor” or “tumor disease” preferably refer to the abnormal growth of cells that form an enlargement or lesion (referred to as neoplastic cells, oncoplastic cells, or tumor cells). “Tumor cells” means abnormal cells that grow by rapid, uncontrolled cell proliferation and continue to grow even after a stimulus that signals the cessation of new growth. Tumors exhibit a partial or complete deficiency of structural organization and functional coordination with normal tissue, forming a distinctive mass of tissue that can usually be benign, provisionally malignant, or malignant.

[0244] In this invention, the terms "cancer" and "cancer disease" are used interchangeably with the terms "tumor" and "tumor disease."

[0245] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process that depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration into the endothelial basement membrane, entry into body cavities and blood vessels, and then, after being transported by the blood, invasion into the target organ. Finally, the growth of a new tumor at the target site, i.e., a secondary tumor or metastatic tumor, depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, because tumor cells or components may remain and cause potential metastasis. In one embodiment, the term "metastasis" according to the present invention refers to "distant metastasis," which is a metastasis far from the primary tumor and the lymph node system in its region.

[0246] The cells of a secondary or metastatic tumor resemble the cells of the original tumor. This means, for example, that if ovarian cancer metastasizes to the liver, the secondary tumor will be made up of abnormal ovarian cells, not abnormal liver cells. In this case, the tumor in the liver is called metastatic ovarian cancer, not liver cancer.

[0247] The term “circulating tumor cells” or “CTC” refers to cells that detach from a primary tumor or tumor metastasis and circulate in the bloodstream. CTCs can constitute the seeds for subsequent growth of additional tumors (metastases) in different tissues. Circulating tumor cells are found at a frequency of order of 1–10 CTCs per mL of total blood in patients with metastatic disease. Research methods have been developed to isolate CTCs. In the field of CTC isolation, several research methods have been described, for example, techniques that utilize the fact that epithelial cells normally express the cell adhesion protein EpCAM, which is not present in normal blood cells. Immunomagnetic bead-based capture involves treating a blood sample with an antibody against EpCAM conjugated with magnetic particles, and then separating the attached cells in a magnetic field. The isolated cells are then stained with antibodies against another epithelial marker, cytokeratin, and a common leukocyte marker, CD45, to distinguish the rare CTCs from contaminated leukocytes. This robust, semi-automated method identifies CTCs with an average yield of approximately 1 CTC / mL and a purity of 0.1% (Allard et al., 2004: Clin Cancer Res 10, pp. 6897-6904). A second method for separating CTCs uses a microfluidics-based CTC capture device, which involves flowing whole blood through a chamber embedded with 80,000 microposts functionalized by coating with an antibody against EpCAM. The CTCs are then stained with a secondary antibody against either cytokeratin or a tissue-specific marker, such as PSA in prostate cancer or HER2 in breast cancer, and visualized by automated scanning of the microposts in multiple planes along a three-dimensional coordinate system. The CTC chip can identify cytokeratin-positive circulating tumor cells in patients with a median yield of 50 cells / ml and a purity ranging from 1-80% (Nagrath et al., 2007: Nature 450, pp. 1235-1239).Another possibility for isolating CTCs is the use of the CellSearch® Circulating Tumor Cell (CTC) Test from Veridex, LLC (Raritan, New Jersey), which captures, identifies, and counts CTCs in a blood tube. The CellSearch® system is a US Food and Drug Administration (FDA) approved method for counting CTCs in whole blood, and it is based on a combination of immunomagnetic labeling and an automated digital microscope. There are also other methods for isolating CTCs described in the literature, all of which can be used in connection with this invention.

[0248] A relapse or recurrence occurs when a person is affected again by a condition they were previously afflicted with. For example, if a patient has had a tumor and successfully been treated for it, and then develops the disease again, the newly developed disease can be considered a relapse or recurrence. However, according to the present invention, a relapse or recurrence of a tumor may or may not occur at the site of the initial tumor. Therefore, for example, if a patient has had an ovarian tumor and successfully been treated for it, a relapse or recurrence may be the development of an ovarian tumor, or it may be the development of a tumor at a site different from the ovary. A tumor relapse or recurrence also includes situations in which the tumor develops at a site different from the site of the initial tumor, as well as at the site of the initial tumor. Preferably, the initial tumor that the patient has been treated for is a primary tumor, and the tumor at a site different from the site of the initial tumor is a secondary or metastatic tumor.

[0249] The term "immunotherapy" refers to the treatment of a disease or condition by inducing, enhancing, or suppressing an immune response. Immunotherapy designed to elicit or amplify an immune response is classified as activating immunotherapy, while immunotherapy that reduces or suppresses an immune response is classified as suppressive immunotherapy. The term "immunotherapy" includes antigen immunization or antigen vaccination, or tumor immunization or tumor vaccination. The term "immunotherapy" also refers to the manipulation of an inappropriate immune response so that it is adjusted to a more appropriate state in relation to autoimmune diseases, such as rheumatoid arthritis, allergic diseases, diabetes, or multiple sclerosis.

[0250] The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual for the purpose of inducing an immune response, for example, for therapeutic or preventive reasons.

[0251] The term “therapeutic treatment” or simply “treatment” refers to any treatment that improves a health condition and / or extends (increases) the lifespan of an individual. Such treatment may also eliminate disease in an individual, inhibit or delay the development of disease in an individual, impede or delay the development of disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or has previously had the disease.

[0252] The terms “preventive measures” or “preventive measures” refer to any measures intended to prevent disease occurring in an individual. The terms “preventive measures” and “preventive measures” are used interchangeably herein.

[0253] The terms “protect,” “prevent,” “prophylactic,” “preventive,” or “protective” relate to the prevention and / or treatment of the development and / or transmission of disease, such as tumors, in an individual. For example, prophylactic administration of immunotherapy by administering the compositions described herein can protect the receiving individual from tumor development. For example, therapeutic administration of immunotherapy by administering the compositions described herein can halt disease development, resulting in, for example, inhibition of tumor progression / growth. This includes slowing of tumor progression / growth, in particular interruption of tumor progression, which preferably leads to tumor elimination. Therapeutic administration of immunotherapy can protect an individual from, for example, the transmission or metastasis of an existing tumor.

[0254] The terms “individual” or “subject” refer to vertebrates, particularly mammals. For example, in the context of this invention, mammals include humans, non-human primates, domesticated mammals such as dogs, cats, sheep, cattle, goats, pigs, horses, and others, laboratory animals such as mice, rats, rabbits, guinea pigs, and others, and animals in captivity, such as zoo animals. The term “subject” refers to non-mammalian vertebrates, such as birds (particularly domesticated birds, such as chickens, ducks, geese, turkeys, etc.) and fish (particularly farmed fish, such as salmon or catfish). As used herein, the term “animal” also includes humans. Preferably, the term “patient” refers to a diseased individual.

[0255] The agents described herein may be administered in the form of any suitable pharmaceutical composition. The term “pharmaceutical composition” refers to a formulation containing a therapeutically effective agent or a salt thereof, preferably together with pharmaceutical excipients, such as buffers, preservatives, and isotonic modifiers. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of the pharmaceutical composition to an individual. Pharmaceutical compositions are also known in the art as pharmaceutical formulations. Pharmaceutical compositions may be administered topically or systemically.

[0256] The term "systemic administration" refers to the administration of a therapeutically effective drug in which the drug is widely distributed in a significant amount throughout the individual's body to exert a biological effect. In this invention, administration is preferably carried out by parenteral administration.

[0257] The term "parenteral administration" refers to the administration of a therapeutically effective drug in a manner that does not involve the drug passing through the intestines. The term "parenteral administration" includes, but is not limited to, intravenous, subcutaneous, intradermal, or intra-arterial administration.

[0258] In one particularly preferred embodiment, the composition according to the present invention is administered to muscle tissue such as skeletal muscle. Therefore, intramuscular administration, such as by intramuscular injection, is a preferred route of administration.

[0259] Administration can be achieved by various methods. In one embodiment, the composition according to the present invention is administered by injection. In a preferred embodiment, the injection is performed with a needle. Needleless injection may also be used as an alternative.

[0260] The pharmaceutical compositions of the present invention may contain at least one adjuvant. The term “adjuvant” refers to a compound that, when administered to an individual in combination with an antigen or antigenic peptide, prolongs, enhances, or promotes an immune response. Adjuvants are presumed to exert their biological activity through one or more mechanisms, including increasing the surface area of ​​the antigen, prolonging the retention of the antigen in the body, delaying antigen release, targeting of the antigen to macrophages, increasing antigen uptake, enhancing antigen processing, stimulating cytokine release, stimulating and activating immune cells such as B cells, macrophages, dendritic cells, and T cells, and nonspecific activation of immune cells. Adjuvants include a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, but are not limited to, saponins, incomplete Freund's adjuvants, complete Freund's adjuvants, tocopherols, or arum.

[0261] The pharmaceutical compositions according to the present invention are generally applied in "pharmaceutically effective amounts" and "pharmaceutically acceptable formulations."

[0262] The term "pharmaceutical effective dose" refers to the amount that achieves the desired response or effect, either alone or in combination with additional doses. In the case of treating a particular disease, the desired response preferably relates to inhibiting the progression of the disease. This includes slowing the progression of the disease, in particular interrupting or reversing its progression. The desired response in the treatment of a disease may be a delay in the onset of the disease or condition, or it may be prevention of the onset. The effective dose of the compositions described herein will depend on the individual parameters of the patient, including the condition to be treated, the severity of the disease, age, physiological state, build and weight, the duration of treatment, the type of therapy being received concurrently (if any), the specific route of administration, and similar factors. Therefore, the dose administered of the compositions described herein may depend on a variety of such parameters. If the response in the patient is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0263] The term "pharmaceutically acceptable" refers to the non-toxicity of a material that does not interact with the active ingredient of a pharmaceutical composition.

[0264] The pharmaceutical composition of the present invention may contain salts, buffers, preservatives, carriers, and optionally other therapeutic agents. Preferably, the pharmaceutical composition of the present invention comprises one or more pharmaceutically acceptable carriers, diluents, and / or additives.

[0265] The term "additive" is intended to refer to all substances in a pharmaceutical composition that are not active ingredients, such as binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavorings, or colorants.

[0266] The term “diluent” refers to a substance that dilutes and / or reduces a substance. Furthermore, the term “diluent” includes any one or more fluids, liquids, or solid suspensions and / or mixed media.

[0267] The term "carrier" refers to one or more suitable solid or liquid fillers or diluents for administration to humans. The term "carrier" also refers to natural or synthetic organic or inorganic components that are combined with an active ingredient to facilitate its application. Preferably, the carrier component is water or a sterile liquid such as mineral oil or oils derived from animals or plants, such as peanut oil, soybean oil, sesame oil, sunflower oil, and others. Salt solutions and aqueous dextrose and glycerin solutions can also be used as aqueous carrier compounds.

[0268] Pharmaceutically acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (edited by A. R. Gennaro, 1985). Examples of suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, sugars, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting-point waxes, and cocoa butter. Examples of suitable diluents include ethanol, glycerol, and water.

[0269] Pharmaceutical carriers, additives, or diluents may be selected in consideration of the intended route of administration and standard pharmaceutical practices. The pharmaceutical compositions of the present invention may include, or in addition to, one or more carriers, one or more additives, or diluents, any suitable binders, lubricants, suspending agents, coating agents, and / or solubilizers. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, and corn sweeteners, natural and synthetic rubbers such as gum arabic and tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Preservatives, stabilizers, dyes, and even flavoring agents may be supplied in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and p-hydroxybenzoic acid esters. Antioxidants and suspending agents may also be used.

[0270] In one embodiment, the composition is an aqueous composition. The aqueous composition may optionally contain a solute, such as a salt. In one embodiment, the composition is in the form of a freeze-dried composition. The freeze-dried composition can be obtained by freeze-drying each aqueous composition.

[0271] The agents and compositions provided herein can be used alone or in combination with other treatment regimens such as surgery, radiation, chemotherapy and / or bone marrow transplantation (autologous, syngeneic, homogeneous, or unrelated).

[0272] The present invention is described in detail and illustrated with figures and examples, which are for illustrative purposes only and are not intended to limit it. Further embodiments similarly included in the present invention are available to those skilled in the art. [Examples]

[0273] The techniques and methods used herein are either described herein or are publicly known, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd The procedure shall be carried out in accordance with the format described in the Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. All methods, including the use of kits and reagents, shall be carried out according to the manufacturer's instructions unless otherwise specified.

[0274] Example 1: Location of a publicly available epitope-containing protein To identify mutant neoepitopes limited to MHC class I ("publicly available epitopes," n=129), the literature was screened and their locations were compared to a random sample of protein-coding genes ("proteome," n=500) (Figure 1). The location of each gene was determined using the gene ontology database (http: / / www.ebi.ac.uk / QuickGO / ). Furthermore, their presence in exosomes was tested using the ExoCarta database (http: / / www.exocarta.org / ). As shown in Figure 1, neoepitope-containing genes were significantly enriched in exosomes and cytosols compared to control genes (Fisher's exact test; p<0.0001).

[0275] In the second step, the presence of genes from epitopes published in the SEREX database (V. Jongeneel, Cancer Immunity, Vol. 1, p.3 (March 30, 2001)) was compared with random control genes (Figure 2). Proteins on the list of the SEREX database shown to be recognized by autoantibodies. Genes of the published epitopes are found in the SEREX database significantly more frequently compared to random peptides (Fisher's exact test; p<0.0001).

[0276] The results shown in Figures 1 and 2 indicate that the presence of mutant genes in exosomes, cytosols or autoantibody databases is a useful parameter for predicting mutant antigens relevant for immunotherapy. <Further Embodiments of the Invention> [Embodiment 1] A method for predicting the usefulness of a protein or fragment thereof expressed by diseased cells for immunotherapy, comprising determining the distribution or localization of a protein or nucleic acid encoding it, or a protein fragment. [Embodiment 2] The method according to Embodiment 1, comprising determining whether a protein or a nucleic acid encoding it, or a protein fragment, is localized or abundant in the cytosol and / or exosome in vivo. [Embodiment 3] The method according to Embodiment 1 or 2, wherein the localization or enrichment of a protein or nucleic acid encoding it, or a protein fragment, in the cytosol and / or exosome indicates that the protein or its fragment is useful for immunotherapy. [Embodiment 4] A method for predicting the usefulness of a protein or fragment thereof expressed by diseased cells for immunotherapy, comprising determining whether the protein or fragment thereof is cross-presented by antigen-presenting cells, preferably professional antigen-presenting cells. [Embodiment 5] The method according to Embodiment 4, wherein cross-presentation of a protein or its fragment by antigen-presenting cells demonstrates that the protein or its fragment is useful for immunotherapy. [Embodiment 6] The method according to Embodiment 4 or 5, wherein determining whether a protein or a fragment thereof is cross-presented by antigen-presenting cells is determining whether the protein or the nucleic acid encoding it, or the protein fragment, is localized in the cytosol and / or exosome in vivo, or is abundant therein. [Embodiment 7] The method according to any one of Embodiments 4 to 6, wherein the localization or enrichment of a protein or nucleic acid encoding it, or a protein fragment, in the cytosol and / or exosome indicates that the protein or its fragment is cross-presented by antigen-presenting cells. [Embodiment 8] The method according to any one of Embodiments 4 to 7, wherein determining whether a protein or a fragment thereof is cross-presented by antigen-presenting cells is equivalent to determining an existing antibody response to the protein or a fragment thereof. [Embodiment 9] The method according to any one of Embodiments 4 to 8, wherein an existing antibody response to a protein or fragment thereof indicates that the protein or fragment is cross-presented by antigen-presenting cells. [Embodiment 10] <0\000929> The method according to any one of Embodiments 4 to 9, comprising determining whether a protein or fragment thereof is cross-presented by antigen-presenting cells, and determining whether a protein or fragment thereof binds to F-actin. [Embodiment 11] The method according to any one of embodiments 4 to 10, wherein the binding of a protein or its fragment to F-actin indicates that the protein or its fragment is cross-presented by an antigen-presenting cell. [Embodiment 12] The method according to any one of embodiments 4 to 11, wherein determining whether a protein or fragment thereof is cross-presented by antigen-presenting cells is equivalent to determining whether a protein or fragment thereof binds to RNA. [Embodiment 13] The method according to any one of embodiments 4 to 12, wherein the binding of a protein or its fragment to RNA indicates that the protein or its fragment is cross-presented by an antigen-presenting cell. [Embodiment 14] The method according to any one of embodiments 2, 3, 6 to 13, wherein the protein fragment is present as an MHC peptide complex within the exosome, preferably on the surface of the exosome. [Embodiment 15] The method according to any one of Embodiments 2, 3, 6 to 13, wherein the localization or enrichment of a protein or nucleic acid encoding it, or a protein fragment in the cytosol, preferably indicates protein processing and presentation in the MHC I pathway of diseased cells. [Embodiment 16] The method according to Embodiment 15, wherein protein processing and presentation in the MHC I pathway lead to recognition by CD8+ T cells of a complex formed by MHC I and protein fragments. [Embodiment 17] The method according to any one of Embodiments 2, 3, 6 to 13, wherein the localization or enrichment of a protein or nucleic acid encoding it, or a protein fragment within an exosome indicates the accumulation of a protein or nucleic acid encoding it, or a protein fragment in an antigen-presenting cell, preferably a professional antigen-presenting cell. [Embodiment 18] The method according to Embodiment 17, wherein the accumulation of a protein or nucleic acid encoding it, or a protein fragment, in antigen-presenting cells preferably indicates protein processing and presentation in the MHC I and / or MHC II pathways of antigen-presenting cells. [Embodiment 19] The method according to Embodiment 18, wherein protein processing and presentation in the MHC I pathway lead to recognition by CD8+ cells of a complex formed by MHC I and protein fragments. [Embodiment 20] A method for predicting the usefulness of a protein or fragment thereof expressed by diseased cells for immunotherapy, the following: (a) Confirming existing antibody responses to proteins or their fragments. (b) Determine whether the protein or its fragments bind to F-actin, and / or (c) Determine whether the protein or its fragments bind to RNA. A method that includes verifying one or more of the following. [Embodiment 21] The method according to Embodiment 20, wherein an existing antibody response to a protein or its fragment indicates that the protein or its fragment is useful for immunotherapy. [Embodiment 22] The method according to Embodiment 20 or 21, wherein the binding of a protein or its fragment to F-actin indicates that the protein or its fragment is useful for immunotherapy. [Embodiment 23] The method according to any one of embodiments 20 to 22, wherein the binding of a protein or fragment thereof to RNA demonstrates that the protein or fragment thereof is useful for immunotherapy. [Embodiment 24] The method according to any one of Embodiments 1 to 23, wherein the protein or fragment thereof comprises a disease-specific amino acid modification. [Embodiment 25] The method according to embodiment 24, wherein the amino acid modification is the result of disease-specific somatic cell mutations. [Embodiment 26] The method according to any one of Embodiments 1 to 25, wherein the disease is cancer and the immunotherapy is anti-cancer immunotherapy. [Embodiment 27] The method according to any one of embodiments 1 to 26, wherein the protein fragment is an MHC-binding peptide or a latent MHC-binding peptide. [Embodiment 28] A method for selecting and / or rating disease-specific amino acid modifications for their usefulness in immunotherapy: (i) A step of identifying proteins expressed by diseased cells, each containing at least one disease-specific amino acid modification, and (ii) A step of confirming the distribution or localization of the protein identified in (i) or the nucleic acid encoding it, or the protein fragment, and (iii) Repeat step (ii) for at least one additional protein identified in (i). A method that includes this. [Embodiment 29] The method according to Embodiment 28, wherein step (ii) is to determine whether a protein or a nucleic acid encoding it, or a protein fragment, is localized or abundant in the cytosol and / or exosome in vivo. [Embodiment 30] The method according to Embodiment 28 or 29, wherein the localization or enrichment of proteins or nucleic acids encoding them, or protein fragments in the cytosol and / or exosomes, demonstrates that disease-specific amino acid modifications are useful for immunotherapy. [Embodiment 31] A method for selecting and / or rating disease-specific amino acid modifications for their usefulness in immunotherapy: (i) A step of identifying proteins expressed by diseased cells, each containing at least one disease-specific amino acid modification, and (ii) A step to determine whether the protein or protein fragment identified in (i) is cross-presented by antigen-presenting cells, preferably professional antigen-presenting cells, and (iii) Repeat step (ii) for at least one additional protein identified in (i). A method that includes this. [Embodiment 32] The method according to embodiment 31, which demonstrates that cross-presentation of proteins or their fragments by antigen-presenting cells is useful for disease-specific amino acid modification for immunotherapy. [Embodiment 33] A method for selecting and / or rating disease-specific amino acid modifications for their usefulness in immunotherapy: (i) A step of identifying proteins expressed by diseased cells, each containing at least one disease-specific amino acid modification, and (ii) For the protein or protein fragment identified in (i), the following: (a) Confirm existing antibody responses to the protein or its fragments. (b) Determine whether the protein or its fragments bind to F-actin, and / or (c) Determine whether the protein or its fragments bind to RNA. To verify one or more of the following, (iii) Repeat step (ii) for at least one additional protein identified in (i). A method that includes this. [Embodiment 34] The method according to Embodiment 33, wherein an existing antibody response to a protein or fragment thereof demonstrates that disease-specific amino acid modification is useful for immunotherapy. [Embodiment 35] The method according to Embodiment 33 or 34, wherein the binding of a protein or fragment thereof to F-actin demonstrates that disease-specific amino acid modification is useful for immunotherapy. [Embodiment 36] The method according to any one of embodiments 33 to 35, wherein the binding of a protein or fragment thereof to RNA demonstrates that disease-specific amino acid modification is useful for immunotherapy. [Embodiment 37] The method according to any one of embodiments 28 to 36, wherein the disease-specific amino acid modification is contained in a protein fragment that is an MHC-binding peptide or a potential MHC-binding peptide. [Embodiment 38] A method according to any one of embodiments 1 to 37, used for the manufacture of a vaccine. [Embodiment 39] The method according to Embodiment 38, wherein the vaccine is derived from one or more proteins or fragments thereof, or one or more disease-specific amino acid modifications, that are predicted to be useful for immunotherapy. [Embodiment 40] A method of providing vaccines: A step of identifying one or more proteins or fragments thereof, or one or more disease-specific amino acid modifications, that are predicted to be useful for immunotherapy according to any one of Embodiments 1 to 39. A method that includes this. [Embodiment 41] A process to provide a vaccine comprising one or more proteins or fragments thereof that are predicted to be useful for immunotherapy, or a peptide or polypeptide containing one or more disease-specific amino acid modifications, or a nucleic acid encoding a peptide or polypeptide. The method according to embodiment 40, further including the method described in embodiment 40. [Embodiment 42] A vaccine manufactured by the method described in any one of embodiments 38 to 41. <Sequence Listing> SEQUENCE LISTING <110> BioNTech RNA Pharmaceuticals GmbH et al. <120> METHODS FOR PREDICTING THE USEFULNESS OF PROTEINS OR PROTEIN FRAGMENTS FOR IMMUNOTHERAPY <130> 674-157 PCT2 <150> PCT / EP2016 / 060897 <151> 2016-05-13 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <220> <221> REPEAT <222> (1)..(3) <223> Portion of sequence repeated a times, wherein a is independently a number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 <220> <221> MISC_FEATURE <222> (1)..(15) <223> a + b + c + d + e are different from 0 and preferably are 2 or more, 3 or more, 4 or more or 5 or more <220> <221> REPEAT <222> (4)..(6) <223> Portion of sequence repeated b times, wherein b is independently a number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 <220> <221> REPEAT <222> (7)..(9) <223> Portion of sequence repeated c times, wherein c is independently a number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 <220> <221> REPEAT <222> (10)..(12) <223> Portion of sequence repeated d times, wherein d is independently a number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 <220> <221> REPEAT <222> (13)..(15) <223> Portion of sequence repeated e times, wherein e is independently a number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 <400> 1 Gly Gly Ser Gly Ser Ser Gly Gly Gly Ser Ser Gly Gly Ser Gly 1 5 10 15 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <400> 2 Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5

Claims

1. A method for predicting the usefulness of neoantigens or their neoepitopes, based on mutations expressed by cancer cells, for immunotherapy, (i) To determine whether neoantigens or their neoepitopes based on mutations expressed by cancer cells are localized or abundant in the cytosol and / or exosomes of cancer cells in vivo, and (ii) Determining whether the neoantigen or its neoepitope is cross-presented by antigen-presenting cells, A method demonstrating that in vivo localization or enrichment of a neoantigen or its neoepitope in the cytosol and / or exosome, and cross-presentation of the neoantigen or its neoepitope by antigen-presenting cells, indicates processing and presentation of the neoantigen in the MHC I pathway, and that the neoantigen or its neoepitope is useful for immunotherapy.

2. The method according to claim 1, wherein the localization or enrichment of a neoantigen or its neoepitope in the cytosol and / or exosome indicates that the neoantigen or its neoepitope is cross-presented by antigen-presenting cells.

3. The method according to claim 1 or 2, wherein determining whether a neoantigen or its neoepitope is cross-presented by antigen-presenting cells is a means of determining an existing antibody response to the neoantigen or its neoepitope.

4. The method according to any one of claims 1 to 3, wherein an existing antibody response to a neoantigen or its neoepitope indicates that the neoantigen or its neoepitope is cross-presented by antigen-presenting cells.

5. The method according to any one of claims 1 to 4, wherein the neoepitope is contained within the exosome as an MHC peptide complex.

6. The processing and presentation of neoantigens in the MHC I pathway is carried out by the CD8 complex formed by the MHC I and the neoepitope of the neoantigen. ＋ The method according to any one of claims 1 to 5, which causes recognition by T cells.

7. The method according to any one of claims 1 to 6, wherein the localization or enrichment of a neoantigen or neoepitope within an exosome indicates the accumulation of a neoantigen or a neoepitope of a neoantigen in a professional antigen-presenting cell.

8. The method according to claim 7, wherein the accumulation of neoantigens or neoepitopes of neoantigens in professional antigen-presenting cells indicates the processing and presentation of neoantigens in the MHC I and / or MHC II pathways of professional antigen-presenting cells.

9. The method according to any one of claims 1 to 8, wherein the neoantigen or its neoepitope comprises cancer-specific amino acid modifications.

10. The method according to claim 9, wherein the amino acid modification is the result of a cancer-specific somatic cell mutation.

11. The method according to any one of claims 1 to 10, wherein the immunotherapy is anti-cancer immunotherapy.

12. The method according to any one of claims 1 to 11, wherein the neoepitope is an MHC-binding peptide or a latent MHC-binding peptide.

13. A method for selecting and / or rating cancer-specific amino acid modifications based on their usefulness in immunotherapy: (i) A step of identifying neoantigens expressed by cancer cells, each based on a mutation containing at least one cancer-specific amino acid modification, and (ii) The neoantigen or neoepitope of the neoantigen based on the mutation identified in (i) (a) whether it is localized or abundant in the cytosol and / or exosomes of cancer cells in vivo, and (b) Whether or not it is cross-presented by professional antigen-presenting cells, The process of determining, (iii) A step in which step (ii) is repeated for at least one further neoantigen identified in (i). Includes, The in vivo localization or enrichment of neoantigens or their neoepitopes in the cytosol and / or exosomes, and the cross-presentation of neoantigens or their neoepitopes by antigen-presenting cells, demonstrate the processing and presentation of neoantigens in the MHC I pathway and indicate that cancer-specific amino acid modifications are useful for immunotherapy. method.

14. The method according to claim 13, wherein the cancer-specific amino acid modification is contained in a neoepitope that is an MHC-binding peptide or a latent MHC-binding peptide.

15. A manufacturing method comprising the method according to claim 13 or 14.

16. The method according to claim 15, wherein the vaccine is derived from one or more neoantigens or neoepitopes thereof, or one or more cancer-specific amino acid modifications, which are predicted to be useful for immunotherapy.

17. Methods of providing vaccines: A step of identifying one or more mutation-based neoantigens or neoepitopes thereof, or one or more cancer-specific amino acid modifications, that are predicted to be useful for immunotherapy according to any one of claims 1 to 16. A method that includes this.

18. A process to provide a vaccine comprising a neoantigen or neoepitope based on one or more mutations predicted to be useful for immunotherapy, or a peptide or polypeptide containing one or more cancer-specific amino acid modifications, or a nucleic acid encoding a peptide or polypeptide. The method according to claim 17, further comprising:

19. A method for producing a vaccine, comprising the method according to any one of claims 15 to 18.

20. (i) A peptide or polypeptide comprising a neoantigen or its neoepitope based on one or more mutations, Each of the mutation-based neoantigens or their neoepitopes is identical to a fragment of the neoantigen or neoepitope expressed by cancer cells, or has an amino acid sequence consisting of the said fragment. The corresponding neoantigen or neoepitope expressed by cancer cells is localized or enriched in vivo within the cytosol and / or exosomes of cancer cells. Here, each neoantigen or its neoepitope, (a) Includes cancer-specific amino acid modifications resulting from cancer-specific somatic mutations, (b) A peptide that binds to an MHC class I molecule, and (c) It can be cross-presented by antigen-presenting cells, and as a result is processed and presented via the MHC class I pathway, and the complex formed by MHC class I and the neoepitope is recognized by CD8+ T cells. Peptides or polypeptides, or (ii) Nucleic acids that comprise a peptide or polypeptide A vaccine containing [this ingredient].

21. A vaccine for use in methods for treating cancer, The cancer contains one or more neoantigens or neoepitopes containing one or more cancer-specific amino acid modifications, Vaccines, (i) A peptide or polypeptide comprising a neoantigen or its neoepitope based on one or more mutations expressed by cancer cells, or one or more cancer-specific amino acid modifications, Each of the mutation-based neoantigens or their neoepitopes is identical to a fragment of the neoantigen or neoepitope expressed by cancer cells, or has an amino acid sequence consisting of the said fragment. The corresponding neoantigen or neoepitope expressed by cancer cells is localized or enriched in vivo within the cytosol and / or exosomes of cancer cells. Each neoantigen or its neoepitope (a) Includes cancer-specific amino acid modifications resulting from cancer-specific somatic mutations, (b) A peptide that binds to an MHC class I molecule, and (c) A peptide or polypeptide that can be cross-presented by antigen-presenting cells, which is then processed and presented via the MHC class I pathway, and which induces recognition by CD8+ T cells of a complex formed by MHC class I and a neoepitope. (ii) Nucleic acids that comprise a peptide or polypeptide Includes, The localization or enrichment of neoantigens or their neoepitopes expressed by cancer cells in the cytosol and / or exosome indicates the processing and presentation of neoantigens via the MHC class I pathway, and Processing and presentation of neoantigens in the MHC class I pathway leads to recognition by CD8+ T cells of a complex formed by the neoepitope of MHC class I and the neoantigen. vaccine.

22. A computer-based analytical process for analyzing and selecting modified peptides for use in vaccines, The process is Determining a modified peptide that is expected to be useful for immunotherapy by the method described in any one of claims 1 to 16, and / or To rate modified peptides according to their predicted usefulness for immunotherapy by the method described in any one of claims 1 to 16. A computer-based analysis process, including...

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